Decrease in Serum Hepatitis C Viral RNA during Alpha-interferon Therapy for Chronic Hepatitis C Michiko Shindo, MD, PhD; Adrian M. Di Bisceglie, MD; Ling Cheung, BA; J. Wai-Kuo Shih, PhD; Karen Cristiano, PhD; Stephen M. Feinstone, MD; and Jay H. Hoofnagle, MD
• Objective: To assess the effect of alpha-interferon therapy on hepatitis C viral RNA in serum of patients with chronic hepatitis C. • Design: Retrospective testing for hepatitis C viral (HCV) RNA and antibody to the hepatitis C virus (antiHCV) of stored serum samples from a randomized, double-blind, placebo-controlled trial of alphainterferon therapy. • Setting: Warren Grant Magnuson Clinical Center of the National Institutes of Health, a tertiary referral center. • Patients: Forty-one patients with chronic non-A, non-B hepatitis were entered in this trial. • Interventions: Twenty-one patients were treated with alpha-interferon, and 20 patients were treated with placebo for 6 months. Seventeen placebo recipients were then treated with alpha-interferon for up to 1 year. • Methods: Samples were tested for anti-HCV by enzyme-linked immunosorbent assay. Hepatitis C viral RNA was detected in serum using the polymerase chain reaction. Titers of both antibody and RNA were determined by serial end-point dilution. • Main Results: At entry into the trial, 37 (90%) of 41 patients had anti-HCV and 39 (95%) had HCV RNA in serum. Anti-HCV titers decreased slightly with treatment. Serum levels of HCV RNA decreased in all patients who responded to alpha-interferon therapy with improvements in serum aminotransferases; in 17 of 21 responders (81%; 95% CI, 58% to 95%) HCV RNA became undetectable. In contrast, in only 2 of 16 (12%; CI, 2% to 38%) patients who did not respond to treatment did HCV RNA become undetectable. In 19 patients treated during the preliminary 6-month period with placebo, HCV RNA remained detectable. Finally, in the 11 patients who relapsed when treatment was stopped, HCV RNA reappeared in the serum, but in 4 of 7 patients with a sustained improvement in serum aminotransferases, HCV RNA remained undetectable. • Conclusions: These results indicate that the clinical and serum biochemical response to alpha-interferon in chronic hepatitis C is associated with a loss of detectable HCV genome from serum.
Annals of Internal Medicine. 1991;115:700-704. From the National Institutes of Health, Bethesda, Maryland; and the Food and Drug Administration, Rockville, Maryland. For current author addresses, see end of text. 700
1 herapy with alpha-interferon has been found to have beneficial effects in chronic non-A, non-B (type C) hepatitis, as shown by a rapid decrease in serum aminotransferase levels into the normal or near-normal range in approximately 50% of cases (1-3). Liver histologic findings, particularly the degree of hepatocellular necrosis and inflammation, also improve with treatment. The mechanisms of the beneficial actions of alpha-interferon in chronic hepatitis C are unknown; it is surmised that interferon inhibits replication of the hepatitis C virus (HCV), which results in amelioration of disease. It was only after most studies of alpha-interferon therapy were begun that the viral agent of hepatitis C was identified. Subsequently, the genome of HCV was cloned and its nucleotide sequence determined (4), which led to development of tests for antibody to HCV (anti-HCV) using recombinant antigen (5-10). Most recently, the highly sensitive technique of polymerase chain reaction was applied successfully to the detection of HCV RNA in serum and liver (11-15). The availability of these new serologic assays led us to reassess a study of the effects of alpha-interferon therapy in chronic hepatitis C (3). The purpose was to evaluate whether alpha-interferon therapy led to inhibition or eradication of HCV and to analyze whether monitoring for the presence of HCV genome in serum could help to predict which patients would respond to therapy and to explain why some patients relapse after treatment is discontinued.
Patients and Methods From 1987 to 1988, 41 patients with chronic non-A, non-B hepatitis were entered in a randomized, double-blind placebo controlled trial of recombinant human alpha-interferon (alfa-2b: "Intron-A," Schering Corporation, Kenilworth, New Jersey). Twenty-one patients were given alpha-interferon at a dose of 2 million units subcutaneously three times a week for 6 months. Twenty patients received placebo on a similar schedule. At the end of 6 months, patients who received placebo were offered therapy with alpha-interferon in a similar dose for 12 months. The details of the patient sample, their treatment, and outcome have been previously reported (3). Initial and final serum samples were tested for anti-HCV by enzyme linked immunoassay (6), and titers of antibody were assessed by testing tenfold dilutions of serum. Initial serum samples were also tested for HCV RNA by polymerase chain reaction. In patients with positive results, follow-up specimens were tested at the end of treatment and 6 months later. Hepatitic C viral RNA was detected in serum samples using the polymerase chain reaction as recently described (11). Viral particles in 50 fjuL of serum were precipitated by the addition of 0.25 mL of 8% polyethylene glycol, overnight incubation at 4 °C, and pelleting for 20 minutes at 1500 g in microfuge tubes. Nucleic acids were extracted from the pellet in guanidiumthiocyanate buffer (4.4 M guanidium-thiocyanate, 25 mM sodium citrate [pH, 7.9], 0.5% sarkosyl, and 0.7% 2-mer-
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Figure 1. Changes in serum alanine aminotransferase and hepatitis C viral RNA. The initial and final serum alanine aminotransferase (ALT) levels ( ) and reciprocal HCV RNA titers ( ) in the 20 patients treated with alpha-interferon and in the 19 patients treated with placebo injections for 6 months. The decreases in ALT and HCV RNA levels during interferon treatment were statistically significant. The dotted area indicates the normal range.
