Germline mutations in BRCA1 and BRCA2 predispose to autosomal-dominant hereditary breast and ovarian cancer, in which determination of carrier status in women presenting with ovarian cancer may affect prognosis and influence treatment. Female carriers can be offered risk-reducing surgeries or can elect for heightened surveillance for breast and ovarian cancer. These implications extend to their families. Morphologic classification of epithelial ovarian carcinoma delineates five major histologic subtypes: high-grade serous, clear cell, endometrioid, mucinous, and low-grade serous. These constitute distinct clinical entities with differing presentation, prognoses, molecular, and immunohistochemistry profiles,1–3 reflecting aberration of specific molecular pathways such as DNA damage repair, cell cycle control, or promotion or control of growth and proliferation.1,4 Seventy percent of epithelial ovarian carcinoma is of the high-grade serous subtype.1 Germline BRCA1 and BRCA2 mutations account for 11.7–15% of invasive epithelial ovarian carcinoma.5–7 Ovarian cancers in BRCA1 and BRCA2 mutation carriers show almost exclusive association with epithelial ovarian carcinoma in contrast to the low likelihood of germ cell or stromal tumors seen in this population.8–10 High-grade serous histology is reported in 76.7–93% of BRCA1 and BRCA2 mutation carriers in population-based series of invasive ovarian carcinoma.5,6,11,12 Our objective was to estimate the incidence of germline BRCA1 and BRCA2 mutation carriers in a consecutive series of women with nonmucinous ovarian cancer unselected for a personal or family history of breast cancer or ovarian cancer in British Columbia.
MATERIALS AND METHODS
From 2004 to 2009, patients were recruited from the Vancouver General Hospital and British Columbia Cancer Agency in Vancouver, British Columbia, Canada. Ethical approval was obtained from the University of British Columbia Ethics Board. All women who had been fully staged and were undergoing debulking surgery (primary or delayed) for cancers of ovarian, peritoneal, or fallopian tube origin were approached for informed consent for the banking of tumor tissue. Pathology review was performed in all cases. Women with borderline ovarian tumors or mucinous histology were excluded and those with endometrioid, clear cell, or serous carcinomas were referred to the British Columbia Cancer Agency Hereditary Cancer Program genetic counselors to discuss and perform germline BRCA1 and BRCA2 testing. Cascade genetic counseling of family members of BRCA1 and BRCA2-positive patients revealed from this study was offered through the same program. Carcinomas of mixed epithelial subtypes were included in the study, and those with a serous component were classified as high-grade serous. Press et al13 previously characterized the BRCA1 and BRCA2 abnormalities in the tumors of 49 of these cases and more recently the detailed immunohistochemical and molecular characterization of the primary tumors and clinical and outcome data related to the full cohort (n=131) has been published.14
The sequence of BRCA1 and BRCA2 was determined from peripheral blood-derived genomic DNA through standard bidirectional dideoxy sequencing of the entire coding and proximal intronic regions. The presence of large genomic rearrangements, conferred by deletions or duplications, was determined by multiplex ligation dependent probe amplification according to the manufacturer's protocol.
Family history acquired at the time of initial consultation by the gynecologic oncology surgical team (nongenetic counselor) and by the genetic counselor at the time of counseling for BRCA1 and BRCA2 mutation testing was reviewed compared with the British Columbia Cancer Agency's referral guidelines for hereditary breast, ovarian cancer, or both (Table 1) (www.bccancer.bc.ca/HPI/CancerManagementGuidelines/HereditaryCancerProgram/referralinformation/hboccriteria.htm). Family histories were considered in first- and second-degree relatives. Whether they would have been referred based on the finding of invasive, nonmucinous epithelial ovarian cancer alone in the proband compared with whether they would have met other criteria for testing was reviewed and compared with germline mutation status. This enabled us to determine the number of mutation carriers that were identified based on their history of ovarian cancer alone.
Statistical analysis was performed using SPSS 20.0.0 and OpenEpi 2.3.1. Ninety-five percent confidence intervals (95% CIs) for mutation frequencies were calculated under the assumption of binomial distributions of the observed numbers of cases using Fisher's exact test. Age comparisons were performed using a Mann-Whitney U test.
