Articles
Peg-interferon plus nucleotide analogue treatment versus
Lancet Gastroenterol Hepatol
2017; 2: 576–84
Published Online
May 15, 2017
http://dx.doi.org/10.1016/
S2468-1253(17)30083-3
See Comment page 543
*Contributed equally
Department of Gastroenterology
and Hepatology, Academic
Medical Center, Amsterdam,
Netherlands (A de Niet PhD,
L Jansen PhD, F Stelma MD,
S B Willemse MD,
R B Takkenberg PhD,
Prof U Beuers MD,
H W Reesink PhD); Department of
Gastroenterology and
Hepatology, OLVG West,
Amsterdam, Netherlands
(S D Kuiken PhD); Department of
Internal Medicine, Medical
Center Zuiderzee, Lelystad,
Netherlands (S Weijer PhD);
Department of Gastroenterology
and Hepatology, VU Medical
Center, Amsterdam, Netherlands
(C M J van Nieuwkerk PhD);
Department of Medical
Microbiology, Academic Medical
Center, Amsterdam, Netherlands
(Prof H L Zaaijer MD,
R Molenkamp PhD); Department
of Blood-borne Infections,
Sanquin Blood Supply
Foundation, Amsterdam,
Netherlands (Prof H L Zaaijer);
Department of Virus Diagnostic
Services, Sanquin Blood Supply
Foundation, Amsterdam,
Netherlands (M Koot PhD); and
Department of Pathology,
Academic Medical Center,
Amsterdam, Netherlands
(J Verheij PhD)
Correspondence to:
Dr Hendrik W Reesink,
Department of Gastroenterology
and Hepatology, Academic
Medical Center, University of
Amsterdam, Meibergdreef 9,
1105 AZ Amsterdam,
Netherlands
h.w.reesink@amc.nl
576 www.thelancet.com/gastrohep Vol 2 August 2017
no treatment in patients with chronic hepatitis B with a low
viral load: a randomised controlled, open-label trial
Annikki de Niet*, Louis Jansen*, Femke Stelma*, Sophie B Willemse, Sjoerd D Kuiken, Sebastiaan Weijer, Carin M J van Nieuwkerk, Hans L Zaaijer,
Richard Molenkamp, R Bart Takkenberg, Maarten Koot, Joanne Verheij, Ulrich Beuers, Hendrik W Reesink
Summary
Background Antiviral treatment is currently not recommended for patients with chronic hepatitis B with a low viral load.
However, they might benefit from acquiring a functional cure (hepatitis B surface antigen [HBsAg] loss with or without
formation of antibodies against hepatitis B surface antigen [anti-HBs]). We assessed HBsAg loss during peg-interferon-
alfa-2a (peg-IFN) and nucleotide analogue combination therapy in patients with chronic hepatitis B with a low viral load.
Methods In this randomised controlled, open-label trial, patients were enrolled from the Academic Medical Center
(AMC), Amsterdam, Netherlands. Eligible patients were HBsAg positive and hepatitis B e antigen (HBeAg) negative
for more than 6 months, could be treatment naive or treatment experienced, and had alanine aminotransferase (ALT)
concentrations less than 5 × upper limit of normal (ULN). Participants were randomly assigned (1:1:1) by a
computerised randomisation programme (ALEA Randomisation Service) to receive peg-IFN 180 µg/week plus
adefovir 10 mg/day, peg-IFN 180 µg/week plus tenofovir disoproxil fumarate 245 mg/day, or no treatment for
48 weeks. The primary endpoint was the proportion of patients with serum HBsAg loss among those who received at
least one dose of study drug or had at least one study visit (modified intention-to-treat population [mITT]). All patients
have finished the initial study of 72 weeks and will be observed for up to 5 years of follow-up. This study is registered
with ClinicalTrials.gov, number NCT00973219.
Findings Between Aug 4, 2009, and Oct 17, 2013, 167 patients were screened for enrolment, of whom 151 were randomly
assigned (52 to peg-IFN plus adefovir, 51 to peg-IFN plus tenofovir, and 48 to no treatment). 46 participants in the
peg-IFN plus adefovir group, 45 in the peg-IFN plus tenofovir group, and 43 in the no treatment group began treatment
or observation and were included in the mITT population. At week 72, two (4%) patients in the peg-IFN plus adefovir
group and two (4%) patients in the peg-IFN plus tenofovir group had achieved HBsAg loss, compared with none of the
patients in the no treatment group (p=0·377). The most frequent adverse events (>30%) were fatigue, headache, fever,
and myalgia, which were attributed to peg-IFN dosing. Two (4%) serious adverse events were reported in the peg-IFN
plus adefovir group (admission to hospital for alcohol-related pancreatitis [week 6; n=1] and pregnancy, which was
electively aborted [week 9; n=1]), three (7%) in the peg-IFN plus tenofovir group (admission to hospital after a suicide
attempt during a severe depression [week 23; n=1], admission to hospital for abdominal pain [week 2; n=1], and an
elective laminectomy [week 40; n=1]), and three (7%) in the no treatment group (admission to hospital for septic
arthritis [week 72; n=1], endocarditis [week 5; n=1], and hyperthyroidism [week 20; n=1]).
Interpretation In patients with chronic hepatitis B with a low viral load, combination treatment (peg-IFN plus adefovir
and peg-IFN plus tenofovir) did not result in significant HBsAg loss compared with no treatment, which does not
support the use of combination treatment in this population of patients.
Funding Roche, Fonds NutsOhra.
Introduction activity or fibrosis, there is currently no indication for
Despite the availability of a safe and effective vaccine for treatment, because viral replication levels are already low,
more than three decades, infection with hepatitis B virus and the progression of liver disease is slower.2,4,5 However,
is still a major public health problem, with an estimated patients with chronic hepatitis B with a low viral load still
240 million chronically infected individuals worldwide.1 have a substantial risk for the development of cirrhosis
The goal of antiviral treatment in patients with chronic and hepatocellular carcinoma.4–11 As shown by the
hepatitis B is to prevent progression to cirrhosis, hepatic REVEAL cohorts,5,7 the risks for development of cirrhosis,
decompensation, and hepatocellular carcinoma by hepatocellular carcinoma, or both, are highest in patients
reducing viral replication.2,3 Treatment is indicated in with the highest viral load, but are already increased at
patients with a high viral load, progressive liver inflam­ lower hepatitis B virus DNA levels. The cumulative
mation, and fibrosis. For patients with chronic hepatitis B incidence of hepatocellular carcinoma in hepatitis B
with a low viral load and no signs of necroinflammatory surface antigen (HBsAg)-positive patients who are

Research in context
Evidence before this study
We searched PubMed for studies of patient cohorts with chronic
hepatitis B treated with peg-interferon alfa (peg-IFN) and
nucleos(t)ide analogues using the terms ‘‘chronic hepatitis B’’,
‘‘combination therapy’’, and ‘’inactive carrier’’ or ‘’low viral
load’’, with no language restrictions. Many results showed data
of combination therapy studies for patients with a high viral
load and an indication for treatment or data for peg-interferon
add-on in patients with chronic hepatitis B with viral
suppression by nucleos(t)ide analogues. Thus far, no studies
have assessed treatment in patients with chronic hepatitis B
with a low viral load. The results of combination treatment with
respect to HBsAg loss (functional cure) are superior to those of
peg-IFN monotherapy in patients with a high viral load.
Furthermore, response to combination treatment was
associated with low baseline HBsAg levels, which is a
characteristic of patients with chronic hepatitis B with a low
viral load. Little T-cell exhaustion, as noted in patients with
chronic hepatitis B with a low viral load, suggests that an
immune modulator could be of value in activating antiviral
immune mechanisms.
Added value of this study
There is an urgent need for treatment options for patients
with chronic hepatitis B that can lead to functional cure,
hepatitis B virus DNA negative and patients with chronic
hepatitis B who have a viral load less than 20 000 IU/mL
ranges from 1·3% to 3·57% at year 13, while the
cumulative incidence of cirrhosis ranges from
4·5% to 9·8%.5,7 Furthermore, due to precore mutants,
patients with chronic hepatitis B with a low viral load can
progress to hepatitis B e antigen (HBeAg)-negative
chronic hepatitis, which could lead to fulminant hepatitis
or cirrhosis.12,13 The risk of hepatitis B virus reactivation in
patients who receive immunosuppression is higher in
patients who are HBsAg positive compared with patients
who are HBsAg negative.14 The most favourable outcome
in the treatment of patients with chronic hepatitis B is
functional cure, defined as the clearance of HBsAg with
or without formation of antibodies against hepatitis B
surface antigen (anti-HBs), which is associated with
improved outcome.15–18 Unfortunately, with a finite course
of pegylated interferon alfa (peg-IFN) or long-term viral
suppression with nucleos(t)ide analogues, this functional
cure is rarely achieved.2
Previous attempts to improve response rates by
combining peg-IFN and nucleos(t)ide analogue therapy
have had varying results.19–21 Nevertheless, combining
peg-IFN with more potent nucleos(t)ide analogues
remains of interest because of the dual effect on both the
innate and adaptive immune responses.22–24 In our
previous study, in which patients with active chronic
hepatitis B with a high viral load were treated with peg-IFN
www.thelancet.com/gastrohep Vol 2 August 2017 577
Articles
defined as HBsAg loss with or without formation of antibodies
against hepatitis B surface antigen (anti-HBs). Achievement of
functional cure has been associated with an improved
outcome in patients with chronic hepatitis B. Our study adds
valuable data to the field of combination treatment for chronic
hepatitis B. Furthermore, because no data are available on
treatment of patients with chronic hepatitis B with a low viral
load, valuable lessons can be learned from our data in which
patients with a low viral load were treated for the first time
with combination therapy. Our findings do not support the
use of combination treatment with peg-IFN and a nucleos(t)-
ide analogue in low viral load patients with chronic hepatitis B.
Implications of all the available evidence
This study shows that treatment with combination therapy
does not lead to a durable functional cure in patients with
chronic hepatitis B with a low viral load. Reporting the data of
treatment intervention studies is paramount for the analysis of
treatment response in different patient groups, even when the
results are not as expected. To the best of our knowledge, this
is the first randomised controlled treatment intervention study
in patients with a low viral load. This study could aid clinicians
in making well informed clinical decisions. Our study underlines
the need for novel treatment options for patients with chronic
hepatitis B.
and adefovir, a relatively high proportion of HBsAg loss
(17%) was noted in HBeAg-negative patients 2 years after
therapy.25 A recent large randomised controlled trial24 in
patients with active chronic hepatitis B showed a higher
proportion of HBsAg loss in patients treated with peg-IFN
and tenofovir than in those receiving either monotherapy.
Most (70–80%) patients with a chronic hepatitis B
infection worldwide have a low viral load. By contrast
with patients with a high viral load, patients with a low
viral load have low HBsAg levels.26 Previous results in
patients with a high viral load suggest that low baseline
HBsAg levels are associated with a response to
combination treatment.25,27 Furthermore, in patients
who have a low viral load as a result of long-term
nucleos(t)ide analogue suppression, low baseline
HBsAg titers were associated with functional cure in
response to peg-IFN add-on therapy.28 Hypo­thetically,
patients with chronic hepatitis B with a low viral load
might be more susceptible to hepatitis B virus treatment
because their antiviral immune response is already
capable of keeping hepatitis B virus DNA and HBsAg at
low levels. This could be related to the finding that
T-cell inhibition is less severe in the presence of low
hepatitis B virus DNA levels.29,30
No randomised controlled studies have yet been done
on the efficacy of peg-IFN or nucleos(t)ide analogues in
patients with chronic hepatitis B with a low viral load.
Therefore, we aimed to compare combination therapy of
 Articles

