Usefulness of HCV-RNA counts by the method of HCV-bDNA probe in interferon retreatment for patients with HCV-RNA positive chronic hepatitis C

International HepatologyCommunications

ELSEVIER

4 (1995) 19-25

Usefulness of HCV-RNA counts by the method of HCV-bDNA probe in interferon retreatment for patients with HCV-RNA positive chronic hepatitis C Yasuji Arase*a, Hiromitsu Kumadaa, Kazuaki Chayamaa, Murashima Naoyaa, Akihito Tsubotaa,Isao Koidaa, Masahiro Kobayashia,Yoshiyuki Suzuki”, Kenji Ikedaa, SatoshiSaitoh”, Mariko Kobayashib aDepartment

of Gastroenterology, bHepatic

Toranomon

Research

Unit,

Hospital, Japan Toranomon

2-2-2 Hospital,

Toranomon, Tokyo,

Minato-ku,

Tokyo

105,

Japan

Received21 November 1994;accepted10 February 1995

Abstract

The objective of this study was to determine how HCV-RNA counts before interferon (IFN) retreatment influence patient response. We retrospectively studied 60 consecutive Japanese patients with chronic hepatitis C. HCV-RNA counts were determined by a new assay; HCV-branched DNA (HCV-bDNA) probe. In this study, 60 patients were retreated with alpha interferon. Two patients were withdrawn because of side effects. In regard to the HCV-RNA level between before the first IFN treatment and before IFN retreatment, HCVRNA level was not significantly different in 74.1% (43/58) of patients. Out of 58 patients, nine (15.5%) had complete responsewith normalization of alanine aminotransferase (ALT) levels and undetectable HCV-RNA more than 6 months after the completion of retreatment. Out of sevenpatients with a HCV-RNA level lessthan 0.5 Meq/ml before IFN retreatment, three (42.9%) had a complete response.On the other hand, out of 17 patients with HCV-RNA level more than 10 Meq/ml before IFN retreatment, there were none with a complete response. These results indicate that a complete responseto readministration of interferon is related to HCV-RNA level before interferon retreatment. Keywords: Chronic hepatitis C; Interferon retreatment; HCV-bDNA * Correspondingauthor. 0928-4346/95/.$.09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI

0928-4346(95)00208-Z

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1. Introduction Interferon (IFN) is effective in reducing serum ALT and HCV-RNA levels in patients with chronic hepatitis C [l-12]. However, many patients remain HCVRNA positive or have abnormal serum transaminase levels after the completion of IFN therapy. In IFN retreatment of non-responders with the first IFN therapy, the rate of complete cures with undetectable HCV-RNA at 6 months after completion of IFN retreatment, is lower than that of first IFN therapy. As IFN retreatment can be associated with more side effects and is costly, selection of patients for IFN retreatment is therefore extremely important. In this study, the usefulness of HCVRNA counts by HCV-bDNA probe in IFN retreatment was examined in patients with chronic hepatitis C. 2. Materials and methods 2.1. Patients

Sixty patients were enrolled in this trial, carried out between May 1992 and April 1993. Enrolment criteria were as follows: (1) average ALT elevations greater than two-fold the upper limits of normal after the first IFN therapy (normal value is 6-50 IU/l or 3-25 KU); (2) liver biopsy (taken within 6 months prior to IFN readministration) showing histological features of chronic active hepatitis; (3) positive HCVRNA at 6 months after the termination of the first IFN therapy; (4) no corticosteroid, immunosuppressive agents, or antiviral agents within 3 months; (5) no hepatitis B surface antigen (HBsAg), hepatitis virus DNA (HBV-DNA), antinuclear antibodies (ANA) or antimitochondrial antibodies (AMA) detectable in serum using radioimmunoassay and spot hybridization, respectively; (6) white blood cell count greater than 3500/~1 and platelet counts greater than 120000/& (7) total dose of first interferon greater than 250 megaunits (MU); (8) HCV-genotype lb. The entire protocol was approved by the hospital ethics committee. The aims, methods and anticipated benefits of the study and side effects of IFN were explained to each patient and written informed consent was obtained from all patients. 2.2. Drug assignment

IFN for retreatment was prepared by Sumitomo Pharmaceutical Co., Tokyo, Japan. IFN vials contained 6 MU of human lymphoblastoid alpha IFN. The patients were given intramuscular injections daily for 8 weeks, followed by IFN three times weekly for another 16 weeks. The total dose of IFN was 624 MU. Clinical evaluation and biochemical and hematological tests were performed at weekly to monthly intervals during IFN retreatment and at monthly intervals during follow up for more than 6 months after the termination of IFN retreatment. A complete response to IFN therapy was defined as a normalization of the serum ALT level and negativity of HCV-RNA more than 6 months after the completion of IFN retreatment. 2.3. Examination of HCV-RNA

by nested RT-PCR

Serum samples were stored at -80°C until analysis for HCV-RNA. Detection was performed blindly in our laboratory. HCV-RNA was assayed using a reverse transcription nested polymerase chain reaction (RT-nested PCR) [ 13- 151.

