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.

Ovarian carcinoma (OC) remains the deadliest gynecologic malignancy.¹ Identifying genetic predisposition offers opportunities for cancer prevention. According to previous studies,^2-4 13% to 18% of OC is associated with germline mutations in BRCA1 and BRCA2. The lifetime risk of developing OC for a woman with a BRCA2 mutation is approximately 20% and approximately 50% for a BRCA1 mutation,⁵ and risk-reducing salpingo-oophorectomy has been shown to significantly reduce the risk of OC and all-cause mortality.^6,7 Other genes from the BRCA-Fanconi anemia pathway such as BRIP1, RAD51C, and RAD51D have been implicated in hereditary OC.^8-13 We previously reported the frequency of these mutations in a small set of unselected patients with OC (n = 360).⁴ Several publications^12-14 reporting on a recent series of unselected OC found lower rates of mutations in all OC genes, perhaps secondary to different sequencing methods. Mutations in other genes within this pathway (PALB2, BARD1, NBN, and CHEK2, among others) have been identified in patients with OC, but it is unknown if these mutations confer an elevated risk of OC.⁴

We sought to determine the frequency of mutations in known or suspected OC susceptibility genes in a large unselected group of patients with ovarian, peritoneal, and fallopian tube carcinoma (collectively, OC) using a comprehensive targeted sequencing method.

Box Section Ref ID

Patients with OC were identified from 3 sources: (1) patients undergoing primary treatment at the University of Washington (UW) Medical Center, and (2) patients who consented for translational research with available DNA from Gynecologic Oncology Group (GOG) protocol 218, and (3) GOG protocol 262. Patients were enrolled at diagnosis and were not selected for age or family history. Pathology was centrally reviewed by gynecologic pathologists, and unsure cases were resolved by consensus. GOG-218 and GOG-262 were large randomized phase III trials studying primary advanced-stage OC. A subset of the UW patients has been previously described.⁴ All patients provided written informed consent on protocols approved by an institutional review board.

Germline DNA extracted from blood was sequenced using BROCA, a targeted capture, massively parallel sequencing test developed at the University of Washington.¹⁵ For this study, we sequenced ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, FAM175A, FANCP, MLH1, MSH2, MSH6, MRE11A, NBN, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, and TP53. Sequencing reads were aligned to the human reference genome (hg19). Variants were identified using GATK37 and Pindel after indel realignment and base quality recalibration. Variants including copy number variations (CNVs) were detected as previously described.^15-17 Missense mutations were only included if proven to be damaging (eg, BRCA1 C61G¹⁸). TP53 missense mutations were classified as deleterious based on available functional data as per the International Agency for Research on Cancer.¹⁹

Two publically available, overlapping, online exome sequencing data sets were used to estimate population mutation frequencies: the European American (EA) data set from the National, Heart, Lung, and Blood Institute Exome Sequencing Project (ESP)²⁰ and the Exome Aggregation Consortium (ExAC).²¹ To account for inaccuracy in indel calling in the ESP, we visually inspected the read data for all coding indel calls in the ESP data set for the genes of interest. Calls that were low quality (<17 Phred score) and low coverage (<5) were excluded. The EA group was used because most OC subjects were white, and the total ESP population overrepresents African Americans. ExAC mutation frequencies were weighted to match the racial distribution in OC cases. Because these exome databases do not include CNVs, CNVs were removed from the OC frequencies for this comparison. Splice mutations were also excluded from OC and population mutation frequencies because they could not all be verified to be damaging.

Clinical information was collected as per the GOG 218 and 262 protocols or extracted from the medical records for UW patients. Race and ethnicity (self-reported in the GOG patients and as noted in the medical records for UW patients) were assessed to determine if they affected mutation rates.

Contingency tables were analyzed with χ² or Fisher exact tests. In GOG patients, progression-free survival (PFS) and overall survival (OS) were defined as the time between enrollment and progression²² or death, respectively. Proportional hazards models were used to provide estimates of relative hazards adjusted for clinical characteristics (further description, as well as the log files of the statistical analyses, are available in the Supplement). The Wald test was used to assess the null hypotheses of equal hazards. All P values are 2-sided.