captoethanol) by phenol-chloroform extraction and ethanol precipitation. Precipitates were washed in 70% ethanol, air dried, and dissolved in 80 /xL of RNase-free water containing 200 units/mL RNAsin (Promega, Madison, Wisconsin). A 10-ttL aliquot of this solution was added to the polymerase chain reaction mixture. The polymerase chain reaction was done by combining the initial reverse transcription step with the first reaction and by using "nested1' primers in a second polymerase chain reaction round to improve both sensitivity and specificity. The polymerase chain reaction primers were synthesized from the highly conserved 5' noncoding region of the HCV genome (16): 5'GGCGACACTCCACCATAGATC-3' (sense; nucleotide numbers 1 to 21) and 5'-GGTGCACGGTCTACGAGACCT-3' (antisense; nucleotide numbers 304 to 324) for the first round, and 5'-CTGTGAGGAACTACTGTCTTC-3' (sense; nucleotide numbers 28 to 48) and 5'-CCCTATCAGGCAGTACCACAA-3' (antisense; nucleotide numbers 264 to 284) for the second round. The reaction mixture consisted of 10 fiL of extracted RNA solution, together with 0.2 /xM of each primer, 60 /JM of the four deoxynucleotides, 2.5 units of Taq DNA polymerase (Perkin Elmer Cetus, Norwalk, Connecticut), 2 units of AMV reverse transcriptase (Promega) and Taq buffer (10 mM Tris-
HC1 (pH, 8.3), 50 mM KC1, 1.5 mM MgCl2, and 0.01% gelatin). The thermocycler was programmed to first incubate the samples for 30 min at 42 °C for the initial reverse transcription step and then to carry out 25 cycles consisting of 95 °C for 30 s, 55 °C for 1 min, and 72 °C for 1 min. For the second reaction, 10 fxL was removed from the first reaction and added to a tube containing the second set of primers, deoxynucleotides, Taq polymerase, and Taq buffer as in the first reaction, but without reverse transcriptase and omitting the initial 30 min incubation at 42 °C. The polymerase chain reaction was then carried out for another 35 cycles as described for the first reaction. The polymerase chain reaction products were analyzed by electrophoresis in 1% agarose gel containing 0.5 /xg/mL ethidium bromide, and DNA was visualized by fluorescence under ultraviolet light. The size of the expected HCV DNA product was 257 bp. For each assay, known positive and negative serum samples were analyzed as controls in parallel with test samples. The relative sensitivity of the assay was determined by comparing HCV RNA detectability in diluted sample extracts to chimpanzee infectivity titers of a human plasma sample known to transmit hepatitis (17, 18). End-point titers of HCV RNA were estimated by testing serial tenfold dilutions of nucleic acids extracted from 50 xtL of serum. Throughout this study, we tried to avoid contamination by strictly applying the measures of Kwok and Higuchi (19); all samples were tested under code. Statistical analyses included comparisons of geometric endpoint titers by paired and unpaired Student /-tests, comparison of group frequencies by chi-square analysis, and computation of 95% confidence intervals (CIs) by the two-sided Fisher exact test. Values are expressed as mean ± SD.
Results Initial Testing At the start of the trial, 37 (90%) of the 41 patients had anti-HCV detectable by first-generation enzymelinked immunoassay and 39 (95%) had HCV RNA in serum as detected by polymerase chain reaction. The two HCV RNA-negative patients both had anti-HCV. Therefore, all 41 patients had serologic evidence of HCV infection: thirty-five had HCV RNA and antiHCV, four had HCV RNA without anti-HCV, and two had anti-HCV without detectable HCV RNA in serum. The initial reciprocal titers of HCV RNA ranged from 101 to 104 per 50 /xL of serum. Simultaneous testing of a standardized inoculum used in chimpanzee transmission studies ( " H " strain) yielded a dilution titer of 107
Figure 2. Changes in hepatitis C viral RNA. The reciprocal titers of HCV RNA in serum of 41 patients with chronic hepatitis C before and after treatment with alpha-interferon or placebo are shown. Left panel. Nine patients who responded to alpha-interferon therapy with a decrease of aminotransferase levels into the normal range. Middle panel. Eleven patients whose aminotransferase levels remained abnormal despite interferon treatment. Right panel. Nineteen placebo recipients. The numbers in boxes indicate the number of patients at each titer. 1 November 1991 • Annals of Internal Medicine • Volume 115 • Number 9
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Figure 3. Changes of hepatitis C viral RNA titer with relapses. The reciprocal titer of HCV RNA in serum of 18 patients with chronic hepatitis C who responded to alpha-interferon therapy with a decrease of aminotransferase levels into the normal range are shown. Eleven patients {left panel) relapsed when interferon was stopped and all 11 were HCV RNA positive 6 months later. Seven patients {right panel) had not relapsed 6 months after interferon was withdrawn, and four remained HCV RNA negative. Patients who received interferon for 6 months ( ); patients who received interferon for 12 months (—).
per mL, which corresponded to the infectivity titer of that sample (18). The interferon-treated and the placebo-treated groups were similar in the percentage of patients with HCV RNA (95% compared with 95%) and in the geometric mean titer of HCV RNA in serum (1: 2733 ± 4321 compared with 1:1426 ± 3050, respectively). There were no correlations found between HCV RNA titer and other clinical serum biochemical or serologic findings in these 41 patients (data not shown).
