10-Year Follow-up After Interferon- Therapy for Chronic Hepatitis C

HEPATOLOGY, October 1998, p. 1121-1127, Vol. 28, No. 4

Daryl T.-Y. Lau1, David E. Kleiner2, Marc G. Ghany1, Yoon Park3, Peter Schmid4, and Jay H.Hoofnagle1

From the 1 Liver Diseases Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD; 2 Laboratory of Pathology, National Cancer Institute, Bethesda MD; 3 Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD; and 4 National Genetics Institute, Los Angeles, CA

http://hepatitis-central.com/

ABSTRACT

Sustained responses to interferon- occur in 10% to 25% of patients with chronic hepatitis C, but the long-term outcome is not well defined. We evaluated the long-term clinical, histological, and virological outcomes of 10 patients with chronic hepatitis C who were treated between 1984 and 1987 with interferon--2b for 52 ± 6 weeks (total doses of 492 ± 116 MU). Before therapy, all 10 had hepatitis C virus (Hepatitis C Virus) RNA, elevations of serum aminotransferases, and chronic hepatitis with fibrosis on liver biopsy. Clinical follow up was 6 to 13 years, and liver biopsies were done 5 to 11 years after initiation of therapy. Hepatitis C Virus RNA was assayed by qualitative and quantitative reverse transcriptase-polymerase chain reaction assays. Among 5 patients who had a 6-month sustained response after therapy, all remained Hepatitis C Virus RNA negative, and at last follow-up, 4 had normal and 1 minimally elevated serum aminotransferase levels. Liver biopsy specimens were nonreactive for Hepatitis C Virus RNA, and all the patients showed improvements in both inflammation and fibrosis and were either normal or had mild, nonspecific inflammatory changes. Among 5 patients without a sustained response, all continued to have Hepatitis C Virus RNA in serum and persistent or intermittent aminotransferase elevations. Liver biopsy specimens showed little or no change in necrosis and inflammation; all except 1 patient had progression of fibrosis scores or cirrhosis. All 5 patients had symptoms of chronic hepatitis, 1 underwent liver transplantation, and another had progressive hepatic decompensation. In conclusion, patients with a 6-month posttreatment virological response have a favorable long-term clinical and histological outcome.(HEPATOLOGY 1998;28:1121-1127.)

INTRODUCTION

Non-A, non-B hepatitis was recognized as the major cause of transfusion-associated hepatitis more than 20 years ago, when the discoverers of the hepatitis A virus showed that cases of viral hepatitis not related to hepatitis B were also not attributable to hepatitis A.1 After more than a decade of intensive research, Houghton and co-workers isolated a fragment of the genome of the hepatitis C virus (Hepatitis C Virus) and subsequently cloned and characterized this agent.2 The development of the diagnostic tests for antibody to Hepatitis C Virus (anti-Hepatitis C Virus)3 and for Hepatitis C Virus RNA4 soon followed. Application of these assays has shown that infection with Hepatitis C Virus affects approximately 4 million Americans5 and that hepatitis C is probably the most common cause of chronic liver disease, cirrhosis, and liver cancer in the Western world. 6,7

Therapy for hepatitis C also has advanced in the past 20 years. Even before the discovery of Hepatitis C Virus, a pilot study conducted at the Clinical Center of the National Institutes of Health (NIH) demonstrated that therapy with interferon- induced clinical and histological remissions in a proportion of patients with chronic non-A, non-B hepatitis.8 Subsequently, a series of randomized clinical trials confirmed the efficacy of interferon as therapy for chronic hepatitis C,9-12 and testing of stored samples showed that successful therapy was usually associated with loss of Hepatitis C Virus RNA from serum and liver. 13,14 A recent NIH Consensus Conference has recommended approaches to diagnosis, prevention, and treatment of hepatitis C.15 A central issue remains whether responses to therapy are sustained and result in permanent remissions or cure of the infection and disease.

