Pentanucleotide Instability Observed in MSI-H Colorectal Cancer

Key Takeaways:

• The authors report higher rates of combined and locus-specific Penta-C/Penta-D discordance in MSI-H versus MSS colorectal cancers.
• Penta-D demonstrated greater instability than Penta-C in MSI-H tumors, highlighting locus-specific variability.
• Discordance was associated with MLH1/PMS2 loss and MLH1 promoter hypermethylation and interpreted within the biologic context of deficient mismatch repair.

Using paired colorectal tumor–matched normal samples, the authors of Pentanucleotide Instability in MSI-High Colorectal Cancer evaluated whether the Penta-C and Penta-D loci—commonly used as identity markers alongside mononucleotide targets in MSI assays—demonstrate allele-size discordance that may complicate interpretation in specimen identity workflows. In their combined-loci analysis, they report that both loci were non-matching more often in MSI-H than in MSS cases (12.3% vs 0.3%, p<0.001). They frame the remainder of the study around locus-specific differences and associated clinicopathologic and mismatch-repair–related features.

The study analyzed 2,609 paired tumor and matched normal DNA samples from participants in the Ohio Colorectal Cancer Prevention Initiative (OCCPI). MSI testing was performed using the Promega MSI Analysis System™ v1.2 with fluorescent fragment analysis by capillary electrophoresis, comparing allele sizes between tumor and matched normal samples. Pentanucleotide “instability” is described as allele-size discordance between tumor and normal beyond expected assay variation. Within this cohort, 16.9% of patients were classified as MSI-H, providing context for MSI-stratified comparisons.

When examined individually, both Penta-C and Penta-D showed higher non-matching frequencies in MSI-H compared with MSS tumors. Among MSI-H samples, non-matching occurred in 27.0% at Penta-C and 39.8% at Penta-D, compared with 2.1% and 2.0% in MSS samples, respectively. These per-locus findings represent a different analytic perspective from the combined-loci analysis and highlight locus-specific variability, with Penta-D demonstrating greater discordance in MSI-H tumors.

To explore correlates, the authors compare pentanucleotide matching status with mismatch-repair–related features, reporting that discordance was more common in tumors with MLH1/PMS2 loss and MLH1 promoter hypermethylation. They also describe electropherogram patterns, noting that triple peaks were more frequent in Penta-D than Penta-C among MSI-H samples (27.7% vs 17.0%). These findings are presented as associations consistent with the biologic context of deficient mismatch repair rather than purely technical artifacts.

In discussion, the authors note that pentanucleotide instability may complicate interpretation in specimen identity workflows when comparing tumor and matched normal samples, as true tumor-associated shifts could resemble non-matching profiles. They further suggest that instability at these loci may reflect broader repeat-length variability in MSI-H tumors and hypothesize that other STR-based identity markers could be affected in similar contexts. They also note potential implications for MSI testing workflows, including situations where discordant identity markers may introduce uncertainty despite MSI positivity based on tumor markers. Overall, the study positions Penta-C/Penta-D discordance as a measurable phenomenon associated with dMMR in MSI-H colorectal cancer.