Pathogenic Variants in BRIP1 , RAD51C , and RAD51D Identified with Multi-Gene Panel Testing for Hereditary Cancers

Background: Professional society guidelines recommend risk-reducing salpingo-oophorectomy (RRSO) for women with pathogenic variants (PVs) in ovarian cancer-risk genes. Personalization of that intervention is based on gene-specific phenotypes; however, the age of ovarian cancer diagnosis in women with PVs in moderate penetrance ovarian cancer-risk genes is not well characterized. Women who had hereditary cancer panel testing from September 2013-May 2019 were included (N=631,950). Clinical/demographic information was compared for women with a PV in BRIP1, RAD51C, or RAD51D versus in BRCA1 or BRCA2. Results: PVs in BRIP1, RAD51C, or RAD51D were identified in 0.5% of all tested women but in 1.6% of women with a history of ovarian cancer (~3-fold increase). PVs in BRCA1 or BRCA2 were identified in 2.4% of all tested women but in 6.1% of women with a history of ovarian cancer (~2.5-fold increase). The proportion of women with a personal or family history of ovarian cancer was similar among women with a PV in BRIP1 , RAD51C , RAD51D , BRCA1 , or BRCA2 . The median age at ovarian cancer diagnosis was 53 years in BRCA1 , 59 years for BRCA2 , 65 years for BRIP1 , 62 years for RAD51C , and 57 years for RAD51D . Conclusions: These data reinforce the importance of identifying PVs in moderate penetrance ovarian cancer-risk genes. The age at ovarian cancer diagnosis was older for women with PVs in BRIP1, RAD51C, or RAD51D , suggesting that it is safe to delay RRSO until age 45-50 in RAD51D PV carriers and possibly, until age 50-55 in BRIP and RAD51C PV carriers.


Background
Ovarian cancer is uncommon yet deadly, with approximately 251,000 cases and 161,000 deaths (4.5% of all deaths in women) occurring each year, globally [1]. The lifetime risk of ovarian cancer is increased among women with Hereditary Breast and Ovarian Cancer syndrome (HBOC). HBOC is associated with pathogenic variants (PVs) in BRCA1 or BRCA2, where BRCA1 PVs are associated with a 39-63% lifetime risk of ovarian cancer and BRCA2 PVs are associated with a 15-27% risk. This is significantly elevated relative to the general population risk of ovarian cancer, which is only 1.3% [2]. More recently, additional genes associated with increased ovarian cancer risk have been identified, including BRIP1, RAD51C, and RAD51D [3,4]. These moderate penetrance genes are associated with lower lifetime risks of ovarian cancer than BRCA1 and BRCA2, but still confer significantly increased risk compared to the general population, with lifetime risks ranging from 6-15%.
Identification of a PV in an ovarian cancer-risk gene may initiate more intensive and personalized medical management that would not be prompted based on family history alone. National Comprehensive Cancer Network (NCCN) guidelines recommend that women with a PV in BRCA1 or BRCA2 consider risk-reducing salpingo-oophorectomy (RRSO) at age 35-45 or earlier, depending on specific family history [5]. These guidelines reflect the level of evidence available regarding the clinical presentation of ovarian cancer in women with PVs in BRCA1 or BRCA2. Specifically, there is robust evidence for a high risk of ovarian cancer at an early age, with BRCA1 PV carriers having an 8-23% risk of ovarian cancer by age 50 [6][7][8][9]. More recent evidence has shown that the risk of early-onset ovarian cancer is lower in BRCA2 PV carriers (0.4-4%). As such, NCCN guidelines now state that it is reasonable to delay RRSO in BRCA2 PV carriers until age 40-45.
For women with PVs in BRIP1, RAD51C, or RAD51D, NCCN guidelines recommend that RRSO be considered at age 45-50 [5]. Although there is sufficient evidence of ovarian cancer risk associated with these three genes to justify consideration of RRSO, the guidelines also state that "the current evidence is insufficient to make a firm recommendation as to the optimal age for this procedure" [5]. This reflects the poor understanding of the exact risk of ovarian cancer and the typical age of onset in women with PVs in BRIP1, RAD51C, and RAD51D. Given the severity of the intervention and associated side-effects, patients and health care providers have strong interest in delaying RRSO until older ages if safe. However, without a better understanding of these parameters, there is uncertainty about the optimal age for surgery and appropriate clinical management of women with PVs in these moderate penetrance ovarian cancer-risk genes.
In order to better characterize the clinical presentation of women with a PV in a moderate penetrance ovarian cancer-risk gene, we evaluated women with a PV in BRIP1, RAD51C, or RAD51D identified during hereditary cancer genetic testing. This includes an assessment of ancestry, personal and family cancer history, and age of ovarian cancer diagnosis. In addition, women with PVs in BRCA1 or BRCA2 were evaluated for comparison.