The aim of the current study was to assess the long-term survival and clinical outcomes of the original cohort of patients with non-A, non-B hepatitis infection who were treated with interferon-at the NIH in the early 1980s.

MATERIALS AND METHODS

Patients. In a pilot dose-finding study conducted at the NIH Clinical Center between 1984 and 1986, 10 patients with chronic non-A, non-B hepatitis were treated with interferon- (-2b; Schering-Plough Corp., Kenilworth, NJ).8 All patients were later confirmed to have chronic Hepatitis C Virus infection on the basis of finding anti-Hepatitis C Virus and Hepatitis C Virus RNA in serum on stored, pretreatment samples. The 10 patients comprised 9 men and 1 woman, were all non-Hispanic whites, and ranged in age from 26 to 62 years (mean, 40 years). The source of Hepatitis C Virus was suspected to be blood transfusion in 6 and injection drug use in 3, and was unknown in 1. When initially evaluated, all patients had mild symptoms of liver disease, persistently elevated serum aminotransferase levels, and liver biopsy specimen changes consistent with chronic hepatitis C. Patients received interferon- subcutaneously in varying dose regimens for 2 to 19 months. In a subsequent reanalysis of this cohort, 6 patients were considered sustained responders, with no Hepatitis C Virus RNA in serum as detected by reverse transcriptase (RT)-polymerase chain reaction (PCR) after several years of follow-up.13 One patient was later reclassified as a virological nonresponder when Hepatitis C Virus RNA was found to be persistently positive in low titers using a more sensitive assay.16

Follow-up and Measurements. After completion of the study, patients were scheduled for at least yearly follow-up visits. Beginning in April 1996, all 9 living patients were contacted and asked to return for reevaluation. The evaluation included complete history and physical examination, a battery of blood tests including serological tests for hepatitis B virus and Hepatitis C Virus infection, abdominal ultrasound, and repeat liver biopsy. These studies were done as a part of clinical research protocols that were approved by the National Institute of Diabetes and Digestive and Kidney Diseases Institutional Review Board, and all patients gave written, informed consent.

Serum Hepatitis C Virus RNA levels were determined by a qualitative RT-PCR assay (Amplicor: Roche Diagnostics Systems, Branchburg, NJ)16 with a lower limit of detection of 400 viral copies/mL serum. The level of Hepatitis C Virus RNA in the final follow-up specimens was measured by a quantitative PCR assay (National Genetics Institutes, Los Angeles, CA) with a lower limit of detection of 100 Hepatitis C Virus copies/mL.17 Hepatitis C Virus genotyping was done using the reverse hybridization assay (line probe assay, LiPA; Innogenetics, Ghent, Belgium)18 and serotyping by immunoassays based on serological differences in antibody to the NS4 region of the genome.19

As a part of the initial study, all patients underwent liver biopsy before treatment and again after 1 year of treatment. Subsequently, follow-up liver biopsies were done on all 10 patients between 5 and 11 years after starting therapy. Frozen liver tissues were available in 7 for hepatic Hepatitis C Virus RNA determination. All liver biopsy specimens were reevaluated under code by a hepatic pathologist (D.E.K.) who was not aware of the sequence of the biopsies, patient's history, or therapy. Liver histology was graded using a modification of the histology activity index (HAI), which comprised the sum of four scores, including periportal necrosis and inflammation (0-10), lobular necrosis and inflammation (0-4), portal inflammation (0-4), and fibrosis (0-4). 20,21 Hepatic Hepatitis C Virus RNA was measured by the qualitative PCR assay (National Genetics Institutes). Liver samples were weighed and total RNA was extracted using guanidinium isothiocyanate and phenol. The tissue RNA was then resuspended to achieve an RNA concentration of 1 µg in 3 µL of solution. A standard RT-PCR procedure was then performed using the same primer pairs as for the serum assay.17

RESULTS

All 10 patients initially had sustained elevations in levels of serum alanine aminotransferase (ALT; range = 148 to 460, mean = 297 ± 35 U/L) and chronic hepatitis with fibrosis on liver biopsy.

