Evaluation of Interferon Treatment in Cirrhotic Patients with Hepatitis C Tatsuya IDE, Michio SATA, Hiroshi SUZUKI, Shiroh MURASHIMA, Ichiroh MIYAJIMA, Miki SHIRACHI and Kyuichi TANIKAWA The Second Department of Internal Medicine, Kurume University School of Medicine (Received: February 22, 1996) (Accepted: April 9, 1996) Key words: liver cirrhosis, hepatitis C, interferon treatment Abstract The aim of this study was to examine the effects of interferon on cirrhotic patients with hepatitis C and the incidence of adverse reactions. The subjects were 35 cirrhotic patients, and 29 chronic active hepatitis patients without cirrhosis (CAH) served as controls. The cirrhotic patients received 3 or 6 million units of human lymphoblastoid interferon daily for one or two weeks and then three times a week for 22 or 23 weeks, while the CAH patients received 6 million units daily for 2 weeks and then three times a week for 14 or 16 weeks. Discontinuation of interferon treatment or dose reduction was required in the 7 cirrhotic patients. The most frequent reason was thrombocytopenia. Dose reduction alone was necessary in two CAH patients. Five cirrhotic patients (14.3%) and nine CAH patients (31.0%) were classified as complete responders to interferon treatment. In all five complete responders with cirrhosis, the hepatitis C virus RNA level before treatment was less than 5 log copies/50 Đl. The results of this study confirm the beneficial effect of interferon in selected patients with cirrhosis on basis of pre-treatment virus levels and platelet count. Introduction Interferons (IFNs) are often used to treat chronic hepatitis C1) `4). Serum hepatitis C virus (HCV) RNA has been reported to be continuously eliminated in about 30-50% of all patients after IFN therapy5) `7). In patients with chronic active hepatitis without cirrhosis (CAH), numerous studies have suggested that high serum HCV RNA levels and HCV genotype II are markers for an unfavorable response to IFN6) `10) Reports on the use of IFN to treat liver cirrhosis (LC) due to hepatitis C have also been published. Many investigators have reported that IFN therapy is less effective for LC than for CAH11)-14). However, there are no widely accepted criteria for determining IFN therapy in patients with cirrhosis. Furthermore, the course of liver function and virologic changes following IFN treatment have not been studied adequately, and have not been sufficiently compared between cirrhotic patients and CAH patients. The present study was undertaken to assess the results of IFN therapy in cirrhotic patients with hepatitis C in order to establish criteria for IFN therapy in cirrhotic patients. Correspondence to: Tatsuya Ide, M. D. The Second Department of Internal Medicine, Kurume University School of Medicine, 67, Asahi-machi, Kurumeshi, Fukuoka-ken 830, Japan
Tatsuya IDE et at Patients and Methods Patients Between October 1992 and December 1993, 35 cirrhotic patients and 29 CAH patients with hepatitis C were enrolled in this study. The diagnoses were based on the result liver function tests, HCV-antibody positivity and histopathological findings in the liver before IFN treatment. According to Pugh's modification of the Child-Turcotte index15), six cirrhotic patients were classified into Child B and the other cirrhotic patients were categorized as Child A. All patients lacked hepatitis B surface antigen (HBsAg) as determined by enzyme immunoassay (EIA; Mizuho Medy Co., Ltd., Tosu, Japan). Patients with ascites, hepatic encephalopathy, and other types of chronic liver disease were excluded. Written informed consent was obtained from all patients. Methods HCV Antibody Anti-HCV was detected by passive hemagglutination using 2nd generation HCV antibody (Abbott HCV PHA 2nd Generation, Dainabot Co., Ltd., Tokyo, Japan). HCV RNA Serum HCV RNA was amplified by reverse transcription- "nested" polymerase chain reaction (RT-PCR) using the method described by Okamoto et al16). The cdna