Participants
The cohort in this retrospective analysis included women who had testing with a multigene hereditary cancer panel (Myriad Genetic Laboratories, Salt Lake City, UT) between September 2013 and May 2019 (N = 631,950). All patients provided informed consent for genetic testing. All patient data was de-identified for analysis. Patients were excluded from this analysis if they were from a state with laws preventing the use of de-identified genetic data for research. Patients were also excluded if they had an unspecified personal cancer history or previous hereditary cancer genetic testing, including founder mutation testing and testing for a known familial mutation. Individuals were also excluded if they were found to have PVs in multiple genes.

Multi-Gene Hereditary Cancer Panel Testing and Variant Classification
Testing was performed in a Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathology (CAP) approved laboratory. The hereditary cancer panel was comprised of 25-28 cancer-predisposition genes, including BRCA1, BRCA2, BRIP1, RAD51C, and RAD51D. This Next Generation Sequencing (NGS) assay has been detailed previously [10,11]. Sequencing and large rearrangement analysis was performed for all genes evaluated here.
Variant classification was based on guidelines from the American College of Molecular Genetics and Genomics and Association for Molecular Pathology using all available functional, statistical, segregation, and literature evidence, as previously described [12,13]. Variants with a laboratory classification of Deleterious or Suspected Deleterious were considered pathogenic. This analysis was based on the classification of all variants as of May 2019, regardless of whether they were classified differently when the test report was issued.

Statistical Analysis
The prevalence of PVs in BRIP1, RAD51C, RAD51D, BRCA1, or BRCA2 was evaluated for the full testing cohort as well as the subset of women who had a personal history of ovarian cancer. The clinical presentation of women with PVs in the moderate penetrance ovarian cancer-risk genes (BRIP1, RAD51C, or RAD51D) was evaluated. This included an evaluation of ancestry, personal and family history of ovarian cancer, and age at diagnosis. Clinical and demographic data were obtained from the provider-completed test request form. Family cancer history was limited to first-and second-degree relatives. History of ovarian cancer included fallopian tube, peritoneal, and ovarian cancer. Analyses were also performed for women with PVs in BRCA1 and BRCA2 and women who were tested and found to carry no PV in any gene (PV-negative) for comparison.
Statistical analyses were performed using SAS® software (SAS Institute Inc., Cary, North Carolina, USA) and R software (R Foundation for Statistical Computing, Vienna, Austria).

Prevalence of Pathogenic Variants in Ovarian Cancer-Risk Genes
Here, we assessed women who carried a PV in one of five ovarian cancer risk-genes. Overall, 0.5% (3,089/631,950) of women tested with the multi-gene panel had a PV in a moderate penetrance ovarian cancerrisk gene. This included 1,779 (0.3%) women with a PV in BRIP1, 855 (0.1%) women with a PV in RAD51C, and 455 (0.1%) women with a PV in RAD51D (Table 1). An additional 15,054 (2.4%) women in the testing cohort had a PV in BRCA1 or BRCA2 (1.1% for BRCA1, 1.3% for BRCA2; Table 1). Most of the testing cohort was negative for a PV in any gene (93.7%, 592,309/631,950). Abbreviations: PV, pathogenic variant *Includes White/Non-Hispanic, Ashkenazi Jewish, and any combination of the two ancestries. **Includes Middle Eastern, Native American, and Other. When the subset of women with a personal history of ovarian cancer was considered, the prevalence of PVs in each ovarian cancer risk gene evaluated here increased, as expected (Table 1). Specifically, 1.6% (456/27,915) of women with a personal history of ovarian cancer had a PV in a moderate penetrance ovarian cancer-risk gene (Table 1). This represents a threefold increase relative to the full testing cohort. PVs in BRIP1 were identified in 0.8% (233/27,915) of women with ovarian cancer. PVs in RAD51C and RAD51D were identified in 0.5% (149/27,915) and 0.3% (74/27,915) of women with ovarian cancer, respectively. The prevalence of PVs in BRCA1 or BRCA2 more than doubled within the subset of tested women with a history of ovarian cancer, with a combined prevalence of 6.1% (3.5% for BRCA1, 2.6% for BRCA2).
The proportion of PV carriers was evaluated for each gene within the most commonly reported ancestries. For the moderate penetrance ovarian cancer-risk genes, the prevalence ranged from 0.1-0.3% with no substantial differences by ancestry (Table 1). In comparison, the PV prevalence for BRCA1 and BRCA2 ranged from 1.0-1.5% in most ancestries (Table 1). Increased prevalence was observed for BRCA1 among individuals of Asian (1.7%) or Hispanic/Latino (1.8%) ancestry and for BRCA2 among individuals of Asian ancestry (1.9%). There were no substantial differences in the proportion of individuals who were PV-negative by ancestry (93.1%-94.4%; Table 2).

