Using target capture sequencing detect microsatellite instability(MSI), tumor mutational burden(TMB) and POLE/POLD1 mutations in colorectal cancer.

Abstract

e15012 Background: Microsatellite instability-High(MSI-H) or mismatch repair protein deficiency (dMMR) is a biomarker to predict immunotherapy response, which is approved by the US Food and Drug Administration in pembrolizumab. Here we describe the comparisons of three methods (IHC\PCR\NGS) in testing MSI or dMMR and also compare the association between MSI, TMB and POLE/POLD1 mutations in colorectal cancer(CRC). Methods: Cases of CRC and matched normal samples were sequenced with 1021 genes panel. TMB was calculated by counting nonsynonymous mutations across a 1Mb coding region. MSI status was analyzed by MSIsensor. TMB-H cutoff was defined more than 20 Muts/Mb referring to the distribution of TMB in MSI-H and MSS pts in cohort 1. Results: 94 samples with MSI status (64 dMMR and 30 pMMR) previously determined by IHC were applied to PCR and NGS. Based on PCR results, the concordance of NGS and IHC was 100% and 91.5%. 8 inconsistent dMMR samples from IHC were proved to be pMMR after review. 1 TMB-H &MSS pts were identified besides 56 TMB-H & MSI-H pts. In 57 TMB-H pts, 32 (56.1%) had POLD1/POLE mutations, and an average of 1.6 mutations was detected per pts. However, none POLD1/POLE mutations were found in 37 TMB-L pts. To verify if POLD1/POLE mutations exist in TMB-H pts specifically, 207 sporadic colorectal cancer samples, including 89 tissues and 118 plasmas, were analyzed. POLD1/POLE mutations appeared in 6 samples: 4 plasma samples (bTMB 20, 29, 57 and 178 Muts/Mb), 2 tissue samples (tTMB 69 and 3 Muts/Mb). The only one found in TMB-L pts was a POLE mutation (p.K2073I, MAF = 4.6%). Conclusions: NGS is a reliable platform to detect MSI status, TMB, and POLD1/POLE mutations. In colorectal cancer, POLD1/POLE mutations have the potential to be an independent biomarker to predict immunotherapy response. Overall, TMB is the biomarker which enables more patients to get benefit from immunotherapy.