Re-Treatment of Patients With Anti-HBe-Positive Chronic Hepatitis B Who Relaps

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Re-Treatment of Patients With Anti-HBe-Positive Chronic Hepatitis B Who Relapsed After an Initial Course of LamivudinePosted 11/20/2003 G. A. Niro, T. Santantonio, R. Fontana, M. Insalata, D. Facciorusso, F. Signorile, F. Perri, A. Guastadisegni, D. Gioffreda, O. Palmieri, G. Pastore, A. Andriulli Summary and IntroductionSummary

Aim: To evaluate the efficacy of a long-term course of lamivudine monotherapy in patients with anti-HBe-positive chronic hepatitis B who relapsed after the first course of either lamivudine/interferon (n = 16; Group 1) or lamivudine (n = 20; Group 2). Methods: Biochemical and virological tests were performed every 3 months. At baseline and breakthrough, the region coding for the YMDD amino acid motif was sequenced. Results: The length of re-treatment averaged 24 months. The virological response peaked at 6 months (94.4%), and declined to 66.7% and 50% at 12 and 24 months, respectively. The rates of breakthrough were 2.9%, 31.4% and 48.6% at 6, 12 and 24 months, respectively. By the second year, responders amounted to 62.5% and 40% in Groups 1 and 2, respectively (P = 0.10). The 18 responders at month 24 are still on therapy after 25-51 months of treatment: 14 still maintain a response, nine from Group 1 and five from Group 2. Conclusions: Re-treatment with lamivudine can control viral replication. This effect is maintained for the initial 12 months in two-thirds of patients, but afterwards the duration of response lessens due to the development of viral resistance.

Introduction

Interferon and lamivudine are the only agents which have been approved for the treatment of chronic hepatitis B. In hepatitis B e antigen (HBeAg)-negative chronic hepatitis B, randomized clinical trials with interferon therapy have shown that the end-of-treatment response is in the range 38-90% in treated patients;[1-5] however, approximately one-half of responders relapsed when therapy was discontinued,[5] so that a sustained response was achieved in 15-25% of patients.[6]

Lamivudine has been shown to be of benefit in patients with HBeAg-negative chronic hepatitis B, providing a 1-year response rate of 70%; however, only 10% of patients maintained the response when the drug was withdrawn.[7-9] The safety of lamivudine has suggested that continuous therapy may be beneficial. The major shortcoming of prolonged therapy is the time-related increasing risk of the emergence of lamivudine-resistant hepatitis B virus (HBV) mutants.[10] Moreover, acute exacerbations of hepatitis may occur after the discontinuation of lamivudine.[11]

In a previous study, we demonstrated that a 12-month course of combination therapy with lamivudine plus interferon was able to prevent the emergence of YMDD mutants;[12] however, the combination regimen failed to prevent both relapse and hepatitis flares when treatment was withdrawn.[12] Rescue therapies for HBeAg-negative chronic hepatitis B patients with worsening liver disease caused by lamivudine-resistant mutants are being evaluated. Recent data have shown that adefovir dipivoxil, a promising anti-HBV agent that has been approved by the Food and Drug Administration, but is not yet licensed in Italy, effectively inhibits replication of YMDD mutants resistant to lamivudine and hence averts the resultant disease.[13] Furthermore, in HBeAg-positive patients, re-treatment with lamivudine was usually effective in controlling exacerbations.[11] Whether this therapeutic option might also be effective for patients with HBeAg-negative chronic hepatitis B remains to be established.

