MEM: An Algorithm for the Reliable Detection of Microsatellite Instability (MSI) on a Small NGS Panel in Colorectal Cancer

SIMPLE SUMMARY: Microsatellite instability (MSI) assessment has become a major issue in the management of colorectal cancer, with the recent approval of anti-PD1 immunotherapies in MSI-metastatic colorectal cancer. The reference PCR method (MSI-PCR) can be costly, time and tissue-consuming. However, NGS could facilitate the assessment of MSI status while simultaneously screening for targetable oncogenic mutations (KRAS, NRAS, BRAF) for any colorectal cancer, but the algorithms developed to date use a large number of microsatellites that have not been approved by international guidelines and which are generally incompatible with small NGS panels. We present the MEM algorithm, which mimics the interpretation of MSI-PCR data by a human operator to reliably assess MSI status using only five validated microsatellites (BAT-25, BAT-26, NR-21, NR-24 and NR-27). We demonstrated that the MEM algorithm was in perfect agreement with MSI-PCR results, in terms of both MSI status and individual microsatellite status, in a cohort of 146 patients. ABSTRACT: Purpose: MEM is an NGS algorithm that uses Expectation-Maximisation to detect the presence of unstable alleles from the NGS sequences of five microsatellites (BAT-25, BAT-26, NR-21, NR-24 and NR-27). The purpose of this study was to compare the MEM algorithm with a reference PCR method (MSI-PCR) and MisMatch Repair protein immunohistochemistry (MMR-IHC). Methods: FFPE colorectal cancer samples from 146 patients were analysed in parallel by MSI-PCR and NGS using the MEM algorithm. MMR-IHC results were available for 133 samples. Serial dilutions of an MSI positive control were performed to estimate the limit of detection. Results: the MEM algorithm was able to detect unstable alleles of each microsatellite with up to a 5% allelic fraction. Of the 146 samples, 28 (19.2%) were MSI in MSI-PCR. MEM algorithm results were in perfect agreement with those of MSI-PCR, at both MSI status and individual microsatellite level (Cohen’s kappa = 1). A high level of agreement was noted between MSI-PCR/MEM algorithm results and MMR-IHC results (Cohen’s kappa = 0.931). Conclusion: the MEM algorithm can determine the MSI status of colorectal cancer samples on a small NGS panel, using only five microsatellites approved by international guidelines, and can be combined with screening for targetable mutations.