Sarah S. Bernards

Inherited Mutations in Women With Ovarian Carcinoma

IMPORTANCE Germline mutations in BRCA1 and BRCA2 are relatively common in women with ovarian, fallopian tube, and peritoneal carcinoma (OC) causing a greatly increased lifetime risk of these cancers, but the frequency and relevance of inherited mutations in other genes is less well characterized. OBJECTIVE To determine the frequency and importance of germline mutations in cancer-associated genes in OC. DESIGN, SETTING, AND PARTICIPANTS A study population of 1915 woman with OC and available germline DNA were identified from the University of Washington (UW) gynecologic tissue bank (n = 570) and from Gynecologic Oncology Group (GOG) phase III clinical trials 218 (n = 788) and 262 (n = 557). Patients were enrolled at diagnosis and were not selected for age or family history. Germline DNA was sequenced from women with OC using a targeted capture and multiplex sequencing assay. MAIN OUTCOMES AND MEASURES Mutation frequencies in OC were compared with the National Heart, Lung, and Blood Institute GO Exome Sequencing Project (ESP) and the Exome Aggregation Consortium (ExAC). Clinical characteristics and survival were assessed by mutation status. RESULTS Overall, the median (range) age at diagnosis was 60 (28-91) years in patients recruited from UW and 61 (23-87) years in patients recruited from the GOG trials. A higher number of black women were recruited from the GOG trials (4.3% vs 1.4%; P = .009); but in patients recruited from UW, there was a higher proportion of fallopian tube carcinomas (13.3% vs 5.7%; P < .001); stage I and II disease (14.6% vs 0% [GOG trials were restricted to advanced-stage cancer]); and nonserous carcinomas (29.9% vs 13.1%, P < .001). Of 1915 patients, 280 (15%) had mutations in BRCA1 (n = 182), or BRCA2 (n = 98), and 8 (0.4%) had mutations in DNA mismatch repair genes. Mutations in BRIP1 (n = 26), RAD51C (n = 11), RAD51D (n = 11), PALB2 (n = 12), and BARD1 (n = 4) were significantly more common in patients with OC than in the ESP or ExAC, present in 3.3%. Race, histologic subtype, and disease site were not predictive of mutation frequency. Patients with a BRCA2 mutation from the GOG trials had longer progression-free survival (hazard ratio [HR], 0.60; 95% CI, 0.45-0.79; P < .001) and overall survival (HR, 0.39; 95% CI, 0.25-0.60; P < .001) compared with those without mutations. CONCLUSIONS AND RELEVANCE Of 1915 patients with OC, 347 (18%) carried pathogenic germline mutations in genes associated with OC risk. PALB2 and BARD1 are suspected OC genes and together with established OC genes (BRCA1, BRCA2, BRIP1, RAD51C, RAD51D, MSH2, MLH1, PMS2, and MSH6) bring the total number of genes suspected to cause hereditary OC to 11.

Mutations in homologous recombination genes and outcomes in ovarian carcinoma patients in GOG 218: An NRG oncology/Gynecologic oncology group study

Purpose: We hypothesized that mutations in homologous recombination repair (HRR) genes beyond BRCA1 and BRCA2 improve outcomes for ovarian carcinoma patients treated with platinum therapy and would impact the relative benefit of adding prolonged bevacizumab. Experimental Design: We sequenced DNA from blood and/or neoplasm from 1,195 women enrolled in GOG-0218, a randomized phase III trial in advanced ovarian carcinoma of bevacizumab added to carboplatin and paclitaxel. Defects in HRR were defined as damaging mutations in 16 genes. Proportional hazards models were used to estimate relative hazards for progression-free survival (PFS) and overall survival (OS). Results: Of 1,195 women with ovarian carcinoma, HRR mutations were identified in 307 (25.7%). Adjusted hazards for progression and death compared with those without mutations were lower for women with non-BRCA HRR mutations [HR = 0.73; 95% confidence interval (CI), 0.57–0.94; P = 0.01 for PFS; HR = 0.67; 95% CI, 0.50–0.90; P = 0.007 for OS] and BRCA1 mutations (HR = 0.80; 95% CI, 0.66–0.97; P = 0.02 for PFS; HR = 0.74; 95% CI, 0.59–0.94; P = 0.01 for OS) and were lowest for BRCA2 mutations (HR = 0.52; 95% CI, 0.40–0.67; P < 0.0001 for PFS; HR = 0.36; 95% CI, 0.25–0.53; P < 0.0001 for OS). A test of interaction showed no difference in the effect of bevacizumab on PFS between cases with and without mutations. Conclusions: HRR mutations, including non-BRCA genes, significantly prolong PFS and OS in ovarian carcinoma and should be stratified for in clinical trials. The benefit of adding bevacizumab was not significantly modified by mutation status. Clin Cancer Res; 24(4); 777–83. ©2017 AACR.

