Low Resistance to Adefovir Combined With Lamivudine: A 3-Year Study of 145 Lamivudine-Resistant Hepatitis B Patients

GASTROENTEROLOGY 2007;133:1445 1451 Low Resistance to Adefovir Combined With Lamivudine: A 3-Year Study of 145 Lamivudine-Resistant Hepatitis B Patients PIETRO LAMPERTICO,* MAURO VIGANÒ,* ELENA MANENTI,* MASSIMO IAVARONE,* ERWIN SABLON, and 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; and Infectious Disease Unit, Innogenetics NV, Ghent, Belgium See editorial on page 1718. 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 adefovirlamivudine 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 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 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 LMVresistant 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. Abbreviations used in this paper: ADV, adefovir dipivoxil; LMV, lamivudine; PCR, polymerase chain reaction. 2007 by the AGA Institute 0016-5085/07/$32.00 doi:10.1053/j.gastro.2007.08.079

1446 LAMPERTICO ET AL GASTROENTEROLOGY Vol. 133, No. 5 Table 1. Demographic, Clinical, and Virologic Features of the 145 LMV-Resistant Patients With Chronic Hepatitis B Enrolled in the Study Patient features No. Age (y), median (range) 56 (19 77) No. of male patients (%) 122 (84) No. HBeAg negative (%) 124 (86) No. with cirrhosis (%) a 106 (73) ALT (IU/L), median (range) 58 (13 2870) HBV DNA (log 10 copies/ml), median (range) 6.0 (3.5 9.3) No. with genotype D of HBV (%) b 124 (92) No. with rtm204v/i mutation (%) 135 (93) No. with rtl180m mutation (%) 96 (66) No. with rtv173i/l mutation (%) 15 (10) No. with rtl80v/i mutation (%) 78 (54) Serum creatinine (mg/dl), median (range) 0.9 (0.6 1.3) a Twelve patients had HCC. b A total of 135 serum samples were available for analysis. 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 LMVresistant 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 log 10 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 log 10 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. Serum Assays Serum aspartate aminotransferase, ALT, albumin, bilirubin, and creatinine levels and international ratio of prothrombin time were measured by standard laboratory procedures. Serum -fetoprotein level was measured using a standard assay (IRMA; Abbott Laboratories, North Chicago, IL). Serum hepatitis B surface antigen, antibody to hepatitis surface antigen, hepatitis B e antigen (HBeAg), and antibody to hepatitis B e antigen were detected by microparticle enzyme immunoassay (AXSYM; Abbott Laboratories). Antibody to hepatitis delta virus was assessed by enzyme-linked immunosorbent assay (Sorin Biomedica, Saluggia, Italy). Antibody to human immunodeficiency virus was detected by human immunodeficiency virus 1 third-generation assay (AXSYM HIV 1/2; Abbott Laboratories) and antibody to hepatitis C virus by a secondgeneration test system (Ortho DS, Raritan, NJ). Serum HBV DNA level was assessed every 2 months by Versant 3.0 (branched DNA; Bayer Corp, Tarrytown, NJ), with a sensitivity limit of 2000 copies/ml or 3.3 log 10 copies/ml. Serum samples lacking detectable HBV DNA by Versant 3.0 ( 2000 copies/ml) at year 1, 2, 3, and 4 were retested using a more sensitive, real-time polymerase chain reaction (PCR) assay, the COBAS TaqMan HBV test (Roche Molecular Systems, Inc, Branchburg, NJ), with a lower limit of quantification of 35 copies/ml (1.5 log 10 copies/ml). LMV-associated (rtv173l, rtl180m, and rtm204v/i/s) and ADV-associated (rta181t/v and rtn236t) mutations were determined using a line probe assay (INNO- LiPA HBV DR v2; Innogenetics NV, Ghent, Belgium). 17 The rti233v mutation was looked for by the recently developed research prototype INNO-LiPA HBV DR v3 assay 18 in patients with HBV DNA levels 2000 copies/ml at year 3. End Points The primary end points of the study were genotypic resistance, virologic breakthrough, and clinical breakthrough to ADV. Genotypic resistance to ADV was defined as the emergence of ADV resistance signature mutations (ie, rta181t/v and rtn236t), which were looked for at yearly intervals by sensitive molecular analysis in all HBV DNA positive serum samples. A virologic breakthrough was defined as 1 log 10 increase in serum HBV DNA level compared with on-treatment nadir. Clinical resistance to ADV was the presence of ADV signature mutations, in the context of a virologic breakthrough and elevation of ALT level. Secondary end points were (1) liver-related complications, such as clinical decompensation (ascites, encephalopathy, jaundice, and gastrointestinal bleeding defined according to internationally agreed criteria) and HCC, and (2) renal toxicity (ie, the proportion of patients with 0.5-mg increase in serum creatinine level compared with baseline values at 2 separate checkpoints). Progres-

