Pharmacological Unmasking Microarray Approach-Based Discovery of Novel DNA Methylation Markers for Hepatocellular Carcinoma

DNA methylation is one of the main epigenetic mechanisms and hypermethylation of CpG islands at tumor suppressor genes switches off these genes. To find novel DNA methylation markers in hepatocellular carcinoma (HCC), we performed pharmacological unmasking (treatment with 5-aza-2'-deoxycytidine or trichostatin A) followed by microarray analysis in HCC cell lines. Of the 239 promoter CpG island loci hypermethylated in HCC cell lines (as revealed by methylation-specific PCR), 221 loci were found to be hypermethylated in HCC or nonneoplastic liver tissues. Thirty-three loci showed a 20% higher methylation frequency in tumors than in adjacent nonneoplastic tissues. Correlation of individual cancer-related methylation markers with clinicopathological features of HCC patients (n = 95) revealed that the number of hypermethylated genes in HCC tumors was higher in older than in younger patients. Univariate and multivariate survival analysis revealed that the HIST1H2AE methylation status is closely correlated with the patient's overall survival (P = 0.022 and P = 0.010, respectively). In conclusion, we identified 221 novel DNA methylation markers for HCC. One promising prognostic marker, HIST1H2AE, should be further validated in the prognostication of HCC patients

Altered expression of thioredoxin reductase-1 in dysplastic bile ducts and cholangiocarcinoma in a hamster model

Thioredoxin reductase 1 (TrxR) is a homodimeric selenoenzyme catalyzing thioredoxin (Trx) in an NADPHdependent manner. With regard to carcinogenesis, these redox proteins have been implicated in cell proliferation, transformation and anti-apoptosis. In the present study, using a hamster cholangiocarcinoma (ChC) model, we evaluated the immunohistochemical expression pattern of TrxR in precancerous lesions and ChCs as well as in normal bile ducts. The goal of this study was to determine the potential role and importance of TrxR in cholangiocarcinogenesis. For the ChC model, we obtained liver tissue specimens with dysplastic bile ducts prior to the development of ChC 8 weeks after initiation of the experiment and ChC samples at 27 weeks. The immunohistochemical analysis showed diffuse cytoplasmic overexpression of TrxR in the dysplastic bile duct epithelial cells as well as in cholangiocarcinoma; this was comparable to the negative or weakly positive in normal and type 1 hyperplastic bile ducts. However, TrxR appeared to be considerably down-regulated in the ChCs when compared to the higher expression observed in the dysplastic bile ducts. Therefore, these results suggest that TrxR overexpression followed by down-regulation might be an important event in cholangiocarcinogenesis, especially at early stages including the cellular transformation of candidate bile ducts. Further studies are however required to determine whether TrxR may be a potential target molecule for chemoprevention against cholangiocarcinogenesis. In addition, the molecular mechanism as well as the importance of the loss of TrxR in the development of cholangiocarcinoma, following dysplastic transformation of bile duct cells, also remains to be clarified.This study was supported by Kangwon National University and Korea Research Foundation Grant (KRF-2004-041- E00324). The authors wish to thank Dr. Min-Ho Choi, College of Medicine, Seoul National University, for providing Clonorchis sinensis

Hepatocellular Carcinoma with Immature T-Cell (T-lymphoblastic) Proliferation

Indolent T-lymphoblastic proliferation has been rarely reported in the upper aerodigestive tract. The lymphoid cells associated with this condition have the morphological and phenotypical features of immature thymocytes. However, their pathogenesis and biology are unknown. We present an unusual type of tumor infiltrating lymphocytes in a case with hepatocellular carcinoma, presumed to be a T-lymphoblastic proliferation. A 58-yr-old female patient presented with indigestion and a palpable epigastric mass. The abdominal computed tomography revealed a mass in the S6 region of the liver. A hepatic segmentectomy was performed. Microscopic examination showed dense isolated nests of monomorphic lymphoid cells within the tumor. Immunohistochemically, the lymphoid cells were positive for CD3, terminal deoxymucleotide transferase (TdT) and CD1a. In addition, they showed dual expression of CD4 and CD8. The polymerase chain reaction used to examine the T-cell antigen receptor gamma gene rearrangement showed polyclonal T-cell proliferation. This is the second case of hepatocellular carcinoma combined with indolent T-lymphoblastic proliferation identified by an unusual tumor infiltrating lymphocytes

