Characteristics of SMAD4 alterations in an immune-cold genomic subtype of biliary tract cancers in the Indian population.

4151 Background: Biliary tract cancers (BTCs), comprising gallbladder cancer (GBC) and cholangiocarcinoma (CCA), are rare but highly aggressive malignancies with limited therapeutic options. While genomic profiling has identified actionable alterations in BTC, data from the Indian population remain underexplored. We characterized the mutational and pathway landscape of BTCs in Indian patients and identified subtype-specific and immune-relevant genomic features. Methods: We retrospectively analyzed 154 BTC tumors, including 69 CCA and 85 GBC cases, using data from targeted next-generation sequencing panels. Somatic single-nucleotide variants, copy-number variations (CNVs), and gene fusions in oncogenic and tumor suppressor genes were curated. Pathway enrichment analysis (Hallmark MSigDB database ( p < 0.05 )), and mutual exclusivity/co-occurrence testing (gene pairs selected based on Benjamini-Hochberg-corrected q-values < 0.05.) were performed to identify biologically relevant genomic signatures. Genomic alterations were correlated with PD-L1 status when available. Results: TP53 was the most frequently mutated gene (53%), followed by KRAS (18%), ARID1A (9%), IDH1 (7%), and PIK3CA (7%). Recurrent amplifications were observed in MYC (12%) and ERBB2 (9%). Pathway enrichment analysis revealed significant dysregulation in the PI3K-AKT-mTOR, Notch, and Wnt/β-catenin signaling pathways (p-value <0.05). IDH1 mutations were predominantly observed in CCA, supporting a clinically actionable subgroup amenable to IDH1-targeted therapy, whereas ERBB2 alterations were enriched in GBC, identifying HER2 as a key therapeutic target in this subtype. Importantly, PD-L1-negative tumors showed a higher frequency of SMAD4 mutations (Fisher’s exact test, p = 0.71), implicating disruption of TGF-β signaling in immune evasion and reduced tumor immune activation. Conclusions: This comprehensive genomic analysis of BTC from the Indian population delineates distinct molecular landscape between GBC and CCA. The subtype-specific actionable alterations, including IDH1 mutations in CCA and ERBB2 alterations in GBC, highlights clinically relevant opportunities for precision therapy. SMAD4 alterations are associated with an immune-cold, poorly immunogenic tumor subtype in BTCs and warrant further investigation as a predictive biomarker for patient stratification in immunotherapy-based treatment strategies. These findings support the utility of molecular stratification in BTC and provide a rationale for integrating targeted and immunotherapy-based approached in Indian patients.