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. 2017 Jan;19(1):84-91.
doi: 10.1016/j.jmoldx.2016.07.010. Epub 2016 Nov 15.

Detection of Mismatch Repair Deficiency and Microsatellite Instability in Colorectal Adenocarcinoma by Targeted Next-Generation Sequencing

Jonathan A Nowak  1 , Matthew B Yurgelun  2 , Jacqueline L Bruce  1 , Vanesa Rojas-Rudilla  1 , Dimity L Hall  1 , Priyanka Shivdasani  1 , Elizabeth P Garcia  1 , Agoston T Agoston  1 , Amitabh Srivastava  1 , Shuji Ogino  3 , Frank C Kuo  1 , Neal I Lindeman  1 , Fei Dong  4
Affiliations

Detection of Mismatch Repair Deficiency and Microsatellite Instability in Colorectal Adenocarcinoma by Targeted Next-Generation Sequencing

Jonathan A Nowak et al. J Mol Diagn. 2017 Jan.

Abstract

Mismatch repair protein deficiency (MMR-D) and high microsatellite instability (MSI-H) are features of Lynch syndrome-associated colorectal carcinomas and have implications in clinical management. We evaluate the ability of a targeted next-generation sequencing panel to detect MMR-D and MSI-H based on mutational phenotype. Using a criterion of >40 total mutations per megabase or >5 single-base insertion or deletion mutations in repeats per megabase, sequencing achieves 92% sensitivity and 100% specificity for MMR-D by immunohistochemistry in a training cohort of 149 colorectal carcinomas and 91% sensitivity and 98% specificity for MMR-D in a validation cohort of 94 additional colorectal carcinomas. False-negative samples are attributable to tumor heterogeneity, and next-generation sequencing results are concordant with analysis of microsatellite loci by PCR. In a subset of 95 carcinomas with microsatellite analysis, sequencing achieves 100% sensitivity and 99% specificity for MSI-H in the combined training and validation set. False-positive results for MMR-D and MSI-H are attributable to ultramutated cancers with POLE mutations, which are confirmed by direct sequencing of the POLE gene and are detected by mutational signature analysis. These findings provide a framework for a targeted tumor sequencing panel to accurately detect MMR-D and MSI-H in colorectal carcinomas.

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Figures

Figure 1
Figure 1
Mismatch repair protein deficiency status compared with mutational burden and single–base pair insertion and deletion mutations in mononucleotide repeat (homopolymer) regions detected by next-generation sequencing in the training (A) and validation (B) data sets. MMR-D, mismatch repair protein deficient; MMR-I, mismatch repair protein intact.
Figure 2
Figure 2
A and B:POLE c.857C>G (p.Pro286Arg) mutations were identified in two colorectal carcinomas with high mutational burdens but without increased indels in homopolymer regions (arrowheads indicate affected nucleotide). C and D: The two high microsatellite instability carcinomas with the highest mutational burden were wild type for POLE mutations.
Figure 3
Figure 3
Mutational signatures of POLE-associated ultramutated colorectal carcinomas (A and B) compared with colorectal carcinomas with high microsatellite instability (C and D).
Figure 4
Figure 4
Commonly mutated colorectal cancer genes in mismatch repair protein deficient (MMR-D) and mismatch repair protein intact (MMR-I) carcinomas. MMR-D carcinomas had a higher frequency of BRAF mutations (P = 0.0007) and lower frequency of TP53 mutations (P < 0.0001).
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