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The Liver Meeting— 54th Annual Meeting of The American Association for the Study of Liver Diseases, October 24 - 28, Boston, MA—HBV Abstracts

Alan Franciscus
Editor-in-Chief, HCV Advocate

Adefovir Dipivoxil 10 mg (ADV) for the Treatment of Chronic Hepatitis B in Patients Pre-liver Transplantation with Lamivudine Resistant Virus

E.Schiff1, C. Lai2, S. Hadziyannis3, P. Neuhaus4, N. Terrault5, P. Lampertico6, H. Tillmann7, D. Samuel8, N. Lama9, C. James9,G. Currie9 and C. Brosgart9 on behalf of the 435 Study Investigator Group 1Center for Liver Disease, University of Miami, FL, USA. 2Queen Mary Hospital, The University of Hong Kong, Hong Kong.3Henry Dunant Hospital, Athens, Greece. 4Department of Surgery, Charite Campus Virchow, Berlin, Germany. 5University ofCalifornia San Francisco, CA, USA. 6Universita Studi Milano e IRCCS, Ospedale Maggiore, Milano, Italy. 7Gastroenterology and Hepatology, Hannover Medical School, Hannover, Germany. 8Hepato-bilary Centre, Paul Brousse Hospital, Villejuif, France.9Gilead Sciences, Inc., Foster City, CA, USA

Background
The incidence of lamuvidine resistance is 24% at one year increasing to 70% with 4 years of therapy. Lamuvidine resistance is a significant clinical problem in patients pre-orthotopic liver transplantation (OLT) and is associated with disease progression and death. High serum HBV DNA pre-OLT may prevent patients being waitlisted for transplantation; and, for patients who go on to transplantation, may result in re-infection of the new graft. ADV has potent in vivo and in vitro activity against wild-type and lamuvidine resistant HBV.

Objective
To evaluate safety and efficacy of ADV and evaluate the impact on waitlist priority in patients receiving ADV 10 mg in an open-label study of pre-OLT patients with lamuvidine-resistant HBV. ADV was added to existing HBV and immunosuppressive therapy.

Methods
One-hundred and twenty-eight pre-OLT patients failing lamuvidine enrolled at 75 centers. The median duration on ADV therapy was 18.7 weeks for the pre-OLT patients. The baseline characteristics were as follows: median serum HBV DNA was 7.4 log10 copies/mL, median ALT was 1.8 xULN (75 IU/L), 40% were Child-Pugh-Turcotte (CPT) Class A, 40% were Class
B and 21% were Class C. The median time on lamivudine therapy until loss of response was 17 months, serum total bilirubin was >ULN in 66%, serum albumin was <LLN in 55%, and prothrombin time was >ULN in 53% of patients. A retrospective analysis was conducted to evaluate the status of the 128 pre-OLT patients while receiving ADV. Physicians completed an additional CRF for 100 of 128 patients (78%) addressing clinical status of waitlisted patients during treatment. The CRF also assessed if ADV treatment allowed transplantation or changed priority on the transplantation waitlist.

Results
At week 48 the median change in serum HBV DNA was –4.1 log10 copies/mL, ALT normalized in 78%, albumin in 81%, bilirubin in 50%, and prothrombin time in 83% of patients. Ninety-two percent had stable or improved CPT scores, the survival rate was 84% (Kaplan-Meier estimate). Of the 45 (45%) pre-OLT patients who subsequently underwent liver trans-plantation, physicians indicated treatment with ADV improved the clinical condition in 38 (84%) patients, thus allowing them to undergo liver transplantation. At the time of follow-up, 55 patients had not been transplanted. In 20 of these patients (36%), treatment with ADV resulted in the clinical condition improving sufficiently to allow removal from the transplantation waitlist or allowed downgrading from high priority to low priority status.

Conclusion
Treatment with ADV 10 mg resulted in significant clinical and laboratory improvements, and improved survival in pre-OLT patients failing LAM therapy. In addition, the clinical condition improved to allow a reduction in their priority status for requiring transplantation or significantly reduced serum HBV DNA and improved clinical status to allow transplantation.

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Poster 953: EPIDEMIOLOGICAL DATA OF HEPATITIS B INFECTION IN FRANCE: RESULTS OF NATIONWIDE SURVEY

Jean-François Cadranel Sr, Centre hospitalier Laennec de Creil, Creil, France; Bruno Lesgourgues, Centre hospitalier du Raincy-Montfermeil, Montfermeil, France; Xavier Causse, Centre hospitalier d'Orléans, Orléans, France; Pierre Lahmek, Centre hospitalier du Raincy-Montfermeil, Montfermeil, France; Guy Bellaïche, Hôpital Robert Ballanger, Aulnay sous Bois, France; Louis Bettan, Centre hospitalier de Villeneuve Saint Georges, Villeneuve Saint Georges, France; Thierry Fontanges, Centre hospitalier de Bourgoin-Jallieu, Bourgoin-Jallieu, France; Arnaud Pauwells, Centre hospitalier de Gonesse, Gonesse, France; Jean Henrion, Centre hospitalier de Jolimont, Jolimont, Belgium; Michel Chousterman, Centre hospitalier intercommunal de Créteil, Créteil, France; Bertrand Condat, Hôpital de Bry sur Marne, Bry sur Marne, France; Claude Eugène, Centre hospitalier de Poissy, Poissy, France; Pascale Hervio, Centre hospitalier de Pontoise, Pontoise, France; Pierre Periac, Centre hospitalier de Saint Denis, Saint Denis, France; Henri Moindrot, Centre hospitalier de Valence, Valence, France; Denis Grasset, Centre hospitalier de Montauban, Montauban, France; Olivier Nouel, Centre hospitalier de Saint Brieuc, Saint Brieuc, France; Jacques Denis, Centre hospitalier de Corbeil-Evry, Corbeil-Evry, France; ANGH (Association Nationale des Gastroentérologues, des Hôpitaux Généraux).

Aims
Epidemiological data about Hepatitis B virus infection in France are scarce ; moreover due to neurological controversial data adverse effects related to hepatitis B vaccine, a dramatic decrease of hepatitis B vaccination has been noted in France since 1996; the aims of this nationwide survey were to describe epidemiological, biological, virological, histological and treatment regimen of HBs antigen (HBs Ag) positive patients followed in non academic hospitals in France.

Methods
From 01/4/2001 to 30/5/2002, the following data of all HBs Ag positive patients followed by gastroentroenterologists from participating centers were anonymously recorded: age, gender, way of discovery HBs antigen positivity, birth area, ALT activity, liver biochemistry, protidogram and prothrombin index. Presence of HBe antigen and/or anti-HBe antibodies, presence of serum HBV-DNA through hybridization tests, presence of Delta Ag and/or antibodies, co-infection with Hepatitis C or H.I.V viruses results of liver biopsy according to Knodell and Metavir classification and therapeutic regimen.

Results
1166 HBs Ag positive patients were followed in 58 non-academic hospitals during the survey period. They were 671 males and 495 females, mean age 40.7± 15 years. 14 (24%) centers followed 30 patients or more, 16 centers (28%) between 10 and 29 patients, 28 (48%) less than 10 patients. Distribution of patient's birth areas were: France 33%, Sub-Saharan Africa 24%, South Europe 12%, Eastern Asia 11% and Maghreb 10%. HBs Ag was discovered during exploration of acute hepatitis in 5% of patients, main other discoveries modalities were: persistent increase of ALT in 26%, familial or sexual contact in 23%, legal screening during pregnancy in 18%, systematic screening in 12%, screening before blood transfusion or blood donation in 5%, examination of patients with either cirrhosis and /or hepatocellular carcinoma in 5%. 29% of patients were HBs Ag "asymptomatic carriers" (repeated normal ALT measures, repeated negativity of serum HBV-DNA with hybridization tests, absence of co-infection with either C, Delta or H.I.V viruses, repeated negativity of serum HBV-DNA with hybridization tests and normal abdominal ultrasonography), 50% of patients had Chronic Hepatitis, 19% had cirrhosis or hepatocellular carcinoma, 4,5% had dual B-C infection. In patients with chronic hepatitis, 43 % were positive for HBe Ag and 57% had HBe antibodies without HBe Ag. Liver biopsy was performed in 558 patients: Median Knodell score was 8 and median activity and fibrosis Metavir scores were 2. Fibrotic lesions according to Metavir score were significantly higher in HBe Ag negative patients (29 % F0 or F1 vs 49%, p<0.002). Percentage of new HBs Ag positive patients was significantly higher after 1996 than before (66.5% vs 1.5%; p<0.0001) suggesting an increase prevalence of HBV infection in our country since this date. 382 patients have been treated: 153 (40%) with alpha interferon, 150 (39%) by lamivudine and 79 (21%) with alpha interferon then, in non-responders, with lamivudine.

Conclusions
This nationwide study suggest an increased prevalence of HBV in our country during the recent years, and the prevalent feature of chronic hepatitis B, mostly of the chronic HBe negative type. After a dramatic decrease programs of vaccination, Hepatitis B neonates vaccination is mandatory.

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Poster 963: HBV GENOTYPE F AND PRECORE STOP CODON MUTATION PREDOMINATE IN AN ARGENTINIAN BLOOD DONOR POPULATION

Paulo H. C. França, UFRJ, Rio de Janeiro, Brazil; Jorge E. González, Silvina Munné, Dr C. G. Malbrán, Buenos Aires, Argentina; Larissa H. Brandão, IDMT, Rio de Janeiro, Brazil; Vera Gouvea, UFRJ, Rio de Janeiro, Brazil; Erwin Sablon, Bart O. M. Vanderborght, Innogenetics, Gent, Belgium.

