Long-term outcome in patients with sustained response is not well known. A few studies of small numbers of patients with follow-up periods of 1 to 4 years have suggested a long-term benefit in some patients; however, late relapse was seen in other studies [7-13]. Little information is available on histologic outcome, and the question of the long-term benefit of interferon- therapy with regard to hepatic viral clearance and histologic improvement remains to be answered. To address this question, we assessed the long-term biochemical, virologic, and histologic outcomes of 80 patients with chronic hepatitis C who had a sustained response during the 6 months after interferon- therapy.

Methods

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Patients

Among 450 patients with chronic hepatitis C who received interferon- therapy in our center between 1987 and 1993, 80 consecutive patients who had a sustained response after therapy were included in our study. Sustained response was defined as 1) strictly normal serum alanine aminotransferase [ALT] levels each month for the first 6 months after the end of therapy and 2) negative results on testing for serum hepatitis C virus (HCV) RNA 6 months after treatment. The 80 patients were followed for at least 1 year after the end of treatment; further follow-up was considered long-term follow-up.

Of these 80 patients, 50 were treated in six randomized, controlled trials of interferon-. Twenty-two received recombinant interferon-2b(Intron A, Schering-Plough, Levallois, France) at a dose of 3 or 5 million U [4, 6, 14]; 19 received lymphoblastoid interferon- (Wellferon, Wellcome, Issy-les-Moulineaux, France) at a dose of 3 or 5 million U [15, 16]; and 9 received recombinant interferon-alpha2a (Roferon-A, Produits Roche, Neuilly, France) at a dose of 3 million U [17]. The six trials were approved by the ethical committee, and all patients gave informed consent. The 30 patients who did not participate in the trials received the licensed standard schedule for interferon- therapy: recombinant interferon-alpha2a or -alpha2b, 3 million U three times a week for 6 months. Treatment schedules are shown in Table 1.

View this table: [in this window] [in a new window]   Table 1. Demographic, Biochemical, Virologic, and Histologic Characteristics before Treatment and Treatment Schedule in 80 Patients with Chronic Hepatitis C and Sustained Response to Interferon- Therapy*

All patients had chronic hepatitis C shown on biopsy within 6 months before treatment; the mean histology score (±SD) was 7.9 ± 2.2 (range, 3 to 13). All patients had serum ALT levels greater than 1.5 times the upper limit of normal for at least 6 months, and all were negative for hepatitis B surface antigen and antibodies to HIV.

Follow-up

Follow-up included clinical assessment every 6 months, measurement of serum ALT levels at least every 3 months, and detection of serum HCV RNA by polymerase chain reaction (PCR) every 6 months.

Antibodies to Hepatitis C Virus

Before treatment, all patients were positive for antibodies to HCV. Antibody testing (done retrospectively for patients treated before 1990) was done by using second-generation enzyme-linked immunoassay and Recombinant Immunoblast Assay (Ortho Diagnostic Systems, Levallois, France).

Serum Hepatitis C Virus RNA

We tested for the presence of serum HCV RNA in all 80 patients on serum specimens that were kept frozen and collected 6 months after withdrawal of interferon- therapy and then every 6 months during the entire follow-up period. Serum HCV RNA was detected by using PCR (Amplicor HCV, Produits Roche) [18].

Serum HCV RNA quantitation was performed with the quantitative branched-DNA signal amplification assay (Quantiplex HCV RNA, Chiron Diagnostics, Eragny, France) before treatment in 62 patients for whom pretreatment serum HCV RNA measurements were available and during follow-up in patients with persistent or fluctuating detectable HCV RNA levels [19].