Clinical characteristics and outcomes of patients with BRCA1 or RAD51C methylated versus mutated ovarian carcinoma

OBJECTIVE In ovarian carcinoma, mutations in homologous recombination DNA repair (HRR) genes, including BRCA1 and RAD51C, are associated with increased survival and specific clinical features. Promoter hypermethylation is another mechanism of reducing gene expression. We assessed whether BRCA1 and RAD51C promoter hypermethylation is associated with similar survival and clinical characteristics. METHODS Promoter methylation of BRCA1 and RAD51C was evaluated using methylation-sensitive PCR in 332 primary ovarian carcinomas. Damaging germline and somatic mutations in 16 HRR genes were identified using BROCA sequencing. RESULTS BRCA1 methylation was detected in 22 carcinomas (6.6%) and RAD51C methylation in 9 carcinomas (2.7%). These small numbers limited the power to detect differences in survival and platinum sensitivity. Mutations in one or more HRR genes were found in 95 carcinomas (29%). Methylation of BRCA1 or RAD51C was mutually exclusive with mutations in these genes (P=0.001). Patients whose carcinomas had BRCA1 methylation (57.7years±2.5) or BRCA1 mutations (54.1years±1.4) were younger than those without (63.3years±0.8; P=0.029, P<0.0001). BRCA1 methylation and germline BRCA1 mutation were associated with high grade serous (HGS) histology (P=0.045, P=0.001). BRCA1 mutations were associated with increased sensitivity to platinum chemotherapy while BRCA1 methylation was not (P=0.034, P=0.803). Unlike HRR mutations, methylation was not associated with improved overall survival compared to cases without methylation or mutation. CONCLUSIONS Patients with BRCA1-methylated carcinomas share clinical characteristics with patients with BRCA1-mutated carcinomas including younger age and predominantly HGS histology. However, unlike mutation, RAD51C and BRCA1 methylation were not associated with improved survival or greater sensitivity to platinum chemotherapy.

Genetic characterization of early onset ovarian carcinoma

OBJECTIVE Ovarian carcinoma (OC) is rare in young women and the fraction of early onset OC attributable to inherited mutations in known OC genes is uncertain. We sought to characterize the fraction of OC that is heritable in women diagnosed with ovarian, fallopian tube, or peritoneal carcinoma at forty years of age or younger. METHODS We sequenced germline DNA from forty-seven women diagnosed with OC at age 40 or younger ascertained through a gynecologic oncology tissue bank or referred from outside providers using BROCA, a targeted capture and massively parallel sequencing platform that can detect all mutation classes. We evaluated 11 genes associated with ovarian carcinoma (BARD1, BRCA1, BRCA2, BRIP1, MLH1, MSH2, MSH6, PALB2, PMS2, RAD51D, and RAD51C) and additional candidate genes in DNA repair (ATM, BAP1, CHEK2, MRE11A, NBN, PTEN, TP53). We counted only clearly damaging mutations. RESULTS Damaging mutations in OC genes were identified in 13 of 47 (28%) subjects, of which 10 (77%) occurred in BRCA1 and one each occurred in BRCA2, MSH2, and RAD51D. Women with a strong family history were no more likely to have an OC gene mutation (8/17, 47%) than those without a strong family history (9/30, 30%, P=0.35). Additionally, damaging mutations in non-OC genes were identified, one in NBN and one in CHEK2. CONCLUSIONS A high proportion of young women with invasive OC have mutations in BRCA1, and a smaller fraction have mutations in other known OC genes. Family history was not associated with mutation status in these early onset cases.