peg-IFN plus adefovir or tenofovir versus no treatment Patients with chronic hepatitis B aged 18–70 years with a
in HBeAg-negative patients with chronic hepatitis B low viral load were enrolled after assessment of eligibility.
with a low viral load, and to investigate the rate of HBsAg Hepatitis B virus DNA below a threshold of 20 000 IU/mL
loss and markers of response to therapy. was chosen as low viral load according to local32 and
Although adefovir is no longer indicated as international2,3 guidelines available at the time of study
monotherapy for viral suppression in patients with a initiation. Inclusion criteria were documented HBsAg
treatment indication, based on our previous results with positivity and HBeAg negativity for more than 6 months.
high rates of functional cure, a treatment group with Exclusion criteria were concurrent infection with hepatitis C
peg-IFN and adefovir was included in the study. Because virus, hepatitis delta virus, or HIV; decompensated liver
tenofovir (which is a stronger suppressor of hepatitis B disease, hepatocellular carcinoma, or a history of bleeding
virus DNA than adefovir, but with unknown immune from oesophageal varices; pregnancy or breastfeeding; and
modulatory effect31) had become available since our alanine aminotransferase (ALT) levels greater than
last study, we also included peg-IFN and tenofovir five times the upper limit of normal (5 × ULN). Patients
combination therapy. The effect of both combination were either treatment naive, or had received peg-IFN or
therapies was compared to an untreated control group nucleos(t)ide analogues more than 6 months before
which is the current standard of practice for chronic inclusion. The full eligibility criteria and screening
See Online for appendix hepatitis B patients with a low viral load. assessments are provided in the appendix (p 1–3).
The study complied with the Declaration of Helsinki
Methods and the principles of Good Clinical Practice and was
Study design and participants approved by a legally instituted ethical committee. All
This investigator-initiated, prospective, open-label, patients gave written informed consent.
randomised controlled trial was done at the Academic
Medical Center (AMC), Amsterdam, Netherlands. Randomisation and masking
Patients were randomly assigned (1:1:1) to receive
pegylated interferon alfa-2a (Pegasys; F Hoffman-La
167 participants screened for Roche, Basel, Switzerland) 180 µg/week, subcutaneously
study
in combination with adefovir dipivoxil (Hepsera; Gilead
Sciences, Foster City, CA, USA) 10 mg orally once daily for
16 excluded 48 weeks; peg-interferon alfa plus tenofovir disoproxil
9 did not meet inclusion criteria
7 withdrawn before randomisation
fumarate (Viread; Gilead Sciences) 245 mg orally once
daily for 48 weeks; or no treatment (current standard
of care) for 48 weeks. Patients were enrolled by the
151 randomised subinvestigators and randomly assigned by a computerised
randomisation programme (ALEA Randomisation
Service). Randomisation was stratified according to
52 assigned to peg-IFN 51 assigned to peg-IFN 48 assigned to no
hepatitis B virus genotype A, non-A (genotype B–G), or
plus adefovir plus tenofovir treatment indeterminable genotype. Patients and investigators were
not masked. After 48 weeks, treatment was discontinued
and all patients were followed up until week 72.
6 did not receive 6 did not receive 5 did not receive
allocated allocated allocated
intervention* intervention* intervention* Procedures
Routine examinations and laboratory tests were done
46 received intervention: 45 received intervention: 43 received intervention: with 2 weekly intervals for the first 2 months and 6 weekly
modified intention-to- modified intention-to- modified intention-to- intervals thereafter (appendix pp 4–7). The viral load was
treat population treat population treat population
assessed before selecting or approaching eligible
patients, if available from the medical chart. Furthermore,
5 discontinued 6 discontinued 1 discontinued viral load was assessed at the time of screening and at
intervention† intervention† intervention† day 1 of treatment, of which the latter was used as
baseline hepatitis B virus DNA level. Due to subsequent
41 completed intervention: 39 completed intervention: 42 completed intervention: changes in expert opinion on the classification of low
per-protocol population per-protocol population per-protocol population viral load, a subgroup analysis on patients with hepatitis B
virus DNA level less than 2000 IU/mL (often referred to
Figure 1: Trial profile as inactive carriers) was done.
*Patients who did not receive allocated intervention; peg-IFN plus adefovir: lost to follow-up (n=3), patient Plasma hepatitis B virus DNA level was determined by
withdrew consent (n=2), worsening of psychiatric symptoms (n=1); peg-IFN plus tenofovir: lost to follow-up
(n=2), patient withdrew consent (n=1), worsening of psychiatric symptoms (n=1), initiation of lamivudine (n=1),
the COBAS TaqMan assay (F Hoffmann-La Roche, Basel,
no health insurance (n=1); no treatment: lost to follow-up (n=2), moved abroad (n=2) and patient withdrew Switzerland). Serum HBsAg level was quantified by using
consent (n=1). †Reasons for treatment discontinuation are specified in the Results. the Abbott HBsAg Architect assay (Abbott Diagnostics,

578 www.thelancet.com/gastrohep Vol 2 August 2017

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Abbott Park, IL, USA). Qualitative detection of serum

Peg-IFN plus Peg-IFN plus No treatment
HBsAg, antibody to HBsAg (anti-HBs), HBeAg, and adefovir (n=46) tenofovir (n=45) (n=43)
antibody to HBeAg (anti-HBe) was done by an enzyme
Sex
immunoassay (AxSYM; Abbott Laboratories, Abbott Park,
Women 18 (39%) 24 (53%) 17 (40%)
IL, USA). ALT levels were expressed as absolute values
Men 28 (61%) 21 (47%) 26 (60%)
(U/L) or relative to the ULN range. ALT reference values
were 45 U/L for men and 34 U/L for women. For Age (years) 44 (12) 43 (12) 41 (10)

histological assessment of liver biopsies the modified Ethnicity

Ishak scoring system was applied, based on a 0–18 score Caucasian 11 (24%) 14 (31%) 17 (40%)
for necroinflammation and a 0–6 score for fibrosis.33 Asian 7 (15%) 11 (24%) 7 (16%)
African 20 (43%) 11 (24%) 14 (33%)
Outcomes South American 8 (17%) 9 (20%) 5 (12%)
The primary objective was the proportion of patients with IFN naive 43 (93%) 42 (93%) 43 (100%)
HBsAg loss. HBsAg loss was defined as undetectable ALT (U/L) 27 (21–42) 25 (19–30) 30 (21–47)
serum HBsAg by AxSYM (<0·05 IU/mL). The secondary HBV genotype
objectives were the proportion of patients with HBsAg Indeterminable 9 (20%) 11 (24%) 8 (19%)
loss who also had anti-HBs seroconversion (defined as A 11 (24%) 10 (22%) 8 (19%)
anti-HBsAg >10 IU/L), and the identification of predictive B 4 (9%) 3 (7%) 2 (5%)
markers of treatment response. Post-hoc analyses C 2 (4%) 1 (2%) 3 (7%)
assessed HBsAg loss at week 48 and HBsAg decline at D 10 (22%) 13 (29%) 12 (28%)
weeks 48 and 72. Patients were considered to be E 10 (22%) 7 (16%) 9 (21%)
non-responders when not meeting the criteria for HBsAg F 0 0 0
loss or HBsAg decline. Adverse events were assessed at G 0 0 1 (2%)
each study visit through anamnesis and laboratory testing. HBV DNA (log10 IU/mL) 2·65 (1·23) 2·79 (1·03) 2·79 (1·04)
HBV DNA <2000 IU/mL 33 (72%) 28 (62%) 30 (70%)
Statistical analysis HBsAg (log10 IU/mL) 3·21 (0·98) 3·31 (0·76) 3·06 (0·88)
The sample size calculation was based on the primary Fibroscans done 41 (89%) 36 (80%) 40 (93%)
endpoint (HBsAg loss at week 72). The assumed response Value (kPa) 5·0 (1·8) 5·4 (1·8) 5·8 (2·0)
rates were 20% for patients treated in each combination Liver biopsies done 40 (87%) 39 (87%) 19 (44%)
group versus 1% for patients in the control group (receiving Median biopsy length (mm) 16 (12–22) 20 (15–26) 13 (11–15)
no treatment).25 A group sample of 44 patients in the control Median portal fields (n) 11 (7–14) 15 (8–18) 9 (7–14)
group was determined to detect a difference with either of Inflammatory score 2 (2–3) 2 (1–3) 2 (2–2)
the treatment arms at the α level of 0·05 (two-sided Fisher’s Ishak fibrosis score 1 (1–1) 1 (1–1) 1 (1–1)
exact test) with a power of 81%. Assuming a 10% drop-out Steatosis, grade 0 (0–1) 0 (0–1) 0 (0–1)
rate, 50 patients or more were needed in each group.
% HBsAg staining 10% (1–35) 25% (5–50) 10% (1–25)
In the primary analysis, differences in the proportion of
patients with HBsAg loss in each group were compared Data are n (%), median (IQR), or mean (SD). Peg-IFN=peg-interferon-alfa-2a. ALT=alanine aminotransferase.
HBV=hepatitis B virus. HBsAg=hepatitis B surface antigen.
with a Pearson χ² test. In this analysis, a modified intention-
to-treat (mITT) model was applied, including all patients Table 1: Baseline characteristics of the modified intention-to-treat population (n=134)
who received at least one dose of study drugs (treatment
groups) or had at least one study visit (no treatment group).
Patients who prematurely discontinued treatment were version 21. All p values are two sided and values below
scored as non-responders. Patients with missing data were 0·05 were considered statistically significant.
considered non-responders. In the secondary analysis on This study is registered with ClinicalTrials.gov, number
HBsAg decline, only patients who completed 48 weeks of NCT00973219.
treatment and 24 weeks of treatment-free follow-up were
included (per-protocol model). Use of the per-protocol Role of the funding source
population for the secondary analysis allowed avoidance of The funder of the study had no role in study design, data
a high proportion of missing data from patients who collection, data analysis, data interpretation, or writing of
discontinued. the report. The corresponding author had full access to all
Baseline and on-treatment variables were compared the data in the study and had final responsibility for the
between study groups using Welch’s t, Students t, decision to submit for publication.
Mann-Whitney U, Kruskall-Wallis, Pearson’s χ², or
Fisher’s exact tests. The associations between variables as Results
potential predictors of HBsAg loss or HBsAg decline Between Aug 4, 2009, and Oct 17, 2013, 167 patients
were examined by multivariable logistic regression were screened for participation in the study, of whom
analysis. Data were analysed with IBM SPSS Statistics, 151 patients were randomly assigned (nine did not

