Tenofovir alafenamide versus tenofovir disoproxil fumarate for the treatment of patients with HBeAg-negative chronic hepatitis B virus infection: a randomised, double-blind, phase 3, non-inferiority trial

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Tenofovir alafenamide versus tenofovir disoproxil fumarate for the treatment of patients with HBeAg-negative chronic hepatitis B virus infection: a randomised, double-blind, phase 3, non-inferiority trial Maria Buti, Edward Gane, Wai Kay Seto, Henry L Y Chan, Wan-Long Chuang, Tatjana Stepanova, Aric-Josun Hui, Young-Suk Lim, Rajiv Mehta, Harry L A Janssen, Subrat K Acharya, John F Flaherty, Benedetta Massetto, Andrea L Cathcart, Kyungpil Kim, Anuj Gaggar, G Mani Subramanian, John G McHutchison, Calvin Q Pan, Maurizia Brunetto, Namiki Izumi, Patrick Marcellin, and the GS-US-320-0108 Investigators*

Summary Background The novel prodrug tenofovir alafenamide delivers the nucleotide reverse transcriptase inhibitor tenofovir to target cells more efficiently at a lower dose than tenofovir disoproxil fumarate, thereby reducing systemic exposure. We compared the efficacy and safety of the two formulations in patients with HBeAg-negative chronic hepatitis B virus (HBV) infection in a non-inferiority study. Methods In this ongoing randomised, double-blind, phase 3, non-inferiority study in 105 centres in 17 countries, patients with HBeAg-negative chronic HBV were randomly assigned (2:1) by a computer-generated allocation sequence (block size six), stratified by plasma HBV DNA concentration and previous treatment status, to receive once-daily oral doses of tenofovir alafenamide 25 mg or tenofovir disoproxil fumarate 300 mg, each with matching placebo. Participants, investigators, and those assessing outcomes were masked to group assignment. Eligible patients were aged at least 18 years with HBeAg-negative chronic HBV infection (with plasma HBV DNA concentrations of >20 000 IU/mL), serum alanine aminotransferase concentrations of greater than 60 U/L in men or greater than 38 U/L in women and at no more than ten times the upper limit of normal, and estimated creatinine clearance of at least 50 mL/min (by the Cockcroft-Gault method). The primary efficacy endpoint was the proportion of patients who had HBV DNA less than 29 IU/mL at week 48 in those who received at least one dose of study drug; the study was powered to show non-inferiority with a 10% efficacy margin of tenofovir alafenamide compared with tenofovir disoproxil fumarate. Bone and renal safety, and key secondary safety endpoints were assessed sequentially. The study will be conducted for a total of 3 years as a double-blind comparison to assess the longer term response to treatment. This study is registered with ClinicalTrials.gov, number NCT01940341. Findings Between Sept 12, 2013, and Oct 31, 2014, 426 patients were randomly assigned (285 assigned to tenofovir alafenamide and 141 assigned to tenofovir disoproxil fumarate; one patient assigned to tenofovir disoproxil fumarate did not receive the treatment. 268 (94%) of 285 patients receiving tenofovir alafenamide had HBV DNA less than 29 IU/mL at week 48 versus 130 (93%) of 140 patients receiving tenofovir disoproxil fumarate (difference 1·8% [95% CI –3·6 to 7·2]; p=0·47), which demonstrates non-inferiority. Patients receiving tenofovir alafenamide had significantly smaller mean percentage declines in bone mineral density than those receiving tenofovir disoproxil fumarate (hip –0·29% [95% CI –0·55 to –0·03] vs –2·16% [–2·53 to –1·79], adjusted percentage difference 1·87% [95% CI 1·42 to 2·32; p<0·0001]; spine –0·88% [–1·22 to –0·54] vs –2·51% [–3·09 to –1·94], adjusted percentage difference 1·64% [95% CI 1·01 to 2·27]; p<0·0001). At week 48, mean change in serum creatinine was small in both groups (tenofovir alafenamide 0·01 mg/dL [95% CI 0·00 to 0·02] vs tenofovir disoproxil fumarate 0·02 mg/dL [0·00 to 0·04], adjusted percentage difference –0·01 mg/dL [95% CI –0·03 to 0·01]; p=0·32), but patients receiving tenofovir alafenamide had a smaller reduction in creatinine clearance (median change in estimated glomerular filtration rate –1·8 mL/min [IQR –7·8 to 6·0] vs –4·8 mL/min [–12·0 to 3·0]; p=0·004). Most adverse events were mild to moderate in severity in the two treatment groups. The most common adverse events overall were headache (tenofovir alafenamide 40 [14%] patients vs tenofovir disoproxil fumarate 14 [10%] patients), nasopharyngitis (30 [11%] vs 15 [11%]), and upper respiratory tract infection (35 [12%] vs ten [7%]). 14 (5%) patients receiving tenofovir alafenamide and nine (6%) patients receiving tenofovir disoproxil fumarate had serious adverse events, none of which was deemed by investigators to be related to study treatment; one patient in the tenofovir disoproxil fumarate group died, but this was not deemed to be related to study treatment. Interpretation In patients with HBeAg-negative chronic HBV, the efficacy of tenofovir alafenamide was non-inferior to that of tenofovir disoproxil fumarate, and had improved bone and renal effects. Longer term follow-up is needed to better understand the clinical impact of these changes. Funding Gilead Sciences. www.thelancet.com/gastrohep Published online September 22, 2016 http://dx.doi.org/10.1016/S2468-1253(16)30107-8

Lancet Gastroenterol Hepatol 2016 Published Online September 22, 2016 http://dx.doi.org/10.1016/ S2468-1253(16)30107-8 See Online/Articles http://dx.doi.org/10.1016/ S2468-1253(16)30024-3 See Online Comment http://dx.doi.org/10.1016/ S2468-1253(16)30083-8 *Members listed in the appendix Liver Unit, Department of Medicine, Hospital General Universitari Vall d’Hebron and Ciberehd del Instituto Carlos III, Barcelona, Spain (Prof M Buti MD); Auckland Clinical Studies, Auckland, New Zealand (E Gane MD); Department of Medicine, Queen Mary Hospital, Hong Kong (W K Seto MD); Department of Medicine and Therapeutics, Institute of Digestive Disease and State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Hong Kong (Prof H L Y Chan MD); Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan (Prof W-L Chuang MD); Modern Medicine Clinic, Moscow, Russian Federation (T Stepanova MD); Department of Medicine, Alice Ho Miu Ling Nethersole Hospital, Hong Kong (A-J Hui MD); Department of Gastroenterology, Asan Medical Center, Seoul, South Korea (Prof Y-S Lim MD); Department of GI Endoscopy, Surat Institute of Medical Sciences (SIDS), Surat, Gujarat, India (R Mehta MD); Toronto Centre for Liver Disease, Toronto, ON, Canada (H L A Janssen MD); All India Institute of Medical Sciences,

