Mitsuhiko Moriyama

Interferon Therapy Reduces the Risk for Hepatocellular Carcinoma: National Surveillance Program of Cirrhotic and Noncirrhotic Patients with Chronic Hepatitis C in Japan

Hepatitis C virus (HCV) infection rarely resolves spontaneously once it becomes chronic (1). Most patients remain asymptomatic for a long period, with liver cirrhosis developing after approximately 30 years (2, 3). Chronic hepatitis C with cirrhosis is a major risk factor for hepatocellular carcinoma (4-7). It has been previously shown that the risk increases with the degree of liver fibrosis (5). Interferon is the only agent known to be effective against HCV infection (8-10). It induces a sustained virologic response in 15% to 30% of patients (11-14). Responders usually show biochemical and histologic improvement (9, 11, 15). Recently, interferon therapy in patients with chronic hepatitis C and cirrhosis was shown to be associated with a reduced incidence of hepatocellular carcinoma (16). Because most patients treated with interferon do not have cirrhosis, we included noncirrhotic as well as cirrhotic patients in our analysis of the effect of interferon therapy on the incidence and prevention of hepatocellular carcinoma. A national surveillance program, the Inhibition of Hepatocarcinogenesis by Interferon Therapy (IHIT) Study, was begun in 1994 as a multicenter, large-scale, retrospective cohort study supported by the Japan Ministry of Health and Welfare as one of the Comprehensive 10-Year Strategy for Cancer Control Projects (17). In this program, patients with chronic hepatitis C who have undergone liver biopsy at one of eight participating institutions are enrolled and followed periodically for development of hepatocellular carcinoma by using several imaging techniques. We analyzed the incidence of hepatocellular carcinoma as of February 1998 by using multivariate proportional hazards regression. Methods Patients The IHIT Study Group approved the design of this study on 21 September 1994. All patients who were positive by a second-generation HCV antibody assay and who had undergone liver biopsy since 1986 at one of the eight participating institutions were enrolled. Patients who were participants in interferon trials for non-A, non-B chronic hepatitis (18-21) and in whom anti-HCV seropositivity was confirmed by using stored sera were also included; these patients had undergone liver biopsy in 1986 or later. Patients were excluded if at the time of liver biopsy they presented with hepatocellular carcinoma or other liver diseases, such as chronic hepatitis B, alcoholic liver disease,autoimmune hepatitis, or primary biliary cirrhosis. The minimum follow-up was established as 1 year for two reasons. First, if hepatocellular carcinoma is detected within 1 year after liver biopsy, the possibility that the cancer was present at the time of liver biopsy cannot be ruled out. Second, interferon therapy must be started within 1year after liver biopsy according to Japanese health insurance rules. By February 1998, 3223 patients who fulfilled the inclusion criteria were registered. Of these patients, 333 were excluded from the analysis: 161 patients (5.0%) transferred to other hospitals without follow-up, and the follow-up period after liver biopsy was less than 1 year for172 patients (5.3%). Thus, 2890 patients were included in the present analysis. Figure 1 shows the schema for patient selection. Figure 1. Schema for patient selection. Interferon therapy was given to 2400 patients; 490patients did not receive treatment (control group). Interferon therapy was initiated within 1 year after liver biopsy (within 6 months in 93% of patients); 84% of patients received interferon-, 14% received interferon-, and 2% received a combination of interferon- and interferon-. The median total dose was 480 MU (first quartile, 324 MU; third quartile, 702 MU), and the median duration of administration was 160 days(first quartile, 94 days; third quartile, 168 days). Once interferon therapy was started, a patient was included in the interferon treatment group even if therapy was discontinued because of adverse events or other reasons. The 490patients who did not receive interferon chose this course of action voluntarily on the basis of concerns about adverse effects; lack of time for therapy; or physician recommendation, which took into account depression, severe diabetes mellitus, or other medical conditions. Serum HCV load was quantitatively determined at the timeof liver biopsy by using various commercial and in-house assays. Because it is difficult to correlate the results of different assay methods, only data obtained with two widely used assays, the branched-DNA probe assay (22) and competitive reverse-transcription polymerase chain reaction (RT-PCR) (23), were used. HCV RNA genotype was determined by RT-PCR using genotype-specific primers (24) or by serologic grouping of serum antibody (25), assuming that genotypes 1a and 1b correspond to serologic group 1 (genotype 1) and genotypes 2a and 2b correspond to serologic group 2 (genotype 