November 2007 LOW RESISTANCE UNDER ADEFOVIR LAMIVUDINE 1447 Table 2. Virologic Response of 145 LMV-Resistant Patients to ADV-LMV Combination Therapy During a Median of 42 Months Months of treatment 12 24 36 48 Virologic response (n 145) (n 112) (n 78) (n 39) HBV DNA undetectable a 89 (61) 78 (70) 62 (79) 32 (82) HBV DNA level 35 to 10 (7) 11 (10) 6 (8) 1 (2) 2000 copies/ml HBV DNA level 2000 46 (32) 23 (20) 10 (13) 6 (15) copies/ml b Virologic breakthrough c 0 0 0 0 NOTE. All values are expressed as n (%). a Lower limit of quantification of real-time PCR: 35 copies/ml (1.5 log copies/ml). b Lower limit of quantification of Versant 3.0: 2000 copies/ml (3.3 log copies/ml). c 1 log increase in HBV DNA level compared with on-treatment nadir, confirmed on 2 occasions. sion to cirrhosis was defined on clinical grounds, that is, albumin level 3.5 g/dl, platelet count 100,000 mm 3, clinical decompensation, and/or ultrasound demonstration of surface nodularity, splenomegaly, and 15-mm portal vein diameter. Statistical Analysis Data were expressed as median and range for discrete variables and as counts and percentages for qualitative variables. Significance of differences in the distribution of qualitative variables was assessed by Fisher exact test. The cumulative probabilities of developing genotype resistance to ADV and HCC were estimated by the Kaplan Meier method. Statistical analysis was performed with Stata (Stata Statistical Software release 7.0; Stata Corp, College Station, TX). Results 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). Serum samples lacking detectable HBV DNA by Versant 3.0 ( 2000 copies/ml) at year 1, 2, 3, and 4 were retested using a more sensitive, real-time PCR assay. The majority of the patients lacking detectable HBV DNA by Versant 3.0 were also HBV DNA negative by real-time PCR (Table 2). The 1-, 2-, 3-, and 4-year rates of negative PCR ( 35 copies/ml HBV DNA) were 61%, 70%, 79%, and 82%, respectively (Table 2). None of the real-time PCR-positive patients with 2000 copies/ml HBV DNA had a virologic breakthrough. 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 wildtype 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 Table 3. Prevalence and Incidence of Genotypic Resistance to ADV in 145 LMV-Resistant Patients Treated With ADV-LMV Combination Therapy During a Median of 42 Months ADV mutants Baseline (n 145) 12 (n 139) Months of treatment 24 (n 112) 36 (n 78) 48 (n 39) rtn236t 0 0 0 0 0 rta181v a 1 (1%) 0 0 0 0 rta181t a 5 (3%) 1 (0.7%) 1 (0.9%) 1 (1.3%) 0 Overall 6 (4%) 1 (0.7%) 1 (0.9%) 1 (1.3%) 0 NOTE. All values are expressed as n (%). a The rta181t and rta181v mutations were detected as a mixed population, together with the wild-type sequence rta181a, in all serum samples. Molecular analysis was performed in patients with detectable serum HBV DNA levels: 145 patients at baseline, 46 at month 12, 23 at month 24, 10 at month 36, and 6 at month 48.