Introduction

HBV-infected individuals carrying HBeAg-negative variants due to HBV precore polymorphisms often present low viremia, are more likely to have silent and persistent infections, and are subsequently at increased risk for cirrhosis and hepatocellular carcinoma. Although distinct precore mutations distributed according to geographic locality and viral genotype have been reported, epidemiological data from South America, and Argentina in particular, concerning precore mutations, genotypes, and polymerase gene polymorphisms are still scarce.

Methods/Patients
We therefore surveyed their prevalence in 75 asymptomatic blood donors (HBsAg and anti-HBc positive individuals) throughout Argentina (15 cities). HBV genotypes were identified by a reverse hybridization genotyping assay (INNO-LiPA HBV Genotyping); precore promotor (1762 and 1764 nucleotides) and codon 28 polymorphisms by INNO-LiPA HBV PreCore; and HBV polymerase gene (codons 108, 204, and 207) by the INNO-LiPA HBV DR assay (all Innogenetics, Gent, Belgium).

Results
The observed prevalence for the HBV genotypes was 64% (48/75) for genotype F, 17.3% (13/75) for each of genotypes A and D, and 1.3% (1/75) for genotype C. Only wild-type polymorphisms at polymerase codons 180, 204, and 207 were found. An extremely high proportion of stop codon mutation (UAG) at the precore codon 28 (66.7%; 50/75) was observed in this symptomless population. Wild-type codon (UGG) was present in 29.3% (22/75) of the samples, and the remaining 4% (3/75) gave a mixed pattern (UGG and UAG in the same sample) by LiPA. The combination of A at 1762nt and G at 1764nt precore promotor was found in 58.7% (44/75) of the samples. The T 1762nt / A 1764nt profile was observed in 28% (21/75) of samples.

Only one sample showed A at both promotor positions examined. Mixed precore promotor polymorphisms were found in 4% (3/75) of samples. A correlation between genotype F and high prevalence of precore stop codon mutation was found (p<0.05). Previously reported associations between genotype A with wild-type codon 28 and genotype D with mutant-type codon 28 were also observed.

Conclusion
In conclusion, HBV genotype F and precore stop codon mutation 28 predominated among a geographically broad Argentinian blood donor population. In countries like Argentina, where genotype F prevails, special attention should be given to the detection of HBeAg-negative variants.

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Resistance Surveillance of Liver Transplantation Patients with Lamivudine-resistant Hepatitis B Virus (HBV) after 96 Weeks of Adefovir Dipivoxil Treatment

S. Xiong1, C. Westland1, C. Brosgart1, M. Wulfsohn1, M. Miller1, J. Villeneuve2, N. Terrault3, F. Zoulim4
1Gilead Sciences, Inc., Foster City, CA, USA, 2Hôpital Saint-Luc, CHUM, Montreal, Canada, 3University of California at San
Francisco, San Francisco, CA, USA, 4INSERM, Lyon, France

Background
Lamivudine (LAM) resistance occurs in approximately 20% after one year, 40% after 2 years and 70% after 3 years. In contrast, resistance to adefovir dipivoxil (ADV) occurs infrequently with 1.6% of CHB patients developing the adefovir resistance mutation rtN236T after 2 years of ADV therapy. Pre-existing LAM resistance mutations or concurrent use of immunosuppressive therapy by LT patients may increase the risk of resistance to ADV.

Objective
To determine the incidence of ADV resistance in a clinical trial of liver transplantation (pre and post) patients with LAMresistant HBV treated with ADV for 96 weeks (LAM therapy was maintained in most patients).

Methods
The HBV reverse transcriptase domain was sequenced for LT patients with detectable HBV DNA by PCR (>1000 copies/ml) after 96 weeks of ADV therapy (n=114). In vitro drug susceptibility was determined following transfection of HepG2 cells with patient-derived HBV clones from baseline and week 96 serum samples as of January 2003.

Results
The rtN236T mutation was observed in 2/114 patients (1.8%) at week 96. LAM had been discontinued at weeks 16 and 28 after initiation of ADV in these two patients respectively. The baseline LAM-resistant YMDD mutation reverted to wildtype prior to week 96 in both patients. Emergence of rtN236T was associated with rebound in serum HBV DNA and ALT elevation in both patients. In vitro phenotypic analysis showed approximately 4-fold reduced susceptibility to adefovir with rtN236T. However, these adefovir-resistant HBV clones were fully susceptible to LAM and entecavir in vitro. LAM therapy was re-initiated, in addition to the ongoing ADV therapy, after emergence of rtN236T in these patients resulting in a > 2.9 log10 copies/mL reduction in serum HBV DNA in both patients. One patient also had a significant reduction (>80%) in ALT within 5 months of LAM treatment. No other mutations potentially associated with adefovir resistance were detected.

Conclusions
Emergence of the adefovir resistance mutation rtN236T was observed infrequently after 2 years in liver transplantation patients (1.8%, 2/114) infected with LAM-resistant HBV, similar to observations in treatment naïve nonliver transplantation patients. The ADV-resistant HBV was sensitive to LAM and addition of LAM resulted in clinical stabilization in both patients.

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Complete Genotypic and Phenotypic Analyses of HBV Mutations Identified in HBeAg- Chronic Hepatitis B Patients Receiving 96 Weeks of Adefovir Dipivoxil (ADV)

W. Delaney1, H. Yang1, C. Westland1, M. Wulfsohn1, C. Gibbs1, M. Miller1, C. Brosgart1, S. Xiong1, P. Angus2, S. Locarnini3, G.
1Gilead Sciences, Inc., Foster City, CA, USA, 2Austin Hospital, Heidelberg, Australia, 3Victorian Infectious Diseases Reference4 Laboratory, North Melbourne, Australia, 4Georgios Papanikolaou Hospital, Thessaloniki, Greece


Background
Lamivudine resistance occurs in 14-32% and 38-50% of CH-B patients after 1 and 2 years of monotherapy, respectively. In contrast, no resistance to ADV was previously identified in patients treated for 48 weeks (n=629).

Objective
To monitor for the emergence of adefovir resistance following 96 weeks therapy in HBeAg- patients participating in a phase 3 trial of ADV (study 438).

Methods
HBV was sequenced for all patients with detectable HBV DNA (>1000 copies/ml, AmplicorTM PCR, ny) at baseline and week 96. In vitro drug susceptibility was determined by transfection of HepG2 cells with patient-derived HBV clones isolated at baseline and week 96.

Results
Novel conserved site substitutions were observed in 5/79 patients (Table 1.) The rtN236T mutation was observed in two patients. Both patients had sub-optimal viral load suppression at week 48 (~1.7 log10) and a slow increase to within 1 log10 of baseline by week 96. In vitro phenotypic analysis indicated that rtN236T conferred a 7- to 14-fold decrease in adefovir susceptibility but remained sensitive to lamivudine and entecavir. The rtA181V mutation was observed in two patients. One patient had viral load rebound to within 1 log10 of baseline by week 96. The second patient had HBV suppressed to below 1000 copies/mL post week 96. In vitro phenotypic analysis indicated that rtA181V conferred a 2- to 3- fold decrease in adefovir susceptibility and a similar change in lamivudine sensitivity. The single patient with the rtK241E + rtK318Q mutations achieved a >5 log10 reduction in serum HBV DNA and remained suppressed to near the limit of Amplicor detection through week 96. HBV isolates from this patient were fully susceptible to adefovir in vitro, suggesting that the rtK241E + rtK318Q mutations do not confer resistance to adefovir.

Conclusions
A novel rtN236T mutation conferring reduced susceptibility to adefovir was identified in 2/79 (2.5%) HBeAgpatients receiving ADV monotherapy for 96 weeks. A second mutation of rtA181V occurred in 2/79 patients, however in vitro phenotypic and clinical data are currently insufficient to conclusively link A181V with adefovir resistance. Resistance monitoring results of this trial are in agreement with those from other adefovir dipivoxil trials and bring the cumulative incidence of rtN236T to 0/629 (0%) patients at 48 weeks and 4/238 (1.7%) patients at 96 weeks.

Table 1. Results of Genotypic and Phenotypic Resistance Analyses from HBeAg- Patients Receiving 96 Weeks of ADV montherapy.

