Colon and endometrial cancers with mismatch repair deficiency can arise from somatic, rather than germline, mutations

Abstract

Background & aims: Patients with Lynch syndrome carry germline mutations in single alleles of genes encoding the mismatch repair (MMR) proteins MLH1, MSH2, MSH6, and PMS2; when the second allele becomes mutated, cancer can develop. Increased screening for Lynch syndrome has identified patients with tumors that have deficiency in MMR, but no germline mutations in genes encoding MMR proteins. We investigated whether tumors with deficient MMR had acquired somatic mutations in patients without germline mutations in MMR genes using next-generation sequencing.

Methods: We analyzed blood and tumor samples from 32 patients with colorectal or endometrial cancer who participated in Lynch syndrome screening studies in Ohio and were found to have tumors with MMR deficiency (based on microsatellite instability and/or absence of MMR proteins in immunohistochemical analysis, without hypermethylation of MLH1), but no germline mutations in MMR genes. Tumor DNA was sequenced for MLH1, MSH2, MSH6, PMS2, EPCAM, POLE, and POLD1 with ColoSeq and mutation frequencies were established.

Results: Twenty-two of 32 patients (69%) were found to have 2 somatic (tumor) mutations in MMR genes encoding proteins that were lost from tumor samples, based on immunohistochemistry. Of the 10 remaining tumors 3 had one somatic mutation in a MMR gene, with possible loss of heterozygosity that could lead to MMR deficiency, 6 were found to be false-positive results (19%), and 1 had only one mutation in a MMR gene and remained unexplained. All of the tumors found to have somatic MMR mutations were of the hypermutated phenotype (>12 mutations/megabase); 6 had mutation frequencies >200/megabase, and 5 of these had somatic mutations in POLE, which encodes a DNA polymerase.

Conclusions: Some patients are found to have tumors with MMR defects during screening for Lynch syndrome, yet have no identifiable germline mutations in MMR genes. We found that almost 70% of these patients acquire somatic mutations in MMR genes, leading to a hypermutated phenotype of tumor cells. Patients with colon or endometrial cancers with MMR deficiency not explained by germline mutations might undergo analysis for tumor mutations in MMR genes to guide future surveillance guidelines.

Keywords: Colon Cancer; Genomic Instability; MSI; dMMR.

Figure 1

Confirmation of two mutations by Sanger sequencing on tumor DNA. Upper chromatogram from patient #688 (MLH1 c.2019delT (heterozygous)) and lower chromatogram from patient #841 (MLH1 c.613dup (hemi/homo)). Samples were sequenced with forward primers (available on request).

Figure 2

PCR testing for MSH2 inversion with positive control (+), patient sample #688 (Pt) and negative control (−) with a 1-kb marker (M). Primers F3 and F4MV amplify a wild-type chromosome 2, F4MV and B3MV detect the 3’ breakpoint for the MSH2 inversion, primers and methods were previously described by Rhees et al. The 12F (exon 12 forward) and 13R (exon 13 reverse) primers were designed as controls to verify DNA integrity.

Figure 3

Total mutation burden per megabase in tumor DNA detected by ColoSeq in exons and introns. White columns represent MSS tumors, black columns MSI tumors and diagonal columns represent tumors with POLE mutations. *Tumors with two somatic MMR mutations explaining screening test findings. #Tumors with MMR mutations possibly explaining screening test findings.