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plus adefovir group, six in the peg-IFN plus tenofovir group,

Peg-IFN plus Peg-IFN plus No treatment p value
adefovir tenofovir and one in the no treatment group (figure 1). Reasons for
treatment discontinuation in the peg-IFN plus adefovir
Intention to treat (n=134)
group were alcohol-related pancreatitis (n=1; at 6 weeks),
Number of participants 46 45 43 ··
dizziness (n=1; at 8 weeks), hair loss (n=1; at 24 weeks), back
HBsAg loss week 48 1 (2%) 3 (7%) 0 0·171*
pain (n=1; at 38 weeks), and laboratory abnormalities with
HBsAg seroconversion at week 48 1 (2%) 2 (4%) 0 0·370*
concomitant alcohol abuse (n=1; at 42 weeks). Treatment in
HBsAg loss at week 72 2 (4%) 2 (4%) 0 0·377*
the peg-IFN plus tenofovir group was discontinued because
HBsAg seroconversion at week 72 1 (2%) 2 (4%) 0 0·370*
of general interferon-related side-effects (n=2; at 1 and
Per protocol (n=122)
8 weeks), nausea and vomiting (n=1; at 12 weeks), depression
Number of participants 41 39 42 ·· (n=2; at 12 and 23 weeks), and hair loss (n=1; at 24 weeks).
Week 48 HBsAg <10 IU/mL 4 (10%) 6 (15%) 1 (2%) 0·122* One patient in the no treatment group was lost to follow-up
Week 48 >0·5 log10 IU/mL decline 15 (37%) 10 (26%) 0 0·0001* after 6 weeks. Thus, the per-protocol population consisted of
Week 48 >1 log10 IU/mL decline 9 (22%) 8 (21%) 0 0·006* 41 patients in the peg-IFN plus adefovir group, 39 in the peg-
HBsAg decline at week 48 (log10 IU/mL) –0·61 (0·92) –0·61 (0·96) –0·06 (0·18) <0·0001† IFN plus tenofovir group, and 42 in the no treatment group.
Week 72 HBsAg <10 IU/mL 5 (12%) 5 (13%) 2 (5%) 0·393* Patient characteristics are shown in table 1. Patients
Week 72 >0·5 log10 IU/mL decline 13 (32%) 14 (35·9%) 3 (7%) 0·005* were predominantly men (75 [56%] of 134), with a mean
Week 72 >1 log10 IU/mL decline 5 (12%) 6 (15%) 0 0·037* age of 43 years (SD 11). Ethnic background was mixed, as
HBsAg decline at week 72 (log10 IU/mL) –0·53 (0·77) –0·59 (0·85) –0·15 (0·22) 0·001† were hepatitis B virus genotypes. Most patients were
Data are n (%) or mean (SD). Differences in the proportion of patients between groups were compared by Pearson
interferon naive, and none of the patients had received a
χ² test* or Welch’s t test†. Peg-IFN=peg-interferon-alfa-2a. HBsAg=hepatitis B surface antigen. nucleos(t)ide analogue within 6 months of entering the
trial. All patients were HBeAg negative, with a low viral
Table 2: HBsAg response rates
load (mean hepatitis B virus DNA 2·74 log10 IU/mL
[SD 1·10]), normal ALT levels (median 27 U/L [IQR 21–37]),
and minimal liver fibrosis (mean Fibroscan value 5·4 kPa
Univariable analysis Multivariable analysis
[SD 1·9]). The modified hepatic activity index (HAI) was
B (SE) p value Model 1 Model 2 low (median 2 [IQR 2–3]) and none of the patients had
B (SE) p value B (SE) p value evidence of liver cirrhosis; the maximum Ishak’s fibrosis
Female sex –1·27 (0·63) 0·041 ·· ·· ·· ·· score was 3 (or a maximum Fibroscan value of 8·1 kPa in
HBV genotype A –0·29 (0·71) 0·683 ·· ·· ·· ·· patients with no liver biopsy available).
Baseline ALT (log10 U/L) –0·02 (0·02) 0·397 ·· ·· ·· ·· 19 patients required peg-IFN dose reductions (11 in the
Baseline HBV DNA (log10 U/L) –0·23 (0·24) 0·341 ·· ·· ·· ·· peg-IFN plus adefovir group and eight in the peg-IFN
Baseline HBsAg (log10 U/L) –0·40 (0·28) 0·160 ·· ·· ·· ·· plus tenofovir group).
Maximum ALT (log10 U/L) 3·30 (1·12) 0·003 ·· ·· 5·02 (1·31) 0·0001 At week 72, two (4%) patients in the peg-IFN plus
Maximum fold-change ALT 0·38 (0·14) 0·006 0·49 (0·16) 0·002 ·· ··
adefovir group, two (4%) patients in the peg-IFN
Week 12 HBV DNA (log10 U/L) –0·44 (0·52) 0·401 ·· ·· ·· ··
plus teno­­fovir group, and no patients in the no treatment
Week 12 HBsAg (log10 U/L) –0·83 (0·27) 0·002 ·· ·· –1·03 (0·31) 0·001
group had HBsAg loss (p=0·377). Of the four patients
who were HBsAg negative at week 72, one patient was
Week 12 HBsAg decline –3·80 (1·12) 0·001 –3·91 (1·20) 0·001 ·· ··
not peg-IFN naive. Patients with HBsAg loss had
Baseline and early on-treatment variables significantly associated with >1 log HBsAg reduction at week 48 in hepatitis B virus genotype A (one patient in the peg-IFN
univariable logistic regression analysis were assessed in two multivariable models. HBV=hepatitis B virus. ALT=alanine
plus adefovir group), genotype B (two patients in the
aminotransferase. HBsAg=hepatitis B surface antigen. B=regression coefficient.
peg-IFN plus tenofovir group), or indeterminable
Table 3: Multivariable analysis of predictors for on-treatment HBsAg decline (one patient in the peg-IFN plus adefovir group). Three
of four patients had anti-HBs higher than 10 IU/L (n=1
from peg-IFN plus adefovir group and n=2 from peg-IFN
meet inclusion criteria and seven withdrew before plus tenofovir group). HBsAg response rates at week 48
randomisation). and week 72 are shown in table 2. At week 48,
52 participants were assigned to peg-IFN plus adefovir, one patient (2%) in the peg-IFN plus adefovir group,
51  were assigned to peg-IFN plus tenofovir, and 48 were three patients (7%) in the peg-IFN plus tenofovir group,
assigned to no treatment (figure 1). 17 patients did not start and no patients in the no treatment group had HBsAg
allocated intervention (reasons stated in figure 1). Between loss (p=0·171). Of four patients with HBsAg loss at week
Sept 7, 2009, and Dec 12, 2013, 134 patients started 48, three had anti-HBs higher than 10 IU/L. During
intervention or no treatment and were included in the follow-up, the patient without anti-HBs conversion
mITT population: 46 of 52 in the peg-IFN plus adefovir (in the peg-IFN plus tenofovir group, indeterminable
group, 45  of 51 in the peg-IFN plus tenofovir group, and genotype) seroreverted to HBsAg positivity to levels
43 of 48 in the no treatment group. Of 134 patients, around the detection limit at week 72. The course of
12 prematurely discontinued the study: five in the peg-IFN virological and biochemical parameters per patient who

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A HBsAg decline B HBV DNA decline

Dosing Follow-up Dosing Follow-up

0
0
Mean decline, log10IU/mL

–1
–0·5

–2
No therapy
–1·0 Peg-IFN plus adefovir
Peg-IFN plus tenofovir
–3
0 12 24 48 72 0 12 24 48 72
Number analysed
No therapy 42 38 39 41 42 42 30 37 40 42
Peg-IFN plus adefovir 42 41 41 41 41 42 33 41 40 41
Peg-IFN plus tenofovir 40 39 40 39 39 40 28 39 40 39

C D
Dosing Follow-up Dosing Follow-up

0
0
Mean decline, log10IU/mL

–1 –1

–2 –2
No therapy
>1 log decline week 48
<1 log decline week 48

0 12 24 48 72 0 12 24 48 72
Week Week
Number analysed
No therapy 42 38 39 41 42 42 30 37 40 41
>1 log decline week 48 17 17 17 17 17 17 11 17 16 17
<1 log decline week 48 63 63 62 63 63 63 50 63 62 63

Figure 2: Post-hoc analysis of HBsAg and HBV DNA decline according to treatment group
Mean log10 IU/mL decline in HBsAg (A) and HBV DNA (B) compared with baseline according to treatment group. Mean log10 IU/mL decline in HBsAg (C) and HBV DNA
(D) compared with baseline in patients with an on-treatment HBsAg decline of >1 log10 IU/mL. Data are for the per-protocol population. HBsAg=hepatitis B surface
antigen. HBV=hepatitis B virus.