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New Delhi, Delhi, India (Prof S K Acharya MD); Gilead Sciences, Foster City, CA, USA (J F Flaherty PharmD, B Massetto MD, A L Cathcart PhD, K Kim PhD, A Gaggar MD, G M Subramanian MD, J G McHutchison MD); Division of Gastroenterology and Hepatology, Department of Medicine, NYU Langone Medical Center, NYU School of Medicine, New York, NY, USA (Prof C Q Pan MD); Hepatology Unit, University Hospital of Pisa, Pisa, Italy (M Brunetto MD); Department of Gastroenterology and Hepatology, Musashino Red Cross Hospital, Tokyo, Japan (N Izumi MD); and Service d’Hépatologie, Hôpital Beaujon, Clichy, France (Prof P Marcellin MD) Correspondence to: Prof Maria Buti, Unidad de Hepatología, Departamento de Medicina Interna, Hospital Universitari Vall d’Hebron, Barcelona 08035, Spain [email protected] See Online for appendix

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Research in context Evidence before this study We searched PubMed using the search terms “HBV”, “hepatitis B virus”, “chronic HBV”, “tenofovir disoproxil fumarate”, “bone toxicity”, and “nephrotoxicity” for clinical trials published from inception to April 30, 2013, restricted to English language publications. Guidelines recommend that patients with active HBeAg-negative chronic HBV infection receive antiviral therapy of indefinite duration. Tenofovir disoproxil fumarate, a first-line therapy for hepatitis B virus (HBV) infection, has a generally favourable side-effect profile, but has been associated with nephrotoxicity and reductions in bone mineral density over long-term use in patients with HBV and HIV infection. Tenofovir alafenamide was developed specifically to deliver the active metabolite of tenofovir to hepatocytes at lower doses than tenofovir disoproxil fumarate. In clinical trials in patients with HIV, those receiving tenofovir alafenamide had lower systemic exposure to tenofovir, and fewer renal and bone effects than those receiving tenofovir disoproxil fumarate. The present study was undertaken to determine if tenofovir alafenamide would

Introduction In patients chronically infected with hepatitis B virus (HBV), the loss of the hepatitis B e antigen (HBeAg) and development of anti-HBe are hallmarks of the inactive carrier state, characterised by low viral replication and normal alanine aminotransferase (ALT) concentrations.1 However, 20–30% of these patients will experience a reactivation of HBV replication with viral populations of precore mutations or basal core promoter mutations that prevent the formation of HBeAg.1–3 HBeAg-negative chronic HBV is increasing in prevalence and is now the most common form of chronic HBV in southern European, Middle Eastern, and north African countries.4,5 Being at a later stage in the natural history of the disease, patients with HBeAg-negative chronic HBV are generally older than those with HBeAg-positive disease and are at increased risk for cirrhosis and liver-related complications, including hepatocellular carcinoma and decompensation.2,6,7 Since very few patients with HBeAg-negative HBV achieve loss of the hepatitis B surface antigen (HBsAg) and almost all experience virological relapse after discontinuation of treatment, most guidelines recommend lifelong therapy, with the goals of viral suppression and ALT normalisation in the absence of HBsAg loss and seroconversion.8–10 The ideal antiviral treatment for this population would have a high barrier to resistance and be safe, effective, and well tolerated without any long-term toxic effects in all patients, including those who have been previously treated for HBV infection. Tenofovir disoproxil fumarate, an orally bioavailable prodrug of the nucleotide analogue reverse transcriptase inhibitor tenofovir, is approved for the treatment of patients with chronic HBV or HIV-1 infection. Tenofovir

offer similar improvements in patients with chronic HBeAg-negative HBV infection with similar antiviral efficacy. Added value of this study Our results show that tenofovir alafenamide is as effective at suppressing HBV replication as tenofovir disoproxil fumarate at a lower dose. Various clinical and laboratory measures suggest that tenofovir alafenamide might have less pronounced adverse effects on bone and renal health than tenofovir disoproxil fumarate. An unexpected finding was that a higher proportion of patients receiving tenofovir alafenamide achieved normalisation of alanine aminotransferase concentrations than those receiving tenofovir disoproxil fumarate; the clinical implications of this finding are unclear. Implications of all the available evidence Although the short-term results of this trial are promising, the possible clinical benefits of tenofovir alafenamide for patients with HBeAg-negative HBV infection will require confirmation in longer term follow-up.

disoproxil fumarate has potent antiviral activity against HBV infection.11 Patients receiving tenofovir disoproxil fumarate for 8 years showed no sign of antiviral resistance, but renal toxic effects are seen in about 2–7% of patients and substantial bone loss can occur in patients with chronic HBV infection.12–14 Tenofovir alafenamide was developed to efficiently deliver the active metabolite to hepatocytes with less systemic exposure.15,16 Circulating concentrations of tenofovir are 90% lower after administration of tenofovir alafenamide than after administration of tenofovir disoproxil fumarate.17,18 In clinical trials, patients infected with HIV-1 who received tenofovir alafenamide had reduced bone and renal effects compared with patients receiving tenofovir disoproxil fumarate.19,20 Our objective in this phase 3 non-inferiority trial was to compare the efficacy, safety, and tolerability of tenofovir alafenamide with that of tenofovir disoproxil fumarate in patients with HBeAg-negative chronic HBV infection.

