Performance of Next Generation Sequencing for the Detection of Microsatellite Instability in Colorectal Cancer with Deficient DNA Mismatch Repair.

ABSTRACT Background & Aims Next generation sequencing (NGS) was recently approved by the FDA to detect microsatellite instability (MSI) arising from defective mismatch repair (dMMR) in patients with metastatic colorectal cancer (mCRC) prior to treatment with immune checkpoint inhibitors (ICI). In this study, we aimed to evaluate and improve the performance of NGS to identify MSI in CRC, especially dMMR mCRC treated with ICI. Methods CRC samples used in this post-hoc study were reassessed centrally for MSI and dMMR status using the reference methods of pentaplex PCR and immunohistochemistry (IHC). Whole exome (WES) was used to evaluate MSISensor, the FDA-approved and NGS-based method for assessment of MSI. This was performed in (i) a prospective, multicenter cohort (C1) of 102 mCRC patients (25 dMMR/MSI, 24 treated with ICI) from clinical trials NCT02840604 and NCT033501260, (ii) an independent retrospective, multicenter cohort of 113 patients (C2, 25 mCRC, 88 non-mCRC, all dMMR/MSI untreated with ICI), (iii) and a publicly available series of 118 CRC patients from the TCGA (C3, 51 dMMR/MSI). A new NGS-based algorithm, namely MSICare, was developed. Its performance for assessment of MSI was compared to MSISensor in C1, C2 and C3 at the exome-level or after downsampling sequencing data to the MSK-ImpactTM gene panel. MSICare was validated in an additional retrospective, multicenter cohort (C4) of 152 new CRC patients (137 dMMR/MSI) enriched in MSH6 and PMS2 deficient tumors (35 dMSH6, 9 dPMS2) following targeted sequencing of samples with an optimized set of microsatellite markers (MSIDIAG). Results At the exome-level, MSISensor was highly specific but failed to diagnose MSI in 16% of MSI/dMMR mCRC from C1 (4/25; sensitivity 84%, 95%CI: 63.9%-95.5%), 32% of mCRC (8/25; sensitivity 68%, 95%CI: 46.5%-85.1%) and 9.1% of nmCRC from C2 (8/88; sensitivity 90.9%, 95%CI: 82.9%-96%), and 9.8% of CRC from C3 (5/51; sensitivity 90.2%, 95%CI: 78.6%-96.7%). Misdiagnosis included 4 mCRCs treated with ICI of which 3 showed an overall response rate without progression at this date. At the exome-level, reevaluation of the MSI genomic signal using MSICare detected 100% of cases with true MSI status amongst C1 and C2. Further validation of MSICare was obtained in CRC tumors from C3, with 96.1% concordance for MSI status. Whereas misdiagnosis with MSISensor even increased when analyzing downsampled WES data from C1 and C2 with microsatellite markers restricted to the MSK-Impact gene panel (sensitivity 72.5%, 95%CI: 64.2-79.7%), particularly in MSH6 deficient setting, MSICare sensitivity and specificity remained optimal (100%). Similar results were obtained with MSICare following targeted NGS of tumors from C4 with the optimized microsatellite panel MSIDIAG (sensitivity 99.3%, 95%CI: 96%-100%; specificity 100%). Conclusions In contrast to MSISensor, the new MSICare test we propose performs at least as efficiently as the reference method, MSI PCR, to detect MSI in CRC regardless of the defective MMR protein under both WES and targeted NGS conditions. We suggest MSICare may become rapidly a reference method for NGS-based testing of MSI in CRC, especially in mCRC where accurate MSI status is required before the prescription of ICI.