Patient Emerging Mutation at Week 96 In Vitro Adefovir Sensitivity (fold resistance) In Vitro Lamivudine Sensitivity (fold resistance) Viral Load Rebound
A rtN236T 13.8 3.5 Yes
B rtN236T 7.3 2.3 Yes
C rtA181V 2.5 3.0 No
D rtA181V 3.0 3.1 Yes
E rtK241E + rtK318Q O 9 Not analyzed No

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48 Weeks of Adefovir Dipivoxil (ADV) Results in a Consistent and Significant Improvement in Liver Histology and Virological Status Regardless of Baseline Knodell Fibrosis Score in Patients with HBeAg- Chronic Hepatitis B

P. Marcellin1, S. Hadziyannis2, N. Tassopoulos3, E. Heathcote4, T. Chang5, G. Kitis6, M. Rizzetto7, Z. Goodman8, S. Chen9, M. Wulfsohn9, M. Wollman9, C. James9, and C. Brosgart9 representing the 438 Study Group 1Hopital Beaujon, Clichy, France 2Henry Dunant Hospital, Athens, 3Western Attica Hospital, Athens, 4Toronto Western, Toronto, 5National Cheng Kung Hospital Taiwan, 6Georgios Papanikolaou Hospital, Thessaloniki, 7Azienda Ospedaliera San Giovanni Battista, Torino, 8Armed Forces Institute of Pathology, Washington DC USA, 9Gilead Sciences, Inc., Foster City, CA, USA

Background
GS-98-438 is a randomized, placebo-controlled clinical trial of ADV for the treatment of HBeAg- chronic hepatitis B. Patients in the trial were randomized to treatment with ADV or placebo (PLB) for 48 weeks. Liver biopsies were performed at baseline, after 48 weeks in all patients. The primary efficacy endpoint was a ¡Ý two-point improvement in the inflammatory score of the Knodell Histology Activity Index (HAI) with no progression of the fibrosis score. This was achieved by 64% (77/121) treated with ADV 10 mg QD and 33% (19/57) PLB treated patients (p<0.001) at 48 weeks.

Objective
The objective of this analysis is to compare the efficacy response between ADV 10 mg and PLB treated patients with regards to baseline Knodell fibrosis scores (F0/F1 = no/portal fibrosis and F3/F4 = bridging fibrosis/cirrhosis) at 48 weeks.

Methods
Evaluable paired baseline and week 48 biopsies were available from 167 patients (91%) enrolled in the study. The biopsies were assessed by a central histopathologist, blinded to treatment assignment and sequence. Biopsies were scored utilizing Knodell HAI scores comparing the baseline to the week 48 biopsy. Change in alanine aminotransferase (ALT), serum HBV DNA (Roche AmplicorTM LLQ 400 copies/mL) and histology were evaluated in patients receiving ADV 10 mg and PLB.

Results
The safety profile of ADV was similar to PLB.

Conclusion
ADV 10 mg results in a consistent and significant biochemical, virological and histological response compared to PLB (ALT normalization, change in ALT, HBV DNA < 400 copies/mL, HBV DNA reduction and histological improvement) regardless of the baseline Knodell fibrosis score.

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Long Term Treatment with Adefovir Dipivoxil (ADV) for Three Years in Patients with Lamivudine resistant (LAM-R) HBV and HIV Co-infection Results in Significant and Sustained Clinical Improvement

Y. Benhamou1, V. Thibault1, P. Vig, Marc Antoine Valantin, Patricia Guyon, Christine Katlama, Biao Lu, Graeme Currie, Carol Brosgart, Thierry Poynard.

Introduction
ADV has demonstrated efficacy and safety in a broad range of populations with chronic hepatitis B, including patients who are treatment-naive and those with LAM-R HBV.

Objective
To evaluate 3 years treatment of ADV (10 mg qd) in patients with LAM-R HBV and HIV co-infection.

Methods
Patients with LAM-R HBV, co-infected with HIV, who previously participated in a one-year pilot study of ADV, continued treatment for a further two years. ADV was added to the pre-existing anti-retroviral therapy including LAM (150 mg bid). Patients were seen every 4 weeks in the first year and every 12 weeks in the 2nd and 3rd years. At each visit, serum ALT, serum HBV DNA (Amplicor Monitor™ƒnRoche, LLQ 2.3 log10 copies/mL), HBV serological markers, plasma HIV RNA (LLQ 2.3 log10 copies/mL) and CD4 cell count were measured.

Results
Of the 35 patients initially enrolled in the one-year study, 29 completed 144 weeks of ADV. Median serum HBV DNA declined from baseline 8.75 (Q1-Q38.41-8.93) log10 copies/mL by 3.98 (3.48-4.79), 4.81 (3.86-5.31) and 5.45 (3.94-6.15) log10 copies/mL at weeks 48, 96 and 144, respectively (p<0.0001). Eight patients (28%) had an undetectable serum HBV DNA (<2.3 log10 copies/mL) at week 144. No patient had a rebound in serum HBV DNA (defined as confirmed ƒ|ƒn1 log10 copies/mL increase from the on treatment nadir). No mutations were identified at conserved sites in either the HBV DNA polymerase or in the HIV RT performed at baseline, weeks 48 and 96. Week 144 genotypic analysis is in progress. Mean ALT at weeks 48 (76.8?9.2 IU/L), 96 (60.4?8.4 IU/L) and 144 (54.0?13.0 IU/L) were significantly decreased from baseline value of 102.3?11.6 IU/L, p =0.04, 0.003, and <0.0001, respectively. Two patients seroconverted to anti-HBe by week 48. There were no serious adverse events related to ADV throughout the study period. There were no significant changes in HIV RNA throughout the study from a mean HIV RNA at baseline of 2.88?0.13 log10 copies/mL. CD4 cell count increased from baseline (423+34 cells/mm3) by 61+30 cells/ mm3 by week 144, p=0.05.

Conclusion
Long-term treatment with ADV (10 mg qd) was well tolerated and resulted in significant and sustained reductions in serum HBV DNA and ALT over the course of 3 years of therapy. The magnitude of the reductions was greater with increasing duration of treatment and there was no loss of suppression seen with 3 years of therapy with ADV.

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Long-term (96 Weeks) Adefovir Dipivoxil in HBeAg- Chronic Hepatitis Results in Significant Virological, Biochemical and Histological Improvement

S. Hadziyannis1, N. Tassopoulos2, J. Heathcote3, T. Chang4, G. Kitis5, M. Rizzetto6, P. Marcellin7, S. Lim8, S. Chen9, M.Wulfsohn9, S. Xiong9, C. James9, G. Currie9, and C. Brosgart9 for the 438 Study Group 1Henry Dunant Hospital, Athens, Greece, 2Western Attica Hospital, Athens, 3Toronto Western, Toronto, 4National Cheng Kung Hospital Taiwan, 5Georgios Papanikolaou Hospital, Thessaloniki, 6Azienda Ospedaliera San Giovanni Battista, Torino, 7Hopital Beaujon, Clichy, 8National University, Singapore, 9Gilead Sciences, Inc., Foster City, CA, USA

Background
Adefovir dipivoxil (ADV) 10 mg has demonstrated significant histological, virological, and biochemical improvement as compared to placebo (PLB) through 48 weeks of therapy in HBeAg positive and HBeAg negative patients. The benefit of 96 weeks of continuous ADV 10 mg therapy was evaluated in this study.

Methods
185 HBeAg negative CHB patients were randomised to receive ADV 10 mg or PLB for 48 weeks in a 2:1 ratio. At 48 weeks, the ADV group was re-randomized (2:1 ratio) to ADV 10 mg (n=180) or PLB (n=40). PLB patients received ADV 10 mg (n=60) in the second 48 weeks. Serum HBV DNA (Roche Amplicor Monitor PCR LLQ <1000 copies /mL) and ALT levels (upper limit normal (ULN) 43 IU/L) were evaluated every 4 weeks. Liver biopsy was performed at baseline and 48 weeks and was optional at 96 weeks. Scoring of biopsies was performed using the Knodell and Ishak scoring systems. Histological improvement was defined as a >2-point improvement in Knodell score with no worsening of fibrosis, with missing biopsies at week 48 considered as failures.

Resistance analysis
The HBV polymerase RT domain was PCR amplified and sequenced for all isolates from patients with detectable HBV DNA at baseline and at week 96.

Results
After 48 weeks of ADV, 64% of patients showed histological improvement, 64% of patients had HBV DNA < 1000 copies /mL and 72% of patients had normalized ALT. 170 patients (92%) completed 96 weeks of the study. In patients receiving ADV 10 mg for 96 weeks, baseline HBV disease characteristics (median) were: serum HBV DNA 7.07 log 10 copies/mL, ALT 98 IU/L (2.3 x ULN), total Knodell HAI score 10, Ishak fibrosis score 2. By 96 weeks, 71 % of patients achieved undetectable levels of serum HBV DNA levels, (< 1000 copies /mL), 81% < 10,000 copies/mL and 88% < 100,000 copies/mL; 50% achieved an undetectable serum HBV DNA within 36 weeks of initiating therapy. Seventy-five percent of patients had normalized ALT, 83% had ALT < 1.2 x ULN and 87% had ALT levels < 1.5 x ULN, with 50% of patients having normalized ALT within 20 weeks of initiating therapy. In the subset of patients with a third biopsy at week 96 (n= 19), histological improvement was seen in 79% at week 96; there was a 5 point median decline in total Knodell score and 53% had a > 1 point decline in the Ishak fibrosis score.

The safety profile of ADV over 96 weeks was similar to that seen in the first 48 week placebo-controlled experience. An adefovir-associated resistance mutation (N236T) was identified in 2/79 patients (2.5%).

