Entecavir treatment for up to 5 years in patients with hepatitis B e antigen-positive chronic hepatitis B
Hepatology Jan 2010 advance pub
Ting-Tsung Chang 1 *, Ching-Lung Lai 2, Seung Kew Yoon 3, Samuel S. Lee 4, Henrique Sergio M. Coelho 5, Flair Jose Carrilho 6, Fred Poordad 7, Waldemar Halota 8, Yves Horsmans 9, Naoky Tsai 10, Hui Zhang 11, Daniel J. Tenney 11, Ricardo Tamez 12, Uchenna Iloeje 11
1Medical College, National Cheng Kung University, Tainan, Taiwan, ROC
2Queen Mary Hospital, Hong Kong, China
3Kangnam St. Mary Hospital, The Catholic University of Korea, Seoul, Korea
4Foothills Provincial General Hospital, University of Calgary, Calgary, Canada
5Hospital Universitario Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
6Department of Gastroenterology, University of S‹o Paulo School of Medicine, S‹o Paulo, SP, Brazil
7Department of Hepatology and Liver Transplantation, Cedars-Sinai Medical Center, Los Angeles, CA
8Department of Infectious Diseases and Hepatology, Nicolaus Copernicus University, Bydgoszcz, Poland
9Department of Hepatology and Gastroenterology, Cliniques St Luc, Brussels, Belgium
10University of Hawaii, Honolulu, HI
11Bristol-Myers Squibb Co. (BMS), Research and Development, Wallingford, CT
12Bristol-Myers Squibb Co., Research and Development, Princeton, NJ
email: Ting-Tsung Chang (firstname.lastname@example.org)
*Correspondence to Ting-Tsung Chang, 138 Sheng-Li Road, Tainan, 70428, Taiwan, Republic of China
Sustained virologic suppression is a primary goal of therapy for chronic hepatitis B (CHB). In study entecavir (ETV)-022, 48 weeks of entecavir 0.5 mg was superior to lamivudine for virologic suppression for hepatitis B e antigen (HBeAg)-positive CHB. A total of 183 entecavir-treated patients from ETV-022 subsequently enrolled in study ETV-901. We present the results after up to 5 years (240 weeks) of continuous entecavir therapy. The entecavir long-term cohort consists of patients who received 1 year of entecavir 0.5 mg in ETV-022 and then entered ETV-901 with a treatment gap 35 days. In ETV-901 the entecavir dose was 1.0 mg daily. For patients with samples available at Year 5, proportions with hepatitis B virus (HBV) DNA <300 copies/mL, normal alanine aminotransferase (ALT) levels, HBeAg loss, and HBeAg seroconversion were determined. In all, 146 patients met criteria for inclusion in the entecavir long-term cohort.
At Year 5, 94% (88/94) had HBV DNA <300 copies/mL and 80% (78/98) had normal ALT levels. In addition to patients who achieved serologic responses during study ETV-022, 23% (33/141) achieved HBeAg seroconversion and 1.4% (2/145) lost hepatitis B surface antigen (HBsAg) during study ETV-901. Through 5 years, entecavir resistance emerged in one patient. The safety profile of entecavir was consistent with previous reports.
Conclusion: Extended therapy with entecavir through 5 years maintained or increased rates of HBV DNA suppression and ALT normalization. Additional patients also achieved HBeAg loss and seroconversion. Entecavir provides sustained viral suppression with minimal resistance during long-term treatment of HBeAg-positive CHB.
Chronic hepatitis B (CHB) affects over 350 million people worldwide. Long-term complications of infection include cirrhosis and hepatocellular carcinoma (HCC), which together cause over 500,000 deaths annually. CHB patients with an elevated viral load (ongoing viral replication) have the highest risk of progressing to these life-threatening complications. To avoid or minimize liver disease progression, CHB treatment recommendations now stress the importance of long-term maintenance of hepatitis B virus (HBV) DNA suppression.[5-7]
Medications currently approved for the treatment of hepatitis B e antigen (HBeAg)-positive CHB include standard interferon-a, pegylated interferon-a, lamivudine, adefovir dipivoxil, entecavir, telbivudine, and tenofovir disoproxil fumarate. Treatment with standard or pegylated interferon has been shown to result in durable serologic responses (HBeAg seroconversion) in HBeAg-positive patients, but these therapies are limited by the need for parenteral administration and a high incidence of adverse events.[8-10] Lamivudine has demonstrated efficacy and safety, but the benefits of treatment have limited durability as resistance reaches about 70% after 4 years of therapy. Current CHB treatment guidelines recommend against the use of lamivudine as first-line therapy due to its high rate of resistance. Although telbivudine demonstrated greater suppression of HBV DNA than lamivudine, monitoring in patients with virologic breakthrough showed that resistance exceeds 20% among HBeAg-positive patients treated for 2 years. Treatment with adefovir for 48 weeks resulted in HBV DNA suppression to <400 copies/mL in only 13% of HBeAg-positive patients, and resistance has been shown to develop in 20% of HBeAg-positive patients after 5 years. Tenofovir treatment for HBeAg-positive CHB achieves high levels of virologic suppression, but at this time, efficacy and resistance data have only been reported through 96 weeks (2 years).
