Liliana Chemello

Natural history of hepatitis C

Ten years after the discovery of the hepatitis C virus (HCV) and its association with NANB hepatitis as a major cause of chronic liver disease worldwide, our knowledge of the natural history of hepatitis C is still limited. The asymptomatic course of the disease in most patients, its slow and silent progression and heterogeneous outcome and the widespread use of interferon therapy during the past decade explain why many questions are still unsolved. The changing epidemiological pattern of HCV and the significant contribution of several cofactors to the severity of liver disease also complicate the development of a general model describing the natural history of hepatitis C. Available data indicate that HCV infection may resolve without any clinical signs of liver disease in individuals exposed to low dose inoculum and that these cases may develop T cell immunity even in the absence of anti-HCV seroconversion. Rates of complete biochemical and virological resolution of acute hepatitis C range between 10 and 50%, and are probably affected by the route of infection, size and type of inoculum and acute phase clinical features. Chronic HCV infection may develop with or without ALT abnormalities and with or without chronic inflammation and increasing fibrosis in the liver. Studies conducted in patients who acquired hepatitis C by blood transfusion 15-25 years ago indicate that 20-30% of them have now progressed to cirrhosis, including 5-10% with end stage liver disease and 4-8% who died of liver-related causes. Similar studies conducted in patients infected by other routes have shown a more benign course of hepatitis C, with little evidence of cirrhosis and no liver-related mortality during the first two decades. Outcomes after longer follow-up need to be assessed. In patients presenting with chronic hepatitis C, fibrosis progression is extremely variable over time and can be partially predicted by the age at infection, disease duration, liver histologic activity and stage of fibrosis and by the ALT profile. However, it is often difficult to predict clinical outcomes in individual cases. In patients who have developed cirrhosis, the 5-year risk of decompensation is between 15 and 20% and that of hepatocellular carcinoma around 10%. Several variables have been shown to influence the natural course of shown C, the most significant being age at infection, alcohol consumption and coinfection with HBV and HIV Studies are being performed to assess the role of host genetics. Viral factors, such as the HCV type and load, seem to have inconsistent or marginal effects.

Serum and liver HCV RNA levels in patients with chronic hepatitis C: correlation with clinical and histological features

Background—Liver disease in chronic hepatitis C virus (HCV) infection ranges from minimal lesions to liver cirrhosis, eventually evolving to hepatocellular carcinoma. Whether and how HCV determines the different clinical and histological manifestations of the disease is not fully understood. Aims—To verify whether the amount of virus in individual patients could be related to the severity of liver injury. Patients and methods—Levels of HCV RNA were measured in serum in 96 consecutive patients with chronic hepatitis type C using a signal amplification assay. The relation between viraemic values and the corresponding viral load in the liver was assessed in a subgroup of 21 patients in whom HCV RNA was measured in serum samples and liver specimens obtained at the same time. Results—A positive correlation was observed between the amount of viral nucleic acid in the two compartments, indicating that levels of viraemia reflect the amount of virus present in the liver. Viral load did not correlate with aminotransferase activities nor with histological diagnosis, and serum and liver levels of HCV RNA were not significantly different in patients infected by the various HCV genotypes. Conclusions—Measurement of HCV replication in serum is a mirror of viral replication in the liver. The extent of replicative activity of HCV does not seem to play a role in the modulation of the associated hepatic disease.

Persistent Hepatitis C Viremia Predicts Late Relapse after Sustained Response to Interferon-α in Chronic Hepatitis C

