Long-Term Interferon+Lamivudine for HBV in HBeAg-Negatives
"Interferon/long-term lamivudine combination therapy in HBeAg-Negative, anti-HBe positive chronic hepatitis B patients"
Journal of Gastroenterology and Hepatology
NIKOLAOS L NIKOLAIDIS*, OLGA I GIOULEME*, KONSTANTINOS A TZIOMALOS*, ADAMOS S SAVERIADIS*, NIKOLAOS GRAMMATIKOS*, PANAGIOTIS DOUKELIS*, ANASTASIOS D VOUTSAS*, THEMISTOKLIS VASSILIADIS*, KALLIOPI PATSIAOURA+, ELENI ORFANOU-KOUMERKERIDOU, AIKATERINI BALASKA* and NIKOLAOS P EUGENIDIS*
*Gastroenterology and Hepatology Section of Second Propaedeutic Department of Internal Medicine, Aristotle University of Thessaloniki, +Department of Pathology, Fourth Department of Internal Medicine, Aristotle University of Thessaloniki, Hippokration General Hospital, Thessaloniki, Greece
--Four patients (11%) cleared HBsAg and developed anti-HBs
-- End-of-treatment responses to IFN in patients with HBeAg-negative chronic hepatitis B range from 40% to 60%. The main advantage of IFN therapy is that a course of finite duration may achieve sustained off-therapy responses in 15-25% of these patients
-- Lamivudine, a nucleoside analog, is a potent inhibitor of HBV replication. The end-point of lack of detectable HBV DNA and normal serum alanine aminotransferase (ALT) levels is reached in 65-79% of patients at the end of 52 weeks of treatment with lamivudine.4 Unfortunately, sustained off-therapy responses to lamivudine are rare; only 11% of treated patients have a sustained response 24 weeks after the end of 12 months of therapy
-- Thirty-five patients (97%) went on to receive lamivudine for a second year, 31 (86%) for a third year, 22 (61%) for a fourth year, nine (25%) for a fifth year and five (14%) for a sixth year
-- The cumulative rates of breakthrough infection at the end of 1, 2, 3 and 4 years of treatment were 0%, 14%, 32% and 59%
-- In this study, 92% of the patients showed combined biochemical and virological response at 12 months, a percentage significantly superior to the reported response rates with either IFN or lamivudine monotherapy.4 This finding provides further support to the assumption of an increased antiviral potential of combination treatment.
-- This is the first study to report the outcome of IFN/long-term lamivudine in HBeAg negative chronic hepatitis B. Our data suggest that this type of combination therapy is effective
Background: Monotherapy with a single antiviral agent is insufficient in controlling hepatitis B virus infection in the majority of patients with anti-HBe positive chronic hepatitis B. Interferon/long-term lamivudine combination therapy was evaluated to determine if this strategy would improve treatment efficacy and reduce the emergence of lamivudine resistance.
Methods: In total, 36 consecutive anti-HBe positive patients were treated with interferon (3 MU subcutaneously three times weekly) and lamivudine (100 mg orally once a day) for 12 months. After completion of the combined treatment, all patients continued to receive lamivudine monotherapy indefinitely.
Results: Overall, 35 patients (97%) showed virological response at 12 months. Four patients (11%) cleared HBsAg and developed anti-HBs. During the follow-up time, after the discontinuation of interferon, of 30 ± 12 months (range: 7-57 months), 13 patients (36%) exhibited breakthrough infection. The cumulative rates of breakthrough infection at the end of 1, 2, 3 and 4 years of treatment were 0%, 14%, 32%, and 59%, respectively.
Conclusions: Combination therapy appears to be effective and may also delay the selection of lamivudine-resistant variants. However, controlled trials are definitely warranted to clarify the potential benefits of combination antiviral treatment over monotherapy.
