Chronic infection with the hepatitis B virus (HBV)

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1 Adding-on Versus Switching-to Adefovir Therapy in Lamivudine-Resistant HBeAg-Negative Chronic Hepatitis B Irene Rapti, 1 Evangelini Dimou, 1,2 Panayota Mitsoula, 2 and Stephanos J. Hadziyannis 1,2 We studied the long-term efficacy of adefovir dipivoxil (ADV) treatment in 42 HBeAgnegative patients with chronic hepatitis B (CHB) who had developed genotypical lamivudine (LAM) resistance with virological and clinical breakthroughs under long-term LAM treatment. Patients were allocated in 2 treatment groups. In the first (n 14), LAM was switched to ADV monotherapy whereas in the second (n 28) ADV was added to LAM. The two groups did not differ in patients characteristics, all of them having HBV genotype D infection with the precore stop codon mutation. Within 12 months from start of ADV treatment, serum HBV DNA became nondetectable and ALT normalized in 71% and 90% of patients, respectively, with no difference between the 2 arms. Patients with baseline HBV DNA levels less than 10 7 copies/ml experienced a significantly earlier and more frequent decline in serum HBV DNA to nondetectable levels as compared with patients with greater than 10 7 HBV DNA copies/ml at baseline (P ) This response has hitherto been maintained (median treatment duration 40 months) in all patients with ADV added to LAM, whereas virological and biochemical breakthroughs due to development of ADV signature resistance mutations occurred in 3 of 14 patients (21%) on ADV monotherapy 15 to 18 months from start of treatment (P ). Conclusion: Adding ADV to LAM in HBeAgnegative CHB patients with LAM resistance effectively suppresses HBV replication in most of them and induces biochemical remission that can be maintained in all of them at least for 3 years without any evidence of development of resistance to ADV. (HEPATOLOGY 2007;45: ) See Editorial on Page 266 Chronic infection with the hepatitis B virus (HBV) represents a global health problem, being a major cause of liver disease, morbidity, and mortality. Effective long-term treatment of chronic viral B liver disease improves significantly patients survival and reduces the risk of development of major complications. 1 Longterm nucleos(t)ide analog administration represents the Abbreviations: ADV, adefovir dipivoxil; CHB, chronic hepatitis B; HBeAg, hepatitis B e antigen; LAM, lamivudine. From the 1 Department of Medicine and Liver Unit, Henry Dunant Hospital, Athens, Greece, and the 2 Hepatitis Research Laboratory at Evgenidion Hospital, Athens University, Athens, Greece. Received August 11, 2006; accepted November 15, Address reprint requests to: Stephanos J. Hadziyannis, Department of Medicine & Liver Unit, Henry Dunant Hospital, 107 Messogion Avenue, Athens, Greece. hadziyannis@ath.forthnet.gr; fax: (30) Copyright 2007 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience ( DOI /hep Potential conflict of interest: Nothing to report. first-line/first-choice therapy for most patients with chronic hepatitis B (CHB), particularly those with the hepatitis B e antigen (HBeAg)-negative type of the disease. 1-5 Lamivudine (LAM) is the first nucleoside analog approved in the treatment of CHB and, because of potency, safety profile, and relatively low cost, it has been and is still widely applied globally as first-choice therapy for CHB patients either HBeAg-positive or HBeAg-negative. 1 However, approximately 70% of CHB patients in long-term LAM therapy develop resistance to the drug within approximately 3 years of treatment and experience clinical relapses that may be severe and occasionally life threatening. 4,6-9 Continuation of LAM therapy in patients who have developed virologic resistance is of no benefit to them. 7,10 LAM-resistant patients have been treated with adefovir dipivoxil (ADV) either in monotherapy or in combination with LAM without significant differences between the two regimens at least during the first year of treatment. 11,12 The recently approved nucleoside analog entecavir is also effective; however, entecavir resistance develops in 9% of LAM-resistant patients within