Abstract AS09: Germline mutations in cancer susceptibility genes in BRCA1 and BRCA2 negative families with ovarian and breast cancer

Abstracts: 10th Biennial Ovarian Cancer Research Symposium; September 8-9, 2014; Seattle, WA Objectives: Germline mutations in cancer susceptibility genes other than BRCA1 and BRCA2 ( BRCA1/2 ) are found in approximately 6% of women with ovarian, fallopian tube, or primary peritoneal cancer. Our objective was to sequence BRCA1/2 -negative ovarian cancer patients with a family history of ovarian or breast cancer to identify inherited mutations that may explain the familial risk. Methods: We used a targeted capture, massively parallel sequencing test called BROCA on ovarian cancer probands with a family history of ovarian or breast cancer, or a personal history of breast cancer. BROCA testing included all known breast and ovarian cancer genes. Only clear loss of function mutations were included. 118 probands were ascertained from a gynecologic oncology tissue bank or outside referrals and provided informed consent. A family history of ovarian cancer was defined as having a first or second degree relative with ovarian cancer. A family history of breast cancer was defined as having a first or second degree relative with pre-menopausal breast cancer, or 2 or more regardless of menopausal status. Subjects were only included in one category. Results: Of 118 ovarian cancer probands, 22 (18.6%) were found to carry deleterious mutations in non- BRCA1/2 cancer susceptibility genes. 8/29 (27.6%) ovarian cancer patients with a personal history of breast cancer had mutations in 7 genes (2 CHEK2 , 2 RAD51D , 1 BRIP1 , 1 TP53 , 1 ATM , and one with both PALB2 and PMS2 ). This included mutations found in 2/5 (40%) who also had a family history of ovarian cancer and 4/10 (40%) who also had a family history of breast cancer. 38 patients had a family history of ovarian cancer with no personal history of breast cancer; 9/38 (23.7%) had mutations in 5 genes (3 BRIP1 , 3 RAD51C , 1 RAD51D , 1 TP53 , and 1 ATM ). Finally, 5/51 (9.8%) ovarian cancer patients with a family history of breast cancer and no personal history of breast cancer had mutations in 5 genes (1 MSH6 , 1 FAM175A , 1 NBN , 1 PALB2 , and 1 CHEK2 ). Conclusions: Germline mutations in DNA-repair genes are present in a substantial fraction of BRCA1/ 2-negative ovarian cancer patients with a personal or family history suggestive of inherited disease. These women may benefit from multiplex gene testing. The detection of inherited mutations in these women may be useful to identify the risk of other cancers, to inform family members of possible risk, and to direct therapy by suggesting candidates for PARP inhibitor therapy. Citation Format: B. Norquist, M. Harrell, T. Walsh, J. Mandell, S. Bernards, K. Agnew, M. Lee, K. Pennington, M.C. King, E. Swisher. Germline mutations in cancer susceptibility genes in BRCA1 and BRCA2 negative families with ovarian and breast cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS09.

Abstract A12: Mutations in homologous recombination genes and response to treatment in GOG 218: An NRG Oncology Study.

