From clusters to clinic: an 8-gene signature combined with mucinous component stratifies benefit of anti-CTLA-4 addition to anti-PD-1 in dMMR/MSI-H metastatic colorectal cancer

Background: Around 15-50% of patients with dMMR/MSI-H metastatic colorectal cancer (mCRC) experience primary and secondary resistance to immune checkpoint inhibitors (ICI).We previously described three transcriptomic clusters (A, B, C) linked to progression-free survival (PFS), with cluster A ("Stromal HIGH -Proliferation LOW" ) associated with the shortest PFS.Here we combine gene signature and mucinous phenotype to predict the benefit from anti-CTLA-4 plus anti-PD-1. Methods:We used 3'RNA-sequencing data from pre-treatment samples of two independent cohorts of patients with dMMR/MSI-H mCRC treated with anti-PD-1anti-LAG-3 ("Mono") or anti-PD-1+anti-CTLA-4 ("Combo").A rank-based k-Top Scoring Pairs (k-TSP) classifier was trained in Cohort 1 (103 patients) to predict assignment to Cluster A versus B/C and validated in Cohort 2 (87 patients).Patients receiving an anti-PD-L1 and with ECOG 2 were excluded.We defined a composite stratum crossing Cluster k-TSP (A vs B/C) with mucinous component (present vs absent).Results: Cluster A was enriched for mucinous tumors and had shorter PFS in both cohorts.The k-TSP selected four gene pairs predictive of Cluster A (YPEL2>H2AZ1, H6PD>COX7A2, STAT5B>SNRPE, ZHX3>TRAPPC4) and was validated in Cohort 2 with 68.1% balanced accuracy.In the pooled population (163 patients), the composite stratum treatment interaction improved model fit (likelihood-ratio p0.04).Patients with Cluster k-TSP A + mucinous (25/163, 15.3%) derived advantage from Combo versus Mono: adjusted 24-month PFS 72.2% vs 13.8% (HR 0.10, 95% CI 0.02-0.39;p<0.001).Conclusions: An 8-Gene signature combined with mucinous component identifies a subgroup that preferentially benefits from anti-PD-1+CTLA-4, warranting prospective validation to guide regimen selection in dMMR/MSI-H mCRC.