table  1.   Clinical, Serum Biochemical, Histological, and Serological Features of Ten Patients With Chronic Hepatitis C Treated With Alpha Interferon
No.	Clinical Features					Initial			Final				Follow-up (yr)	Clinical Outcome
	Resp	Age (yr)	Sex	Source	Genotype	RNA	ALT	HAI	RNA	ALT	HAI	Liver RNA
1	SR	30	M	IDU	1a	+	376	11	<100	20	3		11	Asymptomatic
2	SR	27	M	Tx	2a/2b	+	460	8	<100	52	3		10	Asymptomatic
3	SR	37	M	IDU	2a	+	306	11	<100	21	1		11	Asymptomatic
4	SR	40	M	Tx	3	+	272	11	<100	35	2		10	Asymptomatic
5	SR	29	M	Tx	2a	+	185	11	<100	23	0		10	Asymptomatic
6	NR	51	M	IDU	1	+	219	12	1500	23	11	NA	13	HCC/OLT
7	NR	41	M	Unknown	1b	+	419	14	.18 × 106	181	12	NA	6	Died/Stroke
8	NR	62	F	Tx	1b	+	200	8	.049 × 106	248	16	+	11	Symptoms
9	NR	38	M	Tx	1a	+	148	17	>5 × 106	102	15	+	11	Symptoms
10	NR	46	M	Tx	1b	+	392	15	1.4 × 106	326	12	NA	11	Symptoms
Abbreviations: Resp, virological response; SR, sustained response; NR, non-response; IDU, injection drug use; Tx, transfusion; HAI, histology activity index score; HCC, hepatocellular carcinoma; OLT, orthotopic liver transplantation; NA, not available.

 Two patients had compensated cirrhosis. Testing of stored serum samples revealed that all patients were reactive for both anti-Hepatitis C Virus and Hepatitis C Virus RNA. Two patients had genotype 1a, three 1b, three 2a, and one 3a.18 One patient with low levels of Hepatitis C Virus RNA could not be genotyped but had serotype 1 based on anti-Hepatitis C Virus testing.19

The 10 patients received interferon--2b in varying regimensdaily, every other day, or three times a week in doses ranging from 1 to 5 MU. One patient did not tolerate the side effects of interferon, and therapy was stopped after 8 weeks. The remaining patients received interferon for at least 1 year. The total duration of treatment ranged from 8 to 76 weeks (mean, 52 ± 6 weeks) for a total dose of 196 to 1,408 MU (mean, 492 ± 116 MU). Five patients were classified as sustained responders based upon the lack of Hepatitis C Virus RNA in serum and the presence of normal ALT levels 6 months after stopping therapy. The remaining 5 patients remained Hepatitis C Virus RNA positive and had abnormal ALT levels 6 months after stopping interferon.

The 5 patients with a sustained virological response were on average younger and had a shorter duration of disease than the nonresponders (Table 1). Four of the 5 responders had Hepatitis C Virus genotypes 2 or 3, whereas the nonresponders all had genotype 1. The two groups had similar pretreatment levels of serum aminotransferases and similar HAI scores, but the two patients with cirrhosis were both nonresponders.

The 10 patients were started on treatment between July 1984 and July 1986 and completed treatment between July 1985 and August 1987. All 10 patients have been followed subsequently. One patient (a nonresponder) died of a suspected intracerebral hemorrhage 6.4 years after starting therapy while in the process of undergoing evaluation for liver transplantation; the remaining patients were seen and evaluated in 1996 and 1997. The duration of clinical follow-up from the time of initiation of therapy ranged from 6 to 13 years and averaged 10 years.

At the time of the final follow-up, the 5 sustained responders had no symptoms or physical findings of liver disease. Serum ALT levels were normal in 4 and were minimally elevated in 1 (ALT = 45; normal < 41 U/L). All 5 had no detectable Hepatitis C Virus RNA (<100 copies/mL) in serum, but all 5 were still reactive for anti-Hepatitis C Virus. Liver biopsies, done 10 to 11 years after starting therapy, revealed marked improvement in both the inflammatory and fibrotic components of the HAI scores..