Hepatitis C Virus RNA during Treatment Twenty patients with HCV RNA in serum were randomized to receive a 6-month course of alpha-interferon. During treatment, serum aminotransferases levels fell by an average of 50% in this group (3.42 ± 3.08 jx,kat/L to 1.54 ± 1.19 ^kat/L; P< 0.05) (Figure 1). Concurrently, titers of HCV RNA decreased from a geometric mean of 1:2733 ± 4321 to 1:217 ± 403 at the end of treatment {P < 0.05). Nine of these 20 patients
Figure 4. Hepatitis C viral RNA levels during alpha-interferon treatment and relapse. Course of a patient who responded to interferon therapy with an improvement in serum aminotransferase levels, but who relapsed when interferon was withdrawn. Hepatitis C viral RNA, which was detectable before therapy, was no longer present at the end of therapy, but reappeared when the hepatitis relapsed. 702
(45%) were considered "responders" in that their serum aminotransferases fell into and remained in the normal range during therapy. In these nine patients, HCV RNA titers decreased in eight and became undetectable in six (Figure 2). The geometric mean titer of HCV RNA in these nine patients decreased from 1:3480 ± 4899 to 1:11 ± 33 (P < 0.05). In contrast, serum titers of HCV RNA did not decrease significantly among the 11 patients whose serum aminotransferases did not become and remain normal, although two of these patients became HCV RNA negative. Nineteen patients with HCV RNA in serum were randomized to receive a 6-month course of placebo injections. During placebo treatment, serum aminotransferase levels remained elevated in all patients and the mean values did not change significantly (4.72 ± 2.61 /ikat/L initially and 3.90 ± 2.90 /xkat/L at 6 months; see Figure 1). Overall, the geometric mean titer of HCV RNA decreased in these 19 placebo recipients from 1:1426 ± 3050 to 1:313 ± 424, but this change was not statistically significant. Titers of HCV RNA in serum decreased in 10 patients, remained unchanged in 8, and increased in 1 patient, but no patient became negative for HCV RNA {see Figure 2). Seventeen placebo recipients with HCV RNA in serum were later "crossed over" and treated with alphainterferon for up to 1 year. In this group, serum aminotransferase levels fell into the normal range and remained normal in 12 patients. Serum levels of HCV RNA decreased in all 12 patients, becoming undetectable in 11 patients out of these 12 (data not shown). In the five patients who did not respond to therapy, serum HCV RNA remained detectable. Thus, overall HCV RNA became undetectable in 17 of 21 (81%; 95% CI, 58% to 95%) responders and in only 2 of 16 (12%; CI, 2% to 38%) nonresponders to alpha-interferon therapy compared with none of 19 placebo recipients {P < 0.01).
Hepatitis C Viral RNA in Follow-up Follow-up serum samples were available from 18 patients who had responded to interferon treatment with a
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decline in serum aminotransferase levels into the normal range. Eleven (61%) patients relapsed when therapy was discontinued as marked by the return of elevated serum aminotransferase levels within 1 to 2 months of treatment. Six months after discontinuing alpha-interferon therapy, all 11 patients who relapsed had detectable HCV RNA in serum (Figure 3). Among seven patients who did not relapse, four remained HCV RNA negative. Further, two of the three patients who were HCV RNA positive despite normal aminotransferase levels at 6 months later relapsed, developing elevations in serum aminotransferases 8 months and 2 years after therapy was stopped. Examples of patients who did and did not relapse after a course of alpha-interferon therapy are shown in Figures 4 and 5.
Anti-HCV Titers during Therapy Initially, 37 patients had HCV antibodies. The reciprocal titers of anti-HCV ranged from 10° to 103 (geometric mean titer, 1:30). Among 19 treated patients who had detectable antibody, the mean titer fell from 1:34 to 1:15 after 6 months of alpha-interferon therapy (P < 0.05), and one patient became anti-HCV negative. Among 18 placebo recipients with HCV antibodies, the mean titer fell from 1:28 to 1:17 at the end of placebo treatment, a difference that was not statistically significant. No placebo recipient became anti-HCV negative. Discussion The initial studies of alpha-interferon therapy for chronic hepatitis C were initiated before the identification of HCV, and relied on nonviral indices of response. Nevertheless, several clinical trials showed that interferon therapy led to rapid decreases in serum aminotransferase levels and improvements in hepatic histologic findings (1-3). The subsequent identification of the HCV has allowed a re-evaluation of those studies to examine effects that therapy had on viral markers. We evaluated viral markers among 41 patients entered in a randomized, placebo-controlled trial of alpha-interferon conducted in 1987 and 1988. Anti-HCV was detected in 90% of patients. Titers of this antibody decreased significantly in patients receiving alpha-interferon, but the decrease was slight and only one patient became anti-HCV negative. Further,
anti-HCV titers were low overall and decreased slightly in placebo recipients as well. Thus, a response to interferon could not be reliably monitored or predicted by anti-HCV titers. Change in antibody titers, however, may be delayed, and ultimately loss of anti-HCV may reflect long-term loss of replicating virus (6). Hepatitis C viral RNA was detected in serum of 95% of patients when they entered this controlled trial. Strikingly, serum HCV RNA disappeared in most patients (81%) who were considered responders to alpha-interferon therapy, but the viral genome remained present in most patients who did not respond and in all patients who received placebo. Even in patients who were considered nonresponders, the titers of HCV RNA decreased significantly. These findings suggest that alphainterferon acts by inhibiting viral replication that results in an amelioration of the disease. In some cases, the inhibition appeared to be complete so that interferon therapy could be discontinued without return of the virus. In many patients, however, the inhibition