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Cholangiocarcinoma is a crucial health problem in Northeast Thailand where liver fluke infection is endemic. However, molecular genetic and epigenetic mechanisms involved in carcinogenesis of this cancer remain unclear. We attempted to study genetic and epigenetic alterations of hMLH1 gene in 65 intrahepatic cholangiocarcinoma using polymerase chain reaction (PCR) based microsatellite marker D3S1611 and methylation-specific PCR, respectively. Of 65 cases, 29 (44.6%) showed hypermethylation of hMLH1 promoter. Loss of heterozygosity (LOH) of hMLH1 was detected in 12 of 51 informative cases (23.5%). Five out of 29 (17.2%) methylated cases demonstrated LOH. Aberrant methylation of hMLH1 promoter was significantly associated with poorly differentiated type (P=0.013). Our study suggests that both genetic and epigenetic mechanisms cause the inactivation of hMLH1 where epigenetic is a major event resulting in mismatch repair deficiency and contributing to carcinogenesis of liver fluke related cholangiocarcinoma. Since, gene silencing by methylation is an early event in carcinogenesis, promoter hypermethylation of hMLH1 may be a molecular targeted therapy and prevention of liver fluke related cholangiocarcinoma.

Mutations of p53 are found in the majority of human malignancies. The accumulated mutant p53 can be detected in tumor sections by immunohistochemical methods. The abnormal accumulation of the defective p53 protein can induce the host to develop anti-p53 antibodies in sera of cancer patients. This study aimed to investigate the presence of anti-p53 antibodies in sera of patients with cholangiocarcinoma and to evaluate the correlation between such antibodies and p53 protein accumulation.The presence of serum anti-p53 antibodies in 49 patients with cholangiocarcinoma was determined by ELISA kit (Pharma Cell, France). Immunohistochemical detection of p53 protein expression was examined in available tissue samples of 36 patients.Serum anti-p53 antibodies were detected in 6 of 49 patients with cholangiocarcinoma (12.2%). Immunostaining of p53 was found in 15 of 36 patients (41.6%) and 4 of these 15 patients (26.7%) were positive for anti-p53 antibodies. The association between anti-p53 antibodies and p53 protein expression was statistically significant (P=0.023). No correlation was found between the presence of anti-p53 antibodies and sex, age, histological grade, site and stage of tumor (P0.05).The majority of serum anti-p53 antibodies detected in cholangiocarcinoma were specifically associated with the accumulation of p53 protein in tumor tissues. However, antibody generation against the p53 protein is a relatively uncommon event in cholangiocarcinoma.

Measurement of the activities of alkaline phosphatase isoenzyme has been used for the identification and monitoring of diseases associated with the isoenzyme. Biliary alkaline phosphatase (BALP), an isoform of liver-ALP, has been found in the serum of patients with biliary obstruction and metastatic liver cancer. This study compared the BALP isoform in the serum of patients with cholangiocarcinoma (CCA) with that of non-jaundiced benign hepatobiliary disease, other cancers, and healthy persons. ALP isoforms were separated using cellulose acetate electrophoresis and the activity was demonstrated using indolyl blue reagent. The BALP isoform was demonstrated in 65% of CCA patients independently of jaundice condition or histological grading of the tumor. The level of serum BALP in non-jaundiced CCA was significantly lower than that of jaundiced CCA, and not correlated with serum bilirubin. No BALP was detected in healthy persons. In the patients with high serum ALP (>147U/l), BALP can differentiate non-jaundiced CCA patients from other non-jaundiced carcinoma patients with 85% sensitivity, 79% specificity, and positive and negative predictive values of 81% and 83%, respectively. The demonstration of serum BALP, in particular in non-jaundiced patients with high serum ALP, may indicate the presence of tumor in the bile duct.

Aberrant expression of MUC5AC mucin is obvious in cholangiocarcinoma tissues, however, this mucin has never been detected in the serum. Using immunoblotting marked with antibody vs. MUC5AC core protein, we could detect MUC5AC mucin in the serum of 112 from 179 cholangiocarcinoma patients (62.6% sensitivity), two of the 62 with benign hepatobiliary diseases, six of the 60 with hepato-gastrointestinal cancer, and none in either the 60 active opisthorchiasis or 74 healthy persons. Detection of serum mucin in the serum of cholangiocarcinoma patients corresponded well to the MUC5AC expressed in individual tissues. Serum MUC5AC may be used to enhance the diagnostic accuracy of cholangiocarcinoma.

A 54-year-old male who had no history of previous surgery presented with chronic intermittent constipation. A barium enema showed a 2-cm stenotic lesion at the junction between splenic flexure and descending colon. A colonoscopy revealed extra-luminal compression without mucosal lesion. Laparotomy revealed a parietocolic band compressing the proximal part of the descending colon. The band was lysed. The patient's symptom has improved since the procedure.

