Rintaro Mikata

Methylation Status of Genes Upregulated by Demethylating Agent 5-aza-2′-Deoxycytidine in Hepatocellular Carcinoma

Background/Aims: To determine the clinical significance of gene promoter methylation in hepatocellular carcinoma (HCC), we examined in clinical samples the methylation status of those promoters that showed elevated activity in hepatoma cell lines after 5-aza-2′-deoxycytidine treatment. Methods: Regarding the genes with promoter hypermethylation in the cell lines, their expression levels and methylation status in HCC and non-HCC tissues were assessed by semiquantitive RT-PCR and methylation-specific PCR. To confirm the result, the expression levels and methylation status in 16 additional HCC and non-HCC tissues were assessed. Results: The promoter regions of caveolin 1 (CAV1), cysteine and glycine-rich protein 1 (CSRP1), Kruppel-like factor 6 (KLF6), myosin (light polypeptide 9) (MYL9), and transgelin (TAGLN) were highly methylated in the cell lines. CAV1 and CSRP1 were methylated in HCC more frequently than in non-HCC. KLF6, MYL9, and TAGLN were fully methylated in both HCC and non-HCC. Using additional clinical samples, downregulation of CAV1 and CSRP1 was observed in 38 and 56%, respectively, of the 16 HCC samples and aberrant methylation of CAV1 and CSRP1 was observed in 56% of HCC in both cases. Conclusion:CAV1 and CSRP1 were inactivated in HCC by aberrant methylation and they may serve as important biomarkers of malignancy.

Methylation status of p14ARF, p15INK4b, and p16INK4a genes in human hepatocellular carcinoma.

Abstract: Background: The INK4 locus consisting of three genes involved in the regulation of cell cycle, p16INK4a, p15INK4b, and p14ARF is often disrupted in human neoplasms.

Efficacy of high‐dose corticosteroid in the early stage of viral acute liver failure

Acute liver failure (ALF) is a worldwide problem despite its rare incidence because of its extremely high mortality. There are no beneficial therapies except for emergency liver transplantation for ALF. However, in Japan where the problem of a shortage of donor livers still remains, therapies other than transplantation must be further investigated for patients with ALF. Our aim was to elucidate the efficacy of high‐dose corticosteroid (CS) in decreasing liver enzyme levels in the early stage of ALF.

Ring-like enhancement of focal nodular hyperplasia with hepatobiliary-phase Gd-EOB-DTPA-enhanced magnetic resonance imaging: radiological-pathological correlation.

We report a case of focal nodular hyperplasia in a patient for whom gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI) and histological analysis results were available. Dynamic contrast-enhanced computed tomography showed a well-defined hypervascular lesion 14 mm in diameter with no visible central scars. Gd-EOB-DTPA-enhanced MRI demonstrated strong peripheral enhancement of the lesion during the hepatobiliary phase, resulting in ring-like enhancement. The pathology examination revealed that the lesion was focal nodular hyperplasia (FNH). Immunohistochemistry showed positive expression of OATP8 in the hepatocytes in the peripheral areas of the lesion, whereas expression of OATP8 was lacking in hepatocytes surrounding the central radiating scar. Ring-like enhancement during the hepatobiliary phase of Gd-EOB-DTPA-enhanced MRI may be an important clue for the diagnosis of small FNH.

Analysis of genes upregulated by the demethylating agent 5‐aza‐2′‐deoxycytidine in gastric cancer cell lines