Herein, we have evaluated the benefits of long-term re-treatment with lamivudine monotherapy in HBeAg-negative chronic hepatitis B patients who relapsed after a previous course of either lamivudine monotherapy or combination therapy with lamivudine plus interferon

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Patients and MethodsPatients

This is a follow-up study of our previous controlled trial which evaluated the efficacy of a 12-month course of lamivudine, administered as either monotherapy (100 mg/day), or in combination with interferon (5 MU three times a week; cumulative dose, 780 MU), in Caucasian patients from the Apulia region in southern Italy. All patients were anti-HBe positive, negative for hepatitis C and D virus (HCV and HDV) and human immunodeficiency virus (HIV) infections and similar with respect to age, sex, alanine transaminase and HBV-DNA levels at baseline.[12] In the initial cohort of patients, therapy had to be discontinued per protocol after 1 year of treatment in all cases; of the 50 patients enrolled in the initial trial, five patients who developed a YMDD mutant during the first course of therapy were judged to be unsuitable for lamivudine re-treatment; they showed a virological breakthrough before month 12 and YMDD mutants were detected by sequence analysis. They were excluded because re-treatment in this setting is ineffective due to the prompt reappearance of viral resistance.

Nine more patients from the initial cohort were enrolled in experimental clinical trials using entecavir or adefovir dipivoxil and were also excluded from the present study. The remaining 36 agreed to be re-treated with lamivudine when the disease relapsed after therapy discontinuation; none had experienced a virological breakthrough or had developed YMDD mutants during the previous course of therapy.

Lamivudine Re-Treatment

After an initial evaluation, the 36 patients were administered lamivudine orally at a dose of 100 mg once daily. Patients were seen in the out-patient clinic and had blood drawn at baseline and routinely every 3 months throughout treatment. According to recent guidelines, lamivudine was continued thereafter as long as there was evidence of biochemical and virological response.[6] The initial protocol called for therapy to continue for 5 years. Normal alanine transaminase values and undetectable HBV-DNA (< 0.5 pg/mL) were used as criteria for response. Breakthrough was defined as the reappearance of serum HBV-DNA by molecular hybridization during treatment after an initial virological response, and biochemical breakthrough was defined as an increase in alanine transaminase activity. For patients with virological and biochemical breakthrough, re-treatment had to be stopped whenever alanine transaminase levels reached > 10 times normal, or persistently abnormal alanine transaminase values were found; lamivudine was continued for another 6-9 months in the case of a lower or sporadic increase in alanine transaminase levels.

All patients were treated as part of a two-centre, open-label, prospective trial of long-term therapy of hepatitis B. The protocol was approved by the ethics committees at the two centres, and all patients gave written informed consent.

Laboratory and Virological Testing

Routine laboratory tests, performed on each clinical visit, included serum alanine and aspartate transaminase, serum direct and total bilirubin, albumin and complete blood counts. HBV, HDV, HCV and HIV serological markers were detected by commercial enzyme immunoassays (Abbott Laboratories, North Chicago, IL, USA; Sorin Biomedica, Saluggia, Italy; Ortho Diagnostic Systems, Raritan, NJ, USA). Serum HBV-DNA was detected by a sandwich capture hybridization assay (Digene Diagnostics, Hybrid Capture II, Abbott Laboratories, North Chicago, IL, USA), which has a lower detection limit of 0.5 pg/mL (1.42 x 105 copies/mL). Virological resistance was evaluated by direct sequencing of the polymerase gene at baseline and during treatment in patients with breakthrough, as described previously.[12] With this method, the lower detection limit of a minor viral population is about 20% of the total population.[14] In addition, at baseline, the HBV polymerase region was amplified and analysed by means of the line probe assay (INNO LiPA HBV-DR, Innogenetics, Ghent, Belgium), which identifies 5% of a specific variant within a mixed viral population.[15]

Statistical Analysis

Data were analysed using the SPSS statistical package (version 6.1.3; SPSS Inc., Chicago, IL, USA). Continuous variables were checked for normality using the Shapiro-Wilks test. If normally distributed, Student's t-test was used to evaluate differences between mean values. Otherwise, the Mann-Whitney U-test was carried out to test differences between median values. Pearson's chi-squared test (or Fisher's exact test, when appropriate) was used for categorical variables. A P value of less than 0.05 was considered to be significant. Kaplan-Meier statistics were used to evaluate differences in the cumulative risk of breakthrough occurrence between the two groups of patients, sorted according to the previous course of treatment.