2) (11). Histologic Evaluation Liver biopsy specimens were evaluated by a representative pathologist at each institution (a total of eight pathologists were involved) and were scored for the stage of liver fibrosis and grade of inflammatory activity according to the classification of Desmet and colleagues (26). Stage of fibrosis was assessed from stage F0 (no fibrosis) to stage F4 (cirrhosis), and grade of inflammatory activity was scored from grade A1 (mild) to grade A3 (severe). To confirm interobserver concordance in scoring, a subsequent blind and independent examination of 350randomly selected liver biopsy specimens was conducted by two of the eight pathologists. Definition of Interferon Response Virologic and biochemical criteria were used to define response to interferon therapy. Hepatitis C virus RNA was used as a marker of virologic response and was determined by RT-PCR. A virologic sustained response was defined as HCV RNA negativity more than 6 months after termination of interferon therapy; positivity at the same time point was considered a nonsustained response (27). Patients with nonsustained response included those who had temporary disappearance of viremia followed by relapse. In patients treated before the availability of RT-PCR, virologicresponse was determined by using sera stored at 30 C or collected afterward. The serum alanine aminotransferase (ALT) level was used as a marker of biochemical response to interferon therapy. Sustained biochemical response was defined as persistently normal serum ALT levels more than 6 months after termination of interferon therapy; nonsustained response was defined as elevated serum ALT levels at the same time point. Nonsustained response was subdivided into two categories: mildly elevated for a serum ALT level less than two times the upper limit of normal and highly elevated for a serum ALT level two or more times the upper limit of normal. Screening for Hepatocellular Carcinoma Patients were examined for hepatocellular carcinoma by abdominal ultrasonography at least every 6 months. If hepatocellular carcinoma was suspected on the basis of ultrasonographic results, additional procedures, such as computed tomography, magnetic resonance imaging, abdominal angiography, and ultrasonography-guided tumor biopsy, were used to confirm the diagnosis. Statistical Analysis Statistical analysis was performed by using SAS software, version 6.12 (SAS Institute, Inc., Cary, North Carolina). Interobserver concordance of histologic scoring was evaluated by using the Spearman correlation coefficient. Differences between two groups were evaluated by using the unpaired Student t-test or the Mann-Whitney U-test. Categorical data were compared by using the chi-square test or the Fisher exact probability test. Cumulative incidence curves were determined with the Kaplan-Meier method, and the differences between groups were assessed by using the log-rank test. We used the Cox proportional-hazards regression analysis to examine the effect of interferon therapy on the incidence of hepatocellular carcinoma. Because virologic and biochemical responses were mutually dependent, the risk ratio for hepatocellular carcinoma was calculated separately for these factors. The risk ratio attributable to categorical data, such as stage of liver fibrosis and serum ALT level, was calculated by using dummy variables. A P value less than 0.05 was considered statistically significant. Results Patient Characteristics The demographic and clinical features of patients at the time of their enrollment are summarized in Table 1. The frequency distribution of the stages of liver fibrosis differed between interferon-treated patients and untreated patients. Most laboratory values also differed between the two groups. However, differences in laboratory values between treated patients and untreated patients were not significant at the same stage of fibrosis. This indicated the need to adjust for stage of liver fibrosis, which was done in the following analyses. Table 1. Demographic and Clinical Characteristics Histologic Evaluation The concordance in scores for stage of fibrosis and grade of inflammatory activity determined at each institution and by the two representative pathologists was strong, with Spearman coefficients ranging from 0.897 to 0.918 for stage of fibrosis and from 0.878 to 0.849 for grade of inflammatory activity. The original score was sustained by at least one of the two pathologists in 319 of 350 cases for fibrosis staging and in 320 of 350cases for grading inflammatory activity. Response to Interferon Therapy Response to interferon therapy was determined in 2357(98.2%) of the 2400 interferon-treated patients. Response was not determined in43 patients because of insufficient follow-up (<6 months) after termination of therapy. A sustained virologic response was achieved in 789 patients(33.5%). The response rate was similar regardless of the type of interferon used (32.3%, 34.5%, and 25.6% for interferon-, interferon-, and the combination of the two, respectively). A sustained bioch

