Mutation enrichment in targeted panels flags immunotherapy-responsive POLE-driven hypermutated microsatellite-stable...

Pathogenic mutations in the DNA polymerase ε (POLE) exonuclease domain define a rare but clinically distinct subset of microsatellite-stable (MSS) colorectal cancers (CRCs) characterized by hypermutation and exceptional immune checkpoint blockade sensitivity. Yet POLE testing is not routinely performed, leaving immunotherapy-eligible patients undetected. Because most diagnostic multigene panels do not include POLE, strategies enabling its recognition from routine molecular data are needed. We analyzed 675 CRC cases sequenced using a small targeted NGS panel. Tumors with ≥6 non-synonymous SNVs were flagged as potentially hypermutated. Confirmatory POLE sequencing and comprehensive genomic profiling (CGP) were performed in preselected cases. Findings were validated using two external POLE-mutant CRCs and TCGA-COAD/READ cohorts (>1000 CRCs in total). All POLE-mutant CRCs (n = 5 of 15 flagged cases; two external validation cases) showed exonuclease domain hotspot mutations, pMMR/MSS status, yet MSI-like histopathology. These tumors exhibited predominantly ultra-high TMB, low dbSNP overlap, C>T transition bias, and disrupted co-mutation patterns - canonical POLE-driven hypermutation features. In TCGA, 41/43 POLE-mutant CRCs carried panel-detectable co-mutations. Routine small-panel NGS data can flag candidate POLE-mutant MSS CRCs for confirmatory testing, enabling detection of immunotherapy-responsive tumors otherwise missed. Integrated with AI-based POLE/MSI prediction from H&E slides, this supports multimodal diagnostic workflows enhancing precision immuno-oncology in CRC. Trial registration: NA.