Proteomic Analysis of Human Bile from Malignant Biliary Stenosis Induced by Pancreatic Cancer Annarita Farina,* ,† Jean-Marc Dumonceau, ‡ Jean-Louis Frossard, ‡ Antoine Hadengue, ‡ Denis F. Hochstrasser, †,§ and Pierre Lescuyer †,§ Biomedical Proteomics Research Group, Department of Bioinformatics and Structural Biology, Faculty of Medicine, Geneva University, Geneva CH-1211, Switzerland, Service of Gastroenterology and Hepatology, Geneva University Hospitals, Geneva CH-1211, Switzerland, and Laboratory Medicine Service, Department of Genetics and Laboratory Medicine, Geneva University Hospitals, Geneva CH-1211, Switzerland Received July 2, 2008 Stenosis of the common bile duct may be either due to benign (chronic pancreatitis) or malignant (cholangiocarcinoma, pancreatic adenocarcinoma) conditions. The benign nature of the stricture should be first confirmed in order to ensure appropriate therapy. Therefore, the identification of markers allowing discrimination between malignant and benign biliary stenosis would be very valuable in clinical practice. To this intent, we performed a proteomic analysis of bile samples from patients having a biliary stenosis caused by pancreatic adenocarcinoma. Bile samples were collected during endoscopic retrograde cholangiopancreatography and purified using different methods. The extracted proteins were then analyzed by SDS-PAGE and LC-MS/MS. A total of 127 proteins were identified, 34 of which have not been previously detected in proteomic studies of bile. Among them, several proteins have been described as potential biomarkers of pancreatic cancer. We extended our investigation by studying the expression of some of these pancreatic cancer markers in bile samples collected from patients with various etiologies of biliary stenosis including pancreatic cancer, cholangiocarcinoma, chronic pancreatitis, as well as gallstone-induced stenosis. Our data showed a conspicuous overexpression of CEACAM6 and MUC1 (CA19-9) in pancreatic cancer and cholangiocarcinoma samples, according to the hypothesis that bile fluid collects cancer-associated protein leaking from the tumor microenviron- ment. These results underline the interest of using bile as a source of biomarkers for the diagnosis of malignant biliary stenosis. Keywords: bile • pancreatic cancer • biliary stenosis • proteomics • mass spectrometry • biomarkers • CEACAM6 • MUC1 • CA 19-9. Introduction Stenosis may obstruct the common bile duct, impede the outflow of bile from the liver, and finally cause jaundice. Common bile duct stenosis is most often caused by pancreatic adenocarcinoma, the fourth leading cause of cancer death in the United States. 1 Other etiologies include primary bile duct carcinoma (i.e., cholangiocarcinoma) and benign diseases such as chronic pancreatitis and primary sclerosing cholangitis. 2,3 Tools that are currently available to differentiate between benign and malignant biliary stenosis, including imaging techniques and pathological examination of endoscopic biliary samples, are plagued by a poor sensitivity and/or specificity. 4,5 Resulting diagnostic uncertainties regularly lead to inadequate and potentially harmful disease management. Indeed, 5-10% of duodenopancreatectomies performed for a suspected ma- lignancy reveal benign disease, while this surgical procedure may be associated with a high rate of morbidity and mortality that can reach in referral centers almost 40% and 1-5%, respectively. 6,7 Many investigators, over the years, have sought to find accurate diagnostic markers of pancreatic cancer and other malignant causes of bile duct stenosis. Nevertheless, current standard serum markers, including sialylated Lewis antigen 19-9 (CA 19-9), have not proved of any benefit because of insufficient positive predictive value. 8-10 More reliable markers, to be of useful diagnostic aid, are urgently needed. One potential source of malignant stenosis biomarkers is represented by bile which is the fluid bathing diseased cells. Proteins secreted/shed by tumoral cells of malignant stenoses should be in much higher concentration in bile than in the serum, and therefore, detection of cancer markers in bile might have a greater sensitivity. Recently, bile investigation using proteomic-based approaches has received substantial interest in cancer biomarker discovery. 11 However, up to now, only a few proteomic studies have been performed on bile, probably * To whom correspondence should be addressed: Dr. Annarita Farina, Biomedical Proteomics Research Group, Department of Bioinformatics and Structural Biology, Faculty of Medicine, Geneva University, rue Michel Servet 1, Geneva CH-1211. Telephone, +41.22.3795140; fax, +41.22.3795926; e-mail, Annarita.Farina@medecine.unige.ch. † Geneva University. ‡ Service of Gastroenterology and Hepatology, Geneva University Hospitals. § Department of Genetics and Laboratory Medicine, Geneva University Hospitals. 10.1021/pr8004925 CCC: $40.75  2009 American Chemical Society Journal of Proteome Research 2009, 8, 159–169 159 Published on Web 12/05/2008