Immune-Related Biomarkers and an Exploratory Prognostic Signature in Biliary Tract and Gallbladder Cancer

Gallbladder cancer is an aggressive biliary tract malignancy associated with poor prognosis and limited molecularly guided treatment options. Chronic inflammation and immune dysregulation play central roles in gallbladder carcinogenesis; however, reproducible computational workflows for identifying immune-related biomarkers remain limited. This study describes an integrated bioinformatic workflow for identifying immune-related differentially expressed genes (IRDEGs) and developing an exploratory prognostic gene signature. Differential expression analysis was performed using dataset-specific R-based workflows, and differentially expressed genes were intersected with immune-related gene sets obtained from ImmPort, InnateDB, and the Molecular Signatures Database (MSigDB). Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and gene set enrichment analyses were conducted to characterize biological functions and signaling pathways. Protein-protein interaction networks were constructed using STRING and Cytoscape; immune-cell infiltration was estimated using CIBERSORT; and prognostic genes were identified through sequential univariate, least absolute shrinkage and selection operator (LASSO) Cox, and multivariable Cox regression analyses. The analysis identified 824 consensus differentially expressed genes, including 312 IRDEGs. These genes were significantly enriched in inflammatory and immune-regulatory pathways, including IL6/JAK/STAT3, nuclear factor of kappa light chain enhancer (NF-κB), Toll-like receptor, and chemokine signaling pathways. CXCL8, IL1B, MMP9, TLR2, S100A8, and S100A9 were identified as key hub genes. Immune-cell infiltration analysis revealed a markedly altered tumor immune microenvironment. An exploratory five-gene prognostic signature comprising CXCL8, S100A9, IL16, MMP9, and CD36 successfully stratified patients according to overall survival risk. This integrated workflow provides a reproducible framework for identifying immune-related biomarkers and developing exploratory prognostic signatures in gallbladder cancer. However, independent clinical cohorts and experimental validation are required before these findings can be translated into clinical applications.