Clinical characteristics are provided in Table 1. The median (range) age at diagnosis was 60 (28-91) years for UW patients and 61 (2387) years for GOG patients. There was an increased fraction of black women 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, 182 (9.5%) had mutations in BRCA1 and 98 (5.1%) had mutations in BRCA2. Of BRCA1 and BRCA2 mutations, 38 of 280 (13.6%) were in Ashkenazi Jewish founder mutations BRCA1 185delAG (c.68_69delAG) (n = 18) and 5382insC (c.5266dupC) (n = 14) and BRCA2 6174delT (c.5946delT) (n = 6). Overall, 16 of 182 (8.8%) BRCA1 mutations were large genomic duplications or deletions, also called copy number variants (CNVs). Eight of 1915 patients (0.4%) carried mutations in genes involved in mismatch repair (MMR), including 4 in PMS2, 3 in MSH6, and 1 in MLH1. There were no mutations found in MSH2. Mutation frequencies for BRCA1, BRCA2, and MMR did not differ by ascertainment. Median coverage was 219 fold.

Mutations in BRCA1, BRCA2, BRIP1, PALB2, RAD51C, RAD51D, and BARD1 were all significantly more common in women with OC compared with the ESP or ExAC (Table 2), and using these data, we categorized these genes together with the MMR genes as OC-associated genes. Other putative cancer-associated genes such as CHEK2, NBN, RAD50, FAM175A, and MRE11A were not more frequently mutated in women with OC. Deleterious mutations in ATM and TP53 were more frequent in OC patients when compared with ExAC, but this was not significant when compared with the ESP. Odds ratios for OC are presented in Table 2. There were no mutations found in PTEN or FANCP.

In total, 347 women with OC (18.1%) had 352 mutations in OC-associated genes: 280 (14.6%) in BRCA1 or BRCA2, 64 (3.3%) in another BRCA-Fanconi anemia OC-associated gene (BRIP1, PALB2, RAD51C, RAD51D, or BARD1), and 8 (0.4%) in an MMR gene. Five patients (1.4%) of 347 had more than 1 mutation. Individual mutations and associated clinical data for OC-associated genes other than BRCA1 and BRCA2 are provided in the eTable in the Supplement.

Ovarian cancer–associated mutations were separated into 5 categories: (1) BRCA1, (2) BRCA2, (3) other BRCA-Fanconi anemia OC–associated (BRIP1, PALB2, RAD51C, RAD51D, BARD1), (4) mismatch repair (MSH6, PMS2, MSH2, MLH1), and (5) and no mutation (either a mutation in a gene not clearly associated with OC or no mutation). Women with BRCA1 mutations had a median (range) age at OC onset of 52 (27-77) years, while the median (range) age at OC onset for those with no mutation was 62 (23-91) years (P < .001). The median (range) age of patients with a BRCA2 mutation was 59 (41-83) years; BRCA-Fanconi anemia OC–associated mutation, 60 (34-79) years; and MMR gene mutations, 50 (47-69) years. Table 3 summarizes clinical information by each OC-associated gene. In the 54 of 570 (9.5%) UW patients with a previous history of breast cancer, 28 of 54 (51.8%) had mutations in OC-associated genes, 17 (31.5%) in BRCA1, 9 (16.7%) in BRCA2, and 2 (3.7%) in other OC-associated genes (1, BRIP1 and 1, RAD51D).

Figure 1 summarizes mutation frequency by clinical characteristics. Disease site, race, and ethnicity were not associated with mutation frequency. Overall, 294 patients with high-grade (grade 2-3) serous carcinomas had OC-associated gene mutations, an overall mutation frequency of 19.6% and a combined BRCA1 and BRCA2 frequency of 16.1%. Patients with unspecified carcinomas, endometrioid, carcinosarcoma, and transitional cell histology had similar mutation rates to high-grade serous histology, both for all OC genes and for BRCA1 and BRCA2. Patients with clear cell histology had a lower overall mutation frequency compared with high-grade serous cases (8.6% vs 19.6%; P = .04), but differences in the BRCA1 and BRCA2 mutation frequency were not significant (6.9% vs 16.1%; P = .07). In patients with low-grade (grade 1) serous histology compared with high-grade serous cases, there were fewer mutations overall (5.7% vs 19.6%; P = .003) and for BRCA1 and BRCA2 (5.7% vs 16.1%; P = .02). In 16 women with mucinous histology, no OC-associated mutations were found.