1448 LAMPERTICO ET AL GASTROENTEROLOGY Vol. 133, No. 5 Figure 1. Time course of virologic response and resistance pattern in 6 patients with baseline rta181t/v mutation (cases 1 6). 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

November 2007 LOW RESISTANCE UNDER ADEFOVIR LAMIVUDINE 1449 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. Figure 2. Time course of virologic response and resistance pattern in 3 patients who developed rta181t mutation during combined ADV- LMV treatment (cases 7 9). 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 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) Figure 3. Four-year cumulative rates of clinical decompensation and HCC in 94 cirrhotic patients without HCC at baseline.

1450 LAMPERTICO ET AL GASTROENTEROLOGY Vol. 133, No. 5 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. Discussion 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 LMVresistant patients with chronic hepatitis B that resulted in 25% rates of ADV mutations during 2 years of therapy, 9 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 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

November 2007 LOW RESISTANCE UNDER ADEFOVIR LAMIVUDINE 1451 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 costeffectiveness. 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. References 1. Lok AS, McMahon BJ. Chronic hepatitis B. Hepatology 2007;45: 507 539. 2. Marcellin P, Chang TT, Lim SG, et al, for the Adefovir Dipivoxil 437 Study Group. Adefovir dipivoxil for the treatment of hepatitis B e antigen positive chronic hepatitis B. N Engl J Med 2003;348: 806 814. 3. Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, et al, for the Adefovir Dipivoxil 438 Study Group. Long-term therapy with adefovir dipivoxil for HBeAg-negative chronic hepatitis B for up to 5 years. Gastroenterology 2006;131:1743 1751. 4. Perrillo R, Hann HW, Mutimer D, et al. Adefovir dipivoxil added to ongoing lamivudine in chronic hepatitis B with YMDD mutant hepatitis B virus. Gastroenterology 2004;126:81 91. 5. Peters MG, Hann HW, Martin P, et al. Adefovir dipivoxil alone or in combination with lamivudine in patients with lamivudine-resistant chronic hepatitis B. Gastroenterology 2004;126:91 101. 6. Papatheodoridis GV, Dimou E, Dimakopoulos K, et al. Outcome of hepatitis B e antigen negative chronic hepatitis B on long-term nucleos(t)ide analog therapy starting with lamivudine. Hepatology 2005;42:121 129. 7. Lampertico P, Viganò M, Manenti E, et al. Adefovir rapidly suppresses hepatitis B in HBeAg-negative patients developing genotypic resistance to lamivudine. Hepatology 2005;42:1414 1419. 8. Liaw YF, Lee CM, Chien RN, et al. Switching to adefovir monotherapy after emergence of lamivudine-resistant mutations in patients with liver cirrhosis. J Viral Hepat 2006;13:250 255. 9. Fung SK, Chae HB, Fontana RJ, et al. Virologic response and resistance to adefovir in patients with chronic hepatitis B. J Hepatol 2006;44:283 290. 10. Yeon JE, Yoo W, Hong SP, et al. Resistance to adefovir dipivoxil (ADV) in lamivudine resistant chronic hepatitis B patients treated with ADV. Gut 2006;55:1488 1495. 11. Lee YS, Suh DJ, Lim YS, et al. Increased risk of adefovir resistance in patients with lamivudine-resistant chronic hepatitis B after 48 weeks of adefovir dipivoxil monotherapy. Hepatology 2006;43:1385 1391. 