BRCA1 immunohistochemistry in a molecularly characterized cohort of ovarian high-grade serous carcinomas

Abstract

BRCA1 and BRCA2 dysfunction, frequently seen in high-grade serous ovarian carcinomas, often results from germline mutations, somatic mutations, and promoter methylation. Identification of tumors with BRCA defects has therapeutic and prognostic implications. Identifying germline BRCA mutations is also important given the increased risk for hereditary breast and ovarian carcinoma. Our goal was to assess whether immunohistochemical analysis (IHC) for BRCA1 is an effective method for the detection of BRCA1 dysfunction in molecularly characterized high-grade ovarian serous carcinoma. We identified 43 high-grade ovarian serous carcinomas with known events in BRCA1 and BRCA2 included in The Cancer Genome Atlas Project. BRCA1 stain was first assessed without knowledge of the BRCA status, and a semiquantitative assessment for intensity and amount of staining was performed. The stains were reevaluated and divided into 3 categories (retained, loss, and equivocal) on the basis of correlation with genotyping data. Presence of retained BRCA staining was considered normal, whereas the other patterns, including equivocal staining or loss of staining, were considered abnormal. Two pathologists, blinded to the BRCA status, then scored 2 sets of validation cases selected on the basis of available molecular data-1 with only germline mutation status available (n=31) and 1 with comprehensive genomic data (n=39). The pathologists agreed 88% of the time in the training set and 91% in the validation sets. In the training set, abnormal BRCA staining was seen in 24 cases, of which 21 (87%) showed BRCA1 genetic abnormalities, 1 showed BRCA2 mutations, and 2 showed no BRCA abnormalities. Abnormal BRCA1 staining was noted in all 5 cases with BRCA1 germline mutations, in 3 (60%) of 5 with BRCA1 somatic mutations, and in 13 (93%) of 14 with BRCA1 promoter methylation. The 2 validation sets included 70 additional patients, and all cases with germline BRCA1 mutations (n=11) showed abnormal BRCA1 staining. Tumors with BRCA1 promoter methylation also showed abnormal staining in 6 (86%) of 7 cases. In the entire study, no cases with BRCA1 germline mutation showed intact immunostaining (negative predictive value=100%). This study shows that BRCA1 IHC is well correlated with molecular events in ovarian carcinoma. Considering the high negative predictive value for germline mutations, BRCA1 IHC appears to be an effective approach to stratify patients for germline genetic testing and to detect other mechanisms of BRCA1 dysfunction in high-grade serous ovarian carcinomas.

Figure 1

A. High-grade serous carcinoma lacking nuclear expression of BRCA1. There is a positive internal control. This is interpreted as “BRCA1 loss.” B. High-grade serous carcinoma with very rare BRCA1 positive tumor cell nuclei (example indicated by arrow). The weak staining intensity of positive tumor cell nuclei contrasts with the strong staining seen in tumor infiltrating lymphocytes. This is interpreted as “BRCA1 loss.”

Figure 2

A. High-grade serous carcinoma with numerous tumor cell nuclei that express BRCA1. This is interpreted as retained expression of BRCA1. B. High-grade serous carcinoma with moderate intensity staining in 5–10% of tumor nuclei in a selected intermediate power field. There is a moderately intense internal control. This is interpreted as retained expression of BRCA1.

Figure 3

A. High-grade serous carcinoma with weak staining in 5–10% of tumor cell nuclei, in presence of moderate to strong internal positive control. The thick arrow indicates a positive internal control with moderately intense staining. The thin arrows indicate examples of weakly stained tumor cell nuclei. The density of weakly stained nuclei is higher than in Figure 1B. This is interpreted as an equivocal result B. High-grade serous carcinoma lacking nuclear expression of BRCA1. There is no positive internal control. This is interpreted as an equivocal result. C. High-grade serous carcinoma with equivocal staining results. One pathologist scored this case as showing intact BRCA1 expression whereas the other pathologist scored the case as equivocal. Reasons to explain this discrepancy include differences in estimating the percentage of positive tumor cell nuclei and difficulties distinguishing tumor infiltrating lymphocytes from tumor cells. Compared to Figure 2B, staining intensity of tumor cell nuclei is very weak. Cases with this type of discrepancy were captured as “equivocal” for purposes of the study. The tumor had BRCA1 promoter methylation.

Figure 4

BRCA1 gene expression and promoter methylation values in the training set (n=43). Arrow indicates methylated case with retained immunostaining.

Figure 5

BRCA1 gene expression and promoter methylation values in validation set 2 (n=39). Arrow indicates methylated case with retained immunostaining.