Conclusion
In HBeAg-negative CHB patients, 96 weeks of treatment with ADV 10 mg resulted in continued and sustained reductions in HBV DNA and ALT levels and continued histological improvement, with an adverse-event profile similar to that of PLB. The emergence of resistance to adefovir was delayed and infrequent.
Date: October 28, 2003

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Lack of Ethnic Differences in Reponse to Adefovir Dipivoxil Therapy in HBeAg+ and HBeAg-Patients with Chronic Hepatitis

S. Lim1, P. Marcellin2, N. Tassopoulos3, S. Hadziyannis4, T. Chang5, M. Tong6, W. Sievert7, P. Hu8, G. Currie9, C. James9, G.Choy9, B. Kearney9, S. Chen9, and C.Brosgart9 on behalf of the Investigator Groups for Studies 437 and 438
1National University Hospital, Singapore, Singapore, 2Hopital Beaujon, Paris, France, 3Western Attica General Hospital, Athens,
Greece, 4Henry Dunant Hospital, Athens, Greece, 5National Cheng Kung University Hospital, HK, 6Huntington Memorial, CA, USA, 7Monash Medical Center, Victoria, Australia, 8Peking Union Medical College Hospital, PRC, 9Gilead Sciences, Inc, CA, USA

BACKGROUND
Chronic hepatitis B (CHB) is associated with significant morbidity and mortality. Adefovir dipivoxil (ADV) is a nucleotide analog with in vitro and in vivo activity against HBeAg+, and lamivudine-resistant hepatitis B virus (HBV).

OBJECTIVE
To evaluate response in terms of histological, virological, and biochemical improvement following 48 weeks of treatment with ADV 10 mg in Asians compared to Caucasian patients and to compare the pharmacokinetic (PK) parameters of Asian and Caucasian patients in phase 1 studies.

METHODS
522 CHB patients were treated for 48 weeks in two randomized, double-blind, placebo-controlled studies: 338 HBeAg+ patients (HBV DNA > 106 copies/mL, Roche AmplicorTM PCR, LLQ 400 copies/mL, ALT 1.2–10 x ULN) were randomized in a 1:1 ratio to ADV 10 mg or placebo (PLB); and 184 HbeAg- patients (HBeAg-/HBeAb+, HBV DNA = 105 copies/mL, ALT 1.5–15 x ULN) were randomized in a 2:1 ratio to ADV or PLB. Paired baseline and week 48 liver biopsies were assessed by a single histopathologist blind to treatment assignment and sequence. Improvement was defined as a >2 point decrease in Knodell necroinflammatory score from baseline at week 48 with no worsening of fibrosis, with missing biopsies considered as treatment failures. Virological and biochemical assessments and adverse event monitoring were conducted every 4 weeks. For this analysis, data from the two studies were integrated. Additionally, two PK studies in healthy Asian patients were conducted and PK parameters were compared to data from four PK studies in healthy Caucasians and CHB patients. 12 Asian patients were randomized to receive a single dose of ADV 5 mg, ADV 10 mg, or ADV 30 mg in a cross-over design. In a second study, 24 Asian patients were randomized 5:1 to ADV 10 mg or PLB, respectively for 7 days. Single dose PK parameters for ADV 10 mg in Asian patients were then compared to single dose ADV 10 mg PKs parameters in 83 Caucasians healthy volunteers and 14 CHB patients.

RESULTS
At baseline, 50% of patients enrolled in the treatment studies were Asian and 46% Caucasian. All patient demographic and HBV disease characteristics were well matched. Assessable baseline and week 48 biopsies were available for 88% HBeAg+ and 91% HBeAg patients. At week 48, ADV 10 mg resulted in histological improvement in 60% ADV compared to 26% of PLB (p < 0.001) in Caucasian patients and in 56% ADV compared to 29% of PLB Asian patients (p < 0.001). Change in serum HBV DNA from baseline at week 48 was of -3.9 and -3.7 log10 copies/mL in Caucasian and Asian patients, respectively while 34% of Caucasian patients and 39% of Asian patients had undetectable HBV DNA (< 400 copies/mL) at week 48. Consistently, the reduction in serum HBV DNA was similar across genotypes (A–D). The percentage of patients achieving ALT normalization at week 48 was similar in both groups (Asian 63%, Caucasian 64%). ADV was well tolerated by both ethnic groups with similar frequency of AEs in the ADV and placebo groups. PK parameters (AUC, Cmax, Tmax, and T1/2) were similar in Asian and Caucasian patients in phase 1 clinical trials conducted in the United States and Asia (Figure 1).

Figure 1: Pharmacokinetic Parameters of ADV 10 mg in Asian vs. Caucasian

CONCLUSIONS
In HBeAg+ and HBeAg- CHB patients, 48 weeks of ADV 10 mg once daily demonstrated significant histological, virological, and biochemical improvement compared to PLB, regardless of ethnicity (Asian or Caucasian). The similarity of response was consistent with the PK parameters in Asian and Caucasian patients.

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Analysis of Early Viral Kinetics and T-cell Reactivity in Relation to HBeAg Seroconversion During the First 12 Weeks of Antiviral Therapy for Chronic Hepatitis B

G. Lau1, H. Cooksley2, N. Naoumov2, S. Locarnini3, S. Bowden3, K. Powers4, R. Ribeiro4, S. Lewin4, A. Perelson5, E. Mondou6, C. Brosgart6
1Department of Medicine, Queen Mary Hospital, Hong Kong SAR, China; 2Institute of Hepatology, University College London, London, WC1E, UK; 3Victorian Infectious Diseases Reference Laboratory, University of Melbourne, Parkville, Victoria, Australia
4Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria, Australia; 5Los Alamos National Laboratory, Los Alamos, USA, 6Gilead Sciences, Inc., Durham, NC and Foster City, CA

Background
Optimization of antiviral treatment and long-term control of HBV replication for patients with chronic hepatitis B (CHB) will require a better understanding of HBV kinetics and HBV specific T-cell responses that result in long-term control of HBV replication. Emtricitabine (FTC), an investigational agent, and adefovir dipivoxil (ADV) a licensed agent, have potent in vitro and in vivo activity against HBV.

Aim
To determine whether the kinetics and magnitude of viral suppression and restoration of T-cell responses during the initial 12-weeks of therapy predict HBeAg seroconversion.

Study design and methods
Thirty treatment-naïve CHB patients (ALT>1.3xULN) were randomized in a double-blind, placebo-controlled, single center study to ADV (10 mg qd) or ADV + FTC (200 mg qd) for 48 weeks. Serum samples and peripheral blood mononuclear cells (PBMC) were prospectively collected, for determination of HBV DNA by real-time PCR (dynamic range: 5x102 to 109 copies/mL) and HBV-specific T-cell responses. Mathematical modeling was employed for HBV kinetic analysis. The frequency of IFN-ãproducing CD4+ and CD8+ T-cells in response to HBV Core and Envelope proteins or peptides, respectively were determined by Elispot assays. Definition of restoration of HBV-specific reactivity: greater than 50 specific spot forming cells per 106 PBMC (3 S.D. above baseline). The study is currently ongoing and remains blinded. Preliminary pooled data through week 12 are presented herein.

Results
Pooled data, showed a median reduction in serum HBV DNA of 3.2 log10 copies/mL (range: 1.1-5.9) at week 12 from a median baseline of 6.7 log10 copies/mL (range:3.6-8.6). The kinetics of HBV suppression fitted a 2-phase model, with the first phase ranging from 0.5 to 3.3d (median: 1.2d) and the second phase ranging from 3.6 to 111.9d (median: 16.3d). Restoration of IFN-a T-cell reactivity to Core and Envelope occurred in 4/30 (13%) patients, between week 4 and week 8. HBeAg seroconversion developed in 2 of these 4 patients after 12 weeks of treatment (50%) with, and in 0/26 without increased T-cell reactivity (log rank test, p<0.00005).

Conclusions
Restoration of T-cell reactivity during the first 12 weeks of antiviral treatment was associated with HBeAg seroconversion. Understanding the correlation of early virological response and T-cell reactivity may provide a basis for individualised approach and optimization of therapeutic strategies for treatment of chronic hepatitis B.

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A PHASE III, PARTIALLY DOUBLE-BLINDED STUDY EVALUATING THE EFFICACY AND SAFETY OF PEGINTERFERON ALFA-2a (40KD) (PEGASYS®) ALONE OR IN COMBINATION WITH LAMIVUDINE VS LAMIVUDINE IN 546 PATIENTS WITH HBeAg-NEGATIVE/ANTI-HBe-POSITIVE CHRONIC HEPATITIS B

Patrick Marcellin, Hopital Beaujon, Clichy, France; George K. K. Lau, The University of Hong Kong, Hong Kong, China; Ferruccio Bonino, IRCCS Ospedale Maggiore, Milano, Italy; Patrizia Farci, University of Cagliari, Cagliari, Italy; Stefanos Hadziyannis, Henry Dunant Hospital, Athens, Greece; Rui Jin, Beijing You An Hospital, Beijing, China; Zhi-Meng Lu, Ruijin Hospital, Shanghai, China; Teerha Piratvisuth, Songklanakarin Hospital, Songkla, Thailand; Georgios Germanidis, Papageorgiou General Hospital, Thessalonika, Greece; Cihan Yurdaydin, University of Ankara, Ankara, Turkey; Moises Diago, Hospital General Universitario de Valencia, Valencia, Spain; Selim Gurel, University of Uludag, Bursa, Turkey; Ming-Yang Lai, National Taiwan University, Taipei, Taiwan Republic of China; Peter Button, Roche, Dee Why, Australia; Nigel Pluck, Roche, Welwyn, United Kingdom; For the PEGASYS® Study in HBe-Antigen-Negative CHB.