was subjected to PCR amplification using a set of 5'-non coding region primers, 5'-GGCGACACTCCACCATGAATCACT- 3' (sense primer), and 5'-CACGAATTCAGTCTTCTTTGTCGCGCACACCCAA-3' (antisense primer) for the first for the first round PCR, and 5'-ATAGGATCCACTCCCCTGAGGAACTACTGTC-3' sense, 5'-ATGAATTCATGGTGCACGGTCTACGAGACCTCCCG-3' (antisen seprimer) for the second round PCR17). The PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining. Serum HCV RNA level Serum HCV RNA levels were determined by competitive reverse transcription-polymerase chain reaction (RT-PCR) according to the method described by Kato et al18). Genotyping of HCV HCV genotyping was done using a modification of the method of Okamoto et al.19). The second PCR products were analyzed by electrophoresis in 3% agarose gel, stained with ethidium bromide, and examined under UV light. HCVs were then classified into four types: Type I: 57 pb, Type II: 144 bp, Type III: 174 bp, Type IV: 123 bp DNA fragments and mixed type. Interferon treatment Human lymphoblastoid interferon (natural IFN-cr; Sumitomo Pharmaceutical Co., Ltd, Tokyo, Japan) was used. The Cirrhotic patients received 3 or 6 million units (MU) intramuscularly daily for 1 or 2 weeks and then three times a week for 22 or 23 weeks for a total of 24 weeks of treatment. Before treatment, the patients were randomly assigned to two groups based on the total dose of interferon to be given. The dose of interferon was reduced or the drug was discontinued when severe side effects occurred. The patients in the low-dose group (LD-group) received 168-288 MU, and those in the high-dose group (HD-group) received 432-468 MU. The patients with CAH received 6 MU daily for 2 weeks and then three times a week for 14 or 16 weeks. The total dose in these patients ranged from 336-372 MU. All of the patients were monitored for at least 6 months following treatment. Complete responders (CRs) were defined as patients whose serum alanine aminotransferase (ALT) level remained normal and in whom no serum HCV RNA was detected for at least 6 months after the end of treatment. Non-responders (N Rs) were defined as patients whose HCV RNA reappeared within 6 months after the end of treatment, even though their ALT level fell to within the normal
IFN Treatment in Cirrhotic Patients range. Statistical methods The chi-square test, Fisher's exact probability test, Wilcoxon-signed rank test, and Mann- Whitney U test were used. A p value of 0.05 or less was considered statistically significant. All values are expressed as means }S.E.M. Results 1) Adverse reactions, dose reductions and exclusion. Four patients with cirrhosis were excluded because of severe side effects (Table 1). Two patients suffered general malaise and refused treatment after 8 or 10 weeks of treatment, one developed psoriasis vulgaris after 8 weeks of treatment and was excluded at 15 weeks, and one patient developed severe thrombocytopenia (platelet count; 24 ~109/1) after 3 weeks of treatment and was excluded. This patient's platelet count of before treatment was 42 ~ 109/1. No CAH patients were excluded from this study, The dose of interferon was reduced in three patients with cirrhosis because of thrombocytopenia or leukopenia. Two patients developed thrombocytopenia (platelet count: 39 X 109/1, 30 ~109/1) after 1 and 4 weeks of treatment, respectively, and the dose of interferon was reduced. The platelet counts of these patients before treatment were 79 and 58 ~109/1, respectively. Another patient developed leukopenia (neutrophil count: 0.38 ~109/1) after 4 days of treatment, and the dose of interferon was reduced. The neutrophil count of this patient before treatment was 1.72 ~ 109/1. The dose of interferon was reduced from 6 MU to 3 MU daily in all patients. The mean platelet count in the cirrhotic patients reached a nadir (81 }6 ~109/1) after 4 weeks of treatment. It decreased significantly (p < 0.001) between 2 weeks and the end of treatment, compared with