Methods Study design and participants This randomised, double-blind, non-inferiority study was done at 105 sites (11 in Canada, 14 in the USA, three in the UK, two in France, four in Italy, four in Poland, five in Romania, ten in Russia, one in Spain, five in Turkey, five in Australia, one in New Zealand, ten in India, 11 in Japan, four in Hong Kong, ten in South Korea, and five in Taiwan). Eligible patients were aged at least 18 years with HBeAg-negative chronic HBV infection (with plasma HBV DNA concentrations of >20 000 IU/mL), serum ALT concentrations of greater than 60 U/L in men or

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greater than 38 U/L in women and at no more than ten times the upper limit of normal [ULN], and estimated creatinine clearance of at least 50 mL/min (by the Cockcroft-Gault method). Treatment-naive (less than 12 weeks of previous therapy) as well as treatmentexperienced patients (those who previously received treatment with any oral antiviral agent for HBV infection for ≥12 weeks) were eligible provided they met entry criteria (appendix p 17). Previous interferon use was allowed if stopped at least 6 months before study baseline assessments. We excluded patients with platelet counts of 50 000 cells per μL or less, haemoglobin less than 10 g/dL, albumin less than 3 g/dL, direct bilirubin of greater than 2·5 times the ULN, and aspartate aminotransferase (AST) or ALT greater than ten times the ULN. Patients with evidence of decompensation (ie, clinical ascites, encephalopathy, or variceal haemorrhage) and those with hepatocellular carcinoma were also excluded, as well as patients co-infected with hepatitis C, hepatitis D, or HIV. Full eligibility criteria are in the appendix (p 6). Before enrolment and before any study procedures were done, written informed consent was obtained from all patients. The study was approved by the institutional review board or independent ethics committees at all participating sites as well as the US Food and Drug Administration (FDA) and was done in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice.

Randomisation and masking No more than 45 days after screening, patients were randomly assigned (2:1) to receive tenofovir alafenamide or tenofovir disoproxil fumarate. During the double-blind phase (up to week 96), all patients received placebo tablets matching the alternative treatment (ie, patients assigned to receive tenofovir alafenamide also received a matching tenofovir disoproxil fumarate placebo tablet, and vice versa). Study investigators determined eligibility, obtained a participant number, and received automated treatment assignment via an interactive voice web response system. The computer-generated allocation sequence (with a block size of six) was created by a third party (Bracket, San Francisco, CA, USA). Each patient received a unique subject number during randomisation. Patients, investigators, and all study personnel, including those assessing outcomes, were masked to treatment assignment throughout the double-blind phase. Randomisation was stratified by screening HBV DNA concentrations (<7 log10 IU/mL, ≥7 log10 IU/mL to <8 log10 IU/mL, and ≥8 log10 IU/mL) and previous oral antiviral treatment (treatment naive vs previously treated).

Procedures Patients received tenofovir alafenamide 25 mg orally once daily or tenofovir disoproxil fumarate 300 mg orally once daily for 96 weeks, after which time all patients began

open-label treatment with tenofovir alafenamide 25 mg orally once daily until week 144. Study visits occurred every 4 weeks starting at treatment week 4 until treatment week 48, then every 8 weeks until the end of the double-blind phase at week 96. Laboratory assessments included haematological analysis, serum chemistry tests, fasting lipid parameters, urinalysis, and measures of renal function (urine protein-to-creatinine ratio, urine albumin-to-creatinine ratio, urine retinol-binding-proteinto-creatinine ratio, urine β2-microglobulin-to-creatinine ratio; Covance Laboratories, Indianapolis, IN, USA). Percentage change in bone mineral density was assessed in all patients by dual energy x-ray absorptiometry scans of the lumbar spine and hip at screening, and at weeks 24 and 48 of treatment (and every 24 weeks thereafter). Serum biomarkers of bone turnover were also assessed in the fasted state, including C-type collagen sequence, which is associated with bone resorption, and bone-specific alkaline phosphatase, osteocalcin, and procollagen type 1 N-terminal propeptide, which are all markers associated with bone formation.

Outcomes The primary efficacy endpoint was the proportion of patients with HBV DNA less than 29 IU/mL at week 48 of treatment, as determined by COBAS Taqman HBV test for use with the High Pure System (Roche Molecular Systems, Inc; Pleasanton, CA, USA) with a lower limit of quantitation of 29 IU/mL and a lower limit of detection of 10 IU/mL. The limits of detection and quantification were specified by Roche and confirmed by the central labortaory (Covance) using human plasma samples. Key secondary safety endpoints at week 48 included percentage change in hip bone mineral density, percentage change in spine bone mineral density, and change from baseline in serum creatinine. A prespecified efficacy endpoint was the proportion of patients with ALT normalisation at week 48 (defined as ALT above a ULN of ≤43 U/L for men and ≤34 U/L for women younger than 69 years; ≤35 U/L for men and ≤32 U/L for women older than 69 years and at no more than ten times the ULN by central laboratory normal range, or 30 U/L for men and 19 U/L for women by American Association for the Study of Liver Diseases [AASLD] normal range at baseline but within the normal range at week 48). The entry criteria for ALT (>60 U/L for men and >38 U/L for women) were based on the requirement for ALT concentrations to be at least twice the ULN by AASLD criteria. Resistance surveillance included baseline analysis of all patients by INNOLiPA Multi-DR v2/3 (Immunogenetics; Alpharetta, GA, USA), and population sequencing of the polymerase reverse transcriptase region in patients with virological breakthrough (confirmed HBV DNA of ≥69 IU/mL after a documented concentration <69 IU/mL, or a confirmed log10 IU/mL or more increase from the nadir level), or

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those who discontinued with a concentration of HBV DNA of 69 IU/mL or more on or after week 24. Patients who never achieved HBV DNA less than 69 IU/mL and did not experience virological breakthrough on treatment were not tested for resistance. Other endpoints of interest at week 48 were the proportion of patients with plasma HBV DNA less than 29 IU/mL (target not detected), the proportion of patients with HBsAg loss, the proportion of patients with HBsAg seroconversion to anti-HBs (measured using the Abbott Architect assay with lower limit of quantitation of 0·05 IU/mL), the change from baseline in fibrosis as assessed by FibroTest (BioPredictive, Paris, France), and the incidence of drug-resistant mutations. Adverse events and graded laboratory abnormalities were also assessed.

Statistical analysis Sample sizes of 260 for the tenofovir alafenamide group and 130 for the tenofovir disoproxil fumarate group were

914 patients screened

488 were excluded 470 patients did not meet criteria 14 withdrew consent 4 were outside of visit window

426 randomly assigned

285 assigned to receive tenofovir alafenamide 25 mg for 96 weeks

141 assigned to receive tenofovir disoproxil 300 mg for 96 weeks

1 withdrew consent and did not begin treatment

12 discontinued treatment 4 lost to follow-up 3 withdrew consent 3 due to adverse events 1 at investigator’s discretion 1 due to protocolspecified withdrawal criteria

273 completed double-blind treatment

7 discontinued treatment 2 withdrew consent 2 due to adverse events 1 lost to follow-up 1 due to pregnancy 1 due to non-compliance

133 completed double-blind treatment

1 patient died during follow-up after completing treatment

285 assessed for safety and efficacy

Figure 1: Trial profile

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140 assessed for safety and efficacy

calculated to have 90% power to rule out non-inferiority with a margin of 10% at a one-sided significance level of 0·025. This assumes the expected difference in proportion of patients with HBV DNA <29 IU/mL is 0 and the proportion of patients with HBV DNA <29 IU/mL in the tenofovir disoproxil fumarate group is 91%. The non-inferiority margin was based on results from a phase 3 trial comparing tenofovir disoproxil fumarate with adefovir dipivoxil in patients with chronic HBV infection.21 In that trial, 91% of patients with HBeAg-negative HBV receiving tenofovir disoproxil fumarate had HBV DNA <29 IU/mL at week 48 versus 56% of patients receiving adefovir dipivoxil. A 10% non-inferiority margin, which was agreed upon by regulatory authorities (US FDA), preserves at least 60% of the lower bound of the 95% CI for the difference between tenofovir disoproxil fumarate and adefovir dipivoxil (appendix p 5).21,22 Safety and efficacy were assessed in the full analysis set, which was defined as all patients who were randomly assigned and received at least one dose of study drug. For the primary endpoint and the secondary efficacy endpoints involving proportions, missing data were handled using the missing equals failed approach. The baseline stratumweighted difference in the proportions between the groups and its 95% CI were calculated based on the stratum-adjusted Mantel-Haenszel proportion, where stratification factors included oral antiviral treatment status (treatment naive vs treatment experienced) and baseline HBV DNA (<7 log10 IU/mL, ≥7 log10 IU/mL to <8 log10 IU/mL, ≥8 log10 IU/mL). To control for type I error in the assessment of the primary efficacy endpoint and key secondary safety and efficacy endpoints, the hypothesis testing was done in a sequential order as follows. The primary hypothesis of non-inferiority of tenofovir alafenamide relative to tenofovir disoproxil fumarate was tested first. If non-inferiority was established, multiplicity adjustments were done for the following key secondary safety endpoints with a fallback procedure in the sequential order with prespecified two-sided α levels: percentage change in hip bone mineral density (α=0·02), percentage change in spine bone mineral density (α=0·01), serum creatinine (α=0·02), and treatment-emergent proteinuria (α=0; appendix p 22). We also assessed the primary efficacy endpoint in a per-protocol analysis set, which was prespecified and defined as all patients in the full analysis set except those who did not have week 48 HBV DNA data for any reason other than discontinuation due to lack of efficacy, those who received ongoing therapy with any of the prohibited medications, and those with adherence rate for active study drug up to the week 48 visit below the 2·5th percentile. SAS version 9.2 was used for all analyses. During the study, an independent data monitoring committee reviewed the safety results five times (about every 6 months). This study is registered with ClinicalTrials.gov, number NCT01940341.

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Role of the funding source The study funder oversaw trial management, data collection, statistical analyses, and the writing and review of the report. All authors had access to the study data and reviewed and approved the final manuscript. The corresponding author had full access to all data in the study and had final responsibility for the decision to submit for publication.

Of the 425 patients randomly assigned and treated, 27 did not achieve HBV DNA less than 29 IU/mL at week 48. Of these, 11 did not respond to treatment (HBV DNA ≥29 IU/mL at 48 weeks): seven (2%) patients in the tenofovir alafenamide group and four (3%) in the tenofovir disoproxil fumarate group. Of the other 16 patients who did not have HBV DNA less than 29 IU/mL at week 48, four discontinued treatment due to an adverse

Results Patients were screened from Sept 12, 2013, to Oct 31, 2014. Of the 914 patients screened, 426 were randomly assigned and 425 received treatment: 285 were assigned to receive tenofovir alafenamide and 141 tenofovir disoproxil fumarate (figure 1, appendix p 8). One patient assigned to receive tenofovir disoproxil fumarate withdrew consent and did not begin treatment. The baseline characteristics of the patients in the two groups were generally balanced (table 1), although patients in the tenofovir disoproxil fumarate group were older than those in the tenofovir alafenamide group. Most patients were male and Asian (table 1). About 20% of patients had received previous treatment for HBV with one or more oral nucleos(t)ide antiviral agents (table 1). Of the 91 patients who had previously received treatment with oral nucleos(t)ides, the most common were entecavir (32 patients in the tenofovir alafenamide group and nine patients in the tenofovir disoproxil fumarate group), lamivudine (27 patients and 15 patients), and tenofovir disoproxil fumarate (14 patients and seven patients; appendix p 17). Overall, mean HBV DNA was 5·8 log10 IU/mL (SD 1·33); at baseline 79 (19%) of 425 patients had HBV DNA of at least 7 log10 IU/mL and 17 (4%) of 425 patients had HBV DNA of at least 8 log10 IU/mL. The most common HBV genotypes were C, D, and B (table 1). Median duration of exposure to masked study drug at the time of the present analysis was 56 weeks (IQR 48–64) in both groups. 268 (94%) of 285 patients receiving tenofovir alafenamide had HBV DNA less than 29 IU/mL at week 48 versus 130 (93%) of 140 patients receiving tenofovir disoproxil fumarate (adjusted difference 1·8% [95% CI –3·6 to 7·2; p=0·47]; table 2, figure 2). Because the lower bound of the two-sided 95% CI of the difference in the response rate was greater than the prespecified –10% margin, the tenofovir alafenamide group met the primary endpoint of non-inferiority versus tenofovir disoproxil fumarate. 60 (21%) patients in the tenofovir alafenamide group achieved HBV DNA less than 10 IU/mL (target not detected) at week 48 versus 24 (17%) of those receiving tenofovir disoproxil fumarate (difference 4·0% [95% CI –3·9 to 11·9]). When week 48 results were analysed by a prespecified per-protocol analysis as well as a more conservative imputation rule, the results were consistent with those of the primary analysis in showing that tenofovir alafenamide was non-inferior to tenofovir disoproxil fumarate in antiviral efficacy (appendix p 19).

Tenofovir alafenamide 25 mg (n=285) Age (years)

45 (12)

Tenofovir disoproxil fumarate 300 mg (n=140) 48 (10)

Sex Female

112 (39%)

54 (39%)

Male

173 (61%)

86 (61%)

Asian

205 (72%)

101 (72%)

White

71 (25%)

35 (25%)

Black

5 (2%)

3 (2%)

Native Hawaiian or Pacific Islander

2 (1%)

0

Race

Other BMI (kg/m²)

2 (1%)

1 (1%)

24·6 (4·0)

24·9 (3·8)

5·7 (1·3)

5·8 (1·3)

HBV DNA (log10 IU/mL) HBV DNA by category <7 log10 IU/mL

230 (81%)

116 (83%)

≥7 to <8 log10 IU/mL

42 (15%)

20 (14%)

≥8 log10 IU/mL

13 (5%)

4 (3%)

HBV genotype A

15 (5%)

6 (4%)

B

60 (21%)

40 (29%)

C

115 (40%)

47 (34%)

D

90 (32%)

42 (30%)

E

5 (2%)

2 (1%)

F

0

2 (1%)

Unknown

0

1 (1%)

Previous nucleoside/nucleotide use

60 (21%)

31 (22%)

Previous interferon use

29 (10%)

19 (14%)

ALT (U/L)

67 (44–102)

67 (47–102)

ALT concentration >ULN by central laboratory criteria

236 (83%)

121 (86%)

ALT concentration >ULN by AASLD criteria

276 (97%)

138 (99%)

FibroTest score ≥0·75 eGFR by Cockcroft-Gault (mL/min)

31/280 (11%) 104·7 (27·8)

Proteinuria (dipstick) ≥grade 1

15 (5%)

20/139 (14%) 100·3 (24·2) 5 (4%)

Bone mineral density by dual energy x-ray absorptiometry (g/cm²) Hip

0·95 (0·16)

0·94 (0·14)

Spine

1·05 (0·19)

1·03 (0·18)

Data are n (%), n/N (%), mean (SD), or median (IQR), unless otherwise stated. BMI=body-mass index. HBV=hepatitis B virus. ALT=alanine aminotransferase. ULN=upper limit of normal. AASLD=American Association for the Study of Liver Diseases. eGFR=estimated glomerular filtration rate.

Table 1: Baseline characteristics

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HBV DNA <29 IU/mL

268 (94%)

Normalised ALT by central laboratory normal range

196/236 (83%)

Normalised ALT by AASLD normal range

137/276 (50%)

Loss of HBsAg

0/281

Tenofovir disoproxil fumarate 300 mg (n=140) 130 (93%)

Difference in proportions (95% CI)

0·47

91/121 (75%)

8·0% (–1·3 to 17·2)

0·076

44/138 (32%)

17·9% (8·0 to 27·7) 0% (–2·6 to 2·6)

Tenofovir alafenamide Tenofovir disoproxil fumarate

100

1·8% (–3·6 to 7·2)

0/138

A

p value

0·0005 ··

Data are n (%) or n/N (%), unless otherwise stated. HBV=hepatitis B virus. ALT=alanine transaminase. AASLD=American Association for the Study of Liver Diseases. HBsAg=HBV surface antigen.

80 Proportion of patients (%)

Tenofovir alafenamide 25 mg (n=285)

p=0·47

60

40

20

Table 2: Primary and secondary efficacy endpoints 0

6

B 100

Proportion of patients (%)

80 p=0·076

60

40

20

0

C 100

80 Proportion of patients (%)

event (three [1%] of those receiving tenofovir alafenamide and one [1%] of those receiving tenofovir disoproxil fumarate), two patients had missing data (one [<1%] each in both groups), and ten discontinued for other reasons (eg, withdrawal of consent, loss to follow-up, protocolspecified reason, protocol violation, investigator’s decision: six [2%] and four [3%] patients, respectively). There was no difference in treatment response for all predefined subgroups between the tenofovir alafenamide group and the tenofovir disoproxil fumarate group (appendix p 9). No patient in either group had loss of HBsAg up to week 48. Additionally, mean changes in quantitative HBsAg at week 48 from baseline were minimal in both groups (tenofovir alafenamide –0·09 log10 IU/mL [SD –0·14 to –0·04] vs tenofovir disoproxil fumarate –0·06 log10 IU/mL [–0·12 to 0·00]). 196 (83%) of 236 patients receiving tenofovir disoproxil fumarate with ALT above the ULN at baseline had normal ALT at week 48 of treatment by central laboratory criteria versus 91 (75%) of 121 patients receiving tenofovir disoproxil fumarate (table 2; appendix p 23); the difference (8·0% [95% CI –1·3 to 17·2; p=0·076]) was not significant. However, using criteria recommended by the AASLD (with normal ranges of ≤30 U/L for men and ≤19 U/L for women),23–25 the difference between the proportions of patients achieving normalised ALT in the two groups was significant: 137 (50%) of 256 patients receiving tenofovir alafenamide versus 44 (32%) of 138 patients receiving tenofovir disoproxil fumarate (difference 17·9% [95% CI 8·0 to 27·7; p=0·0005]; table 2; appendix p 24). At week 48, patients receiving tenofovir alafenamide had a mean percentage decrease in hip bone mineral density of –0·29% (95% CI –0·55 to –0·03) from a mean baseline value of 0·95 g/cm² (SD 0·156), whereas patients receiving tenofovir disoproxil fumarate had a decrease of –2·16% (–2·53 to –1·79) from a mean baseline value of 0·94 g/cm² (SD 0·144); adjusted percentage difference 1·87% (95% CI 1·42 to 2·32; p<0·0001; figure 3). Likewise, the mean percentage change in spine bone mineral density from baseline to week 48 was –0·88% (95% CI –1·22 to –0·54) from a mean baseline value of 1·05 g/cm² (SD 0·19) for patients receiving tenofovir alafenamide versus –2·51%

60

40

p=0·0005

20

0 0

8

16

24 Time (weeks)

32

40

48

Figure 2: Viral suppression and alanine aminotransferase (ALT) normalisation by study week (A) Proportion of patients with HBV DNA less than 29 IU/mL by study week. Bars are 95% CIs. (B) Proportion of patients achieving ALT normalisation by central laboratory (Covance) criteria by study week (≤43 U/L for men and ≤34 U/L for women <69 years of age; ≤35 U/L for men and ≤32 U/L for women >69 years of age). (C) Proportion of patients achieving ALT normalisation by American Association for the Study of Liver Diseases (AASLD) criteria (≤19 U/L for women and ≤30 U/L for men) by study week.

(–3·09 to –1·94) from a mean baseline value of 1·03 g/cm² (SD 0·18) for patients receiving tenofovir disoproxil fumarate (adjusted percentage difference 1·64% [95% CI 1·01 to 2·27; p<0·0001]). In an exploratory analysis,

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A Tenofovir alafenamide Tenofovir disoproxil fumarate

4

Mean change (%)

2

0

–0·29

–2

–2·16

p<0·0001

–4

–6

B 4

Mean change (%)

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0 –0·88 p<0·0001

–2

–2·51

–4

–6 0

24

48

Time (weeks)

Figure 3: Changes in bone mineral density (A) Mean percentage change in hip bone mineral density at weeks 24 and 48 of treatment. (B) Mean percentage change in spine bone mineral density at weeks 24 and 48 of treatment. Bars are 95% CIs.

0·10

Mean change in serum creatinine (mg/dL)

27 (10%) of 270 patients in the tenofovir alafenamide group had a greater than 3% decrease in hip bone mineral density at week 48 versus 44 (33%) of 133 in the tenofovir disoproxil fumarate group (p<0·0001). Similarly, 60 (22%) of 271 patients in the tenofovir alafenamide group had a greater than 3% decrease in spine bone mineral density at week 48 versus 52 (39%) of 133 in the tenofovir disoproxil fumarate group (p=0·0004). Biomarkers associated with bone resorption (C-type collagen sequence) and formation (procollagen type 1 N-terminal propeptide, bone-specific alkaline phosphatase, and osteocalcin) showed significantly smaller changes from baseline at week 48 in patients receiving tenofovir alafenamide than those receiving tenofovir disoproxil fumarate (p<0·001 for all biomarkers; appendix pp 12–16). Fractures occurred in three patients receiving tenofovir alafenamide, of which two were associated with trauma: a 37-year-old white woman with normal baseline bone density fractured her tibia on day 57 of treatment, and a 65-year-old Asian woman with osteoporosis at baseline fractured a finger on day 350 of treatment. One patient had a spinal compression fracture that was identified incidentally on a CT scan for evaluation of ureteric calculus (this was judged by the radiologist to be an old fracture). No patients receiving tenofovir disoproxil fumarate had a bone fracture event. Both groups had small mean increases in serum creatinine from baseline to week 48 (tenofovir alafenamide 0·01 mg/dL [95% CI 0·00–0·02] vs tenofovir disoproxil fumarate 0·02 mg/dL [0·00–0·04]); the difference between the two groups was not statistically or clinically significant (adjusted percentage difference –0·01 mg/dL [95% CI –0·03 to 0·01]; p=0·32; figure 4). Treatment-emergent proteinuria was similar in both groups: 54 (19%) of 285 patients receiving tenofovir alafenamide versus 26 (19%) of 140 patients receiving tenofovir disoproxil fumarate; most events were mild (grade 1). At week 48, patients receiving tenofovir alafenamide had a lower median decrease in estimated glomerular filtration rate (by Cockcroft-Gault) than patients receiving tenofovir disoproxil fumarate (–1·8 mL/min [IQR –7·8 to 6·0] vs –4·8 mL/min [–12·0 to 3·0]; p=0·004; appendix p 25). Percentage increases from baseline to week 48 in the markers of proximal tubular dysfunction, urine retinol-binding-protein-to-creatinine ratio and urine β2-microglobulin-to-creatinine ratio, were significantly smaller in patients receiving tenofovir alafenamide than in those receiving tenofovir disoproxil fumarate (p<0·001 for the differences at week 48; appendix p 20). No significant differences were observed between groups in percentage change in the ratios of urine protein to creatinine and urine albumin to creatinine. At baseline, 389 (92%) patients had wild-type virus (tenofovir alafenamide 260 [91%] of 285 patients, tenofovir disoproxil fumarate 129 [92%] of 140 patients; 13 (3%) patients had primary resistance mutations

Tenofovir alafenamide 25 mg Tenofovir disoproxil fumarate 300 mg

0·05 p=0·32

0

0

8

16

24 Time (weeks)

32

40

48

Figure 4: Mean change from baseline in serum creatinine (mg/dL) by study week Bars are 95% CIs.

present (eight patients in the tenofovir alafenamide group and five patients in the tenofovir disoproxil fumarate group). Primary resistance mutations were more common in treatment-experienced patients (eight [9%] of 90 patients) with the most common being lamivudine (five [6%] patients) and entecavir (two [2%]

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Patients with any adverse event

Tenofovir alafenamide 25 mg (n=285)

Tenofovir disoproxil fumarate 300 mg (n=140)

210 (74%)

99 (71%)

Deaths

0

1 (<1%)*

Patients with adverse events leading to study drug discontinuation

3 (1%)

2 (1%)

Patients with a grade 3 or 4 adverse event

12 (4%)

6 (4%)

Patients with serious adverse events

14 (5%)

9 (6%)

Adverse events occurring in ≥5% of patients in any treatment group Headache

40 (14%)

Nasopharyngitis

30 (11%)

15 (11%)

Upper respiratory tract infection

35 (12%)

10 (7%)

Cough

18 (6%)

8 (6%)

Fatigue

16 (6%)

9 (6%)

Arthralgia

11 (4%)

10 (7%)

Nausea

15 (5%)

9 (6%)

Back pain

14 (5%)

7 (5%)

82/282 (29%)

30/140 (21%)

Patients with grade 3–4 laboratory abnormalities†

14 (10%)

Grade 3–4 laboratory abnormalities in ≥1% of patients‡ Alanine aminotransferase >5×ULN

8 (3%)

4 (3%)

Aspartate aminotransferase >5×ULN

8 (3%)

4 (3%)

14 (5%)

3 (2%)

Amylase >2×ULN γ-glutamyl transferase >5×ULN

0

3 (2%)

Fasting glucose >250 mg/dL

4/280 (1%)

0

3/280 (1%)

0

Total cholesterol >300 mg/dL Fasting LDL cholesterol >300 mg/dL Creatine kinase ≥10×ULN Non-fasting glucose >250 mg/dL

14/277 (5%)

1/135 (1%)

7 (2%)

3 (2%)

10 (4%)

2 (1%)

Occult blood

17 (6%)

7 (5%)

Urine erythrocytes

17/252 (7%)

9/127 (7%)

Urine glucose

15 (5%)

2 (1%)

Data are n (%) or n/N (%). ULN=upper limit of normal. *An Asian man aged 51 years with cirrhosis died from hepatocellular carcinoma at week 56. †Laboratory results are based on 282 patients for tenofovir alafenamide 25 mg and 140 patients for tenofovir disoproxil fumarate 300 mg, unless otherwise stated. ‡Values are n (%) of patients with any given laboratory abnormality.

Table 3: Adverse events

patients) mutations (appendix p 18). Four patients qualified for resistance testing during the study, two in each treatment group. In the two patients receiving tenofovir alafenamide who had virological breakthrough, the virus showed no sequence changes from baseline. Of the two patients receiving tenofovir disoproxil fumarate who qualified for resistance testing, one had virological breakthrough and the other was viraemic when discontinued from the study; in both patients we were unable to sequence the virus. One patient in the tenofovir alafenamide group and one in the tenofovir disoproxil fumarate group who had virological breakthrough were found to be non-adherent to treatment by testing for serum drug concentrations of tenofovir. No resistance was detected in either treatment group. 8

Both treatments were well tolerated; most adverse events were mild to moderate in severity (table 3). Discontinuation of treatment due to adverse events was uncommon: three (1%) patients receiving tenofovir alafenamide and two (1%) patients receiving tenofovir disoproxil fumarate stopped treatment because of one or more adverse events (appendix p 10). The most common adverse events overall were headache, nasopharyngitis, and upper respiratory tract infection. 14 (5%) patients receiving tenofovir alafenamide and nine (6%) patients receiving tenofovir disoproxil fumarate had serious adverse events, none of which was deemed by investigators to be related to study treatment (table 3). Serious adverse events that occurred in more than one patient were hepatocellular carcinoma, which occurred in one patient receiving tenofovir alafenamide and three patients receiving tenofovir disoproxil fumarate, ureteric calculus in two patients receiving tenofovir alafenamide, and cellulitis in two patients receiving tenofovir disoproxil fumarate. No patient died during treatment, but one patient, an Asian man aged 51 years with HBV genotype C and cirrhosis who received tenofovir disoproxil fumarate discontinued after 54 weeks of treatment after being diagnosed with hepatocellular carcinoma and pulmonary embolism, and died 9 days later. Hepatocellular carcinoma was listed as the cause of death. No patients experienced a renal-related serious adverse event or adverse event leading to study drug discontinuation, and no patients experienced an adverse event of proximal tubulopathy, including Fanconi syndrome. Three patients had confirmed renal laboratory abnormalities during treatment: one Asian man aged 52 years with a medical history of diabetes and hypertension receiving tenofovir alafenamide had an increase of at least 0·5 mg/dL from baseline in serum creatinine and creatinine clearance less than 50 mL/min (both at week 44) coinciding with worsening of his diabetes control; one white man aged 55 years receiving tenofovir alafenamide had serum phosphorus less than 2 mg/dL at weeks 32 and 36; and one white man aged 30 years receiving tenofovir disoproxil fumarate had serum phosphorus less than 2 mg/dL at weeks 4 and 8 of treatment. The incidence of grade 3 or 4 laboratory abnormalities was similar between treatment groups (82 [29%] of 282 patients receiving tenofovir alafenamide vs 30 [21%] of 140 patients receiving tenofovir disoproxil fumarate); among the most common abnormalities were raised ALT (eight patients in the tenofovir alafenamide group vs four patients in the tenofovir disoproxil fumarate group) and AST (eight patients vs four patients), raised amylase (14 patients vs three patients), and increased LDL cholesterol (14 patients vs one patient; table 3). Two patients receiving tenofovir alafenamide had ALT flare during the first 4 weeks of treatment. Higher percentages of patients receiving tenofovir alafenamide had hyperglycaemia (4%) and glycosuria (5%) than those receiving tenofovir disoproxil fumarate (1% for both).

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Most patients receiving tenofovir alafenamide who had increased LDL cholesterol had either a history of dyslipidaemia, a raised value at baseline, or both. There were no differences between treatment groups in patients initiating lipid-modifying agents.

Discussion This randomised, phase 3 clinical trial shows that the antiviral efficacy of tenofovir alafenamide at 48 weeks of treatment is non-inferior to that of tenofovir disoproxil fumarate in patients with HBeAg-negative chronic HBV infection. Both groups achieved high rates of viral suppression overall and in prespecified subgroups. Rates of ALT normalisation at 48 weeks were not significantly higher in patients receiving tenofovir alafenamide than in those receiving tenofovir disoproxil fumarate, but were significantly higher when assessed by AASLD criteria.7,24,25 No patient in either group experienced HBsAg loss until 48 weeks, which was consistent with results from similar studies.21,26–28 Given the similar rates of viral suppression between the two treatment groups, the higher rate of ALT normalisation among patients receiving tenofovir alafenamide than among those receiving tenofovir disoproxil fumarate was unexpected. The consistency of this finding at all study timepoints (appendix pp 23, 24) and the fact that the same effect was observed in a similarly designed trial of 873 patients with HBeAg-positive chronic HBV29 suggest that it is not a chance result. Further study will be needed to determine the mechanism for this effect. Overall, both treatments were well tolerated and safe, with similar rates of adverse events, serious adverse events, and laboratory abnormalities. In both groups, only 1% of patients discontinued treatment because of adverse events. However, important differences between the treatment groups were noted in various bone and renal variables after only 48 weeks of therapy. These differences are relevant for patients with HBeAg-negative chronic HBV, who are older and have more comorbidities than patients with HBeAg-positive chronic HBV, and therefore at higher risk for progressive renal dysfunction and osteoporosis. Tenofovir alafenamide was designed to provide liver-targeted antiviral therapy for patients with chronic HBV infection while avoiding the bone and renal toxic effects associated with the use of tenofovir disoproxil fumarate by reducing systemic exposure to tenofovir. Reductions in hip and spine bone mineral density have been noted in patients infected with HIV receiving long-term treatment with tenofovir disoproxil fumarate,30–32 and have more recently been described in patients receiving tenofovir disoproxil fumarate for chronic HBV infection.33 Such changes are of particular relevance in light of results from a large longitudinal cohort study34 in Taiwan done over an 11-year period, which showed that patients with chronic HBV are at greater risk for developing osteoporosis than are matched non-infected controls, even after correcting for

confounding factors, including age, sex, and presence of comorbidities. In our study, patients receiving tenofovir alafenamide had significantly smaller declines in hip and spine bone mineral density at 48 weeks than did patients receiving tenofovir disoproxil fumarate. Furthermore, a significantly lower percentage of patients receiving tenofovir alafenamide had bone loss of more than 3% at both hip and spine at week 48. The reduced effect of tenofovir alafenamide on bone is further supported by its consistently minimal effect on several markers of bone turnover: C-type collagen sequence, a marker of resorption; and the formation markers procollagen type 1 N-terminal propeptide, osteocalcin, and bone-specific alkaline phosphatase (appendix). However, it cannot be ruled out that the slightly older age of patients receiving tenofovir disoproxil fumarate can at least partly account for these differences in bone parameters. Long-term treatment with tenofovir disoproxil fumarate is also associated with kidney injury in some patients, including cases of acute renal failure, proximal tubulopathy, and in rare instances, Fanconi syndrome.35–40 Treatment with tenofovir disoproxil fumarate also causes modest declines in glomerular filtration rate, which might be the result of subclinical tubular injury.41,42 In our study, patients receiving tenofovir alafenamide had a smaller decline in estimated glomerular filtration rate compared with those receiving tenofovir disoproxil fumarate, although change in serum creatinine was not significantly different at week 48. Although rates of proteinuria by dipstick and the quantitative markers of urine protein-to-creatinine and urine albumin-tocreatinine ratios did not differ between treatments, patients receiving tenofovir alafenamide had smaller changes in urine retinol-binding-protein-to-creatinine ratio and β2-microglobulin-to-creatinine ratio, both of which are considered more sensitive and specific indicators of renal tubular dysfunction.43 Again, these differences might be partly due to the slightly older age of patients randomly assigned to receive tenofovir disoproxil fumarate. This study has several limitations. Although our sample size was large enough to show non-inferiority in efficacy of tenofovir alafenamide to tenofovir disoproxil fumarate, the 48-week duration might not be long enough to demonstrate potential differences in the incidences of clinically important renal and bone events between patients receiving tenofovir alafenamide and those receiving tenofovir disoproxil fumarate. Longer follow-up (up to 8 years) is planned to determine whether the short-term improvements we observed in bone and renal parameters will translate to a reduced incidence of bone and renal events over the long term. Additionally, patients with advanced liver disease or clincially significant renal impairment (eGFR <50 mL/min) were not assessed in this study. Studies are planned to determine the use of tenofovir alafenamide in these important populations.

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In conclusion, the high rate of viral suppression, lack of resistance, and favourable safety profile of tenofovir alafenamide shown in this phase 3 study support long-term therapy in patients with HBeAg-negative chronic HBV. Analysis of the planned long-term follow-up will determine the durability of these outcomes. Contributors MBu, JFF, AG, GMS, and JGM contributed to the study design. MBu, EG, WKS, HLYC, W-LC, TS, A-JH, Y-SL, RM, HLAJ, SKA, CQP, MBr, NI, and PM served as investigators in this study. JFF, BM, ALC, KK (study statistician), and AG contributed to the data interpretation. All authors contributed to the writing and review of the report. Declaration of interests MBu serves on advisory boards for Gilead Sciences, Merck, Bristol-Myers Squibb, Janssen, and AbbVie, and is a speaker for Gilead Sciences, Merck, Bristol-Myers Squibb, Janssen, and AbbVie. EG receives a grant or research funding from Gilead Sciences, serves on advisory boards for AbbVie, Boehringer Ingelheim, Gilead Sciences, Janssen, Novartis, Roche, and Tibotec, and is a speaker for Gilead Sciences, Novartis, Roche, and Tibotec. WKS serves on advisory boards for Gilead and Bristol-Myers Squibb, and is a speaker for Gilead, AbbVie, Bristol-Myers Squibb, and Novartis. HLYC serves on advisory boards for Gilead Sciences, Abbvie, Bristol-Myers Squibb, and Roche, is a speaker for Gilead Sciences, Abbvie, Bristol-Myers Squibb, Roche, Novartis, and Echosens, and receives grant funding from Roche. W-LC serves on advisory boards for Gilead Sciences, AbbVie, BMS, Roche, and PharmaEssentia, and is a speaker for Gilead Sciences, Bristol-Myers Squibb, Merck, and Roche. Y-SL serves on advisory boards for Bayer, Bristol-Myers Squibb, and Gilead Sciences, receives grant funding from Bayer, Bristol-Myers Squibb, Gilead Sciences, and Novartis, and is a speaker for Bayer and Gilead Sciences. HLAJ receives grant funding from AbbVie, Bristol-Myers Squibb, Gilead Sciences, Innogenetics, Janssen, Medimmune, Medtronic, Merck, Novartis, and Roche, and is a consultant for AbbVie, Benitec, Bristol-Myers Squibb, Eiger Bio, Gilead Sciences, GlaxoSmithKline, Innogenetics, ISIS Pharmaceuticals, Janssen, Medimmune, Medtronic, Merck, Novartis, Roche, and Tekmira. CQP is a consultant for Gilead Sciences, Johnson & Johnson, and Bristol-Myers Squibb, serves on advisory boards for Gilead Sciences, Bristol-Myers Squibb, and AbbVie, and receives grant funding from Gilead Sciences and Merck. MBr serves on advisory boards for Gilead Sciences, Merck, AbbVie, and Janssen, and is a speaker for Bristol-Myers Squibb. PM is a consultant for Roche, Gilead Sciences, Bristol-Myers Squibb, Vertex, Novartis, Janssen, Merck, AbbVie, Alios BioPharma, Idenix, and Akron, receives grant funding and research support from Roche, Gilead Sciences, Bristol-Myers Squibb, Novartis, Janssen, Merck, and Alios BioPharma, and is a speaker for Roche, Gilead Sciences, Bristol-Myers Squibb, Vertex, Novartis, Janssen, Merck, Boehringer, Pfizer, and AbbVie. JFF, BM, ALC, KK, AG, GMS, and JGM are employees and stockholders of Gilead Sciences. TS, A-JH, RM, SKA, and NI declare no competing interests. Acknowledgments This study was sponsored by Gilead Sciences. Writing assistance was provided by David McNeel of Gilead Sciences. References 1 Trépo C, Chan HLY, Lok A. Hepatitis B virus infection. Lancet 2014; 384: 2053–63. 2 Hadziyannis SJ. Natural history of chronic hepatitis B in Euro-Mediterranean and African countries. J Hepatol 2011; 55: 183–91. 3 Bonino F, Brunetto MR, Rizzetto M, Will H. Hepatitis B virus unable to secrete e antigen. Gastroenterology 1991; 100: 1138–41. 4 Fattovich G, Bortolotti F, Donato F. Natural history of chronic hepatitis B: special emphasis on disease progression and prognostic factors. J Hepatol 2008; 48: 335–52. 5 Manesis EK. HBeAg-negative chronic hepatitis B: from obscurity to prominence. J Hepatol 2006; 45: 343–46. 6 Pungpapong S, Kim R, Poterucha JJ. Natural history of hepatitis B virus infection: an update for clinicians. Mayo Clin Proc 2007; 82: 967–75.

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www.thelancet.com/gastrohep Published online September 22, 2016 http://dx.doi.org/10.1016/S2468-1253(16)30107-8

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