Background
Conventional interferon alfa (IFNa) is one of the preferred first-line treatments for
chronic hepatitis B (CHB), but its pharmacokinetic characteristics (high peak-to-trough ratio, rapid elimination, thrice-weekly administration) represent a major limitation. Peginterferon alfa-2a (40KD) (PEGASYS®) offers the advantage of once-weekly administration while ensuring uniform therapeutic drug concentrations throughout the entire dosing interval. Peginterferon alfa-2a (40KD) has been shown to be superior to conventional IFNa in the treatment of HBeAg positive CHB (Cooksley et al, J Viral Hep 2003).

Objective
To compare the efficacy and safety of peginterferon alfa-2a (40KD) with and without lamivudine (LAM) to LAM alone in the treatment of HBeAg-negative/anti-HBe-positive patients with CHB.

Methods
This is a multinational, phase III study (PEGASYS® Study in HBe-Antigen-Negative CHB) conducted in 13 countries in accordance with international and local principles of Good Clinical Practice. All included patients were adults with CHB, documented by presence of HBsAg for >6 months and confirmed by liver biopsy. Patients were required to be HBeAg-negative and to have a positive anti-HBe test for at least 6 months, with detectable HBV DNA (>100,000 copies/mL by PCR) and an ALT value >ULN but <10 x ULN.

Patients with decompensated liver disease, patients co-infected with HCV, HDV or HIV, and patients who had received therapy for hepatitis B in the prior 6 months were excluded.

Patients were randomized using a 1:1:1 ratio to one of the following treatments:
1) Peginterferon alfa-2a (40KD) 180 µg once weekly + oral placebo once daily for 48 weeks;
2) Peginterferon alfa-2a (40KD) 180 µg once weekly + LAM 100 mg once daily for 48 weeks;
3) LAM 100 mg once daily for 48 weeks.

Patients in all three arms were followed for an additional 24 weeks after completion of the 48-week treatment course. Primary study endpoints were ALT normalization and reduction in HBV DNA to <20,000 copies/mL (by Roche AMPLICOR HBV MONITOR™ PCR) at end-of-follow-up. In addition, the study will explore changes in histology, based on paired biopsy comparison; loss of HBsAg and presence of anti-HBs; as well as safety and tolerability aspects.

Results
In total, 546 patients have been treated in the study. The baseline demographics of all patients in the study are as follows:
Gender: male = 85%, female = 15%;
Race: Oriental = 60%, Caucasian = 39%, Other = 1%;
Age (mean +/- SD): 40.2 +/- 11.2 years;
Body weight (mean +/- SD): 70.5 +/- 12.5 kg;
Baseline ALT (mean+/-SD): 96.9 +/- 99.1 IU/L;
HBV DNA (mean+/-SD): 7.2+/-1.9 log10 copies/mL;
HBV genotype: A = 5%, B = 24%, C = 34%, D = 34%, mixed/other = 3%;
HBV mutations: at nucleotide 1896 (pre-core stop codon) = 450/546 (82%); at nucleotide 1764 (basal core promoter region) = 386/523 (74%); both mutations = 304/520 (58%)
Histology: advanced fibrosis or cirrhosis = 27.5%

Conclusions
This is one of the largest studies ever conducted in patients with HBeAg-negative/anti-HBe-positive chronic hepatitis B. The study will answer two important questions of clinical relevance: 1) is peginterferon alfa-2a (40KD) (PEGASYS®), administered for a defined treatment period superior to LAM in achieving sustained viral response; 2) is a combination comprising peginterferon alfa-2a (40KD) plus LAM better than either agent alone? All patients in the study have reached the treatment-free follow-up period and full data will be available for presentation at the meeting.

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RESULTS OF A ONE-YEAR INTERNATIONAL PHASE IIB COMPARATIVE TRIAL OF TELBIVUDINE, LAMIVUDINE, AND THE COMBINATION, IN PATIENTS WITH CHRONIC HEPATITIS B

Ching-Lung Lai, Univ Hong Kong, Hong Kong, China; Nancy W. Y. Leung, Chinese Univ Hong Kong, Hong Kong, China; Eng-Kiong Teo, Changi Gen Hosp, Singapore, Singapore; Myron Tong, Huntington Mem Hosp, Pasadena, CA; Florence Wong, Toronto Gen Hosp, Toronto, ON, Canada; Hie-Wan Hann, Jefferson Med Coll, Philadelphia, PA; Steven Han, UCLA Sch Medicine, Los Angeles, CA; Thierry Poynard, Hosp Pitie-Salpetriere, Paris, France; Maureen Myers, George Chao, Deborah Lloyd, Nathaniel Brown, Idenix Pharmaceuticals, Cambridge, MA.

Introduction
Current antiviral therapy for chronic hepatitis B (CHB) is suboptimal with regard to durable suppression of HBV replication, and some therapies have safety and tolerance issues. A completed phase I/II trial of telbivudine (L–deoxythymidine; LdT) in patients with CHB indicated marked antiviral effects with no identified safety issues, prompting the present 1-year comparative trial of LdT and LdT+lamivudine vs. standard lamivudine (LAM) therapy.

Methods
This randomized, multicenter, international clinical trial evaluated the efficacy and safety of one year of treatment with standard LAM monotherapy (100 mg/day) compared to 4 investigational treatment regimens:
1. telbivudine 400 mg/day,
2. telbivudine 600 mg/day,
3. telbivudine 400 mg/day plus lamivudine (comb400), and
4. telbivudine 600 mg/day plus lamivudine (comb600).

Patients were HBeAg-positive adults with compensated CHB and baseline ALT >1.3 xULN. The primary efficacy measure was serum HBV DNA reduction over time, assessed by the COBAS Amplicor PCR assay. Key secondary endpoints included serum ALT normalization, HBeAg loss or seroconversion, and safety.

Results
104 eligible patients with compensated CHB were randomized (1:1:1:1:1). Median serum HBV DNA reductions at Week 52, in log10 copies/ml for the five treatment groups were: 4.66 for standard lamivudine therapy, 6.43 for telbivudine 400 mg/day, 6.09 for telbivudine 600 mg/day, 6.40 for lamivudine plus telbivudine 400 mg/day combo 400, and 6.05 for the lamivudine plus telbivudine 600 mg/day.

Conversely, residual HBV viremia at Week 52 was highest in lamivudine monotherapy recipients (median HBV DNA level at week 52 = 2.9 log10 copies/ml), while the median HBV DNA level in telbivudine monotherapy recipients was non-detectable by PCR <2 log10 copies/ml). For the combined LdT-containing treatment arms, antiviral efficacy at Week 52 was similar for Asian and non-Asian patients, with median HBV DNA reductions of 6.3 and 6.0 log10 copies/ml, respectively.

Key efficacy results at Week 52 were 61% of patients receiving telbivudine , 49% of patients receiving the combination of telbivudine and 32% of patients receiving lamivudine monotherapy.

Consistent with previous studies of anti-HBV therapies, mean pre-treatment ALT level was higher (212 vs 168 IU/mL) and mean pretreatment HBV DNA level was lower (8.7 vs 9.4 log10 copies/mL) among patients who lost HBeAg by Week 52, compared to those who remained HBeAg seropositive.

All study treatments were well-tolerated, with no treatment- or dose-related pattern of adverse events or laboratory abnormalities. Viral breakthrough and genotyping data will be available by the time of the meeting.

Conclusions
After one year of treatment, antiviral activity was significantly (p<0.05) greater for telbivudine compared to lamivudine, and ALT normalization was greatest for telbivudine monotherapy.

Combination treatment was not better than telbivudine alone in this study. These data support telbivudine as a promising safe and potent anti-HBV drug candidate, which appears likely to afford improved anti-HBV efficacy. Phase III trials of telbivudine are underway internationally.

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EFFECTS OF LAMIVUDINE ON DISEASE PROGRESSION AND DEVELOPMENT OF LIVER CANCER IN ADVANCED CHRONIC HEPATITIS B: A PROSPECTIVE DOUBLE-BLIND PLACEBO-CONTROLLED CLINICAL TRIAL

Yun-Fan Liaw, Chang Gung University, Taipei, Taiwan Republic of China; Joseph J. Y. Sung, The Chinese University of Hong Kong, Hong Kong, Hong Kong Special Administrative Region of China; Wan Cheng Chow, Singapore General Hospital, Singapore, Singapore; Kelly Shue, GlaxoSmithKline Asia Pacific, Singapore, Singapore; Oliver Keene, GlaxoSmithKline R&D, Greenford, United Kingdom; Geoff Farrell, The Storr Liver Unit, Sydney, Australia.

Introduction
Cirrhosis due to chronic hepatitis B is a common cause of liver failure, hepatocellular carcinoma (HCC) and a major cause of mortality in the Asia-Pacific region. Lamivudine (LAM) markedly suppresses viral replication and this confers histological improvement and improved liver function. However, longer term use leads to viral breakthrough in some patients due to emergence of YMDD variants, so the net impact on clinical disease progression in HBV related cirrhosis has been unclear.

Objective
To compare the long term efficacy and safety of lamivudine (LAM) and placebo (PLB) in the progression of HBV related cirrhosis.

Methods
Patients with histologically confirmed compensated HBV related cirrhosis were randomised 2:1 to LAM 100mg/day or PLB for up to 5yrs. The primary endpoint was time to disease progression defined as Child-Pugh score increase > 2, development of HCC, SBP, bleeding upper GI varices or liver-related death. Following blinded treatment, open label lamivudine was offered to eligible patients (eg. those reaching a clinical endpoint). An independent data safety monitoring board (DSMB) monitored study progress at interim analyses using prospectively defined stopping criteria.