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ResultsBaseline Features

The 36 patients enrolled in the present study included 16 (44%) patients initially treated with lamivudine-interferon combination therapy (Group 1) and 20 patients previously treated with lamivudine monotherapy (Group 2) (Table 1). The majority of patients in both groups were males (86%), with an age range of 18-64 years (mean, 47 years). The average age and sex were similar in the two patient groups.

The time elapsed between the initial treatment and current re-treatment was 8.1 ± 7.2 months (range, 2-23 months), and was shorter in Group 1 (3.3 ± 1.8 months) than in Group 2 (12.7 ± 7.4 months). At the start of re-treatment, the median alanine transaminase level was 340 IU/L; 39%, 19% and 42% of patients had alanine transaminase levels below five times normal values, 5-10 times normal values and more than 10 times normal values, respectively. Alanine transaminase levels more than eight times the norm were more frequent in patients from Group 1 (P = 0.04). HBV-DNA was positive in all patients, with a mean value of 157 pg/mL; at baseline, 58.3% of patients had HBV-DNA levels of less than 100 pg/mL and 41.6% had HBV-DNA levels of more than 100 pg/mL. The numbers of patients with median HBV-DNA levels of < 100 pg/mL were significantly different in the two patient groups, and values of > 100 pg/mL were more frequently observed in Group 1 (P = 0.02).

At the time of initiation of lamivudine re-treatment, no drug-resistant HBV variant was identified by sequence analysis. Negative results were also obtained by the INNO LiPA HBV-DR assay in all but one patient, in whom a mixture of predominant wild-type and mutated virus strains was detected (wt + L528M/YVDD + L528M/YIDD).

Virological Changes During Therapy

At the time of this analysis, the total treatment duration ranged from 12 to 51 months, and averaged 24 months. The percentages of virological response at different time points during lamivudine re-treatment are provided in Figure 1: serum HBV-DNA levels decreased rapidly in all but one patient and were no longer detectable by hybridization assay within 3 months in 32 patients (88.9%). Of the four non-responders at 3 months, three were HBV-DNA negative at 6 months, whereas the remaining patient was still HBV-DNA positive at 6 months and was considered as a primary non-responder. The virological response peaked at 6 months of re-treatment with a value of 94.4%; thereafter, the percentages of responders began to decline and were 66.7% at 12 months, 55.6% at 18 months and 50.0% at 24 months. The decline in responders was counterbalanced by the number of patients with a virological breakthrough; of the 35 patients with an initial response to re-treatment (32 patients at 3 months and three patients at 6 months), the rates of breakthrough at 6, 12, 18 and 24 months were 2.9%, 31.4%, 42.9% and 48.6%, respectively.

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Figure 1. (click image to zoom) Virological response during long-term re-treatment of 36 anti-HBe-positive patients with chronic hepatitis B who relapsed after an initial course of lamivudine with or without interferon

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The frequencies of virological response and breakthrough during long-term lamivudine re-treatment are reported separately for the 16 patients initially treated with lamivudine-interferon combination therapy (Group 1) and the 20 patients previously treated with lamivudine monotherapy (Group 2) (Figure 2). At 3 and 6 months of re-treatment, responders amounted to 94% and 100%, respectively, in Group 1 patients, and 85% and 90%, respectively, in Group 2 patients. After 1 year of treatment, 12 patients (75%) from Group 1 were still in full remission, whereas the proportion of responders from Group 2 had decreased to 60% (12 of 20); this difference was not statistically significant (P = 0.48). By the second year of re-treatment, the number of responders had declined further to 10 (62.5%) and eight (40%) in Group 1 and Group 2, respectively (P = 0.10). These 18 responders are still on therapy; at present, the re-treatment duration is in the range 25-51 months; 14 of the 18 patients have maintained a therapeutic response, nine in Group 1 and five in Group 2.

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Figure 2. (click image to zoom) Percentage of response (R) and breakthrough (BR) during long-term re-treatment with lamivudine. Patients were divided according to the previous course of therapy: combination therapy with lamivudine and interferon (Group 1) and lamivudine monotherapy (Group 2).