Tumoral LINE-1 hypomethylation is associated with poor survival of patients with intrahepatic cholangiocarcinoma

ECC: Extrahepatic cholangiocarcinoma; ICC: Intrahepatic cholangiocarcinoma; IG: Intraductal growth; LINE-1: Long interspersed element-1; MF: Mass-forming; PI: Periductal infiltrative; TNM: Tumor, node, and metastasisAbstract Background DNA methylation changes occurring in cancer cells are featured with both promoter CpG island hypermethylation and diffuse genomic hypomethylation. Long interspersed element-1 (LINE-1) is repeated in an interspersed manner with an estimated 500,000 copies per genome. LINE-1 has its CpG sites of the 5′ untranslated region methylated heavily in normal cells and undergoes demethylation in association with cancerization. However, little information is available regarding LINE-1 hypomethylation and its prognostic implication in intrahepatic cholangiocarcinomas. Methods A total of 172 cases of intrahepatic cholangiocarcinomas were analyzed for their methylation levels at four CpG sites of LINE-1 using bisulfite pyrosequencing. We examined the relation between tumoral LINE-1 methylation level and clinicopathological features, including survival. Results Tumor differentiation, lymphatic invasion, and T stage were associated with a low average methylation level of LINE-1 at the four CpG sites; LINE-1 methylation level tended to be lower in high-grade differentiation, lymphatic emboli, and higher T stage. LINE-1 hypomethylation was significantly linked with lower cancer-specific survival in patients with intrahepatic cholangiocarcinoma and was found to be an independent prognostic parameter. Conclusions Our findings suggest that tumoral LINE-1 hypomethylation could be a molecular biomarker heralding poor prognosis of patients with intrahepatic cholangiocarcinoma. Our findings need to be validated in further study.This work was supported by a grant from the National Research Foundation (NRF) grants funded by the Korean Ministry of Science, ICT and Future Planning (2011–0030049 and 2016M3A9B6026921), a grant from the Priority Research Centers Program through the NRF (2009–0093820), and a grant from the Korea Health Technology R & D Project through the Korea Health Industry Development Institute funded by the Korean Ministry of Health and Welfare (HI14C1277). The funding bodies had no role in the design of the study, the collection, analysis, and interpretation of the data, or in the writing of the manuscript

Tumoral LINE-1 hypomethylation is associated with poor survival of patients with intrahepatic cholangiocarcinoma

