Discovery and Qualification of Serum Protein Biomarker Candidates for Cholangiocarcinoma Diagnosis Kassaporn Duangkumpha, †,‡ Thomas Stoll, § Jutarop Phetcharaburanin, †,‡ Puangrat Yongvanit, ‡ Raynoo Thanan, † Anchalee Techasen, ‡,∥ Nisana Namwat, †,‡ Narong Khuntikeo, ‡,⊥ Nittaya Chamadol, ‡,# Sittiruk Roytrakul, ∇ Jason Mulvenna, § Ahmed Mohamed, § Alok K. Shah, § Michelle M. Hill,* ,§ and Watcharin Loilome* ,†,‡ † Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand ‡ Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand § QIMR Berghofer Medical Research Institute, Herston, Queensland 4006, Australia ∥ Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand ⊥ Department of Surgery, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand # Department of Radiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand ∇ Proteomics Research Laboratory, Genome Institute, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani 12120, Thailand * S Supporting Information ABSTRACT: 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 40 protein candidates selected using multiple reaction 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. KEYWORDS: cholangiocarcinoma, proteomics, mass spectrometry, multiple reaction monitoring, serum biomarker discovery pipeline ■ INTRODUCTION Cholangiocarcinoma (CCA) is an aggressive cancer of the bile duct epithelium with a poor survival rate due to lack of specific clinical symptoms leading to late diagnosis. 1 CCA is a major public health problem in the northeast of Thailand where it shows the highest incidence in the world. 2 Chronic inflammation of the biliary tract caused by the liver fluke (Opisthorchis viverrini, Ov) is the principal mechanism that drives cholangiocarcinogenesis in the Mekong area of South- east Asia. 2,3 Oxidative stress induced by Ov infection leads to DNA damage, abnormal tissue remodeling, and the alteration of gene expression, all of which have been implicated in carcinogenesis. 4,5 Interestingly, a number of molecules have been reported to be differentially abundant during Ov- associated cholangiocarcinogenesis that could, therefore, be used as biomarkers for the assessment and chemoprevention of liver fluke-associated cholangiocarcinoma. 6 Moreover, the chronic injury affecting the bile duct epithelial cells during Ov infection leads to periductal fibrosis (PDF), which is believed to be an intermediate pathological condition leading to CCA. We recently reported histological confirmation using ultrasound-based diagnosis of PDF and CCA in a cohort of CCA patients. 7 Although ultrasonography is a potentially Received: April 13, 2019 Published: July 16, 2019 Article pubs.acs.org/jpr Cite This: J. Proteome Res. XXXX, XXX, XXX-XXX © XXXX American Chemical Society A DOI: 10.1021/acs.jproteome.9b00242 J. Proteome Res. XXXX, XXX, XXX−XXX Downloaded via KHON KAEN UNIV on August 19, 2019 at 05:04:07 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.