Huang, Huaizhi

Abstract Purpose: To determine the relationship between germline pathogenic variants (PV) in cancer predisposition genes and the risk of ductal carcinoma in situ (DCIS). Experimental Design: Germline PV frequencies in breast cancer predisposition genes (ATM, BARD1, BRCA1, BRCA2, CDH1, CHEK2, PALB2, RAD51C, and RAD51D) were compared between DCIS cases and unaffected controls and between DCIS and invasive ductal breast cancer (IDC) cases from a clinical testing cohort (n = 9,887), a population-based cohort (n = 3,876), and the UK Biobank (n = 2,421). The risk of contralateral breast cancer (CBC) for DCIS cases with PV was estimated in the population-based cohort. Results: Germline PV were observed in 6.5% and 4.6% of women with DCIS in the clinical testing and population-based cohorts, respectively. BRCA1, BRCA2, and PALB2 PV frequencies were significantly lower among women with DCIS than those with IDC (clinical cohort: 2.8% vs. 5.7%; population-based cohort: 1.7% vs. 3.7%), whereas the PV frequencies for ATM and CHEK2 were similar. ATM, BRCA1, BRCA2, CHEK2, and PALB2 PV were significantly associated with an increased risk of DCIS (OR > 2.0), but only BRCA2 PV were associated with high risk (OR > 4) in both cohorts. The cumulative incidence of CBC among carriers of PV in high-penetrance genes with DCIS was 23% over 15 years. Conclusions: The enrichment of PV in ATM, BRCA1, BRCA2, CHEK2, and PALB2 among women with DCIS suggests that multigene panel testing may be appropriate for women with DCIS. Elevated risks of CBC in carriers of PV in high-penetrance genes with DCIS confirmed the utility of testing for surgical decision-making.

Abstract Germline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to breast, ovarian, prostate and pancreatic cancer. However, variants of uncertain significance (VUS) (n>4000) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants. Here we report on comprehensive saturation genome editing-based functional characterization of 97% of all possible single nucleotide variants (SNVs) in the BRCA2 DNA Binding Domain hotspot for pathogenic missense variants that is encoded by exons 15 to 26. The assay was based on deep sequence analysis of surviving endogenously targeted haploid cells. A total of 7013 SNVs were characterized as functionally abnormal (n=955), intermediate/uncertain, or functionally normal (n=5224) based on 95% agreement with ClinVar known pathogenic and benign standards. Results were validated relative to batches of nonsense and synonymous variants and variants evaluated using a homology directed repair (HDR) functional assay. Breast cancer case-control association studies showed that pooled SNVs encoding functionally abnormal missense variants were associated with increased risks of breast and ovarian cancer (odds ratio (OR) 3.89, 95%CI: 2.77-5.51). In addition, 86% of tumors associated with abnormal missense SNVs displayed loss of heterozygosity (LOH), whereas 26% of tumors with normal variants had LOH. The functional data were added to other sources of information in a ClinGen/ACMG/AMP-like model and 700 functionally abnormal SNVs, including 220 missense SNVs, were classified as pathogenic or likely pathogenic, while 4862 functionally normal SNVs, including 3084 missense SNVs, were classified as benign or likely benign. These classified variants can now be used for risk assessment and clinical care of variant carriers and the remaining functional scores can be used directly for clinical classification and interpretation of many additional variants.