was not complete, and HCV RNA became detectable again within a few months after therapy was discontinued. Although the disappearance of HCV RNA from serum correlated with a response to alpha-interferon therapy, it did not help in predicting responsiveness to alpha-interferon or whether the response would be sustained when interferon was withdrawn. When therapy was discontinued, however, the return of HCV RNA in the serum usually predicted eventual relapse in disease and, in several cases, preceded development of elevated aminotransferases. Interestingly, a higher rate of loss of HCV RNA and a higher rate of sustained response to alpha-interferon was found with a 12-month course of therapy than with a 6-month course, suggesting that longer periods of therapy are more effective in inducing a permanent suppression of replication. This increase in sustained responses with more prolonged courses of interferon will need to be confirmed in future controlled trials that compare different regimens of therapy. Testing for HCV in serum can be helpful in monitoring therapy for chronic hepatitis C and shows that alpha-interferon therapy leads to suppression of viral replication. The disappearance of detectable HCV RNA, however, does not provide absolute evidence of longterm clearance of virus. Use of more sensitive assays for HCV RNA or testing for HCV RNA on liver tissue may ultimately provide the necessary serologic markers
Figure 5. Hepatitis C viral RNA levels during and after alpha-interferon treatment. Course of a patient who had a sustained response to alpha-interferon therapy after a 6-month period of placebo treatment. Hepatitis C viral RNA, which was detected during the placebo treatment, was not detectable during and after interferon therapy. 1 November 1991 • Annals of Internal Medicine • Volume 115 • Number 9
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to identify clearance of this virus and to predict longterm remission in disease. Acknowledgments: The authors thank Ms. Jeanne Waggoner for technical assistance, Ms. Yoon Park and Linda Murray for nursing assistance, and Dr. Jay Everhart for help in statistical analysis. Requests for Reprints: Michiko Shindo, MD, PhD, Liver Diseases Section, Building 10, Room 4D52, National Institutes of Health, Bethesda, MD 20892. Current Author Addresses: Drs. Shindo, Di Bisceglie, and Hoofnagle: Liver Diseases Section, Building 10, Room 4D52, National Institutes of Health, Bethesda, MD 20892. Ms. Cheung and Dr. Shih: Department of Transfusion Transmitted Virus Research, Building 10, Room ID46, National Institutes of Health, Bethesda, MD 20892. Drs. Cristiano and Feinstone: Center for Biologic Evaluation and Research, Hepatitis Research Laboratory, Food and Drug Administration, Rockville, MD 20895. References 1. Hoofnagle JH, Mullen KD, Jones DB, Di Bisceglie A, Peters M, Waggoner JG, et al. Treatment of chronic non-A, non-B hepatitis with recombinant human alpha-interferon. A preliminary report. N Engl J Med. 1986;315:1575-8. 2. Davis GL, Balart LA, Schiff ER, Lindsay K, Bodenheimer HC Jr, Perrillo RP, et al. Treatment of chronic hepatitis C with recombinant interferon alfa. N Engl J Med. 1989;321:1501-6. 3. Di Bisceglie AM, Martin P, Kassianides C, Lisker-Melman M, Murray L, Waggoner J, et al. Recombinant interferon alfa therapy for chronic hepatitis C. A randomized, double-blind, placebo-controlled trial. N Engl J Med. 1989;321:1506-10. 4. Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science. 1989;244:359-62. 5. Kuo G, Choo QL, Alter HJ, Gitnick GL, Redecker AG, Purcell RH, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science. 1989;244:362-4. 6. Alter HJ, Purcell RH, Shih JW, Melpolder JC, Houghton M, Choo QL, et al. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N Engl J Med. 1989;32:1494-500.
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7. van der Poel CL, Reesink HW, Lelie PN, Leentvaar-Kuypers A, Choo QL, Kuo G, et al. Anti-hepatitis C antibodies and non-A, non-B post-transfusion hepatitis in The Netherlands [Letter]. Lancet. 1989;2:297-8. 8. Esteban JI, Esteban R, Viladomiu L, Lopez-Talavera JC, Gonzalez A, Hernandez JM, et al. Hepatitis C virus antibodies among risk groups in Spain. Lancet. 1989;2:294-7. 9. Kuhnl P, Seidel S, Stangel W, Beyer J, Sibrowski W, Flik J. Antibody to hepatitis C virus in German blood donors [Letter]. Lancet. 1989;2:324. 10. Roggendorf M, Deinhardt F, Rasshofer R, Eberle J, Hopf U, Moller B, et al. Antibodies to hepatitis C virus [Letter]. Lancet 1989,2: 324-5. 11. Weiner AJ, Kuo G, Bradley DW, Bonino F, Saracco G, Lee C, et al. Detection of hepatitis C viral sequences in non-A, non-B hepatitis. Lancet. 1990;335:1-3. 12. Ulrich PP, Romeo JM, Lane PK, Kelly I, Daniel LJ, Vyas GN. Detection, semiquantitation, and genetic variation in hepatitis C virus sequences amplified from the plasma of blood donors with elevated alanine aminotransferases. J Clin Invest. 1990;86:1609-14. 13. Kato N, Yokosuka O, Omata M, Hosoda K, Ohto M. Detection of hepatitis C virus ribonucleic acid in the serum by amplification with polymerase chain reaction. J Clin Invest. 1990;86:1764-7. 14. Garson JA, Tedder RS, Briggs M, Tuke P, Glazebrook JA, Trute A, et al. Detection of hepatitis C viral sequences in blood donations by "nested" polymerase chain reaction and prediction of infectivity. Lancet. 1990;335:1419-22. 15. Kanai K, Iwata K, Nakao K, Kako M, Okamoto H. Suppression of hepatitis C virus RNA by interferon-alpha [Letter]. Lancet 1990; 336:245. 16. Okamoto H, Okuda S, Sugiyama Y, Yotsumoto S, Tanaka T, Yoshizawa H, et al. The 5'-terminal sequence of the hepatitis C virus genome. Jpn J Exp Med. 1990;60:167-77. 17. Feinstone SM, Alter HJ, Dienes HP, Shimizu Y, Popper H, Blackmore D, et al. Non-A, non-B hepatitis in chimpanzees and marmosets. J Infect Dis. 1981;144:588-98. 18. Cristiano K, Baker B, Di Bisceglie A, Feinstone S. Detection of hepatitis C viral RNA by the polymerase chain reaction. In: Hollinger FB, Lemon SM, Margolis HS; eds. Viral Hepatitis and Liver Disease. Baltimore: Williams and Wilkins; 1991: [In press]. 19. Kwok S, Higuchi R. Avoiding false positives with PCR. Nature. 1989;339:237-8.
