Tetsuro Urashima

Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues

MicroRNAs (miRNAs) are a non-coding family of genes involved in post-transcriptional gene regulation. These transcripts are associated with cell proliferation, cell differentiation, cell death and carcinogenesis. We analysed the miRNA expression profiles in 25 pairs of hepatocellular carcinoma (HCC) and adjacent non-tumorous tissue (NT) and nine additional chronic hepatitis (CH) specimens using a human miRNA microarray. Targets and references samples were co-hybridized to a microarray containing whole human mature and precursor miRNA sequences. Whereas three miRNAs exhibited higher expression in the HCC samples than that in the NT samples, five miRNAs demonstrated lower expression in the HCC samples than in the NT samples (P<0.0001). Classification of samples as HCC or NT by using support vector machine algorithms based on these data provided an overall prediction accuracy of 97.8% (45/46). In addition, the expression levels of four miRNAs were inversely correlated with the degree of HCC differentiation (P<0.01). A comparison of CH and liver cirrhosis samples revealed significantly different pattern of miRNA expression (P<0.01). There were no differences, however, between hepatitis B-positive and hepatitis C-positive samples. This information may help clarify the molecular mechanisms involved in the progression of liver disease, potentially serving as a diagnostic tool of HCC.

Identification of human cancer-related genes by naturally occurring Hepatitis B Virus DNA tagging.

Proviral tagging has been used in animals as a powerful tool for cancer genetics. We show that a similar approach is possible in patients with hepatocellular carcinoma (HCC) infected by Hepatitis B Virus (HBV), a human pararetrovirus which may act by insertional mutagenesis. In this work, the HBV genome is used as a probe to identify cancer-related genes. By using HBV-Alu-PCR, we obtained 21 HBV/cellular DNA junctions from 18 different patients. In six of 21, we found the HBV DNA integrated into a cellular gene: (1) Sarco/Endoplasmic Reticulum Calcium ATPase1 Gene; (2) Thyroid Hormone Receptor Associated Protein 150 alpha Gene; (3) Human Telomerase Reverse Transcriptase Gene; (4) Minichromosome Maintenance Protein (MCM)-Related Gene; (5) FR7, a new gene expressed in human liver and cancer tissues; and (6) Nuclear Matrix Protein p84 Gene. Seven junctions contained unique cellular sequences. In the remaining eight, the HBV DNA was next to repetitive sequences, five of them of LINE1 type. The cellular genes targeted by HBV are key regulators of cell proliferation and viability. Our results show that studies on HBV-related HCCs allow to identify cellular genes involved in cancer. We therefore propose this approach as a valuable tool for functional cancer genomic studies in humans.

Integration of hepatitis B virus DNA into the myeloid/lymphoid or mixed-lineage leukemia (MLL4) gene and rearrangements of MLL4 in human hepatocellular carcinoma.

Integration of hepatitis B virus (HBV) DNA into host DNA is detected in about 90% of HBV‐related hepatocellular carcinoma (HCC), but the preferential sites of the viral integration etiologically relevant to oncogenesis have been controversial. By using an adaptor‐ligation/suppression‐PCR, we identified four integrations into the myeloid/lymphoid or mixed‐lineage leukemia 4 (MLL4) gene from 10 HCC patients with positive HBV surface antigen (HBsAg). Determination of the cellular‐virus DNA junction demonstrated that various lengths of the virus were integrated within 300 bp of intron 3 flanked by the Alu element of MLL4. Chimeric hepatitis B virus X gene (HBx)/MLL4 transcripts and the HBx fusion proteins were detected. DNA microarray revealed that HBx/MLL4 fusion proteins suppressed unique genes in HepG2 cells. Finally, chromosomal translocations of intron 3 of MLL4 to the specific region of chromosome 17p11.2 in 22 out of 32 HCC patients were observed, showing that the intron 3 region of MLL4 gene would be a target of translocation breakpoint. In conclusion, the present data suggest that the translocation breakpoint of MLL4 gene is one of the preferential targets for HBV DNA integration into the MLL4 gene and the HBV DNA integration may be involved in liver oncogenesis. Hum Mutat 29(5), 703–708, 2008.

