Liver Fluke Related Cholangiocarcinoma Research Articles

ARID1A alterations and their clinical significance in cholangiocarcinoma.

BackgroundARID1A is a member of the SWI/SNF chromatin remodeling complex. It functions as a tumor suppressor and several therapeutic targets in ARID1A-mutated cancers are currently under development, including EZH2. A synthetic lethal relationship between ARID1A and EZH2 has been revealed in several tumor entities. Although genomic alterations of ARID1A have been described in various cancers, no study has examined correlations between ARID1A gene mutation and protein expression with clinicopathologic parameters and prognosis, particularly in liver fluke-related cholangiocarcinoma (Ov-CCA). Here, we investigated the clinical significance of ARID1A mutations and protein expression in CCA tissues and determined whether there is a correlation with EZH2 protein expression.MethodsWe evaluated ARID1A and EZH2 immunoreactivity using immunohistochemistry in 98 Ov-CCA with a wide range of clinicopathological features. Somatic mutations of ARID1A were analyzed using the ICGC sequencing data in 489 of Ov and non Ov-CCA and assessed prognostic values.ResultsWhile detecting a loss or reduction of ARID1A expression in 54 cases (55%) in Ov-CCA, ARID1A expression was associated with ARID1A mutations (p < 0.001, adjusted p-value < 0.001). We observed that 12 of 13 tumors (92%) with loss of ARID1A expression had truncating mutations. There were nine of 13 tumors (69%) with loss of ARID1A expression and 25 of 41 tumors (61%) with low ARID1A expression exhibited distant metastasis (p = 0.028, adjusted p-value = 0.168). ARID1A was predominantly mutated in Ov-CCA compared to non Ov-CCA (24% and 14% in Ov-CCA and non Ov-CCA, respectively, p = 0.027). There were 36 of 72 (50%) and 52 of 79 (66%) tumors with ARID1A mutation showed tumor stage IV and T3/T4, respectively. The significant mutual exclusivity and co-occurrence between ARID1A and TP53/KRAS mutations were not found in ICGC cohort. In addition, high EZH2 expression, a potential synthetic lethal target in ARID1A-mutated tumors, was detected in 49 of 98 Ov-CCA (50%). Importantly, neither ARID1A expression nor ARID1A mutations correlated with EZH2 expression in this cohort.ConclusionWe found that ARID1A inactivation, by somatic mutation or by loss of expression, frequently occurs in Ov-CCA. Reduction of ARID1A expression and/or somatic mutation was shown to be associated with CCA progression. These findings suggest that ARID1A may serve as a prognostic biomarker, and thus may be a promising therapeutic target for CCA.

Expected rate of liver fluke-related cholangiocarcinoma in endemic area of liver fluke with antiparasitic drug distribution program for disease control in Thailand.

Parasitic-induced cancer is an important problem in tropical oncology. Liver fluke-related biliary tract cancer of cholangiocarcinoma is an important kind of endemic cancer in Southeast Asia. This cancer is related to the liver fluke infection. The local public health policies for disease control include antiparasitic drug, praziquantel, distributing aiming at getting rid of risk factor, and opisthorchiasis. In the present report, the authors reappraise on the local epidemiological data on liver fluke infection among the local people in endemic area Thailand under antiparasitic drug distribution program for further assessment for expected rate of cholangiocarcinoma. The present study is a clinical mathematical modeling study. First, the retrospective reappraisal on the available local data on liver fluke infection among the local people in endemic area of Thailand, a tropical country in Indochina, with a different history of previous exposure to antiparasitic drug is done. Then, a mathematical model based on predictive modeling and probability assignment technique is developed for the assessment of estimated rate of cholangiocarcinoma. According to the present study, the expected rate of liver fluke-related cholangiocarcinoma is equal to 0.0564%. There is a strong relationship between expected rate of liver fluke-related cholangiocarcinoma and number of previous exposure to praziquantel (r = 0.99, P = 0.02). According to the study, the high expected rare of cholangiocarcinoma is observed despite the use of general antiparasitic drug distribution policy. It implies finding for new adjusted public health manipulation for disease prevention and management.

Contribution of RIZ1 to Regulation of Proliferation and Migration of a Liver Fluke-Related Cholangiocarcinoma Cell

Retinoblastoma-interacting zinc finger gene (RIZ1) is a tumor suppressor gene which is highly inactivated by promoter hypermethylation in patients with liver fluke-related cholangiocarcinoma (CCA). Epigenetic aberration of this gene might withdraw the ability to restrain tumor cell proliferation and migration. We aimed to define the role of RIZ1 on cell proliferation and migration in CCA cell line. Small interference RNA (siRNA) was used to knock down the expression of RIZ1 in a CCA-derived cell line in which cell proliferation and cell migration were performed. A predominant nuclear localization of RIZ1 was observed. Reduction of RIZ1 by siRNA augmented cell proliferation and migration. The result suggested that RIZ1 might play a role in regulating cell proliferation and migration in CCA. Reduction of RIZ1 expression may aggravate the progression of CCA.

