Real-World Evaluation of Microsatellite Instability Detection via Targeted NGS Panels in Routine Molecular Diagnostics

Microsatellite instability (MSI) is a clinically important biomarker for predicting responses to immune checkpoint inhibitors and identifying individuals with Lynch syndrome. Although MSI detection has been incorporated into Illumina's next-generation tumor sequencing workflows, interpretation of the results remains challenging due to the absence of standardized thresholds and reporting criteria. In this retrospective study, we assessed the performance of MSI detection using Illumina's targeted NGS panels-TruSight Tumor 170 and TruSight Oncology 500. The NGS-based MSI results were compared to those obtained by the reference method, MSI-PCR, across multiple tumor types in a real-world cohort of 331 cancer patients. The NGS method demonstrated high concordance overall (AUC = 0.922), though sensitivity was lower in colorectal cancers (AUC = 0.867) due to broader score variability and overlapping distributions. Our findings support the clinical utility of Illumina's NGS-derived MSI scores for identifying MSI-H tumors, with a recommended MSI score cut-off value of ≥13.8%. Additionally, a borderline group was introduced, defined by an MSI score ranging from ≥8.7% to <13.8%. Within this range, the integration of TMB into the MSI classification workflow significantly improves diagnostic accuracy. For samples that remain inconclusive, orthogonal confirmation using MSI-PCR is advised to ensure accurate MSI classification.