Chronic infection with the hepatitis C virus (HCV) is a major cause of cirrhosis and hepatocellular carcinoma worldwide. In many countries, interferon- therapy is the only licensed method for treating this condition. It has been licensed on the basis of randomized, controlled trials that have shown its effectiveness in reducing disease activity and virus replication [1, 2]. However, few treated patients achieve a complete response that is maintained after therapy, and many others have a transient response or no response at all [3, 4]. Serum alanine aminotransferase activity is the conventional marker used to monitor response to therapy, and sustained biochemical response is defined as normalization that lasts for 6 to 12 months after cessation of interferon- therapy. More recently, serum HCV RNA testing has been recognized as an important component of assessing response to treatment; patients who respond at the biochemical level and maintain normal alanine aminotransferase levels after therapy may remain viremic, indicating that interferon- has suppressed liver disease activity but has not eradicated the virus [5]. There is increasing agreement that patients with biochemical response should be tested for serum HCV RNA to more accurately define the effect and efficacy of treatment. However, little is known of the clinical significance and long-term outcome of persistent viremia with normal alanine aminotransferase activity after interferon- therapy. It is still unclear whether this profile reflects delayed virus clearance or persistence of a noncytopathic type or load of HCV or whether it should instead be regarded as a predictor of reactivation of liver damage and progression of disease. To clarify these issues, we investigated the long-term outcome of a large series of consecutive patients with chronic hepatitis C who had persistently maintained normal alanine aminotransferase activity for at least 1 year after cessation of interferon- therapy. Methods Patients Between January 1990 and November 1993, 440 consecutive patients with chronic hepatitis C were treated with interferon- therapy in three consecutive randomized trials [6, 7] using different schedules of treatment. Inclusion and exclusion criteria were identical for the three studies, and the clinical characteristics of patient subgroups were similar [8]. Enrolled patients were between 18 and 65 years of age and had a persistent (lasting more than 6 months) elevation of alanine aminotransferase levels of more than twice the upper limit of normal positive result on testing for antibody to HCV, and histologic evidence of chronic hepatitis with or without Child grade A cirrhosis. Patients who were also infected with hepatitis B virus or human immunodeficiency virus who had autoimmunity or comorbid conditions were not included. The total interferon- dose ranged from 234 MU to 740 MU, administered for 6 or 12 months. Alanine aminotransferase levels were tested monthly during therapy. Complete biochemical response was defined as normalization of liver enzyme levels during therapy, maintained until withdrawal of therapy. After cessation of interferon- therapy, patients who had responded were monitored for serum alanine aminotransferase at 3-month intervals; sustained biochemical response was defined as enzyme levels that were normal for as long as 12 months after therapy. Of 440 treated patients, 259 (59%) showed complete biochemical response during treatment, but 141 (54%) of the 259 had relapse within 12 months after therapy was discontinued. Of the 118 patients with alanine aminotransferase levels that were normal for as long as 12 months after therapy, 107 could be tested for serum HCV RNA and followed prospectively for an additional 6 to 36 months (mean follow-up after therapy, 35 18 months). In the remaining 11 patients who had responded to interferon-, serum samples were not available for HCV RNA testing 1 year after therapy. Testing Serum HCV RNA was tested using nested polymerase chain reaction [9]; the sensitivity limit of the assay was between 103 and 104 copies/mL. The virus genotype was identified using spot hybridization with genotype-specific oligonucleotide probes [9] and was classified according to the system of Simmonds and colleagues [10]. Liver biopsies were done during the 6 months before therapy. Sixty-three of the 107 patients with persistently normal alanine aminotransferase levels consented to have liver biopsy, done 8 to 12 months after cessation of interferon therapy. The histologic evaluation was made by a pathologist who was blinded to clinical data and to the sequence in which biopsy specimens had been obtained. Continuous variables were compared by using the t-test, and proportions were compared by using the chi-square test. The probability of maintaining a sustained response to therapy in different groups was analyzed by using the Kaplan-Meier method and was compared by using the log-rank test. Results One year after therapy, serum HCV RNA was detectable in 27 (25%) of the 107 patients with sustained biochemical response; 80 patients were negative for HCV RNA. Retrospective analysis of stored serum samples showed that 13 of 27 (48%) patients positive for HCV RNA were viremic at completion of therapy. In the remaining 14 patients (52%), HCV RNA had reappeared either during the first 6 months (6 patients) or the second 6 months (8 patients) after therapy. Patients who were positive for HCV RNA differed substantially from those who were negative for HCV RNA; the former were older and were treated with a smaller total dose of interferon-. Furthermore, a statistically significant difference was seen in the distribution of virus genotypes; HCV RNA-positive patients had a higher prevalence of HCV genotype 2 and a lower prevalence of HCV genotype 3 than did HCV RNA-negative patients. After treatment, a liver biopsy could be done while alanine aminotransferase levels were normal in 14 HCV RNA-positive patients and 49 nonviremic patients. Chronic hepatitis with piecemeal necrosis was seen in 8 of 14 (57%) HCV RNA-positive patients; the remaining patients had chronic hepatitis with portal inflammation but no periportal necrosis (4 patients) or minimal changes (2 patients). In contrast, active histologic findings were seen in only 6 of 49 (12%) HCV RNA-negative patients (P = 0.01); the remaining patients had chronic hepatitis with portal inflammation but no piecemeal necrosis (25 patients) or either normal liver histologic findings or minimal changes (18 patients). When the 107 patients were followed prospectively beyond the first year after therapy had been discontinued, the outcomes in viremic and nonviremic patients clearly differed. All HCV RNA-negative patients maintained normal alanine aminotransferase levels and remained negative for HCV RNA, whereas a biochemical relapse was seen in 8 of the 27 (30%) viremic patients. The estimated probability of a return of elevated alanine aminotransferase levels 4 years after cessation of treatment was 53% in viremic patients and 0% in the negative patients (P < 0.001) (Figure 1). No characteristics could be used to distinguish patients with early (within 12 months) or late (beyond 12 months) relapse after therapy. However, when all patients who had relapse were compared with all patients who had sustained biochemical and virologic long-term responses, the former were older (P < 0.01), had a longer duration of disease (P < 0.01), and had a different distribution of HCV genotype (P < 0.05) with increased prevalence of genotype 1 and decreased prevalences of genotypes 2 and 3. Figure 1. Probability of maintaining normal serum alanine aminotransferase levels at long-term follow-up (Kaplan-Meier curves). P Discussion Our study shows that serum HCV RNA testing is clinically useful when done 1 year after interferon- therapy in patients with chronic hepatitis C who have responded to therapy with persistently normal aminotransferase levels. If the test result is negative 1 year after therapy, the response is permanent; in patients who are still viremic 1 year after therapy, liver disease is often still histologically active and the risk for a late reactivation of hepatitis is substantial (53% of patients in our study had probability of reactivation by 4 years after therapy). Our results are consistent with previous reports on the discrepancy between biochemical and virologic responses to interferon- in chronic hepatitis C and on the possibility of late relapse after a prolonged biochemical response [11, 12]. Only one HCV RNA test was done 1 year after therapy, and we cannot exclude the possibility of some false-negative results caused by intermittent viremia or HCV-RNA levels below the sensitivity of our assay. Furthermore, viremia may not accurately reflect the presence or absence of the virus in the liver. Despite these limitations and the fact that not all of our patients who responded to interferon could have a liver biopsy done after treatment, our results clearly indicate that serum HCV RNA should be tested in all patients who have developed a sustained biochemical response to interferon- to establish whether the response is complete and permanent. Patients who are negative for HCV RNA may be considered cured. Viremic patients with normal alanine amino-transferase levels should be followed carefully well beyond the first year after cessation of therapy, because many will eventually relapse. Studies must be done to determine whether treatment with larger interferon- doses could reduce the prevalence of residual viremia after therapy and whether patients who remain positive for HCV RNA after interferon- therapy could benefit from early retreatment. Dr. Belussi: Divisione Malattie Infettive, O. Le Grazie, 5501 San Marco, 30124 Venezia, Italy. Dr. Martinelli: Divisione Medica, O.C. Cittadella (PD), via Circonvallazione, 35013 Cittadella (PD), Italy.