In gastric cancer, increasing numbers of genes have been reported to be silenced by aberrant methylation. However, global analysis of epigenetic inactivation in cancer cells has rarely been performed. For screening the genes upregulated by the demethylating agent 5‐aza‐2′‐deoxycytidine (DAC), cDNA microarray analysis (AceGene®, containing 30,000 genes) was performed using gastric cancer cell lines (AGS, MKN74, MKN1, MKN45 and Kato3) treated with DAC. The candidate upregulated genes were confirmed by real‐time PCR, and the methylation status of 5′CpG islands was determined by bisulfite DNA sequencing or methylation‐specific PCR. Among the upregulated genes considered to have CpG island in their promoter regions, we selected 5 genes (BCL2L10, DKK1, DNAJD1, GAGED2 and NMU) that exhibited a greater than 3‐fold increase in at least 2 cell lines. Of these, we could determine the methylation status of 5′CpG islands of BCL2L10, DKK1 and DNAJD1. 5′CpG of BCL2L10 and DNAJD1 was hypermethylated in 4 of 5 gastric cancer cell lines, whereas 5′CpG of DKK1 was hypermethylated in only 1 cell line. MSP analysis for BCL2L10 revealed that the CpG island was demethylated after DAC treatment. In addition, we observed that overexpression of BCL2L10 could promote apoptosis and growth‐inhibitory effect in gastric cancer cell lines. In conclusion, some of the genes upregulated by DAC treatment may be transcriptionally repressed by promoter hypermethylation. These genes might be related to gastric carcinogenesis. In particular, the suppression of BCL2L10, which could induce apoptosis and inhibit proliferation of cancer cells, might be one of the underlying mechanisms for gastric carcinogenesis.

Quantification of hepatitis C virus in patients treated with peginterferon-alfa 2a plus ribavirin treatment by COBAS TaqMan HCV test.

Summary.  Extremely low levels of serum hepatitis C virus (HCV) RNA can be detected by COBAS TaqMan HCV test. To investigate whether the COBAS TaqMan HCV test is useful for measuring rapid virological response (RVR) and early virological response (EVR) to predict sustained virological response (SVR), we compared the virological response to PEG‐IFN‐alfa 2a plus RBV in 76 patients infected with HCV genotype 1 when undetectable HCV RNA by the COBAS TaqMan HCV test was used, with those when below 1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test was used, which corresponded to the use of traditional methods. Among the 76 patients, 28 (36.8%) had SVR, 13 (17.1%) relapsed, 19 (25.0%) did not respond, and 16 (21.0%) discontinued the treatment due to side effects. The positive predictive values for SVR based on undetectable HCV RNA by COBAS TaqMan HCV test at 24 weeks after the end of treatment [10/10 (100%) at week 4, 21/23 (91.3%) at week 8 and 26/33 (78.7%) at week 12] were superior to those based on <1.7 log IU/mL HCV RNA [17/19 (89.4%) at week 4, 27/38 (71.0%) at week 8, and 27/43 (62.7%) at week 12]. The negative predictive values for SVR based on <1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test [46/57 (80.7%) at week 4, 37/38 (97.3%) at week 8, and 32/33 (96.9%) at week 12] were superior to those based on undetectable HCV RNA [48/66 (72.7%) at week 4, 46/53 (86.7%) at week 8, and 41/43 (95.3%) at week 12]. The utilization of both undetectable RNA and <1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test is useful and could predict SVR and non‐SVR patients with greater accuracy.

Use of F-18 Fluorodeoxyglucose Positron Emission Tomography With Dual-Phase Imaging to Identify Intraductal Papillary Mucinous Neoplasm

BACKGROUND & AIMS We investigated the usefulness of dual-phase F-18 fluorodeoxyglucose positron emission tomography with computed tomography (FDG-PET/CT) to differentiate benign from malignant intraductal papillary mucinous neoplasms (IPMNs) and to evaluate branch-duct IPMNs. METHODS We used FDG-PET/CT to evaluate IPMNs in 48 consecutive patients who underwent surgical resection from May 2004 to March 2012. IPMNs were classified as benign (n = 16) or malignant (n = 32) on the basis of histology analysis. The ability of FDG-PET/CT to identify branch-duct IPMNs was compared with that of the International Consensus Guidelines. RESULTS The maximum standardized uptake value (SUVmax) was higher for early-phase malignant IPMNs than that for benign IPMNs (3.5 ± 2.2 vs 1.5 ± 0.4, P < .001). When the SUVmax cutoff value was set at 2.0, early-phase malignant IPMNs were identified with 88% sensitivity, specificity, and accuracy. The retention index values for malignant and benign IPMNs were 19.6 ± 17.8 and -2.6 ± 12.9, respectively. When the SUVmax cutoff was set to 2.0 and the retention index value to -10.0, early-phase malignant IPMNs were identified with 88% sensitivity, 94% specificity, and 90% accuracy. In identification of branch-duct IPMNs, when the SUVmax cutoff was set to 2.0, the sensitivity, specificity, and accuracy values were 79%, 92%, and 84%, respectively. By using a maximum main pancreatic duct diameter ≥7 mm, the Guidelines identified branch-duct IPMNs with greater specificity than FDG-PET/CT. The Guidelines criteria of maximum cyst size ≥30 mm and the presence of intramural nodules identified branch-duct IPMNs with almost equal sensitivity to FDG-PET/CT. CONCLUSIONS Dual-phase FDG-PET/CT is useful for preoperative identification of malignant IPMN and for evaluating branch-duct IPMN.