Genotype and Serotype of Hepatitis C Virus

We did HCV genotyping in 62 patients for whom pretreatment serum measurements were available. Genotyping was done by using the reverse hybridization assay (LiPA, InGeN, Rungis, France) after amplification with the PCR assay [20]. The classification system of Simmonds and colleagues was used [21]. In 18 patients who had no pretreatment serum aliquots available and whose post-treatment serum specimens were negative on PCR, HCV genotyping was not possible and HCV serotyping was done. Serotyping was performed with an immunoenzymatic assay that shows antibodies directed to peptides encoded by the NS4 region of the HCV genome, which are specific for serotypes 1, 2, 3, 4, 5, and 6 (Murex Diagnostics, Chatillon, France) [22]. These serotypes are classified according to the respective genotypes in the classification system of Simmonds and colleagues [21].

Liver Histologic Studies

Liver biopsy was done at the end of interferon- therapy or 6 or 12 months after therapy, according to the relevant protocol, in patients who participated in the randomized trials. Another biopsy was done 1 to 6 years after therapy in all patients. Biopsy specimens obtained before and after treatment were assessed for fibrosis (score, 0 to 4) and activity (score, 0 to 18) according to the scoring system of Knodell and coworkers [23]. Chronic hepatitis C was defined as mild if the total histology score was less than 6, moderate if the score was between 6 and 9, and severe if the score was greater than 9. Biopsy specimens obtained by using a percutaneous Menghini needle were at least 10 mm long. Specimens were coded and were assessed by one pathologist.

After interferon- therapy, 109 liver biopsies were done in 69 of the 80 patients (32 patients had 2 biopsies, and 4 patients had 3 biopsies); 22 were done at the end of treatment and 87 were done during follow-up. In 48 of the 69 patients, the last post-treatment biopsy was done at least 1 year after the end of interferon- treatment (2.2 ± 1.3 years [range, 1 to 6.2 years]). In these 48 patients, histologic outcome was assessed by comparing the pretreatment biopsy specimen with the last biopsy specimen obtained after treatment. Histologic outcome was defined as "improved" if the total histology score was at least 2 points lower in the post-treatment specimen, as "no change" if the score was 1 point lower in the post-treatment specimen or was the same in the two specimens, and as "deterioration" if the score was higher in the post-treatment specimen.

Liver Hepatitis C Virus RNA

A liver tissue specimen was collected 1 to 5 years after treatment in 27 patients (2 years after treatment in 10 patients); 13 of the 27 had a liver tissue specimen collected before treatment. All tissue samples were immediately frozen in liquid nitrogen and kept at –80°C.

All tubes and tissue grinders used for the liver HCV RNA extraction procedure were disposable and sterile and were autoclaved. Liver RNA was extracted with the guanidium isothiocyanate procedure (Trizol, Appligen, Illkirch, France), and PCR was performed in the 5'NC region of the HCV genome according to the methods of Martinot-Peignoux and associates [24]. The quality of the extracted liver RNA was ascertained with the amplification of the cellular 28S ribosomal messenger RNA (rRNA) [25]. The 28S rRNA represents 80% of the total cellular RNA; the presence of 28S rRNA attested to the good preservation of the liver specimens. Specimens without amplifiable 28S rRNA were excluded from our study. All PCR assays were done in the presence of negative and positive controls, which were liver tissue specimens collected during the period in which the study specimens were collected. Negative controls were obtained from patients without antibodies to HCV and patients without detectable serum HCV RNA who had chronic liver disease. Positive controls were obtained from patients with untreated chronic hepatitis C. At each step of the procedure, a negative specimen without nucleic acid was added. All PCR procedures on liver and serum were done with strict application of the measures described by Kwok and Higuchi [26].

Role of the Funding Source

The funding source did not influence the analysis or interpretation of the data or the decision to submit the paper for publication.

Results

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In our 80 patients with chronic hepatitis C, a sustained response was defined by 1) a persistently normal serum ALT level throughout the 6-month post-treatment follow-up period and 2) negative results on serum HCV RNA testing 6 months after treatment. Patients were followed for 1 to 7.6 years after the end of interferon- therapy (mean ±SD, 4.0 ± 2.0 years). The characteristics of the 80 persons with sustained response are shown in Table 1. The numbers of patients contributing data at each time point during follow-up are shown in Table 2.