Results
651 patients were randomised at 41 sites in 9 Asia-Pacific countries - 85% male, 98% Asian, mean age 44.3yrs. At the recommendation of the DSMB, the study was terminated at the second interim analysis, due to a significant difference in the number of endpoints between the two treatment groups, and patients were offered open label lamivudine. Median treatment exposure was 32.4 (range 0.0-42.1) months. 72 patients reached clinical endpoints - 34/436 (8%) of patients on LAM and 38/215 (18%) on PLB (hazard ratio 0.45, 95% CI 0.28 to 0.73, p=0.001). Kaplan-Meier estimates of proportions without disease progression after 3yrs were 91% on LAM and 79% on PLB. Increase in Child-Pugh score occurred in 15 (3%) of patients on LAM and 19 (9%) on PLB (hazard ratio 0.45, 95% CI 0.22 to 0.90, p=0.023), while HCC occurred in 17 (4%) on LAM and 16 (7%) on PLB (hazard ratio 0.49, 95% CI 0.25 to 0.99, p=0.047). Covariate modeling of time to disease showed that the factors to significantly impact the outcome, other than treatment, were baseline Child-Pugh score and baseline Ishak fibrosis score. In both cases, higher scores were associated with a greater frequency of endpoints. Treatment was well tolerated. 12% of patients on LAM and 18% on PLB reported serious adverse events during double-blind treatment. There were 16 deaths, 2 occurred during double-blind therapy and 14 after a clinical endpoint had been reached (7 during off-treatment follow-up and 7 during open label lamivudine).

Conclusions
Continuous long-term lamivudine treatment delays clinical progression in advanced stages of CHB by significantly reducing the rate of liver complications, and reducing the risk of HCC development.

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Resistance Surveillance of Liver Transplantation Patients with Lamivudine-resistant Hepatitis B Virus (HBV) after 96 Weeks of Adefovir Dipivoxil Treatment
S. Xiong1, C. Westland1, C. Brosgart1, M. Wulfsohn1, M. Miller1, J. Villeneuve2, N. Terrault3, F. Zoulim4
1Gilead Sciences, Inc., Foster City, CA, USA, 2Hôpital Saint-Luc, CHUM, Montreal, Canada, 3University of California at San Francisco, San Francisco, CA, USA, 4INSERM, Lyon, France.

Background
Lamivudine (LAM) resistance occurs in approximately 40% of chronic hepatitis B (CHB) patients after 2 years of LAM monotherapy. In contrast, resistance to adefovir dipivoxil (ADV) occurs infrequently with 1.6% of CHB patients developing the adefovir resistance mutation rtN236T after 2 years of ADV therapy. Pre-existing LAM resistance mutations or concurrent
use of immunosuppressive therapy by LT patients may increase the risk of resistance to ADV.

Objective
To determine the incidence of ADV resistance in a clinical trial of liver transplantation (pre and post) patients with LAM resistant HBV treated with ADV for 96 weeks (LAM therapy was maintained in most patients).

Methods
The HBV reverse transcriptase domain was sequenced for LT patients with detectable HBV DNA by PCR (>1000 copies/ml) after 96 weeks of ADV therapy (n=114). In vitro drug susceptibility was determined following transfection of HepG2 cells with patient-derived HBV clones from baseline and week 96 serum samples.

Results
The rtN236T mutation was observed in 2/114 patients (1.8%) at week 96. LAM had been discontinued at weeks 16 and 28 after initiation of ADV in these two patients respectively. The baseline LAM-resistant YMDD mutation reverted to wildtype prior to week 96 in both patients. Emergence of rtN236T was associated with rebound in serum HBV DNA and ALT elevation in both patients. In vitro phenotypic analysis showed approximately 4-fold reduced susceptibility to adefovir with rtN236T. However, these adefovir-resistant HBV clones were fully susceptible to LAM and entecavir in vitro. LAM therapy was re-initiated, in addition to the ongoing ADV therapy, after emergence of rtN236T in these patients resulting in a > 2.9 log10 copies/mL reduction in serum HBV DNA in both patients. One patient also had a significant reduction (>80%) in ALT within 5 months of LAM treatment. No other mutations potentially associated with adefovir resistance were detected.

Conclusions
Emergence of the adefovir resistance mutation rtN236T was observed infrequently after 2 years in liver transplantation patients (1.8%, 2/114) infected with LAM-resistant HBV, similar to observations in treatment naïve nonliver transplantation patients. The ADV-resistant HBV was sensitive to LAM and addition of LAM resulted in clinical stabilization in both patients.

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Poster 1146: RESPONSE TO ALPHA-INTERFERON PROLONGS SURVIVAL AND REDUCES THE RISK OF HEPATOCELLULAR CARCINOMA IN CHRONIC HEPATITIS B
Monika van Zonneveld, Hubert G M Niesters, Robert A de Man, Solko W Schalm, Harry L A Janssen, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands

Introduction
Data on the long-term effects of alpha-interferon (IFN) treatment on disease progression and mortality in patients with chronic hepatitis B(CHB) are limited.

Patients/Methods
To evaluate factors influencing clinical outcome and survival, we performed a follow-up study on 165 HBeAg-positive CHB patients treated with IFN. Forty-eight patients received two or more courses of IFN. The median dose of IFN was 30 MU/week (range 2-70 MU/week), the median duration of therapy was 16 weeks (range 1-92 weeks).

Eighty-seven patients (53%) were additionally treated with nucleoside analogues. Response to treatment was defined as HBeAg seroconversion within 12 months after the end of IFN therapy. Of the 165 patients 72% were male and 75% were Caucasian. Median follow-up was 8.8 years (range 0.3-24 years). Fifty-four patients (33%) responded to IFN treatment. Of the responders 52% lost HBsAg as compared to 9 % of the non-responders (p<0.001).

Results
Liver histology showed a decreased necroinflammatory activity and less progression of fibrosis in responders. Twenty-six patients died during follow-up, 16 of liver-related complications. Hepatocellular carcinoma was found in 8 patients, of which 6 were non-responders and one a responder who relapsed. The baseline factors age, albumin level and presence of liver cirrhosis were independent predictors of survival in multivariate analysis. Multivariate analysis showed a significantly improved survival (RR 0.25; 95% CI 0.09-0.70) and decreased risk of developing hepatocellular carcinoma in responders (RR 0.08; 95% CI 0.01-0.74). Responders also had a significantly improved survival if only cirrhosis was taken into account.

Conclusion
We conclude that response to IFN therapy results in a prolonged virological remission with a increased rate of HBsAg seroconversion and improved liver histology. Response to IFN therapy increases survival and reduces the risk of developing hepatocellular carcinoma.

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Poster 1153: THE IMPACT OF INTERFERON-ALFA (IFNa) THERAPY ON LIVER HISTOLOGY IN PATIENTS WITH HBeAg-NEGATIVE CHRONIC HEPATITIS B [HBeAg(-)CHB]
George V Papatheodoridis, Evangelos Cholongitas, Efi Kanta, Kalliopi Petraki, Ioannis Ketikoglou, Emanuel K. Manesis, Hippokration Hospital, Athens, Greece

Introduction
IFNa therapy has been shown to improve liver histology and to reduce the rate of fibrosis progression in chronic hepatitis C, but such an effect of IFNa has not been evaluated in chronic hepatitis B.

Patients/Methods
We studied 147 patients with HBeAg(-)CHB who had 2 or more liver biopsies during the last 10 years at our Department and had been treated with IFNa or had remained untreated. Patients treated with other antiviral or immuno-modulatory agents were excluded. All liver biopsies were evaluated blindly by a single liver histopathologist according to the classification of Ishak et al, 1995. IFNa induced sustained biochemical response in 30 (SRs), response at the end of therapy (EOT) and subsequent relapse in 57 (RRs) and no response in 33 (NRs) of the 120 treated patients, while 27 patients remained untreated. Median interval between the 2 biopsies was 24 (12-160) months.

Results
Grading score improved by >2 points in 40% of treated (SRs:77%, RRs:32%, NRs:21%) and 30% of untreated patients and worsened by >2 points in 21% (SRs:0%, RRs:26%, NRs:30%) and 48% of cases respectively (P=0.01).

In particular, among the 57 RRs, improvement and worsening in grading score were observed in 50% and 8% of the 24 cases with biopsies within the first 6 months and in 18% and 39% of the 33 cases with biopsies after the first 6 months following EOT (P=0.008).

Fibrosis score improved by >1 point in 17.5% of treated (SRs:40%, RRs:9%, NRs:12%) and 4% of untreated patients and worsened by >1 point in 34% (SRs:7%, RRs:40%, NRs:48%) and 70% of cases respectively (P=0.002).

In particular, among the 57 RRs, improvement and worsening in fibrosis score were observed in 13% and 23% of the 31 cases with biopsies within the first 12 months and in 4% and 62% of the 26 cases with biopsies after the first 12 months following EOT (P=0.01).

The fibrosis progression rate per year (mean±SE) was worse in the untreated (0.427±0.119) than treated patients (0.067±0.052, P=0.001) (SRs:-0.294±0.111, P<0.001; RRs:0.167±0.063, P=0.04; NRs:0.224±0.010, P=0.09 compared to untreated cases). The fibrosis progression rate in the untreated patients was not significantly different than the net fibrosis progression rate (after subtraction of duration of IFNa therapy) in NRs (0.457±0.175, P=0.23) or in RRs (0.221±0.125, P=0.13). In multivariate analysis, greater FPR was significantly associated with older age (P=0.01), worse change in grading score (P<0.001), lower baseline fibrosis (P=0.035) and type of response to IFNa (P=0.032), but not with IFNa therapy per se or any other epidemiological, biochemical and virologic patient characteristic.