In the GOG patients, hazard ratios (HRs) for progression and death were adjusted for protocol, study treatment, stage, residual disease, and initial performance status. The GOG patients were followed for 5 years. The median PFS for GOG patients without a BRCA2 mutation was 21.9 months, and the median PFS for those with no mutation was 14.2 months (HR, 0.60; 95% CI, 0.45-0.79; P < .001). The median OS for GOG patients with a BRCA2 mutation had not yet been reached, and the median OS for GOG patients with a BRCA2 mutation was 44 months (HR, 0.39; 95% CI, 0.25-0.60; P < .001) (Figure 2). The median OS in patients with a BRCA1 mutation was 56 months and was intermediate between patients with a BRCA2 mutation and women without mutations. Relative to patients without mutations, the HR for BRCA1 mutations was 0.75 (95% CI, 0.56-1.00; P = .05) (Figure 2B). In the UW group, OS for women with stage-III and stage-IV OC was longer in both patients with a BRCA2 mutation (median, 70 months; P = .004, log-rank) and BRCA1 mutation (median, 46 months; P = .04, log-rank) when compared with women without mutations (median, 36 months). Exploratory analyses of the individuals from the GOG studies suggest that compared with those without mutations, the PFS event rates for those individuals with BRCA1 (P = .009) or BRCA2 (P < .001) mutations are time-dependent (Figure 2A). Specifically, the PFS event rate is initially lower for those with BRCA1 or BRCA2 mutations but this advantage declines over time. There is a similar time-dependent decline in the relative death rate for those with BRCA1 mutations compared with those without mutations (P < .001) (Figure 2B). In both GOG (Figure 2) and UW patients (data not shown), survival was similar for women with mutations in BRCA1 and other OC-associated genes in the BRCA-Fanconi anemia pathway, but the analysis was limited due to low non-BRCA mutation frequency.

BRCA1 and BRCA2 are part of the BRCA-Fanconi anemia DNA repair pathway that controls DNA repair via homologous recombination.^23-25 It is plausible that damaging mutations in other genes in this pathway may also confer a risk of OC. Indeed, our data demonstrate that mutations in the BRCA-Fanconi anemia OC–associated genes BRIP1, RAD51C, and RAD51D together account for an additional 2.5% of unselected OC. Previous studies of high-risk families or specific founder mutations have suggested that damaging mutations in BRIP1, RAD51C, and RAD51D confer a relative risk increase for OC of 6 to 8 fold and an absolute lifetime risk of 10% to 15%.^8-11,26,27 Our data from unselected patients with OC confirm BRIP1, RAD51C, and RAD51D as important OC-associated genes. The Fanconi anemia gene BRIP1 (FANCJ), mutated in 1.4% of women with OC, is the next most commonly mutated gene in OC after BRCA1 and BRCA2.

In addition to identifying mutations in genes already implicated in OC, we identified mutations in the BRCA-Fanconi anemia genes PALB2 and BARD1 more frequently in women with OC compared with population frequencies in the ESP and ExAC (Table 2). PALB2 (FANCN) is a Fanconi anemia gene whose protein binds BRCA1 and BRCA2 at sites of DNA damage.²⁸ Mutations in PALB2 are associated with an elevated risk of breast cancer and have been identified in families with both breast cancer and OC^29-31 but have not been clearly associated with OC risk.^28,29 An analysis of germline mutations in high-grade serous OC from the Cancer Genome Atlas project identified PALB2 as the only gene other than BRCA1 and BRCA2 to be significantly more frequently mutated compared with a subset of ESP controls from the Women’s Health Initiative. The PALB2 mutation frequency in our larger series was significant compared with population rates and was associated with similar odds ratios for OC as RAD51C, RAD51D, and BRIP1 (Table 2). The corrected frequency of PALB2 mutations in the ESP is consistent with PALB2 mutation rates from other previously published control sets^30,32 where full sequencing of PALB2 was performed supporting the use of the ESP as a comparison population.