Personal and Family History of Ovarian Cancer by Gene
The proportion of PV carriers with a personal history of ovarian cancer was evaluated by gene ( The proportion of PV carriers who had a first-or second-degree family member with a history of ovarian cancer was also evaluated by gene (

Age of Ovarian Cancer by Gene
The age at ovarian cancer diagnosis was evaluated according to gene (Table 2). Women with a PV in BRCA1 had the lowest median age at ovarian cancer diagnosis, at 53 years of age. Women with a PV in BRCA2 had a median age at diagnosis of 59 years. Similar to BRCA2, the median age of ovarian cancer diagnosis was older for women with a PV in BRIP1 (65 years), RAD51C (62 years), or RAD51D (57 years). For comparison, PV-negative women with ovarian cancer had a median age at diagnosis of 59 years.
Because the age at ovarian cancer diagnosis may inform management, we also looked at the overall distribution of the age at ovarian cancer diagnosis by gene. Overall, the distribution of age at ovarian cancer diagnosis was skewed to younger ages for women with PVs in BRCA1 (Fig. 1). This is the only gene where the interquartile range overlapped with age 50 (a proxy for the average age of menopause). Overall, 60.7% (564/919) of women with a PV in BRCA1 and a personal history of ovarian cancer were diagnosed after age 50.
In comparison, the distribution of age at diagnosis was skewed to older ages for the other genes evaluated (Fig. 1 1). In addition, there were very few women with PVs in the moderate penetrance ovarian cancer-risk genes who were diagnosed at very young ages (Fig. 1). The percentage of PV-negative women who had a diagnosis of ovarian cancer after the age of 50 was 69.2% (16,383/23,685).