2 308 RAPTI ET AL. HEPATOLOGY, February 2007 months of therapy. 13,14 No data exist on the long-term efficacy of switching from LAM to ADV versus adding ADV in HBeAg-negative LAM-resistant patients. In our center, an open-label study of long-term LAM treatment in HBeAg-negative CHB patients has been running since When ADV became first available in an expanded access program, a two-arm study comparing the efficacy and safety of adding ADV in LAM-failing patients versus switching from LAM to ADV was initiated. We report on the long-term efficacy of up to 4 years duration in the two arms of this ongoing study. Patients and Methods Study Population. Adult patients with HBeAg-negative HBV chronic liver disease participating in an ongoing, previously described protocol of long-term LAM monotherapy 7,8 were included in the current randomized controlled study if they had developed genotypical HBV resistance plus virological and biochemical breakthroughs to LAM. Patients included in the source protocol of longterm LAM treatment were hepatitis B surface antigen (HBsAg)-positive, HBeAg-negative/anti-HBe positive for 6 months or longer, had elevated ALT values in three separate monthly occasions, HBV-DNA greater than 10 5 copies/ml within the last month before starting LAM therapy, and all had compensated liver disease with histological findings of chronic hepatitis either with or without histological evidence of cirrhosis. Patients were excluded if they had antibody to hepatitis C or D virus or to human immunodeficiency virus (HIV), or had received a liver transplant or any antiviral drug other than IFN- in the past. All patients gave written informed consent to participate in these studies. Both study protocols conformed to the ethical guidelines of the 1975 Declaration of Helsinki. Long-Term ADV Treatment in Patients Who Developed LAM Resistance. Forty-two patients who developed virological and clinical resistance to LAM were included in this two-arm treatment protocol of ADV at a 10-mg daily dose. In arm A, LAM treatment was switched to ADV whereas in arm B ADV was added on LAM. Virological Assays. Serum HBV-DNA levels were determined by a commercially available quantitative PCR assay (Amplicor HBV Monitor Test, Roche Diagnostics GmbH, Mannheim, Germany) with a sensitivity, according to the manufacturer, of approximately 1,000 copies/ml (cp/ml) as well as by a real time in-house PCR of similar sensitivity. All serum samples with HBV DNA nondetectable by or close to the cutoff level of these PCR assays, were retested by the Cobas TaqMan PCR assay supplied by Roche. 15,16 Sequencing of the HBV genome for YMDD and ADV mutations was performed at baseline and on any occasion of virological breakthrough under therapy. HBV-DNA extraction, PCR amplification, and sequencing were performed as described in a previous study of our group, 7 using Dye Primer Cycle Sequencing Kit and the Open Gene automated DNA sequencing system (Visible Genetics Inc., Toronto, Canada) according to manufacturer s instructions and primers S1 (5 -ATGGAGAA- CATCACATCAGGA, nt ), POL3 (5 - AAGGATCCAGTTGGC, nt ), POL14 (5 -AGTCCCCAACCTCCA-3, nt ), POL2 (5 -ACGGGGTAAAGGTTC-3, nt ) and S3 (5 -CAAGGTATGTTGCCCGTTTG, nt ). The amplified rt region was approximately 200 codons long covering domains B to D. HBV precore sequences were determined in serum samples drawn at baseline using primers AC1 (5 -CAC- CTCTGCACGTCGCATGG, nt ) and BC1 (5 -GGAAAGAAGTCAGAAGGCAA, nt ) both for amplification and sequencing. HBV genotypes were also determined at baseline by comparing the obtained S gene nucleotide sequence (nt ) with published sequences. 17 Follow-up and Definitions. All patients were followed with monthly clinical examinations and routine laboratory tests. The upper limit of normal for ALT was 49 IU/l. Initial virological response was considered as nondetectable serum HBV-DNA by PCR and initial biochemical response as the decline of ALT within normal range in two consecutive determinations during therapy. Virological breakthrough was considered as the reappearance of detectable serum HBV-DNA by PCR after an initial period of nondetectability, and biochemical breakthrough was defined as the increase of ALT activity above 1.5 upper limit of normal after an initial return to normal levels. Statistical Analysis. All data were analyzed using the statistical package SPSS (version 10.0, SPSS Inc., Chicago, IL). The Mann-Whitney test was used for comparisons of quantitative variables between groups, Wilcoxon matched-pairs signed-ranks test for evaluation of changes of variables within the same group, and the corrected chi-square or 2-tailed Fisher s exact test for qualitative data. The Kaplan-Meier method was used to estimate both virologic and biochemical remission rates during the study period and Cox regression analysis to evaluate the association of several characteristics with the maintenance of remission. In all cases, a 2-tailed P 0.05 was considered statistically significant.

