Gotaro Yamada

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

Distribution of Hepatitis B Virus Genotypes among Patients with Chronic Infection in Japan Shifting toward an Increase of Genotype A

ABSTRACT Acute hepatitis B virus (HBV) infection has been increasing through promiscuous sexual contacts, and HBV genotype A (HBV/A) is frequent in patients with acute hepatitis B (AHB) in Japan. To compare the geographic distribution of HBV genotypes in patients with chronic hepatitis B (CHB) in Japan between 2005 and 2006 and between 2000 and 2001, with special attention to changes in the proportion of HBV/A, a cohort study was performed to survey changes in genotypes of CHB patients at 16 hospitals throughout Japan. Furthermore, we investigated the clinical characteristics of each genotype and examined the genomic characteristics of HBV/A isolates by molecular evolutionary analyses. Of the 1,271 patients, 3.5%, 14.1%, and 82.3% were infected with HBV/A, -B, and -C, respectively. In comparison with our previous survey during 2000 and 2001, HBV/A was twice as frequent (3.5% versus 1.7%; P = 0.02). The mean age was lower in the patients with HBV/A than in those with HBV/B or -C. Based on phylogenetic analyses of 11 full-length genomes and 29 pre-S2/S region sequences from patients, HBV/A isolates were imported from Europe and the United States, as well as the Philippines and India. They clustered with HBV/A from AHB patients and have spread throughout Japan. HBV/A has been increasing in CHB patients in Japan as a consequence of AHB spreading in the younger generation through promiscuous sexual contacts, aided by a tendency of HBV/A to induce chronic hepatitis. The spread of HBV/A infection in Japan should be prevented by universal vaccination programs.

Immunohistochemical Evaluation of Oxidative Stress Markers in Chronic Hepatitis C

Oxidative stress (OS) plays a major role in chronic hepatitis C. Various OS markers have been found to be elevated in hepatitis C virus (HCV)-related liver disease. This study detected the presence of OS in serum and liver biopsy specimens of HCV patients. Reactive oxygen molecules (ROM) in sera of 54 HCV patients were compared with 23 controls. OS markers 8-hydroxydeoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal, malondialdehyde, and thioredoxin were measured in liver biopsy specimens of 18 HCV patients with fibrosis staging F1 (six); F2 (two), F3 (four), and F4 (six). The interferon (IFN) response and hepatocellular carcinoma (HCC) occurrence in the presence of OS markers were also evaluated. The level of ROM in HCV patients was 318 +/- 56.7 Carr compared with 248 +/- 40.8 Carr in controls (p=0.032). Multivariate analysis found age (p=0.0236) to be the only independent variable associated with increase in ROM in sera. In liver biopsy specimens, OS markers were found mainly around the area of piecemeal necrosis or the periportal area. The presence of OS markers seemed to increase with fibrosis staging, although not significantly. The OS DNA damage marker 8-OHdG was detected in the nucleus of hepatocytes. Thirteen patients received IFN therapy. During the 4-year follow-up period, HCC developed in four nonresponders to IFN and in one untreated patient. OS markers were stained in both HCC cells and non-HCC cells in HCC patients. OS markers were found in serum and liver specimens of HCV-associated liver disease and in HCC tissue. Detection of OS markers may be important for monitoring disease progression in HCV patients. Antioxidant therapy in combination with antiviral therapy may minimize liver damage and aid in the prevention and subsequent development of HCC.

Effect of a Moderately Energy-Restricted Diet on Obese Patients With Fatty Liver

The effects of a moderately energy-restricted (25 kcal/kg) diet on liver-function tests, anthropometric measurements, mononuclear-cell phospholipid fatty acid, lymphocyte blastogenesis, and plasma prostaglandin E2 and alpha-tocopherol levels were observed at weeks 0, 8, and 24 in 14 obese patients with fatty liver. Serum aminotransferase levels were improved significantly, with decreases in the body mass index and waist circumference. Decreases in energy intake from carbohydrate and increases in intake of vitamin A, vitamin C, and vegetables were observed at week 24. In mononuclear-cell phospholipids, linoleic acid (18:2omega 6), which was significantly lower in patients than in controls at week 0, was increased at week 24. In contrast, arachidonic acid was decreased. Plasma prostaglandin E2 levels were significantly lower in patients than in controls at week 0 and increased at week 24. The mononuclear-cell response for phytohemagglutinin correlated with 18:2omega 6 in mononuclear-cell phospholipids (r = 0.692, P < 0.01). Improvement of the serum alanine-aminotransferase level correlated with an increase in the plasma alpha-tocopherol level (r = -0.667, P < 0.01) and increases in consumption of vitamin A, omega 3 polyunsaturated fatty acids, and vegetables. These findings suggest that a hypoenergetic diet rich in omega 3 polyunsaturated fatty acids and antioxidants might be beneficial for obese patients with fatty liver.

Epidemiology of genotypes of hepatitis C virus in Japanese patients with type C chronic liver diseases: A multi-institution analysis

Sixteen medical institutions in Japan collaborated in this study of the epidemiology of hepatitis C virus (HCV) genotypes. A total of 4176 patients with type C chronic liver disease, from the four main islands of Japan, were evaluated. Of those evaluated, 2794 had chronic hepatitis, 727 had liver cirrhosis and 655 had hepatocellular carcinoma. The HCV genotype of the patients was determined by an enzyme‐linked immunosorbent assay based on serological genotype 1‐ and 2‐specific recombinant peptides (SG‐1 and SG‐2, respectively) of the NS4 region. The prevalence of SG‐1 and SG‐2 HCV was similar in the four main islands of Japan. SG‐1 HCV predominated in each disease category (69–76%). The percentage of patients with SG‐1 HCV increased by 7%, while that of patients with SG‐2 HCV decreased by 7%, as liver disease progressed in severity from chronic hepatitis to carcinoma (P < 0.001). Patients with either SG‐1 or SG‐2 had a similar mean age and history of blood transfusion. In conclusion, SG‐1 HCV was found to predominate in Japan, and the HCV genotype was found to be related to the stage of hepatitis C disease.

Virion-like structures in HeLa G cells transfected with the full-length sequence of the hepatitis C virus genome

Abstract Background & Aims: The process and the site of hepatitis C virus (HCV) particle formation in cells after infection remain unknown. The aim of this study was to create an in vitro model for the study of HCV particle formation. Methods: HeLa G cells were transfected with the full-length sequence of the HCV genome. Viral protein expression was analyzed using immunoblotting. The cells were examined using immunoelectron and conventional electron microscopy. Results: Core, E2, NS3, NS5a, and NS5b proteins were identified using immunoblotting. Immunoelectron microscopy showed that the core antigen was located along the membrane of the endoplasmic reticulum (ER) and occasionally in its cisternae. Core antigen-positive particles of 30 nm in diameter were found in the cytosol and in the cisternae of the ER. The particles in the cisternae were coated with an outer membrane that was connected to the ER membrane. Conventional electron microscopy revealed particles of 45 nm in diameter with electrondense cores in the cisternae of the ER. The outer membrane of the particles was occasionally connected to the ER membrane. Conclusions: The findings suggest that HCV core proteins are synthesized and assembled into particles in the cytosol and that they bud into the cisternae of the ER to form coated particles.

Influence of viral load and genotype in the progression of Hepatitis B-associated liver cirrhosis to hepatocellular carcinoma.

Abstract: Aim/Background: Hepatitis B virus (HBV) is an important factor in the development of hepatocellular carcinoma (HCC). We studied the influence of HBV viral load on HCC occurrence in HBV related liver cirrhosis (LC).

Effect of Vitamin E on Serum Aminotransferase and Thioredoxin Levels in Patients with Viral Hepatitis C

Objectives: Oxidative stress induces cellular responses such as cell death, gene activation and cell proliferation, in the liver. Vitamin E (Vit. E) has been found to protect the liver against oxidative stress in animal experiments. Thioredoxin (TRX) is a stress inducible, multifunctional protein, secreted during oxidative stress. This study evaluated effects of Vit. E on serum TRX and aminotransferase levels in hepatitis C virus (HCV) patients, partly non-responsive to initial interferon (IFN), with higher than average level of serum alanine aminotransferase (ALT) after receiving anti-inflammatory drug treatment. Methods: Seventeen HCV patients (male=3; female=14) of age 62±7.65 years receiving anti-inflammatory drug therapy, at least 6 months prior to Vit. E administration, were given d-α´-tocopherol 500 mg/day, orally, for a period of 3 months. ALT, aspartate aminotransferase (AST), TRX and Vit. E were measured at 0, 1, 2 and 3 months and 1 month after end of treatment. As controls, the same patients biochemical data, 3 months from the start of therapy were used. Patients were divided into three categories: total patients “T”, low ALT group “L” (ALT<70 IU/l) and high ALT group “H”(ALT>70 IU/l), respectively.Results: The ALT level was lowered, significantly in group H, in the 1st, 2nd, 3rd and 1-month post therapy, compared to the initial value. But group L showed little or no change in ALT. Post Vit. E therapy, in groups T and H, the TRX level was elevated but remained below initial levels, whereas in group L, TRX level remained significantly lower than the pretreatment value. Groups T and L, showed significant reduction (p<0.05) in serum TRX levels in the 2nd and 3rd month. Group H showed a tendency towards TRX reduction, but not significantly. Serum Vit. E levels increased significantly (p<0.0001) from the 1st to 3rd month in all three T, H and L groups. Conclusion: Oxidative stress induced liver damage is reduced by Vit. E in patients with viral hepatitis C, particularly those with initial ALT levels >70 IU/l. Vit. E treatment causes reduction of oxidative stress markers as TRX and ALT in sera. Therefore, Vit. E can act as a supportive therapy to combat liver damage caused by oxidative stress, in such patients with continuously high levels of ALT even after anti-viral and anti-inflammatory drug therapy.

Comparison of outcomes between patients with alcoholic cirrhosis and those with hepatitis C virus-related cirrhosis

Background and Aim:  The natural history of alcoholic cirrhosis, especially in Asian countries, has not been completely understood thus far.

Virological Response in Patients with Hepatitis C Virus Genotype 1b and a High Viral Load

Background and objective:In Japan the prevalence of the hepatitis C virus (HCV) antibody is highest in the elderly population. Therefore, it is important for elderly patients to undergo interferon (IFN) therapy. In patients with HCV genotype 1b and a high viral load, the sustained virological response (SVR) rate is lower in older compared with younger patients receiving combination antiviral therapy. In addition, inadequate adherence to combination therapy is often seen in elderly patients, and is associated with reduced response rates. The aim of this retrospective analysis was to evaluate the effects of host-related factors (i.e. sex, age, baseline HCV RNA level, bodyweight and fibrosis stage) and peginterferon (PEG IFN)-α-2a plus ribavirin dose reductions on SVR rates.Methods:A total of 192 treatment-naive patients with a HCV genotype 1b infection and a high viral load were included in the analysis. Patients had been enrolled into a phase III trial of 48 weeks of treatment with PEG IFN-α-2a plus ribavirin or PEG IFN-α-2a plus placebo. All patients were evaluated for effect of drug exposure on SVR. In addition, the impact of host-related factors or dose reductions on SVR was assessed.Results:Approximately 30% of patients were considered elderly (≥60 years of age). The overall SVR rate was significantly higher in patients treated with combination therapy versus monotherapy (59.4% vs 24.0%, p < 0.001). Attainment of an SVR following combination therapy was not influenced by any factor evaluated in the analysis, although elderly males were associated with decreased SVR rates. Younger age (odds ratio [OR] 1.081; 95% CI 1.125, 1.034; p = 0.0009), lower baseline HCV RNA levels (OR 1.003; 95% CI 1.006, 1.001; p = 0.006) and a severe fibrosis stage (F3/4) [OR 6.194; 95% CI 1.037, 37.000; p = 0.0455] significantly increased the likelihood of achieving an SVR with monotherapy. In the combination therapy group, patients maintaining a full dosage schedule of PEG IFN-α-2a and ribavirin and those requiring dose reductions of either study drug had similar SVR rates (64.5% vs 61.9%). However, the SVR rate was reduced to 33.3% among patients who discontinued combination therapy. Three out of the 31 patients who received the full dosage schedule were elderly patients. In addition, of the 15 patients who discontinued combination therapy, three were <50 years of age and six were ≥60 years of age. The SVR rate was reduced in patients with cumulative PEG IFN-α-2a and ribavirin doses of <60%; the majority of these patients were elderly.Conclusion:The attainment of an SVR following PEG IFN-α-2a plus ribavirin combination therapy was not influenced by any of the host-related factors evaluated in this analysis, although elderly males were associated with a decreased SVR rate. Younger age, male sex and lower baseline HCV RNA levels significantly increased the likelihood of achieving an SVR with monotherapy. In addition, dose reductions appeared to have a negative impact on SVR in elderly patients. Therefore, it is important to minimize PEG IFN-α-2a and ribavirin dose reductions by effectively managing treatment-related adverse events in elderly patients.