Clinical Outcomes

At the end of follow-up, 79 of the 80 persons with sustained response were fully active and alive; 1 had died of peritoneal carcinomatosis related to colon carcinoma. Of the 5 patients with cirrhosis, none developed decompensated liver disease or hepatocellular carcinoma as assessed by abdominal ultrasonography done every 6 months. Of the 75 patients who did not have cirrhosis before receiving interferon- therapy, none developed cirrhosis. All patients who had fatigue before treatment (60%) said that the fatigue completely disappeared after treatment.

Serum Alanine Aminotransferase Levels

By definition, all patients had normal serum ALT levels during the 6-month follow-up period after treatment. During the subsequent long-term follow-up period, 74 of the 80 patients studied (93% [95% CI, 84% to 97%]) maintained persistently normal serum ALT levels. Six patients (7% [CI, 3% to 16%]) had ALT levels that fluctuated between normal and twice the upper limit of normal. No reactivation of liver disease with ALT levels above twice the upper limit of normal was seen.

Serum Hepatitis C Virus RNA

The proportion of low, moderate, and high HCV RNA levels before treatment is shown in Table 1. By definition, no patients had detectable serum HCV RNA 6 months after treatment. During the long-term follow-up period, serum HCV RNA was persistently undetectable in 72 of the 75 patients tested (96% [CI, 89% to 99%]) and was intermittently detectable in 3 patients (4% [CI, 1% to 11%]). Two of these 3 patients had transient detectable HCV RNA (one positive result on PCR with HCV RNA level less than 3.5 x 10⁵ genome equivalents/mL) with normal serum ALT levels; the other had late relapse with reappearance of HCV RNA at 24 months and a slight increase in serum ALT level (1.3 times the upper limit of normal) 38 months after the end of treatment.

Genotype and Serotype of Hepatitis C Virus

In 62 patients, HCV genotype was identified by using the reverse hybridization assay. The HCV serotype was identified in 7 additional patients. Neither genotype nor serotype could be identified in 11 patients. The distribution of HCV genotypes or serotypes is shown in Table 1. Of the 5 patients with cirrhosis who had sustained response, 1 had genotype 1b and 1 had genotype 3a; genotype or serotype could not be determined in the other 3 patients.

Liver Histologic Findings

Before treatment, liver histologic studies showed chronic hepatitis C to be mild in 10 patients (12%), moderate in 46 patients (58%), and severe in 24 patients (30%).

The 109 liver biopsies done after treatment were classified into five groups according to the delay after the end of therapy. Analysis of the biopsy specimens showed progressive and continuous histologic improvement with decreases in the histology score in biopsy specimens obtained at the end of treatment; during the first, second, and third year after treatment; and more than 3 years after treatment (Figure 1). The decrease was more marked for lobular and periportal necrosis than for portal inflammation (Figure 2). No significant change was seen in fibrosis (Figure 2).

View larger version (13K): [in this window] [in a new window]   Figure 1. Total Knodell score before, at the end of, and after interferon- (IFN) therapy. Liver biopsies were done during the first, second, or third year after therapy or later than the third year after therapy.

View larger version (20K): [in this window] [in a new window]   Figure 2. Knodell score for fibrosis (top left), portal inflammation (bottom left), periportal necrosis (top right), and lobular necrosis (bottom right) before, at the end of, and after interferon- (IFN) therapy. Liver biopsies were done during the first, second, or third year after therapy or later than the third year after therapy.

Liver histologic studies showed no cirrhosis in any of the patients who had not had cirrhosis before receiving interferon- treatment. In particular, none of the 24 patients who had had severe chronic hepatitis C before treatment (mean follow-up, 4.5 ± 1.8 years [range, 1 to 6.6 years]) developed cirrhosis. Five patients had had cirrhosis before treatment; post-treatment liver biopsy done in 4 of these patients 0.5 to 3.6 years after treatment showed a marked decrease of activity in 2 patients and a deterioration in 2 others (one had a 1-point increase in the histology score and the other had a 2-point increase).