achieved HBsAg loss during treatment, or follow-up, or
both (n=5), is shown in the appendix (p 8).
In a post-hoc per-protocol analysis, mean HBsAg level
had declined significantly in all study groups at week 48
compared with at baseline: mean –0·61 log10 IU/mL
(SD 0·92) reduction for the peg-IFN plus adefovir group
(p=0·0001), –0·61 log10 IU/mL (0·96) reduction for
the peg-IFN plus tenofovir group (p=0·0003), and
–0·06 log10 IU/mL (0·18) reduction for the no treatment
group (p=0·042; table 3). No difference in HBsAg decline
was noted between the two treatment groups (p=0·990).
However, HBsAg declined more strongly in the peg-IFN
plus adefovir group (p=0·0005) and the peg-IFN plus
tenofovir group (p=0·001) than in the no treatment
group (figure 2A, B). An HBsAg decline of more than
1·0 log10IU/mL was noted in 17 (21%) treated patients
(nine [22%] in the peg-IFN plus adefovir group and eight
[21%] in the peg-IFN plus tenofovir group), but in none of
the no treatment patients (p=0·001).
During follow-up, HBsAg levels remained significantly
lower than pretreatment levels in all groups: mean
reduction –0·53 log10 IU/mL (SD 0·77) at week 72 for the
peg-IFN plus adefovir group (p<0·0001), mean reduction
–0·59 (0·85) log10 IU/mL reduction at week 72 for the
peg-IFN plus tenofovir group (p=0·0001), and mean
reduction –0·15 (0·22) log10 IU/mL reduction at week 72
for the no treatment group (p<0·0001; table 3; figure 2A,B).
Despite the slight increase in mean HBsAg levels during
treatment-free follow-up of treated patients, the decline in
HBsAg remained significantly larger in the peg-IFN plus
adefovir group (p=0·004) and peg-IFN plus tenofovir
group (p=0·004) than in the no treatment group.
The increase in HBsAg level during the treatment-
free follow-up (HBsAg rebound) was particularly
pronounced in patients with an HBsAg decline of
more than 1·0 log10 IU/mL at week 48, and 13 patients in
this group remained HBsAg-positive at this timepoint
(figure 2C). Of these, 12 (92%) of 13 had an HBsAg

www.thelancet.com/gastrohep Vol 2 August 2017 581

 Articles

between patients with or without more than 1·0 log10 IU/mL

Peg-IFN Peg-IFN No
plus plus treatment HBsAg decline (figure 2D).
adefovir tenofovir (n=43) Baseline and early on-treatment variables were
(n=46) (n=45) compared between treated patients with more than
Serious adverse events* 2 (4%) 3 (7%) 3 (7%) 1·0 log10 IU/mL reduction in HBsAg level at week 48 and
General those with less than 1·0 log10 IU/mL reduction (appendix
Fatigue 23 (50%) 28 (62%) 2 (5%) p 9). Because the decline in HBsAg between the peg-IFN
Headache 18 (39%) 19 (42%) 2 (5%) plus adefovir group and peg-IFN plus tenofovir group
Fever 16 (35%) 18 (40%) 0 were similar, these were combined for this analysis.
Myalgia 15 (33%) 16 (36%) 0 Baseline HBsAg levels were not significantly lower in
Other flu-like symptoms 9 (20%) 9 (20%) 2 (5%) treated patients with a strong HBsAg decline. However,
Dizziness 8 (17%) 7 (16%) 5 (12%) significant predictors of HBsAg decline in univariable
Dyspnoea 3 (7%) 1 (2%) 0 analysis were male sex (p=0·041), higher maximum
Back pain 2 (4%) 7 (16%) 3 (7%) on-treatment ALT level (p=0·003), and lower week 12
Cough 2 (4%) 3 (7%) 0 HBsAg level (p=0·002). Both on-treatment ALT increase
Change in menstrual pattern 4 (9%) 4 (9%) 1 (2%)
and HBsAg level at week 12 were independent predictors
Digestive tract
of HBsAg decline at week 48, in different multivariable
Abdominal pain 4 (30%) 10 (22%) 7 (16%)
logistic regression models (table 3).
Most patients had a baseline hepatitis B virus DNA
Change in stool consistency 10 (22%) 6 (13%) 1 (2%)
level less than 2000 IU/mL (33 [72%] patients in the
Nausea 17 (37%) 3 (7%) 1 (2%)
peg-IFN plus adefovir group, 28 [62%] in the peg-IFN
Loss of appetite 7 (15%) 13 (29%) 0
plus tenofovir group, and 30 [70%] in the no treatment
Dysgeusia 4 (9%) 3 (7%) 1 (2%)
group; table 1), including all four patients with HBsAg
Gingivitis 1 (2%) 5 (11%) 0
loss at week 72. In this subgroup analysis, HBsAg
Dermatological
declined more on average in the peg-IFN plus adefovir
Skin rash 7 (15%) 9 (20%) 1 (2%)
group (p=0·010) and peg-IFN plus tenofovir group
Pruritus 9 (20%) 7 (16%) 0
(p=0·003) than in the no treatment group at week 72
Alopecia 8 (17%) 3 (7%) 0
(appendix p 10).
Dry mucous membranes 3 (7%) 5 (11%) 0
The most frequent adverse events (>30%) were fatigue,
Dry skin 2 (4%) 4 (9%) 0
headache, fever, and myalgia, which were attributed to
Psychiatric peg-IFN dosing (table 4). Two (4%) serious adverse events
Depression, including mood 10 (22%) 12 (27%) 1 (2%) were reported in the peg-IFN plus adefovir group
changes
(admission to hospital for alcohol-related pancreatitis
Concentration problems 2 (4%) 6 (13%) 0
[week 6; n=1] and pregnancy, which was electively aborted
Insomnia 5 (11%) 5 (11%) 0
[week 9; n=1]), three (7%) in the peg-IFN plus tenofovir
Laboratory
group (admission to hospital after a suicide attempt
Anaemia (<6·0 mmol/L) 4 (9%) 7 (16%) 0
during a severe depression [week 23; n=1], admission to
Neutropenia (<0·75 × 10⁹ cells/L) 15 (33%) 7 (16%) 1 (2%)
hospital for abdominal pain [week 2; n=1], and an elective
Thrombocytopenia 5 (11%) 0 0
laminectomy [week 40; n=1]), and three (7%) in the no
(<75 × 10⁹ cells/L)
treatment group (admission to hospital for septic arthritis
Thyroid abnormalities 2 (4%) 5 (11%) 0
[week 72; n=1], endocarditis [week 5; n=1], and
On-treatment increase in ALT† 24 (52%) 24 (53%) 3 (7%)
hyperthyroidism [week 20; n=1]). During treatment,
Off-treatment increase in ALT† 4 (9%) 3 (7%) 4 (9%)
48 (53%) of 91 patients in the intervention groups
Peg-IFN=peg-interferon-alfa-2a. ALT=alanine aminotransferase. *Serious adverse (24 [52%] of 46 in the peg-IFN plus adefovir group and
events: peg-IFN plus adefovir: admission to hospital for alcohol-related pancreatitis 24 [53%] of 45 in the peg-IFN plus tenofovir group) had
(week 6; n=1), pregnancy, which was electively aborted (week 9; n=1); peg-IFN plus
tenofovir: admission to hospital after a suicide attempt during a severe depression
ALT levels greater than 2 × ULN compared with three (7%)
(week 23; n=1), admission to hospital for abdominal pain (week 2; n=1), and an of 43 patients in the no treatment group (p<0·0001).
elective laminectomy (week 40; n=1); no treatment: admission to hospital for septic During follow-up, the rate of ALT levels greater than
arthritis (week 72), endocarditis (week 5), and hyperthyroidism (week 20). †Increase in
ALT of more than 2 × the upper limit of normal (45 U/L for men, 34 U/L for women).
2 × ULN was similar in the intervention groups (four [9%]
of 46 in the peg-IFN plus adefovir group and three [7%] of
Table 4: Overview of adverse events in the modified intention-to-treat 45 in the peg-IFN plus tenofovir group) versus the no
population treatment group (four [9%] of 43; p=0·751).

rebound during the treatment-free follow-up (mean Discussion

increase 0·84 log10 IU/mL at week 72 compared with Our results show no differences in functional cure, defined
week 48). The decline in hepatitis B virus DNA level and as HBsAg loss with or without formation of antibodies
subsequent hepatitis B virus DNA rebound did not differ against anti-HBs between the treated and untreated patient

