Cholangiocarcinoma (CCA) - bile duct cancer - is associated with late presentation, poses challenges for diagnosis and has high mortality, features that highlight the need for biomarkers than can be measured early and in accessible samples such as plasma. However, despite extensive investigations, efforts have failed to yield biomarkers with adequate diagnostic accuracy and utility for CCA. We will address previous limitations in the discovery of CCA biomarker(s) by undertaking a biomarker program in the global epicenter of CCA, Khon Kaen province, Thailand, which has highest incidence of intrahepatic CCA in the world. A key factor in the success of this proposal is that, while the causative agent for CCA in the West remains obscure, the single most important risk factor for intrahepatic CCA in Thailand has long been established - infection with the liver fluke Opisthorchis viverrini (OV). As determined by the WHO's IARC, no stronger link between a human malignancy and a eukaryotic pathogen exists than between CCA and infection with OV. We will utilize this well-established link between a parasite infection and cancer for the discovery and verification of biomarkers for CCA in plasma. Using a quantitative proteomic approach, we propose to scan 30 frozen, resected liver tumor tissues from confirmed OV-induced CCA cases to assemble a suite of proteins (candidate biomarkers) proximal to the disease site. We will complement this analysis with a scan of CCA cell lines which, unlike the frozen liver sections, measure the expression of secreted/membrane proteins (the secretome). Potential biomarkers identified from these two sources will be verified in plasma samples paired with the 30 frozen, resected liver tissues from confirmed OV-induced CCA patients. The candidate biomarkers will then be verified in the plasma of OV- infected individuals at risk for CCA in a case control study from our NIAID-sponsored longitudinal study that traces cholangiocarcinogenesis from chronic O. viverrini infection to CCA. The use of this exceptional set of samples will enable us to address previous limitations to CCA biomarker discovery as follows. First, we can reliably measure risk for exposure by determining infection with OV. Second, the Khon Kaen study site has the highest incidence of CCA in the world, giving us access to large numbers of CCA samples. Third, using our NIAID-sponsored longitudinal study, we can trace pathogenesis along the entire continuum: from infection with OV to diagnosis with CCA. Fourth, we plan biomarker discovery in banked tissue proximal to the tumor (resected liver) followed by verification in plasma from these same CCA patients and then in "at risk" patients in our cohort study. Hence, the overarching innovation of this proposal is that we will utilize a model of human carcinogenesis in which the major risk factor, as well as many of the intermediate stages on the pathway, to CCA have been well-defined.