DNA mismatch repair gene variant classification: evaluating the utility of somatic mutations and mismatch repair deficient colonic crypts and endometrial glands

Germline pathogenic variants in the DNA mismatch repair (MMR) genes (Lynch syndrome) predispose to colorectal (CRC) and endometrial (EC) cancer. Lynch syndrome specific tumor features were evaluated for their ability to support the ACMG/InSiGHT framework in classifying variants of uncertain clinical significance (VUS) in the MMR genes. Twenty-eight CRC or EC tumors from 25 VUS carriers (6x MLH1 , 9x MSH2 , 6x MSH6 , 4x PMS2 ), underwent targeted tumor sequencing for the presence of microsatellite instability/MMR-deficiency (MSI-H/dMMR) status and identification of a somatic MMR mutation (second hit). Immunohistochemical testing for the presence of dMMR crypts/glands in normal tissue was also performed. The ACMG/InSiGHT framework reclassified 7/25 (28%) VUS to likely pathogenic (LP), three (12%) to benign/likely benign, and 15 (60%) VUS remained unchanged. For the seven re-classified LP variants comprising nine tumors, tumor sequencing confirmed MSI-H/dMMR (8/9, 88.9%) and a second hit (7/9, 77.8%). Of these LP reclassified variants where normal tissue was available, the presence of a dMMR crypt/gland was found in 2/4 (50%). Furthermore, a dMMR endometrial gland in a carrier of an MSH2 exon 1-6 duplication provided further support for upgrade of this VUS to LP. Our study confirmed that identifying these Lynch syndrome features can improve MMR variant classification, enabling optimal clinical care. Simple Summary Lynch syndrome is caused by germline pathogenic variants in the DNA mismatch repair (MMR) genes predisposing carriers to colorectal and endometrial cancer. Genetic testing for Lynch syndrome, in the form of multigene panel testing, frequently identifies variants of uncertain clinical significance (VUS). These VUS have limited clinical actionability and create uncertainty for patients and clinicians regarding their risk of cancer. In this study, we tested carriers of germline VUS for features consistent with Lynch syndrome, namely 1) tumor microsatellite instability/MMR-deficiency, 2) the presence of a somatic second hit in the MMR gene harboring the VUS by tumor sequencing and 3) the presence of MMR-deficiency in normal colonic mucosa crypts or normal endometrial glands. Our findings showed that microsatellite instability/MMR-deficiency status and somatic second hits were consistent with MMR variant classifications as determined by the ACMG/InSiGHT framework. In addition to this, the presence of MMR-deficient crypts/glands were consistent with pathogenic variant classification. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This project was funded by a National Health and Medical Research Council of Australia (NHMRC) project grant GNT1125269 (PI- Daniel Buchanan). DDB is supported by an NHMRC Investigator grant (GNT1194896) and University of Melbourne Dame Kate Campbell Fellowship. RW is supported by the Margaret and Irene Stewardson Fund Scholarship and by the Melbourne Research Scholarship. PG is supported by the University of Melbourne Research Scholarship. MAJ is supported by an NHMRC Investigator grant (GNT1195099). The Colon Cancer Family Registry (CCFR, [www.coloncfr.org][1]) is supported in part by funding from the National Cancer Institute (NCI), National Institutes of Health (NIH) (award U01 CA167551). Support for case ascertainment was provided in part from the Surveillance, Epidemiology, and End Results (SEER) Program and the following U.S. state cancer registries: AZ, CO, MN, NC, NH; and by the Victoria Cancer Registry (Australia) and Ontario Cancer Registry (Canada). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All studies were approved by the Human Research Ethics Committees at The University of Melbourne (HREC#1750748) and hospitals governing participating family cancer clinics. Written informed consent was obtained from all participants. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors [1]: http://www.coloncfr.org