582 www.thelancet.com/gastrohep Vol 2 August 2017


groups. Our study is the first, to our knowledge, to analyse
the effect of antiviral treatment in patients with chronic
hepatitis B with a low viral load. The rationale for the study
was based on the hypothesis that combining peg-IFN with
a nucleos(t)ide analogue results in increased rates of
HBsAg loss and anti-HBs seroconversion. Due to the
relatively high rate of HBsAg loss in our cohort of patients
with chronic hepatitis B with a high viral load treated with
peg-IFN and adefovir, particularly in HBeAg-negative
patients with low HBsAg levels,25 we explored the effect of
this combination. In our previous study in patients with a
high viral load, HBsAg loss was associated with low
baseline HBsAg levels. Because HBeAg-negative patients
with a low viral load generally have low HBsAg levels, we
hypothesised that this patient group could benefit from
combination treatment. Furthermore, we hypothesised
that immuno­ modulation might be effective in patients
with a low viral load, because they have residual hepatitis B
virus-specific T-cell activity, possibly sensitive to a boost
with immuno­modulatory agents. However, despite the low
baseline HBsAg levels26 and the significant residual T-cell
function in this group,29,30 combination therapy did not lead
to the anticipated increased levels of functional cure.
Because the definition of patients with a low viral load
(inactive carriers) changed over time, a subanalysis was
done, taking into account only those patients with
hepatitis B virus DNA less than 2000 IU/mL. Although all
four patients with HBsAg loss were included in the group
of patients with hepatitis B with a virus viral load less than
2000 IU/mL, no statistical difference was noted between
treated and untreated patients with hepatitis B with virus
DNA levels less than 2000 IU/mL.
When quantitative HBsAg level was taken into account,
the decline in HBsAg was significantly greater in the
treatment groups than in the no treatment group. This is
in line with the findings of Bourlière and colleagues28
who reported a significant decline in HBsAg levels in
nucleos(t)ide analogue suppressed patients treated with
peg-IFN add-on compared with no peg-IFN add-on,
while no difference in functional cure was observed
between the groups. Although the exact mechanism
might differ, boosting the immune system in patients
with a low viral load and residual T-cell function does not
seem to be effective in acquiring functional cure in either
of these patient groups. Furthermore, Bourlière and
colleagues28 showed no increase in the proportion of
patients with HBsAg loss during long-term follow-up.
Whether a strong HBsAg decline can lead to future
HBsAg loss in patients with chronic hepatitis B with a
low viral load will be determined in our subsequent
5-year follow-up study. Here, the outcome of HBsAg
decline was associated with an on-treatment ALT increase
and with low HBsAg level at week 12, indicating that the
observed decline occurred early. Association of HBsAg
loss and ALT flare has previously been observed in
patients treated with combination therapy.24 However,
because of low numbers and a rebound in HBsAg levels
www.thelancet.com/gastrohep Vol 2 August 2017 583
Articles
at week 72 in a proportion of patients, these results
should be interpreted with caution. Furthermore, this
study was done in a heterogeneous population. Although
this accurately reflects the mixed European population,
associations with ethnicity or hepatitis B virus genotype
could have been underestimated.
Peg-IFN treatment is known to be associated with
severe side-effects and safety issues. In our study, despite
initial consent, 17 patients withdrew consent after
randomisation. Even though these patients were equally
distributed among the three randomisation groups, the
acceptability rate of peg-IFN treatment was low, and
similar to previous studies.28 Furthermore, judging
by the high rate of adverse events and treatment
discontinuation, peg-IFN was not well tolerated.
In this study, we have included two groups of combination
therapy. In addition to its antiviral effect, adefovir has been
shown to enhance innate immune functions, suggesting a
possible synergistic effect when combined with peg-IFN.34,35
In our previous study, peg-IFN plus adefovir combination
therapy resulted in high rates of HBsAg loss. Because
adefovir has largely been replaced by tenofovir, which has a
similar mechanism of action but is more potent in
suppressing hepatitis B virus DNA, we also included a
group with peg-IFN plus tenofovir combination.31 Because
there are no previous data for treatment of patients with a
low viral load, comparison of our results with peg-IFN or
nucleos(t)ide analogue monotherapy data is not possible.
However, we aimed to investigate the difference between
combination therapy and no treatment. Based on natural
history reports, HBsAg loss in untreated patients with
chronic hepatitis B is 0·8–1·0% per year, which makes a
control group indispensable.36,37
In conclusion, to our knowledge, this is the first treatment
intervention study done in patients with chronic hepatitis B
with a low viral load. Although HBsAg clearance rates did
not differ significantly between the treatment and control
groups, HBsAg decline was significantly greater in patients
treated with peg-IFN and nucleos(t)ide analogue
combination therapy at week 72 than in untreated patients.
Although follow-up data will show whether a strong
HBsAg decline could lead to higher rates of HBsAg loss in
the treated patients in the long term, our findings do not
support the use of combination treatment with peg-IFN
and nucleos(t)ide analogue in patients with chronic
hepatitis B with a low viral load. Pursuing functional cure
for patients with chronic hepatitis B with a low viral load is
supported by various benefits for the patient, including the
discontinuation of close monitoring, a decreased risks of
hepatitis B virus reactivation, and overcoming a stigma.
The develop­ ment of new antiviral hepatitis B virus
compounds gives hope for future treatment for this large
group of patients with chronic hepatitis B.
Contributors
AdN, RBT, and HWR designed the original study protocol. AdN, LJ, FS,
and SBW enrolled patients, did the clinical trial, designed the database,
and analysed the data. AdN, LJ, and FS wrote the report. All remaining
 Articles
authors critically revised the report. SDK, SW, and CMJdN assisted in
patient recruitment. HLZ, RM, and MK supervised virological analyses.
FS and JV did the histological analyses. UB and HWR assisted in patient
inclusion and supervised clinical activities.
Acknowledgments
We would like to express great appreciation to Martine W Peters,
Jeltje Helder, and Hadassa Heidsieck for patient follow-up and sampling.
We thank Marjan J Sinnige for handling patient material and doing
additional immunological analyses, as well as Meike H van der Ree for
critical revision of the manuscript.
Declaration of interests
HWR is a consultant for AbbVie, BMS, Boehringer Ingelheim, ENYO,
Gilead Sciences, Janssen-Cilag, Merck, PRA Health Sciences, Regulus,
Roche, and R-Pharm, and received grant and research support from
AbbVie, Bristol Myers Squibb, Boehringer Ingelheim, ENYO, Gilead
Sciences, Janssen-Cilag, Merck, PRA Health Sciences, Regulus, Replicor,
and Roche. RBT provided sponsored lectures for Bristol Myers Squibb
and Gilead Sciences. SBW provided sponsored lectures for Gilead, Roche,
BMS, and Janssen. All other authors declare no competing interests.
References
1 Ott JJ, Stevens GA, Groeger J, Wiersma ST. Global epidemiology of
hepatitis B virus infection: new estimates of age-specific HBsAg
seroprevalence and endemicity. Vaccine 2012; 30: 2212–29.
2 European Association for the Study of the Liver. EASL clinical
practice guidelines: management of chronic hepatitis B virus
infection. J Hepatol 2012; 57: 167–85.
3 Lok ASF, McMahon BJ. Chronic hepatitis B: update 2009.
Hepatology 2009; 50: 661–62.
4 Chen C, Lee W, Yang H, et al. Changes in serum levels of HBV
DNA and alanine aminotransferase determine risk for
hepatocellular carcinoma. Gastroenterology 2011; 141: 1240–48.
5 Iloeje U, Yang H, Su J, Jen C-L, You S, Chen C. Predicting cirrhosis
risk based on the level of circulating hepatitis B viral load.
Gastroenterology 2006; 130: 678–86.
6 Tai D-I, Lin S-M, Sheen I-S, Chu C-M, Lin D-Y, Liaw Y-F.
Long-term outcome of hepatitis B e antigen-negative hepatitis B
surface antigen carriers in relation to changes of alanine
aminotransferase levels over time. Hepatology 2009; 49: 1859–67.
7 Chen C-J, Yang H-I, Su J, et al. Risk of hepatocellular carcinoma
across a biological gradient of serum hepatitis B virus DNA level.
JAMA 2006; 295: 65–73.
8 Kumar M, Sarin SK, Hissar S, et al. Virologic and histologic features
of chronic hepatitis B virus-infected asymptomatic patients with
persistently normal ALT. Gastroenterology 2008; 134: 1376–84.
9 Evans AA, Chen G, Ross EA, Shen F, Lin W, London WT.
Eight-year follow-up of the 90,000-person Haimen City cohort:
I. Hepatocellular carcinoma mortality, risk factors, and gender
differences. Cancer Epidemiol Biomarkers Prev 2002; 11: 369–76.
10 Beasley RP. Hepatitis B virus. The major etiology of hepatocellular
carcinoma. Cancer 1988; 61: 1942–56.
11 Invernizzi F, Viganò M, Grossi G, Lampertico P. The prognosis and
management of inactive HBV carriers. Liver Int 2016; 36: 100–04.
12 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.
13 Papatheodoridis GV, Chrysanthos N, Hadziyannis E, Cholongitas E,
Manesis EK. Longitudinal changes in serum HBV DNA levels and
predictors of progression during the natural course of
HBeAg-negative chronic hepatitis B virus infection. J Viral Hepat
2008; 15: 434–41.
14 Perrillo RP, Gish R, Falck-Ytter YT. Exam 3: American
Gastroenterological Association Institute Technical Review on
Prevention and Treatment of Hepatitis B Virus Reactivation During
Immunosuppressive Drug Therapy. Gastroenterology 2015; 148: e16–17.
15 Chen Y, Sheen IS, Chu C, Liaw Y. Prognosis following
spontaneous hbsag seroclearance in chronic hepatitis B patients
with or without concurrent infection. Gastroenterology 2002;
123: 1084–89.
16 Yuen MF, Wong DKH, Sablon E, et al. HBsAg seroclearance in
chronic hepatitis B in the Chinese: virological, histological, and
clinical aspects. Hepatology 2004; 39: 1694–701.
584 www.thelancet.com/gastrohep Vol 2 August 2017
17 Durantel D, Zoulim F. New antiviral targets for innovative treatment
concepts for hepatitis B virus and hepatitis delta virus. J Hepatol 2016;
64: S117–31.
18 Moucari R, Mackiewicz V, Lada O, et al. Early serum HBsAg drop:
a strong predictor of sustained virological response to pegylated
interferon alfa-2a in HBeAg-negative patients. Hepatology 2009;
49: 1151–57.
19 Janssen HLA, van Zonneveld M, Senturk H, et al. Pegylated interferon
alfa-2b alone or in combination with lamivudine for HBeAg-positive
chronic hepatitis B: a randomised trial. Lancet 2005; 365: 123–29.
20 Marcellin P, Bonino F, Lau GKK, et al. Sustained response of
hepatitis B e antigen-negative patients 3 years after treatment with
peginterferon Alfa-2a. Gastroenterology 2009; 136: 2169–79.
21 Viganò M, Invernizzi F, Grossi G, Lampertico P. Review article:
the potential of interferon and nucleos(t)ide analogue combination
therapy in chronic hepatitis B infection. Aliment Pharmacol Ther
2016; 44: 653–61.
22 Thimme R, Dandri M. Dissecting the divergent effects of
interferon-alpha on immune cells: time to rethink combination
therapy in chronic hepatitis B? J Hepatol 2013; 58: 205–09.
23 Tan AT, Hoang LT, Chin D, et al. Reduction of HBV replication
prolongs the early immunological response to IFNα therapy.
J Hepatol 2014; 60: 54–61.
24 Marcellin P, Ahn SH, Ma X, et al. Combination of tenofovir disoproxil
fumarate and peginterferon α-2a increases loss of hepatitis B surface
antigen in patients with chronic hepatitis B. Gastroenterology 2016;
150: 134–44.
25 Takkenberg RB, Jansen L, de Niet A, et al. Baseline hepatitis B
surface antigen (HBsAg) as predictor of sustained HBsAg loss in
chronic hepatitis B patients treated with peginterferon alfa-2a and
adefovir. Antivir Ther 2013; 18: 895–904.
26 Jaroszewicz J, Calle Serrano B, Wursthorn K, et al. Hepatitis B surface
antigen (HBsAg) levels in the natural history of hepatitis B virus
(HBV)-infection: a European perspective. J Hepatol 2010; 52: 514–22.
27 Brunetto MR, Oliveri F, Colombatto P, et al. Hepatitis B surface
antigen serum levels help to distinguish active from inactive hepatitis B
virus genotype D carriers. Gastroenterology 2010; 139: 483–90.
28 Bourlière M, Rabiega P, Ganne-Carrie N, et al. Effect on HBs
antigen clearance of addition of pegylated interferon alfa-2a to
nucleos(t)ide analogue therapy versus nucleos(t)ide analogue
therapy alone in patients with HBe antigen-negative chronic
hepatitis B and sustained undetectable plasma hepatitis B virus
DNA: a randomised, controlled, open-label trial.
Lancet Gastroenterol Hepatol 2017; 2: 177–88.
29 Boni C, Fisicaro P, Valdatta C, et al. Characterization of hepatitis B
virus (HBV)-specific T-cell dysfunction in chronic HBV infection.
J Virol 2007; 81: 4215–25.
30 de Niet A, Stelma F, Jansen L, et al. Restoration of T cell function in
chronic hepatitis B patients upon treatment with interferon based
combination therapy. J Hepatol 2015; 64: 539–46.
31 Marcellin P, Heathcote EJ, Buti M, et al. Tenofovir disoproxil
fumarate versus adefovir dipivoxil for chronic hepatitis B.
N Engl J Med 2008; 359: 2442–55.
32 Buster EHCJ, van Erpecum KJ, Schalm SW, et al. Treatment of
chronic hepatitis C virus infection—Dutch national guidelines.
Neth J Med 2008; 66: 292–306.
33 Ishak K, Baptista A, Bianchi L, et al. Histological grading and
staging of chronic hepatitis. J Hepatol 1995; 22: 696–99.
34 Murata K, Asano M, Matsumoto A, et al. Induction of IFN-λ3 as an
additional effect of nucleotide, not nucleoside, analogues: a new
potential target for HBV infection. Gut 2016; published online Oct 26.
DOI:10.1136/gutjnl-2016-312653.
35 Villani N, Caliò R, Balestra E, et al. 9-(2-Phosphonylmethoxyethyl)
adenine increases the survival of influenza virus-infected mice by an
enhancement of the immune system. Antiviral Res 1994; 25: 81–89.
36 Alward W, McMahon B, Hall D, Heyward W, Francis D, Bender T.
The long-term serological course of asymptomatic hepatitis B virus
carriers and the development of primary hepatocellular carcinoma.
J Infect Dis 1985; 151: 604–09.
37 Liaw Y, Sheen I, Chen T, Chu C, Pao C. Incidence, determinants
and significance of delayed clearance of serum HBsAg in chronic
hepatitis B virus infection: a prospective study. Hepatology 1991;
13: 627–31.
Supplementary appendix
This appendix formed part of the original submission and has been peer reviewed.
We post it as supplied by the authors.