206 Background: Cholangiocarcinoma in Thailand and Laos is associated with the liver fluke Opisthorchis viverrini (OV). Molecular differences between OV-CCA and Non-OV CCA remain to be defined. MiRNAs are regulators of mRNA that result in transcriptional repression or gene silencing. MiRNA profiles of OV vs. non-OV CCA have not yet been studied. Methods: 50 OV-CCA and 16 non-OV CCA pathological samples were obtained from Liver Fluke and Cholangiocarcinoma Center, Khonkaen University, Thailand and University of Texas, MD Anderson Cancer Center (MDACC), USA, respectively. Macrodissected common bile duct samples from MDACC were used as control groups. Global miRNA expression profiling was performed using the miRCURY LNA microRNA Array (Exiqon).qRT-PCR and in situ hybridization (ISH) were used to validate the miRNAs noted to be significant on the microRNA array. Results: Ten samples of OV and non-OV CCA were analyzed for global miRNA expression; 46 were used for validation. MiRNA microarray identified 71 miRNAs that were differentially expressed between OV-CCA and non-OV CCA (p

Opisthorchis viverrini is considered among the most important of the food-borne trematodes due to its strong association with advanced periductal fibrosis and bile duct cancer (cholangiocarcinoma). We investigated the relationship between plasma levels of Interleukin (IL)-6 and the risk of developing advanced fibrosis and bile duct cancer from chronic Opisthorchis infection. We show that IL-6 circulates in plasma at concentrations 58 times higher in individuals with advanced fibrosis than age, sex, and nearest-neighbor matched controls and 221 times higher in individuals with bile duct cancer than controls. We also observed a dose-response relationship between increasing levels of plasma IL-6 and increasing risk of advanced fibrosis and bile duct cancer; for example, in age and sex adjusted analyses, individuals with the highest quartiles of plasma IL-6 had a 19 times greater risk of developing advanced periductal fibrosis and a 150 times greater risk of developing of bile duct cancer than individuals with no detectable level of plasma IL-6. Finally, we show that a single plasma IL-6 measurement has excellent positive predictive value for the detection of both advanced bile duct fibrosis and bile duct cancer in regions with high O. viverrini transmission. These data support our hypothesis that common mechanisms drive bile duct fibrosis and bile duct tumorogenesis from chronic O. viverrini infection. Our study also adds a unique aspect to the literature on circulating levels of IL-6 as an immune marker of hepatobiliary pathology by showing that high levels of circulating IL-6 in plasma are not related to infection with O. viverrini, but to the development of the advanced and often lethal pathologies resulting from chronic O. viverrini infection., Author Summary O. viverrini is among the few parasites considered a Class 1 carcinogen because of its strong association with bile duct cancer (cholangiocarcinoma). Currently, more than 40 million people are infected with O. viverrini worldwide. Thailand has the highest prevalence of O. viverrini at 10 million people infected and also the highest incidence of Opisthorchis-associated bile duct cancer (cholangiocarcinoma) in the world. In the current study, we also show that levels of IL-6 in plasma are associated in a dose-dependent fashion with Opisthorchis-induced bile duct fibrosis (periductal fibrosis) and cholangiocarcinoma. More importantly, we show that O. viverrini infection alone does not elevate IL-6 levels in the plasma. It is only in the presence of these advanced pathologies (advanced fibrosis or cholangiocarcinoma) from chronic O. viverrini infection that significantly elevates plasma levels of IL-6 are observed. Moreover, we show that plasma IL-6 is an easily accessible biomarker for the detection of advanced periductal fibrosis and cholangiocarcinoma, which would be a critical advance for this region of Thailand and other countries in Southeast Asia, where the prevalence of O. viverrini infection can reach as high as 80% and the incidence of bile duct cancer is the highest in the world.