the pretreatment level. At the 4th weeks after the end of treatment, the mean platelet count had recovered to the pre-treatment level. Two CAH patients developed leukopenia, and the dose of interferon was reduced. Discontinuance or reduction of treatment was more frequent in the cirrhotic patients than in the CAH patients, but this was not significant (7 of 35 [20%] vs 2 of 29 [7%], P = Table 1 Number of Patients with Adverse Reactions and Complete Responders to Interferon Treatment The figures in parenthses are percentages * Cirrhosis vs. chronic active hepatitis without cirrhosis, Fisher's exact probability test t Pugh's modification of the Child-Turcotte index. õ
Tatsuya IDE et al 0.13). Thirty-two cirrhotic patients and 29 CAH patients completed 16 or more weeks of treatment and could be included in the evaluation interferon's efficacy. Table 2 shows the characteristics of the patients enrolled in this study. Only the platelet count was significantly lower in patients with cirrhosis than in CAH patients (p < 0.0001). 2) Assessment of the efficacy of interferon. Five (14.3%) of the 35 cirrhotic patients were CRs (Table 1). One patient had a low serum albumin level (3.2 g/dl) with marked thrombocytopenia (58 ~109/1) and was categorized as Child B before treatment. Three of the five patients received low-dose interferon therapy (total dose 168, 225, 261 MU). Nine (31%) of the 29 patients with CAH were CRs. The difference in response rate between the cirrhotic patients and the CAH patients was not significant (P=0.10). Table 3 shows the number of patients with normal ALT levels before, during and after interferon treatment. The number of patients with normal ALT levels between 4 weeks of treatment and the end of treatment was significantly lower in the cirrhotic patients than in CAH patients. There were also significantly fewe patients with normal ALT levels in the HD-group compared to the CAH group between 2 and 12 weeks of treatment. ALT levels returned to normal within 4 weeks after the start of IFN treatment in eight patients with cirrhosis. None of these eight patients were classified as CRs to IFN therapy. All patients classified as CRs showed normalization of ALT levels 5 or more weeks after the start Table 2 Patient characteristics Values are means }S.E.M., Normal reference range : <30 IU/L for ALT, N.T., not tested. HD, High dose. LD, Low dose., *Chi-square test, **Mann-Whitney U test. Table 3 Numbers of Patients with Normal ALT Levels Before, During and After Interferon Treatment Percentages in parenthese. Cirrhosis vs chronic active hepatitis without cirrhosis, ap < 0.05, bp <0.01, cp <0.001 by the chi square test or Fisher's exact probability test.
IFN Treatment in Cirrhotic Patients of IFN treatment, where as all 8 of the CR of the patients with CAH showed normalization of ALT levels within the first 4 weeks of IFN therapy. Mean serum albumin levels in the cirrhotic patients reached a nadir (3.6 } 0.1 g/dl) after 2 weeks of treatment, but returned to the pre-treatment level after 4 weeks of treatment (Fig. 1). At 8th and 24th week after the end of treatment, the albumin level was significantly higher than the pretreatment level (p <0.01). Fig. 2 shows serum HCV RNA levels during and after interferon treatment in genotype II groups of both cirrhotic patients treated with high-dose IFN and patients with CAH. After 12 weeks of treatment, HCV RNA had become undetectable in 5 of the 8 (62.5%) patients with CAH, and in 4 of the 9 (44.4%) patients with cirrhosis, although the differences were not significant (P =0.40). By the end of treatment, HCV RNA had become undetectable in 6 of the 8 (75%) patients with CAH, and in 2 of the 8 (25%) patients with cirrhosis, although again the differences were not significant (P=0.07). The pre-treatment HCV RNA levels in the cirrhotic patients were significantly higher in the NRs than in the CRs (p <0.01) (Fig. 3). None of the 22 patients with HCV RNA levels of 5 or