Assessment of microsatellite instability (MSI) status in colorectal cancer tissue using a next generation sequencing-based tumor profiling assay

Background: MSI status, along with PD-L1 expression and tumor mutational burden (TMB), is an important predictive biomarker for immune checkpoint inhibitors. Recent studies demonstrated that next-generation sequencing (NGS)-based MSI assays can simultaneously detect MSI status and variants in other target genes. We developed and validated an NGS-based MSI assay that utilizes data from a 49 gene panel used for solid tumor profiling. Here we report the concordance of this NGS-MSI assay with 2 commonly used PCR fragment-based methods for MSI status. We also evaluated the relationship between NGS-MSI status and TMB as assessed with the 49-gene solid-tumor profiling assay. Method: This study used de-identified, formalin-fixed, paraffin embedded tumor tissue and adjacent normal tissue from 55 patients with colorectal cancer. All specimens were tested with a hybrid capture-based 49-gene NGS assay on the Illumina NextSeq500 platform. Components of the MSIsensor software v0.2 and a Fisher’s Exact test were used to discriminate the repeat distributions between the tumor and normal specimens on the following MSI markers: BAT25, BAT26, NR21, NR24, and NR27. Specimens demonstrating instability in 2 or more the 5 markers were considered MSI positive (MSI-H), and others were considered MSI negative (MSI-L/MSS).NGS-MSI results were compared to those of the Bethesda NCI panel (BAT25, BAT-26, D5S346, D2S123 and D17S250) (55 patients) and the Promega MSI test (BAT25, BAT26, NR21, NR24 and Mono27) (52 patients). TMB was compared between NGS-MSI-positive and NGS-MSI-negative specimens. Results: The NGS-MSI assay showed strong concordance with both the Bethesda NCI panel and the Promega MSI test. Concordance between the NGS-MSI and Bethesda-MSI test was 97% (53/55): 26 specimens were concordant for MSI positive and 27 were concordant for MSI negative, while 2 were MSI negative by NGS-MSI but MSI-H by Bethesda-MSI. Concordance between the NGS-MSI and Promega-MSI tests was 98% (51/52): 25 specimens were MSI positive/MSI-H and 26 were MSI negative/MSS or MSI-l. The discordant specimen was MSI-negative by NGS-MSI and MSI-H by Promega-MSI. The mean TMB was higher in MSI-positive (11.3 mutations/specimen) than in NGS-MSI-negative specimens (2.5 mutations/specimen). The mutations and its relationship with MSI status in 4 DNA mismatch repair genes (MLH1, PMS2, MSH2 and MSH6) were under analysis. Conclusion: The NGS-based MSI assay, performed as part of a 49-gene NGS tumor profiling test, demonstrated strong concordance with the Bethesda and Promega MSI tests. As expected, MSI-positive tumor specimens had higher TMB than did MSI-negative samples. Citation Format: Kevin Z. Qu, Suzzette Arnal, Sirisha Sunkara, Mohammad R. Sheikholeslami, Zhong J. Zhang, Felicitas L. Lacbawan, Charles Ma, Sugganth Daniel. Assessment of microsatellite instability (MSI) status in colorectal cancer tissue using a next-generation sequencing-based tumor profiling assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4026.