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Recently, we demonstrated von Hippel-Lindau gene product (pVHL) was expressed in normal pancreatic ducts but absent in pancreatic ductal adenocarcinoma (PDA). Previous studies have suggested the diagnostic value of S100P, S100A4, and S100A6 in PDA. In this study, we evaluated pVHL, S100P, S100A4, and S100A6 as potential markers for PDA, pancreatic intraepithelial neoplasia (PanIN), ampullary adenocarcinoma (AAD), and cholangiocarcinoma (CC). Immunostains were performed on 56 PDA cases, 20 AAD cases, and 28 CC cases using antibodies against pVHL, S100P, S100A6, and S100A4. Western blots were also performed on 2 cases of PDA and the matching non-neoplastic pancreatic tissues. Of the 56 PDA cases, immunoreactivity for S100P, S100A6, and S100A4 was observed in 56, 55, and 41 cases, respectively. Non-neoplastic ductal epithelium was negative for S100P in all cases. Ninety percent of PanINs were also positive for S100P. pVHL was not detected in all PDAs and 96% of PanINs by immunohistochemistry. S100P, S100A4, and S100A6 were present in a significant number of AADs and CCs; and pVHL expression was observed in 25% of AADs and 21% of CCs. Our data indicate that (1) S100P and pVHL are a pair of sensitive and specific markers for identifying primary PDA and PanIN; (2) up-regulation of S100P and down-regulation of pVHL may play a role in early tumorigenesis in PDA; and (3) the 4 markers studied have limited value in differentiating among PDA, AAD, and CC.