3 HEPATOLOGY, Vol. 45, No. 2, 2007 RAPTI ET AL. 309 Table 1. Baseline Characteristics of the Patients in the Two Treatment Arms ADV Monotherapy ADV LAM Combination All Patients P Value Age (y) Median Range (min-max) Male sex (%) 14/14 (100%) 25/28 (89.3%) 39/42 (92.8%) Cirrhosis (%) 4/14 (28.5%) 12/28 (42.9%) 16/42 (38.1%) YMDD mutations M204I or M204I/L180M 8/14 (57.1%) 16/28 (57.1%) 24/42 (57.1%) M204V/L180M 6/14 (42.8%) 12/28 (42.9%) 18/42 (42.9%) Prior LAM therapy (mo) Median Range (min-max) Treatment duration Median Range (min-max) Serum ALT (IU/L) Median Range (min-max) HBV-DNA (copies/ml) Median 1.5E Range (min-max) E E E 08 Results Biochemical and Virologic Response. Forty-two LAM-resistant patients were included in the study and were randomly assigned to ADV monotherapy (arm A) or an ADV-LAM combination scheme (arm B) in a 1 to 2ratio. The baseline characteristics of the studied patients are shown in Table 1. There were no differences in patients characteristics between the two arms of the study. All were white, had HBV genotype D infection, and all were harboring the precore stop codon mutation with G1896A. Sixteen of the patients had histological evidence of cirrhosis. Patients had been on LAM treatment for 12 to 84 months (median, 32 months) before starting ADV and were positive for LAM-resistant signature YMDD mutations (Table 1), whereas none of them had such changes detectable at start of LAM therapy. Serum ALT levels declined under treatment in both arms of the study, with median ALT values declining from 135 IU/l at baseline to 39, 34, 22, and 24 IU/l at months 3, 6, 12, and 24, respectively, in the ADV monotherapy arm and from 108 IU/l at baseline in the combination arm to 39, 35, 28, and 24.5 IU/l at the same times, respectively. Return of serum ALT to normal levels was observed in 57%, 78.6%, 92.9%, and 72.7% of the patients in arm A at 3, 6, 12, and 24 months of treatment, respectively, versus 60.7%, 70.4%, 88%, and 91% of patients in arm B, for the same points in time, respectively. In Fig. 1, the cumulative probability of return of ALT to normal in the 2 arms of treatment is depicted. There were no statistically significant differences between groups. However, biochemical relapse after return of ALT to normal was observed only in the ADV monotherapy arm. No ALT flares were manifested after either switching to or adding ADV to LAM. Serum HBV DNA levels declined quickly under therapy in both arms without significant differences between ADV monotherapy and combination therapy, at least up to month 12. The cumulative probability of HBV DNA becoming nondetectable under therapy in both arms of the study is shown in Fig. 2. Further analysis of the virological response to treatment according to the baseline HBV DNA levels showed a significantly earlier and more frequent decline to nondetectable levels by PCR among Fig. 1. Cumulative probability of abnormal ALT in HBeAg-negative patients with lamivudine (LAM) resistance included in the two treatment arms of the study. Adefovir (ADV) alone (arm A) is shown by the medium-weight line; the combination of ADV with LAM (arm B) is shown by the dotted line; and both arms are the dashed line (P between the two arms of treatment).

