Cholangiocellular carcinoma (CCC) and pancreatic ductal adenocarcinoma (PDAC) are two highly aggressive cancer types that arise from epithelial cells of the pancreatobiliary system. Owing to their histological and morphological similarity, differential diagnosis between CCC and metastasis of PDAC located in the liver frequently proves an unsolvable issue for pathologists. The detection of biomarkers with high specificity and sensitivity for the differentiation of these tumor types would therefore be a valuable tool. Here, we address this problem by comparing microdissected CCC and PDAC tumor cells from nine and eleven cancer patients, respectively, in a label-free proteomics approach. The novel biomarker candidates were subsequently verified by immunohistochemical staining of 73 CCC, 78 primary, and 18 metastatic PDAC tissue sections. In the proteome analysis, we found 180 proteins with a significantly differential expression between CCC and PDAC cells (p value < 0.05, absolute fold change > 2). Nine candidate proteins were chosen for an immunohistochemical verification out of which three showed very promising results. These were the annexins ANXA1, ANXA10, and ANXA13. For the correct classification of PDAC, ANXA1 showed a sensitivity of 84% and a specificity of 85% and ANXA10 a sensitivity of 90% at a specificity of 66%. ANXA13 was higher abundant in CCC.It presented a sensitivity of 84% at a specificity of 55%. In metastatic PDAC tissue ANXA1 and ANXA10 showed similar staining behavior as in the primary PDAC tumors (13/18 and 17/18 positive, respectively). ANXA13, however, presented positive staining in eight out of eighteen secondary PDAC tumors and was therefore not suitable for the differentiation of these from CCC. We conclude that ANXA1 and ANXA10 are promising biomarker candi- The majority of malignant neoplasms located in the liver are metastases originating from primary tumor sites in other organs, most commonly the colon or the pancreas (1). In many cases, a histological or immunohistological examination by an experienced pathologist can specify the type and origin of the underlying cancer. Hepatocellular carcinoma or hepatic metastasis from primaries such as pulmonary adenocarcinoma, colorectal adenocarcinoma, and breast carcinoma are usually easily distinguishable by morphology and means of known immunohistochemical markers. For primary cholangiocellular carcinoma (CCC) 1 and metastases of pancreatic ductal adenocarcinoma (PDAC), however, the distinction in a liver biopsy is basically an unsolvable task because of their high similarity.From the ‡Medizinisches Proteom-Center, Ruhr-Universitä t Bochum, Germany; §Institut fü r Pathologie, Universitä tsklinikum Essen, Universitä t-Duisburg-Essen, Germany; ¶Klinik fü r Allgemeinchirurgie, Viszeral-und Transplantationschirurgie, Universitä tsklinikum Essen, Universitä t-Duisburg-Essen, Germany; ʈInstitut fü r Pathologie und 1 The abbreviations used are: CCC, cholangiocellular carcinoma; ANXA, annexin; AUC, area under the curve; BH, Benjamini-Hochberg; CKM...