Antibodies to interferon (IFN) in hepatitis C patients relapsing while continuing recombinant IFN-α2 therapy

A number of trials have demonstrated that IFN‐α is effective in chronic hepatitis C virus infection. It is known, however, that a number of chronic hepatitis C patients experience, after an initial response to IFN, disease reactivation or relapse (also called ‘breakthrough’) while IFN therapy is still ongoing. Since in a number of clinical conditions a significant correlation between development of antibodies to IFN and failure of therapy has been established, we addressed the possibility that the development of antibodies to IFN may take part in the relapse occurring in hepatitis C patients during recombinant IFN‐α (rIFN‐α) therapy. The prevalence of neutralizing (NA) and binding antibodies (BA) to rIFN‐α2 has been evaluated in 45 patients with chronic hepatitis C treated with rIFN‐α2a who first normalized aminotransferase (ALT) levels, and subsequently showed disease reactivation while on treatment. The presence of NA and BA was tested before therapy, during the response to IFN treatment, and at the time when ALT started to rise again to abnormal levels. The results showed that no patients had detectable antibodies to IFN before therapy and during the period of response to the therapy, while most of them (88.9%) developed NA and/or BA to IFN‐α2 concomitantly with disease reactivation. In particular, in 29 of the 45 patients (64.4%) ALT normalized on treatment and rose to abnormal levels when NA appeared in their serum, while in 11 of the 16 (68.8%) remaining patients the relapse was associated with BA development. The frequency of seroconversion in these patients is significantly higher than that observed in the control group. These data indicate that antibodies to IFN may be responsible for breakthrough in the majority of patients showing disease reactivation while rIFN‐α therapy is still ongoing.