Supplement to: de Niet A, Jansen L, Stelma F, et al. Peg-interferon plus nucleotide

analogue treatment versus no treatment in patients with chronic hepatitis B with a low
viral load: a randomised controlled, open-label trial. Lancet Gastroenterol Hepatol 2017;
published online May 15. http://dx.doi.org/10.1016/S2468-1253(17)30083-3.
Appendix to:
Peg-interferon plus nucleotide analogue treatment versus
no treatment in patients with chronic hepatitis B with a low
viral load: a randomised controlled, open-label, trial.
Supplementary Information S1: Detailed study design and study population

Study design
This is a three arm open-label prospective randomized controlled trial. (n=150) The trial is designed to compare
the efficacy of treatment with Peg-IFN and ADF or Peg-IFN and TDF versus no treatment for clearance of the
HBsAg in chronic hepatitis B patients with low viral load.

Patients will be enrolled into the study after assessment of eligibility. Group 1 will consist of patients treated
with Peg-IFN 180µg once weekly and ADF 10mg once daily (standard doses), group 2 will consist of patients
treated with Peg-IFN 180µg once weekly and TDF 300mg (equivalent to 245 mg tenofovir disoproxil) once daily
(standard doses) and group 3 will consist of untreated patients as negative controls.

The trial consists of a screening period of approximately 4 weeks, a 48 week treatment period, a 24 week
follow-up period and a long term follow up period of 5 year. A schematic overview of the study design is
presented in table 1 and 2 (see appendix).

150 patients will be randomized in a 50:50:50 ratio to one of the treatment groups. Randomization will be
stratified to genotype A to optimize balance between treatment groups. All patients will receive medication
for the period of 48 weeks. For enrolment into the study a liver biopsy at time of enrolment is compulsory for
patients selected in the treatment groups. For enrolment of patients that are selected in the non-treatment
group a liver biopsy is optional but not compulsory for participation. In patients selected in the treatment
group a second liver biopsy at week 48 is recommended for evaluation of therapeutic responses and to guide
any further possible therapy. Due to the burden of undergoing a liver biopsy, the second liver biopsy is not
compulsory to take part into the study. Patients that are selected in the non treatment group will not undergo
a biopsy at week 48. Patients will be clinically monitored intensively according to the assessment schedule
(table 1). In the long term follow up period patients will be clinically monitored each year.

Study population
Population (base)
Patients will be enrolled into the study after assessment of eligibility. Target population will consist of adult
patients (≥ 18 years) with chronic hepatitis B ( HBsAg positivity ≥ 6 months), HBeAg negativity, HBV DNA <
*
20,000 IU/ml and ALT < 5 upper limit of normal.
Inclusion criteria
1. Male and female patients > 18 and ≤ 70 years of age
2. Positive HBsAg for more than 6 months.
3. Negative for HBeAg for more than 6 months.
4. HBV DNA < 20,000 IU/ml
5. Patients with chronic hepatitis B who are either naive to antiviral treatment, or have received either
interferon (IFN) or nucleoside/nucleotide analogues in the past but are still positive for HBsAg.
*
6. Serum ALT ≤ 5 ULN as determined by two values taken >14 days apart during the six months before the
first dose of study drug with at least one of the determinations obtained during the screening period.
7. Negative urine or serum pregnancy test (for women of childbearing potential) documented within the 24-
hour period prior to the first dose of test drug.
Exclusion criteria
1. Patients co-infected with HCV, HIV or who have decompensated liver disease, hepatocellular carcinoma,
significant cardiac disease, significant renal disease, seizure disorders or severe retinopathy.

1
2. Patients who have received nucleos(t)ide analogues for their chronic hepatitis B within 6 weeks before
enrollment or have received Peg-IFN within 3 months before enrollment.
3. Patients must not have received any other systemic anti-viral, anti-neoplastic or immuno-modulatory
treatment (including supraphysiologic doses of steroids or radiation) ≤3 months prior to the first dose of
study drug or the expectation that such treatment will be needed at any time during the study.
4. Positive test at screening for anti-HAV IgM, anti-HIV, HCV RNA. (Patients that have cleared the hepatitis C virus can
be included in the study)
5. Patients who are expected to need systemic antiviral therapy other than that provided by the study at any
time during their participation in the study are also excluded. Exception: patients who have had a limited
(≤7 day) course of acyclovir for herpetic lesions more than 1 month prior to the first administration of test
drug are not excluded.
6. Evidence of decompensated liver disease (Child pugh B-C)
7. Serum total bilirubin > twice the upper limit of normal at screening
8. History or other evidence of bleeding from esophageal varices or other conditions consistent with
decompensated liver disease.
9. History or other evidence of a medical condition associated with chronic liver disease other than HBV (e.g.,
hemochromatosis, autoimmune hepatitis, metabolic liver diseases including Wilson’s disease and alfa1-
antitrypsin deficiency, alcoholic liver disease, toxin exposures, thalassemia).
10. Women with ongoing pregnancy or who are breast feeding.
3 3
11. Neutrophil count <1500 cells/mm or platelet count <80,000 cells/mm at screening.
12. Hemoglobin < 7.1 mmol/L (< 11.5 g/dL) for females and < 7.8 mmol/L (< 12.5 g/dL) for men at screening.
13. Serum creatinine level >1.5 times the upper limit of normal at screening.
14. Unstable ongoing severe psychiatric disease, especially depression (stable patients can be included).
15. History of immunologically mediated disease (e.g., inflammatory bowel disease, idiopathic
thrombocytopenic purpura, lupus erythematosus, autoimmune hemolytic anemia, scleroderma, severe
psoriasis, rheumatoid arthritis).
16. History or other evidence of chronic pulmonary and cardiac disease associated with functional limitation.
Severe cardiac disease (e.g., NYHA Functional Class III or IV, myocardial infarction within 6 months,
ventricular tachyarrhythmias requiring ongoing treatment, unstable angina or other significant
cardiovascular diseases).
17. History of a severe seizure disorder or current anticonvulsant use and clinically unstable disease.
18. Evidence of an active or suspected cancer or a history of malignancy where the risk of recurrence is ≥20%
within 5 years. Patients with a lesion suspicious of hepatic malignancy on a screening imaging study will
only be eligible if the likelihood of carcinoma is ≤10% following an appropriate evaluation.
19. Major organ transplantation. (patients with skin, cornea or bone transplantation are allowed to be
included into the study)
20. Thyroid disease with thyroid function poorly controlled on prescribed medications. Patients with elevated
thyroid stimulating hormone or T4 concentrations, with elevation of antibodies to thyroid peroxidase and
any clinical manifestations of thyroid disease that are not stable on prescribed medication are excluded.
Stable patients can be included.
21. History or other evidence of severe retinopathy (e.g. CMV retinitis, macula degeneration) or clinically
relevant ophthalmological disorder due to diabetes mellitus or hypertension.
22. Inability or unwillingness to provide informed consent or abide by the requirements of the study.
23. History or other evidence of severe illness or any other conditions which would make the patient, in the
opinion of the investigator, unsuitable for the study.
24. Patients with a value of alfa-fetoprotein >100 ng/mL are excluded, unless stability (less than 10% increase)
has been documented over at least the previous 3 months.
25. Evidence of current hard drug(s) (i.e. cannabis products are allowed) and/or alcohol abuse (20g/day for
women and 30g/day for men).
26. Patients included in another trial or having been given investigational drugs within 12 weeks prior to
screening.

2
Supplementary Table S2: Screening Assessments
Medical History And Physical Includes body weight, vital signs
Examination
Eye examination Eye examination by an ophthalmologist at screening will be done on indication.Patients with
preexisting ophthalmologic disorders (eg, diabetic or hypertensive retinopathy) should receive
periodic ophthalmologic exams during Peg-IFN therapy. Peg-IFN treatment should be
discontinued in patients who develop new or worsening ophthalmologic disorders.
Clinical Chemistry ASAT, ALAT, bilirubin, creatinine, urea, sodium, chloride, potassium, calcium, phosphorus,
prothrombin time, alkaline phosphatase, total protein, albumin, BUN, uric acid, cholesterol,
triglycerides, glucose
Hematology Leukocyte count, differential WBC, red blood count, platelets
Immunology and Special Chemistry HBeAg, anti-HBe, HBsAg, anti-HBs
anti-HIV, anti-HCV, anti-HDV, Alfa-fetoprotein, ceruloplasmin,
alfa1-antitrypsin, ferritin
Only for patients at risk without previous data being available: AMA, ANA, ASMA, thyroid
peroxidase antibodies.
Virology Quantitative HBV-DNA measurement
Thyroid Function Tests TSH, free T4
Urinalysis Dipstick with subsequent microscopic evaluation if positive for hemoglobin

Chest X-Ray Will be done during screening (Not necessary if (1) a chest X-ray available to the investigator has
been obtained within the past 12 months and (2) the patient’s pulmonary disease has remained
clinically stable.)
Electrocardiogram Only for anyone with a history of pre-existing cardiac disease.
Liver Biopsy (a) Performed at start of the study and repeated after 48 weeks.

(b)
Fibroscan Performed at start of the study and repeated after 48 weeks.
Liver Imaging (ultrasound) Performed at screening or start of the study, repeated after 48 weeks and on indication.
Liver Imaging (CT or MRI) Only for patients suspected to have hepatic neoplasia.
HCG Pregnancy Test For women of childbearing potential a negative urine (or serum) HCG test needs to be
documented within 24 hours prior to the first dose.