4 310 RAPTI ET AL. HEPATOLOGY, February 2007 Fig. 2. Cumulative probability of serum HBV-DNA to remain detectable by real-time PCR in the two treatment arms of the study and among all treated patients. ADV alone (arm A) is represented by the mediumweight line; the combination of ADV with LAM (arm B) is shown by the dotted line; and both arms by the dashed line (P between the two arms of treatment). patients with fewer than 10 7 copies/ml at baseline compared with greater than 10 7 copies/ml. The difference was statistically significant in each arm separately (P and for arms A and B, respectively) with the P value reaching in the statistical analysis of all patients in the study (Fig. 3). Biochemical and virological responses in both arms of treatment are depicted in Table 2. In both arms, optimal or suboptimal virological responses at month 12 (undetectable HBV DNA or 10 4 copies/ml, respectively) were more frequent in patients with lower than 10 7 copies/ml at baseline. Moreover, the hitherto end of treatment virological response rates were found to be statistically different according to this cutoff: HBV-DNA level of 10 4 copies/ml at month 12 (P 0.04 for arm A, P for arm B and P for all patients). Neither ALT at baseline (P for arm A, P for arm B, and P for all patients) nor duration of prior LAM treatment (P 0.83 for arm A, P 0.77 for arm B, and P for all patients) affected optimal or suboptimal response at month 12. All patients achieving nondetectability of HBV-DNA also had their ALT levels return to normal, except 1 patient in arm B who, despite complete virologic response with undetectable HBV-DNA from month 15 of treatment, remained with persistently increased ALT activity. This individual underwent a second liver biopsy in the third year of therapy that showed typical changes of steatohepatitis. HBV Resistance to ADV. Sequencing of the HBV- DNA under therapy disclosed that LAM-resistant HBV mutations disappeared under ADV monotherapy as well as under combination therapy, except in patients in arm B with suboptimal response to combination therapy not achieving undetectability by PCR of HBV-DNA. Such patients continued to harbor LAMresistant HBV strains up to the time that HBV-DNA reached nondetectability even after increasing the ADV dose (ADV dose was doubled in two patients). In Table 3, 4,7,18,19 serial genotypic HBV resistance mutation in patients with positive PCR samples during therapy are shown. All patients with suboptimal response to ADV in arm A(n 3) had their LAM-resistant HBV mutants reversed to wild-type HBV. All three of them developed subsequently genotypical resistance to ADV at months 15, 18, and 18, respectively, with selection of rtn236t in the first, rta181v in the second, and a mixed population of rtn236t and rta181t in the third. Genotypic resistance was followed by both virological and biochemical breakthrough in 2 of them and only by virological breakthrough in the third (mutation A181T N236T). In all 3 patients, LAM was added as soon as the breakthrough was identified, because adefovir-resistant HBV mutants, particularly rtn236t, have been found to be sensitive to LAM. 18,19 No evidence of ADV resistance could be detected in arm B with maximal hitherto duration of therapy up to 53 months. Although the number of patients in this study is relatively small, the difference in the cumulative probability of ADV resistance between the 2 treatment arms was found to be statistically significant (P ) (Fig. 4). Side Effects. Long-term ADV treatment in these LAM-resistant HBeAg( ) patients, either alone or in combination with LAM, was generally well tolerated. No patient in group A has discontinued the drug or reduced Fig. 3. Cumulative probability of serum HBV-DNA to remain detectable by real-time PCR according to baseline values among all patients with LAM-resistant HBeAg-negative chronic hepatitis B treated with ADV either alone or in combination with LAM. Dashed line: baseline HBV DNA 7 log (arm I), dotted line: 7 log (arm II) (P ).

