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Attapol Titapun,1,2 Vor Luvira,1,2 Tharatip Srisuk,1,2 Apiwat Jareanrat,1,2 Vasin Thanasukarn,1,2 Malinee Thanee,2,3 Prakasit Sa-Ngiamwibool,2,3 Sureerat Padthaisong,2,4 Kassaporn Duangkumpha,2,4 Manida Suksawat,2,4 Watcharin Loilome,2,4 Paiboon Sithithaworn,2,5 Anchalee Techasen,2,6 Bandit Thinkhamrop,2,7 Alexa Dzienny,8 Ayse Caglayan,9 David Park,9 Simran Mahmud,9 Narong Khuntikeo1,2 1Department of Surgery, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; 2Cholangiocarcinoma Research Institute (CARI), Khon Kaen University, Khon Kaen, Thailand; 3Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; 4Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; 5Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; 6Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand; 7Department of Epidemiology and Biostatistics, Faculty of Public Health, Khon Kaen University, Khon Kaen, Thailand; 8School of Medicine and Health Sciences, George Washington University, Washington, DC, USA; 9Faculty of Medicine, Imperial College London, London, UKCorrespondence: Attapol TitapunDepartment of Surgery, Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandTel +66-43-363252Email attati@kku.ac.thBackground: Opisthorchis viverrini (OV)-associated cholangiocarcinoma (CCA) has a high immune response with chronic inflammation and oxidative stress. CD44 and Nestin, two cancer stem cell (CSC) markers, play major roles in cancer cell survival. Effects of immune response and expression CSC markers on survival of patients with CCA remain unclear.Objective: To investigate the effects of level of OV IgG together with CSC marker expression and also the combination of these markers on survival of CCA patients after curative resection.Methods: All serum specimens from CCA patients who underwent curative surgery from 2005 to 2015 were examined for IgG for OV antigen by ELISA. Tissue specimens were studied for CD44 and Nestin expression. Survival analysis by Cox proportional hazard model was used for estimating hazard ratio (HR) with a 95% confidence interval (CI).Results: In this study, 122 (69.3%) of 176 were positive for OV IgG, and 35 (19.9%) were considered to have high-positive OV IgG. CD44s positive expression was found in 54 (40%), CD44v6 high expression in 96 (69.6%), CD44v8-10 high expression in 87 (63.5%) and Nestin high expression in 21 (16.1%). Multivariate survival analysis found that high-positive OV IgG and late stage tumor were independent prognostic factors with the adjusted HR of 2.24 (95% CI 1.27– 3.93) and 2.78 (95% CI 1.46– 5.29), respectively. Subgroup analysis in early and late stage CCA showed that a combined positive OV IgG and CD44s expression with the high expression of CD44v8-10 had the significantly poorest prognosis with HR of 3.75 (95% CI 1.61– 8.72) and HR of 1.76 (95% CI 1.02– 3.03), respectively.Conclusion: A high level of OV IgG as well as a high level of CSC markers resulted in an aggressive CCA. OV IgG level together with CSC markers can be used as the prognostic markers for CCA patients’ survival. The study of the CD44 pathway is promising for adjuvant treatment.Keywords: CCA, OV‐associated CCA, survival, OV IgG antibody, CD44s, CD44v8-10

Background Serum antibody for Opisthorchis viverrini (OV) is strong evidence for a history of OV infection in people. Currently, no studies have examined whether varying cholangiocarcinoma (CCA) prevalence levels are linked to previous OV infection nor have they provided comprehensive assessment and characterization of OV-associated CCA. Objective Our study examined the prevalence of serum IgG antibodies for OV-positive CCA cases and determined whether there were correlations of IgG antibodies with histopathologic features, HER2, PD-L1, and FGFR2 expression, as well as their roles on the patients' survival. Methods The study involved 221 CCA surgical patients at Khon Kaen University Hospital, Thailand, from 2005 to 2017. Serum specimens were tested for OV IgG by ELISA. CCA tissue microarrays were used to examined for HER2, PD-L1, and FGFR2 expression. Logistic regression was used to investigate an association between factors and IgG. Cox regression was used to determine factors that affected CCA patient survival. Results IgG for OV were positive in 162 cases, and the prevalence was 73.3% (95% CI=68.0-78.7). About three quarters (78.3%) had large duct type tumors and concomitant intraductal papillary neoplasm of bile ducts (IPNB) occurred in 92 (50%) cases. HER2 expression was positive in 94 (61.4%) cases. Positive PD-L1 and FGFR2 expression occurred in 125 (83.9%) and 100 (67.1%) cases. IgG for OV had no significant correlation to any histological feature but had significant correlation with HER2 overexpression with adjusted OR=2.32 (95% CI=1.09-4.96, P=0.03). Cases of CCA with OV IgG positive had a significantly poor prognosis with adjusted HR=1.66 (95% CI=1.13-2.43, P=0.01). Conclusion We found a high prevalence of serum IgG for OV-positive CCA patients and a correlation with overexpression of HER2. Moreover, IgG for OV and HER2 expression indicated poor survival of CCA. Therefore, future clinical studies for anti-HER2 treatments should focus on OV-associated CCA.