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At 6 months of re-treatment, all Group 1 patients were still responding, whereas a virological breakthrough was detected in one of the 19 initial responders (5.3%) in Group 2. At 12 and 24 months of re-treatment, a virological breakthrough had occurred in four (25%) and six (37.5%) patients from Group 1, and in seven (31.8%) and 11 (57.9%) patients from Group 2; these differences were not statistically significant (P = 0.49 and P = 0.31, respectively). By Kaplan-Meier analysis, at 42 months of continuous re-treatment with lamivudine monotherapy, the cumulative proportions of patients who were lamivudine resistant were 82% in Group 2 and 44% in Group 1 (log-rank test: P = 0.154) (Figure 3).

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Figure 3. (click image to zoom) Kaplan-Meier analysis of virological breakthrough in anti-HBe-positive patients with chronic hepatitis B re-treated with lamivudine monotherapy. Patients were divided according to the previous course of therapy: combination therapy with lamivudine and interferon (IFN + LAM; Group 1) and lamivudine monotherapy (LAM; Group 2).

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The predictors of long-term response to re-treatment are evaluated in Table 2. Responders did not differ from those who experienced a breakthrough with regard to gender, alanine transaminase values, HBV-DNA levels, presence of cirrhosis and previous treatment with lamivudine-interferon or lamivudine monotherapy. However, they differed in age [responders being older than those who developed an HBV mutant (P = 0.04)] and in the time interval between initial treatment and re-treatment [responders being re-treated after a shorter period (P = 0.01)]. Two features of responders approached statistical significance: higher baseline alanine transaminase levels and previous treatment with lamivudine-interferon.

Biochemical Changes During Therapy

Serum alanine transaminase levels declined during lamivudine therapy, generally after the decrease in HBV-DNA levels. Alanine transaminase levels were normal in 68% of patients at 3 months, in 83% at 6 months, in 61% at 1 year and in 44% at 2 years. The rise in alanine transaminase levels during treatment followed the development of resistance. The alanine transaminase deviation pattern at the time of virological breakthrough differed: alanine transaminase reached values of acute hepatitis (10 times normal values) in nine patients, five of whom demonstrated an alanine transaminase peak which coincided with the reappearance of HBV-DNA and the detection of YMDD variants; in the remaining four patients, the peak occurred within 8 months from breakthrough. Apart from general malaise and asthenia, no patient became icteric or developed signs of liver decompensation. These nine patients were maintained on lamivudine for another 6 months; alanine transaminase levels were persistently elevated (five times normal) during treatment. In the remaining 12 breakthrough patients, milder elevations of alanine transaminase levels were observed.

Viral Resistance

The detection of YMDD mutants was demonstrated in all 21 patients at breakthrough. The YIDD mutant was detected in eight patients; YVDD mutants were found in nine patients in combination with L528M; YIDD mutants in combination with L528M were detected in four patients.

Characterization of Non-Responder

A 58-year-old man with histologically demonstrated liver cirrhosis agreed to re-treatment for biochemical and virological relapse which occurred 2 months after the first course of lamivudine monotherapy. At baseline before re-treatment, the patient had high serum alanine transaminase levels and was HBV-DNA positive by hybridization assay; a wild-type YMDD sequence was detected by direct sequencing. The virological and biochemical profile of this patient on re-treatment is reported in Figure 4. After 2 months of re-treatment, the YVDD/L528M mutant replaced the wild-type virus and, subsequently, the YIDD mutant replaced the YVDD mutant. Lamivudine was withdrawn at month 16 and interferon therapy was initiated on a compassionate basis. After a transient hepatitic flare, during which the re-emergence of L528M/YVDD as the dominant mutant viral population was observed, alanine transaminase normalized and HBV-DNA tested negative. The baseline serum sample was also analysed by the INNO LiPA HBV-DR test, and a mixture of wild-type virus, L528M/YVDD and L528M/YIDD mutants was found.

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Figure 4. (click image to zoom) Biochemical, virological and molecular analysis of an anti-HBe-positive chronic hepatitis B patient treated with lamivudine and interferon (IFN). ALT, alanine transaminase; HBV-DNA, hepatitis B virus DNA; WT, wild type.