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• Objective: To assess the effect of alpha-interferon therapy on hepatitis C viral RNA in serum of patients with chronic hepatitis C. • Design: Retrospective testing for hepatitis C viral (HCV) RNA and antibody to the hepatitis C virus (antiHCV) of stored serum samples from a randomized, double-blind, placebo-controlled trial of alphainterferon therapy. • Setting: Warren Grant Magnuson Clinical Center of the National Institutes of Health, a tertiary referral center. • Patients: Forty-one patients with chronic non-A, non-B hepatitis were entered in this trial. • Interventions: Twenty-one patients were treated with alpha-interferon, and 20 patients were treated with placebo for 6 months. Seventeen placebo recipients were then treated with alpha-interferon for up to 1 year. • Methods: Samples were tested for anti-HCV by enzyme-linked immunosorbent assay. Hepatitis C viral RNA was detected in serum using the polymerase chain reaction. Titers of both antibody and RNA were determined by serial end-point dilution. • Main Results: At entry into the trial, 37 (90%) of 41 patients had anti-HCV and 39 (95%) had HCV RNA in serum. Anti-HCV titers decreased slightly with treatment. Serum levels of HCV RNA decreased in all patients who responded to alpha-interferon therapy with improvements in serum aminotransferases; in 17 of 21 responders (81%; 95% CI, 58% to 95%) HCV RNA became undetectable. In contrast, in only 2 of 16 (12%; CI, 2% to 38%) patients who did not respond to treatment did HCV RNA become undetectable. In 19 patients treated during the preliminary 6-month period with placebo, HCV RNA remained detectable. Finally, in the 11 patients who relapsed when treatment was stopped, HCV RNA reappeared in the serum, but in 4 of 7 patients with a sustained improvement in serum aminotransferases, HCV RNA remained undetectable. • Conclusions: These results indicate that the clinical and serum biochemical response to alpha-interferon in chronic hepatitis C is associated with a loss of detectable HCV genome from serum.
Annals of Internal Medicine. 1991;115:700-704. From the National Institutes of Health, Bethesda, Maryland; and the Food and Drug Administration, Rockville, Maryland. For current author addresses, see end of text. 700
1 herapy with alpha-interferon has been found to have beneficial effects in chronic non-A, non-B (type C) hepatitis, as shown by a rapid decrease in serum aminotransferase levels into the normal or near-normal range in approximately 50% of cases (1-3). Liver histologic findings, particularly the degree of hepatocellular necrosis and inflammation, also improve with treatment. The mechanisms of the beneficial actions of alpha-interferon in chronic hepatitis C are unknown; it is surmised that interferon inhibits replication of the hepatitis C virus (HCV), which results in amelioration of disease. It was only after most studies of alpha-interferon therapy were begun that the viral agent of hepatitis C was identified. Subsequently, the genome of HCV was cloned and its nucleotide sequence determined (4), which led to development of tests for antibody to HCV (anti-HCV) using recombinant antigen (5-10). Most recently, the highly sensitive technique of polymerase chain reaction was applied successfully to the detection of HCV RNA in serum and liver (11-15). The availability of these new serologic assays led us to reassess a study of the effects of alpha-interferon therapy in chronic hepatitis C (3). The purpose was to evaluate whether alpha-interferon therapy led to inhibition or eradication of HCV and to analyze whether monitoring for the presence of HCV genome in serum could help to predict which patients would respond to therapy and to explain why some patients relapse after treatment is discontinued.
Patients and Methods From 1987 to 1988, 41 patients with chronic non-A, non-B hepatitis were entered in a randomized, double-blind placebo controlled trial of recombinant human alpha-interferon (alfa-2b: "Intron-A," Schering Corporation, Kenilworth, New Jersey). Twenty-one patients were given alpha-interferon at a dose of 2 million units subcutaneously three times a week for 6 months. Twenty patients received placebo on a similar schedule. At the end of 6 months, patients who received placebo were offered therapy with alpha-interferon in a similar dose for 12 months. The details of the patient sample, their treatment, and outcome have been previously reported (3). Initial and final serum samples were tested for anti-HCV by enzyme linked immunoassay (6), and titers of antibody were assessed by testing tenfold dilutions of serum. Initial serum samples were also tested for HCV RNA by polymerase chain reaction. In patients with positive results, follow-up specimens were tested at the end of treatment and 6 months later. Hepatitic C viral RNA was detected in serum samples using the polymerase chain reaction as recently described (11). Viral particles in 50 fjuL of serum were precipitated by the addition of 0.25 mL of 8% polyethylene glycol, overnight incubation at 4 °C, and pelleting for 20 minutes at 1500 g in microfuge tubes. Nucleic acids were extracted from the pellet in guanidiumthiocyanate buffer (4.4 M guanidium-thiocyanate, 25 mM sodium citrate [pH, 7.9], 0.5% sarkosyl, and 0.7% 2-mer-
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Figure 1. Changes in serum alanine aminotransferase and hepatitis C viral RNA. The initial and final serum alanine aminotransferase (ALT) levels ( ) and reciprocal HCV RNA titers ( ) in the 20 patients treated with alpha-interferon and in the 19 patients treated with placebo injections for 6 months. The decreases in ALT and HCV RNA levels during interferon treatment were statistically significant. The dotted area indicates the normal range.