Conclusions
In HBeAg(-)CHB patients, IFNa significantly improves the necroinflammatory activity and reduces the rate of fibrosis progression, but such an effect is mainly observed in patients with sustained biochemical responses. In patients with biochemical relapses, the timing of follow-up liver biopsy is extremely important, since severity of histological lesions significantly worsens after the first 6-12 months following the end of IFNa therapy. The strongest factor associated with the fibrosis progression rate is the change in liver necroinflammatory activity.

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Poster 1162: LY582563 (MCC-478), A 2-AMINOPURINE NUCLEOTIDE ANALOGUE, INHIBITS PACKAGING AND REPLICATION OF HBV GENOME IN VITRO
Tim Shaw, Danni Colledge, Vitina Sozzi, Stephen A. Locarnini, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Vic, Australia

Background
LY582563, a new synthetic nucleotide analogue developed jointly by Mitsubishi Pharmaceutical Company and Eli Lilly, is a potent inhibitor of replication of both wild type (wt) and lamivudine (LMV) resistant HBV strains. Prior experiments indicate that its mechanism(s) of action differs from those of LMV and adefovir.

Aims
To investigate the mechanisms by which LY582563 inhibits HBV replication, in vitro.

Methods
Compound LY58263 was provided by Eli Lilly. The following viral parameters were investigated:

(1) Endogenous HBV polymerase activity was measured in cell free assays using isolated, partly purified virus particles as the enzyme source.
(2) Biosynthesis of viral antigens and replicative intermediates including HBV ccc DNA were studied in HepG2 cells transduced with wt HBV using a recombinant baculovirus vector.
(3) Three derivatives of the Huh-7 cell line, in which the expression of HBV core protein is controlled by a tetracycline responsive element (pTRE) were used to study packaging in trans.
These were: pTRE (control), PC47 (precore producing; no rescue control), C4B (core producing; will rescue).
(4) AD-38 HepG2 cells, in which expression of an integrated HBV genome occurs in response to tetracycline withdrawal, were used to compare the antiviral effects of LY582563 and interferon-alpha (IFN).

Production of HBV RNA, intra- and extra- cellular DNA and nuclear ccc DNA was monitored by Northern blotting, quantitative RT-PCR (QPCR), and Southern blotting respectively. Viral antigens were detected and quantified by SDS-PAGE and immunoblotting. Southern blotting, QPCR, immunoblotting and electron microscopy (EM) were used to study capsid formation and encapsidation of pregenomic RNA (pgRNA).

Results
Phosphorylated metabolites of LY582563 did not inhibit endogenous HBV DNA polymerase activity. LY582563 had no significant effect on generation of HBV cccDNA, RNA, or the expression of viral antigens (HBe, HBs or HBc) in the recombinant baculovirus system. Inhibition of packaging of the pgRNA in cis, but not in trans, was observed in both baculovirus and pTRE based assays. EM examination revealed that the effect on packaging was not due to inhibition of core dimerisation or production of nucleocapsids. Furthermore, the antiviral effect of LY582563 in AD38 HepG2 cells did not appear to be mediated by an IFN-like mechanism.

Conclusions
LY582563 interfered with the packaging of HBV pgRNA into nucleocapsids. Since correctly assembled nucleocapsids are a prerequisite for efficient HBV replication, interference with the packaging of HBV pgRNA by LY582563 may be the primary mechanism by which it inhibits HBV replication. Further studies are required to determine whether LY582563 specifically or differentially inhibits priming of reverse transcription or RNase H activity. Based on this novel mechanism of action and its activity against wt and LMV-resistant HBV, further clinical studies of LY582563 appear warranted.

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ALT FLARES IN PATIENTS WITH HBeAg-POSITIVE CHRONIC HEPATITIS B AND TREATED WITH EITHER CONVENTIONAL INTERFERON ALFA-2a OR PEGINTERFERON ALFA-2a (40KD) (PEGASYS®) ARE NOT NECESSARY FOR A SIGNIFICANT RESPONSE TO THERAPY
W. Graham E. Cooksley, Royal Brisbane Hospital, Herston, Australia; Ming-Yang Lai, National Taiwan University, Taipei, Taiwan Republic of China; Teerha Piratvisuth, Songklanakarin Hospital, Songkla, Thailand; Yuan-Jen Wang, Taipei Veterans General Hospital, Taipei, Taiwan Republic of China; Varocha Mahachai, Chulalongkorn Hospital, Bangkok, Thailand; You-Chen Chao, Tri-Service General Hospital, Taipei, Taiwan Republic of China; Tawesak Tanwandee, Siriraj Hospital, Bangkok, Thailand; Anuchit Chutaputti, Pramongkutklao Hospital, Bangkok, Thailand; Wen-Yu Chang, Kaohsiung University Hospital, Kaohsiung, Taiwan Republic of China; Friederike E. Zahm, Roche, Basel, Switzerland; Nigel Pluck, Roche, Welwyn, United Kingdom.

Introduction
Transaminase flares are an inherent aspect of the natural course and progression of chronic hepatitis B (CHB) and are considered to be a reflection of host immune activity against infected hepatocytes. Furthermore, prominent ALT flares occurring during, or shortly after, interferon treatment of CHB have been associated with HBeAg seroconversion. Nucleoside/nucleotide analogues lead to suppression of viral replication and concomitant reduction of ALT flares. The antiviral activity of interferon also suppresses viral replication but at the same time modulation of the host immune system may provoke cytotoxic elimination of hepatocytes.

Objective
The use of peginterferon alfa-2a (40KD) (PEGASYS®) has previously been shown to exert greater antiviral activity than conventional interferon as measured by PCR testing of HBV DNA during therapy (Cooksley et al, J Viral Hep 2003). The objective here is to explore the incidence of significant ALT flares during the same comparative clinical trial of conventional interferon alfa-2a and peginterferon alfa-2a (40KD) therapy in HBeAg positive CHB, and relate them to the combined viral HBeAg status, HBV DNA and ALT outcomes.

Methods
A total of 194 adults with HBeAg-positive CHB and screening ALT >2 times the upper limit of normal (ULN), were randomized to treatment with conventional interferon alfa-2a 4.5 MIU, or peginterferon alfa-2a (40KD) 90 µg, 180 µg, or 270 µg for 24 weeks and then monitored for an additional 24 weeks. The most discriminating measure of efficacy was made using a combined response, which was determined at the end of the follow up period and required: loss of HBeAg, HBV DNA suppression to <500,000 copies/mL (Roche COBAS AMPLICOR HBV MONITOR™ Test, sensitivity of 200 copies/mL) and ALT normalization. Various levels of ALT rises have been used to define a significant flare and the definition used here is a peak ALT in excess of 10xULN.

Results
ALT flares (>10xULN) were seen in 22% of patients receiving conventional interferon alfa-2a, and 27%, 28% and 19% of patients receiving 90,180 and 270 µg peginterferon alfa-2a (40KD), respectively. Data on the frequency of on-therapy and follow-up flares in responders and non-responders are summarized below.

TREATMENT IFNa-2a Peg
90µg
Peg
180µg
Peg
270µg
All Peg
doses
No. of patients 51 49 46 48 143
Patients with an ALT Flare 11
(22%)
13
(27%)
13
(28%)
9
(19%)
35
(24%)
- Flare during therapy 5
(10%)
10
(20%)
9
(20%)
5
(10%)
24
(17%)
- Flare during follow up 6
(12%)
3
(6%)
4
(9%)
4
(8%)
11
(8%)
           
PATIENTS WITH
COMBINED RESPONSE
6
(12%)
13
(27%)
13
(28%)
9
(19%)
35
(24%*)
- Flare during therapy 1 3 1 0 4
- Flare during follow up 1 0 0 0 0
           
PATIENTS WITH NO
COMBINED RESPONSE
45 36 33 39 108
- Flare during therapy 4 7 8 5 20
- Flare during follow up 5 3 4 4 11

Flare is a peak ALT>10xULN.
Combined response is comprised: HBeAg loss and HBV DNA <500,000 copies/ml and ALT normal.
* p=0.036 in comparison with conventional IFNa-2a

Conclusions
Flares were observed in almost a quarter of all patients, but they occurred less frequently in patients who showed a significant therapeutic response (6 patients, 15%) than in those who did not (40 patients, 26%). This suggests that a marked ALT flare in patients treated with either interferon alfa-2a or peginterferon alfa-2a (40KD) (PEGASYS®) is not necessary for a significant response to therapy to occur. It is noteworthy that in the patients not showing a combined response, ALT flares were seen more frequently in those treated with peginterferon alfa-2a (40KD). However, these flares occurred more often during therapy than after therapy. Further implications of these observations will be explored in large ongoing comparative studies of peginterferon alfa-2a (40KD) at 180 µg in combination with either placebo or lamivudine vs lamivudine alone.