Systemic manifestations and liver disease in patients with chronic hepatitis C and type II or III mixed cryoglobulinaemia

The aim of this study was to evaluate the prevalence of cryoglobulins in patients with chronic hepatitis B and C virus infection and to investigate the association of type II and type III mixed cryoglobulinaemia with systemic manifestations and liver disease stage and outcome in hepatitis C virus (HCV)‐positive patients. We analysed the prevalence of cryoglobulinaemia in a cohort of patients with chronic liver disease and compared the systemic manifestations and liver involvement in HCV‐positive patients with type II or type III mixed cryoglobulinaemia. The prevalence of serum cryoglobulins was significantly higher in HCV‐positive patients than in hepatitis B surface antigen (HBsAg)‐positive patients (55.4 vs 20.6%). In HCV‐positive patients, stage of liver disease correlated with the prevalence of cryoglobulinaemia. Patients with type II cryoglobulins showed a significantly higher risk of cirrhosis and of extrahepatic manifestations while patients with type III cryoglobulins had a significantly higher prevalence of hepatocellular carcinoma. During follow‐up the former had an odds ratio of 11.9 of death from extrahepatic complications while the latter had an odds ratio of 3.4 of dying from hepatic disease. Our study confirms the high frequency of mixed cryoglobulinaemia in patients with chronic hepatitis C virus infection. The presence and type of cryoglobulins seem to be associated with different clinical manifestations and outcome.

Liver stellate cells in chronic viral hepatitis: the effect of interferon therapy

BACKGROUND/AIMS Liver stellate cell proliferation and differentiation into myofibroblast-like cells is related to the development of liver fibrosis. Several cytokines, including interferons, regulate liver stellate cell proliferation and phenotypic modulation. Recent studies indicate that human liver stellate cells express the alpha-isotype of actin, specific to smooth muscle cell differentiation. We aimed to evaluate the expression of alpha-smooth muscle actin-positive liver stellate cells in patients with chronic viral hepatitis and to evaluate whether and how such expression can be modified by alpha-interferon treatment. METHODS Using immunohistochemistry, and a semi-quantitative scoring method, we evaluated alpha-smooth muscle actin expression in liver stellate cells before and after alpha-interferon therapy in a series of liver biopsies from 44 patients with chronic viral hepatitis. RESULTS Before therapy, a large number of liver stellate cells expressing alpha-smooth muscle actin were present throughout all acinar zones. A significant reduction in alpha-smooth muscle actin expression by liver stellate cells was demonstrated in biopsies performed after suspending the interferon treatment. The drop in the number of alpha-smooth muscle actin-labelled cells after therapy correlated closely with the improvement in the histological index of activity. CONCLUSIONS The results suggest a specific effect of interferon on liver stellate cells, possibly related to its anti-inflammatory action.

Treatment with interferon(s) of community-acquired chronic hepatitis and cirrhosis type C

Two hundred and thirty-four patients with chronic non-A, non-B hepatitis, 86% positive for anti-HCV by ELISA, were treated with recombinant interferon-alpha 2a or with natural (human-leukocytes-derived) interferon-alpha using different dosage and periods of administration. Interim analysis of follow-up data indicate that 65-70% of patients treated initially with 6 MU, thrice weekly, of recombinant interferon-alpha 2a achieved a complete biochemical response (normalization of alanine aminotransferase: ALT) during therapy compared to 56-58% of those treated with 3 MU, thrice weekly, of recombinant or natural interferon-alpha. A 12-month schedule of interferon administration appeared superior to a 6-month schedule in reducing the probability of reactivation of liver disease after therapy withdrawal, although further data are needed to confirm such a conclusion. The probability of response to interferon in terms of maintaining normal ALT after withdrawal did not appear to be influenced by sex, while it was significantly higher in patients aged below 45 years and in those without cirrhosis.