Identification of hepatitis B virus integration in hepatitis C virus-infected hepatocellular carcinoma tissues

BACKGROUND/AIMS The integration of HBV DNA is thought to be involved in the initial stage of hepatocarcinogenesis, and it has been reported that transactivating factors encoded by the X and preS2/S genes stimulate transcription of multiple viral and cellular genes. We assessed the possible contributions of hepatitis B virus integration to the occurrence of hepatocellular carcinoma in hepatitis C virus-infected as well as in hepatitis B virus-infected patients by identifying the integrated HBV DNA sequence, and the X and preS2/S regions were further investigated in HBV DNA-integrated cases. METHODS Southern blot hybridization for detecting HBV DNA in tumor tissues from 28 hepatocellular carcinoma patients was carried out with full-length HBV DNA, and then with X and preS2/S regions as probes. We also carried out reverse transcription-polymerase chain reaction for detecting HCV RNA to confirm hepatitis C virus-infection in liver tissues. RESULTS Clonally integrated HBV DNA sequences were demonstrated in 16 of 28 patients (57.1%), including five HBsAg seropositive and 11 HBsAg seronegative patients. Of these 11 HBsAg seronegative patients, 10 were also positive for anti-HCV in their sera, and all nine examined cases had HCV RNA in liver. Furthermore, the X region was identified in 14 of 16 HBV DNA integrated cases (87.5%), and the preS2/S region in 6/16 (37.5%). CONCLUSIONS The present Southern blot analysis demonstrates that clonally integrated HBV DNA sequences were identified even in hepatitis C virus-infected hepatocellular carcinoma patients at a high rate (10/18, 55.6%), and suggests that integrated hepatitis B virus, whose major component is the X gene, may play an important role in hepatocarcinogenesis in hepatitis B virus-integrated cases with and without hepatitis C virus infection.

Aggressive surgical resection for hilar-invasive and peripheral intrahepatic cholangiocarcinoma.

The clinicopathology and surgical outcome of intrahepatic cholangiocarcinomas are not fully understood. The objective of this study was to clarify the clinicopathologic features of intrahepatic cholangiocarcinoma and evaluate prognostic factors influencing survival. Forty consecutive patients with intrahepatic cholangiocarcinomas undergoing surgical resection at Chiba University Hospital between October 1981 and October 1997 were analyzed retrospectively. Intrahepatic cholangiocarcinomas were classified as hilar-invasive type (n = 26) or peripheral type (n = 14). Patients with peripheral-type tumors had a significantly (p = 0.005) better 5-year survival rate (43%) than those with the hilar-invasive type (4%). Hilar-invasive-type tumors had perineural invasion (100%) and nodal involvement (85%) more frequently than did peripheral-type tumors. Despite aggressive surgical resection, the surgical margin was positive in 88% of patients with hilar-invasive type tumors (23/26) and 29% of patients with peripheral-type tumors (4/14). There was no evidence of a survival benefit of vascular resection for patients with a hilar-invasive intrahepatic cholangiocarcinoma. Patients with lymph node metastasis had a significantly worse prognosis (p = 0.0004). No patients with nodal involvement survived more than 38 months. Negative perineural invasion (p = 0.008) and a negative microscopic margin (p = 0.008) were significantly associated with improved survival. Better survival results could be achieved by curative resection with a free margin for hilar-invasive and peripheral intrahepatic cholangiocarcinoma.

Hepatitis B virus-related insertional mutagenesis implicates SERCA1 gene in the control of apoptosis

We have used the Hepatitis B Virus DNA genome as a probe to identify genes clonally mutated in vivo, in human liver cancers. In a tumor, HBV-DNA was found to be integrated into the gene encoding Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA), which pumps calcium, an important intracellular messenger for cell viability and growth, from the cytosol to the endoplasmic reticulum. The HBV X gene promoter cis-activates chimeric HBV X/SERCA1 transcripts, with splicing of SERCA1 exon 11, encoding C-terminally truncated SERCA1 proteins. Two chimeric HBV X/SERCA1 proteins accumulate in the tumor and form dimers. In vitro analyses have demonstrated that these proteins localize to the ER, determine its calcium depletion and induce cell death. We have also shown that these biological effects are related to expression of the SERCA, rather than of the viral moiety. This report involves for the first time the expression of mutated SERCA proteins in vivo in a tumor cell proliferation and in vitro in the control of cell viability.

