Integrative multi-omics identifies cholangiolocarcinoma as a distinct molecular subtype of intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (iCCA) exhibits profound heterogeneity, complicating its clinical management. This study aims to define the molecular features underlying its histological subtypes to better understand their biology and identify potential therapeutic opportunities. METHODS: We performed an integrated multi-omics analysis on a cohort of 283 histologically defined iCCA samples, utilizing targeted DNA sequencing (n=207) and transcriptomic profiling (n=281). RESULTS: Cholangiolocarcinoma (64/283, 22.6%), conventional small-duct-type (SD-type, 124/283, 43.8%), and large-duct-type (LD-type, 95/283, 33.6%) iCCA exhibited distinct molecular features. Cholangiolocarcinoma was characterized by lower frequencies of KRAS and TP53 mutations, enrichment of oxidative phosphorylation-related transcriptional programs, and a relatively immune-cold microenvironment with reduced immune-related signatures and tertiary lymphoid structure (TLS)-related scores. In contrast, LD-type tumors exhibited higher KRAS and TP53 mutation rates and more aggressive features, while SD-type tumors displayed intermediate characteristics. Subtype-dependent associations were observed between metabolic pathway activity and immune features. In an independent validation cohort (n=150), cholangiolocarcinoma was associated with favorable overall survival. CONCLUSIONS: This study supports cholangiolocarcinoma as a biologically distinct subtype of iCCA and highlights subtype-specific genomic, metabolic, and immune features. These findings provide a refined framework for histomolecular classification of iCCA and may inform future subtype-aware research. IMPACT AND IMPLICATIONS: This multi-omics study provides an integrated molecular characterization of the three major histological subtypes of intrahepatic cholangiocarcinoma (iCCA), linking histopathologic classification with distinct genomic, transcriptomic, immune, and metabolic features. Our findings support cholangiolocarcinoma (CLC) as a molecularly distinct subtype, while highlighting divergent biological programs across CLC, small-duct type, and large-duct type tumors. Rather than relying on morphology alone, this framework provides a biologically informed basis for subtype-specific investigation of iCCA. These findings establish a molecular framework for future subtype-aware translational studies in iCCA and provide a resource for generating biologically informed hypotheses regarding subtype-specific therapeutic vulnerabilities. Further validation in independent cohorts and functional studies will be required before clinical translation.