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. 2025 Jun 3;163(6):909-919.
doi: 10.1093/ajcp/aqaf026.

Highly sensitive and specific markers for detection of mismatch repair deficiency by next-generation sequencing

Ming-Tseh Lin  1 , Eric S Christenson  2 , Aparna Pallavajjala  1 , James R Eshleman  1   2
Affiliations

Highly sensitive and specific markers for detection of mismatch repair deficiency by next-generation sequencing

Ming-Tseh Lin et al. Am J Clin Pathol. .

Abstract

Objective: To identify exonic markers that could improve analytic performance characteristics of next-generation sequencing (NGS) in detecting mismatch repair deficiency (dMMR) using colorectal cancer (CRC) as a model.

Methods: Coding sequences of a target NGS panel (~1.13 megabase) were compared between dMMR CRC and mismatch repair-proficient (pMMR) CRC in a training cohort (41 dMMR CRCs and 213 pMMR CRCs) and a validation cohort (33 dMMR CRCs and 307 pMMR CRCs) with documented mismatch repair status by immunohistochemical and/or microsatellite instability assays.

Results: The dMMR CRC cases showed significantly higher insertion/deletion (indel) mutations within exonic homopolymers (homo-indels), occurring predominantly within longer repeats of 5 to 10 nucleotides (92%, P < .0001), rather than shorter repeats of 2 to 4 nucleotides seen in pMMR CRC (62%). Homo-indels in dMMR CRC were not random. Hotspot loci were consistent between the training and validation cohorts. The dMMR defined by indels within homopolymers of 5 or more nucleotides, homopolymers of 7 or more nucleotides, or a panel of hotspots all showed 100% sensitivity and specificity with a range of cutoffs.

Conclusions: We propose that this approach allows one to identify highly sensitive and specific markers for detecting dMMR CRC by NGS alone. Further studies are warranted to test whether these markers are applicable to non-CRC neoplasms.

Keywords: CRC; homopolymer; hotspots; microsatellite instability; mismatch repair deficiency; next-generation sequencing.

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