Persistent infection of hepatitis E virus transmitted by blood transfusion in a patient with T-cell lymphoma.

Aim:  With advent of reverse‐transcription (RT)/polymerase chain reaction (PCR) for detection of the hepatitis E viral genome, we carried out retrospective examinations.

Hepatitis C Virus Core Protein Interacts with 14-3-3 Protein and Activates the Kinase Raf-1

ABSTRACT Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver dysfunction in humans and is epidemiologically closely associated with the development of human hepatocellular carcinoma. Among HCV components, core protein has been reported to be implicated in cell growth regulation both in vitro and in vivo, although mechanisms explaining those effects are still unclear. In the present study, we identified that members of the 14-3-3 protein family associate with HCV core protein. 14-3-3 protein bound to HCV core protein in a phosphoserine-dependent manner. Introduction of HCV core protein caused a substantial increase in Raf-1 kinase activity in HepG2 cells and in a yeast genetic assay. Furthermore, the HCV core–14-3-3 interaction was essential for Raf-1 kinase activation by HCV core protein. These results suggest that HCV core protein may represent a novel type of Raf-1 kinase-activating protein through its interaction with 14-3-3 protein and may contribute to hepatocyte growth regulation.

Serum concentrations of human hepatocyte growth factor is a useful indicator for predicting the occurrence of hepatocellular carcinomas in C-viral chronic liver diseases

Numerous reports have examined the relationship between hepatocyte growth factor (HGF) and either the facilitation or suppression of the occurrence of hepatocellular carcinoma (HCC).

Efficacy and safety of entecavir in lamivudine-refractory patients with chronic hepatitis B: randomized controlled trial in Japanese patients.

Background and Aim:  Entecavir is a potent inhibitor of both wild‐type and lamivudine‐resistant hepatitis B virus (HBV) with proven clinical efficacy. We conducted a randomized, double‐blind, multicenter study in Japan (ETV‐052) evaluating the efficacy and safety of two doses of entecavir in adult patients with lamivudine‐refractory chronic hepatitis B infection.

Influence of occult hepatitis B virus coinfection on the incidence of fibrosis and hepatocellular carcinoma in chronic hepatitis C.

We examined prospectively the influence of occult hepatitis B virus (HBV) infection on the histopathological features and clinical outcome of HCV RNA-positive chronic hepatitis (CH-C) and detected hepatitis B core (HBc) particles in hepatocytes. The subjects were 468 patients with CH-C or liver cirrhosis (LC) who were negative for serum hepatitis B surface antigen (HBsAg) by enzyme-linked immunosorbent assay. HBV DNA was detected in serum by nested PCR. HBsAg and HBc antigen (HBcAg) in liver were investigated using immunohistochemical techniques and light (LM) and electron microscopy (EM). Serum HBV DNA was detected in 43.6% of the patients studied. There were no significant differences between HBV DNA-positive and DNA-negative patients in terms of their clinical profiles. For HBV DNA-positive patients, the degree of inflammatory cell infiltration and irregular regeneration of hepatocytes was significantly greater than for HBV DNA-negative patients. The cumulative probability of development of hepatocellular carcinoma (HCC) was significantly higher for HBV DNA-positive patients than for HBV DNA-negative patients. HBV DNA positivity was a risk factor for the occurrence of HCC according to multivariate analysis. HBsAg and HBcAg were detected in 8.5 and 72.3%, respectively, of the livers of serum HBV DNA-positive individuals. Core particles were detected in the nuclei of the hepatocytes by IEM. The histopathological features and long-term outcome of CH-C or LC could be affected by occult HBV infection.

The Clinical Features of Chronic Hepatitis C Are Not Affected by the Coexistence of Hepatitis B Virus DNA in Patients Negative for Hepatitis B Surface Antigen

Hepatitis B virus (HBV) DNA has been detected in the sera of hepatitis patients who are negative for hepatitis B surface antigen (HBsAg) by polymerase chain reaction (PCR). The purpose of the present study was to clarify the clinical characteristics of patients with chronic hepatitis C who are negative for serum HBsAg and positive for HBV DNA. The subjects included 49 patients with chronic hepatitis C who were negative for serum HBsAg and 119 blood donors who served as healthy controls. Serum samples were tested for the presence of HBV DNA by the nested PCR method. Serum HBV DNA was detected in 18 (37.7%) of the 49 chronic hepatitis C patients and in none (0%) of the 119 blood donors. Among the hepatitis C patients, HBV DNA was detected in 20.7% of those who were negative for all HBV-associated markers and in 57.1% of those who were positive for one or more HBV-associated marker. The HBV DNA-positive rate among those in each F stage did not significantly differ. The liver function parameters of the HBV DNA-positive and the HBV DNA-negative chronic hepatitis C patients did not significantly differ. These results suggest that hepatitis C virus is frequently coinfected with serum HBsAg-negative HBV, and that the incidence of HBV infection in blood donors is low. However, it is considered that HBsAg-negative HBV infection does not modify the blood biochemical features of chronic hepatitis C.

