Long-term histologic improvement and loss of detectable intrahepatic HCV RNA in patients with chronic hepatitis C and sustained response to interferon-alpha therapy

Ann Intern Med 1997 Nov 15;127(10):875-81
Marcellin P, Boyer N, Gervais A, Martinot M, Pouteau M, Castelnau C, Kilani A, Areias J, Auperin A, Benhamou JP, Degott C, Erlinger S Hopital Beaujon, Clichy, France.

In summary, in patients with chronic hepatitis C, the persistence of normal serum ALT levels (monitored every month during the 6 months after interferon-a 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-alpha therapy have a low risk for further relapse of viral infection and a low risk for development of cirrhosis or hepatocellular carcinoma.

BACKGROUND: Less than 20% of patients with chronic hepatitis C have a sustained response to interferon-alpha therapy. The long-term benefit of interferon-alpha with regard to hepatic viral clearance and histologic improvement remains unknown.

OBJECTIVE: To determine the long-term biochemical, virologic, and histologic outcomes in patients with chronic hepatitis C who have a sustained response to interferon-alpha therapy.

DESIGN: Prospective cohort study.
SETTING: University hospital.
PATIENTS: 80 patients who had chronic hepatitis C, had a sustained biochemical and virologic response to interferon-alpha therapy, and were followed for at least 12-months.

MEASUREMENTS: Serum hepatitis C virus (HCV) RNA detected by polymerase chain reaction (PCR); HCV genotyping determined by line probe assay; liver histologic studies; liver HCV RNA detected by PCR on frozen liver tissue samples (in 27 patients); and repeated measurements of serum alanine aminotransferase (ALT) levels. Liver biopsy was done before treatment in all 80 patients, and at least one biopsy was done in 69 patients 1 to 6 years after treatment. RESULTS: The 80 patients had follow-up 1 to 7.6 years (mean +/- SD, 4.0 +/- 2.0 years) after interferon-alpha treatment. The follow-up period was 1, 2, 3, 4, 5, 6, and more than 6 years in 11, 13, 14, 18, 10, 12, and 2 patients, respectively, after the end of therapy. During the entire follow-up period, 93% (95% CI, 84% to 97%) of patients had persistently normal serum ALT levels. Serum HCV RNA remained undetectable in 96% (CI, 89% to 99%) of patients. A comparison of liver histologic findings before and 1 to 6.2 years after interferon-alpha treatment showed a clear improvement in 94% (CI, 83% to 99%) of patients. In 62% of patients, the last biopsy done showed normal or nearly normal histologic findings. Liver HCV RNA was detectable before treatment in all 13 patients tested and was undetectable 1 to 5 years after treatment in all 27 patients tested.

CONCLUSIONS: In patients with chronic hepatitis C who have persistently normal serum ALT levels and no detectable serum HCV RNA 6 months after interferon-alpha therapy, a long-term sustained biochemical and virologic response is generally seen. This response is associated with an absence of detectable intrahepatic HCV RNA and marked histologic improvement.

Comment in: Ann Intern Med 1997 Nov 15;127(10):918-20
Comment in: Ann Intern Med 1998 Jun 1;128(11):956

Here is a more detailed discussion of the study.

Results-
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-alpha 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-alpha 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)

Liver histologic studies showed no cirrhosis in any of the patients who had not had cirrhosis before receiving interferon-a 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-alpha 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 less than or equal to 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 3 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 13patients 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-
Our study shows that most patients with chronic hepatitis C who have 1) persistently normal serum ALT levels during the 6 months after interferon-alpha 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-alpha 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-alpha 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-alpha 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-alpha 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-alpha 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-alpha 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-alpha 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-a 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-alpha therapy have a low risk for further relapse of viral infection and a low risk for development of cirrhosis or hepatocellular carcinoma.