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Background: DNA genetic testing is commonly used to inform treatment decisions for breast cancer patients. In recent years, RNA genetic testing has shown promise for increasing the detection of disease-causing variants and decreasing inconclusive results. Here we describe the impact of concurrent DNA and RNA genetic testing on identifying breast cancer patients with germline cancer predisposition who may have been missed by DNA-only testing. Methods: We performed a retrospective review of breast cancer patients who received concurrent DNA and RNA hereditary cancer panel testing between March 2019 and February 2020 at Ambry Genetics. Patients underwent DNA and RNA genetic testing of up to 18 hereditary cancer genes at the discretion of the ordering healthcare provider (APC, ATM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MLH1, MSH2, MSH6, MUTYH, NF1, PALB2, PMS2 exons 1-10, PTEN, RAD51C, RAD51D, and TP53). Breast cancer patients with positive results, defined as the presence of a pathogenic or likely pathogenic variant in any of these 18 genes, were selected for inclusion in this study (n=946). Results: Concurrent DNA and RNA genetic testing led to the identification of 23 breast cancer patients with positive results who would have otherwise received inconclusive or negative results with DNA-only testing. These cases represented 2.4% of all positive results reported in the 18 genes studied (n=23/946). The majority of RNA-related positive results occurred in either ATM (n=14) or BRCA1/2 (n=7). The remaining two cases involved alterations in NF1 and PMS2. Guidelines for risk-reducing breast surgery (BRCA1/2) and breast imaging surveillance (ATM, BRCA1/2, NF1) were relevant for 30.4% and 95.7% of patients with RNA-dependent positive results, respectively. In addition, treatment options such as PARP inhibitors or clinical trial eligibility were potentially implicated for 91.3% of RNA-dependent positives (BRCA1/2, ATM). In 16 of the 23 cases, variants would have been detected by DNA-only testing but would have remained inconclusive without supporting RNA data. In the remaining 7 cases, abnormal RNA results led to the identification of pathogenic/likely pathogenic intronic variants beyond the analytical range of DNA testing. Thus, these seven patients would have received negative results from DNA-only testing. Four of these cases involved pathogenic/likely pathogenic intronic variants in ATM and three involved pathogenic intronic variants in BRCA1. Conclusions: One in 41 breast cancer patients who test positive on concurrent DNA and RNA genetic testing would have received negative or inconclusive results from DNA-only testing. These findings demonstrate the impact of a comprehensive diagnostic testing approach that includes concurrent DNA and RNA analysis and highlights the important implications for the personalized management of these breast cancer patients including potential missed opportunities for early detection and prevention of additional cancer and familial testing in the absence of RGT. Citation Format: Holly LaDuca, Lily Hoang, Carolyn Horton, Jessica Profato, Tameron Harvell, Jill S Dolinsky, Tina Pesaran, Rachid Karam. Rna genetic testing improves detection of patients with hereditary breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD10-07.

BACKGROUND: Germline genetic testing is routinely incorporated into clinical care for breast cancer patients to inform management decisions and reduce risk for developing subsequent cancers. While the diagnostic yield of cancer genetic testing has increased over the years due to adoption of multigene panels, a substantial portion of breast cancer patients remain without a molecular diagnosis yet are suspected to have a genetic mutation that could not be detected and/or classified with standard DNA testing techniques. We assessed the ability of a novel genetic testing approach involving simultaneous DNA and RNA analysis to increase the diagnostic yield and decrease the number of variants of unknown significance (VUS). METHODS: Women with a personal history of breast cancer were ascertained from a larger cohort of patients referred for concurrent RNA sequencing alongside DNA hereditary cancer panel testing by ordering clinicians from 18 collaborating medical centers across the United States. Test result classifications were evaluated for women whose testing included sixteen clinically-actionable hereditary breast and/or ovarian cancer (HBOC) genes (ATM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MLH1, MSH2, MSH6, NF1, PALB2, PMS2, PTEN, RAD51C, RAD51D, and TP53). RESULTS: In this cohort of 746 breast cancer patients, the addition of RNA sequencing increased the pathogenic variant detection rate from 8% to 9% across sixteen HBOC genes. These RNA-related positive results included two pathogenic variants in BRCA1 occurring outside the standard