Clinical testing for mismatch repair (MMR) deficiency often entails serial testing of tumor and constitutional DNA using multiple assays. To minimize cost and specimen requirements of MMR testing, we developed an integrated targeted sequencing protocol (termed MultiMMR) that tests for promoter methylation, mutations, copy number alterations, copy neutral loss of heterozygosity, and microsatellite instability from a single aliquot of DNA. Hybrid capture of DNA-sequencing libraries constructed with methylated adapters was performed on 142 samples (60 tumors and 82 constitutional samples) from 82 patients with MMR-associated colorectal, endometrial, and brain cancers as well as a synthetic DNA mix with 11 known mutations. The captured material was split to enable parallel bisulfite and conventional sequence analysis. The panel targeted microsatellite regions and 13 genes associated with MMR, hypermutation, and hereditary colorectal cancer. MultiMMR recapitulated clinical testing results in 23 of 24 cases, was able to explain MMR loss in an additional 29 of 48 patients with incomplete or inconclusive testing, and identified all 11 MMR variants within the synthetic DNA mix. Promoter methylation and microsatellite instability analysis found 95% and 97% concordance with clinical testing, respectively. We report the feasibility for amalgamation of the current stepwise and complex clinical testing workflow into an integrated test for hereditary and somatic causes of MMR deficiency.
We thank all study participants for providing specimens for analysis within this study, the staff at the Princess Margaret Genomics Centre (Troy Ketela, Neil Winegarden, Julissa Tsao, and Nick Khuu) and Bioinformatics Services (Carl Virtanen and Zhibin Lu) for their expertise in generating the sequencing data used within this study, the Advanced Molecular Diagnostics Laboratory at the Princess Margaret Cancer Centre for performing MS-MLPA on a subset of our cases (Suzanne Kamel-Reid, Tracy Stockley, and Sylvie Grenier), and Michael Hoffman for his advice and editing of the manuscript.