Pretreatment and post-treatment liver biopsy specimens were compared for the 48 patients who had had their last post-treatment liver biopsy done at least 1 year after the end of interferon- therapy. Before therapy, the mean histology score of these 48 patients was 8.2 ± 2.0 (range, 4 to 13). The last liver biopsy, done 1 to 6.2 years after treatment, showed a mean histology score of 3.6 ± 2.4 (range, 0 to 12). Thirty patients (62%) had normal or nearly normal histologic findings (histology score 2), 11 (23%) had mild chronic hepatitis C, 6 (13%) had moderate chronic hepatitis C, and 1 (2%) had severe chronic hepatitis C. Comparison of histology scores for the pretreatment biopsy specimen and the last biopsy specimen showed improvement (decrease 2) in 94% (CI, 83% to 99%) of patients, no change in 2% (CI, 0% to 11%), and deterioration in 4% (CI, 1% to 14%). Of the 2 patients who had deterioration, one had slight intermittent elevation of serum ALT levels without detectable serum HCV RNA and one had persistently normal serum ALT levels with detectable serum HCV RNA (detected once, 6 months after the end of treatment), which disappeared during the long-term follow-up period. These patients had 1-point and 2-point increases, respectively, in histology score.

Liver Hepatitis C Virus RNA

Before treatment, HCV RNA was detectable in the liver specimens of all 13 patients tested. One to 5 years after treatment, liver HCV RNA was detectable in none of the 27 patients tested. The 28S mRNA was detectable in all specimens tested, which ascertained the good quality of liver RNA. Among the 27 patients with no detectable liver HCV RNA, serum HCV RNA test results were fluctuating in 2 patients (the patients were negative for HCV RNA at the time of the liver biopsy) and negative in 25 patients.

Discussion

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Our study shows that most patients with chronic hepatitis C who have 1) persistently normal serum ALT levels during the 6 months after interferon- therapy and 2) no detectable serum HCV RNA with PCR 6 months after treatment have long-term biochemical and histologic improvement. The absence of detectable HCV RNA in the serum and liver several years after interferon- therapy suggests the possible eradication of HCV infection.

Although 19 patients had severe chronic hepatitis C without cirrhosis before treatment, no new cases of cirrhosis appeared; this suggests that interferon- treatment has long-term benefit in patients with chronic hepatitis C. However, follow-up of these patients was too short to allow definitive conclusions about the potential effect of the sustained response to interferon- therapy on prevention of progression to cirrhosis. In our study, five patients with cirrhosis had a sustained biochemical and virologic response with histologic improvement, showing that the long-term response in patients with cirrhosis is rare but can be complete. Furthermore, no decompensation and no hepatocellular carcinoma occurred in these patients. It has been suggested [27] that interferon- treatment might decrease the risk for hepatocellular carcinoma in patients with chronic hepatitis C. In our study, the number of patients with cirrhosis was too small to allow us to draw conclusions about the effect of interferon- therapy on risk for liver decompensation and hepatocellular carcinoma.

The 7% rate of late increase in serum ALT levels (the increase was mild in all cases) seen in our 80 patients is lower than the rates of 25% to 79% found in other studies [7-12]. The discrepancy might be related to our patient selection process, which was based on our stringent definition of sustained response (that is, normal serum ALT levels each month for 6 months after therapy and undetectable serum HCV RNA 6 months after therapy); the same definition was used in the study by Reichard and colleagues [13], in which the relapse rate was also 7%. In the study by Chemello and coworkers [12], no relapse was seen in 80 patients who had a sustained response defined by normal ALT levels and undetectable serum HCV RNA 1 year after therapy.