Entecavir demonstrated superior histologic, virologic, and biochemical benefit compared to lamivudine after 48 weeks in entecavir (ETV)-022, a study conducted in nucleoside-naïve HBeAg-positive CHB patients. In a blinded extension of this study, which evaluated continued entecavir or lamivudine treatment through 96 weeks, increasing numbers of entecavir-treated patients experienced virologic, biochemical, and HBeAg serologic responses, with a safety profile comparable to that of lamivudine. The current report presents efficacy and safety results for HBeAg-positive patients who received a minimum of 52 weeks of blinded entecavir treatment in study ETV-022 (www.clinicaltrials.gov identifier: NCT00036608), and subsequently received open-label entecavir in rollover study ETV-901 for a cumulative total duration of up to 5 years (240 weeks).
This analysis provides data on long-term treatment with entecavir in nucleoside-naïve, HBeAg-positive patients with CHB, and demonstrates that long-term entecavir therapy in this population achieved and maintained HBV DNA suppression. At Year 5, 94% of patients in the entecavir long-term cohort had HBV DNA <300 copies/mL. The importance of maintaining prolonged HBV DNA suppression to avoid or minimize the long-term complications of CHB has been recognized in several long-term studies of disease progression and outcome. Patients with persistently elevated viral load are at the greatest risk of developing liver disease progression and adverse outcomes. It has also been shown that even patients with low-level HBV DNA viremia (below 104 to 105 copies/mL) are at risk of fibrosis, cirrhosis, and HCC. Current CHB treatment recommendations advocate sustained suppression of HBV DNA as the primary goal of antiviral therapy. A key requirement for maintaining long-term HBV DNA suppression is the avoidance of resistance to the antivirals.
The current cohort analysis followed entecavir-treated patients from ETV-022 who continued to receive entecavir in ETV-901 through up to 5 years of therapy. Most patients who did not enter ETV-901 were responders in ETV-022, the majority of whom achieved HBV DNA <300 copies/mL through 2 years of entecavir therapy. Despite high proportions of the previous nonresponders and only one previous responder in the entecavir long-term cohort, high rates of HBV DNA to <300 copies/mL (94%) and normal ALT levels (80%) were achieved or maintained at 5 years.
Patients in ETV-901 received entecavir 1.0 mg daily and in some cases had a period of treatment with both entecavir and lamivudine. Most patients in the entecavir long-term cohort with HBV DNA <300 copies/mL had already achieved this endpoint after 2 years of treatment with entecavir 0.5 mg daily. This result suggests that the dosage increase from 0.5 mg (in ETV-022) to 1.0 mg (in ETV-901) had minimal contribution to the virologic suppression noted in the long-term cohort. Further evidence that entecavir at a dose of 0.5 mg daily achieves and maintains HBV DNA suppression during long-term therapy is suggested by a recent study of entecavir in Japanese patients with CHB. In that study, 87% of patients achieved HBV DNA <400 copies/mL after 3 years of continuous treatment with entecavir 0.5 mg daily.
Treatment with entecavir beyond 2 years resulted in incremental benefits for serologic response. Most patients who had previously undergone HBeAg seroconversion after 2 years of entecavir therapy in study ETV-022 were categorized as responders and did not enroll in study ETV-901. Therefore, HBeAg seroconversion occurring in 23% (33/141) of the entecavir long-term cohort during study ETV-901 represents an incremental improvement in serologic response in a difficult-to-treat population, in addition to the 31% of patients with HBeAg seroconversion through 2 years in study ETV-022. Continued treatment with entecavir beyond 2 years also resulted in incremental benefit of HBsAg loss: in addition to the 18 (5%) patients who lost HBsAg during ETV-022, two more patients (1.4%, 2/145) in the entecavir long-term cohort lost HBsAg during study ETV-901. It should be noted that HBV serology assays during study ETV-901 were performed at local laboratories using variable methodologies, in contrast to the uniform assay performed in a central laboratory during study ETV-022.