12. Chen CH, Wang JH, Lee CM, et al. Virological response and incidence of adefovir resistance in lamivudine-resistant patients treated with adefovir dipivoxil. Antiviral Ther 2006;11:771 778. 13. Schiff E, Lai CL, Hadziyannis S, et al. Adefovir dipivoxil for waitlisted and post-liver transplantation patients with lamivudineresistant hepatitis B: final long-term results. Liver Transpl 2007;13:349 360. 14. Locarnini S, Mason WS. Cellular and virological mechanisms of HBV drug resistance. J Hepatol 2006;44:422 431. 15. Zoulim F. In vitro models for studying hepatitis B virus drug resistance. Semin Liver Dis 2006;26:171 180. 16. Benhamou Y, Thibault V, Vig P, et al. Safety and efficacy of adefovir dipivoxil in patients infected with lamivudine-resistant hepatitis B and HIV-1. J Hepatol 2006;44:62 67. 17. Hussain M, Fung S, Libbrecht E, et al. Sensitive line probe assay that simultaneously detects mutations conveying resistance to lamivudine and adefovir. J Clin Microbiol 2006;44:1094 1097. 18. Doutreloigne J, Van Assche E, Sablon E. Analysis of drug resistance mutations associated with entecavir, tenofovir and adefovir dipivoxil treatment with a novel line probe assay (LiPA) (abstr). J Hepatol 2007;46:S196. 19. Schildgen O, Sirma H, Funk A, et al. Variant of hepatitis B virus with primary resistance to adefovir. N Engl J Med 2006;354: 1807 1812. 20. Curtis M, Zhu Y, Borroto-Esoda K. HBV rti233v polymerase variant remains sensitive to adefovir (abstr). J Hepatol 2007;46: S26. 21. Villet S, Pichoud C, Villeneuve JP, et al. Selection of a multiple drug-resistant hepatitis B virus strain in a liver-transplanted patient. Gastroenterology 2006;131:1253 1261. 22. Liaw YF, Sung JJY, Chow WC, et al, for the Cirrhosis Asian Lamivudine Multicentre Study Group. Lamivudine for patients with chronic hepatitis B and advanced liver disease. N Engl J Med 2004;351:1521 1531. 23. Di Marco V, Marzano A, Lampertico P, et al, for the Italian Association for the Study of the Liver (AISF). Clinical outcome of HBeAg-negative chronic hepatitis B in relation to virological response to lamivudine. Hepatology 2004;40:883 891. 24. van Bömmel F, Zöllner B, Sarrazin C, et al. Tenofovir for patients with lamivudine-resistant hepatitis B virus (HBV) infection and high HBV-DNA level during adefovir therapy. Hepatology 2006; 44:318 325. 25. Hezode C, Chevaliez S, Bouvier-Alias M, et al. Efficacy and safety of adefovir dipivoxil 20mg daily in HBeAg-positive patients with lamivudine-resistant hepatitis B virus and a suboptimal virological response to adefovir dipivoxil 10 mg daily. J Hepatol 2007; 46:791 796. 26. Rapti I, Dimou E, Mitsoula P, et al. Adding-on versus switching-to adefovir therapy in lamivudine-resistant HBeAg-negative chronic hepatitis B. Hepatology 2007;45:307 313. Received April 18, 2007. Accepted August 29, 2007. Address requests for reprints to: Massimo Colombo, MD, 1st Division of Gastroenterology, Fondazione IRCCS Maggiore Hospital, Policlinico, Mangiagallii, Regina Elena, University of Milan, Via F. Sforza 35-20122 Milan, Italy. e-mail: massimo.colombo@unimi.it; fax: (39) 0250320410. Supported in part by Consorzio Interuniversitario Trapianti d Organo, Rome, and FIRST 2006, University of Milan. The authors report no conflict of interest. The authors thank Caterina M. Puricelli for her expert secretarial assistance and Roche Molecular Diagnostics (Rotkreuz, Switzerland) for kindly providing COBAS TaqMan HBV test kits.