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by bDNA signal amplification

The sandwich assay with signal amplification using bDNA was performed by the method of Johnson (Lau et al. [16]). This assay is based upon specific hybridisation of virus RNA in the sample by synthetic oligonucleotides to the highly conserved 5’-untranslated region and core gene of HCV-RNA immobilised on the surface of a microtiter plate well. In the HCV-bDNA probe method, 50 ~1 of serum sample was added to 150 ~1 of the mixture (protenase K, extraction buffer, extender probe and capture probe) on a microtiter plate and allowed to stand at 63°C for 16 h. Amplifier probe (50 ~1) was added to the plate and second reaction was produced at 53°C for 30 min. Labeling was then produced by adding 50 ~1 of label probe and processing at 53°C for 15 min. After that, the chemiluminescence from dioxysetane, which represents HCV-RNA counts, was measured at 37°C and the data were converted into HCV-RNA counts on the basis of the control into megaequivalents/ml (Meq/ml). The patients were classified into four groups based on the HCV-RNA counts as follows: (1) 10.5, (2) >0.5 and I 1, (3) > 1 and I 10, (4) > 10. HCV genotypes were classified by a PCR using a mixture of primers for the five genotypes known to exist in Japan, as reported previously (13,141. 2.5. Statistical analysis

Statistical analysis was performed by the X*-test to examine the relationship between complete response rate and HCV-RNA level by the HCV-bDNA probe.

3. Resdts In this study two patients were withdrawn because of interferon side effects. One patient had a retinal bleeding at 6 weeks after IFN retreatment and another had a hypo-thyroidism at 4 months after IFN retreatment. Hence, the results reported are from the remaining 58 patients. Demographic and laboratory data for these 58 paTable 1 Clinical profiles of patients with chronic hepatitis C retreated with interferon Clinical features Age (years) Sex Male Female Liver histology CHZA CHZB Serum ALT Serum AST Total dose of first IFN therapy Treatment schedule of first IFN therapy Daily Intermittent Combined Figures in parentheses are medians.

23-60 (45) 44

14 32 26 62-289 (135) W/I 31-206 (93) IU/I 256-480 (336) MU 39 12 7

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tients are shown in Table 1. In the first IFN therapy, 39 patients had been treated with 6 MU of beta IFN daily for 6 or 8 weeks, 12 patients treated with 6MU of alpha IFN three times a week for 24 or 28 weeks and 7 patients treated with 6MU of alpha IFN daily for 2 or 4 weeks plus two or three times a week for 16 or 24 weeks. 3.1. Changes in HCV-RNA counts between befbre the first IFN treatment and before the retreatment Changes in HCV-RNA counts between before the first IFN treatment and before the retreatment are shown in Figs. 1 and 2. As shown in Fig. 1 and Fig. 2, 73.5% (36/49) in an incomplete response group and 77.8% (7/9) in a complete response group had unchanged HCV-RNA counts. In patients with more than 10 Meq/ml of HCV-RNA before first IFN treatment or retreatment, none showed complete response after IFN retreatment.

HCV-RNA (Meq/ml)

before the first IFN treatment

before the IFN retreatment

10s

19

17

l$


22

23

0.5s


<0.5

4

4

w

4

Fig. 1. Changes in HCV-RNA counts between before the first IFN treatment and before the retreatment in patients with an incomplete response.

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before the first IFN treatment

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before the IFN retreatment 4

4

7 0.55

<1

1

2

<0.5

Unchanged Decreased

7/9(77.8%) 2/9(22.25/o)

Fig. 2. Changes in HCV-RNA counts between before the first IFN treatment and before the retreatment in patients with a complete response.

3.2. Efficacy of interferon with respect to HCV-RNA (Table 2)

level by the HCV-bDNA

probe

Patients were divided into four groups based on the HCV-RNA level. Data on efficacy of IFN retreatment are given in Table 2. Of patients showing a HCV-RNA level of less than 0.5 Meq/ml, 42.9% (3/7) had a complete response to retreatment. The complete response rate after IFN retreatment decreased based on an increase in HCV-RNA level before retreatment. Of 17 patients with a HCV-RNA level greater than 10 Meq/ml, there were none with a complete response (P < 0.05). Table 2 Complete response to IFN retreatment with respect to HCV-RNA level by the method of HCV-bDNA probe HCV-RNA (Meq/ml)

Complete response rate

co.5

317 (42.9%) 2i7 (28.6%) 4127 (14.8%) o/17 (0%)

