Citations

Abstract Background: Germline genetic testing of individuals with a diagnosis of triple negative breast cancer, young age at diagnosis, or a family history of breast and/or ovarian cancer has led to the identification of many unique BRCA2 missense variants of uncertain significance (VUS). VUS in BRCA2 are predominantly missense mutations that have unclear relevance to breast, ovarian, and other cancers. Thus, patients found to have a germline BRCA2 VUS do not know if the variant is associated with high risks of these cancers similar to truncating mutations, intermediate risks more similar to CHEK2 mutations, or low risks of no clinical significance. Furthermore, it is unclear if germline BRCA2 VUS, or somatic VUS identified by tumor sequencing, are associated with hypersensitivity to selected DNA damaging and cross-linking agents. Methods: We have used a homology directed repair (HDR) cell-based assay to characterize missense variants in the DNA binding domain (DBD) of BRCA2. The method has been validated using known pathogenic and known non-pathogenic BRCA2 missense variants and has 100% sensitivity (95% confidence interval (CI): 75.3%–100%) and 100% specificity (95% CI: 81.5%–100%) for pathogenic BRCA2 variants. A classifier of variant pathogenicity based on the mean and variances of the distributions of the HDR results of the known pathogenic and neutral variants has been established. We have also developed PARP inhibitor and cisplatin drug response assays for BRCA2 missense variants. Results: Assessment of 207 BRCA2 missense variants, identified in public databases such as BRCA exchange and ClinVar, by the HDR assay identified 71 deleterious variants with >99% probability of pathogenicity, 116 neutral variants with >99% probability of neutrality, and 20 with hypomorphic activity and potentially intermediate risk. A combination of the functional data and sequence-based predictors of protein activity in a Bayesian prediction model resulted in classification of the deleterious variants as pathogenic cancer predisposing variants and the neutral variants as non-pathogenic with low clinical significance. The influence of the deleterious/pathogenic variants on PARPi and cisplatin response was also assessed. Conclusion: The HDR assay is effective for characterization of BRCA2 VUS. The combination of functional data and in silico prediction models provides a robust tool for clinical annotation of BRCA2 VUS. HDR function of BRCA2 missense variants is strongly correlated with response to targeted therapy. Citation Format: Couch FJ, Shimelis H, Hart SN, Moore RM, Thomas A, Lipton GB, Iversen E. Cancer risks and response to targeted therapy associated with BRCA2 variants of uncertain significance [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS4-06.

Abstract Breast cancer is the most common cancer among women in the United States, with up to 30% of those diagnosed displaying a family history of breast cancer. To date, 18% of the familial risk of breast cancer can be explained by SNPs. This review summarizes the discovery of risk-associated SNPs using candidate gene and genome-wide association studies (GWAS), including discovery and replication in large collaborative efforts such as The Collaborative Oncologic Gene-environment Study and OncoArray. We discuss the evolution of GWAS studies, efforts to discover additional SNPs, and methods for identifying causal variants. We summarize findings associated with overall breast cancer, pathologic subtypes, and mutation carriers (BRCA1, BRCA2, and CHEK2). In addition, we summarize the development of polygenic risk scores (PRS) using the risk-associated SNPs and show how PRS can contribute to estimation of individual risks for developing breast cancer. Cancer Epidemiol Biomarkers Prev; 27(4); 380–94. ©2018 AACR. See all articles in this CEBP Focus section, “Genome-Wide Association Studies in Cancer.”

