Comparative analysis of microsatellite instability by next-generation sequencing, MSI PCR and MMR...

Checkpoint inhibitor approval for microsatellite instability-high (MSI-H) tumours has made MSI as a therapeutically important biomarker. Next-generation sequencing (NGS)-based MSI detection is being widely used for assessing MSI. However, MSI tumours detected using NGS and their relevance to MSI-polymerase chain reaction (PCR) and mismatch repair deficiency (dMMR) are unclear. In 1942 solid cancer cases tested using NGS-based comprehensive cancer panel with 523 genes (1.94 mb), the MSI score, tumour mutation burden (TMB; ≥ 10 mutations/mb), and frameshift mutations were analysed. GeneScan analyses of five mononucleotide markers (MSI-PCR) and MMR protein immunohistochemistry (IHC) were compared with the NGS-MSI results. With a ≥ 12% MSI score as a cut-off for MSI-H, two MSS cases were classified as MSI-H. With a ≥ 20% cut-off, 10 cases categorised as MSS by NGS were MSI-H/dMMR by MSI-PCR and MMR IHC. To avoid discrepant cases, we adopted a high MSI cut-off and a borderline MSI category. Finally, MSI-H (≥ 20%), borderline MSI (≥ 7% and < 20%), and MSS (< 7%) were found in 35 (1.8%), 24 (1.2%), and 1883 (97%) cases, respectively. All MSI-H cases by NGS were MSI-H/dMMR by MSI-PCR and MMR IHC. Of the 24 borderline MSI cases by NGS, MSI-H/dMMR was 9 (37.5%) cases, MSS/dMMR was 1 (4.2%) case, and 11 (45.8%) of them had high TMB. All MSS cases by NGS were MSS/pMMR by MSI-PCR/IHC, and 257 (13.6%) had high TMB. With those arbitrary cut-off points, 10 (0.5%) MSS cases using NGS were discrepant with MSI-PCR or MMR IHC, and all were borderline MSI cases. The mean number of frameshift mutations was significantly higher in the MSI-H group (28.3) than in the borderline MSI (7.7) or MSS (1.3) groups (p < 0.001). In conclusion, to facilitate therapeutic decision-making for NGS, cut-off points for MSI can be defined based on MSI-PCR/dMMR confirmation.