Net closes in on genetics of liver fluke-associated cholangiocarcinoma

Findings published in Nature Genetics provide the first insights into the mutational landscape of liver fluke-related cholangiocarcinoma (CCA).

CpG-island methylation study of liver fluke-related cholangiocarcinoma.

Background:Genetic changes have been widely reported in association with cholangiocarcinoma (CCA), while epigenetic changes are poorly characterised. We aimed to further evaluate CpG-island hypermethylation in CCA at candidate loci, which may have potential as diagnostic or prognostic biomarkers.Methods:We analysed methylation of 26 CpG-islands in 102 liver fluke related-CCA and 29 adjacent normal samples using methylation-specific PCR (MSP). Methylation of interest loci was confirmed using pyrosequencing and/or combined bisulfite restriction analysis, and protein expression by immunohistochemistry.Results:A number of CpG-islands (OPCML, SFRP1, HIC1, PTEN and DcR1) showed frequency of hypermethylation in >28% of CCA, but not adjacent normal tissues. The results showed that 91% of CCA were methylated in at least one CpG-island. The OPCML was the most frequently methylated locus (72.5%) and was more frequently methylated in less differentiated CCA. Patients with methylated DcR1 had significantly longer overall survival (Median; 41.7 vs 21.7 weeks, P=0.027). Low-protein expression was found in >70% of CCA with methylation of OPCML or DcR1.Conclusion:Aberrant hypermethylation of certain loci is a common event in liver fluke-related CCA and may potentially contribute to cholangiocarcinogenesis. The OPCML and DcR1 might serve as methylation biomarkers in CCA that can be readily examined by MSP.

Genetic and epigenetic alterations of RIZ1 and the correlation to clinicopathological parameters in liver fluke-related cholangiocarcinoma

The retinoblastoma interacting zinc finger (RIZ1) gene is adjacent to D1S228 where microsatellite instability has been associated with poor patient survival in liver fluke-associated cholangiocarcinoma (CCA). An understanding of the molecular mechanisms underlying the carcinogenesis and pathogenesis of CCA is necessary to improve patient survival. Therefore, we determined the genetic and epigenetic alterations of RIZ1 in 81 CCA samples and 69 matched non-tumor tissues. Methylation was found in 31 of 81 (38%) tumor samples and in 5 of 69 (7%) matched non-tumor tissues. Frameshift mutations (2 of 81) and loss of heterozygosity (LOH) (14 of 81) were not common. Statistical analysis found no significant correlation between RIZ1 alterations and clinicopathological features, but RIZPro704 LOH was associated with patient survival in the multivariate analysis. RIZ1 hypermethylation may be one of the crucial molecular events contributing to cholangiocarcinogenesis, and RIZPro704 LOH may adversely impact patient survival. The biological function of RIZ1 in CCA should be further investigated in order to verify its potential role in regulating this cancer.

Preferentially different mechanisms of inactivation of 9p21 gene cluster in liver fluke–related cholangiocarcinoma

Cholangiocarcinoma in northeast Thailand is associated with liver fluke infection. Mechanisms of inactivation of the p15(INK4b), p16(INK4a), and p14(ARF) have been reported in many human cancers but have not hitherto been studied in liver fluke-related cholangiocarcinoma, particularly genetic and epigenetic effects on protein expression. We investigated loss of heterozygosity and microsatellite instability and performed fine mapping of the chromosomal region 9p21-pter in 94 microdissected cholangiocarcinoma samples using polymerase chain reaction based-microsatellite markers. Methylation and protein expression of p14(ARF), p15(INK4b), and p16(INK4a) was determined using methylation-specific polymerase chain reaction and immunohistochemistry, respectively. Genetic and epigenetic alterations, including loss of protein expression, were correlated with clinicopathological data. Fine mapping at 9p21-pter showed a distinctive region between D9S286 and D9S1752 of common loss. Methylation frequency was 40.2% for p14(ARF), 48.9% for p15(INK4b), and 28.3% for p16(INK4a). Loss of protein expression of p14(ARF), p15(INK4b), and p16(INK4a) was 30.9%, 58%, and 81.5%, respectively. Both p14(ARF) methylation and allelic loss at 9p21 were associated with loss of p14(ARF) expression. Poor prognosis was associated with loss of p16(INK4a) expression. In conclusion, mechanisms of inactivation of p14(ARF), p15(INK4b), and p16(INK4a) in liver fluke-related cholangiocarcinoma are preferentially different, by which epigenetic event being the main mechanism of p14(ARF), whereas p16(INK4a) and p15(INK4b) inactivation occurs through genetic and both genetic and epigenetic events, respectively.

