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Discovery and Qualification of Serum Protein Biomarker Candidates for Cholangiocarcinoma Diagnosis.

Cholangiocarcinoma (CCA) is a major health problem in northeastern Thailand. The majority of CCA cases are clinically silent and difficult to detect at an early stage. Although abdominal ultrasonography (US) can detect pre-malignant periductal fibrosis (PDF), this method is not suitable for screening populations in remote areas. With the goal of developing a blood test for detecting CCA in the at-risk population, we carried out serum protein biomarker discovery and qualification. Label-free shotgun proteomics was performed on depleted serum samples from 30 participants (n=10 for US-normal, US-PDF and CCA groups). Of 42 protein candidates selected using multiple reactions monitoring on 90 additional serum samples (n=30 per group), 11 discriminatory proteins were obtained using supervised multivariate statistical analysis. We further evaluated 3 candidates using ELISA and immunohistochemistry (IHC). S100A9, thioredoxin (TRX) and cadherin-related family member 2 (CDHR2) were significantly different between CCA and normal, and CCA and PDF groups when measured in an additional 247 serum samples (p<0.0001). By IHC, TRX and CDHR2 were detected in the cytoplasm and nucleus of CCA and inflammatory cells. S100A9 was detected in the infiltrating tumor stroma immune cells. Proteomics discovery and qualification in depleted sera revealed promising biomarker candidates for CCA diagnosis.