Of 131 women who participated in genetic counseling, 26 women (20%) were found to harbor germline mutations in either BRCA1 (n=19) or BRCA2 (n=7) (Fig. 1). Pathology review revealed that all 26 of these women had high-grade serous ovarian cancer (Table 2). Therefore, if we consider only the 103 cases of high-grade serous, referral to the Hereditary Cancer Program for genetic counseling and the option of BRCA1 and BRCA2 testing based on the criteria of high-grade serous histology alone would have revealed a BRCA1 and BRCA2 germline mutation rate of 25% in this cohort.
Current guidelines for referral to the British Columbia Cancer Agency Hereditary Cancer Program include the criterion of offering genetic counseling and testing to individuals with nonmucinous ovarian cancer diagnosed at any age. The importance of this criterion is evident, as even with a comprehensive family history obtained by a genetic counselor, germline BRCA1 and BRCA2 mutation carriers with high-grade serous histology and without a personal or family history suggestive of hereditary breast and ovarian cancer would not be identified. By showing the number of germline BRCA1 and BRCA2 mutation carriers with high-grade serous epithelial ovarian carcinoma meeting only the criterion of ovarian cancer in the proband, Figure 2 outlines which cases would be captured by family histories (by a genetic counselor or nongenetic counselor) as compared with histology-driven referral. Figure 2A–B shows the difference between family history ascertained by the gynecologic oncology surgeon (denoted as nongenetic counselor) and the genetic counselor. If the first family history was taken by another health care provider other than the gynecologic surgeon (eg, general practitioner), this was also recorded as a nongenetic counselor. Restricting BRCA1 and BRCA2 testing to women with family histories of hereditary breast and ovarian cancer, as ascertained by the surgeon, missed 14 mutation carriers, lowering detection rates to 9% (12/131) or 11.6% (12/103) if only considering the patients with high-grade serous histology. This improved to 16% (21/131) or 20.4% (21/103) when ascertained by the genetic counselor. Therefore, on reviewing the histories as obtained by the nongenetic counselor compared with those ascertained by the genetic counselor, it was apparent that 35% (9/26, 95% CI 17–56%) of BRCA1 and BRCA2 mutation carriers with high-grade serous epithelial ovarian carcinoma would have been missed if referrals for testing had only been based on having a family history suggestive of hereditary breast and ovarian cancer (Fig. 2). Even with the more extensive history obtained by a genetic counselor, 19% (5/26, 95% CI 6–39%) of mutation carriers did not meet the familial component of hereditary breast and ovarian cancer criteria and before the inclusion of the criterion addressing singleton cases of nonmucinous epithelial ovarian carcinoma in the proband would not have been offered testing. Capture of these probands was secondary to our research study protocol in which counseling and testing was offered to all patients and what was an emerging practice change to consider referral for all nonmucinous ovarian carcinomas. If considering only cases with high-grade serous epithelial ovarian carcinoma and without a family history of hereditary breast and ovarian cancer, 12% of women (5/42, 95% CI 4–26%) were germline BRCA1 and BRCA2 mutation carriers. Furthermore, the number of mutation-positive women without an apparent family history increased to 14 individuals when the family history was taken by a nongenetic counselor. The average age of ovarian cancer diagnoses in women with high-grade serous epithelial ovarian carcinoma who did not have a family history of hereditary breast and ovarian cancer was 47.4 years in mutation-positive cases (ranging from 42 to 54 years) and 62 years in mutation-negative cases (ranging from 37 to 84 years). This difference was significant when compared using a Mann-Whitney U test (P=.003). Of the two mutation carriers that harbored the recurrent c.185 delAG mutation, only one was reported to be of Ashkenazi Jewish descent. Two unrelated individuals carried exon 13 duplications.