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Poster 1171: MOLECULAR EPIDEMIOLOGY OF HEPATITIS B VIRUS IN PATIENTS WITH HBEAG-NEGATIVE/ANTI-HBE-POSITIVE CHRONIC HEPATITIS B FROM DIFFERENT GEOGRAPHICAL AREAS DATA FROM THE PEGASYS® STUDY IN HBE-ANTIGEN-NEGATIVE CHB
Maurizia Brunetto, AnnaMaria Maina, Filippo Oliveri, Azienda Ospedaliera Pisana, Pisa, Italy; Ferruccio Bonino, IRCCS Ospedale Maggiore, Milano, Italy; Giuseppe Colucci, Roche Molecular Diagnostics, Rotkreuz, Switzerland; Patrick Marcellin, Hopital Beaujon, Clichy, France; George K K Lau, The University of Hong Kong, Hong Kong, China; Patrizia Farci, University of Cagliari, Cagliari, Italy; Stefanos Hadziyannis, Henry Dunant Hospital, Athens, Greece; Nigel Pluck, Roche, Welwyn, United Kingdom; For the PEGASYS® Study in HBe-Antigen-Negative CHB

Background
Anti-HBe positive chronic hepatitis B (CHB) patients of the Mediterranean area are typically infected with HBV genotype D mutants bearing the G to A switch at nucleotide (nt) 1896 of the pre-core (PC) region. In these patients, mutations at nt 1764 of the basic core promoter (BCP) have also been identified, but these do not appear to be a distinctive feature of the disease. Reports demonstrate the worldwide prevalence of anti-HBe positive CHB and therefore studies are needed to compare virological and clinical features of this disease in different geographic areas.

Objective

To characterize the genotype and genetic profile of HBV, as well as baseline viral load, in patients with anti-HBe positive CHB who participated in a multicenter study of peginterferon alfa-2a (40KD) (PEGASYS®) in combination with either placebo or lamivudine vs lamivudine alone.

Methods
This is a multinational, phase III study (PEGASYS® Study in HBe-Antigen-Negative CHB) conducted in 13 countries in accordance with international and local principles of Good Clinical Practice. All patients were anti-HBe positive for >6 months, and had elevated ALT, serum HBV DNA >105 copies/mL, and necroinflammation at histology (<2 years). Assays utilised were: COBAS AMPLICOR™ HBV Monitor (Roche Molecular Systems) for HBV DNA, INNO-Lipa HBV Genotyping test (Innogenetics), and Affigen HBV mutant VL19 Test (Sangtec Molecular Diagnostic AB) for the nt 1764 and 1896 genetic profile with a semiquantitative evaluation of wild-type/mutant HBV relative proportions (0 Affigene Mutant Units (AMU) = 100% of wild-type, 20 AMU = 100% of mutant virus).

Results
Fifty-nine percent of the patients enrolled in the study were Asians and 41% Europeans; 85% were males. Asians and Europeans had mean ages of 38.5 and 44.4 years, respectively (p<0.001). Viral load had a bimodal distribution, with 2 peaks at 106 and 108 copies/ml, and mean baseline HBV DNA values were 7.33 log10 copies/mL for Asians and 6.99 log10 copies/mL for Europeans (p=0.023). HBV genotypes were A in 5%, B in 24%, C in 34% and D in 34%; 3% of patients had other or mixed genotypes. Mutations at nucleotides 1764 and 1896 were characterized in 523 and 546 patients, respectively. The 1764 mutant was absent (0 AMU) or at low levels (1–5 AMU) in 163 patients (31.2%), at moderate levels (6–10 AMU) in 20 (3.8%) and at high levels (11–20 AMU) in 340 (65%); high levels of the 1764 mutation were more frequent in Europeans (p=0.003) and older patients (p<0.001). The 1764 mutant was inversely proportional to viral load (p<0.001).

In the multivariate analysis, older age (p<0.001) and lower viremia levels (p=0.008), and not the geographical origin, were independent factors significantly associated with the 1764 mutant. The 1896 mutant was absent (0 AMU) or at low levels (1–5 AMU) in 121 patients (22.2%), at moderate levels (6–10 AMU) in 37 (6.8%) and at high levels (11–20 AMU) in 388 (71%); high levels of the 1896 mutation were more frequent in Europeans (p=0.001).

The prevalence of the 1896 mutant was not influenced by viral load or age. In the multivariate analysis, geographic area (p=0.001) was the only independent factor associated with the 1896 mutant.

The 1764 mutation at high levels (11–20 AMU) was found in >70% of patients with genotypes A, C or D, but was much less common in those with genotype B (37%). The 1896 mutant was most common in patients with genotypes B and D (>75%).

Conclusions
These data suggest that the 1764 mutant appears to be selected over the course of chronic HBV infection. In contrast, the 1896 mutant appears as a specific virological feature of a subset of chronic hepatitis B patients influenced by host (race or environment) and/or viral (genotype) factors. Our data provide the basis for exploring the correlation of HBV genetic profile with therapeutic outcome. These aspects will be investigated in the PEGASYS® Study in HBe-Antigen-Negative CHB.

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Poster 246A: PEGINTERFERON ALFA-2B AND LAM IVUDINE COMBINATION THERAPY COMPARED WITH PEGINTERFERON ALFA-2b FOR CHRONIC HBEAG- POSITIVE HEPATITIS B: A RANDOMIZED CONTROLLED TRiAL IN 307 PATIENTS
HLA Janssen. H Senlurk, S Zeuzem, U Akarca, Y Cakaloglu, K Simon, T So Man
Kit, G Gerken, SW Schelm for the HBV 99-01 Study Group.Erasmus Medical Center, Rotterdam, The Netherlands, 2 Cerrehpasa Medical School, Istanbul, Turkey, University Hospital Homburg/Saar, Germany 4 Egé University, Izmir, Turkey. Istanbul University Medical School, Istanbul. Turkey, Medical University Wroclaw, Poland, 7 Princess Margaret Hospital, Hang Kong, S University Hospital Essen, Germany

Introduction
Interferon alfa, lamivudine or its combination lead to HBeAg seroconversion in less than one-third of patients. We investigated whether long-term combination therapy with peginterterorn alfa-2b (PEG-IFN) and lamivudine enhances response rate in comparison PEG-IFN monotherapy.

Three-hundred-seven patients were originally enrolled. One entire study center (25 patients) was dropped for protocol issues, 11 patients were HBeAg neagative at the start of therapy and 7 patients did not received therapy (no drug) so the modified intent to treat analysis was reduced to 266 patients. Study participants were randomized to receive PEG-IFN plus lamivudine (100 mg/day) or placebo for 1 year. The PEG-IFN dose was 100 µg per week s.c from month 0-8 and 50 µg from month 9 to 12. Patients were followed until 6 months after end of therapy.

The primary endpoint was defined as serum HBeAg negativity. The secondary endpoints were defined as HBV DBA <200,000 copies/ml, HBV DNA PCR negative (detection limit 400 copies/ml), ALT normalization and HBsAg loss. All patients were HBV-DNA positive (hybridization assay) and had ALT values more than twice ULN at baseline.

Key inclusion criteria included: HBeAg positive, serum ALT > 2 x ULN, age > 16 yo, compensated liver disease, not antiviral therapy 6 months prior to study entry, no coinfection with HIV, HCV or HDV.

Patient Demography

 
PEG-IFN
N=136
PEG-IFN/LAM
N = 130
Male
79%
75%
Age* (yr)
33
32
Orientals
21%
19%
Weight* (kg)
73
73
Prior IFN (%)
21%
21%
Prior Lamivudine (%)
12%
13%
ALT (ULN)
3.2
3.3
Cirrhosis
10%
12%
HBV DNA* (log 10)
9.2
9.2
Genotype A
35%
33%
Genotype B
9%
9%
Genotype C
16%
14%
Genotype D
38%
40%

*mean

The end of follow-up results:

 
PEG-IFN
PEG-INF PLUS LAM
P Value
HBeAg loss
36%
35%
P=0.91
HBV DNA response
27%
32%
P=0.44
HBV DNA PCR negative
7%
9%
P=0.43
ALT normalization
32%
35%
P=0.60
HBsAg loss
7%
7%

It should be noted that at the end of the 52-week treatment period, more patients had become HBeAg negative when treated with PEG-Intron plus lamivudine that with PEG-Intron alone (44 % vs. 29%, respectively). However, at the end of the 26-week follow-up period, the sustained response in the two study groups was statistically the same (35% and 36%, respectively). Similarly, at the end of the follow-up period the two groups showed equivalent responses to secondary endpoints: the percent of patients who achieved reduced levels of HBV DNA (32 % and 27%), seronegativity for HBV DNA (9% and 7%), seronegativity for HBsAg (7% for both groups), and normal serum levels of the standard liver enzyme marker ALT (35% and 32%, respectively). Sustained response rates varied by HBV genotype: 47 %, 44%, 27% and 25% for genotypes A, B, C and D, respectively.

Safety
Serious adverse events occurred in 32 patients (12%) – 17 probably related to therapy and were all reversible. The side effect profile was similar to conventional IFN with no difference in the side effects between PEG-IFN or combination of PEG-IFN and lamivudine. No deaths were reported as a result of therapy.

Conclusion
The authors concluded:

1. Long-term PEG-Intron monotherapy is effective for HBeAg positive chronic hepatitis B;
2. Combination with PEG-Intron and lamivudine leads to higher end-of-treatment response, but equal sustained response as PEG-Intron monotherapy;
3. PEG-Intron and its combination with lamivudine is well tolerated;
4. Peginterferon monotherapy should be the standard of care to achieve sustained response in HBeAg positive chronic hepatitis B.

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