(a) When a participant has objections against a second liver biopsy at week 48, he or she can refuse it. The
participant will than not be excluded from the study.
(b) A fibroscan measures the liver stiffness, e

3
Supplementary Information S3: Schedule of Assessments

Assessment/ Procedure Screening Study treatment Period Peg-INF and ADF, Peg-IFN and tenofovir or no treatment Follow-up
(weeks (weeks) (weeks)

Study weeks -4 to 0 B Day 3 1 2 4 6 8 12 18 24 30 36 42 48 2 4 8 12 16 20 24

Informed consent, medical history X

Physical examination X X X X X X X X X X X X X

Liver biopsy (preceding 12 months) X X

Urine or serum pregnancy test a X x X X X X X X X X X X X X X X

Chest X-ray, selected patients b X

Ultrasound of the liver X

Fibroscan X X

CT or MRI, selected patients c

Electrocardiogram, selected patients d X

Anti-HAV IgM, anti-HIV, anti-HCV, X

HCV-RNA, anti-HDV

Ophthalmoloic examination e (on indication) X

4
Assessment/ Procedure Long term Follow-up
(years)
Years 1 2 3 4 5
Physical examination X X X X X
g
Complete hematology with differential WBC, and platelets
(on indication)
Chemistry 1 g X X X X X
g
Chemistry 2 (on indication)
Chemistry 3 (on indication)
Fibroscan f X X X
HBeAg, anti-HBe (frozen for central processing)HBV-DNA X X X X X
(frozen for central processing) iHBsAg, anti-HBs (frozen for
central processing)

Ultrasound, CT or MRI (on indication) c

PBMC’s (frozen for central processing) X X X X X
TM RNA (k)
Paxgene X X X X X
Plasmatube (proteomics profiling) (l) X X X X X
(m)
Quality of life X X X X X

5
Supplementary Information S3 (cont.): Schedule of Assessments

Assessment / Procedure Screening Study treatment Period Follow-up

(weeks) (weeks) (week)
Study weeks -4 to 0 B day 3 1 2 4 6 8 12 18 24 30 36 42 48a 50a 52a 56a 60a 64 68 72a
g
Complete hematology with differential WBC, and platelets X X X X X X X X X X X X X X X X X X X X
Chemistry 1 g X X X X X X X X X X X X X X X X X X
Chemistry 2 g X X X X X X X X X
Chemistry 3 X X X
Urinalysis, dipstick (on indication) g X X X X X X X X X X X
Ceruloplasmin, alfa-1-antitrypsin X
Alfa-fetoprotein X X X X
AMA, ANA, ASMA, thyroid peroxidase antibodies, X X
selected patients h
HBeAg, anti-HBe (frozen for central processing) X X X
HBsAg, anti-HBs (frozen for central processing) X X X X X X X X X X X X X X X X X X X X X X
HBV-DNA (frozen for central processing) i X X X X X X X X X X X X X X X X X X X X X X
Alpha-2 macro globulin, Haptoglobin, Apolipoprotein A1 X
Viral sequencing X X X X X X X X X X X X X X X X X X X X
PBMC’s (frozen for central processing) X X X X X X X X X X X X
PAXgeneTM DNA (j) (human genomics) X
PAXgeneTM RNA (k) (human genomics) X X X X X X
Plasma tube (proteomics profiling ) (l) X X X X X X
Adverse events X X X X X X X X X X X X X X X X X X X X X
(m)
Quality of life X X X X X

Chemistry 1 : ASAT, ALAT,

Chemistry 2: sodium, chloride, potassium, bilirubin, creatinine, urea, prothrombin time, APTT, antitrombine III, alkaline phosphatase, calcium, phosphorus, total protein,
albumin, uric acid, TSH, free T4 and glucose.
Chemistry 3: cholesterol, triglycerides, sober glucose

B: baseline

(a) For females of child bearing potential only, a pregnancy test will be performed within 24 hours prior to first dose. A pregnancy test must be done at any time a
secondary amenorrhea of more than 1 week occurs.
(b) Only for patients with pre-existing pulmonary disease (Not necessary if (1) a chest X-ray available to the investigator has been obtained within the past 12 months
and (2) the patient’s pulmonary disease has been clinically stable)

6
(c) Patients with cirrhosis or marked fibrosis on liver biopsy or raised AFP need to have a liver imaging study during the screening period to rule out hepatic neoplasia.
(d) For anyone with preexisting cardiac disease.
(e) Eye examination will be done on indication. Any patient complaining of decrease or loss of vision must have a prompt and complete eye examination. Patients with
preexisting ophthalmologic disorders (eg, diabetic or hypertensive retinopathy) should receive periodic ophthalmologic exams during Peg-IFN therapy. Peg-IFN
treatment should be discontinued in patients who develop new or worsening ophthalmologic disorders.
(f) non-invasive measurent of the liver to detect fibrosis and cirrhosis
(g) Only for patients with decreased kidney function. If there are clinically significant laboratory abnormalities, repeat no less frequently than every 2 weeks or as
clinically indicated, with appropriate toxicity management, until they return to normal or baseline values. Urinalysis to be performed via dipstick, with subsequent
microscopic evaluation if positive for hemoglobin at the discretion of the investigator.
(h) Only for certain patients at risk, don’t repeat if data from previous assessments are available
®
(i) Roche TaqMan ; when HBV DNA is more then the maximal detection limit, ten or hundred time dilution is proceded.
(j) Paxgene TM DNA sample (tube) will be collected. The DNA samples will be used exclusively for exploratory DNA research (genotyping of hostgenomics) to evaluate
drug disposition genes.(e.g. metabolic enzymes and drug transporters), to expore possible underlying genetic variants involved in therapy response in HBV
chronically infected.Samples will only be processed in a number of subjects. No other testing will be performed on these samples.
(k) Paxgene TM RNA sample (tube) will be collected to assess the expression of RNA in peripheral blood using whole genome microarray technology. These samples
ares collected as part of an effort to better understand the effects of medication. Samples will only be processed in a number of subjects. No other testing will be
performed on these samples.
(l) plasma tube will be collected for proteomics profiling. These samples are collected as part of a study to better understand the effects of medication on liver activity.
Samples will only be processed in a number of subjects. No other testing will be performed on these samples. Possible protein candidates include: SR-B1(CD36),
CD81, certain Claudins, STAT-1 and 2, ApoE and more.
(m) During the study a quality of life index according to SF36 standardized questionnaires will be administered

7
Supplementary Figure S1. Individual plots of virological and biochemical characteristics in patients with
HBsAg negativity during or after treatment.

M a le , H B V g e n o ty p e B M a le , H B V g e n o ty p e in d e te rm in a b le

250 250
P e g -IF N a n d T D F P e g -IF N a n d T D F
1 0 ,0 0 0 1 0 ,0 0 0

H B V D N A ( IU /m L )
H B V D N A ( IU /m L )

a n ti-H B s (IU /m L )
200
a n ti-H B s (IU /m L )

200

H B s A g ( IU /m L )
H B s A g ( IU /m L )

1 ,0 0 0 1 ,0 0 0

A L A T (U /L )
A L A T (U /L )
150 150
100 100

LLQ 100 LLQ 100

LLD LLD
50 50

LLD LLD
0 0
START 12 24 36 48 72 START 12 24 36 48 72

A xS Y M : P O S NEG NEG NEG A xS Y M : P O S PO S PO S NEG

M a le , H B V g e n o ty p e A F e m a le , H B V g e n o ty p e B
250
P e g -IF N a n d A D V P e g -IF N a n d T D F
1 0 ,0 0 0 1 0 ,0 0 0 400
H B V D N A ( IU /m L )

H B V D N A ( IU /m L )
a n ti-H B s (IU /m L )

a n ti-H B s (IU /m L )
200
H B s A g ( IU /m L )

1 ,0 0 0
H B s A g ( IU /m L ) 1 ,0 0 0
A L A T (U /L )

A L A T (U /L )
300
150
100 100

200
LLQ 100 LLQ

LLD LLD 100

50

LLD LLD
0 0
START 12 24 36 48 72 START 12 24 36 48 72

A xS Y M : P O S NEG NEG NEG A xS Y M : P O S NEG NEG NEG

M a le , H B V g e n o ty p e in d e te rm in a b le
250
P e g -IF N a n d T D F
1 0 ,0 0 0
H B V D N A ( IU /m L )
a n ti-H B s (IU /m L )

200
H B s A g ( IU /m L )

1 ,0 0 0
A L A T (U /L )

150
100

LLQ 100

LLD
50

LLD
0
START 12 24 36 48 72

A xS Y M : P O S NEG NEG P O S (± )

Quantitative HBsAg levels (red line) were determined by Architect at regular intervals, whereas qualitative HBsAg signals (below
individual plots) were determined by AxSYM at 24 week intervals.

8
Supplementary Table S4. Baseline and early on-treatment variables in treated patients with >1·0 log reduction in
HBsAg level at Week 48 and <1 log reduction.

HBsAg response at Week 48 (end-of-treatment)

> 1 log decline < 1 log decline p
Demographics
c
Female, n (%) 4 (24) 33 (52) .034
a
Mean age, years (SD) 40.6 (3.0) 44.9 (1.5) .696
c
Ethnicity .778
Caucasian, n (%) 4 (24) 16 (25)
Asian, n (%) 6 (29) 22 (19)
African, n (%) 5 (43) 12 (35)
South American, n (%) 2 (12) 13 (21)
c
IFN naïve, n (%) 15 (88) 59 (94) .452
Laboratory
b
Median baseline ALT, U/L (iqr) 25 (20-32) 24 (19-37) .986
b
Median zenith ALT, U/L (iqr) 106 (86-204) 72 (45-103) .002
a
Mean baseline ALT, log10 U/L (SD) 1.42 (.12) 1.44 (.24) .625
a
Mean zenith ALT, log10 U/L (SD) 2.11 (.27) 1.87 (.26) .001
c
HBV genotype .176
Indeterminable, n (%) 5 (29) 14 (22)
A, n (%) 3 (18) 14 (22)
B, n (%) 4 (24) 3 (5)
C, n (%) 0 (0) 3 (5)
D, n (%) 3 (18) 14 (22)
E, n (%) 2 (12) 15 (24)
F, n (%) 0 (0) 0 (0)
G, n (%) 0 (0) 0 (0)
a
Mean baseline HBV-DNA*, (SD) 2.51 (.26) 2.81 (.14) .345
a
Mean HBV DNA week 12, (SD) .84 (.22) 1.03 (.09) .407
a
HBV DNA decline week 12, (SD) -1.42 (.41) -1.78 (.14) .328
a
Mean baseline HBsAg*, (SD) 2.96 (.20) 3.31 (.11) .148
a
Mean HBsAg week 12, (SD) 2.13 (.34) 3.23 (.12) .000
a
HBsAg decline week 12, (SD) -.83 (.19) -.08 (.03) .001
Fibroscan
a
Mean value, kPa (SD) 5.0 (1.8) 5.1 (.2) .419
Liver biopsy
b
Median inflammatory score, (iqr) 2 (1-3) 2 (2-2) .335
b
Median Ishak fibrosis score, (iqr) 1 (1-1) 1 (1-1) .978
b
Median % HBsAg staining, (iqr) 15 (5-40) 15 (2-35) .757

* Differences between variables were assessed by Student’s t (a), Mann-Whitney U (b), or Pearson’s Chi-squared
test (c).