Objective: Bevacizumab is an anti-vascular endothelial growth factor monoclonal antibody with activity in ovarian cancer. GOG 218 was a phase III, randomized trial in advanced primary ovarian, fallopian tube, and peritoneal carcinoma (collectively, OC), examining the role of adding bevacizumab to a regimen of q21 day carboplatin and paclitaxel. Our objective was to examine whether mutations in homologous recombination (HR) genes affect response to treatment. Methods: We sequenced germline (from blood) and/or somatic (from neoplastic tissue) DNA from 1195 women enrolled in GOG 218 using the targeted capture and multiplex sequencing assay BROCA-HR, focusing on 16 DNA repair genes. Defects in HR were defined as germline or somatic mutations in genes predicted to affect HR, including BRCA1, BRCA2, and others. Proportional hazards models were used to provide estimates of relative hazards for progression free survival (PFS) and overall survival (OS) adjusted for clinical characteristics. Results: Of 1195 women with OC, germline or somatic mutations were identified in 147 (12.3%) in BRCA1, 78 (6.5%) in BRCA2, and 81 (6.8%) in other, non-BRCA HR genes. Total mutation frequency (all genes combined) in those with high-grade serous histology was 26.9% (261/971), but this was not significantly higher than the mutation frequency in endometrioid histology (10/42, 23.8%), clear cell histology (6/28, 21.4%), or unspecified carcinoma (20/90, 22.2%). Mutation frequency also did not differ by disease site. Median PFS and OS by group were as follows: BRCA1: 15.6 and 53.7 months, BRCA2: 21.6 and 75.2 months, non-BRCA HR: 16 and 56 months, and no mutation: 12.6 and 42 months. Adjusting for study treatment, stage, residual disease, and initial performance status, hazards for progression and death compared to those without mutations were significantly lower for those with mutations, specifically with BRCA1 mutations (hazard ratio (HR) 0.80, 95% CI 0.66 – 0.97, p=0.02 for PFS; HR 0.75, 95% CI 0.60 – 0.95, p=0.02 for OS), with BRCA2 mutations (HR 0.52, 95% CI 0.40 – 0.67, p Conclusions: Women with OC with either germline or somatic mutations affecting HR have significantly longer PFS and OS than those without mutations. Disease site and histology were not predictive of mutation status. The effect of mutation status on response by treatment arm will be reported at presentation. Citation Format: Barbara M. Norquist, Mark F. Brady, Maria I. Harrell, Tom D. Walsh, Ming K. Lee, Suleyman Gulsuner, Sarah S. Bernards, Silvia Casadei, Robert A. Burger, Susan A. Davidson, Robert S. Mannel, Paul A. DiSilvestro, Heather A. Lankes, Nilsa C. Ramirez, Mary Claire King, Michael J. Birrer, Elizabeth M. Swisher. Mutations in homologous recombination genes and response to treatment in GOG 218: An NRG Oncology Study. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A12.

Abstract POSTER-CTRL-1206: The mutational landscape of fallopian tube carcinoma

Abstracts: 10th Biennial Ovarian Cancer Research Symposium; September 8-9, 2014; Seattle, WA Fallopian tube (FT) carcinoma is a rare gynecological malignancy, whose incidence is most likely underestimated due to misdiagnoses as ovarian cancer. Recent advances in pathology with more thorough serial sectioning techniques have led to better identification. We undertook to define germline and somatic mutations in 98 FT carcinoma cases enrolled at diagnosis in IRB-approved studies at the University of Washington, excluding cases with carcinoma identified at the time of risk-reducing surgery. We sequenced DNA from blood and neoplastic tissue using BROCA, a targeted capture and massively parallel genomic sequencing approach that detects all classes of mutation in 64 genes. The fraction of all primary ovarian, peritoneal and FT carcinoma defined as a FT primary increased over time in our tissue bank: FT diagnoses accounted for 23% of cases in the past five years compared to 5% in the previous 10 years (p=0.002). Of 98 subjects with FT carcinoma, 32 (33%) had loss of function germline mutations in 9 genes. Mutations in BRCA1 were identified in 18 (19%), making up the largest number of germline mutations, and in BRCA2 in 4 (4%). Germline mutations were also identified in 6 other known breast and/or ovarian cancer susceptibility genes including 2 in BRIP1 (2%), 2 in CHECK2 (2%), and 1 each in BARD1, BLM, MRE11A , and ATM (1%). One mutation was found in the FA gene FANCL . Additionally, we found 1 mosaic mutations in TP53 (1%) and 2 in PPM1D (2%). Together germline and mosaic mutations were identified in 34 (35%) of cases. We sequenced neoplastic DNA with BROCA in 30 FT carcinomas and found 27 somatic mutations in TP53 (90%), 1 in PTEN (3%) and 1 in CHEK2 (3%). The germline mutation rate was higher in FT carcinoma compared to our previous studies in ovarian carcinoma (35% versus 22%), though the identified genes were similar. Recently identified ovarian cancer susceptibility genes such as BRIP1, BARD1 , and PPM1D are also associated with hereditary fallopian tube cancer. Known breast cancer genes (i.e. CHEK2, MRE11A, ATM ) and other cancer associated genes ( BLM, FANCL ) require further study to prove their association with FT or ovarian cancer risk. An increased pathological recognition of the FT as a primary site of disease is changing the relative distribution of FT, ovarian, and peritoneal carcinoma in some centers. Citation Format: M.I. Harrell, T. Walshb, K.J. Agnew, B. Norquist, M.K. Lee, S. Bernards, K.P. Pennington, E.M. Swisher. The mutational landscape of fallopian tube carcinoma [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-CTRL-1206.