Discussion
Ovarian cancer represents 3.7% of all female cancers and is usually diagnosed in advanced stages with a poor prognosis, with overall survival being the worst of all gynecologic malignancies. Professional society guidelines include gene-specific risk reducing recommendations [5]. While these guidelines incorporate decades of evidence for BRCA1 and BRCA2, guidelines are not as clear for other, less well-characterized genes associated with increased ovarian cancer risk. In this analysis, we evaluated the clinical presentation of over 3,000 women with PVs in BRIP1, RAD51C, or RAD51D identified by a multigene hereditary cancer panel. To our knowledge, this is the largest published study evaluating the ovarian cancer risk and age of onset associated with pathogenic variants in these moderate penetrance ovarian cancer-risk genes.
The data presented here for BRIP1, RAD51C, and RAD51D supports previous research demonstrating an increased risk of ovarian cancer for women with PVs in these genes. The prevalence of a personal or family history of ovarian cancer among women with PVs in BRIP1, RAD51C, or RAD51D was similar to that observed for women with PVs in BRCA1 or BRCA2 in this cohort. This supports a recent study that utilized a large clinical cohort to quantify gene-specific ovarian cancer risk. In this study, Kurian et al. demonstrated that BRIP1, RAD51C, and RAD51D are all significantly associated with ovarian cancer [14]. Furthermore, the relative risk of ovarian cancer associated with RAD51C and RAD51D was comparable to BRCA2, with odds ratios for all three genes of approximately five [14]. In addition, there was a substantial enrichment of PVs in these three genes among women with ovarian cancer compared to PV-negative women in this cohort. Collectively, this reiterates the importance of pan-cancer panel testing in women with ovarian cancer. Given the poor prognosis associated with this disease, identifying PVs in genes that confer an increased risk for ovarian cancer outside of BRCA1 and BRCA2 is critical for appropriate patient management.
NCCN guidelines recommend that women with PVs in ovarian cancer-risk genes consider RRSO [5]. Given the psychological and medical complications of premature menopause, patients and providers must balance the timing of RRSO with the risk of ovarian cancer. For BRCA1, the risk of ovarian cancer at an early age has been well established. This was also observed here, where women with PVs in BRCA1 had the youngest median age at diagnosis. The median age at ovarian cancer diagnosis of women with a PV in BRIP1, RAD51C, or RAD51D was much older and more than three quarters of women with a PV in one of these three genes and a history of ovarian cancer was diagnosed after the age of 50. For BRIP1 and RAD51C, the median age at ovarian cancer diagnosis was after 60 years. This is comparable to what is seen in the general population, where about half of the women who are diagnosed with ovarian cancer are 63 years or older [3].
At the individual gene level in this cohort, one may determine that it is reasonable to delay RRSO until age 45-50 for women with a PV in a moderate penetrance ovarian cancer-risk gene. In addition, it may be reasonable to delay RRSO until age 50-55 for women with a BRIP1 or RAD51C PV, which is at a time when natural menopause typically occurs. Delayed RRSO in these women may minimize the vasomotor symptoms and cardiovascular risk associated with a premature menopause as well as its negative effect on bone metabolism, and possibly, cognition and longevity [15][16][17]. Overall, these data aid in supporting providers and their patients in the clinical decision-making process based on a more refined risk of ovarian cancer.
The data presented here also spur an interesting possible application of panel testing among women with ovarian cancer as a method to tailor treatment. Women with defects in the homologous recombination repair (HRR) pathway are more likely to benefit from DNA-damaging therapies, such as PARP inhibitors or platinumbased regimens [18,19]. Previous research has shown that the presence of germline or tumor PVs in BRCA1 or BRCA2 predict benefit from such therapies among women with ovarian cancer [20,21]. The presence of PVs in other genes in the HRR pathway, including BRIP1, RAD51C, or RAD51D, express a phenotype similar to BRCArelated HRR defects [22]. This suggests that panel testing may help guide treatment selection for women with ovarian cancer by identifying PVs in BRIP1, RAD51C, or RAD51D [23].
While this study is informative, it is not without limitations. First, family history information was obtained from provider completed test request forms and may not be comprehensive. Given the size of this cohort, it was not feasible to confirm the reported family and personal history. In accordance with other data and to help minimize the impact of inaccuracies [24], family history was only considered for first-and second-degree relatives. In addition, our population was composed of women referred for genetic testing and is therefore enriched for individuals with a personal and family history of ovarian cancer. In order to avoid over-interpretation of the data for BRIP1, RAD51C, and RAD51D, we evaluated PV-negative women to provide an appropriate baseline for this elevated risk population. This characteristic should be considered when generalizing this study's results.

Conclusion
As hereditary cancer risk assessment is increasingly incorporated into clinical care, clinicians may identify more patients who carry mutations in ovarian cancer risk genes beyond BRCA1 and BRCA2. The data presented here refines our understanding of the ovarian cancer risk and the typical age of diagnosis in women with PVs in moderate penetrance ovarian cancer-risk genes to inform safe and appropriate medical management. Our findings suggest that it is safe to delay RRSO until age 45-50 for women with PVs in RAD51D and possibly later for women with PVs in BRIP1 or RAD51C. Overall, these data reiterate the importance of identifying PVs in these genes, given the elevated risk of ovarian cancer in the proband and the family. By more precisely understanding gene-specific ovarian cancer risk, patients and providers can better personalize preventative and treatment interventions.

Declarations
Ethics approval and Consent to Participate: All information was collected in the course of clinical genetic testing. Patients consented for clinical testing and all information was de-identified prior to analysis.

Figure 1
Distribution of age at ovarian cancer diagnosis by gene.