The Efficacy and Tolerability of a Triple Therapy Containing a Potassium-Competitive Acid Blocker Compared With a 7-Day PPI-Based Low-Dose Clarithromycin Triple Therapy.

OBJECTIVES:This study evaluated the efficacy and tolerability of potassium-competitive acid blocker (P-CAB), a new class of gastric acid inhibitory agents, as first-line H. pylori eradication treatment compared with 7-day proton pump inhibitor (PPI)-based triple therapy.METHODS:We retrospectively reviewed the medical records of 661 consecutive patients who received first-line H. pylori eradication treatment between January 2013 and October 2015. Patients who received 7-day P-CAB therapy (vonoprazan 20 mg+amoxicillin 750 mg+clarithromycin 200 mg twice/day; n=181) were compared with those who received 7-day PPI therapy (lansoprazole 30 mg/rabeprazole 20 mg+amoxicillin 750 mg+clarithromycin 200 mg twice/day; n=480) using propensity score matching analysis. The successful eradication and adverse event rates were compared between the two groups.RESULTS:The propensity score matching analysis yielded 175 matched pairs. Adjusted comparisons between the two groups showed a significantly higher eradication rate for the P-CAB than the PPI group in both intention-to-treat (89.1 vs. 70.9%; P<0.001) and per-protocol analyses (91.2 vs. 71.7%; P<0.001). There was no significant difference in the incidence of adverse events between the two therapies except skin rash. No patients discontinued H. pylori eradication treatment because of adverse events.CONCLUSIONS:Seven-day P-CAB-based triple therapy was more effective than 7-day PPI-based triple therapy as a first-line H. pylori eradication treatment. Seven-day P-CAB-based triple therapy was generally well-tolerated.

Detection of precore/core-mutant hepatitis B virus genome in patients with acute or fulminant hepatitis without serological markers for recent HBV infection

To confirm the possibility that some hepatitis B virus (HBV) variants do not induce HB s antigen (HBsAg), anti-HB core antibody (anti-HBc) and anti-HBc IgM in a transient infection, polymerase chain reaction (PCR) was performed in 20 patients with acute hepatitis and 7 patients with fulminant hepatitis. Patients were diagnosed with non-A, non-B hepatitis by serological markers at admission. PCR successfully amplified the precore/core gene in 5 (25%) of the patients with acute hepatitis and 2 (29%) of the patients with fulminant hepatitis. Subsequent sequencing revealed frequent mutations including precore-defects in the precore/core gene.

Detection of Serum and Intrahepatic Human Hepatocyte Growth Factor in Patients with Type C Liver Diseases

We determined hepatocyte growth factor (HGF) levels in the serum and liver of patients with hepatitis C and assessed the relationship to histological findings of the liver and hepatitis C virus-related markers in the serum in patients with type C liver diseases. The subjects were 108 patients with chronic hepatitis C (CH), 70 patients with liver cirrhosis C (LC), 38 patients with hepatocellular carcinoma (HCC) and 20 patients with acute hepatitis (AH). As normal controls 20 subjects were studied. The serum HGF levels were measured using an enzyme-linked immunosorbent assay kit. Intrahepatic HGF was investigated by immunoperoxidase staining using monoclonal HGF antibody. The serum HGF level was highest in patients with AH. The serum HGF levels tended to be higher in patients with LC and HCC than those with CH. Further, the serum HGF level was related to the degree of intrahepatic inflammatory cell infiltration and fibrosis, and intrahepatic HGF was noted primarily in the cell membrane of mesenchymal cells in focal necrosis. The degree of intrahepatic HGF expression tended to be higher in patients with high serum HGF levels. In patients with HCC, however, HGF showed little localization in cancer cells, but was noted in infiltrating mesenchymal cells in both cancerous and noncancerous regions. In conclusion, the measurement of serum HGF levels may be useful for estimating the degree of intrahepatic inflammatory reaction and fibrosis. Although further study is necessary, the high serum level of HGF revealed high carcinogenic states in chronic hepatitis and liver cirrhosis type C.