Genotypes of hepatitis C virus in Italian patients with chronic hepatitis C

Abstract To assess the prevalence of different hepatitis C virus genotypes in an European population of patients with chronic hepatitis C, 79 consecutive Italian patients were studied. After having cloned and sequenced part of the 5′ untranslated region of the virus in 21 patients, oligonucleotide probes were synthesized to be used in a more rapid dot-blot hydridization test. Using this method, 42% of patients were found infected by HCV type 1, 45% by HCV type 2 and 4% by HCV type 3, while seven patients remained unclassified. Patients infected by HCV type 3 were significantly younger and had a milder form of liver disease, compared to those infected by HCV type 1 or HCV type 2. Patients with HCV type 2 or HCV type 3 responded to interferon therapy much better than those with HCV type 1. These results provide information on the prevalence of different HCV genotypes in our region, and indicate the usefulness of the dot-blot hybridization procedure for rapid screening of HCV genotypes.

Antibody to Hepatitis C Virus and Liver Disease in Volunteer Blood Donors

OBJECTIVE To evaluate the specificity of antibodies to hepatitis C virus (anti-HCV) and their relation to liver disease in blood donors. DESIGN Case series of consecutive blood donors found positive for anti-HCV by enzyme-linked immunosorbent assay (ELISA). Patients were evaluated for antibody specificity using a recombinant immunoblotting assay (RIBA) and were evaluated for biochemical evidence of liver disease. Patients showing increased alanine aminotransferase (ALT) levels had a liver biopsy. SETTING University hospital. PARTICIPANTS Fifty consecutive blood donors found to be anti-HCV positive on both an initial and repeat ELISA. Inclusion criteria were as follows: an absence of hepatitis B surface antigens and non-organ-specific autoantibodies; a daily alcohol intake of less than 50 g; no history of recent hepatotoxic drug use; and normal serum levels of alpha 1 antitrypsin, ceruloplasmin, and copper. MAIN RESULTS Anti-HCV positivity was confirmed by RIBA in only 13 of 50 donors (26%) who had positive ELISA results. These 13 donors had an elevated ALT level and histologic evidence of chronic hepatitis, which was active in 8 patients (62%) and had already produced cirrhosis in 2 patients (15%). In contrast, the 17 donors with an intermediate RIBA pattern had only mild and often nonspecific histologic liver abnormalities. The 20 patients with a negative RIBA result had normal ALT levels. CONCLUSION In blood donors, the anti-HCV RIBA is not only more specific than the anti-HCV ELISA, but is also useful in identifying patients who have an underlying chronic liver disease.

Progressive increase of SCCA-IgM immune complexes in cirrhotic patients is associated with development of hepatocellular carcinoma.

About 3–4% of cirrhotic patients develop primary liver cancer every year. Specific serologic markers have not yet been identified for screening of high risk patients. The serpin squamous cell carcinoma antigen (SCCA) is overexpressed in liver cancer and circulating SCCA‐IgM complexes have been described in patients with hepatocellular carcinoma (HCC). The aim of the present study was to assess the behavior of SCCA‐IgM in relation to HCC development in patients with cirrhosis. A retrospective, longitudinal study was conducted in a cohort of prospectively followed cirrhotic patients. Two groups with similar clinical profile at presentation were studied : group A included 16 patients who developed HCC during a median follow up of 4 years; group B included 17 patients who did not develop HCC during the same time interval. Circulating SCCA‐IgM immune complexes were determined using a recently standardized ELISA assay. At presentation similar levels of SCCA‐IgM complexes [mean ± SD: 267.40 ± 382.25 U/ml vs. 249.10 ± 446.90 U/ml, p = 0.9006] and of alpha‐fetoprotein [AFP; 24.11 ± 59.04 IU/ml vs. 10.91 ± 23.34 IU/ml, p = 0.3995] were detected in group A and in group B. The increase over time (ϕ) of SCCA‐IgM, assessed within at least one year before clinical diagnosis of HCC, was remarkably higher in group A than in group B (mean ± SD = 280.05 ± 606.71 (U/ml)/year vs. −37.92 ± 95.94 (U/ml)/year, p = 0.0408), while AFP increase was not significantly different (11.89 ± 23.27 (IU/ml)/year vs. 3.67 ± 11.46 (IU/ml)/year, p = 0.2179). Receiver operating characteristic (ROC) curves were plotted for the rate of change in the levels of both markers and the diagnostic accuracy measured as AUROC was higher for SCCA‐IgM ϕ (0.821) than for AFP ϕ (0.654). In conclusion, the progressive increase of SCCA‐IgM over time was associated with liver tumor development, suggesting that monitoring the behavior of SCCA‐IgM might become useful to identify cirrhotic patients at higher risk of HCC development.