Abstract
INTRODUCTION: Cholangiocarcinoma (CCA) is an aggressive malignancy with poor prognosis and limited treatment options. Molecular heterogeneity and the tumor immune microenvironment play crucial roles in CCA progression and therapeutic response, but comprehensive stratification remains insufficient for guiding treatment decisions. METHODS: This study performed integrated multi-omics profiling as well as immunohistochemical validation on 424 CCA patients. Multidimensional data, including genomic, epigenomic, transcriptomic, and immune profiling, were analyzed to identify clinically relevant molecular subtypes. RESULTS: Four distinct molecular subtypes of CCA were identified, each exhibiting unique clinical and immunological features: C1 (proliferation): driven by TP53/KRAS mutations and CpG island methylator phenotype (CIMP+) hypermethylation, showing T helper 17 cells infiltration and poor outcomes; C2 (Immune-Suppressed Macro_LYVE1 [ISM_LYVE1]): stroma-rich with LYVE1(+) macrophages and epithelial-mesenchymal transition (EMT) activation; C3 (immune-activated Macro_C1QC (IAM_C1QC)): enriched in C1QC(+) macrophages, CD8(+) T-cells, and metabolic pathways, highly responsive to immune checkpoint blockade (ICB; 75% overall response rate); C4 (immune exclusion): FGFR2-altered and IDH1-mutant, with an immunologically cold phenotype. CONCLUSION: We validated ATP2B1 as a novel prognostic biomarker and developed a 160-gene classifier for subtype prediction. The C3 subtype's exceptional ICB response, independent of conventional biomarkers (PD-L1/microsatellite instability/tumor mutational burden), highlights the clinical utility of this classification system in guiding precision immunotherapy for CCA.