captoethanol) by phenol-chloroform extraction and ethanol precipitation. Precipitates were washed in 70% ethanol, air dried, and dissolved in 80 /xL of RNase-free water containing 200 units/mL RNAsin (Promega, Madison, Wisconsin). A 10-ttL aliquot of this solution was added to the polymerase chain reaction mixture. The polymerase chain reaction was done by combining the initial reverse transcription step with the first reaction and by using "nested1' primers in a second polymerase chain reaction round to improve both sensitivity and specificity. The polymerase chain reaction primers were synthesized from the highly conserved 5' noncoding region of the HCV genome (16): 5'GGCGACACTCCACCATAGATC-3' (sense; nucleotide numbers 1 to 21) and 5'-GGTGCACGGTCTACGAGACCT-3' (antisense; nucleotide numbers 304 to 324) for the first round, and 5'-CTGTGAGGAACTACTGTCTTC-3' (sense; nucleotide numbers 28 to 48) and 5'-CCCTATCAGGCAGTACCACAA-3' (antisense; nucleotide numbers 264 to 284) for the second round. The reaction mixture consisted of 10 fiL of extracted RNA solution, together with 0.2 /xM of each primer, 60 /JM of the four deoxynucleotides, 2.5 units of Taq DNA polymerase (Perkin Elmer Cetus, Norwalk, Connecticut), 2 units of AMV reverse transcriptase (Promega) and Taq buffer (10 mM Tris-
HC1 (pH, 8.3), 50 mM KC1, 1.5 mM MgCl2, and 0.01% gelatin). The thermocycler was programmed to first incubate the samples for 30 min at 42 °C for the initial reverse transcription step and then to carry out 25 cycles consisting of 95 °C for 30 s, 55 °C for 1 min, and 72 °C for 1 min. For the second reaction, 10 fxL was removed from the first reaction and added to a tube containing the second set of primers, deoxynucleotides, Taq polymerase, and Taq buffer as in the first reaction, but without reverse transcriptase and omitting the initial 30 min incubation at 42 °C. The polymerase chain reaction was then carried out for another 35 cycles as described for the first reaction. The polymerase chain reaction products were analyzed by electrophoresis in 1% agarose gel containing 0.5 /xg/mL ethidium bromide, and DNA was visualized by fluorescence under ultraviolet light. The size of the expected HCV DNA product was 257 bp. For each assay, known positive and negative serum samples were analyzed as controls in parallel with test samples. The relative sensitivity of the assay was determined by comparing HCV RNA detectability in diluted sample extracts to chimpanzee infectivity titers of a human plasma sample known to transmit hepatitis (17, 18). End-point titers of HCV RNA were estimated by testing serial tenfold dilutions of nucleic acids extracted from 50 xtL of serum. Throughout this study, we tried to avoid contamination by strictly applying the measures of Kwok and Higuchi (19); all samples were tested under code. Statistical analyses included comparisons of geometric endpoint titers by paired and unpaired Student /-tests, comparison of group frequencies by chi-square analysis, and computation of 95% confidence intervals (CIs) by the two-sided Fisher exact test. Values are expressed as mean ± SD.
Results Initial Testing At the start of the trial, 37 (90%) of the 41 patients had anti-HCV detectable by first-generation enzymelinked immunoassay and 39 (95%) had HCV RNA in serum as detected by polymerase chain reaction. The two HCV RNA-negative patients both had anti-HCV. Therefore, all 41 patients had serologic evidence of HCV infection: thirty-five had HCV RNA and antiHCV, four had HCV RNA without anti-HCV, and two had anti-HCV without detectable HCV RNA in serum. The initial reciprocal titers of HCV RNA ranged from 101 to 104 per 50 /xL of serum. Simultaneous testing of a standardized inoculum used in chimpanzee transmission studies ( " H " strain) yielded a dilution titer of 107
Figure 2. Changes in hepatitis C viral RNA. The reciprocal titers of HCV RNA in serum of 41 patients with chronic hepatitis C before and after treatment with alpha-interferon or placebo are shown. Left panel. Nine patients who responded to alpha-interferon therapy with a decrease of aminotransferase levels into the normal range. Middle panel. Eleven patients whose aminotransferase levels remained abnormal despite interferon treatment. Right panel. Nineteen placebo recipients. The numbers in boxes indicate the number of patients at each titer. 1 November 1991 • Annals of Internal Medicine • Volume 115 • Number 9
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Figure 3. Changes of hepatitis C viral RNA titer with relapses. The reciprocal titer of HCV RNA in serum of 18 patients with chronic hepatitis C who responded to alpha-interferon therapy with a decrease of aminotransferase levels into the normal range are shown. Eleven patients {left panel) relapsed when interferon was stopped and all 11 were HCV RNA positive 6 months later. Seven patients {right panel) had not relapsed 6 months after interferon was withdrawn, and four remained HCV RNA negative. Patients who received interferon for 6 months ( ); patients who received interferon for 12 months (—).
per mL, which corresponded to the infectivity titer of that sample (18). The interferon-treated and the placebo-treated groups were similar in the percentage of patients with HCV RNA (95% compared with 95%) and in the geometric mean titer of HCV RNA in serum (1: 2733 ± 4321 compared with 1:1426 ± 3050, respectively). There were no correlations found between HCV RNA titer and other clinical serum biochemical or serologic findings in these 41 patients (data not shown).