analytical range of DNA testing and three VUS (one each in ATM, BRCA2, and PMS2) that were reclassified as likely pathogenic as a result of additional information provided by RNA sequencing. In addition, two VUS were reclassified to benign/likely benign (one each in MSH2 and BRCA2). Together, these five variant reclassifications contributed to a 3% relative decrease in the number of unique VUS classifications (reduced from 182 to 177 unique VUS). In addition, 31 previously-tested patients received reclassification reports. CONCLUSIONS: Concurrent DNA and RNA genetic testing has shown immediate promise in this pilot study, leading to the identification of five breast cancer patients with mutations in clinically actionable genes that would otherwise have received inconclusive or negative results with DNA testing alone. By increasing the detection of germline pathogenic variants and reducing VUS classifications, concurrent DNA and RNA genetic testing increases the diagnostic yield and clinical impact of hereditary cancer testing for breast cancer patients. Citation Format: Holly LaDuca, Lily Hoang, Jill Dolinsky, Jessica Profato, Amal Yussuf, Carolyn Horton, Cara Dresbold, Cassie Garcia, Catherine Koptiuch, Danielle Dondanville, Danielle McKenna, Danielle Menashe, Deborah Wham, Deepika Nathan, Diane Samad, Elizabeth Hoodfar, Gayle Patel, Jen Moore, Jennifer Geurts, John Lee, Kara Milliron, Khateriaa Pyrtel, Meagan Farmer, Meredith Seidel, Morgan Depas, Nichole Morman, Olivia Tan, Rebekah Krukenberg, Rob Pilarski, Samantha Stachowiak, Sandra Jenkinson, Sara Pirzadeh-Miller, Shraddha Gaonkar, Tiffani Demarco, Brigette Tippin Davis, Elizabeth C Chao, Rachid Karam. Concurrent DNA and RNA genetic testing identifies more patients with hereditary breast cancer than DNA testing alone [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-08-08.

Genetic testing for hereditary breast and ovarian cancer (HBOC) is becoming increasingly widespread in the era of precision medicine. The implementation of next-generation sequencing (NGS) has resulted in an explosion of genetic data. While the majority of patients receive definitive results, germline unclassified variants with unknown function are regularly detected in thousands of patients. In particular, variants of unknown significance (VUS) in the HBOC susceptibility genes BRCA1 and BRCA2 pose a quandary to medical providers and patients because these genes are clinically actionable. A large percentage of VUS in BRCA1/2 are predicted to affect splicing. Previous efforts have focused on interrogating splicing VUS using low-throughput and/or imprecise techniques. Therefore, we developed and validated a method for using RT-PCR NGS to accurately and efficiently characterize germline splicing defects. The ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium recommends capillary electrophoresis and Sanger sequencing of subcloned transcripts as the gold standard to identify the BRCA1/2 transcripts that are present in patient samples. We followed the recommendations of the ENIGMA consortium and were able to largely replicate their results when we analyzed the cDNA of lymphoblastoid cell lines (LCLs) generated by the kConFab consortium from carriers of BRCA1 or BRCA2 variants known to be associated with splicing defects. In addition to LCLs, we analyzed RNA extracted from normal blood controls and normal breast tissue for the entire BRCA1 gene. We compared our capillary electrophoresis and Sanger sequencing results to our newly developed RT-PCR NGS assay. For this assay, we used RT-PCR NGS coupled to our custom in-house bioinformatics analysis to identify splicing events including exon skipping, novel intron insertion, and alternative 5’ donor and 3’ acceptor splicing sites. Capillary electrophoresis allowed us to roughly visualize the various transcripts present in our samples, though sequencing was needed to confidently identify the exact splicing event. Similar to Sanger sequencing, our RT-PCR NGS assay detected all major splicing events. Interestingly, we found that RT-PCR NGS allowed us to visualize more minor splicing events present in the samples due to our high coverage. We sequenced tens of thousands of reads with RT-PCR NGS, as opposed to Sanger sequencing, which was limited to low-throughput sequencing of single colonies containing subcloned RT-PCR products. In conclusion, RT-PCR NGS is a reliable high-throughput alternative to the gold-standard splicing assays. Our assay will greatly improve the interpretation of splicing VUS detected by clinical genetic tests for BRCA1/2. Citation Format: Suzette Farber-Katz, Vickie Hsuan, Jayne Hoo, Sitao Wu, Huy Vuong, Dong Xu, Hsiao-Mei Lu, Phillip Gray, Aaron Elliott, Rachid Karam. Alternative splicing analysis identifies mutation hotspots in hereditary breast and ovarian cancer genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1450. doi:10.1158/1538-7445.AM2017-1450