Ten-year long-term results after non-surgical management of hepatolithiasis, including cases with choledochoenterostomy.

Long‐term follow‐up of non‐surgical procedures for the management of hepatolithiasis has been reported, but risk factors for mortality have not been properly evaluated.

Identification of a novel serum biomarker for pancreatic cancer, C4b-binding protein α -chain (C4BPA) by quantitative proteomic analysis using tandem mass tags

Background:Pancreatic ductal adenocarcinoma (PDAC) remains a devastating disease due to the lack of specific early diagnostic markers. To improve the outcomes, proteomic approaches are being developed for the discovery of novel biomarkers of PDAC.Methods:Using tandem mass tag labelling and LC-MS/MS, we performed comparative analyses of pre- and postoperative sera from PDAC patients to identify specific serum biomarkers for PDAC. In validation studies, we evaluated the discriminatory power of candidate proteins.Results:Among the 302 proteins analysed, 20 were identified as potential biomarkers, with C4b-binding protein α-chain (C4BPA) and polymeric immunoglobulin receptor (PIGR) being selected for further analysis. The sera levels of C4BPA and PIGR were significantly higher in the preoperative PDAC patients than in the postoperative ones (P<0.008, P<0.036, respectively). In addition, serum C4BPA levels, but not PIGR, in patients with PDAC were significantly higher than those in healthy controls as well as in patients with pancreatitis and other malignancies including biliary tract cancers (BTC) (P<0.001). The respective area under the receiver operator characteristics (ROC) curve (AUC) was 0.860 for C4BPA, 0.846 for CA19-9 and 0.930 for the combination of C4BPA and CA19-9 in PDAC vs non-cancer individuals. The respective AUC was 0.912 for C4BPA, 0.737 for CA19-9 in Stages I and II of PDAC, 0.854 for C4BPA and 0.264 for CA19-9 in PDAC vs BTC.Conclusions:We have demonstrated that C4BPA is a novel serum biomarker for detecting early stage PDAC, as well as for distinguishing PDAC from other gastroenterological cancers. Further analysis in large cohort studies will warrant C4BPA as a promising biomarker of PDAC in clinical use.

Analysis of the complete hepatitis B virus genome in patients with genotype C chronic hepatitis and hepatocellular carcinoma

Hepatitis B virus (HBV) genotype C and the basic core promoter (BCP) mutations were reported to be associated with the development of hepatocellular carcinoma (HCC). In this study the full sequences of HBV genomes were analyzed in order to find the other predictors of HCC development. We determined the full sequences of HBV genomes in 24 genotype C carriers who developed HCC (HCC group) at the beginning of follow‐up and at the time of HCC diagnosis, and 20 patients who did not develop HCC (non‐HCC group) served as a control. The number of nucleotide and amino acid substitutions in most regions was higher in the HCC group than in the non‐HCC group, and the following substitutions and deletions were found more frequently in the HCC group than in the non‐HCC group: G1317A and T1341C/A/G in the X promoter region were detected in 13 and six of the HCC cases, four and none of the non‐HCC cases, respectively; and pre‐S2 deletion was detected in eight HCC and none of the non‐HCC cases. Compared with the wild type X promoter, the mutant type X promoters, M1 (G1317A), M2 (T1341C), and M4 (T1341G) showed increases in activity of 2.3, 3.8, and 1.4 times, respectively, in HepG2 cells. Substitutions and deletion of nucleotides of the HBV genome, especially the pre‐S2 deletion and G1317A and T1341C/A/G mutations may be useful markers for predicting the development of HCC. (Cancer Sci 2007; 98: 1921–1929)