Hepatitis C Virus RNA during Treatment Twenty patients with HCV RNA in serum were randomized to receive a 6-month course of alpha-interferon. During treatment, serum aminotransferases levels fell by an average of 50% in this group (3.42 ± 3.08 jx,kat/L to 1.54 ± 1.19 ^kat/L; P< 0.05) (Figure 1). Concurrently, titers of HCV RNA decreased from a geometric mean of 1:2733 ± 4321 to 1:217 ± 403 at the end of treatment {P < 0.05). Nine of these 20 patients
Figure 4. Hepatitis C viral RNA levels during alpha-interferon treatment and relapse. Course of a patient who responded to interferon therapy with an improvement in serum aminotransferase levels, but who relapsed when interferon was withdrawn. Hepatitis C viral RNA, which was detectable before therapy, was no longer present at the end of therapy, but reappeared when the hepatitis relapsed. 702
(45%) were considered "responders" in that their serum aminotransferases fell into and remained in the normal range during therapy. In these nine patients, HCV RNA titers decreased in eight and became undetectable in six (Figure 2). The geometric mean titer of HCV RNA in these nine patients decreased from 1:3480 ± 4899 to 1:11 ± 33 (P < 0.05). In contrast, serum titers of HCV RNA did not decrease significantly among the 11 patients whose serum aminotransferases did not become and remain normal, although two of these patients became HCV RNA negative. Nineteen patients with HCV RNA in serum were randomized to receive a 6-month course of placebo injections. During placebo treatment, serum aminotransferase levels remained elevated in all patients and the mean values did not change significantly (4.72 ± 2.61 /ikat/L initially and 3.90 ± 2.90 /xkat/L at 6 months; see Figure 1). Overall, the geometric mean titer of HCV RNA decreased in these 19 placebo recipients from 1:1426 ± 3050 to 1:313 ± 424, but this change was not statistically significant. Titers of HCV RNA in serum decreased in 10 patients, remained unchanged in 8, and increased in 1 patient, but no patient became negative for HCV RNA {see Figure 2). Seventeen placebo recipients with HCV RNA in serum were later "crossed over" and treated with alphainterferon for up to 1 year. In this group, serum aminotransferase levels fell into the normal range and remained normal in 12 patients. Serum levels of HCV RNA decreased in all 12 patients, becoming undetectable in 11 patients out of these 12 (data not shown). In the five patients who did not respond to therapy, serum HCV RNA remained detectable. Thus, overall HCV RNA became undetectable in 17 of 21 (81%; 95% CI, 58% to 95%) responders and in only 2 of 16 (12%; CI, 2% to 38%) nonresponders to alpha-interferon therapy compared with none of 19 placebo recipients {P < 0.01).
Hepatitis C Viral RNA in Follow-up Follow-up serum samples were available from 18 patients who had responded to interferon treatment with a
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decline in serum aminotransferase levels into the normal range. Eleven (61%) patients relapsed when therapy was discontinued as marked by the return of elevated serum aminotransferase levels within 1 to 2 months of treatment. Six months after discontinuing alpha-interferon therapy, all 11 patients who relapsed had detectable HCV RNA in serum (Figure 3). Among seven patients who did not relapse, four remained HCV RNA negative. Further, two of the three patients who were HCV RNA positive despite normal aminotransferase levels at 6 months later relapsed, developing elevations in serum aminotransferases 8 months and 2 years after therapy was stopped. Examples of patients who did and did not relapse after a course of alpha-interferon therapy are shown in Figures 4 and 5.
Anti-HCV Titers during Therapy Initially, 37 patients had HCV antibodies. The reciprocal titers of anti-HCV ranged from 10° to 103 (geometric mean titer, 1:30). Among 19 treated patients who had detectable antibody, the mean titer fell from 1:34 to 1:15 after 6 months of alpha-interferon therapy (P < 0.05), and one patient became anti-HCV negative. Among 18 placebo recipients with HCV antibodies, the mean titer fell from 1:28 to 1:17 at the end of placebo treatment, a difference that was not statistically significant. No placebo recipient became anti-HCV negative. Discussion The initial studies of alpha-interferon therapy for chronic hepatitis C were initiated before the identification of HCV, and relied on nonviral indices of response. Nevertheless, several clinical trials showed that interferon therapy led to rapid decreases in serum aminotransferase levels and improvements in hepatic histologic findings (1-3). The subsequent identification of the HCV has allowed a re-evaluation of those studies to examine effects that therapy had on viral markers. We evaluated viral markers among 41 patients entered in a randomized, placebo-controlled trial of alpha-interferon conducted in 1987 and 1988. Anti-HCV was detected in 90% of patients. Titers of this antibody decreased significantly in patients receiving alpha-interferon, but the decrease was slight and only one patient became anti-HCV negative. Further,
anti-HCV titers were low overall and decreased slightly in placebo recipients as well. Thus, a response to interferon could not be reliably monitored or predicted by anti-HCV titers. Change in antibody titers, however, may be delayed, and ultimately loss of anti-HCV may reflect long-term loss of replicating virus (6). Hepatitis C viral RNA was detected in serum of 95% of patients when they entered this controlled trial. Strikingly, serum HCV RNA disappeared in most patients (81%) who were considered responders to alpha-interferon therapy, but the viral genome remained present in most patients who did not respond and in all patients who received placebo. Even in patients who were considered nonresponders, the titers of HCV RNA decreased significantly. These findings suggest that alphainterferon acts by inhibiting viral replication that results in an amelioration of the disease. In some cases, the inhibition appeared to be complete so that interferon therapy could be discontinued without return of the virus. In many patients, however, the inhibition was not complete, and