Simple SummaryGallbladder cancer (GBC) is a significant health problem in Asia and Latin America, particularly in Chile. The limited therapeutic options for GBC demand the identification of targetable proteins with predictive value. We evaluated the prognosis impact of 18 different protein markers in primary lesions from 241 GBC patients using automated immunohistochemistry, a semi-automatic method for image analysis, univariate and multivariate statistical analyses, and machine learning algorithms. Our results show a significant association between the expression of the MRP2/CXCR4/PD-L1 cluster of markers and prolonged patient survival. Hence, our results suggest that this cluster could be a valuable prognostic tool for GBC.AbstractGallbladder cancer (GBC) is a rare pathology in Western countries. However, it constitutes a relevant health problem in Asia and Latin America, with a high mortality in middle-aged Chilean women. The limited therapeutic options for GBC require the identification of targetable proteins with prognostic value for improving clinical management support. We evaluated the expression of targetable proteins, including three epithelial tumor markers, four proteins associated with multidrug and apoptosis resistance, and eleven immunological markers in 241 primary gallbladder adenocarcinomas. We investigated correlations between tumor marker expression, the primary tumor staging, and GBC patients’ survival using automated immunohistochemistry, a semi-automatic method for image analysis, univariate and multivariate statistical analyses, and machine learning algorithms. Our data show a significant association between the expression of MRP2 (p= 0.0028), CXCR4 (p= 0.0423), and PD-L1 (p= 0.0264), and a better prognosis for patients with late-stage primary tumors. The expression of the MRP2/CXCR4/PD-L1 cluster of markers discriminates among short-, medium-, and long-term patient survival, with an ROC of significant prognostic value (AUC = 0.85,p= 0.0012). Moreover, a high MRP2/CXCR4/PD-L1 co-expression is associated with increased survival time (30 vs. 6 months,p= 0.0025) in GBC patients, regardless of tumor stage. Hence, our results suggest that the MRP2/CXCR4/PD-L1 cluster could potentially be a prognostic marker for GBC.