ECC: Extrahepatic cholangiocarcinoma; ICC: Intrahepatic cholangiocarcinoma; IG: Intraductal growth; LINE-1: Long interspersed element-1; MF: Mass-forming; PI: Periductal infiltrative; TNM: Tumor, node, and metastasisAbstract Background DNA methylation changes occurring in cancer cells are featured with both promoter CpG island hypermethylation and diffuse genomic hypomethylation. Long interspersed element-1 (LINE-1) is repeated in an interspersed manner with an estimated 500,000 copies per genome. LINE-1 has its CpG sites of the 5′ untranslated region methylated heavily in normal cells and undergoes demethylation in association with cancerization. However, little information is available regarding LINE-1 hypomethylation and its prognostic implication in intrahepatic cholangiocarcinomas. Methods A total of 172 cases of intrahepatic cholangiocarcinomas were analyzed for their methylation levels at four CpG sites of LINE-1 using bisulfite pyrosequencing. We examined the relation between tumoral LINE-1 methylation level and clinicopathological features, including survival. Results Tumor differentiation, lymphatic invasion, and T stage were associated with a low average methylation level of LINE-1 at the four CpG sites; LINE-1 methylation level tended to be lower in high-grade differentiation, lymphatic emboli, and higher T stage. LINE-1 hypomethylation was significantly linked with lower cancer-specific survival in patients with intrahepatic cholangiocarcinoma and was found to be an independent prognostic parameter. Conclusions Our findings suggest that tumoral LINE-1 hypomethylation could be a molecular biomarker heralding poor prognosis of patients with intrahepatic cholangiocarcinoma. Our findings need to be validated in further study.This work was supported by a grant from the National Research Foundation (NRF) grants funded by the Korean Ministry of Science, ICT and Future Planning (2011–0030049 and 2016M3A9B6026921), a grant from the Priority Research Centers Program through the NRF (2009–0093820), and a grant from the Korea Health Technology R & D Project through the Korea Health Industry Development Institute funded by the Korean Ministry of Health and Welfare (HI14C1277). The funding bodies had no role in the design of the study, the collection, analysis, and interpretation of the data, or in the writing of the manuscript

Hepatocellular carcinoma in liver transplantation candidates: detection with gadobenate dimeglumine-enhanced MRI

The purpose of this study was to retrospectively evaluate the diagnostic performance of dynamic gadobenate dimeglumine-enhanced MRI with explant pathologic correlation in the detection of hepatocellular carcinoma (HCC) in patients undergoing liver transplantation. MATERIALS AND METHODS: Forty-seven patients (28 men, 19 women; mean age, 49 years) underwent dynamic gadobenate dimeglumine-enhanced MRI within 3 months before primary liver transplantation. Dynamic imaging was performed before (unenhanced) and after (hepatic arterial, portal venous, equilibrium, and 1-hour delayed phases) IV bolus administration of gadobenate dimeglumine at 0.1 mmol/kg body weight. Retrospective image analysis to detect HCC nodules was performed independently by two abdominal radiologists who had no pathologic information. On a per-nodule basis, the sensitivity and positive predictive value were calculated for the two observers. Sensitivity and specificity in the diagnosis of HCC also were evaluated. Fisher's exact test was performed to determine whether there was a detection difference between HCC nodules 1 cm in diameter or larger and nodules smaller than 1 cm and to evaluate the differences in causes of false-positive MRI findings based on lesion size (>or= 1 cm vs < 1 cm). RESULTS: Twenty-seven patients had 41 HCCs. In HCC detection, gadobenate dimeglumine-enhanced MRI had a sensitivity of 85% (35 of 41 HCCs) and a positive predictive value of 66% (35 of 53 readings) for observer 1 and a sensitivity of 80% (33 of 41 HCCs) and a positive predictive value of 65% (34 of 52 readings) for observer 2. For both observers, sensitivity in the detection of HCCs 1 cm in diameter and larger (91-94%) was significantly different (p < 0.05) from that in detection of HCCs smaller than 1 cm (29-43%). Nonneoplastic arterial hypervascular lesions more often caused false-positive diagnoses of lesions smaller than 1 cm in diameter (80-86%) on MR images than of those 1 cm in diameter and larger (0-25%). The difference was statistically significant (p < 0.05) for both observers. In diagnosis, gadobenate dimeglumine-enhanced MRI had a sensitivity of 87% (20 of 23 patients) and a specificity of 79% (19 of 24 patients) for both observers. CONCLUSION: Dynamic gadobenate dimeglumine-enhanced MRI has a sensitivity of 80-85% and a positive predictive value of 65-66% in the detection of HCC. The technique, however, is of limited value for detecting and characterizing lesions smaller than 1 cm in diameter

Primary Biliary Lymphoma Mimicking Cholangiocarcinoma: A Characteristic Feature of Discrepant CT and Direct Cholangiography Findings

