['To investigate biological differences and prognostic indicators of different ampullary cancer (AC) subtypes.', 'AC is associated with a favorable prognosis compared with other periampullary carcinomas. Aside from other prognostic factors, the histological origin of AC may determine survival. Specifically, the pancreatobiliary subtype of AC displays worse prognosis compared with the intestinal subtype. However, knowledge of inherent molecular characteristics of different periampullary tumors and their effects on prognosis has been limited.', 'Gene expression profiling was used to screen for differential gene expression between 6 PDAC cases and 12 AC cases. Among others, hepatocyte nuclear factor 4α (HNF4α) mRNA overexpression was observed in AC cases. Nuclear HNF4α protein expression was assessed using tissue microarrays consisting of 99 individual AC samples. The correlation of HNF4α expression with clinicopathological data (n = 99) and survival (n = 84) was assessed.', 'HNF4α mRNA is 7.61-fold up-regulated in AC compared with that in PDAC. Bioinformatics analyses indicated its key role in dysregulated signaling pathways. Nuclear HNF4α expression correlates with histological subtype, grading, CDX2 positivity, MUC1 negativity and presence of adenomatous components in the carcinoma. The presence of HNF4α is a univariate predictor of survival in AC mean survival (50 months versus 119 months, P = 0.002). Multivariate analysis revealed that HNF4α negativity (HR = 17.95, 95% CI: 2.35-136.93, P = 0.005) and lymph node positivity (HR = 3.33, 95% CI: 1.36-8.18, P = 0.009) are independent negative predictors of survival.'] Does hepatocyte nuclear factor ( HNF ) 4α expression distinguish ampullary cancer subtypes and prognosis after resection?