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

The aim of the study is to discover and qualify serum protein biomarkers for cholangiocarcinoma diagnosis. Serum samples from normal, periductal fibrosis and CCA groups were screened using LC-MS/MS and MRM/MS as described in manuscript.

All depleted serum samples were spiked with iRT and processed as described in manuscript