Hepatitis B virus (HBV) infection is a global public health problem and is the leading cause of cirrhosis and hepatocellular carcinoma (HCC) worldwide. It is estimated that there are at least 400 million HBV carriers in the world and that up to 1 million die annually due to hepatitis B associated liver disease.1
Pre-core mutant (HBeAg-negative) chronic hepatitis B infection represents a potentially severe and progressive form of chronic liver disease with very rare spontaneous remissions, frequent progression to cirrhosis and increased risk of the development of HCC.2 It accounts for 7-30% of patients with chronic HBV infection worldwide and affects 10-15% of patients with chronic hepatitis B in the USA and Northern Europe, as many as 50%-80% of those in Southern Europe and the Middle East, and 40-55% of those in Asia.3 Therefore, pre-core mutant infection makes a substantial contribution to hepatitis B infection across the world and its treatment is an important component in the control of hepatitis infection and associated disease.
Two forms of therapy are currently available for chronic hepatitis B: interferon (IFN) alpha and nucleoside analogs. End-of-treatment responses to IFN in patients with HBeAg-negative chronic hepatitis B range from 40% to 60%. The main advantage of IFN therapy is that a course of finite duration may achieve sustained off-therapy responses in 15-25% of these patients.4
Lamivudine, a nucleoside analog, is a potent inhibitor of HBV replication. The end-point of lack of detectable HBV DNA and normal serum alanine aminotransferase (ALT) levels is reached in 65-79% of patients at the end of 52 weeks of treatment with lamivudine.4 Unfortunately, sustained off-therapy responses to lamivudine are rare; only 11% of treated patients have a sustained response 24 weeks after the end of 12 months of therapy.5 Therefore, long-term continuous therapy rather than a limited course of treatment with lamivudine might be a more appropriate approach to therapy in chronic hepatitis B. Nevertheless, long-term treatment is associated with a high rate of viral resistance. These mutants have changes in the conserved YMDD motif of the catalytic domain of the polymerase enzyme that confer resistance to lamivudine.6 Lamivudine resistance increases from 10 to 27% at 1 year to 40-56% at 2 years and 67% at 3 years.5,7-10 Although several questions were initially raised about the clinical significance of viral resistance to lamivudine, it has now become clearer that the emergence of resistance has a negative impact on the efficacy of therapy in HBeAg-negative chronic hepatitis B patients.9,10
It is apparent that monotherapy with a single antiviral agent is insufficient in controlling HBV infection in the majority of patients. An obvious question is whether combination therapy utilizing therapeutic agents with additive or synergistic activity and a capacity to prevent development of drug resistance is a more appropriate treatment for this disease. Promising results from the combination of IFN and lamivudine have been reported in some studies, but not in others, and thus no definite conclusions can be drawn.11-14 Thus, in this study, IFN/long-term lamivudine combination therapy was evaluated in patients with chronic anti-HBe positive hepatitis B in order to determine if this strategy would improve treatment efficacy and reduce the emergence of lamivudine resistance.
Between April 1998 and October 2002, a total of 36 consecutive anti-HBe positive patients (28 males, 77.8%; mean age 49.8 years; range: 28-67 years) were enrolled in this prospective study. The study was performed in Hippokration General Hospital, Thessaloniki, a tertiary teaching hospital. To be eligible, the patients had to fulfill the following criteria: (i) age greater that 18 years; (ii) detectable HBsAg for at least 6 months; (iii) HBeAg negativity; (iv) anti-HBeAg positivity with signs of viral replication (serum HBV-DNA positive); and (v) serum ALT levels above the normal range on at least two separate occasions in the previous 6 months. Amongst the 36 patients enrolled, 24 (66.7%) were naive to antiviral treatments for hepatitis B (group A) and 12 (33.3%) had been previously treated with IFN 5 MU subcutaneous (s.c) three times weekly for at least 12 months (group B); eight of the latter had showed no response and four had relapsed after discontinuing therapy.
