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Low Resistance to Adefovir Combined With Lamivudine: A 3-Year Study of 145 Lamivudine-Resistant Hepatitis B Patients
  Gastroenterology Nov 2007
Pietro Lampertico_, Mauro Vigano_, Elena Manenti_, Massimo Iavarone_, Erwin Sablon, Massimo Colombo_ _ "A. M. and A. Migliavacca" Center for Liver Disease, Division of Gastroenterology, Department of Medicine, Fondazione Policlinico, Mangiagalli e Regina Elena, University of Milan, Milan, Italy Infectious Disease Unit, Innogenetics NV, Ghent, Belgium
"In conclusion, in LMV-resistant patients, combined ADV-LMV therapy attenuated the risk of genotypic resistance to ADV, preventing virologic and clinical breakthrough during a 3-year period."

Background & Aims: Adefovir monotherapy is an established treatment modality for lamivudine-experienced patients with chronic hepatitis B, but it carries a significant risk of resistance in the long term. We assessed whether this risk could be overcome by adefovir-lamivudine combination therapy.
Methods: A total of 145 lamivudine-resistant patients with chronic hepatitis B (73% cirrhotics, 86% hepatitis B e antigen negative, 92% genotype D) were treated with adefovir 10 mg in addition to lamivudine 100 mg. Liver function tests and hepatitis B virus (HBV) DNA (Versant 3.0) were assessed bimonthly, whereas adefovir-related mutations were searched by INNO-LiPA assay at baseline and at yearly intervals.
Results: During 42 months (range, 12-74), 116 patients (80%) cleared serum HBV DNA, 67 (84%) had normalized alanine aminotransferase levels, and 145 (100%) remained free of virologic and clinical breakthroughs, independently of the degree of HBV suppression. The rtA181V/T was the only adefovir-related mutation detected, which occurred in 6 patients at baseline (4%; 1 rtA181V and 5 rtA181T) and in an additional 3 patients (2%; all rtA181T) during treatment. In all these 9 patients, HBV DNA levels progressively declined during therapy to become undetectable in 7 (78%). The 1-, 2-, 3-, and 4-year cumulative rates of de novo rtA181T were 1%, 2%, 4%, and 4%, respectively. None of the cirrhotic patients clinically decompensated, but 11 (12%) developed hepatocellular carcinoma.
Conclusions: Under prolonged adefovir-lamivudine therapy, patients with lamivudine-resistant hepatitis B were unlikely to develop genotypic resistance to adefovir and had durable prevention of virologic and clinical breakthrough.
The paradigm of chronic hepatitis B therapy is achieving a persistent suppression of hepatitis B virus (HBV), aimed at halting progression of liver damage and preventing development of liver-related complications.1 Nowadays, either limited treatment with interferon alfa or indefinite therapy with nucleos(t)ide analogues is the mainstay therapeutic strategy against chronic HBV infection.1 Adefovir dipivoxil (ADV) has gained popularity as a first-line treatment modality for patients with compensated hepatitis B, by virtue of its satisfactory efficacy coupled with an excellent record of safety.2, 3 ADV is also a first-option therapy for lamivudine (LMV)-resistant patients with chronic hepatitis B, although it is still controversial whether LMV should be continued or not in these patients.4, 5, 6, 7, 8, 9, 10, 11, 12, 13 The preliminary studies, in fact, which suggested ADV monotherapy to be as effective as combination with LMV in the treatment of LMV-resistant patients, independently of disease severity,4, 5, 8 were later contradicted by 4 studies demonstrating rapid emergence (25% in 2 years) of ADV resistance and virologic breakthrough in patients so treated.9, 10, 11, 12 Combination therapy, therefore, is now being reconsidered as a therapeutic option for LMV-experienced patients, its superiority versus ADV monotherapy in these patients being biologically plausible, as shown by in vitro studies that demonstrated no cross-resistance between these 2 nucleos(t)ide analogues.14, 15 However, at present there are no data on the long-term efficacy of combination therapy in LMV-resistant patients except for a small study in human immunodeficiency virus-coinfected patients with chronic hepatitis B who remained ADV mutant-free during 3 years of ADV-LMV combination therapy.16
The present study assesses the efficacy of combined ADV-LMV therapy in a large series of white patients with LMV-resistant hepatitis B followed up for a median of 42 months with frequent measurements of both serum HBV DNA levels and genotypic resistance profile by means of sensitive assays.
