Mutation Burden and I Index for Detection of Microsatellite Instability in Colorectal Cancer by Targeted Next-Generation Sequencing

Abstract

Next-generation sequencing (NGS) panels are widely used for defining tumor mutation profiles and determining treatment approaches. We performed targeted NGS with 382 colorectal cancer genes with known microsatellite instability (MSI). After exclusion of germline alterations, the load of somatic mutations and small insertion/deletion (indel) alterations were determined. In the test set, 79 patients with 41 microsatellite-stable (MSS) and 38 MSI tumors were included. There were 120 MSS and eight MSI-high tumors in the validation set. The number of somatic mutations of whole samples were distinguished into three groups: mutant functional polymerase epsilon catalytic subunit, MSI, and MSS tumors. The median numbers of somatic and indel mutations in MSI tumors were higher. The indel mutation to whole mutation ratio (I index) was higher in MSI tumors. Hypermutation and low I index of polymerase epsilon catalytic subunit mutant tumors, a somatic mutation load cutoff of 40, and an I index of 9% were selected as the criteria for detecting MSI tumors with high sensitivity and specificity. With the analysis of alteration patterns of homopolymer genes, a higher median number of homopolymer mutations in MSI tumors was observed. Mutated homopolymer 5 was selected as the criterion for detecting MSI tumors. MSI in colorectal cancer can be detected by targeted NGS panels with high sensitivity and specificity using somatic mutation load and I index.

Spontaneous mutations in the single TTN gene represent high tumor mutation burden

“…A high correlation was typically observed in the tumors known as hypermutated tumor types, such as SKCM, CRC, UCEC, LUAD, BLCA, that frequently possess more than 10 mutations per Mb. 21 , 30 However, TTN-TMB revealed low correlation coefficients in tumor types that possessed low mutation rates, including breast, kidney, and thyroid tumors. Therefore, TTN-TMB may possess limitations in regard to those tumor types.…”

“…For the validation cohort, 24 patients with CRC were selected from our previous study 21 according to the following criteria: (1) tumors were evaluated using PCR-based analysis of microsatellite loci and (2) tNGS data were available. Of the 24 CRCs, 14 (58.3%) were the MSI-H phenotype, as confirmed by MSI PCR analysis, and the remaining 10 (41.7%) were microsatellite stable (MSS), with a POLE pathogenic mutation in one case.…”

“…BAM files from the tNGS for the AMC cohort ( n = 24 CRCs) were obtained from our previous study. 21 Detailed information on the targeted sequencing method has been described previously. 21 Briefly, the targeted NGS panel, OncoPanel AMC version 3 (OP_AMCv3), was designed at AMC using SureDesign (Agilent Technologies, USA) and Genome Reference Consortium Human Build 37 (GRCh37) as the reference genome.…”

Comprehensive routine diagnostic screening to identify predictive mutations, gene amplifications, and microsatellite instability in FFPE tumor material

“…MSI in a non-MSIprone tumor or a KIT mutation in melanoma, which might be beneficial for treatment options [5,51,52]. Previously, simultaneous detection of mutations with either amplifications or MSI using targeted NGS was described [10,13,19,[53][54][55]. To our knowledge this is the first small smMIP-based NGS panel that allows concurrent analysis of mutations, amplifications, and MSI by numerous microsatellite loci.…”

Mismatch Repair Pathway, Genome Stability and Cancer

“…Such inappropriate number of repeats suggests the dysfunctional DNA mismatch repair in tumor cells. Several papers (Nowak et al, 2017;Zhu et al, 2018;Kim et al, 2019;Pang et al, 2019;Yamamoto and Imai, 2019;Rosenthal et al, 2020) report on comparisons between the classical diagnostic methods, and next generation sequencing (NSG) or targeted next generation sequencing. They show that next generation sequencing and targeted next generation sequencing can also accurately detect MSI.…”