Baseline liver histology was assessed in 33 patients at entry (unless performed within the previous year); three patients from group B had undergone liver biopsy prior to the first course of IFN but denied to undergo a biopsy at the beginning of this study. A single pathologist evaluated all biopsy specimens and scored them according to the Knodell histological activity index (HAI).15
Patients were considered not eligible for the study if they had any of the following: (i) anti-hepatitis C virus (HCV) positive; (ii) hepatitis delta or human immunodeficiency virus (HIV) infection; (iii) decompensated chronic liver disease (bilirubin level > 42.75 Ámol/L; INR > 1.2; serum albumin < 35 g/L) or ascites, variceal bleeding or hepatic encephalopathy, hematological abnormalities (hemoglobin level < 1.7 mmol/L; white cell count < 3.5 x 109/L; platelets count < 100 x 109/L); (iv) anti nuclear antibody, anti smooth muscle antibody, or anti liver kidney microsomal antibody positive (titre > 1:80 IU/L); and (v) clinical history positive for other chronic liver disease. Pregnant and breast-feeding women were also excluded. The baseline characteristics of the patients are presented in Table 1.
All patients were scheduled to receive INF alpha-2b (Intron A, Schering Plough, Kenilworth, NJ, USA) (3 MU s.c. three times weekly) and lamivudine (100 mg orally once a day) for 12 months. We chose this particular dosing scheme of IFN because sustained response rates to IFN treatment are doubled with prolonged treatment (12 vs 5-6 months); on the contrary, the dose of IFN has little effect in sustained response rates but increases the risk of treatment discontinuation due to side-effects.4 After the completion of the combined treatment, all patients continued to receive indefinite lamivudine monotherapy. If breakthrough infection occurred, lamivudine was not discontinued; all these patients received in addition either IFN or adefovir dipivoxil. Patients were included in the analysis for a given year if they had taken lamivudine for at least 1 month during that year.
Patients were evaluated on entry, then monthly until the end of the therapy and every 6 months thereafter for vital signs, alanine and aspartate transaminase, direct and total bilirubin, albumin, complete blood counts, creatinine, amylase, creatine kinase and thyroid stimulating hormone. At 6-month intervals, blood was also tested for HBsAg, anti-HBs and for HBV-DNA.
Routine biochemical and hematological tests were performed using automated techniques. HBsAg and antibodies against HBsAg were measured using routine, commercially available enzyme immunoassays. Before January 2001, HBV DNA was detected due to cost restrictions with an in-house qualitative polymerase chain reaction (PCR) assay that had an undefined sensitivity.16 From January 2001 onward, HBV DNA levels were measured with the use of a commercially available quantitative PCR assay (Amplicor HBV-DNA Monitor Test; Roche Diagnostics, Branchburg, NJ, USA) with a sensitivity of 400 copies/mL. Breakthrough infection was defined as reappearance of serum HBV DNA after its initial disappearance.
Primary efficacy end-points included biochemical (decrease of serum ALT to within the normal range) and virological response (decrease of serum HBV DNA to undetectable levels) at 12 months, sustained suppression of HBV DNA levels (defined as the absence of detectable levels on consecutive measurements during follow-up), sustained return of serum ALT levels to normal, and loss of detectable levels of HBsAg and appearance of antibody to HBsAg (referred to as HBsAg seroconversion). Secondary end-points included incidence of breakthrough infection and safety.
Written informed consent was obtained from each patient. The study protocol conformed to the ethical guidelines of the Declaration of Helsinki.
All data were analyzed using the statistical package SPSS (version 10.0; SPSS, Chicago, IL, USA). The Mann-Whitney test was used for comparisons of quantitative variables between groups, Wilcoxon matched-pairs signed rank test for evaluation of changes of variables within the same group, and the chi-squared test for comparisons of qualitative data. In all cases, a two-tailed P-value less than 0.05 was considered statistically significant.
Demographic and clinical features of the patients at entry are shown in Table 1; patients in groups A and B did not differ significantly in any of these characteristics.