Four patients had mild degrees of inflammation, but none had fibrosis above what was considered a normal or insignificant amount. Results of testing liver tissue samples for Hepatitis C Virus RNA by PCR were negative in all 5 patients. An example of pretreatment, end-of-treatment, and 10-year follow-up liver histological findings on a sustained responder is shown in

Fig. 2. Light microscopic findings on liver biopsies from a patient (number 5) who had a sustained response to interferon- therapy. (A) Before therapy: HAI score = 16. (B) At the end of 12 months of therapy: HAI score = 4. (C) Ten years after starting therapy: HAI score = 0. (Hematoxylin-eosin; original magnification ×20.)

The 5 nonresponder patients continued to have symptoms of chronic hepatitis as well as abnormal levels of serum aminotransferases and Hepatitis C Virus RNA in serum. At the time of the final follow-up clinical evaluation (6.4 to 12.6 years after initiation of therapy), serum Hepatitis C Virus RNA was detectable at titers between 1,500 and greater than 5 million copies/mL. Liver biopsies done 5 to 11 years after starting therapy revealed no change in average total HAI scores compared with pretreatment biopsies (mean: 13.2 pretreatment, 13.2 posttreatment). Fibrosis scores increased in two of the three without preexisting cirrhosis.

An example of pretreatment, end-of-treatment, and 10-year follow-up liver histological findings on a nonresponder is shown in

Fig. 4. Light microscopic findings on liver biopsies from a patient (number 9) who had a transient response to therapy with subsequent relapse. (A) Before therapy: HAI score = 17. (B) At the end of 12 months of therapy: HAI score = 10. (C) Eleven years after starting therapy: HAI score = 15. (Hematoxylin-eosin; original magnification ×20.)

One patient with cirrhosis underwent liver transplantation for hepatocellular carcinoma 5 years after a 1-year course of therapy. During the 7 years since transplantation, he has had no evidence of cancer recurrence and has had normal ALT levels despite the presence of Hepatitis C Virus RNA in serum. A second nonresponder developed decompensated liver disease and died of an intracerebral bleed while under evaluation for liver transplantation.

DISCUSSION

The current analysis provides a 10-year follow-up of the first cohort of patients with chronic hepatitis C who received interferon- for this disease. Five patients had a 6-month posttreatment biochemical and virological response. During the ensuing 10 years, all 5 were without clinical or virological evidence of disease. These findings suggested that interferon- therapy eradicated Hepatitis C Virus infection and that these 5 patients were cured of the chronic hepatitis C. Liver biopsies were done on all 5 patients more than 10 years after starting therapy and showed a resolution of hepatic fibrosis in all, suggesting that with eradication of the chronic viral infection, liver fibrosis can regress. Liver biopsy tissue was normal in 1 patient, but showed nonspecific inflammatory changes in 4 others. The presence of occasional inflammatory cells in liver biopsies and the presence of a slightly elevated ALT level in 1 patient may be indicative of low levels of ongoing hepatitis in these patients. However, it is important that sensitive PCR assays for Hepatitis C Virus RNA revealed no evidence for residual virus in biopsy tissue. These results are quite different from similar studies done on patients with hepatitis B who respond to interferon treatment, who typically do not have resolution of fibrosis and who continue to have a low-grade hepatitis and hepatitis B virus DNA detectable by PCR in liver biopsies despite the loss of hepatitis B virus DNA and even hepatitis B surface antigen with treatment.22