Long-term surgical outcome of noninvasive and minimally invasive intraductal papillary mucinous adenocarcinoma of the pancreas

AbstractThe objective of this study was to clarify the long-term outcome after surgical resection in patients with noninvasive and minimally invasive intraductal papillary mucinous adenocarcinoma. We performed a retrospective review of the clinicopathological features and outcome in patients who underwent pancreatic resection for noninvasive and minimally invasive intraductal papillary mucinous adenocarcinoma between November 1982 and December 1997 at Chiba University Hospital. Minimally invasive structures were pathologically observed in five cases. The mean age of patients with either noninvasive (n = 16) or minimally invasive n = 5) adenocarcinoma was 61 years. Of the patients with minimally invasive adenocarcinoma, 4 had abdominal pain. Conversely, 7 patients with noninvasive adenocarcinoma had no complaint. The mean size of noninvasive and minimally invasive tumors was 2.5 cm (range 0.8 to 4.0) and 3.3 cm (range 2.5 to 4.5), respectively. The overall 5-year and 10-year survival rates for all 21 patients were 89% and 47%, respectively. Disease recurred in 3 patients; 2 patients with minimally invasive adenocarcinoma and 1 with noninvasive adenocarcinoma. Recurrence sites were peritoneumn = 2) and main pancreatic duct of the remnant pancreas (n = 1); 5 disease-free patients died of unrelated causes. The remaining 13 patients are alive and disease free 3 to 12 years after surgery. Noninvasive and minimally invasive intraductal papillary mucinous adenocarcinoma had a favorable prognosis after surgical treatment.

P21WAF1/CIP1 messenger RNA expression in hepatitis B, C virus-infected human hepatocellular carcinoma tissues.

The primary objective of this study was to clarify the significance of p21WAF1/CIP1(p21) gene expression in the tumorgenicity of hepatitis B virus (HBV) and hepatitis C virus (HCV) infected human hepatocelluar carcinoma (HCC).

A case of gallbladder adenomyomatosis with pancreaticobiliary maljunction and an anomaly of the cystic duct joined the common channel

A 46-yr-old woman was admitted to our hospital with mild epigastric pain. Ultrasonography and computed tomography revealed an extremely thickened gallbladder wall. Endoscopic retrograde cholangiopancreatography demonstrated that the main pancreatic duct joined the nondilated common bile duct at the outer point of the duodenal wall (P-C type of pancreaticobiliary maljunction), and the cystic duct joined the common channel directly. The intraoperative amylase levels of the bile juices both in the common bile duct and the cystic duct were high. A cholecystectomy was performed. The wall of the gallbladder was markedly thick, yellowish, elastic, and soft. Histologically, Rokitansky-Aschoff sinus proliferation, hypertrophy of smooth muscles, and fibrosis were seen. The diagnosis was a generalized type of adenomyomatosis. The pathogenesis of the adenomyomatosis was believed to result from chronic stimulation as a result of pancreatic juice reflux. The etiology of this unusual type of junction was considered to be the result of the combination of pancreaticobiliary maljunction and an anomaly of lower junction of the cystic duct.

Characterization of hyperplastic foci observed in surgical specimens of hepatocellular carcinoma

By reviewing previous surgical specimens of hepatocellular carcinoma, 17 cases with hyperplastic foci (HPF) characterized by discernible increase in nuclear densities, could be histologically selected. Nuclear densities of HPF and control hepatic parenchyma were assessed quantitatively by counting the nuclear number of hepatic cells, and proliferating cell nuclear antigen labeling index was measured. HPF occurred multifocally, confined within a lobular unit, smoothly merging into surrounding hepatic parenchyma. Nuclear densities of HPF were 1.71 times greater than those of control hepatic parenchyma. The hepatocytes of HPF also showed significantly higher proliferative activities than those of control parenchyma. In addition, noticeable structural distortions, such as focal trabecular thickening or microacinar formation of hepatocytes, were sometimes observed in HPF. However, these HPF seemed to be distinguished from minute de novo hepatocellular carcinoma (HCC) or intrahepatic HCC metastasis, because of paucity of distinctive atypical changes, and intimate correlation with neighboring hepatocytes. Several adjacent HPF were aggregated to form a much larger unit of a hyperplastic area with loss of fibrous septa of liver cirrhosis. It was suggested that grossly detectable large regenerative nodules are produced via fusion of several adjacent HPF.