Baseline liver histology showed mild hepatitis in 12 patients (36%; eight in group A), moderate/severe hepatitis in 19 patients (58%; 14 in group A) and active cirrhosis in two (6%; both in group A).
Overall, 33 patients (92%) showed combined biochemical and virological response at 12 months. We found no differences in response rates between patients in groups A and B as well as between IFN responders and non-responders within group B.
Serum ALT normalized in all patients after a median time of 5 months (range: 1-24 months). Two patients, one from each group, showed late biochemical response at 18 and 24 months, respectively. After 12 months of therapy, mean ALT values were significantly lower than before treatment (29 IU/L vs 142 IU/L; P < 0.0005). The rate of biochemical response (96% in group A vs 92% in group B) and mean ALT values (29 IU/L in group A vs 28 IU/L in group B) after 12 months of therapy were similar in both groups.
HBV-DNA was undetectable in 35 patients (97%) at 12 months; one patient from group A exhibited breakthrough infection at 12 months after an initial (at 6 months) combined biochemical and virological response.
Four patients (11%) cleared HBsAg and developed anti-HBs; this occurred 3, 18, 18 and 36 months after the initiation of treatment, respectively. Three of these patients belonged in group A and one relapsed after an initial response to IFN. These patients were significantly younger and had significantly lower ALT levels at baseline compared to those who did not develop anti-HBs (P < 0.05 for both comparisons).
Except for patients who developed anti-HBs, all others (32/36) are still receiving lamivudine. Thirty-five patients (97%) went on to receive lamivudine for a second year, 31 (86%) for a third year, 22 (61%) for a fourth year, nine (25%) for a fifth year and five (14%) for a sixth year (Table 2). The mean ± SD duration of treatment was 39 ± 16 months (range: 3-69 months).
During a follow-up time of 30 ± 12 months (range: 7-57 months), 13 patients (36%) exhibited breakthrough infection, 10 from group A and three from group B; all the latter were IFN non-responders. The mean interval from onset of treatment to the occurrence of breakthrough infection was 24 months (range: 12-42 months). The proportion of patients that had a breakthrough infection per year during years 1, 2, 3 and 4 was 0%, 14%, 16% and 14%, respectively; no patient developed breakthrough infection thereafter (Table 2). The cumulative rates of breakthrough infection at the end of 1, 2, 3 and 4 years of treatment were 0%, 14%, 32% and 59%, respectively. Viral breakthrough was followed by an increase in ALT levels that exceeded the baseline values in five cases; values exceeding 1000 IU/L were observed in only one subject. None of the patients showed alterations of bilirubin values or hepatic decompensation during breakthrough. None of the baseline demographic and clinical features predicted for the development of lamivudine resistance. Patients who did not exhibit breakthrough infection (23/36) received lamivudine for 34 ± 3 months (range: 3-61 months).
Five patients required discontinuation of the administration of IFN after a median of 6 months (range: 4-9 months) due to adverse events such as flu-like symptoms (fatigue, low-grade fever, arthralgia and headache). None of these patients were cirrhotic and all of them continued to receive lamivudine monotherapy and showed virological and biochemical response at 12 months. After a duration of treatment of 44 ± 13 months (range: 27-61 months) one of these patients exhibited breakthrough that occurred 18 months after the initiation of combined treatment.
The current approach to therapy for HBV infection involves relatively short-term therapy with either IFN or nucleoside analogs, allowing recovery of the immune response to an extent which then allows control of the infection in the absence of further antiviral drug administration. Nevertheless, in some patients, the immune system is unable to recover and control re-emergence of HBV. This is the case in the majority of HBeAg negative viremic patients. INF has only mild to moderate virus-suppressive activity but has immunomodulatory effects and can induce an effective host immune response in susceptible patients resulting in sustained responses.17 Lamivudine has marked virus-suppressive activity in the majority of patients and enhances, in vitro, the activity of IFN against HBV.17,18 It is therefore logical to explore the efficacy of combination therapy of lamivudine and INF in the hope of increasing the virus-suppressive activity and to induce host immunity in all treated patients.