5 HEPATOLOGY, Vol. 45, No. 2, 2007 RAPTI ET AL. 311 Table 2. Biochemical and Virologic Response in the 2 Treatment Arms ADV Monotherapy ADV LAM Combination Treatment P Value Median ALT (min-max) 6 months 34 IU/l (17 104) 35 IU/l (19 131) months 22 IU/l (9 87) 28 IU/l (14 146) months 24 IU/l (15 55) 24.5 IU/l (12 69 IU/l) % of patients with ALT 49 IU/l 6 months 78.6% 70.4% months 92.9% 88% months 72.7% 91% Median HBV-DNA (min-max) 6 months 2,617 copies/ml (1, ,300) 1,000 copies/ml (1,000 13,000,000) months 1,000 copies/ml (1,000 2,929,000) 1,000 copies/ml (1,000 12,000,000) months 1,000 copies/ml (1,000 4,957,000) 1,000 copies/ml (1,000 58,190) HBV-DNA 1000 cp/ml 6 months 45.5% 57.1% months 78.6% 68% months 75% 82.6% the dosage, and none has developed decompensation of cirrhosis or HCC. As far as serious adverse events are concerned, one of the patients underwent gastrectomy due to gastric cancer but remains in good clinical condition with undetectable HBV-DNA. In the 28 patients in the combination arm, the hitherto median duration of treatment is 40 months (range, 9-53). All patients are still under therapy. In 2 of the 28 patients in the combination arm, both with cirrhosis, the ADV dose had to be reduced to 10 mg every other day, because of a decrease of creatinine clearance from 114 to 50 ml/ min and from 71 ml/min to 36 ml/min, respectively. As far as serious adverse events are concerned, 3 of the 16 patients with cirrhosis (all in group B) developed HCC. Two of them had an optimal and the third had a suboptimal virological response to treatment. In 1 of these patients, HCC was diagnosed in the second year and in the other 2 during the third year of treatment. No statistically significant difference was seen in the rate of HCC development between the 2 arms (P 0.545). One of these patients underwent chemoembolization and is currently in a transplantation waiting list. The second patient underwent hepatectomy and is in good condition, free of cancer, while continuing combination therapy. The third patient refused any therapeutic modality for HCC but continues antiviral treatment. Discussion The results of the current study clearly show that a daily 10-mg dose of ADV in LAM-resistant patients with HBeAg( ) CHB when added to ongoing LAM treat- Table 3. Lamivudine-Resistant and Adefovir-Resistant HBV Mutations in PCR-Positive Products at Baseline and During Treatment* Patient Treatment Arm Genotypic Resistance Baseline Mo 12 Mo 18 Mo 24 Mo 36 1 A M204I 180L/204M 180L/204M 180L/204M Not applicable 181Thr/N236T 181Thr/N236T 2 A L180M/M204V 180L/204M 180L/204M 180L/204M 180L/204M A181V A181V A181V 3 A L180M/M204I L180M/M204I 180L/204M 180L/204M 180L/204M N236T N236T N236T 4 B L180M/M204V L180M/M204V L180M/M204V L180M/M204V L180M/M204V 5 B L180M/M204V L180M/M204V Non-detectable Non-detectable Non-detectable 6 B L180M/M204I L180M/M204I L180M/M204I Non-detectable Non-detectable 7 B L180M/M204V L180M/M204V L180M/M204V L180M/M204V L180M/M204V 8 B L180M/M204V L180M/M204V Non-detectable Non-detectable Non-detectable 9 B L180M/M204V L180M/M204V L180M/M204V L180M/M204V Not applicable 10 B M204I M204I M204I Non-detectable Non-detectable 11 B L180M/M204I L180M/M204I Non-detectable Non-detectable Non-detectable *Wild-type virus is:180l/204m, Lamivudine-resistant HBV mutations are: M204I, L180M/M204V, L180M/M204I, and adefovir-resistant mutations are: A181V, N236T, 181Thr/N236T. 4,7,18,19

6 312 RAPTI ET AL. HEPATOLOGY, February 2007 Fig. 4. ADV resistance development on the basis of LAM combination treatment. Dashed line: combination with LAM (arm B), dotted line: ADV monotherapy (arm A) (P ). ment achieves effective virological suppression associated with biochemical remission in more than 80% without emergence of ADV resistance for up to 3 to 4 years. The efficacy of HBV-DNA suppression in terms of decline to nondetectable levels by sensitive PCR assays at month 12 of therapy was found to be practically the same as in the arm of patients treated with ADV alone. However, after the first year of therapy, 21% of the patients in the monotherapy arm compared with 0% in the combination arm developed genotypic ADV resistance with virological breakthroughs. Despite the relative small number of patients in arm A, the difference is clearly statistically significant (P , Fig. 4). ADV resistance in HBeAg( ) treatment-naïve patients with CHB has been observed in less than 2% of such individuals during the second year of ADV monotherapy. 