Methods

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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

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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.

View larger version (25K): [in this window] [in a new window]   Figure 1. Probability of maintaining normal serum alanine aminotransferase levels at long-term follow-up (Kaplan-Meier curves). The line with asterisks represents patients who were negative for hepatitis C virus RNA (HCV-RNA), and the solid line represents patients who were positive and had a sustained biochemical response to interferon- as long as 12 months after discontinuation of therapy. In viremic patients, the estimated probability of hepatitis relapse is 53% 4 years after therapy; in HCV RNA-negative patients, the probability is 0% (log-rank test; P < 0.001).

Discussion

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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.