Three recent publications^12-14 by the Ovarian Cancer Association Consortium (OCAC) have examined rates of mutations in BRCA1, BRCA2, MMR genes, RAD51C, RAD51D, BRIP1, PALB2, and BARD1 compared with controls. The sequencing methodology reported by OCAC is different from our study. As the authors acknowledged, their sequencing methods resulted in relatively low coverage, were unable to detect genomic rearrangements, and were likely to lead to underestimation of mutation frequencies.¹² A recent OCAC study¹⁴ demonstrates a less sensitive sequencing method with a BRCA1 mutation rate of 3.8% that is significantly lower than our BRCA1 mutation rate of 9.5% (84 of 2222 vs 182 of 1915; P < .001, Fisher exact test) and lower than that reported in other population-based series.^2,3 Even if we exclude genomic rearrangements, which OCAC could not detect, our 8.7% BRCA1 mutation rate remains significantly higher (166 of 1915 vs 84 of 2222; P < .001, Fisher exact test). Despite a mutation rate for RAD51C and RAD51D that was approximately 30% lower than ours, OCAC identified an odds ratio similar to our calculation for these genes. OCAC did not find a difference in mutation frequency for PALB2 or BARD1 in OC cases vs controls,¹³ but their lower mutation rates in cases and an unexpectedly high PALB2 mutation rate in controls affected their power to detect such a difference.

BARD1 forms a heterodimer with BRCA1 mediated by their homologous ring finger motifs.³³ This heterodimerization is critical to several tumor suppressor functions of BRCA1, and mutations in BRCA1 that affect binding to BARD1 are associated with increased cancer risk.³⁴BARD1 mutations were rare in OC patients but significantly more common than in the general population, leading to wide confidence intervals for the generated odds ratio for OC (Table 2). However, the significant P value and the shared homology to BRCA1 support BARD1 as a rare OC susceptibility gene. These results are interpreted with some caution as 2 of the BARD1 mutation carriers also had mutations in BRCA1 (eTable in the Supplement). Mutations in BARD1 have also been identified in women with triple negative breast cancer, another phenotype associated with BRCA1 mutations.³⁵ Additional studies are needed to define the absolute risk of BARD1 for both breast cancer and OC. The addition of BARD1 and PALB2 to other known OC-associated genes (BRCA1, BRCA2, RAD51C, RAD51D, BRIP1, PALB2, MSH2, MLH1, MSH6, and PMS2) brings the total suspected hereditary OC genes to 11.

In contrast to PALB2 and BARD1, mutations in other genes in the BRCA-Fanconi anemia pathway previously implicated in breast cancer risk but not OC risk (CHEK2, NBN, RAD50, FAM175A, and MRE11A) were not more commonly mutated in patients with OC. Mutations in TP53 that causes early onset breast cancer and Li Fraumeni syndrome and mutations in ATM were slightly more frequent in OC than in population controls, which appeared significant when compared with the larger ExAC database. Given that mutations in all of these genes are rare, confidence intervals are wide, and we cannot fully exclude that some of these genes, in particular ATM and TP53, are associated with some risk for OC. However, a relatively high risk of OC is unlikely.

Mutations in MMR genes (MSH2, MLH1, MSH6, and PMS2), which cause Lynch syndrome are often cited as the other major cause of hereditary OC in addition to BRCA1 and BRCA2.³⁶ However, MMR mutations were infrequent (n = 8; 0.4%) in this series. Interestingly, 7 of 8 (88%) MMR mutations in women with OC occurred in PMS2 or MSH6, in contrast to patients with colon cancer, in which MSH2 and MLH1 mutations predominate.³⁷ Notably, 2 of 3 OCs with MSH6 mutations were endometrioid and low stage, but all 4 PMS2-mutated cases were advanced-stage high-grade serous carcinomas. Our data are consistent with a population-based study³⁸ of 1638 women with invasive OC who were sequenced for mutations in MLH1, MSH2, and MSH6, where 9 of 1638 (0.5%) had mutations (5 of 9 in MSH6 [PMS2 not assessed]).

The National Comprehensive Cancer Network recommends genetic testing for all women affected by OC, but some authors have proposed limiting BRCA1 and BRCA2 testing to high-grade serous OC and suggested that BRCA1 and BRCA2 mutations found in nonserous cases represent pathological misclassification.³⁹ In our study, all cases were centrally reviewed by a panel of gynecologic pathologists, minimizing pathological misclassification. The overall mutation rate for high-grade serous histology was not significantly different from the mutation frequency in undifferentiated carcinoma, endometrioid, or carcinosarcoma histologies. While clear cell and low-grade serous OC did have fewer mutations compared with high-grade serous OC, the mutation rates (5 of 58 patients [8.6%] and 4 of 70 patients [5.7%]) are probably still high enough to warrant genetic testing. Therefore, our data do not support the restriction of genetic testing to women with high-grade serous OC. Race and ethnicity also did not predict mutation status and should not be used to guide testing. Consistent with previous studies, OC survival was correlated with mutation status, with BRCA2 mutation carriers having the longest PFS and OS.^2,40 This study is unique in that cancer treatments were standardized within the confines of clinical trials, reducing potential bias in how mutation status affected survival. Mutation status should be considered when analyzing outcomes of OC clinical trials.