20 A similarly high prevalence of ADV resistance under monotherapy with this drug has recently been reported in LAM-resistant patients with CHB from Asia, most of them HBeAg ( ) In view of these findings, one may reasonably assume that HBV mutants with changes conferring resistance to LAM develop earlier and more frequent ADV resistance than wild-type HBV strains, similar to what has been found in the case of entecavir therapy for the treatment of LAM-resistant patients. 14 Conversely, the potency of ADV in achieving nondetectability of serum HBV-DNA evaluated at month 12 of therapy does not differ whether the drug is given alone or in combination with LAM and is mainly determined in both groups by HBV-DNA levels at baseline (Fig. 3). Similar observations have recently been reported in Italian CHB patients with LAM resistance 24 as well as in treatment-naïve patients under ADV monotherapy with maximal HBV-DNA suppression achieved at month 12 of treatment and being again determined by baseline HBV-DNA levels On the basis of this evidence one may propose that in HBeAg-negative patients, both LAM-resistant and treatment-naïve baseline HBV- DNA levels less than 10 7 copies/ml are used as a strong determinant and excellent predictor of response to ADV treatment and achievement of HBV-DNA negativity at month 12. In particular, in clinical practice patients under LAM monotherapy should be strictly monitored for HBV resistance, and ADV should be added on LAM before serum HBV DNA increases to such high levels. Development of ADV resistance in treatment-naïve patients has already been found to be determined by HBV-DNA levels at month 12 of therapy. 27 The same observation has been made in the current study, because all patients in the monotherapy arm who developed ADV resistance had detectable HBV-DNA levels at month 12 (ranging from 9,710 to 2,929,000 copies/ml), whereas all of the other monotherapy patients had undetectable HBV-DNA at the same time. Conversely, although eight patients from the combination arm had also detectable HBV-DNA levels at month 12, none of them developed subsequent ADV resistance. These findings are compatible with the hypothesis that the concomitantly ongoing LAM administration hampers the emergence of ADVresistant strains and is consistent with in vitro and in vivo data that ADV-resistant HBV mutants are sensitive to LAM. 28,29 Despite the fact that no ADV-resistant mutations were identified in LAM-resistant patients treated by the combination of ADV with LAM, clearly the efficacy of this therapy was not sufficient to achieve virological and biochemical remission in all patients. In all such patients, however, continuing detectability of HBV DNA was always found to be due to persistence of the LAM-resistant strains of HBV (Table 3). The most probable explanation for this appears to be the relative low potency of the 10-mg daily dose of ADV, which was actually selected not for its robustness but because of safety reasons. 26 Therefore, to deal with inadequate potency of ADV, either its dose should be increased or another drug of similar resistance profile but of higher potency against LAM-resistant mutants should be substituted. Tenofovir fumarate, to which LAM-resistant HBV strains are also sensitive and which is much more potent than ADV, 31,32 is an excellent candidate for such cases. The optimal time for a change to this compound could be month 12 of treatment. In the current study, in those patients with persistent HBV DNA detectability beyond year 3, the ADV dose was increased to 20 mg/day with good response, without side effects, followed by significant decline of HBV DNA levels to close to nondetectability.