more log copies/50,ul were CRs. Five (50%) of the 10 patients with low HCV RNA levels (less than 5) were CRs and 5 were NRs. Only one patient, an NR, developed hepatocellular carcinoma, 3 months after treatment with- Fig. 2 Changes in serum HCV RNA levels during and after interferon treatment in genotype II cirrhotic patients treated with high-dose IFN (a) and genotype II patients with chronic active hepatitis without cirrhosis (b). (a) Cirrhosis, High Dose IFN, Genotype II (n=10) Fig. 1 Changes in serum albumin levels during and after interferon treatment of cirrhotic patients. (n=32, *p < O.01 by Wilcoxon-signed rank test) (b) Chronic Active Hepatitis without Cirrhosis, Genotype II (n =8)
Tatsuya IDE et al Fig. 3 Pretreatment serum HCV RNA level in cirrhotic patients. (p <0.01 by Mann-Whitney U test) drawal. Abdominal ultrasound scanning was performed in 5 CR patients received an every three months for 14.7 }2.7 (9-22) months following therapy, and none of the patients developed hepatocellular carcinoma. Discussion This study analyzed changes in ALT levels and serum HCV RNA levels in cirrhotic patients during and after IFN therapy and compares them with those of CAH patients. Twenty percent of the cirrhotic patients required discontinuation or dose reduction of IFN, often because of thrombocytopenia. None of the cirrhotic patients with a pretreatment platelet count of 80 ~109/1 or more required discontinuation or dose reduction. When IFN therapy is used to treat for cirrhotic patients with pre-treatment platelet counts below 80 ~109/1, careful selection of dosages and adequate follow-up after treatment are necessary. The ALT levels returned to normal during IFN treatment in fewer cirrhotic patients receiving high doses of IFN than in CAH patients. Early normalization of ALT levels (within the first 4 weeks of IFN therapy) was uncommon in the cirrhotic patients, even in the patients ultimately classified as CRs. This finding indicates that IFN therapy should be continued in cirrhotic patients even when ALT levels do not normalize soon. It has been reported that HCV genotypes are related to the response of chronic hepatitis patients to IFN therapy, and that there is a smaller percentage of CRs in the genotype II patients6)'). HCV RNA disappeared after the start of treatment and remained absent until the end of treatment in 75% of with genotype II CAH patients, but HCV RNA disappeared in only 25% of the cirrhotic patients. In some patients, it reappeared before the end of treatment. It is unknown why fewer cirrhotic patients treated with IFN experience normalization of ALT and disappearance of HCV RNA. This may be (1) because the transfer of IFN to the liver is inadequate in the presence of liver fibrosis or altered hepatic blood flow, or (2) because IFN-resistant clones proliferate as the disease develop into LC, even in patietns with the same HCV genotype. Although the percentage of CRs to IFN therapy among the cirrhotic patients was not high, IFN therapy was markedly effective in some patients with advanced LC or receiving low doses of IFN. Therefore, it is important to establish criteria for selecting appropriate cirrhotic patients for IFN therapy. The complete response rate in CAH patients was 31.0%. Although CRs were more common
IFN Treatment in Cirrhotic Patients among the CAH patients, the difference between the response rates in the cirrhotic patients and the CAH patiens was not significant. This finding may have been due to (1) the longer duraion of IFN therapy in the cirrhotic patients than in the CAH patients, (2) the development of severe fibrosis20) in eleven CAH patients (38%), and (3) small number of patients. The HCV RNA levels of all of the CRs was less than 5 log copies/50 Đl, and 50% of all patients with levels less than 5 log copies/50 ill showed complete responses to IFN therapy. This suggests that cirrhotic patients are likely to respond to IFN therapy completely if their HCV RNA levels