In this study, 92% of the patients showed combined biochemical and virological response at 12 months, a percentage significantly superior to the reported response rates with either IFN or lamivudine monotherapy.4 This finding provides further support to the assumption of an increased antiviral potential of combination treatment.
Long-term suppression of HBV replication is necessary until the infected cells containing covalently closed circular DNA (cccDNA) have been eliminated. One year of lamivudine does not reduce the number of infected cells to less than 10% of its initial value; furthermore, the half-life of these cells may be 10 or more years.19,20 Lamivudine has no effect on intrahepatic levels of cccDNA and on transcription of HBV-specific RNA from the cccDNA, which may explain the rapid reappearance of serum HBV DNA after cessation of antiviral therapy.21 Sustained off-therapy responses to lamivudine are rare, regardless of the duration of treatment. These findings indicate that lamivudine should be used as long-term therapy aimed at inducing a long-term remission or cure. Nevertheless, the virus-suppressive activity of lamivudine is limited in time by the progressive emergence of drug-resistant mutants. Resistance to lamivudine is clinically expressed by the virological breakthrough phenomenon, characterized by increasing levels of active viral replication, which trigger the immune response sooner or later, culminating in episodes of biochemical breakthroughs and inevitably, active liver histological lesions which usually persist thereafter with variable progression rates.9,10 As was first documented with the chemotherapy for tuberculosis and has been confirmed for oncology and infectious diseases over the decades, most recently with HIV, combination therapy to completely suppress ongoing replication is required to arrest the emergence of resistance in chronic processes.22 The synergistic antiviral activity provided by combination therapy could more rapidly decrease the viral load, thereby reducing the possibility that YMDD mutants emerge and might be selected during lamivudine treatment. In our study, during a median follow-up time of 29 months only 36% of our patients exhibited breakthrough infection. The cumulative rates of breakthrough infection at the end of 1, 2, 3 and 4 years of treatment were 0%, 14%, 32% and 59%, respectively. These rates compare favorably with those reported this far in patients receiving long-term lamivudine monotherapy for pre-core mutant chronic hepatitis B infection.5,7-10
It could be hypothesized that elimination of infected hepatocytes due to either the natural turnover of liver cells (e.g. by apoptosis) or to the HBV-specific T cell response restored by lamivudine therapy and stimulated by IFN on one hand, and hepatocellular regeneration without viral infection provided by continuing therapy on the other, might lead to the eradication of HBV infection.23,24 This has been reported in 10% of patients receiving IFN in a median time of 2.9 years after the end of IFN treatment.25,26 Whether lamivudine monotherapy will be followed by late losses of HBsAg still remains to be seen, but appears to be an unusual phenomenon.27 Loss of HBsAg and development of anti-HBs occurred in 11% of our patients. It is noteworthy that seroconversion happened in a relatively short interval after the initiation of the antiviral therapy in the majority of these patients and this can be attributed to the above-mentioned synergistic mechanism of action of IFN and lamivudine. Recently, it has been shown that, in patients with HBeAg-negative chronic hepatitis B, a sustained biochemical remission after antiviral therapy usually accompanied by low residual viremia levels and sometimes by HBsAg loss, is associated with a reduced risk of the development of liver decompensation and/or HCC and with improved survival.25 Thus, the loss of HBsAg and development of anti-HBs observed in our patients is of paramount significance. In addition, the delay in the emergence of YMDD resulting from combination treatment maintained prolonged inhibition of viral replication in our patients (HBV-DNA less than 400 copies/mL in sequential measurements) and will possibly lead to substantial long-term benefits as well.
This is the first study to report the outcome of IFN/long-term lamivudine in HBeAg negative chronic hepatitis B. Our data suggest that this type of combination therapy is effective and may also delay the selection of YMDD variants allowing the maintenance of the antiviral effect of lamivudine; it thus appears to represent an attractive approach for the antiviral treatment of these patients.