There are currently no published guidelines for managing unaffected women found to have mutations in BRIP1, PALB2, RAD51C, RAD51D, and BARD1. We found similar odds ratios for OC conferred by damaging mutations in these genes to the previously identified 6- to 8- fold relative risk in BRIP1, RAD51C, and RAD51D^8-11,26,27 and a similar age distribution of OC diagnosis for women with mutations in these genes compared with BRCA2 (Table 2) (Table 3). If a lifetime risk of 10% to 15% is confirmed for these genes, it would be reasonable to consider a risk-reducing salpingo-oophorectomy by age 45 years.

There are several limitations to this study. This was not a population-based study, and the GOG cases were limited to stage-III andstage-IV cancers. However, all patients were enrolled at the time of diagnosis, which reduces survival bias, and all patients were also unselected for age or family history. The ESP is thought to reflect US population mutation rates as opposed to cancer-free controls because cancer status is not fully characterized. ExAC has the advantage of sequencing data on a large number of people characterized by racial and ethnic group, but ExAC also includes patients with known malignancies such as those in the Cancer Genome Atlas studies. The inclusion of known patients with cancer in ExAC could falsely lower our calculated odds ratio for OC. Ideally, we would use a larger, well-characterized control group with matching age and race and known cancer status. However, it is reassuring that mutation frequencies in the ESP for these genes were similar to previously published controls.^8,9,30,32,41 Further research is needed to clarify the roles of inherited mutations in PALB2 and BARD1 in ovarian cancer risk.

We present data from a large, comprehensively sequenced group of unselected OC patients and provide data to implicate 2 new suspected hereditary OC genes, BARD1 and PALB2. Overall, 347 of 1915 (18.1%) OC patients had 352 germline mutations in 11 OC genes (BRCA1, BRCA2, BRIP1, RAD51C, RAD51D, PALB2, BARD1, PMS2, MSH6, MLH1, and MSH2). Mutations were identified in all histologies of OC with the exception of mucinous carcinoma, and 72 of 352 (20.5%) OC-associated mutations occurred in a gene other than BRCA1 or BRCA2.

Corresponding Author: Barbara Norquist, MD, Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Washington, Seattle, WA 98195-6460 (bnorquis@uw.edu).

Accepted for Publication: November 5, 2015.

Published Online: December 30, 2015. doi:10.1001/jamaoncol.2015.5495.

Author Contributions: Drs Swisher and Birrer contributed equally to this project and are considered cosenior authors. Dr Norquist had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Norquist, Harrell, Mannel, King, Swisher, Birrer.

Acquisition, analysis, or interpretation of data: Norquist, Harrell, Brady, Walsh, Lee, Gulsuner, Bernards, Casadei, Yi, Burger, Chan, Davidson, Mannel, DiSilvestro, Lankes, Ramirez, King, Swisher, Birrer.

Drafting of the manuscript: Norquist, Brady, Lankes, Ramirez, Swisher, Birrer.

Critical revision of the manuscript for important intellectual content: Norquist, Harrell, Walsh, Lee, Gulsuner, Bernards, Casadei, Yi, Burger, Chan, Davidson, Mannel, DiSilvestro, Ramirez, King, Swisher, Birrer.

Statistical analysis: Norquist, Brady, Lee, Gulsuner, Yi, King.

Obtained funding: Swisher, Birrer.

Administrative, technical, or material support: Norquist, Harrell, Walsh, Bernards, Chan, Mannel, DiSilvestro, Lankes, King, Swisher.

Study supervision: Norquist, Swisher, Birrer.

Histopathological review of biospecimens and support from the GOG tissue bank: Ramirez.

Interpretation of genetic data: King.