The long-term beneficial outcome of patients with chronic hepatitis C who have a biochemical and virological response to interferon- has been documented in a number of studies from Europe and Asia.23-26 In these studies, which provided an average follow-up of 2 to 5 years, relapses in disease were uncommon among patients who remained Hepatitis C Virus RNA negative after therapy, the reported rate of relapses ranging from 4% to 6% in the two largest series. 25,26 Possible explanations for the occurrence of late relapses despite lack of Hepatitis C Virus RNA 6 months after stopping therapy include reinfection caused by repeated exposure to hepatitis C, intermittent viremia combined with infrequency of testing, and unreliability of the PCR assay used to detect viral RNA at 6 months. Indeed, in the current study, one patient was initially thought to be a virological responder. During follow-up, however, he developed hepatocellular carcinoma, required liver transplantation, and was subsequently found to be Hepatitis C Virus RNA positive. Testing of stored serum samples by sensitive assays showed that this patient had been Hepatitis C Virus RNA positive shortly after stopping therapy, but that levels were persistently low, in the range of 100 to 1.000 Hepatitis C Virus copies/mL, and thus not detected using early generations of PCR assays. These observations suggested that the standard definition of a response to interferon- therapy (i.e., lack of Hepatitis C Virus RNA in serum by a sensitive technique at least 6 months after stopping therapy) is a reliable standard to use in evaluating antiviral therapies of hepatitis C and may indicate cure of disease.

This pilot study was somewhat unusual for the high rate of response to interferon-. In recent controlled trials in chronic hepatitis C, the response rates to interferon- have usually ranged from 6% to 14%,27-30 significantly below the 50% rate in this cohort. The reasons for these differences are probably in the selection of patients in this study, who were generally young, without evidence of other disease. Furthermore, most of the responders in this study happened to have only mild or moderate degrees of fibrosis on liver biopsy and Hepatitis C Virus genotypes 2 and 3, factors that are known to be associated with high rates of response to interferon. 12,18,30

The 5 nonresponders in this study were also distinctive in demonstrating evidence of progressive disease, the two with preexisting cirrhosis being referred for liver transplantation and the rest having continued symptoms with restrictions on physical activities and progression of fibrosis on liver biopsy. Studies of the natural history of hepatitis C starting with the onset of infection suggest that the progression of this disease is slow and insidious.31 However, reports on the course of hepatitis C in patients seen at referral centers indicate that this disease frequently leads to end-stage liver disease within a few years of evaluation.32 In this initial study of interferon- therapy for non-A, non-B hepatitis, patients were selected on the basis of having symptoms of disease, high levels of serum aminotransferases, and significant degrees of inflammation, necrosis, and fibrosis on liver biopsyfeatures that may predict more rapid progression of disease.

In summary, 5 of 10 patients with chronic hepatitis C who were treated with interferon- between 1984 and 1987 had a virological response and all 5 demonstrated a sustained clinical, biochemical, and virological resolution of disease. In all 5, hepatic fibrosis detected on pretreatment liver biopsies had resolved. Mild hepatic inflammation found on liver biopsy in some responders was of unclear significance but did not appear to be associated with progression of liver disease.

References

Footnotes

Acknowledgement: The authors thank the many nurses, technicians and physicians who were responsible for caring for the patients in this study, including Jeanne Waggoner, John Vergalla, Kevin Mullen, D. Brian Jones, Vinod Rustgi, Adrian Di Bisceglie, Marion Peters, E. Anthony Jones, Harvey J. Alter, and Michiko Shindo. The authors also thank Dr. Peter Simmonds for serotyping of anti-Hepatitis C Virus and Dr. J. W. Shih for performing Amplicor assays.

Abbreviations: Hepatitis C Virus, hepatitis C virus; anti-Hepatitis C Virus, antibody to Hepatitis C Virus; NIH, National Institutes of Health; RT, reverse transcriptase; PCR, polymerase chain reaction; HAI, histology activity index; ALT, alanine aminotransferase.

Received April 2, 1998; accepted June 1, 1998.

Address reprint requests to: Dr. Daryl Lau, Liver Diseases Section, NIDDK, Building 10, Room 9B16, NIH, Bethesda, MD 20892. Fax: (301) 402-0491; e-mail: daryll@bdg10.niddk.nih.gov.

0270-9139/98/2804-0030$3.00/0