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Mutation enrichment in targeted panels flags immunotherapy-responsive POLE-driven hypermutated microsatellite-stable
Nic Gabriel Reitsam1,2,3, Kathrin Anna Schneider4,5, Bianca Grosser4,5,6
1Pathology, Faculty of Medicine, University of Augsburg, Augsburg, Germany. nic.reitsam@uka-science.de.
Abstract:
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.
Insights
Pathogenic mutations in DNA polymerase ε (POLE) can cause hypermutated, microsatellite-stable colorectal cancers (CRCs) that respond to immunotherapy. Routine molecular data can identify these POLE-mutant CRCs for targeted testing and treatment.
Area of Science:
- Oncology
- Genetics
- Immunotherapy
Background:
- Pathogenic mutations in the DNA polymerase ε (POLE) exonuclease domain characterize a distinct subset of microsatellite-stable (MSS) colorectal cancers (CRCs).
- These POLE-mutant CRCs are hypermutated and highly sensitive to immune checkpoint blockade therapy.
- Current diagnostic approaches often miss POLE mutations as they are not routinely tested, leading to missed opportunities for immunotherapy in eligible patients.
Purpose of the Study:
- To develop a strategy for identifying POLE-mutant MSS CRCs using routine molecular data from small targeted next-generation sequencing (NGS) panels.
- To enable the detection of immunotherapy-eligible patients with POLE-mutant CRCs who might otherwise be missed by standard testing protocols.
Main Methods:
- Analysis of 675 CRC cases sequenced with a small targeted NGS panel, flagging tumors with ≥6 non-synonymous single nucleotide variants (SNVs) as potentially hypermutated.
- Confirmatory POLE sequencing and comprehensive genomic profiling (CGP) were performed on preselected cases.
- Validation using external POLE-mutant CRC cohorts and The Cancer Genome Atlas (TCGA) colorectal adenocarcinoma (COAD/READ) datasets (>1000 CRCs).
Main Results:
- All identified POLE-mutant CRCs (n=7) exhibited exonuclease domain hotspot mutations, proficient mismatch repair (pMMR)/MSS status, and MSI-like histopathology.
- These tumors displayed ultra-high tumor mutational burden (TMB), low dbSNP overlap, a C>T transition bias, and disrupted co-mutation patterns.
- Routine small-panel NGS data successfully flagged candidate POLE-mutant MSS CRCs, with 41/43 POLE-mutant CRCs in TCGA carrying panel-detectable co-mutations.
Conclusions:
- Routine small-panel NGS data can effectively flag potential POLE-mutant MSS CRCs, facilitating confirmatory testing and identification of patients responsive to immunotherapy.
- This approach can significantly improve the detection rate of immunotherapy-eligible CRC patients.
- Integrating this strategy with AI-based predictions from H&E slides can enhance multimodal diagnostic workflows for precision immuno-oncology in colorectal cancer.
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