These results suggest that germline BRCA1 and BRCA2 mutations are confined to the high-grade serous histologic subtype, reflecting our own and other's experiences of frequent aberration of the BRCA1 and BRCA2 pathways within this subtype, but not in nonhigh-grade serous subtypes. The same series reported here was also analyzed for somatic mutations in BRCA1 and BRCA2 with five additional women discovered to have mutations of clinical significance in their tumors (germline DNA normal). BRCA1 and BRCA2 germline or somatic mutation frequency of high-grade serous cancers was therefore 30% (31/103) and similar to the reported findings by Hennessy et al15 (somatic and germline mutations in BRCA1 and BRCA2 in 23% of high-grade serous epithelial ovarian carcinoma). Methylation of BRCA1 was found in an additional 20% of high-grade serous tumors bringing the total percentage of high-grade serous tumors showing BRCA1 and BRCA2 abnormalities to 50%.14
Population-based studies have shown the incidence of germline BRCA1 and BRCA2 mutations associated with high-grade serous histology to be 16.5–18%.7,11 Although studies have identified BRCA1 and BRCA2 mutations in subtypes other than high-grade serous, the classification of tumors was based on pathology reports without review and use of current diagnostic criteria.5,6,11 In the Cancer Genome Atlas, germline BRCA1 and BRCA2 mutations were found in only 15% of high-grade serous carcinomas (47/316 cases). The lower mutation rate may have related to case selection and the technical aspects of the sequencing and mutation analysis.16 Recently, Arnold et al17 reported a 22.2% frequency of germline BRCA1 and BRCA2 mutations in patients with high-grade epithelial ovarian carcinoma with a serous component and no Ashkenazi Jewish heritage. Walsh et al18 reported a one in four BRCA1 and BRCA2 mutation frequency in patients with high-grade serous epithelial ovarian carcinoma, although also identified mutations in undifferentiated carcinomas, endometrioid and clear cell subtypes, possibly relating to the method and extent of pathology review, which was not specified. With the evolution of histopathologic subtype diagnosis for ovarian carcinoma, and the appreciation of defining molecular abnormalities, specific subtypes can now be very reproducibly diagnosed with high interobserver agreement using a combination of morphologic appearance and immunophenotyping.2,19,20 In our cohort, we found germline BRCA1 and BRCA2 mutations to be exclusive to the high-grade serous subtype, suggesting that nonhigh-grade serous epithelial ovarian carcinomas are at very low risk of hereditary breast and ovarian cancer and thus may not require referral to the Hereditary Cancer Program for genetic counseling. These results will need to be validated in a larger population-based series that also includes careful pathologic review of the primary tumors; however, if the association holds true, histology-based referral to a Hereditary Counseling Program should be incorporated into the current referral schema. This would result in cost savings and improved use of resources, preventing unnecessary referrals in nonhigh-grade serous cases in which the incidence of BRCA1 and BRCA2 mutations of clinical significance is low or nil. It may also, as outlined, reduce the likelihood of missed probands when referral is based on family history alone. Thorough pathology assessment, using morphologic, molecular, and immunohistochemical methodology,2,3 is essential to the success of this strategy. Although clinical criteria for referral for hereditary breast and ovarian cancer syndrome exist and include referral of women with epithelial ovarian, fallopian tube, or primary peritoneal cancer (National Comprehensive Cancer Network Guidelines Version 1.2011),21 also with the serous subtype being specified in the 2009 American College of Obstetricians and Gynecologists guidelines,22 currently there is no mechanism to ensure that they are being referred for genetic counseling regarding their risk for germline BRCA1 and BRCA2 mutations. One way to address this would be the inclusion of reflex recommendations on the pathology report that recommend that genetic assessment be offered to all women with high-grade serous ovarian, fallopian tube, or peritoneal carcinoma. This uniform referral strategy may lead to an increased opportunity for determination of BRCA1 and BRCA2 carrier status in women belonging to patient populations that may be underreferred for genetic counseling. Further education in the community for referring family practitioners and general gynecologists regarding the importance of referral for genetic counseling for patients with high-grade serous ovarian carcinoma will also help ensure patients and families potentially at high risk for breast, ovarian, and other related cancers are not missed.
In view of the strong association and high incidence (25%) of underlying BRCA1 and BRCA2 mutations in women with high-grade serous ovarian (pelvic) carcinoma, genetic assessment for consideration of BRCA1 and BRCA2 germline testing should be offered to all women diagnosed with this histologic subtype of ovarian cancer regardless of age or family history.
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