Background Missense (amino-acid changing) variants found in cancer predisposition genes often create difficulties when clinically interpreting genetic testing results. Although bioinformatics has developed approaches to predicting the impact of these variants, many of these approaches have not been readily applicable in the clinical setting. Bioinformatics approaches for predicting the impact of these variants have not yet found their footing in clinical practice because 1) interpreting the medical relevance of predictive scores is difficult; 2) the relationship between bioinformatics “predictors” (sequence conservation, protein structure) and cancer susceptibility is not understood. Methodology/Principal Findings We present a computational method that produces a probabilistic likelihood ratio predictive of whether a missense variant impairs protein function. We apply the method to a tumor suppressor gene, BRCA2, whose loss of function is important to cancer susceptibility. Protein likelihood ratios are computed for 229 unclassified variants found in individuals from high-risk breast/ovarian cancer families. We map the variants onto a protein structure model, and suggest that a cluster of predicted deleterious variants in the BRCA2 OB1 domain may destabilize BRCA2 and a protein binding partner, the small acidic protein DSS1. We compare our predictions with variant “re-classifications” provided by Myriad Genetics, a biotechnology company that holds the patent on BRCA2 genetic testing in the U.S., and with classifications made by an established medical genetics model [ 1 ]. Our approach uses bioinformatics data that is independent of these genetics-based classifications and yet shows significant agreement with them. Preliminary results indicate that our method is less likely to make false positive errors than other bioinformatics methods, which were designed to predict the impact of missense mutations in general. Conclusions/Significance Missense mutations are the most common disease-producing genetic variants. We present a fast, scalable bioinformatics method that integrates information about protein sequence, conservation, and structure in a likelihood ratio that can be integrated with medical genetics likelihood ratios. The protein likelihood ratio, together with medical genetics likelihood ratios, can be used by clinicians and counselors to communicate the relevance of a VUS to the individual who has that VUS. The approach described here is generalizable to regions of any tumor suppressor gene that have been structurally determined by X-ray crystallography or for which a protein homology model can be built.

Gene‐panel sequencing allows comprehensive analysis of multiple genes simultaneously and is now routinely used in clinical mutation testing of high‐risk breast and ovarian cancer patients. However, only BRCA1 and BRCA2 are often analyzed also for large genomic changes. Here, we have analyzed 10 clinically relevant susceptibility genes in 95 breast or ovarian cancer patients with gene‐panel sequencing including also copy number variants (CNV) analysis for genomic changes. We identified 12 different pathogenic BRCA1, BRCA2, TP53, PTEN, CHEK2, or RAD51C mutations in 18 of 95 patients (19%). BRCA1/2 mutations were observed in 8 patients (8.4%) and CHEK2 protein‐truncating mutations in 7 patients (7.4%). In addition, we identified a novel duplication encompassing most of the RAD51C gene. We further genotyped the duplication in breast or ovarian cancer families (n = 1149), in unselected breast (n = 1729) and ovarian cancer cohorts (n = 553), and in population controls (n = 1273). Seven additional duplication carries were observed among cases but none among controls. The duplication associated with ovarian cancer risk (3/590 of all ovarian cancer patients, 0.5%, P = .032 compared with controls) and was found to represent a large fraction of all identified RAD51C mutations in the Finnish population. Our data emphasizes the importance of comprehensive mutation analysis including CNV detection in all the relevant genes.

AbstractThe purpose of this study is to identify regions of spinal column in which more than three contiguous vertebrae can be reliably and quickly aligned within 1 mm using a 6‐degree (6D) couch and full body immobilization device. We analyzed 45 cases treated over a 3‐month period. Each case was aligned using ExacTrac x‐ray positioning system with integrated 6D couch to be within 1° and 1 mm in all six dimensions. Cone‐Beam computed tomography (CBCT) with at least 17.5 cm field of view (FOV) in the superior–inferior direction was taken immediately after ExacTrac positioning. It was used to examine the residual error of five to nine contiguous vertebrae visible in the FOV. The residual error of each vertebra was determined by expanding/contracting the vertebrae contour with a margin in millimeter integrals on the planning CT such that the new contours would enclose the corresponding vertebrae contour on CBCT. Submillimeter initial setup accuracy was consistently achieved in 98% (40/41) cases for a span of five or more vertebrae starting from T2 vertebra and extending caudally to S5. The curvature of spinal column along the cervical region and cervicothoracic junction was not easily reproducible between treatment and simulation. Fifty‐seven percent (8/14) of cases in this region had residual setup error of more than 1 mm in nearby vertebrae after alignment using 6D couch with image guidance. In conclusion, 6D couch integrated with image guidance is convenient and accurately corrects small rotational shifts. Consequently, more than three contiguous vertebrae can be aligned within 1 mm with immobilization that reliably reproduces the curvature of the thoracic and lumbar spinal column. Ability of accurate setup is becoming less a concern in limiting the use of stereotactic radiosurgery or stereotactic body radiation therapy to treat multilevel spinal target.