7 HEPATOLOGY, Vol. 45, No. 2, 2007 RAPTI ET AL. 313 In conclusion, the high rate of HBV-DNA nondetectability by the combination of ADV and LAM therapy in month 12 in LAM-resistant HBeAg( ) patients in this study, combined with the absence of ADV resistance for up to 4 years of ongoing therapy, makes reasonable the recommendation of adding ADV while continuing LAM, as the first-line, first-choice therapy for this group of patients. Acknowledgment: We thank pharmaceutical company Gilead Sciences Inc. for the supply of ADV capsules of 10 mg on a compassionate basis during the first years of the study and Roche Analytical for the donation of Cobas TaqMan HBV reagents. References 1. Lok AS, McMahon BJ. Chronic hepatitis B: update of recommendations. HEPATOLOGY 2004;39: Papatheodoridis GV, Hadziyannis SJ. Review article: current management of chronic hepatitis B. Aliment Pharmacol Ther 2004;19: Santantonio T, Mazzola M, Iacovazzi T, Miglietta A, Guastadisegni A, Pastore G. Long-term follow-up of patients with anti-hbe/hbv DNApositive chronic hepatitis B treated for 12 months with lamivudine. J Hepatol 2000;32: Tassopoulos NC, Volpes R, Pastore G, Heathcote J, Buti M, Goldin RD, et al. Efficacy of lamivudine in patients with hepatitis B e antigen-negative/hepatitis B virus DNA-positive (precore mutant) chronic hepatitis B. Lamivudine Precore Mutant Study Group. HEPATOLOGY 1999;29: Hadziyannis SJ, Vassilopoulos D. Hepatitis B e antigen-negative chronic hepatitis B. HEPATOLOGY 2001;34: Chen CH, Lee CM, Lu SN, Wang JH, Tung HD, Hung CH, et al. Comparison of clinical outcome between patients continuing and discontinuing lamivudine therapy after biochemical breakthrough of YMDD mutants. J Hepatol 2004;41: Hadziyannis SJ, Papatheodoridis GV, Dimou E, Laras A, Papaioannou C. Efficacy of long-term lamivudine monotherapy in patients with hepatitis B e antigen-negative chronic hepatitis B. HEPATOLOGY 2000;32: Papatheodoridis GV, Dimou E, Laras A, Papadimitropoulos V, Hadziyannis SJ. Course of virologic breakthroughs under long-term lamivudine in HBeAg-negative precore mutant HBV liver disease. HEPATOLOGY 2002; 36: Di Marco V, Marzano A, Lampertico P, Andreone P, Santantonio T, Almasio PL, et al. Clinical outcome of HBeAg-negative chronic hepatitis B in relation to virological response to lamivudine. HEPATOLOGY 2004;40: Liaw YF, Chien RN, Yeh CT. No benefit to continue lamivudine therapy after emergence of YMDD mutations. Antivir Ther 2004;9: Peters M, Hann H, Martin P, Heathcote J, Buggisch P, Rubin R, et al for the GS study group. Adefovir dipivoxil alone or in combination with lamivudine in patients with lamivudine-resistant chronic hepatitis B. Gastroenterology 2004;126: Schiff E, Lai CL, Hadziyannis S, Neuhaus P, Terrault N, Colombo M, et al on behalf of the adefovir dipivoxil study 435 international investigators group. Adefovir dipivoxil therapy for lamivudine-resistant hepatitis B in pre- and post-liver transplantation patients. HEPATOLOGY 2003;38: Sherman M, Yurdaydin C, Sollano J, Silva M, Liaw Y-F, Cianciara J, et al. Entecavir for treatment of lamivudine-refractory, HBeAg-positive chronic hepatitis B. Gastroenterology 2006;130: Colonno R, Rose R, Levine S, Baldick J, Pokornowski K, Plym M, et al. Entecavir two year resistance update: no resistance observed in nucleoside naïve patients and low frequency resistance emergence in lamivudine-refractory patients. HEPATOLOGY 2005;42:573A. 15. Sum