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Despite advances in genetic testing for Lynch syndrome, nearly one quarter of mismatch repair-deficient (MMRd) colorectal and endometrial cancers remain unexplained. When added to germline DNA testing, RNA sequencing can increase diagnostic yield, improve variant classification and reduce variants of uncertain significance. Here, we describe two cases where RNA sequencing uncovered likely pathogenic

Summary Determination of the clinical relevance of rare germline variants of uncertain significance (VUSs) in the BRCA2 cancer predisposition gene remains a challenge as a result of limited availability of data for use in classification models. However, laboratory-based functional data derived from validated functional assays of known sensitivity and specificity may influence the interpretation of VUSs. We evaluated 252 missense VUSs from the BRCA2 DNA-binding domain by using a homology-directed DNA repair (HDR) assay and identified 90 as non-functional and 162 as functional. The functional assay results were integrated with other available data sources into an ACMG/AMP rules-based classification framework used by a hereditary cancer testing laboratory. Of the 186 missense variants observed by the testing laboratory, 154 were classified as VUSs without functional data. However, after applying protein functional data, 86% (132/154) of the VUSs were reclassified as either likely pathogenic/pathogenic (39/132) or likely benign/benign (93/132), which impacted testing results for 1,900 individuals. These results indicate that validated functional assay data can have a substantial impact on VUS classification and associated clinical management for many individuals with inherited alterations in BRCA2.

Purpose Despite the rapid uptake of multigene panel testing (MGPT) for hereditary cancer predisposition, there is limited guidance surrounding indications for testing and genes to include. Methods To inform the clinical approach to hereditary cancer MGPT, we comprehensively evaluated 32 cancer predisposition genes by assessing phenotype-specific pathogenic variant (PV) frequencies, cancer risk associations, and performance of genetic testing criteria in a cohort of 165,000 patients referred for MGPT. Results We identified extensive genetic heterogeneity surrounding predisposition to cancer types commonly referred for germline testing (breast, ovarian, colorectal, uterine/endometrial, pancreatic, and melanoma). PV frequencies were highest among patients with ovarian cancer (13.8%) and lowest among patients with melanoma (8.1%). Fewer than half of PVs identified in patients meeting testing criteria for only BRCA1/2 or only Lynch syndrome occurred in the respective genes (33.1% and 46.2%). In addition, 5.8% of patients with PVs in BRCA1/2 and 26.9% of patients with PVs in Lynch syndrome genes did not meet respective testing criteria. Conclusion Opportunities to improve upon identification of patients at risk for hereditary cancer predisposition include revising BRCA1/2 and Lynch syndrome testing criteria to include additional clinically actionable genes with overlapping phenotypes and relaxing testing criteria for associated cancers.

PURPOSE The current diagnostic testing algorithm for Lynch syndrome (LS) is complex and often involves multiple follow-up germline and somatic tests. We aimed to describe the results of paired tumor/germline testing performed on a large cohort of patients with colorectal cancer (CRC) and endometrial cancer (EC) to better determine the utility of this novel testing methodology. MATERIALS AND METHODS We retrospectively reviewed a consecutive series of patients with CRC and EC undergoing paired tumor/germline analysis of the LS genes at a clinical diagnostic laboratory (N = 702). Microsatellite instability, MLH1 promoter hypermethylation, and germline testing of additional genes were performed if ordered. Patients were assigned to one of five groups on the basis of prior tumor screening and germline testing outcomes. Results for each group are described. RESULTS Overall results were informative regarding an LS diagnosis for 76.1% and 60.8% of patients with mismatch-repair–deficient (MMRd) CRC and EC without and with prior germline testing, respectively. LS germline mutations were identified in 24.8% of patients in the group without prior germline testing, and interestingly, in 9.5% of patients with previous germline testing; four of these were discordant with prior tumor screening. Upon excluding patients with MLH1 promoter hypermethylation and germline mutations, biallelic somatic inactivation was seen in approximately 50% of patients with MMRd tumors across groups. CONCLUSION Paired testing identified a cause for MMRd tumors in 76% and 61% of patients without and with prior LS germline testing, respectively. Findings support inclusion of tumor sequencing as well as comprehensive LS germline testing in the LS testing algorithm. Paired testing offers a complete, convenient evaluation for LS with high diagnostic resolution.

Despite advances in genetic testing for Lynch syndrome, nearly one quarter of mismatch repair-deficient (MMRd) colorectal and endometrial cancers remain unexplained. When added to germline DNA testing, RNA sequencing can increase diagnostic yield, improve variant classification and reduce variants of uncertain significance. Here, we describe two cases where RNA sequencing uncovered likely pathogenicMSH2variants in families with MMRd tumours that were initially unexplained following comprehensive genetic testing for Lynch syndrome.