are low. It also suggests that total IFN dose should be reduced in patients with low HCV RNA levels but in whom adverse reactions such as thrombocytopenia or leukopenia are anticipated. Our subjects showed a significant increase in serum albumin levels 2 and 6 months following the end of IFN therapy. This suggests that IFN therapy reduces the severity of hepatic inflammation in LC, which leads to improved hepatic reserve capacity, even when it is ineffective in virus elimination. Patients with hepatitis C-associated LC to be treated with IFN must be selected patients on the basis of pre-treatment platelet count, HCV genotype, and HCV RNA levels, and they must be checked for disappearance of HCV RNA during IFN therapy. Since cirrhotic patients have a low probability of complete response to any regimen of treatment currently used, it is also necessary to define the goal of treatment and establish a new regiment when using IFN to treat this disease. References 1) Hoofnagle JH, Di BA.: Treatment of chronic type C hepatitis with alpha interferon. Semin Liver Dis 1989; 9: 259-263. 2) Davis GL, Balart LA, Schiff ER et al.: Treatment of chronic hepatitis C with recombinant interferon alfa. A multicenter randomized, controlled trial. Hepatitis interventional Therapy Group. N Engl J Med 1989; 321: 1501-1506. 3) Causse X, Godinot H, Chevallier M et al.: Comparison of 1 or 3 MU or interferon alfa-2b and placebo in patients with chronic non-a, non-b hepatitis. Gastroenterology 1991; 101: 497-502. 4) Macellin P, Boyer N, Giostra E et al.: Recombinant human alpha-interferon in patients with chronic non-a, non-b hepatitis: a multicenter randomized controlled trial from france. Hepatology 1991; 13: 393-397. 5) Omata M, Ito Y, Yokosuka 0 et al.: Randomized, double-blind, placebo-controlled trial of eight-week course of recombinant alpha-interferon for chronic non-a, non-b hepatitis. Dig Dis Sci 1991; 36: 1217-1222. 6) Tsubota A, Chayama K, Ikeda K et al.: Factors predictive of response to interferon-alpha therapy in hepatitis C virus infection. Hepatology 1994; 19: 1088-1094. 7) Hayashi J, Ohmiya M, Kishihara Y et al.: A statistical analysis of predictive factors of response to human lymphoblastoid interferon in patients with chronic hepatitis C. Am J Gastroenterol 1994; 89: 2151-2156. 8) Yoshioka K, Kakumu S, Wakita T et al.: Detection of hepatitis C virus by polymerase chain reaction and response to interferon-alpha therapy: relationship to genotypes of hepatitis C virus. Hepatology 1992; 16: 293-299. 9) Magrin S, Craxi A, Fabiano C et al.: Serum hepatitis C virus (HCV)-RNA and response to alpha-interferon in anti-hcv positive chronic hepatitis. J Med Virol 1992; 38: 200-206. 10) Hagiwara H, Hayashi N, Mita E et al.: Quantitative analysis of hepatitis C virus RNA in serum during interferon alfa therapy. Gastroenterology 1993; 104: 877-883. 11) Alberti A, Chemello L, Bonetti P et al.: Treatment with interferon(s) of community-acquired chronic hepatitis and cirrhosis type C. The TVVH Study Group. J Hepatol 1993; s123-s126. 12) Saito T, Shinzawa H, Kuboki M et al.: A randomized, controlled trial of human lymphoblastoid interferon in patients with compensated type C cirrhosis. Am J Gastroenterol 1994; 89: 681-686. 13) Jouet P, Roudot TF, Dhumeaux D & Metreau JM.: Comparative efficacy of interferon alfa in cirrhotic and noncirrhotic patients with non-a, non-b, C hepatitis. Gastroenterology. 1994; 106: 686-690. 14) Serfaty L, Giral P. Loria A, Andreani T, Legendre C & Poupon R.: Factors predictive of the response to interferon in patients with chronic hepatitis C. J Hepatol 1994; 21: 12-17. 15) Pugh RNH, Murray-Lyon IM, Dawson JL, Pietroni MC, Williams R.: Transection of the oesophagus for bleeding oesophageal varices. Br J Surg 1973; 60: 646-649. 16) Okamoto H, Okada 5, Sugiyama Y et al.: The 5'-terminal sequence of the hepatitis C virus genome. Jpn J Exp Med 1990; 60: 167-177.
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