Patient drop-out and missing data are common during long-term studies. In this study, 47 patients had discontinued treatment prior to Year 5, and five patients who were still on-treatment at Year 5 had missing HBV DNA measurements. An LOCF analysis was conducted as a sensitivity analysis and 88% of all patients in this long-term cohort had HBV DNA <300 copies/mL either at Year 5 or at last observation prior to Year 5. Interpretation of this sensitivity analysis should be done with some caution, as it makes two major assumptions by definition: 1) it assumes that patients who discontinued treatment without achieving HBV DNA <300 copies/mL would not have achieved it with longer treatment; and 2) it assumes that patients who achieved this endpoint prior to discontinuing would have maintained it over time. Given the results of the primary analysis and entecavir's antiviral potency, it is likely that this is a conservative analysis; however, the 88% response rate in the sensitivity analysis is consistent with the results of the primary analysis.
All the patients in this cohort were monitored as part of the entecavir resistance cohort; for the whole cohort through 5 years, only one patient (who received concurrent entecavir and lamivudine early in ETV-901) developed substitutions associated with entecavir resistance (during Year 3). The rate of entecavir resistance remains rare over long-term therapy and distinguishes it from other HBV antivirals with long-term data. Entecavir's resistance profile is believed to result from its potent viral suppression and high genetic barrier to resistance.
Through 5 years of therapy in this cohort, entecavir maintained a safety and tolerability profile consistent with that reported in previous studies. No patient discontinued therapy due to adverse events. One patient experienced an ALT flare and one case of HCC (diagnosed during the first year of treatment in study ETV-022) was reported.
In summary, the results from this entecavir long-term cohort show that among HBeAg-positive patients, therapy with entecavir for 5 years achieves and maintains high rates of HBV DNA suppression and normal ALT levels, with minimal development of resistance. Entecavir was also well tolerated through 5 years of dosing. With its safety, viral suppression, and resistance profile, entecavir is now considered a preferred choice for treatment of nucleoside-naïve HBeAg-positive CHB patients.
Of the 354 patients who were randomized to treatment with entecavir 0.5 mg in the ETV-022 study, 183 enrolled in the ETV-901 study (11 responders, 151 virologic responders, and 21 nonresponders; Fig. 1). Of these, 146 patients (one responder, 126 virologic responders, and 19 nonresponders) had a treatment gap of =35 days between the last study dose in ETV-022 and the first study dose in ETV-901 and were considered continuously treated. These 146 patients constituted the nucleoside-naïve HBeAg-positive entecavir long-term cohort.
Among the 146 patients in the entecavir long-term cohort, 68% (99/146) received entecavir through 5 years. Forty-seven patients discontinued treatment prior to the Year 5 visit. The reasons for treatment discontinuation were: completion of treatment in the opinion of the investigator (12), progression of CHB (1); death (5); loss to follow-up (2); patient noncompliance (1); withdrawal of consent (14); minimal virologic response (3); and other (9). Mean time on therapy for the entecavir long-term cohort (n = 146) through studies ETV-022 and ETV-901 was 248 weeks. Of the 146 patients, 132 received entecavir in ETV-022 and entecavir together with lamivudine in study ETV-901, and 14 received only entecavir through both studies. Of the 132 patients who received entecavir with lamivudine in study ETV-901, 12 received the combined regimen only (mean exposure to lamivudine was 26.4 weeks) and 120 received entecavir without lamivudine after initially receiving both (mean exposure to entecavir and lamivudine were 169 and 25.5 weeks, respectively).
Baseline (pretreatment) demographic and disease characteristics for the entecavir long-term cohort are presented in Table 1. The majority of patients in the cohort were male (80%) and Asian (64%), with a mean age of 36 years. Mean baseline levels of HBV DNA and ALT were 9.9 log10 copies/mL and 122 IU/L, respectively. Infection with HBV genotype A (26%), B (27%), or C (30%) accounted for most patients; 4% were infected with HBV genotype D.
HBV DNA was suppressed early in therapy and extended treatment increased or maintained viral suppression through Year 5 (Fig. 2). Mean change from baseline in HBV DNA at Year 5 was -7.2 log10 copies/mL. Fifty-five percent of patients in the cohort had achieved HBV DNA <300 copies/mL at Year 1 of the Phase III study (ETV-022; Fig. 3). The proportion of patients in the entecavir long-term cohort achieving HBV DNA <300 copies/mL increased from 55% in Year 1 to 83% in Year 2. Among 116 patients who had HBV DNA <300 copies/mL at Year 2, 109 (94%) achieved this response while receiving entecavir 0.5 mg daily in study ETV-022 and the other seven achieved the endpoint while receiving entecavir 1.0 mg ± lamivudine (in study ETV-901). Continuous treatment through Years 3, 4, and 5 resulted in increasing proportions of patients achieving and maintaining HBV DNA <300 copies/mL, with 94% (88/94) of patients achieving or maintaining this endpoint at Year 5. Figure 4 shows the distribution of patients according to HBV DNA level at Year 5; only one patient had HBV DNA >105 copies/mL.
Five additional patients who remained on-treatment at Year 5 had missing HBV DNA measurements: four had HBV DNA <300 copies/mL at their last observation prior to the Year 5 analyses; one had detectable HBV DNA and had ETV resistant virus emerge during Year 3, as described below. Of the 47 patients in the cohort who discontinued treatment during study ETV-901 prior to Year 5, 79% (37/47) had HBV DNA <300 copies/mL on their last HBV DNA measurement. The sensitivity analysis was conducted based on the intention-to-treat (ITT) population. The last observed HBV DNA levels for all patients who were either still on study but had a missing PCR test at Year 5 (n = 5) or who had discontinued prior to Year 5 (n = 47) were carried forward; this maintained the total number of patients in this cohort intact (n = 146). When the Year 5 HBV DNA endpoint is calculated using this method, 88% (129/146) of patients had HBV DNA <300 copies/mL at Year 5.
As with virologic response, results for ALT normalization among patients in the entecavir long-term cohort at Year 1 were consistent with results of the overall ETV-022 patient population (Fig. 5). Sixty-five percent (95/146) of patients in the entecavir long-term cohort achieved ALT = 1 x ULN at Year 1. Treatment in Year 2 resulted in increasing proportions achieving the endpoint (78%, 109/140), and continuous treatment through Years 3, 4, and 5 resulted in maintenance of ALT normalization (80% 78/98 at Year 5; Fig. 5). At Year 5, the mean ALT level for the entecavir long-term cohort was 33 IU/L, a decrease from the mean level of 122 IU/L at baseline.
During study ETV-022, 31% (110/354) and 5% (18/354) of patients achieved HBeAg seroconversion and HBsAg loss, respectively, through up to Year 2 of treatment plus up to 24 weeks of posttreatment follow-up. Due to protocol-defined management criteria, most patients who achieved HBeAg loss or HBeAg seroconversion during ETV-022 discontinued study therapy (as responders), did not enroll in ETV-901, and thus were not part of the entecavir long-term cohort. Among 146 patients in the entecavir long-term cohort, two achieved HBeAg seroconversion during ETV-022 but did not meet the virologic criterion for response (HBV DNA <0.7 MEq/mL), and three experienced seroreversion after HBeAg seroconversion during ETV-022 and therefore enrolled in ETV-901. Continued treatment in ETV-901 resulted in 33 additional patients (23%, 33/141) achieving HBeAg seroconversion on-treatment. One patient in the entecavir long-term cohort achieved HBsAg loss during ETV-022 and two additional patents (1.4%, 2/145) achieved HBsAg loss during continued treatment in ETV-901.
Resistance and Virologic Breakthrough.
Genotypic testing of isolates from patients with HBV DNA >/= 300 copies/mL at Years 1, 2, 3, 4, and 5 identified one patient (1/146) with entecavir resistance that emerged during Year 3, and has been reported. This patient received 16 weeks of lamivudine plus entecavir prior to being switched to entecavir alone (per protocol amendment), showed the simultaneous emergence of L180M/M204V (associated with lamivudine resistance) and S202G (associated with entecavir resistance) at Week 139, and had virologic breakthrough at Week 148. Of the 47 patients who discontinued treatment prior to Year 5, 10 had HBV DNA >/= 300 copies/mL at the last on-treatment measurement. Genotypic testing of isolates from these 10 patients found no evidence of entecavir resistance.
The safety profile for this cohort during treatment with open-label entecavir (study ETV-901) is summarized in Table 2. During ETV-901, no patient in this cohort discontinued entecavir due to an adverse event (Table 2). Adverse events occurring in >/=10% of patients are shown in Table 3. The most common serious adverse events were increased ALT and liver abscess, both occurring in two (1%) patients. One patient, who stopped study medication 172 weeks after initially starting on entecavir, experienced an ALT flare that was associated with a >/= 2-log increase in HBV DNA. This patient was subsequently lost to follow-up at Week 176. The safety profile for the entecavir long-term cohort during study ETV-901 was consistent with the safety profile reported for all entecavir-treated patients through 2 years in study ETV-022. Within study ETV-901, there was no observed difference between the cumulative safety profile of the entecavir long-term cohort (n = 146) and that of the larger patient population treated in the rollover study (ETV-901). Through 5 years of entecavir treatment and posttreatment follow-up, one patient (of 146) in the entecavir long-term cohort developed HCC (described below).
For the entecavir long-term cohort, five deaths were reported during study ETV-901, including off-treatment follow-up. No death was attributed to study medication. The investigator-assigned causes of death were liver failure (1), motor vehicle accident (3), and unknown (1). The patient who died from liver failure was diagnosed with HCC at Week 51 of study ETV-022, and completed 2 years of dosing in that study. The patient subsequently enrolled in study ETV-901, was treated for 40 weeks and died during Week 136 (total entecavir treatment time) of liver failure secondary to progression of HCC.
Patients and Methods
Study ETV-022 was a randomized, double-blind comparison of entecavir and lamivudine for up to 96 weeks in nucleoside-naïve patients with HBeAg-positive CHB. A total of 715 patients were enrolled at 137 centers worldwide between December 2001 and September 2002. Patients were randomized to receive entecavir 0.5 mg or lamivudine 100 mg once daily for a minimum of 52 weeks. Patients classified as responders (HBV DNA <0.7 MEq/mL and HBeAg loss) or nonresponders (HBV DNA >/= 0.7 MEq/mL) at Week 48 discontinued therapy at Week 52. Responders were followed off-treatment for 24 weeks and nonresponders were offered enrollment into rollover study ETV-901 or, at the discretion of the investigator, alternative off-study anti-HBV therapy. Patients who achieved a protocol-defined virologic response (serum HBV DNA <0.7 MEq/mL [about 700,000 copies/mL] by branched DNA [bDNA] assay [Bayer Diagnostics, formerly Chiron Diagnostics], but remained HBeAg-positive) at Week 48 were offered continued blinded treatment through Week 96 or until loss of HBeAg. During the second year of treatment (Weeks 52-96) any virologic responders (HBV DNA <0.7 MEq/mL) who became responders or nonresponders discontinued study therapy.
Study ETV-901 is an ongoing multinational rollover study designed to provide open-label entecavir to patients from 10 Phase II or Phase III entecavir studies. For patients in the ETV-022 study, the following subgroups of patients could enter ETV-901: 1) virologic responders at Week 48 (Year 1) who opted not to continue to a second year in study ETV-022; 2) virologic responders at Week 96 (Year 2); 3) nonresponders from either the first or second year of blinded treatment; and 4) responders from either the first or second year of blinded treatment who experienced virologic relapse (defined as serum HBV DNA >/= 0.7 MEq/mL and/or detection of HBeAg on two occasions >/= 2 weeks apart) during off-treatment follow-up. The nucleoside-naïve HBeAg-positive entecavir long-term cohort (hereafter called the entecavir long-term cohort) consists of entecavir-treated patients from study ETV-022 who had = 35-day off-treatment gap between the last entecavir dose in study ETV-022 and the first entecavir dose in study ETV-901, and includes all patients who satisfy this definition regardless of treatment response achieved in ETV-022.
Initially, due to ongoing blinding of Phase II/III studies, patients enrolling in ETV-901 received entecavir plus lamivudine. The protocol was subsequently amended so that patients received only entecavir (1.0 mg daily). Duration of treatment in study ETV-901 was at the discretion of the investigator; patients could continue until study closure if the investigator judged that continued treatment was in the patient's best interest or discontinue at any time. All patients who discontinued treatment in ETV-901 were required to be followed for at least 24 weeks postdosing to assess safety.
The studies were conducted in accordance with the ethical principles of the Declaration of Helsinki and in adherence to the laws and regulatory requirements of all participating countries. Written informed consent was obtained from all study participants.
Complete inclusion criteria for enrollment in the ETV-022 study have been described. Eligible patients were 16 years or older and had HBeAg-positive CHB and compensated liver function. Patients were required to have detectable hepatitis B surface antigen (HBsAg) for at least 6 months and evidence of chronic hepatitis on a baseline liver biopsy completed within 1 year of randomization. At screening, patients were required to have serum HBV DNA >/= 3 MEq/mL (about 3 million copies/mL) by bDNA assay, and alanine aminotransferase (ALT) 1.3-10 times the upper limit of normal (ULN). Exclusion criteria included coinfection with hepatitis C virus, hepatitis D virus, or human immunodeficiency virus (HIV), prior treatment with lamivudine for >12 weeks, and exposure to other antiviral agents within 6 months of randomization. Patients treated in study ETV-022 who were eligible to enter study ETV-901 are described above in Study Design.
Time on Treatment.
For the HBeAg-positive entecavir long-term cohort, time on treatment for efficacy analyses was defined as the total duration in weeks from the first dose in ETV-022 to the last date of dosing up to Week 240 (Year 5) in ETV-901. Safety analyses were based on exposure during ETV-901.
In ETV-901, serum HBV DNA was determined by polymerase chain reaction (PCR) assay at 12-week intervals during the first year and at 24-week intervals thereafter while treatment continued. HBV serologies were obtained every 12 weeks during the first and second year of open-label treatment. Efficacy assessments for the cohort include proportions of patients at 48, 96, 144, 192, and 240 weeks (Years 1, 2, 3, 4, and 5, respectively) who met the following endpoints: HBV DNA <300 copies/mL, HBeAg loss, HBeAg seroconversion, and normal ALT (1.0 x ULN). Mean serum levels of HBV DNA and ALT for the cohort were also determined at baseline and at 24-week intervals through Year 5.
Safety analyses for the HBeAg-positive entecavir long-term cohort included the following events occurring on-treatment during study ETV-901: adverse events, serious adverse events, treatment discontinuations due to adverse events or laboratory abnormalities, and ALT flares. Deaths that occurred on-treatment in study ETV-901 or during off-treatment follow-up are also described. The results of safety analyses for study ETV-022 have been reported.
Genotypic analyses were performed to assess and characterize any emerging entecavir resistance substitutions among HBV isolates from patients who had HBV DNA >/= 300 copies/mL at Years 1, 2, 3, 4, and 5, and from patients who discontinued treatment for any reason prior to Year 5 (end of dosing) and had HBV DNA >/= 300 copies/mL at the time of treatment discontinuation. Genotypic and phenotypic analyses were also performed on isolates from all patients experiencing virologic breakthrough (>/= 1 log10 increase from nadir) at the time breakthrough occurred.
All data analyses are descriptive. Tabulations by treatment group are presented for each of the efficacy and safety variables. Continuous variables are summarized using the mean, median, minimum, and maximum values. Binary variables are summarized by counts and percentages. Efficacy endpoints were assessed among patients with available samples. An additional sensitivity analysis using the last observation carried forward method (LOCF) was conducted for the endpoint of HBV DNA <300 copies/mL at Week 240 (Year 5). In this analysis, the last observed HBV DNA levels were carried forward for those patients without Year 5 measurements, i.e., patients who had either discontinued prior to Year 5 or who were still on study but had a missing HBV DNA measurement at Year 5.
Safety analyses for the cohort include all adverse events that occurred on-treatment in study ETV-901 and all deaths that occurred on-study (during treatment in ETV-901 or during off-treatment follow-up).
Serum HBV DNA was quantified by a central laboratory using the Roche COBAS Amplicor PCR assay (v. 2.0; lower limit of quantification 300 copies/mL [57 IU/mL]; Pleasanton, CA). In study ETV-022, HBV serologies (HBsAg, anti-HBs, HBeAg, anti-HBe) were assessed in a central laboratory using the Abbott AxSYM microparticle enzyme immunoassay (Abbott Laboratories, North Chicago, IL) and DiaSorin enzyme immunoassay. In study ETV-901, HBV serologies were assessed in local laboratories using available methodologies. ALT was assessed by local laboratories in studies ETV-022 and ETV-901. Genotyping of HBV DNA involved PCR amplification of the HBV reverse transcriptase domain, followed by nucleotide sequence analysis. In phenotypic analyses, substitutions that emerged on-treatment were cloned into HBV expression vectors, which were transfected into HepG2 hepatoma cells in the presence of entecavir. The amounts of replicated, encapsulated HBV DNA was immunocaptured from the culture media and quantified to determine the susceptibility to entecavir.[20-22]
Role of the Funding Source.
The study was designed by the sponsor in collaboration with expert hepatologists. The sponsor collected the data, carried out the statistical analyses, and coordinated the writing of the article with all authors.