Conflict of Interest Disclosures: Dr Burger previously served as a consultant to Genentech/Roche during advisory board meetings. Dr Mannel reports participation in advisory boards for Endocyte, AstraZeneca, MedImmune, Oxigene, Advaxis, and Amgen, and his institution was reimbursed for his time. No other conflicts were reported.

Funding/Support: This study was supported by National Cancer Institute grants to the Gynecologic Oncology Group Administrative Office (grant No. CA 27469), the Gynecologic Oncology Group Statistical and Data Center (grant No. CA 37517), the Gynecologic Oncology Group Tissue Bank (grants U10 CA27469, U24 CA114793, and U10 CA180868); NRG Oncology (grants No. 1 U10 CA180822, R01CA131965 [Dr Swisher], R01CA157744 [Dr King], RO1CA175716 [Dr Walsh, Dr King], and P50CA083636 [Dr Swisher]); and the Ovarian Cancer Research Foundation, The Breast Cancer Research Foundation, the Department of Defense Ovarian Cancer Research Program (grants No. OC093285 [Dr Walsh] and OC120312 [Dr Swisher]), and the Wendy Feuer Research Fund for Prevention and Treatment of Ovarian Cancer. This study was also funded by the Women’s Reproductive Health Research Career Development Award (5K12HD001264-13 [Dr Norquist]), the Liz Tilberis Early Career Award from the Ovarian Cancer Research Foundation (Dr Norquist). Dr Birrer also received grants RC4CA156551, RO1CA142834, and financial support from the Julie Fund to support this study.

Role of the Funder/Sponsor: The funders/sponsors had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Additional Contributions: The authors would like to thank Barry E. Storer, PhD, Department of Biostatistics, University of Washington School of Public Health, Seattle, Washington, for reviewing the manuscript, and Kim M. Blaser with NRG Oncology for providing assistance with manuscript preparation. The authors would like to thank the NHLBI GO ESP and its ongoing studies that produced and provided exome variant calls for comparison: the Lung GO Sequencing Project (HL-102923), the WHI Sequencing Project (HL-102924), the Broad GO Sequencing Project (HL-102925), the Seattle GO Sequencing Project (HL-102926) and the Heart GO Sequencing Project (HL-103010). The authors would like to thank the ExAC and the groups that provided exome variant data for comparison. A full list of contributing groups can be found at http://exac.broadinstitute.org/about.

Additional Information: The following institutions participated in GOG 218 and 262: Roswell Park Cancer Institute, University of Alabama at Birmingham, Duke University Medical Center, Abington Memorial Hospital, Walter Reed Army Medical Center, Wayne State University, University of Minnesota Medical School, Mount Sinai School of Medicine, Northwestern Memorial Hospital, University of Mississippi Medical Center, Colorado Gynecologic Oncology Group PC, University of California at Los Angeles, University of Washington, University of Pennsylvania Cancer Center, Milton S. Hershey Medical Center, University of Cincinnati, University of North Carolina School of Medicine, University of Iowa Hospitals and Clinics, University of Texas Southwestern Medical Center at Dallas, Indiana University School of Medicine, Wake Forest University School of Medicine, University of California Medical Center at Irvine, Rush-Presbyterian-St Luke's Medical Center, Magee Women's Hospital, SUNY Downstate Medical Center, University of Kentucky, University of New Mexico, The Cleveland Clinic Foundation, State University of New York at Stony Brook, Washington University School of Medicine, Memorial Sloan-Kettering Cancer Center, Cooper Hospital/University Medical Center, Columbus Cancer Council, MD Anderson Cancer Center, University of Massachusetts Medical School, Fox Chase Cancer Center, Women's Cancer Center, University of Oklahoma, University of Virginia Health Sciences Center, University of Chicago, Mayo Clinic, Case Western Reserve University, Tampa Bay Cancer Consortium, Yale University, GOG Japan-Saitama Medical University International Medical Center, University of Wisconsin Hospital, Cancer Trials Support Unit, University of Texas - Galveston, Women and Infants Hospital, Korean Gynecologic Oncology Group, The Hospital of Central Connecticut, Georgia Core, GYN Oncology of West Michigan, PLLC, Aurora Women's Pavilion of West Allis Memorial Hospital, Fred Hutchinson Cancer Research Center, UCSF - Mount Zion, St Joseph’s Hospital and Medical Center, Carolinas Medical Center/Levine Cancer Institute and Community Clinical Oncology Program.