To determine whether expression of certain enzymes related to 5-fluorouracil (5-FU) metabolism predicts 5-FU chemosensitivity in cholangiocarcinoma (CCA).The histoculture drug response assay (HDRA) was performed using surgically resected CCA tissues. Tumor cell viability was determined morphologically with hematoxylin and eosin- and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling-stained tissues. The mRNA expression of thymidine phosphorylase (TP), orotate phosphoribosyl transferase (OPRT), thymidylate synthase (TS), and dihydropyrimidine dehydrogenase (DPD) was determined with real-time reverse transcriptase-polymerase chain reaction. The levels of gene expression and the sensitivity to 5-FU were evaluated.Twenty-three CCA tissues were obtained from patients who had been diagnosed with intrahepatic CCA and who underwent surgical resection at Srinagarind Hospital, Khon Kaen University from 2007 to 2009. HDRA was used to determine the response of these CCA tissues to 5-FU. Based on the dose-response curve, 200 μg/mL 5-FU was selected as the test concentration. The percentage of inhibition index at the median point was selected as the cut-off point to differentiate the responding and non-responding tumors to 5-FU. When the relationship between TP, OPRT, TS and DPD mRNA expression levels and the sensitivity of CCA tissues to 5-FU was examined, only OPRT mRNA expression was significantly correlated with the response to 5-FU. The mean expression level of OPRT was significantly higher in the responder group compared to the non-responder group (0.41 ± 0.25 vs 0.22 ± 0.12, P0.05).OPRT mRNA expression may be a useful predictor of 5-FU chemosensitivity of CCA. Whether OPRT mRNA could be used to predict the success of 5-FU chemotherapy in CCA patients requires confirmation in patients.

A group of 87 adults from a small village in north-east Thailand was chosen to undergo ultra-sound investigation based on their intensity of infection with the liver fluke, Opisthorchis viverrini, or clinical status (history of jaundice, current hepatomegaly). From this group, 8 cases of suspected early cholangiocarcinoma were found, and the diagnostic features of 6 of the 8 were confirmed by computerized tomography scan and endoscopic retrograde cholangiopancreatography. In addition, several cases of mild gall-bladder disease, chronic cholecystitis, cholelithiasis and parenchymal liver disease were detected. A highly significant positive relationship between the intensity of liver fluke worm burden and the severity of biliary tract disease within individuals is reported. These results indicate that Opisthorchis is associated with moderate to severe hepatobiliary disease in a considerable proportion of infected individuals.

AIM: To determine allelic imbalance on chromosomal region 21q22-qter including trefoil factor family genes (TFF) in cholangiocarcinoma (CCA) patients and analyze the correlation between allelic imbalances and clinicopathological parameters. METHODS: Quantitative PCR amplification was performed on four microsatellite markers and trefoil factor family genes (TFF1, TFF2, and TFF3) using a standard curve and SYBR Green I dye method. The relative copy number was determined by DNA copy number of tested locus to reference locus. The relative copy number was interpreted as deletion or amplification by comparison with normal reference range. Associations between allelic imbalance and clinicopathological parameters of CCA patients were evaluated by χ2-tests. Kaplan-Meier method was used to analyze survival. RESULTS: The frequencies of amplification at D21S1890, D21S1893, and TFF3 were 32.5%, 30.0%, and 28.7%, respectively. Patients who had amplification at regions covering D21S1893, D21S1890, and TFF showed poor prognosis, whereas patients who had deletion showed favorable prognosis (mean: 51.7 wk vs 124.82 wk, P = 0.012). Multivariate Cox regression analysis revealed that amplification of D21S1893, D21S1890 and TFF, blood vessel invasion, and staging were associated with poor prognosis. CONCLUSION: D21S1893-D21S1890 region may harbor candidate genes especially TFF and serine protease family, which might be involved in tumor invasion and metastasis contributing to poor survival. The amplification in this region may be used as a prognostic marker in the treatment of CCA patients.

Nitrative and oxidative DNA damage such as 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation has been implicated in initiation and/or promotion of inflammation-mediated carcinogenesis. The aim of this study is to clarify whether these DNA lesions participate in the progression of intrahepatic cholangiocarcinoma.We investigated the relation of the formation of 8-nitroguanine and 8-oxodG and the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) with tumor invasion in 37 patients with intra-hepatic cholangiocarcinoma.Immunohistochemical analyses revealed that 8-nitroguanine and 8-oxodG formation occurred to a much greater extent in cancerous tissues than in non-cancerous tissues. HIF-1alpha could be detected in cancerous tissues in all patients, suggesting low oxygen tension in the tumors. HIF-1alpha expression was correlated with inducible nitric oxide synthase (iNOS) expression (r = 0.369 and P = 0.025) and 8-oxodG formation (r = 0.398 and P = 0.015). Double immunofluorescence study revealed that iNOS and HIF-1alpha co-localized in cancerous tissues. Notably, the formation of 8-oxodG was correlated significantly with lymphatic invasion (r = 0.386 and P = 0.018). Moreover, 8-nitroguanine and 8-oxodG in non-cancerous tissues were associated significantly with neural invasion (P = 0.042 and P = 0.026, respectively). These results suggest that reciprocal activation between HIF-1alpha and iNOS mediates persistent DNA damage, which induces tumor invasiveness via mutations, resulting in poor prognosis.The formation of 8-nitroguanine and 8-oxodG plays an important role in multiple steps of genetic changes leading to tumor progression, including invasiveness.