Differential expression of Trefoil factor family reflects their different roles in liver fluke related Cholangiocarcinoma

Differential expression of Trefoil factor family reflects their different roles in liver fluke related Cholangiocarcinoma

368 POSTER Genetic instability at 9p21 and its significance as prognostic indicators in liver fluke related cholangiocarcinoma

368 POSTER Genetic instability at 9p21 and its significance as prognostic indicators in liver fluke related cholangiocarcinoma

394 POSTER Inhibition of thymidine phosphorylase decreases tumour aggressiveness but reduces chemosensitivity in liver fluke related cholangiocarcinoma

Mouse embryonic stem (mES) cells will give rise to all of the cells of the adult mouse, but they failed to rejoin half of the DNA double-strand breaks (dsb) produced by high doses of ionizing radiation. A deficiency in DNA-PKcs appears to be responsible since mES cells expressed <10% of the level of mouse embryo fibroblasts (MEFs) although Ku70/80 protein levels were higher than MEFs. However, the low level of DNA-PKcs found in wild-type cells appeared sufficient to allow rejoining of dsb after doses <20 Gy even in G1 phase cells. Inhibition of DNA-PKcs with wortmannin and NU7026 still sensitized mES cells to radiation confirming the importance of the residual DNA-PKcs at low doses. In contrast to wild-type cells, mES cells lacking H2AX, a histone protein involved in the DNA damage response, were radiosensitive but they rejoined double-strand breaks more rapidly. Consistent with more rapid dsb rejoining, H2AX−/− mES cells also expressed 6 times more DNA-PKcs than wild-type mES cells. Similar results were obtained for ATM−/− mES cells. Differentiation of mES cells led to an increase in DNA-PKcs, an increase in dsb rejoining rate, and a decrease in Ku70/80. Unlike mouse ES, human ES cells were proficient in rejoining of dsb and expressed high levels of DNA-PKcs. These results confirm the importance of homologous recombination in the accurate repair of double-strand breaks in mES cells, they help explain the chromosome abnormalities associated with deficiencies in H2AX and ATM, and they add to the growing list of differences in the way rodent and human cells deal with DNA damage.

Amplification of D22S283 as a favorable prognostic indicator in liver fluke related cholangiocarcinoma

To analyze the DNA copy number of target genes NF2, TIMP3, ST13, TOB2, BIK, and TP and the reference microsatellite markers D22S283, D22S423, and D22S274 mapped on 22q12-qter in liver fluke related cholangiocarcinoma (CCA) and define its correlation with clinical parameters. Quantitative real time PCR (qPCR) was used for determining allelic imbalances in 65 liver fluke related CCA tissues. Statistical correlations between allelic imbalances and clinicopathological parameters, i.e. age, sex, tumor stage, histological type, blood vessel invasion, nerve invasion and lymphatic invasion were evaluated by means of the chi2 test. Cox regression analysis was used for determining patient's survival. Amplifications of the TP (22q13.33), TOB2 (22q13.2-13.31), D22S283 (22q12.3), TIMP3 (22q12.3) and NF2 (22q12.2) were found in 35 (53.8%), 28 (43.1%), 27 (41.5%), 24 (36.9%), and 24 (36.9%), respectively. Losses at the D22S423 (22q13.1-13.2) and BIK (22q13.31) were detected in 26 (40%) and 23 (35.4%), respectively. Significant correlations were observed between lymphatic invasion and allelic losses of BIK (P = 0.025) and D22S283 (P = 0.041). Univariate and multivariate Cox regression analysis revealed D22S283 amplification as an independent predictor of good prognosis (P = 0.006, death hazard ratio = 0.411, 95% CI = 0.217-0.779) and blood vessel invasion as an independent poor prognostic factor (P = 0.042, death hazard ratio = 1.911, 95% CI = 1.022-3.571) in CCA patients. This study provides evidence for the involvement of gene amplification and deletion on chromosome 22q in liver fluke related CCA. This is the first report of D22S283 amplification as an independent indicator of favorable prognosis in liver fluke related CCA.

Amplification of chromosome 21q22.3 harboring trefoil factor family genes in liver fluke related cholangiocarcinoma is associated with poor prognosis

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. 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 chi(2)-tests. Kaplan-Meier method was used to analyze survival. 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. 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.

Promoter hypermethylation is a major event of hMLH1 gene inactivation in liver fluke related cholangiocarcinoma

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.

599 Promoter hypermethylation of hMLH1 gene in liver fluke related cholangiocarcinoma

599 Promoter hypermethylation of hMLH1 gene in liver fluke related cholangiocarcinoma

Microsatellite alterations in liver fluke related cholangiocarcinoma are associated with poor prognosis

We have characterized the role of genetic alterations in the development of liver fluke related cholangiocarcinoma. We analyzed the loss of heterozygosity (LOH) and microsatellite instability (MSI) of hMSH2, hMLH1, and p53 genes in 55 patients with intrahepatic cholangiocarcinoma by using polymerase chain reaction based microsatellite markers D2S119, D3S1611, and TP53, respectively and determined the association between microsatellite alterations and patient survival. A total of 27 (49.1%) out of 55 cases exhibited microsatellite alterations in one locus or more. Of 55 samples, 11 (20%) demonstrated MSI at D2S119 and four (7%) showed MSI at D3S1611. LOH was shown in seven out of 36 (19%) informative cases for D3S1611 and 16 out of 50 (32%) for TP53. Microsatellite alterations at loci studied were significantly associated with poor survival ( P=0.0098). This study suggests that genetic alterations of DNA mismatch repair genes and tumor suppressor gene p53 may be involved in cholangiocarcinogenesis and these alterations may be of value as prognostic indicators for liver fluke related cholangiocarcinoma.