Materials and Methods
Study Profile

Starting in 2001, when ADV became available in our center for the rescue treatment of patients with LMV-resistant strains of HBV, 282 patients were enrolled into a prospective, open-label study aimed at assessing the long-term efficacy and safety of ADV added to ongoing LMV. Here we present the first 145 consecutive LMV-resistant patients with chronic hepatitis or cirrhosis due to HBV who completed at least 1 year of treatment with ADV and LMV. These patients were followed up during treatment for a median of 42 months (range, 12-74 months); all were followed up for 1 year, 112 for 2 years, 78 for 3 years, and 39 for 4 or more years. The present study includes 69 patients who were previously described in the 24-month follow-up study.7 LMV resistance was defined as >1 log10 increase in serum HBV DNA level compared with on-treatment nadir and confirmed by molecular analysis. Table 1 summarizes the demographic, clinical, and virologic features of the patients at baseline. Hepatocellular carcinoma (HCC) was already present in 12 cirrhotic patients. Eighty patients (55%) received ADV in addition to LMV at the time of clinical resistance to LMV, that is, when HBV DNA level peaked above 6 log10 copies/mL and serum alanine aminotransferase (ALT) level was higher than the upper normal limit; 65 patients (45%) were given ADV at the time of virologic breakthrough, that is, when HBV DNA level was between 3 and 6 logs and ALT level was less than the upper normal limit. Ten patients were undergoing immunosuppressive therapy because of liver (n = 5), kidney (n = 3), or bone marrow transplantation (n = 2). All patients gave their written informed consent. Details of the study were approved by the local institutional review committee.
HBV DNA Response

Serum HBV DNA became undetectable (<2000 copies/mL) in 88 patients (61%) by month 6, in an additional 11 patients (8%) by year 1, in 10 (7%) by year 2, and in 7 (5%) additional patients by year 3 (overall, 116 [80%]). The 1-, 2-, 3-, and 4-year rates of HBV DNA clearance were 68% (99/145), 79% (89/112), 87% (68/78), and 85% (33/39), respectively. Twenty-nine patients (20%) remained viremic despite long-term combined treatment, showing, however, a median decrease in HBV DNA levels from the baseline values of 8.2 log (range, 3.9-9.3) to 4.4 log (range, 3.3-6.1) of the last available serum sample.
During surveillance with Versant 3.0, none of the patients with a complete (n = 116) or incomplete (n = 29) HBV DNA clearance had a virologic breakthrough during the study period (Table 2).
Genotypic Resistance to ADV and LMV
Genotypic resistance to ADV was looked for in all patients at baseline (n = 145) and in 85 serum samples from 46 patients with persistent viremia (>2000 copies/mL) while on treatment. Among these 85 on-treatment serum samples, 46 were collected at year 1, 23 at year 2, 10 at year 3, and 6 at year 4.
The rtN236T mutation was not found in any baseline or on-treatment serum sample (Table 3). By converse, 6 patients (4%) circulated at baseline the rtA181T/V mutation as a mixed viral population with the rtA181A wild-type sequence; 5 patients had the rtA181T and 1 the rtA181V mutation. Figure 1 shows the time course of the virologic response and ADV resistance profile during combined treatment in these 6 patients (cases 1-6). Three patients (cases 1, 2, and 5) rapidly cleared HBV DNA, whereas the remaining 3 (cases 3, 4, and 6) had the pattern of ADV resistance confirmed in the year 1 serum sample, in the context of a progressively declining viremia, which became undetectable in 2 of them. The only patient (case 4) who did not clear HBV DNA had only 6 months of follow-up after the molecular identification of mutated strains. Among the 139 patients devoid of ADV resistance at baseline, none developed the rtN236T or the rtA181V mutation but 3 (2%) circulated the rtA181T mutation as a mixed viral population with wild-type strains rtA181A after 12, 24, and 36 months of combined therapy (cases 7-9; Figure 2). Despite emergence of this mutation, serum HBV DNA levels continued to decline progressively in all 3 patients, becoming undetectable in 2 (cases 7 and 8) after 24 and 28 months of combined treatment (Figure 2). The rates of de novo genotypic resistance to rtA181T were 0.7% (1/139) at month 12, 0.9% (1/112) at month 24, 1.3% (1/78) at month 36, and 0% (0/39) at month 48 (Table 2), with yearly estimated cumulative rates of 1%, 2%, 4%, and 4%, respectively.
