Immunohistochemical profiling of ampullary carcinoma: Molecular subtypes, prognostic markers, and the role of Claudin-18.

Ampullary cancer is a rare malignancy, histologically divided into intestinal and pancreatobiliary subtypes based on histomorphological and immunophenotypic features as they arise from different epithelial origins and exhibit distinct biological behavior, treatment responses, and clinical outcomes. However, current histomorphological and immunohistochemical classification often remains ambiguous, complicating accurate subtype assignment to guide therapeutic approaches. In this retrospective study of 125 ampullary carcinoma cases, we performed immunohistochemical profiling using a panel of markers including CDX2, CK20, mucins (MUC1, MUC2, MUC5AC), and claudins (CLDN1-4, CLDN7, CLDN18) to refine subtype classification and identify prognostic biomarkers. Hierarchical clustering based on marker expression H-scores revealed two molecular subtypes corresponding to intestinal and pancreatobiliary differentiation. While CDX2 and CK20 are routinely used in combination for diagnosis, other markers, particularly CLDN3, CLDN7, and MUC5AC, demonstrated superior diagnostic and prognostic performance to CDX2. CK20, by contrast, remained a robust marker of intestinal differentiation and favorable outcome. Pancreatobiliary-type tumors exhibited elevated MUC1, MUC5AC, and CLDN18 expression, and were associated with advanced stage, invasive features, and significantly worse survival. Multivariate analysis showed high MUC5AC and apical MUC1 expression to be associated with adverse outcomes, whereas CLDN1, CLDN3 and CLDN4 expression were linked to improved survival. Membranous CLDN18 expression was more common in pancreatobiliary-type ampullary cancer, making it a potential therapeutic target. These findings underscore the diagnostic and prognostic value of multiplex immunohistochemical profiling. We propose retaining CK20 but replacing CDX2 with a more informative marker, namely CLDN3, to enhance classification and therapeutic stratification.