Background: Microsatellite stable colorectal cancer (MSS-CRC) in advanced-stage disease remains a major therapeutic challenge due to its molecular heterogeneity. While classification frameworks such as CMS (Consensus Molecular Subtypes) and IMF (Intrinsic CMS, MSI, Fibrosis) have improved understanding of localized CRC, they offer limited insight into the biology of metastatic tumors. To address this gap, we implemented a multi-omic study of a cohort of treatment-naïve MSS-CRC patients with synchronous liver metastases (CRLM) and a large MSS-CRC Real World Dataset (RWD).

Methods: Paired primary CRC and CRLM specimens from 20 patient underwent comprehensive profiling, including Whole Exome, Transcriptome: bulk and single-nucleus RNA sequencing (snRNA-seq) analysis. The intrinsic CMS framework (iCMS) was applied to characterize tumor-intrinsic states across primary CRC and CRLM. iCMS scores were inferred using previously published templates. Key findings were validated in a real-world dataset (TEMPUS MSS-CRC) profiled using multi-omics approaches.

Results: Analysis of 388,018 single nuclei revealed 6 epithelial clusters associated with specific iCMS subtypes, supported by concordant bulk RNA-seq calls. Subtype composition at snRNA-seq level revealed that approximately one-third of tumors exhibited a hybrid phenotype with admixture of iCMS2 and iCMS3 epithelial cells. Tumors that were iCMS-unclassified in bulkRNAseq consistently displayed hybrid composition in snRNA-seq. Similarity metrics (cosine distance) from bulk data correlated with admixture proportions in snRNAseq.
Genomic analysis revealed mutations in APC and KRAS as the predominant genetic events in non-hybrid iCMS2 and iCMS3 tumors, aligned with their differential WNT and MAPK pathway activity, respectively. In contrast, iCMS-hybrid tumors were significantly enriched for clonal co-mutations in WNT and MAPK drivers, with overrepresentation of specific KRAS variants (G12D, G13D). These iCMS-hybrid tumors exhibited intermediate WNT-MAPK co-activation. In the metastatic setting, iCMS2 and iCMS3 tumors maintained their subtype identity, whereas iCMS-hybrid primary tumors showed a shift toward iCMS2 dominance in liver, suggesting that the liver microenvironment promotes expansion of the iCMS2 state.

Conclusions: Our study identifies a novel iCMS-hybrid phenotype with transcriptomic plasticity and co-activation of WNT and MAPK pathways, offering refined molecular resolution and potential to guide precision therapy in advanced MSS-CRC.

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