HCV RNA became detectable again within a few months after therapy was discontinued. Although the disappearance of HCV RNA from serum correlated with a response to alpha-interferon therapy, it did not help in predicting responsiveness to alpha-interferon or whether the response would be sustained when interferon was withdrawn. When therapy was discontinued, however, the return of HCV RNA in the serum usually predicted eventual relapse in disease and, in several cases, preceded development of elevated aminotransferases. Interestingly, a higher rate of loss of HCV RNA and a higher rate of sustained response to alpha-interferon was found with a 12-month course of therapy than with a 6-month course, suggesting that longer periods of therapy are more effective in inducing a permanent suppression of replication. This increase in sustained responses with more prolonged courses of interferon will need to be confirmed in future controlled trials that compare different regimens of therapy. Testing for HCV in serum can be helpful in monitoring therapy for chronic hepatitis C and shows that alpha-interferon therapy leads to suppression of viral replication. The disappearance of detectable HCV RNA, however, does not provide absolute evidence of longterm clearance of virus. Use of more sensitive assays for HCV RNA or testing for HCV RNA on liver tissue may ultimately provide the necessary serologic markers
Figure 5. Hepatitis C viral RNA levels during and after alpha-interferon treatment. Course of a patient who had a sustained response to alpha-interferon therapy after a 6-month period of placebo treatment. Hepatitis C viral RNA, which was detected during the placebo treatment, was not detectable during and after interferon therapy. 1 November 1991 • Annals of Internal Medicine • Volume 115 • Number 9
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to identify clearance of this virus and to predict longterm remission in disease. Acknowledgments: The authors thank Ms. Jeanne Waggoner for technical assistance, Ms. Yoon Park and Linda Murray for nursing assistance, and Dr. Jay Everhart for help in statistical analysis. Requests for Reprints: Michiko Shindo, MD, PhD, Liver Diseases Section, Building 10, Room 4D52, National Institutes of Health, Bethesda, MD 20892. Current Author Addresses: Drs. Shindo, Di Bisceglie, and Hoofnagle: Liver Diseases Section, Building 10, Room 4D52, National Institutes of Health, Bethesda, MD 20892. Ms. Cheung and Dr. Shih: Department of Transfusion Transmitted Virus Research, Building 10, Room ID46, National Institutes of Health, Bethesda, MD 20892. Drs. Cristiano and Feinstone: Center for Biologic Evaluation and Research, Hepatitis Research Laboratory, Food and Drug Administration, Rockville, MD 20895. References 1. Hoofnagle JH, Mullen KD, Jones DB, Di Bisceglie A, Peters M, Waggoner JG, et al. Treatment of chronic non-A, non-B hepatitis with recombinant human alpha-interferon. A preliminary report. N Engl J Med. 1986;315:1575-8. 2. Davis GL, Balart LA, Schiff ER, Lindsay K, Bodenheimer HC Jr, Perrillo RP, et al. Treatment of chronic hepatitis C with recombinant interferon alfa. N Engl J Med. 1989;321:1501-6. 3. Di Bisceglie AM, Martin P, Kassianides C, Lisker-Melman M, Murray L, Waggoner J, et al. Recombinant interferon alfa therapy for chronic hepatitis C. A randomized, double-blind, placebo-controlled trial. N Engl J Med. 1989;321:1506-10. 4. Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science. 1989;244:359-62. 5. Kuo G, Choo QL, Alter HJ, Gitnick GL, Redecker AG, Purcell RH, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science. 1989;244:362-4. 6. Alter HJ, Purcell RH, Shih JW, Melpolder JC, Houghton M, Choo QL, et al. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N Engl J Med. 1989;32:1494-500.
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7. van der Poel CL, Reesink HW, Lelie PN, Leentvaar-Kuypers A, Choo QL, Kuo G, et al. Anti-hepatitis C antibodies and non-A, non-B post-transfusion hepatitis in The Netherlands [Letter]. Lancet. 1989;2:297-8. 8. Esteban JI, Esteban R, Viladomiu L, Lopez-Talavera JC, Gonzalez A, Hernandez JM, et al. Hepatitis C virus antibodies among risk groups in Spain. Lancet. 1989;2:294-7. 9. Kuhnl P, Seidel S, Stangel W, Beyer J, Sibrowski W, Flik J. Antibody to hepatitis C virus in German blood donors [Letter]. Lancet. 1989;2:324. 10. Roggendorf M, Deinhardt F, Rasshofer R, Eberle J, Hopf U, Moller B, et al. Antibodies to hepatitis C virus [Letter]. Lancet 1989,2: 324-5. 11. Weiner AJ, Kuo G, Bradley DW, Bonino F, Saracco G, Lee C, et al. Detection of hepatitis C viral sequences in non-A, non-B hepatitis. Lancet. 1990;335:1-3. 12. Ulrich PP, Romeo JM, Lane PK, Kelly I, Daniel LJ, Vyas GN. Detection, semiquantitation, and genetic variation in hepatitis C virus sequences amplified from the plasma of blood donors with elevated alanine aminotransferases. J Clin Invest. 1990;86:1609-14. 13. Kato N, Yokosuka O, Omata M, Hosoda K, Ohto M. Detection of hepatitis C virus ribonucleic acid in the serum by amplification with polymerase chain reaction. J Clin Invest. 1990;86:1764-7. 14. Garson JA, Tedder RS, Briggs M, Tuke P, Glazebrook JA, Trute A, et al. Detection of hepatitis C viral sequences in blood donations by "nested" polymerase chain reaction and prediction of infectivity. Lancet. 1990;335:1419-22. 15. Kanai K, Iwata K, Nakao K, Kako M, Okamoto H. Suppression of hepatitis C virus RNA by interferon-alpha [Letter]. Lancet 1990; 336:245. 16. Okamoto H, Okuda S, Sugiyama Y, Yotsumoto S, Tanaka T, Yoshizawa H, et al. The 5'-terminal sequence of the hepatitis C virus genome. Jpn J Exp Med. 1990;60:167-77. 17. Feinstone SM, Alter HJ, Dienes HP, Shimizu Y, Popper H, Blackmore D, et al. Non-A, non-B hepatitis in chimpanzees and marmosets. J Infect Dis. 1981;144:588-98. 18. Cristiano K, Baker B, Di Bisceglie A, Feinstone S. Detection of hepatitis C viral RNA by the polymerase chain reaction. In: Hollinger FB, Lemon SM, Margolis HS; eds. Viral Hepatitis and Liver Disease. Baltimore: Williams and Wilkins; 1991: [In press]. 19. Kwok S, Higuchi R. Avoiding false positives with PCR. Nature. 1989;339:237-8.
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