Primary non-Hodgkin's lymphoma arising from the bile duct is extremely rare and the reported imaging features do not differ from those of cholangiocarcinoma of the bile duct. We report a case of a patient with extranodal marginal zone B-cell lymphoma of mucosa associated lymphoid tissue (MALT), who presented with obstructive jaundice and describe the distinctive radiologic features that may suggest the correct preoperative diagnosis of primary lymphoma of the bile duct. Primary MALT lymphoma of the extrahepatic bile duct should be considered in the differential diagnosis when there is a mismatch in imaging findings on computed tomography or magnetic resonance imaging and cholangiography

Altered expression of thioredoxin reductase-1 in dysplastic bile ducts and cholangiocarcinoma in a hamster model

Thioredoxin reductase 1 (TrxR) is a homodimeric selenoenzyme catalyzing thioredoxin (Trx) in an NADPH-dependent manner. With regard to carcinogenesis, these redox proteins have been implicated in cell proliferation, transformation and anti-apoptosis. In the present study, using a hamster cholangiocarcinoma (ChC) model, we evaluated the immunohistochemical expression pattern of TrxR in precancerous lesions and ChCs as well as in normal bile ducts. The goal of this study was to determine the potential role and importance of TrxR in cholangiocarcinogenesis. For the ChC model, we obtained liver tissue specimens with dysplastic bile ducts prior to the development of ChC 8 weeks after initiation of the experiment and ChC samples at 27 weeks. The immunohistochemical analysis showed diffuse cytoplasmic overexpression of TrxR in the dysplastic bile duct epithelial cells as well as in cholangiocarcinoma; this was comparable to the negative or weakly positive in normal and type 1 hyperplastic bile ducts. However, TrxR appeared to be considerably down-regulated in the ChCs when compared to the higher expression observed in the dysplastic bile ducts. Therefore, these results suggest that TrxR overexpression followed by down-regulation might be an important event in cholangiocarcinogenesis, especially at early stages including the cellular transformation of candidate bile ducts. Further studies are however required to determine whether TrxR may be a potential target molecule for chemoprevention against cholangiocarcinogenesis. In addition, the molecular mechanism as well as the importance of the loss of TrxR in the development of cholangiocarcinoma, following dysplastic transformation of bile duct cells, also remains to be clarified

Suppression of osteopontin inhibits chemically induced hepatic carcinogenesis by induction of apoptosis in mice

Previous clinical reports have found elevated osteopontin (OPN) levels in tumor tissues to be indicative of greater malignancy in human hepatocellular carcinoma (HCC). However, the role of OPN on carcinogenesis and its underlying mechanism remain unclear. In the present study, we investigated the oncogenic role of OPN in diethylnitrosamine (DEN)-induced hepatic carcinogenesis in mice. The overall incidence of hepatic tumors at 36 weeks was significantly lower in OPN knockout (KO) mice than in wild-type (WT) mice. Apoptosis was significantly enhanced in OPN KO mice, and was accompanied by the downregulation of epidermal growth factor receptor (EGFR). In the in vitro study, OPN suppression also led to lower mRNA and protein levels of EGFR associated with the downregulation of c-Jun in Hep3B and Huh7 human HCC cells lines, which resulted in increased apoptotic cell death in both cell lines. Moreover, a positive correlation was clearly identified between the expression of OPN and EGFR in human HCC tissues. These data demonstrate that the OPN deficiency reduced the incidence of chemically induced HCC by suppressing EGFR-mediated antiapoptotic signaling. An important implication of our findings is that OPN positively contributes to hepatic carcinogenesis.OAIID:RECH_ACHV_DSTSH_NO:T201614207RECH_ACHV_FG:RR00200001ADJUST_YN:EMP_ID:F001304CITE_RATE:5.008DEPT_NM:의학과EMAIL:[email protected]_YN:YCONFIRM: