Shigetoshi Fujiyama

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

Glypican-3, overexpressed specifically in human hepatocellular carcinoma, is a novel tumor marker

With the global pandemic of hepatitis B and C infections, the incidence of Hepatocellular carcinoma (HCC) is rapidly increasing world wide. We identified glypican-3 (GPC3), a novel oncofetal gene over-expressed specifically in human HCC, as based on data of cDNA microarrays. As GPC3 is a GPI-anchored membrane protein and could be secreted, we attempted to detect secreted GPC3 protein in sera from HCC patients using Western blotting and ELISA. GPC3 protein was positive in sera of 40.0% (16/40) of HCC patients, and negative in sera from subjects with liver cirrhosis (LC) (0/13), chronic hepatitis (CH) (0/34), and healthy donors (0/60). All subjects were Japanese. Although 12 of 40 HCC patients were negative for both alpha-fetoprotein (AFP) and PIVKA-II well known tumor markers of HCC, four of these were GPC3-positive in the sera. We also observed vanishing GPC3 protein in the sera of three patients after the surgical treatment for HCC. On the other hand, immunohistochemical analysis revealed that HCC expressed GPC3 protein in all 14 HCC patients tested. In conclusion, GPC3, as defined in this study was shown to be a useful tumor marker for cancer-diagnosis for large numbers of patients with HCC.

Coinfection of hepatitis C virus in patients with chronic hepatitis B infection

Enzyme-linked immunosorbent assays for detecting antibodies against hepatitis C virus and the polymerase chain reaction were tested in 82 chronic hepatitis B surface antigen carriers for their accuracy in diagnosing patients coinfected with hepatitis B and C viruses. To clarify the role of each virus in chronic hepatitis, serologic assays against hepatitis B virus were also tested. Thirteen (14.9%), 14 (17.1%) and 15 (18.3%) patients were anti-HCV positive using C100 (HCV1), JCC, and a second generation test (HCV2), respectively. HCV RNA was detected by polymerase chain reaction in 9 of 18 anti-HCV-positive cases. Although HCV1 assays were not sufficient, either the JCC or HCV2 assay detected all polymerase chain reaction-positive cases. Fifteen of 18 specimens that were positive in at least one of the three ELISA were seronegative for the hepatitis B e antigen. As judged by HBV DNA polymerase activity, titers of hepatitis B surface antigen and immunoglobulin A antibody against hepatitis B core antigen (IgA anti-HBc), activity of hepatitis B virus replication and immune response against hepatitis B virus in patients with coinfection was decreased to the level of hepatitis B virus asymptomatic carriers. These results show that hepatitis C virus appears to be the primary cause of active hepatitis in most patients with hepatitis B and hepatitis C virus coinfection.

Hepatic arterial injection chemotherapy with cisplatin suspended in an oily lymphographic agent for hepatocellular carcinoma

A method to prepare cisplatin suspended in an oily lymphographic agent, Lipiodol (LPS), has been established to deliver cisplatin to hepatocellular carcinoma (HCC) by the hepatic artery. Seventy‐one patients, one Stage I, 16 Stage II, 16 Stage III, and 38 Stage IV, were treated with LPS therapy. A partial response was obtained in 33 cases (46.5%), a minor response in 20 cases (28.2%), and no change in 18 cases (25.3%). In 34 patients whose serum alpha‐fetoprotein (AFP) levels were greater than 400 ng/ml, the serum AFP levels decreased in 31 patients (91.2%). The AFP decreased by more than 50% in 25 cases (73.5%) and more than 75% in 19 cases (55.9%). The plasma des‐γ‐carboxy prothrombin (DCP) levels decreased in all of the 26 DCP‐positive patients. The survival rate was 77% at 6 months and the 1‐year survival rate was estimated to be 55%. The patients treated with LPS therapy survived longer compared with patients given Lipiodol containing neocarzinostatin by the hepatic artery. Complications such as acute gastroduodenal mucosal lesions (24%), cholecystitis (2.8%), pancreatitis (7%), delayed jaundice (7%), and hepatic encephalopathy (4.2%) were observed after therapy. The peak plasma platinum (Pt) concentrations determined as ultrafilterable Pt occurred 5 to 20 minutes, and 5 to 60 minutes as total Pt after the end of LPS injection. The Pt concentrations in the tumor tissues were 42 times higher in four operated cases and 7.1 times higher in six autopsy cases than those in the nontumorous tissue. These results suggest that LPS selectively accumulates in the HCC, is long‐lasting and gradually releases the drug. In addition it is effective as a new anti‐cancer therapy for hepatocellular carcinoma.

Tumor Markers in Early Diagnosis, Follow-Up and Management of Patients with Hepatocellular Carcinoma

The mainstay for the diagnosis for hepatocellular carcinoma (HCC) includes serological tumor markers, such as alpha-fetoprotein, the L3 fraction thereof and PIVKA-II, in addition to imaging modalities. They do not correlate, but complement each other. Hence, a combination of them designed on the basis of their characteristics needs to be worked out. First, it is necessary to identify the patients at high risk for developing HCC, such as those with chronic hepatitis or liver cirrhosis, and in the follow-up conduct regular check-ups for serological tumor markers. Those testing positive for any marker are at the highest risk for developing HCC, even when imaging fails to disclose any space-occupying lesions. Following high-risk patients for serological tumor markers, in concert with imaging, makes accurate evaluation of the efficacy of therapies for HCC possible. Since serological tumor markers can signal the development of HCC earlier than any other laboratory tests, they offer excellent means of identifying relapsing HCC. Equally important in the management of patients with HCC are biological indicators for malignancy, the selection of therapeutic interventions and the prediction of the outcome.

Calcium influx triggers the sequential proteolysis of extracellular and cytoplasmic domains of E-cadherin, leading to loss of β-catenin from cell – cell contacts

Cadherins are major cell – cell adhesion molecules in both tumor and normal tissues. Although serum levels of soluble E-cadherin have been shown to be higher in the cancer patients than in healthy volunteers, the detail mechanism regulating release of soluble E-cadherin remains to be elucidated. Here we show that the ectodomain of E-cadherin is proteolytically cleaved from some cancer cells by a membrane-bound metalloprotease to yield soluble form, and the residual membrane-tethered cleavage product is subsequently degraded by intracellular proteolytic pathway. Futhermore, we show that extracellular calcium influx, that is induced by mechanical scraping of cells or ionomycin treatment, enhances the metalloprotease-mediated E-cadherin cleavage and the subsequent degradation of the cytoplasmic domain. Immunocytochemical analysis demonstrates that the sequential proteolysis of E-cadherin triggered by the calcium influx results in translocation of β-catenin from the cell – cell contacts to cytoplasm. Our data suggest that calcium influx-induced proteolysis of E-cadherin not only disrupts the cell – cell adhesion but also activates β-catenin-mediated intracellular signaling pathway, potentially leading to alterations in motility and proliferation activity of cells.

Risk factors for development of hepatocellular carcinoma in patients with chronic hepatitis C after sustained response to interferon.

BackgroundInterferon (IFN) is expected to prevent the progression of hepatitis C virus infection to cirrhosis and the development of hepatocellular carcinoma (HCC), but there have been several reports of the development of HCC after a sustained response to IFN. Our aim was to elucidate the incidence and clinical features of, and risk factors for, HCC in sustained responders to IFN, taken for the treatment of chronic hepatitis C.MethodsWe designed a retrospective cohort study conducted at 16 major Hospitals. The subjects were a total of 1056 patients showing sustained responses, 29 of whom developed HCC.ResultsThe incidence of HCC per 100 person-years was 0.56 (95% confidence interval, 0.35–0.76) in sustained responders. By the Cox proportional hazard model, we found that older age, higher serum aspartate aminotransferase level, and lower platelet count before IFN therapy were independent risk factors associated with the development of HCC. A risk index of HCC development, based on the coefficients of these risk factors, was used to classify patients into three groups, with low, intermediate, and high risk. The incidence rates of HCC for these three groups were 0.11, 0.44, and 1.98 per 100 person-years, respectively. The median period to the development of HCC was 4.6 years (range, 1.4–9.0 years), and there were no other specific clinical features of the HCC that developed in these patients.ConclusionsThis study suggests that the risk of development of HCC is not completely eliminated in sustained responders to IFN. These findings may be useful in determining a follow-up strategy after a sustained response to IFN.

Plasma abnormal prothrombin (Des‐γ‐carboxy prothrombin) as a marker of hepatocellular carcinoma

Des‐γ‐carboxy prothrombin [DCP], a protein induced by vitamin K absence or antagonist‐II and also abbreviated PIVKA‐II, was evaluated as a serologic marker for hepatocellular carcinoma (HCC). Its plasma levels were measured by enzyme immunoassay (E‐1023) using an anti‐DCP monoclonal antibody in 514 patients with various diseases. Of 120 patients with HCC, 76 (63%) had abnormal DCP levels greater than 0.1 arbitrary unit (AU)/ml and 58 (48%) showed levels greater than 0.3 AU/ml. When a diagnostic minimum level of 0.3 AU/ml was applied for DCP, false‐positive cases of HCC were virtually eliminated. In some patients with HCC, plasma DCP levels normalized after surgical resection of the tumor. However, they rose again later with recurrence of the disease. The sensitivity of DCP in the diagnosis and monitoring of HCC was increased by serial and simultaneous determinations of alpha‐fetoprotein (AFP), because high DCP levels were observed more often in low AFP‐producing HCC patients. Elevated plasma DCP levels were not related to low vitamin K concentration in the serum. In fact, in many patients vitamin K administration resulted in only a moderate reduction of DCP levels. These results suggested strongly that DCP was synthesized by the hepatoma cells.

Determination of R(+)- and S(–)-Lansoprazole Using Chiral Stationary-Phase Liquid Chromatography and Their Enantioselective Pharmacokinetics in Humans

AbstractPurpose. Stereoselective and sensitive methods employing chiral stationary phase columns for HPLC determination of enantiomers of lansoprazole in the human serum were developed and pharmacokinetic behaviors of the enantiomers were evaluated in seven subjects. Methods. Five chiral stationary phase columns: Chiralcel OD (cellulose tris(3,5-dimethyl-phenylcarbamate)), OF (cellulose tris(4-chloro-phenylcarbamate)), OG (cellulose tris(4-methylphenylcarbamate)) and OJ (cellulose tris(4-methylbenzoate)), and Chiralpak AS (amylose tris ((S)-1 -phenylethylcarbamate)) were investigated. Results. Chiralcel OD and Chiralpak AS columns gave a good resolution of R(+)- and S(–)-enantiomers from racemic lansoprazole, but Chiralcel OF, OG, and OJ did not. The mean Cmax and the AUC values of R(+)-enantiomer were 3–5 times greater than those of S(–)-enantiomer following oral administration of 30 mg of racemic lansoprazole. The CLtot values of R(+)-enantiomer were significantly smaller than those of S(–)-enantiomer. Binding of R(+)-enantiomer to human serum proteins was significantly greater than that of S(–)-enantiomer. The mean metabolic ratio (metabolites/parent compound) in human liver microsomes of S(–)-enantiomer was significantly greater than that of R(+)-enantiomer. Conclusions. The stereoselective pharmacokinetics of lansoprazole enantiomers is likely due to its Stereoselective protein binding and/ or metabolism.

Transcatheter arterial chemotherapy with and without embolization in patients with hepatocellular carcinoma.

Objective: This study compared the antitumor effect, adverse effects and survival between transcatheter arterial embolization (TAE) and transcatheter arterial infusion chemotherapy (TAI) in patients with hepatocellular carcinoma (HCC). Methods: The study population consisted of 168 consecutive patients with advanced HCC treated with transcatheter arterial treatments using cisplatin suspended in lipiodol. Among these, 74 patients were treated with TAE, and the remaining 94 patients were treated with TAI. Results: There were no significant differences in any baseline characteristics except hemoglobin, platelets, albumin, and glutamic pyruvic transaminase. Complete or partial tumor response was achieved in 54 patients (73%) in the TAE group and in 48 patients (51%) in the TAI group (p < 0.01). There were two treatment-related deaths caused by acute hepatic failure and acute renal failure in the TAE group. Nausea and deterioration of serum transaminase after TAE were significantly more severe than after TAI. Median survival time and survival rates at 5 years were 3.1 years and 25% in the TAE group, and 2.5 years and 18% in the TAI group (p = 0.37). Conclusion: TAE has a higher antitumor effect than TAI, but does not significantly improve the survival of patients with HCC.