By the end of the study, only 2 of the 9 ADV-resistant patients failed to achieve undetectable HBV DNA, however, in the context of a continuous tapering down of a mixed viral population of mutated and wild-type sequences at position 181 of the pol gene. Both wild-type and mutated strains at position rt181 were quantified by densitometric comparison of the specific bands on the INNO-LiPA strips. In 1 patient (case 4), mutated rtA181V prevailed both at baseline (80%) and at month 12 (60%). In the second patient (case 9), the rtA181T was detected at month 36 only, representing 10% of the overall strain population.
In 46 patients who remained viremic during combination therapy, the pattern of LMV resistance was assessed at yearly intervals and compared with baseline. LMV-resistant mutations were persistently detected in 43 patients (94%).
To define factors associated with persistent viremia at year 3, virologic, clinical, and demographic features at baseline of the 10 patients with >2000 copies/mL HBV DNA were compared with those of the 68 patients with <2000 copies/mL HBV DNA. Viremic patients were more likely to have HBeAg (50% vs 7%; P < .001), genotype non-D (30% vs 6%; P = .03), and >8 logs copies/mL HBV DNA (80% vs 24%; P < .001). None of the 10 patients with persistent viremia at year 3 circulated the rtI233V mutation, either at baseline or year 3.
ALT and Serologic Response
Sixty-seven patients (84%) with high baseline levels of ALT showed ALT normalization during treatment, at rates of 84% (67/80), 87% (49/62), and 89% (39/45) after 1, 2, and 3 years, respectively. Among the 65 patients with normal ALT levels at baseline, only 1 (1.5%) had an elevated ALT level during treatment without any change in viremia. Overall, none of the 145 patients had a clinical breakthrough during therapy.
Eight patients (38%) lost HBeAg and 5 (24%) seroconverted to antibody to hepatitis B e antigen after 6-50 months of treatment (median, 23 months). Two of these patients cleared serum hepatitis B surface antigen. One, who later seroconverted to antibody to hepatitis surface antigen, withdrew from antiviral therapy, remaining hepatitis B surface antigen and HBV DNA negative and antibody to hepatitis surface antigen positive for the following 36 months (last serum sample available). The other patient cleared serum hepatitis B surface antigen without seroconverting to antibody to hepatitis surface antigen; therefore, this patient continued to receive antiviral treatment.
Progression of Hepatitis B and Survival
None of the 39 patients with chronic hepatitis progressed to cirrhosis or developed HCC. Two patients died of HBV-unrelated causes (ie, recurrent hemangioendothelioma and recurrent hematologic disease). Of the 12 cirrhotic patients with HCC at the onset of the study, 2 underwent liver transplantation and 2 died of HCC progression. Among 94 HCC-free cirrhotic patients, 11 (12%) developed de novo HCC after 3-38 months of treatment (median, 12 months). In 6 patients, HCC developed 3-27 months (median, 13 months) following serum HBV DNA clearance; in the other 5 patients with persistent viremia, HCC developed 3-38 months (median, 10 months) after addition of ADV, and median HBV DNA level at the time of HCC development was 85,200 copies/mL (range, 5000-231,000 copies/mL). The 4-year cumulative probability of developing HCC was 15% (Figure 3). Four patients underwent liver transplantation, and 4 patients died because of liver failure due to HCC progression (n = 2) or causes unrelated to HBV (ie, lung cancer and cholangiocarcinoma). None of the 94 HCC-free cirrhotic patients developed clinical decompensation during the study period (Figure 3).
Safety and Tolerability
Median serum creatinine level was 0.9 mg/dL (range, 0.6-1.3 mg/dL) at baseline and 0.9 mg/dL (range, 0.4-1.9 mg/dL) at the end of study (not significant). Ten patients (7%) had to reduce ADV dosing from 10 mg/day to 10 mg every other day because of a >0.5-mg/dL increase in serum creatinine level compared with baseline, which occurred after a median of 8 months (range, 5-17 months) of treatment. Renal function stabilized or improved in all patients after dose adjustment, and none of the patients with reduced ADV dosing had a virologic breakthrough during 6-19 months (median, 12 months) of follow-up.