SS, Wong DK, Yuen JC, Lai CL, Yuen MF. Comparison of the COBAS Taqman HBV test with the COBAS Amplicor monitor test for measurement of hepatitis B virus DNA in serum. J Med Virol 2005;77: Hadziyannis AS, Mitsoula P, Hadziyannis E, Panopoulou E, Colucci G, Hadziyannis SJ. Monitoring serum HBV-DNA levels with the TaqMan technique during treatment of HBeAg (-) chronic hepatitis B with lamivudine. HEPATOLOGY 2005;42:726A. 17. Chu CJ, Hussain M, Lok AS. Quantitative serum HBV DNA levels during different stages of chronic hepatitis B infection. HEPATOLOGY 2002;36: Villeneuve J-P, Durantel D, Durantel S, Westland C, Xiong S, Brosgart C, et al. Selection of a hepatitis B virus strain resistant to adefovir in a liver transplantation patient. J Hepatol 2003;39: Angus P, Vaughan R, Xiong S, Yang H, Delaney W, Gibbs C, et al. Resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the HBV polymerase. Gastroenterology 2003;125: Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, Chang TT, Kitis G, Rizzetto M, et al. Long-term therapy with adefovir dipivoxil for HBeAgnegative chronic hepatitis B. N Engl J Med 2005;352: Lee YS, Suh DJ, Lim YS, Jung SW, Kim KM, Lee HC, 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: Fung S, Chae HB, Fontana R, Conjeevaram H, Marrero J, Oberhelman K, et al. Virologic response and resistance to adefovir in patients with chronic hepatitis B. J Hepatol 2006;44: Yeon JE, Yoo W, Hong SP, Chang YJ, Yu SK, Kim JH, et al. Resistance to adefovir dipivoxil (ADV) in lamivudine-resistant chronic hepatitis B patients treated with ADV. Gut 2006;55: Lampertico P, Marzano A, Levrero M, Santantonio T, Andreone P, Brunetto M, et al. A multicenter Italian study of rescue adefovir dipivoxil therapy in lamivudine resistant patients: a 2-year analysis of 604 patients. HEPATOLOGY 2005;42:591A. 25. Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, Chang TT, Kitis G, Rizzetto M, et al. Adefovir dipivoxil for the treatment of hepatitis B e antigen-negative chronic hepatitis B. N Engl J Med 2003;348: Marcellin P, Chang T, Lim S, Tong M, Sievert W, Shiffman M, et al. Adefovir dipivoxil for the treatment of hepatitis B e antigen-positive chronic hepatitis B. N Engl J Med 2003;348: Locarnini S, Qi X, Arterburn S, Snow A, Brosgart C, Currie G, et al. Incidence and predictors of emergence of adefovir resistant HBV during four years of adefovir dipivoxil (ADV) therapy for patients with chronic hepatitis B (CHB). J Hepatol 2005;42:S Westland C, Delaney W 4th, Yang H, Chen SS, Marcellin P, Hadziyannis S, et al. Hepatitis B virus genotypes and virologic response in 694 patients in phase III studies of adefovir dipivoxil. Gastroenterology 2003;125: Westland CE, Yang H, Delaney WE 4 th, Wulfsohn M, Lama N, Gibbs CS, et al. Activity of adefovir dipivoxil against all patterns of lamivudine-resistant hepatitis B viruses in patients. J Viral Hepatol 2005;12: Suzuki F, Kumada H, Nakamura H. Changes in viral loads of lamivudineresistant mutants and evolution of HBV sequences during adefovir dipivoxil therapy. J Med Virol 2006;78: van Bommel F, Wunsche T, Mauss S, Reinke P, Bergk A, Schurmann D, et al. Comparison of adefovir and tenofovir in the treatment of lamivudineresistant hepatitis B virus infection. HEPATOLOGY 2004;40: Delaney WE 4th, Ray AS, Yang H, Qi X, Xiong S, Zhu Y, et al. Intracellular metabolism and in vitro activity of tenofovir against hepatitis B virus. Antimicrob Agents Chemother 2006; 50: