Long-term efficacy and safety of emtricitabine plus tenofovir DF vs. tenofovir DF monotherapy in adefovir-experienced chronic hepatitis B patients

Research Article

Long-term efficacy and safety of emtricitabine plus tenofovir DF vs. tenofovir DF monotherapy in adefovir-experienced chronic hepatitis B patients Thomas Berg1,⇑, Fabien Zoulim2, Bernd Moeller3, Huy Trinh4, Patrick Marcellin5, Sing Chan6, Kathryn M. Kitrinos7, Phillip Dinh7, John F. Flaherty Jr.7, John G. McHutchison7, Michael Manns8 1

Sektion Hepatologie, Klinik und Poliklinik für Gastroenterologie und Rheumatologie, Universitätsklinikum Leipzig, Leipzig, Germany; INSERM U1052 and Hospices Civils de Lyon, Lyon, France; 3Private practice, Berlin, Germany; 4Private practice, San Jose, CA, USA; 5 Service d’Hepatologie and INSERM CRB3, University of Paris, Hopital Beaujon, Pavillon Abrami, Paris, France; 6Private practice, Flushing, NY, USA; 7Gilead Sciences, Inc., Foster City, CA, USA; 8Department of Gastroenterology, Hepatology and Endocrinology, Medical School of Hannover, Hannover, Germany

2

Background & Aims: Suboptimal virologic response to nucleos(t)ide analogs may represent a significant risk factor for resistance development in patients with chronic hepatitis B virus infection; treatment options have not been well studied. We evaluated long-term efficacy and safety of tenofovir alone and in combination with emtricitabine in a prospective, placebo-controlled trial in patients who remained viremic on adefovir therapy. Methods: Hepatitis B e antigen-positive and -negative patients with hepatitis B virus DNA P1000 copies/ml despite up to 96 weeks of adefovir were randomized to double-blind tenofovir or emtricitabine/tenofovir for 168 weeks. Patients with hepatitis B virus DNA P400 copies/ml (P69 IU/ml) at or after week 24 could switch to open-label emtricitabine/tenofovir. Results: Overall, 90/105 (86%) patients (46/53 tenofovir and 44/ 52 emtricitabine/tenofovir) completed the 168-week study period, including 74/105 (70%) patients (35/53 tenofovir and 39/52 emtricitabine/tenofovir) who completed the study on their initial randomized treatment. Long-term viral suppression (hepatitis B virus DNA <400 copies/ml) was maintained at week 168 in 84% and 82% of patients receiving either emtricitabine/tenofovir combination or tenofovir monotherapy, respectively (non-completer equal to failure analysis). Baseline viral load as well as the presence of lamivudine and/or adefovir resistance-associated

Keywords: Adefovir; Emtricitabine; Hepatitis B; Monotherapy; Combination treatment; Mutations; Resistance; Tenofovir. Received 6 December 2012; received in revised form 18 November 2013; accepted 25 November 2013; available online 1 December 2013 ⇑ Corresponding author. Address: Sektion Hepatologie, Klinik und Poliklinik für Gastroenterologie und Rheumatologie, Universitätsklinikum Leipzig, Liebigstr. 20, 04103 Leipzig, Germany. Tel.: +49 341 97 12330; fax: +49 341 97 12339. E-mail address: [email protected] (T. Berg). Abbreviations: HBV, hepatitis B virus; HBeAg, hepatitis B e antigen; TDF, tenofovir disoproxil fumarate; FTC, emtricitabine; ALT, alanine aminotransferase; ULN, upper limit of normal; FTC/TDF, emtricitabine/tenofovir disoproxil fumarate; HBsAg, hepatitis B surface antigen; pol/RT, polymerase/reverse transcriptase; NC = F, non-completer equal to failure; NC/S = F, non-completer or switch equal to failure; AE, adverse event.

mutations at baseline had no impact on long-term treatment response. No resistance to tenofovir was observed through 168 weeks. Both treatments had a favorable safety profile. Conclusions: Tenofovir monotherapy is as effective as emtricitabine/tenofovir combination therapy in maintaining long-term viral suppression in patients with a suboptimal response to adefovir, and is well tolerated in this population. Ó 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Introduction Adefovir dipivoxil was approved for the treatment of chronic hepatitis B virus (HBV) infection in 2002 in the US and 2003 in Europe. Although initially recommended as a first-line therapy, the incidence of adefovir resistance during long-term treatment was found to be relatively high – 20% of hepatitis B e antigen (HBeAg)-positive and 29% of HBeAg-negative patients had developed adefovir resistance mutations after 5 years of treatment [1]. In addition, many patients without evidence of resistance fail to fully suppress HBV DNA while on adefovir monotherapy, which can result in continued disease progression and possible resistance development during long-term treatment [2]. Current guidelines [3,4] favor switching to a single agent with a more potent activity such as entecavir or tenofovir disoproxil fumarate (TDF). Entecavir is effective as monotherapy in treatment-naïve patients with low rates of resistance (0.5% to 1.2%) with up to 6 years of treatment [5–7]. However, nearly three-fourths of patients with suboptimal response to adefovir and prior resistance to lamivudine develop resistance to entecavir after 36 months of therapy, suggesting that it is not an optimal treatment in this population [8]. TDF is a nucleotide analogue currently recommended as firstline treatment for chronic HBV infection [1,3,9] based on results from two prospective, randomized, double-blind trials in mostly treatment-naïve HBeAg-positive and HBeAg-negative patients.

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Research Article In these trials, TDF demonstrated superior efficacy compared with adefovir dipivoxil at 48 weeks, and, in the open-label TDF extension phase, TDF provided potent, long-term suppression of HBV replication through 5 years with no evidence of resistance [2,10–13]. TDF has been evaluated as a rescue strategy for patients with suboptimal responses or virologic breakthrough during adefovir therapy, with variable results [14–18]. Importantly, none of these studies followed patients for longer than 96 weeks. We previously reported 48-week results from a prospective, randomized, controlled evaluation of the efficacy and safety of TDF monotherapy vs. a fixed combination of emtricitabine (FTC) plus TDF in patients who had a suboptimal response to adefovir treatment. At 48 weeks, both of these approaches were found to provide profound viral suppression and were well tolerated [19]. The present report describes the final long-term results of this study through 168 weeks.

Patients and methods This was a prospective, randomized, double-blind, double-dummy, 168-week clinical trial conducted in compliance with all regulatory obligations and the institutional review board and informed consent regulations at each investigational site (ClinicalTrials.gov Identifier NCT00307489). The protocol, methods, and statistical analyses have been described in detail [19]. The first patient was screened on March 30, 2006, and the last week 168 observation occurred on October 5, 2010. Details on patient inclusion and exclusion criteria have been previously described [19]. All patients were required to be naïve to TDF and entecavir; prior lamivudine therapy (alone or in combination with adefovir)

was allowed, and previous interferon therapy was allowed provided it was discontinued 6 months before study entry. Study treatment Briefly, patients were randomized (1:1) to receive TDF 300 mg once daily as monotherapy or the fixed-dose combination of FTC 200 mg and TDF 300 mg once daily (FTC/TDF). Patients in either treatment arm with confirmed plasma HBV DNA P400 copies/ml (P69 IU/ml) at or after 24 weeks were eligible to switch to open-label FTC/TDF or could discontinue the study at the discretion of the investigator. Outcome measures The primary endpoint was the proportion of patients with plasma HBV DNA 400 copies/ml (<69 IU/ml) at week 48 [19]. Secondary endpoints at week 168 included the proportion of patients with plasma HBV DNA <400 copies/ml (<69 IU/ml), the proportion of patients with normal or normalized serum ALT, HBeAg and hepatitis B surface antigen (HBsAg) loss and seroconversion, safety and tolerability, and surveillance for HBV-polymerase/reverse transcriptase (pol/RT) resistance mutations. Virologic breakthrough was defined as two consecutive 1.0-log10 copies/ml or greater increases in HBV DNA from nadir, or two consecutive values P400 copies/ml after being <400 copies/ml. Genotypic analyses were conducted by population sequencing and HBV DR v2/v3 INNO-LiPA assay (Innogenetics NV, Ghent, Belgium) [19] and phenotypic analyses were conducted as previously described [20]. Data analysis and statistical methods Details have been previously reported [19]. Efficacy analyses were performed on the full analysis set, which included all randomized patients to whom at least one dose of study drug was dispensed. Efficacy comparisons (the primary comparison at 48 weeks as well as the 168-week

Randomized and treated n = 105

TDF n = 53

FTC/TDF n = 52

Switched to open-label FTC/TDF, n = 16 Discontinued early, n = 4:

Discontinued early, n = 2:

• Withdrew consent, n = 3 • Lost to follow-up, n = 1

• Investigator discretion, n = 1 • Seroconversion, n = 1

Discontinued early, n = 5:

Discontinued early, n = 4:

• Safety/tolerability/efficacy, n = 3 • Investigator discretion, n = 1 • Withdrew consent, n = 1

Completed week 168 on randomized treatment, n = 35

Switched to open-label FTC/TDF, n = 9

Completed week 168 on open-label FTC/TDF, n = 11

• Safety/tolerability/efficacy, n = 2 • Lost to follow-up, n = 1 • Pulmonary cancer (death), n = 1

Completed week 168 on randomized treatment, n = 39

Completed week 168 on open-label FTC/TDF, n=5

Fig. 1. Patient disposition. FTC/TDF, emtricitabine/tenofovir disoproxil fumarate; TDF, tenofovir disoproxil fumarate. Overall, 87% and 85% of patients completed 168 weeks of treatment in the TDF and FTC/TDF groups, respectively. Eleven of 16 (69%) in the TDF arm who switched to open-label FTC/TDF and five of nine (56%) in the FTC/TDF arm who switched to open-label FTC/TDF completed 168 weeks of treatment.

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A 100 80 60 40 TDF FTC/TDF

20

96 10 8 12 0 13 2 14 4 15 6 16 8

84

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Proportion of patients achieving HBV DNA <400 copies/ml (%)

comparison) were made using a ‘‘non-completer equal to failure’’ analysis wherein any patient not completing the study for any reason was considered a failure regardless of whether or not the patient was switched to open-label FTC/TDF (NC = F). In the NC = F analysis, patients were considered failures at all time points after the time of discontinuation from the study. Additionally, patients who remained on study but with random missing DNA results at a given study visit (e.g., samples not analyzable, patient missed the visit, or sample obtained outside the visit window) were excluded from the denominator in the NC = F analysis. A second, more conservative analysis was also performed wherein patients who discontinued the study for any reason or were switched to open-label FTC/TDF were considered failures (NC/S = F). The Cochran-Mantel-Haenszel test, controlling for randomization strata (lamivudine experience and baseline HBeAg status), was used to evaluate betweengroup differences in categorical variables, the van Elteren test, controlling for randomization strata, to evaluate between-group differences and changes from baseline in continuous variables, and the Fisher’s exact test to evaluate treatment group differences in the percentages of patients with laboratory abnormalities. For the post hoc analyses of proportion with increases in serum creatinine of 0.2 or 0.3 mg/dl from baseline [21,22], the Clopper-Pearson exact method was used to construct 95% confidence intervals.

Weeks on study Wk of study

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96 10 8 12 0 13 2 14 4 15 6 16 8

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A total of 106 patients were randomized and 105 were treated (TDF [n = 53] and FTC/TDF [n = 52]) from 28 study sites in the United States and Europe (Fig. 1). Seventy-four patients, 35 (66%) in the TDF group and 39 (75%) in the FTC/TDF group, completed 168 weeks on their initially randomized treatment. Six patients (2 TDF and 4 FTC/TDF) discontinued before week 168, including 3 patients (1 TDF and 2 FTC/TDF) who discontinued prior to week 48 [19]. Twenty-five (16 TDF and 9 FTC/TDF) of 34 patients meeting protocol-defined eligibility were switched to open-label FTC/ TDF at or after week 24 (19/25 switched at week 24 and 6/25 switched before week 48). Among the 25 patients who switched, 16 (11 TDF and 5 FTC/TDF) completed 168 weeks of treatment. Overall, 87% and 85% completed 168 weeks of treatment in the TDF and FTC/TDF groups, respectively. Baseline demographic and disease characteristics have been previously described [19]. The two groups were similar, with the majority (76%) being male and with equal proportions (42% each) of Asian and white patients. Mean baseline HBV DNA was 6.0 log10 copies/ml; in addition to adefovir, 58% also received prior or concurrent lamivudine.

100

0 4 8 12

Patient population

Proportion of patients achieving HBV DNA <400 copies/ml (%)

B

Results

Weeks on study Wk of study

0

4

8

12 24 36 48 60 72 84 96 108 120 132 144 156 168

FTC/TDF

N 52 52 50 52 52 51 52 52 52 51 52 51 51 51 52 52 50

TDF

N 53 53 52 53 53 53 53 52 52 51 53 52 53 52 51 49 51

Fig. 2. Virologic response. (A) Results of the primary efficacy analysis, proportion of patients who achieved HBV DNA levels <400 copies/ml (non-completer equals failure analysis; NC = F). (B) Results of the secondary efficacy analysis, proportion of patients who achieved <400 copies/ml (non-completer or switch equals failure analysis; NC/S = F).

the lower limit of quantification from week 48 through week 168 in both groups. No statistically significant between-group difference was observed at any follow-up visit.

Long-term virologic response Results at 168 weeks were generally consistent with the 48-week findings, with patients in each treatment group maintaining similar treatment responses through 168 weeks [19]. In the primary analysis (NC = F), 82% (42/51) and 84% (42/50) of patients in the TDF and FTC/TDF groups, respectively, achieved complete viral suppression defined as HBV DNA <400 copies/ml (<69 IU/ml) at week 168 (p = 0.781) (Fig. 2A). In the alternative analysis in which patients who switched to FTC/TDF were regarded as treatment failures (NC/S = F), 65% (33/51) in the TDF group and 74% (37/50) in the FTC/TDF group achieved HBV DNA <400 copies/ ml at week 168 (p = 0.324) (Fig. 2B). In an analysis based on observed data (missing = excluded), 100% of patients who completed blinded treatment (33/33 TDF and 37/37 FTC/TDF) had HBV DNA <400 copies/ml at week 168 (data not shown). The viral kinetic curves were nearly identical for both treatments (data not shown); mean HBV DNA levels remained near

Impact of added FTC on virologic response to TDF There were 18/53 (34%) patients randomized to TDF who had HBV DNA P400 copies/ml at or after week 24, with 16 patients switching to open-label FTC/TDF and two patients remaining on TDF monotherapy. Although patients meeting the switch criterion took longer to achieve HBV DNA <400 copies/ml (week 72) compared with those who did not meet the switch criterion (week 12), complete viral suppression was maintained in similar proportions of both groups through week 168 (Fig. 3). Virologic response by resistance profile and baseline disease characteristics The presence of baseline resistance-associated mutations was determined by INNO-LiPA (v2/v3): 16 patients had adefovir resistance-associated mutations, 14 had lamivudine resistance-associated mutations, and 13 had both adefovir and lamivudine resistance-associated mutations (Supplementary Table 1). The

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Research Article Median HBV DNA (log10 copies/ml)

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Switch status TDF Mono

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Added FTC

6 5 4 3 2

LLOQ

1 0 4 8 12

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Fig. 3. Median HBV DNA values over 168 weeks for patients in the TDF arm who did or did not meet criteria for an early switch to FTC/TDF. Sixteen patients with confirmed plasma HBV DNA P400 copies/ml at 24 weeks had treatment switched to open-label FTC/TDF.

presence of adefovir and/or lamivudine resistance-associated mutations at baseline had no demonstrable effect on long-term treatment response; mean HBV DNA levels remained at or below 400 copies/ml from week 48 through week 168 for most patients regardless of baseline resistance profile (Fig. 4A and B). When the adefovir-resistant subgroup (n = 29) was further analyzed by prior lamivudine use (lamivudine naïve, no lamivudine resistance mutations [n = 4], lamivudine experienced, no lamivudine resistance mutations [n = 12], and lamivudine experienced with lamivudine resistance mutations [n = 13]), neither prior lamivudine exposure nor the presence of lamivudine resistance-associated mutations had an impact on HBV DNA responses through 168 weeks (data not shown). Previous analyses demonstrated that baseline viral load appeared to impact early viral decay kinetics, with rapid responders (patients who achieved HBV DNA <400 copies/ml before week 12) having lower median baseline viral loads (4.5 log10 copies/ml) compared to slow responders (patients who did not achieve HBV DNA <400 copies/ml by week 24) (7.2 log10 copies/ml) (p <0.05) (Supplementary Table 2) [23]. Baseline viral load had no impact on long-term treatment response in this study; the median HBV DNA for both rapid and slow responders was <400 copies/ ml from week 48 through week 168. Resistance surveillance Resistance surveillance through week 48 has been previously reported with no tenofovir resistance-associated mutations identified [19]. Of the 102 patients who completed the first 48 weeks of treatment, 11 qualified for genotypic testing between weeks 48 and 168 (Supplementary Fig. 1). Five patients qualified due to persistent viremia (HBV DNA never below 400 copies/ml), five experienced virologic breakthrough (one with two independent episodes), and one had persistent viremia at an earlier time point followed by an episode of virologic breakthrough at a later time point. Genotypic testing was performed on samples from all 13 qualifying time points. Three time points 718

had no change from baseline, two had unique polymorphic changes, one had a reversion at a conserved site, one had conserved site changes, including an adefovir resistance-associated mutation (rtY158F + rtN236T), and six could not be genotyped. The rtY158F conserved site change remained phenotypically sensitive to TDF. Overall, no resistance to TDF was detected in any patient. The patient with detectable rtN236T during on-treatment genotypic analysis had rtN236T detected at baseline by INNOLiPA but not by population sequencing. This patient had a decline from baseline in HBV DNA of 3.8 log10 copies/ml at weeks 48 and 96; however, because the patient remained viremic at weeks 48 and 96, genotyping was performed. Population sequencing detected rtN236T at weeks 48 and 96 as well as the conserved site change of rtY158F at week 96. Despite the presence of the rtN236T mutation in this patient, HBV DNA levels declined to <400 copies/ml by week 144, and viral suppression was maintained through week 168. Biochemical response There were no significant differences between treatment arms in biochemical endpoints at any follow-up visits. At week 168, 74% of patients in both treatment groups had normal ALT levels; 68% who received TDF and 71% who received FTC/TDF achieved ALT normalization (NC = F analysis). At week 168, mean (standard deviation) decrease in ALT from baseline was 27 (60) U/L in the TDF group and 54 (142) U/L in the FTC/TDF group. Serologic response Progressively increasing rates of HBeAg loss and seroconversion to anti-HBe were seen in the subset of HBeAg-positive patients with similar results observed between treatment groups. At week 168, 22% of patients in the TDF group and 24% in the FTC/TDF group experienced HBeAg loss and 14% in each group had seroconversion to anti-HBe antibody (NC = F analysis). Three

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JOURNAL OF HEPATOLOGY A Resistance group

Mean HBV DNA (log10 copies/ml)

7

FTC/TDF, ADV-R FTC/TDF wild-type, LAM-R TDF, ADV-R TDF wild-type, LAM-R FTC/TDF wild-type TDF wild-type

6 5 4 3 2 LLOQ 1

0 4 8 12

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Mean HBV DNA (log10 copies/ml)

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FTC/TDF, ADV-R + LAM-R FTC/TDF wild-type TDF, ADV-R + LAM-R TDF wild-type

6 5 4 3 2 LLOQ 1

0 4 8 12

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Fig. 4. Changes in HBV DNA over 168 weeks by baseline resistance pattern and treatment group. The baseline resistance patterns were determined by INNO-LiPA. (A) Shows the HBV DNA changes over time for patients with ADV-R or LAM-R compared to wild type; (B) compares patients with ADV-R + LAM-R to wild type. ADV, adefovir; FTC, emtricitabine; FTC/TDF, emtricitabine/tenofovir disoproxil fumarate; LAM, lamivudine; R, resistant; TDF, tenofovir disoproxil fumarate.

HBeAg-positive patients (all in the TDF group) experienced HBsAg loss with seroconversion during the study. All three patients with HBsAg loss also experienced HBeAg loss with only one patient also showing confirmed HBeAg seroconversion.

Safety Both TDF and FTC/TDF were well tolerated through 168 weeks; no new safety signals were identified between weeks 48 and

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Research Article Table 1. Summary of adverse events.

TDF, n = 53 n (%) Any adverse event 49 (92) Grade 2, 3, or 4 adverse events 37 (70) Grade 3 or 4 adverse events 1 (2) Serious adverse events* 6 (11) 1 (2) Adverse events resulting in study discontinuation† Grade 3 or 4 laboratory 10 (19) abnormalities 0 ALT flare‡

p value

FTC/TDF, n = 52 n (%) 42 (81) 33 (63) 4 (8)* 10 (19) 1 (2)

0.092 0.539 0.205 0.290 -

12 (23)

0.638

2 (4)

-

⁄

One patient (FTC/TDF group) experienced an ALT flare (ALT >10  ULN and >2  baseline) that was considered related to study drug.   Grade 2 erectile dysfunction (TDF arm); death due to pulmonary cancer with osseous metastasis (FTC/TDF arm). Neither adverse event was considered by the investigator to be study drug related. à Between-group difference not statistically significant; p = 0.205. ALT, alanine aminotransferase; FTC/TDF, emtricitabine/tenofovir disoproxil fumarate; TDF, tenofovir disoproxil fumarate; ULN, upper limit of normal.

168 (Table 1). No differences were observed between the treatment groups in the overall incidence of adverse events (AEs) or in the incidence of any specific AE, serious AEs, AEs that resulted in study discontinuation, or laboratory abnormalities. The most frequent AEs in both treatment groups were nasopharyngitis (27%), headache (24%), and fatigue (17%). No patient experienced an on-treatment hepatic flare between weeks 48 and 168. No renal AEs occurred that were considered by the investigator to be related to the study drug. No patient experienced a grade 2 or greater increase in serum creatinine or grade 3 or greater decrease in serum phosphorus, or had a confirmed (upon retest) renal endpoint (increase in serum creatinine concentration of P0.5 mg/dl, creatinine clearance <50 ml/min, or serum phosphorus <2 mg/dl). Given several recent publications suggesting that changes in renal function smaller than those we employed for study endpoints could be clinically meaningful [21,22], we performed post hoc analyses of changes in serum creatinine in this study. Over 168 weeks, confirmed increases from baseline of P0.2 and P0.3 mg/dl in serum creatinine occurred in 9.5% (95% confidence interval [CI] 4.7% to 16.8%) and 2.9% (95% CI 0.6% to 8.1%) of patients, respectively (results for both groups combined).

Discussion In this prospective, controlled, long-term trial, TDF monotherapy provided highly effective viral suppression that was safe and well tolerated in patients with suboptimal response to adefovir. TDF alone was as effective as the combination of FTC/TDF, even in patients with lamivudine- and/or adefovir-associated resistance mutations at baseline. These data confirm and build upon the preliminary findings from other studies, which suggest that TDF monotherapy is an effective option in patients with suboptimal response to adefovir, including those with resistance [14–19]. The HBV DNA suppression rates we observed compare favorably with those reported in prospective clinical trials of TDF in treatment-naïve patients [2,9,10]. In the present study, 83% of patients achieved HBV DNA <400 copies/ml (<69 IU/ml) by week

720

168 with TDF monotherapy, which is comparable to the 81% of treatment-naïve patients (87% HBeAg-negative and 72% HBeAgpositive) who achieved HBV DNA <400 copies/ml after 144 weeks of TDF monotherapy [10]. Most published studies, including the present trial, indicate that the antiviral response to TDF is not impacted by baseline adefovir resistance-associated mutations [15,17,19]. In contrast, van Bömmel and colleagues [18] reported that only 33% of patients with baseline adefovir resistance compared to 90% of patients without adefovir resistance achieved HBV DNA levels below the level of quantification after 12 months of TDF treatment [18]. Interpretation of their findings is confounded by differences in baseline viral load; namely, adefovir-resistant patients with HBV DNA levels >107 copies/ml were less likely to achieve complete viral suppression during TDF treatment compared with adefovir-resistant patients having HBV DNA <107 copies/ml [18]. Median HBV DNA values in the present study were approximately 3 logs lower than those reported by van Bömmel and colleagues [18], which suggests that with longer follow-up a higher percentage of patients in their cohort might have achieved complete viral suppression. This point is illustrated by patient 1051, who was randomized to the TDF arm and entered our study with the rtN236N/T mutation. This patient had HBV DNA of 8.75 log10 copies/ml at baseline and did not achieve full virologic suppression until week 120 despite having switched to open-label FTC/TDF at week 24 (Supplementary Fig. 1). Another recent study by Patterson and colleagues [17] suggests that the adefovir resistance mutation rtN236T is capable of attenuating the treatment response to TDF. Although few cases of documented adefovir resistance (including rtN236T) were seen in the present study, our findings support the concept that baseline HBV DNA level, rather than the presence of resistance mutations, has the greatest impact on early viral load decay kinetics, but neither had an effect on long-term HBV DNA suppression. When the impact of the rtN236T mutation was also evaluated in our study using an rtN236T allele-specific polymerase chain reaction assay, our results demonstrated that TDF equally suppressed rtN236T and wild-type populations through the first 24 weeks of treatment [24]. Furthermore, there was no observed impact of the presence of rtA181T/V mutations on the suppression of rtN236T populations [24]. Although the publication by Patterson and colleagues [17] suggested that rtN236T might attenuate treatment response, we observed HBV DNA levels to decline with further treatment, suggesting that other factors, including baseline viral load and/or other mutations in the HBV genome, may have impacted treatment response. We continued to see no evidence of TDF resistance-associated mutations over 168 weeks of continuous treatment, even in patients harboring lamivudine and/or adefovir resistance-associated mutations at baseline. There were seven episodes of confirmed virologic breakthrough occurring between week 48 and week 168, and poor adherence to treatment (as determined by no tenofovir detectable in the plasma at the time of resistance testing) was determined to be the causative factor in at least three of these episodes. Similar results have been reported for TDF in treatment-naïve patients after up to 5 years of treatment [11]. Treatment with TDF and FTC/TDF was well tolerated through 168 weeks, consistent with other prospective clinical trials in treatment-naïve patients and with safety data collected over 5 years [25]. There were no significant differences in the rates

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JOURNAL OF HEPATOLOGY of clinical and laboratory AEs in the two treatment groups. Through 168 weeks, the rate of serious AEs was low, there was no increase in treatment-related serious AEs, and no new safety signals were detected. Importantly, although adefovir is known to be associated with renal events, there was no evidence of an increased risk of renalrelated AEs or abnormalities in renal laboratory parameters in either group [10]. In the present trial and others evaluating TDF in patients with chronic hepatitis B [10,19,25], an increase of P0.5 mg/dl from baseline in serum creatinine was included as a pre-specified threshold for renal toxicity. No patients met this endpoint in our study. However, recent authors have suggested using a lower threshold to detect smaller changes in renal function that could also be clinically meaningful [21,22]. In a post hoc analysis, confirmed increases from baseline in serum creatinine of P0.2 and P0.3 mg/dl were observed in 9.5% and 2.9% of patients, respectively. These findings are notably lower than those of Gish et al. [21], who in a retrospective, community-based cohort study reported 18.8% of non-transplant patients treated with TDF to show a confirmed change of P0.2 mg/dl from baseline in serum creatinine. These authors also reported a similar incidence (20.9%) of serum creatinine increases in entecavir-treated patients [21]. An important limitation of this study was that patients with an inadequate initial response to TDF monotherapy could receive open-label treatment with FTC/TDF at or after 24 weeks, thus limiting a direct comparison of TDF monotherapy vs. combination therapy. However, when efficacy analyses were conducted that considered patients who switched to FTC/TDF as failures, similar efficacy was demonstrated for the TDF monotherapy arm compared with the combination FTC/TDF treatment arm [26]. In conclusion, our long-term results demonstrate that TDF effectively maintains viral suppression in patients having a suboptimal response to adefovir, and that baseline resistance profile does not negatively impact response to treatment. Furthermore, treatment for 168 weeks was safe and well tolerated. Importantly, in this treatment-experienced population we observed no evidence of an additional treatment benefit from combination therapy with FTC and TDF.

Huy Trinh has received financial compensation for consultancy and lecture activities from Bristol-Myers Squibb, Gilead Sciences, Inc., and Vertex Pharmaceuticals and has received research grants from Bristol-Myers Squibb and Gilead Sciences, Inc. and is a stockholder of Gilead Sciences, Inc. Patrick Marcellin has received research grants from HoffmanLa Roche, Gilead Sciences, Inc., Bristol-Myers Squibb, Vertex Pharmaceuticals, Novartis Pharmaceuticals, Janssen/Tibotec, Merck, Boehringer Ingelheim, Abbott Laboratories, and Pfizer and has received financial compensation for consultancy and/or lecture activities from Hoffman-La Roche, Gilead Sciences, Inc., BristolMyers Squibb, Novartis Pharmaceuticals, Janssen/Tibotec, and Merck. Sing Chan is a stockholder of Gilead Sciences, Inc. Kathryn M. Kitrinos, Phillip Dinh, John F. Flaherty, Jr., and John G. McHutchison are employees and stockholders of Gilead Sciences, Inc. Michael Manns has received financial compensation for consultancy and/or lecture activities from Hoffman-La Roche Inc., Bristol-Myers Squibb, Gilead Sciences, Inc., Novartis Pharmaceuticals, Merck Pharmaceuticals, and GlaxoSmithKline and has received research grants from Hoffman-La Roche Inc., Gilead Sciences, Inc., Novartis Pharmaceuticals, and Bristol-Myers Squibb. Acknowledgement Financial support for medical editorial assistance was provided by Gilead Sciences, Inc. We thank Mariana Ovnic, PhD, and Evelyn Albu, PhD, of Percolation Communications LLC for their medical editorial assistance.

Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2013.11. 024.

References Financial support This study was sponsored by Gilead Sciences, Inc.

Conflict of interest Thomas Berg has received financial compensation for consultancy and/or lecture activities from Hoffman-La Roche Inc., Boehringer Ingelheim, Bristol-Myers Squibb, Gilead Sciences, Inc., Novartis Pharmaceuticals, Merck Pharmaceuticals, and Janssen and has received research grants from Hoffman-La Roche Inc., Gilead Sciences, Inc., Novartis Pharmaceuticals, and Bristol-Myers Squibb. Fabien Zoulim has received financial compensation for consultancy and/or lecture activities from Gilead Sciences, Inc., Hoffman-La Roche, and Bristol-Myers Squibb and has received research grants from Hoffman-La Roche, Gilead Sciences, Inc., and Bristol-Myers Squibb.

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