A sustained virologic response was seen in 72 of the 75 patients tested in our study (96%). Although the sustained disappearance of detectable serum HCV RNA might be not sufficient to rule out a persistent low-level viral infection, the persistence of normal serum ALT levels in 68 of the 72 patients who were negative for HCV RNA and were followed for 1 to 7.6 years suggests the sustained inhibition of viral replication. Thus, normal serum ALT levels and the absence of detectable serum HCV RNA during the 6-month post-treatment follow-up period seem to be reliable indicators of sustained response.

In a few patients (4%), serum HCV RNA levels remained detectable at fluctuating low levels for more than 6 months after treatment. One of the three patients who were positive for serum HCV RNA had relapse 38 months after treatment. Thus, the persistence of detectable serum HCV RNA despite normal serum ALT levels probably reflects the persistence of HCV replication with the risk for late relapse, as previously shown [12].

Previous studies have shown some characteristics to be associated with sustained response. Of note, 62% of the persons with sustained response in our study were 40 years of age or younger, 60% had had HCV infection for 10 years or less, 48% were infected by HCV genotype or serotype 3, 16% were infected with HCV genotype or serotype 2, and 71% had low serum HCV RNA levels (<3.5 x 10⁵ genome equivalents/mL) before treatment. Among the patients with sustained response, genotypes 1a and 1b were less common and genotype 3a was more common than in the general population of patients with chronic hepatitis C at our center (3% compared with 15% for genotype 1a, 27% compared with 48% for genotype 1b, and 50% compared with 21% for genotype 3a) [28]. This result is in accordance with the view that HCV genotypes 1a and 1b are associated with a poor rate of sustained response to interferon- therapy.

One to 6 years after treatment, about two thirds of patients had normal or nearly normal histologic findings. The range of histology scores, both before therapy (4 to 13) and after therapy (0 to 12), were wide. However, most patients (88%) had moderate or severe chronic hepatitis before therapy and most (85%) had no chronic hepatitis C or mild chronic hepatitis C after therapy. Histologic improvement was gradual and continued for several years after the end of therapy, as suggested by smaller studies [11, 13]. However, mild liver inflammation persisted for up to 6 years in some patients despite persistently normal serum ALT levels and negative results on testing for serum and liver HCV RNA. The persistence of inflammatory infiltrates may be due to continuing low-level HCV replication in the liver and without detectable liver HCV RNA by the method used. It is also possible that some degree of intrahepatic cellular immune response to HCV persists and takes many years to disappear.

The absence of detectable liver HCV RNA 1 to 5 years after treatment is consistent with the view that HCV infection may be cleared with interferon- therapy in patients with chronic hepatitis C. It confirms, in more patients and with a longer follow-up period, the results of previous studies, in which 86% to 91% of persons with sustained response had no detectable liver HCV RNA 1 to 2 years after therapy [11, 13]. The disappearance of liver HCV RNA seems to be correlated with the disappearance of serum HCV RNA. The presence of a low level of HCV RNA in the liver, not detected on PCR, cannot be ruled out. In our study, however, no relapse occurred among the 27 patients without liver HCV RNA during follow-up; this suggests that these patients may be cured of disease.

In summary, in patients with chronic hepatitis C, the persistence of normal serum ALT levels (monitored every month during the 6 months after interferon- therapy) and the absence of detectable serum HCV RNA 6 months after treatment seem to be reliable indicators of long-term biochemical and virologic remission with histologic improvement. Clearance of serum HCV RNA is associated with clearance of intrahepatic HCV RNA, and this suggests that viral infection might be eradicated and patients might be cured of disease. This supports the hypothesis that persons with sustained responses to interferon- therapy have a low risk for further relapse of viral infection and a low risk for development of cirrhosis or hepatocellular carcinoma.

Dr. Degott: Service d'Anatomie et Cytologie Pathologiques, Hopital Beaujon, 100 Boulevard du General Leclerc, 92118 Clichy, France.