0.5 d
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4. Discussion IFN is effective in decreasing ALT and HCV-RNA level against chronic hepatitis C [l-l l] and in a recent study the therapeutic efficacy was shown to increase with a higher total dose of IFN and prolonged treatment [12]. By prolonged IFN therapy of 8 weeks continuous plus 16 weeks intermittent administration, complete response rate attains about 40-50%. Remainder cases however, have abnormal ALT levels. The rate of complete response in IFN retreatment is lower than that of the first IFN therapy and IFN retreatment can be associated with more side effects. On the other hand, IFN is an expensive drug, therefore selection of patients for IFN retreatment is extremely important. In this study, we investigated IFN retreatment in 60 patients who did not show normalization of ALT after termination of the first IFN treatment. None of the patients had a complete response when HCV-RNA level was more than 10 Meq/ml at the starting point of retreatment. On the other hand, IFN retreatment was effective when HCV-RNA level was lower than 1 Meq/ml. Our results show that determination of HCV-RNA level by bDNA probe before retreatment is one of the important factors to consider when selecting patients for IFN retreatment. HCV-RNA counts by competitive PCR can detect slight traces of HCV-RNA and aid in estimating effect during IFN therapy. But HCV-RNA counts by bDNA probe are detectable only in patients with more than 0.5 Meq/ml and negative in those with less than 0.5 Meq/ml. In fact, according to HCV-RNA counts by bDNA probe, most of the patients had negative counts during IFN therapy. Therefore, we have little information on the changes of HCV-RNA counts by DNA probe during IFN therapy. HCV-RNA counts by bDNA probe however, can be measured easily and inexpensively. We had reported that patients with transient negative HCV-RNA up to 1 month from starting point of the first IFN therapy appear to have a higher chance of complete response with IFN retreatment [17]. In the present study, we conclude that determination of HCV-RNA level by bDNA probe will assist the physician in selecting patients for IFN retreatment. References [l] Hoofnagle JH, Mullen KD, Jones DB et al. Treatment of chronic non-A, non-B hepatitis with recombinant human alpha interferon. N Engl J Med 1986; 315: 1575-8. [2] Thompson BJ, Doran M, Lever AML et al. Alpha interferon therapy for non-A, non-B hepatitis transmitted by gamma-globulin replacement therapy. Lancet 1987; 1: 539-41. [3] Di Bisceghe AM, Martin P, Kassiandies C et al. Recombinant interferon-alpha therapy for chronic hepatitis C. N Engl J Med 1989; 321: 1506-10. [4] Kiyosawa K, Sodeyama S, Oike Y et al. Treatment of chronic non-A, non-B hepatitis with human interferon-beta. In: Zuckerman A, ed. Viral Hepatitis and Liver Disease. New York: Alan R. Liss, 1988; 898-901. [5] Fujioka S, Hino K, Fukuhara A et al. Voluminal comparative tests with human interferon-beta for non-A, non-B chronic hepatitis. Acta Hepatol Jpn 1989; 30: 516--21. [6] Kakumu S, Arao M, Yoshioka E et al. Recombinant human interferon therapy for chronic non-A, non-B hepatitis: second report. Am J Gastroenterol 1989; 85: 655-9. [7] Omata M, Ito Y, Yokosuka 0 et al. Histological changes of liver by the treatment of chronic non-

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A, non-8 hepatitis with recombinant leucocyte interferon alpha: comparison with histological changes in chronic hepatitis B. Dig Dis Sci 1989; 34: 330-7. [S] Kumada H, Arase Y, Chayama K et al. Usefulness of HCV antibody titer in the interferon therapy. Acta Hepatol Jpn 1990; 3 1: 902-6. [9] Chayama K, Saito S, Arase Y et al. Sensitive and specific detection of hepatitis C virus RNA with nested polymerase chain reaction and digoxigenin-labelled cDNA probe. Acta Hepatol Jpn 1991; 32: 135-40.

[IO] Arase Y, Kumada H, Chayama K et al. Alanine aminotransferase and HCV-RNA responses following interferon therapy HCV-RNA positive chronic hepatitis. Gastroenterol Jpn 1992; 33: 805-6.

[l 11 Hagiwara H, Hayashi N, Mita E et al. Quantitative analysis of hepatitis C virus in serum during interferon-alpha therapy. Gastroenterology 1993; 1041 877-83. [12] Arase Y, Chayama K, Akihito T et al. Interferon therapy for HCV-RNA positive chronic hepatitis. Acta Hepatol Jpn 1993; 34: l-7. [13] Kubo Y, Takeuchi K, Boonmar S et al. A cDNA fragment of hepatitis C virus isolated from an implicated donor or post-transfusion non-A, non-B hepatitis in Japan. Nucleic Acids Res 1989; 17: 10367.

Chayama K, Tsubota A, Arase Y et al. Genotypical classification of hepatitis C virus genome with polymerase chain reaction using a mixed primer set. Acta Hepatol Jpn 1992; 33: 805-6 [ISI Okamoto H, Sagiyama Y, Qkada S et al. Typing hepatitis C virus by polymerase chain reaction with type-specific primers: application to clinical survey and tracing infectious sources. J Gen Viral

[14]

1990, 60: 167-77.

Lau J, Davis G, Kniffen J et al. Significance of serum hepatitis C virus RNA levels in chronic hepatitis C. Lancet 1993; 341: 1501-4. [17] Arase Y, Kumada H, Chayama K et al. Interferon retreatment of non-responders with HCV-RNApositive chronic hepatitis C. J Gastroenterol 1994; 29: 299-304. [16]