Abstract Pathogenic protein-truncating variants of RAD51C, which plays an integral role in promoting DNA damage repair, increase the risk of breast and ovarian cancer. A large number of RAD51C missense variants of uncertain significance (VUS) have been identified, but the effects of the majority of these variants on RAD51C function and cancer predisposition have not been established. Here, analysis of 173 missense variants by a homology-directed repair (HDR) assay in reconstituted RAD51C−/− cells identified 30 nonfunctional (deleterious) variants, including 18 in a hotspot within the ATP-binding region. The deleterious variants conferred sensitivity to cisplatin and olaparib and disrupted formation of RAD51C/XRCC3 and RAD51B/RAD51C/RAD51D/XRCC2 complexes. Computational analysis indicated the deleterious variant effects were consistent with structural effects on ATP-binding to RAD51C. A subset of the variants displayed similar effects on RAD51C activity in reconstituted human RAD51C-depleted cancer cells. Case–control association studies of deleterious variants in women with breast and ovarian cancer and noncancer controls showed associations with moderate breast cancer risk [OR, 3.92; 95% confidence interval (95% CI), 2.18–7.59] and high ovarian cancer risk (OR, 14.8; 95% CI, 7.71–30.36), similar to protein-truncating variants. This functional data supports the clinical classification of inactivating RAD51C missense variants as pathogenic or likely pathogenic, which may improve the clinical management of variant carriers. Significance: Functional analysis of the impact of a large number of missense variants on RAD51C function provides insight into RAD51C activity and information for classification of the cancer relevance of RAD51C variants.

PURPOSE To estimate the risk of contralateral breast cancer (CBC) among women with germline pathogenic variants (PVs) in ATM, BRCA1, BRCA2, CHEK2, and PALB2. METHODS The study population included 15,104 prospectively followed women within the CARRIERS study treated with ipsilateral surgery for invasive breast cancer. The risk of CBC was estimated for PV carriers in each gene compared with women without PVs in a multivariate proportional hazard regression analysis accounting for the competing risk of death and adjusting for patient and tumor characteristics. The primary analyses focused on the overall cohort and on women from the general population. Secondary analyses examined associations by race/ethnicity, age at primary breast cancer diagnosis, menopausal status, and tumor estrogen receptor (ER) status. RESULTS Germline BRCA1, BRCA2, and CHEK2 PV carriers with breast cancer were at significantly elevated risk (hazard ratio > 1.9) of CBC, whereas only the PALB2 PV carriers with ER-negative breast cancer had elevated risks (hazard ratio, 2.9). By contrast, ATM PV carriers did not have significantly increased CBC risks. African American PV carriers had similarly elevated risks of CBC as non-Hispanic White PV carriers. Among premenopausal women, the 10-year cumulative incidence of CBC was estimated to be 33% for BRCA1, 27% for BRCA2, and 13% for CHEK2 PV carriers with breast cancer and 35% for PALB2 PV carriers with ER-negative breast cancer. The 10-year cumulative incidence of CBC among postmenopausal PV carriers was 12% for BRCA1, 9% for BRCA2, and 4% for CHEK2. CONCLUSION Women diagnosed with breast cancer and known to carry germline PVs in BRCA1, BRCA2, CHEK2, or PALB2 are at substantially increased risk of CBC and may benefit from enhanced surveillance and risk reduction strategies.

Abstract Purpose: The identification of variants of uncertain significance (VUS) in the BRCA1 and BRCA2 genes by hereditary cancer testing poses great challenges for the clinical management of variant carriers. The ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) variant classification framework, which incorporates multiple sources of evidence, has the potential to establish the clinical relevance of many VUS. We sought to classify the clinical relevance of 133 single-nucleotide substitution variants encoding missense variants in the DNA-binding domain (DBD) of BRCA2 by incorporating results from a validated functional assay into an ACMG/AMP-variant classification model from a hereditary cancer–testing laboratory. Experimental Design: The 133 selected VUS were evaluated using a validated homology-directed double-strand DNA break repair (HDR) functional assay. Results were combined with clinical and genetic data from variant carriers in a rules-based variant classification model for BRCA2. Results: Of 133 missense variants, 44 were designated as non-functional and 89 were designated as functional in the HDR assay. When combined with genetic and clinical information from a single diagnostic laboratory in an ACMG/AMP-variant classification framework, 66 variants previously classified by the diagnostic laboratory were correctly classified, and 62 of 67 VUS (92.5%) were reclassified as likely pathogenic (n = 22) or likely benign (n = 40). In total, 44 variants were classified as pathogenic/likely pathogenic, 84 as benign/likely benign, and 5 remained as VUS. Conclusions: Incorporation of HDR functional analysis into an ACMG/AMP framework model substantially improves BRCA2 VUS re-classification and provides an important tool for determining the clinical relevance of individual BRCA2 VUS.