Whether monoallelic MUTYH mutations increase female breast cancer risk remains controversial. This study aimed to determine if monoallelic MUTYH mutations are associated with increased breast cancer risk in women undergoing multigene panel testing (MGPT). The prevalence of monoallelic MUTYH mutations was compared between Non-Hispanic white female breast cancer cases (n = 30,456) and cancer-free controls (n = 12,289), all of whom underwent MGPT that included MUTYH. We tested breast cancer associations with MUTYH alleles using Fisher’s exact test, followed by multivariate logistic regression adjusted for age at testing and MGPT type ordered. Frequencies of the two most common MUTYH founder mutations, p.G396D and p.Y179C, were compared independently between the breast cancer cases and MGPT controls, as well as the healthy UK10K control population (n = 2640). Comparing cases to MGPT controls, no association was observed between female breast cancer and any monoallelic MUTYH carrier status (OR 0.86–1.36, p = 0.21–0.96). Similarly, comparisons to UK10K controls revealed no significant increase in breast cancer risk associated with p.G396D (OR 1.20, p = 0.44) or p.Y179C (OR 1.71, p = 0.24). This study did not find a significant increase in breast cancer risk associated with monoallelic MUTYH mutations.

We explored the germline mutation spectrum and prevalence among 1650 women with breast and uterine cancer (BUC) who underwent multi-gene hereditary cancer panel testing at a single commercial laboratory.The combined frequency of mutations in 23 BC and/or UC genes was compared between BUC cases and control groups with (1) no personal cancer history; (2) BC only; and (3) UC only using logistic regression.Fourteen percent (n = 231) of BUC cases tested positive for mutations in BC and/or UC genes and were significantly more likely to test positive than individuals with BC only (P 0.001), UC only (P 0.01), or unaffected controls (P 0.001). Analysis of gene-specific mutation frequencies revealed that MSH6, CHEK2, BRCA1, BRCA2, ATM, PMS2, PALB2 and MSH2 were most frequently mutated among BUC cases. Compared to BC only, BRCA1, MLH1, MSH2, MSH6, PMS2 and PTEN mutations were more frequent among BUC; however, only ATM mutations were more frequent among BUC compared to UC only. All of the more commonly mutated genes have published management guidelines to guide clinical care. Of patients with a single mutation in a gene with established testing criteria (n = 152), only 81.6% met their respective criteria, and 65.8% met criteria for multiple syndromes.Women with BUC are more likely to carry hereditary cancer gene mutations than women with breast or uterine cancer alone, potentially warranting expanded genetic testing for these women. Most mutations found via multi-gene panel testing in women with BUC have accompanying published management guidelines and significant implications for clinical care.

e13109 Background: Many multigene panel (MGP) options are available for hereditary cancer testing. Previously, management guidelines were only published for a few high-risk genes, but now such guidelines exist for most genes on MGPs. Many clinicians question which MGP(s) may be most appropriate for their patients. We examined the likelihood of a positive result for patients diagnosed with breast cancer (BC) or colorectal cancer (CRC) across several MGPs and assessed the potential impact of these results on patient care. Methods: Positive results, defined by the presence of at least one pathogenic or likely pathogenic variant, were assessed for patients with BC or CRC across various MGPs ordered 6/2012-6/2016 at one commercial laboratory. For BC, the MGPs used for comparison were a high-risk BC panel (up to 6 genes), a comprehensive BC panel (up to 17 genes), and a multi-cancer panel (up to 32 genes). For CRC, a comprehensive CRC panel (up to 17 genes) was compared to the 32 gene multi-cancer panel. Results: For both BC and CRC patients, the utilization of a MGP with more genes led to more positive results than a MGP with fewer genes, with the vast majority of additional findings occurring in genes with published management guidelines (Table). Conclusions: The majority of positive results occurring in genes on larger MGPs, but not smaller MGPs, are in genes with published management guidelines.These results can impact treatment decisions and cancer risk management, including earlier and/or more frequent screening, chemoprevention, and/or other measures. In some cases, positive results occurred in genes not associated with the patient’s cancer diagnosis, but may still impact risk management for other cancer(s). Further studies are needed to determine the impact of larger MGPs on clinical outcomes for patients and their families. [Table: see text]

1549 Background: The purpose of this study is to explore the mutation spectrum and prevalence among women with breast and uterine cancer (BUC) who were clinician-referred for multi-gene panel testi...