This is the first study in a large cohort of white patients with LMV-resistant hepatitis B infection to assess the long-term efficacy of ADV-LMV therapy. More than two thirds of LMV-resistant patients achieved and maintained a virologic response during a median of 42 months of therapy with ADV-LMV. More importantly, none of the patients developed virologic or clinical breakthrough, including those with incomplete viral suppression. An additional interesting finding was that none of the patients developed genotypic resistance for rtN236T and for rtA181V, the 2 clinically relevant molecular signature mutations associated with ADV resistance. Finally, all 3 patients (2%) who developed the rtA181T mutation achieved an antiviral response on continuation of therapy, which could also suppress ADV-resistant strains in those few patients (4%) who had these mutants present at the onset of combined treatment.
In comparison with ADV monotherapy in LMV-resistant patients with chronic hepatitis B that resulted in 25% rates of ADV mutations during 2 years of therapy,9, 10, 11, 12 our "add on" strategy was superior to a "switch to" strategy in the treatment of LMV-resistant patients, because it caused very low rates of mutated ADV strains. The possible explanation for this is the ability of ADV and LMV in combination to cross-inhibit the corresponding drug-related HBV mutants, thereby preventing accumulation of mutated strains with adequate fitness for replication.14, 15
The low rates of ADV resistance found in our patients receiving combination therapy are unlikely biased by an underestimation effect. We have, in fact, consecutively investigated a large number of patients with chronic hepatitis B who were followed up for an average of 42 months and were monitored with the most sensitive molecular tools available for the detection of ADV resistance, like INNO-LiPA HBV DR v2.17 With this assay, in fact, we could identify minor populations of mutated HBV strains that emerged at each annual follow-up point. Because we acknowledge, however, that the risk of developing drug resistance may go beyond the time frame covered by the present study, we believe that surveillance of our patients should be prolonged further to establish the long-term efficacy of the combination regimen. The lesson from HBeAg-negative, LMV-naive patients under ADV monotherapy, in fact, was the delayed emergence of resistant virus strains, from 11% at year 3 to 29% at year 5 of follow-up.3
In our study, there were 9 patients (11%) who failed to clear serum HBV DNA despite 3 years of combination therapy and absence of known resistant ADV strains of HBV. Although suboptimal responses to combination therapy in these patients were likely due to high pretreatment levels of HBV DNA, confirming the importance of baseline viremia in the outcome of ADV therapy,7 we cannot exclude other interpretations, such as pol gene polymorphisms other than the rtI233V.19, 20 The latter was not present in any of our suboptimal responders. Complete suppression of viral replication, in fact, is the only approach to prevent evolutionary changes in the HBV polymerase gene activity, which may later erode the clinical efficacy of treatment by development of multiple drug-resistant strains.21, 22, 23 From a practical point of view, patients who unsatisfactorily responded to combination therapy might achieve a sustained suppression of HBV with more active drugs like tenofovir or higher ADV dosing.24, 25 However, because the clinical experience with 20 mg ADV is too limited, we acknowledge that increased dosing with ADV should be applied under strict surveillance of renal function.
While none of the cirrhotic patients in our study developed clinical decompensation during 3 years of ADV-LMV combination, 11 (12%) developed HCC. These findings confirm that the risk of neoplastic transformation is maintained despite long-term suppressive therapy, as also reported in patients on LMV or ADV monotherapy.3, 22, 23 These important changes in the natural course of HBV-associated liver disease following therapy with anti-HBV analogues has made HCC the dominant indication for liver transplantation and the prime cause of death in patients with chronic hepatitis B in Italy.
Our data provide some preliminary information on how to define an optimal algorithm for the management of LMV-resistant patients with chronic hepatitis B. The current guidelines suggest that these patients should undergo surveillance at 3-month intervals with sensitive HBV DNA assays with the goal of intercepting as early as possible a loss of virologic response to nucleos(t)ides, thereby improving the outcome of rescue treatments with ADV.1 Our findings suggest that patients achieving a complete virologic response to combination therapy could safely be enrolled in a prospective study of relaxed intervals of serum HBV DNA monitoring. One point of controversy, in fact, is whether ADV-LMV combined therapy is cost-effective in the treatment of LMV-resistant patients with chronic hepatitis B, that is, whether incremental costs of maintaining LMV are outbalanced by substantial therapeutic benefits. The only study that compared monotherapy with combination therapy did confirm the clinical advantage of the latter regimen over the former, but it was underpowered to assess cost-effectiveness.26 A randomized controlled study would probably help to settle this issue.
In conclusion, in LMV-resistant patients, combined ADV-LMV therapy attenuated the risk of genotypic resistance to ADV, preventing virologic and clinical breakthrough during a 3-year period.
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