9
Supplementary Table S5: HBsAg response rates in patients with baseline HBV-DNA <2,000 IUmL and > 2,000 IUmL

Peg-IFN plus Peg-IFN plus No treatment

Week 72 HBsAg response
adefovir n=46 tenofovir n=45 n=43
Baseline HBV-DNA <2,000 IU/mL n = 33 n = 28 n = 30
HBsAg negative, n (%) 2 (6) 2 (7) 0 (0)
HBsAg < 10 IU/mL, n (%) 4 (12) 5 (18) 2 (7)
Mean HBsAg decline,
-0.52 (.78) -0.78 (.98) -0.13 (0.24)
Log10 IU/mL (SD)
>1 log10 IU/mL decline, n (%) 3 (9) 6 (21) 0 (0)
>0.5 log10 IU/mL decline, n (%) 11 (34) 13 (46) 2 (7)
Baseline HBV-DNA >2,000 IU/mL n = 13 n = 17 n = 13
HBsAg negative, n (%) 0 (0) 0 (0) 0 (0)
HBsAg < 10 IU/mL, n (%) 1 (8) 0 (0) 0 (0)
Mean HBsAg decline,
-0.54 (.67) -0.22 (.22) -0.21 (0.16)
Log10 IU/mL (SD)
>1 log10 IU/mL decline, n (%) 2 (15) 0 (0) 0 (0)
>0.5 log10 IU/mL decline, n (%) 4 (31) 1 (6) 1 (8)

10
 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

‘A randomized prospective open-label trial for comparing combination therapy

Peg-Interferon alfa-2a
(PEGASYS®) and Adefovir dipivoxil (Hepsera®) and
combination therapy Peg-Interferon alfa-2a (PEGASYS®)
and Tenofovir disoproxil fumarate (Viread®) versus no treatment in HBeAg
negative chronic hepatitis B patients with low viral load’
 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

PROTOCOL TITLE: ’ A randomized prospective open-label trial for comparing combination

therapy Peg-Interferon alfa-2a (40 KD) (PEGASYS®) and Adefovir dipivoxil (Hepsera®) and
combination therapy Peg-Interferon alfa-2a (PEGASYS®)
and Tenofovir disoproxil fumarate (Viread®) versus no treatment in HBeAg negative chronic
hepatitis B patients with low viral load’

Short title HBsAg loss in HBV patients with low HBV-DNA

load, comparing ADV/PegIFN or TDF/PegIFN with
no treatment. A 3-arm randomized open-label
prospective trial.
Version 4
Date 16-07-2013
Protocol number TTM16002
MEC nummer MEC 09/139
Principal investigator(s) Dr. H.W. Reesink
Dept. of Gastroenterology and Hepatology
Academic Medical Center, University of
Amsterdam
Room G4-214 /mailbox C2-331
Meibergdreef 9
1105 AZ Amsterdam
Email: h.w.reesink@amc.nl

Sponsor Dr. H.W. Reesink

Dept. of Gastroenterology and Hepatology

Academic Medical Center, University of
Amsterdam
Room G4-214 /mailbox C2-331
Meibergdreef 9
1105 AZ Amsterdam
Email: h.w.reesink@amc.nl
 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

Independent physician(s) Prof. dr. U.H. Beuers

Dept. of Gastroenterology and Hepatology
Academic Medical Center, University of
Amsterdam
Room G4-213
Meibergdreef 9
1105 AZ Amsterdam
Email: u.h.beuers@amc.nl

Laboratory sites Academic Medical Center, University of

Amsterdam
 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

PROTOCOL SIGNATURE SHEET

Name Signature Date

Project leader/Principal Investigator: Dr. H.W. Reesink
Dept. of Gastroenterology
and sponsor and Hepatology
Academic Medical Center,
University of Amsterdam
Room G4-215 /mailbox C2-
331
Meibergdreef 9
1105 AZ Amsterdam
Email: h.w.reesink@amc.nl
 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

Coordinating Investigator Drs. F. Stelma

Dept. of Gastroenterology
and Hepatology
Academic Medical Center,
University of Amsterdam
Room G4-214 /mailbox C2-
331
Meibergdreef 9
1105 AZ Amsterdam
Email: f.stelma@amc.nl
+31 20 5667805
Coordinating Investigator Drs. L.J. Jansen
Dept. of Gastroenterology
and Hepatology
Academic Medical Center,
University of Amsterdam
Room G4-214 /mailbox C2-
331
Meibergdreef 9
1105 AZ Amsterdam
Email: l.jansen@amc.nl
+31 20 5665383
 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

TABLE OF CONTENTS

1. INTRODUCTION AND RATIONALE ................................................................................11

2. OBJECTIVES ...................................................................................................................14
2.1 Primary Objective:.......................................................................................................15
2.2. Secondary Objective: .................................................................................................15
3 STUDY DESIGN ...............................................................................................................17
4. STUDY POPULATION .....................................................................................................17
4.1 Population (base) ........................................................................................................17
4.2 Inclusion criteria ..........................................................................................................17
4.3.Exclusion criteria .........................................................................................................17
4.4 Sample size calculation...............................................................................................19
5. TREATMENT OF SUBJECTS ..........................................................................................20
5.1 Investigational product/treatment ................................................................................20
5.2 Use of co-intervention .................................................................................................21
5.3 Escape medication (if applicable)................................................................................21
6. INVESTIGATIONAL MEDICINAL PRODUCT ...................................................................22
7. METHODS .......................................................................................................................34
7.1 Study parameters/endpoints .......................................................................................35
7.1.1. Main study parameter/endpoint ...........................................................................35
7.1.2. Secondary study parameters/endpoints...............................................................35
7.1.3. Other study parameters .......................................................................................35
7.2. Randomisation, blinding and treatment allocation .....................................................34
7.3. Study procedures ......................................................................................................34
7.4. Withdrawal of individual subjects ..............................................................................34
7.4.1.Specific criteria for withdrawal ..............................................................................35
7.5. Replacement of individual subjects after withdrawal ..................................................35
7.6.Follow-up of subjects withdrawn from treatment..........................................................35
7.7Premature termination of the study ..............................................................................35
8.0 SAFETY REPORTING ...................................................................................................36
8.1Safety Assessments ....................................................................................................36
8.2 Section 10 WMO event ...............................................................................................36
8.3 Adverse and serous adverse events ...........................................................................36
8.4 Follow-up of adverse events .......................................................................................39
9 STATISTICAL ANALYSIS .................................................................................................40
9.1 Descriptive statistics ...................................................................................................41
9.2.Interim analysis ..........................................................................................................42
10 ETHICAL CONSIDERATIONS ........................................................................................43
10.1 Regulation statement ................................................................................................43
10.2 Recruitment and consent ..........................................................................................43
10.3 Compensation for injury ............................................................................................44
10.4 Incentives..................................................................................................................44
11. ADMINISTRATIVE ASPECTS AND PUBLICATION .......................................................44

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 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

11.1 Handling and storage of data and documents ...........................................................44

11.2 Amendments.............................................................................................................44
11.3 Annual progress report..............................................................................................44
11.4 End of study report....................................................................................................44
11.5 Public disclosure and publication policy ....................................................................44
12. REFERENCES ...............................................................................................................45
13. APPENDIX .....................................................................................................................48

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 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS

ABR ABR form (General Assessment and Registration form) is the application
form that is required for submission to the accredited Ethics Committee
(ABR = Algemene Beoordeling en Registratie)
AE Adverse Event
AR Adverse Reaction
CA Competent Authority
CCMO Central Committee on Research Involving Human Subjects
CV Curriculum Vitae
DSMB Data Safety Monitoring Board
EU European Union
EudraCT European drug regulatory affairs Clinical Trials GCP Good Clinical Practice
IB Investigator’s Brochure
IC Informed Consent
IMP Investigational Medicinal Product
IMPD Investigational Medicinal Product Dossier
METC Medical research ethics committee (MREC); in Dutch: medisch ethische
toetsing commissie (METC)
(S)AE Serious Adverse Event
SPC Summary of Product Characteristics (in Dutch: officiële productinfomatie
IB1-tekst)
Sponsor The sponsor is the party that commissions the organisation or performance
of the research, for example a pharmaceutical
company, academic hospital, scientific organisation or investigator. A party
that provides funding for a study but does not commission it is not
regarded as the sponsor, but referred to as a subsidising party.
SUSAR Suspected Unexpected Serious Adverse Reaction
Wbp Personal Data Protection Act (in Dutch: Wet Bescherming Persoonsgevens)
WMO Medical Research Involving Human Subjects Act (Wet Medisch-
wetenschappelijk Onderzoek met Mensen

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 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

SUMMARY

Rationale:
Worldwide, approximately 400 million people are chronically infected with hepatitis B virus
(HBV). Chronic HBV infection increases the risk of developing cirrhosis, hepatic
decompensation and hepatocellular carcinoma (HCC).(1, 2) The risk of developing
hepatocellular carcinoma is highest in HBeAg positive patients with high HBV DNA levels,
but still the relative risk remains 10 for HBeAg negative patients. (3) Furthermore it has been
shown that when HBsAg is cleared before cirrosis has developed, the prognosis is excellent
(4, 5). Recently we have shown that HBeAg negative patients with high HBV-DNA load and
low baseline HBsAg levels had a significantly higher HBsAg clearance (positive predictive
value of 85%) after combination therapy with peginterferon alfa2a (Peg-IFN) and adefovir. (6)
Based on these results, a trial was designed to investigate whether combination of a
nucleos(t)ide analogue combined with PegIFN, could also provoke a high rate of HBsAg
clearance in chronic hepatitis B patients with low (HBV DNA <20,000 IU/mL) viral load.

Study design:
This is a three arm open-label prospective randomized controlled trial. 150 patients will be
enrolled into the study after assessment of elegibility. All patients must have documented
HBsAg positivity for longer than 6 months, HBeAg negativity, anti-HBe positivity, HBV DNA <
20,000 IU/mL and ALT < 5 * upper limit of normal. Patients with a Child Pugh class B or C
will be excluded. Group 1 will consist of patients treated with Peg-IFN and adefovir, group 2
will consist of patients treated with Peg-IFN and tenofovir and group 3 will consist of
untreated controls. Patients in group 1 and 2 will receive medication for the period of one
year. For enrolment into the study a liverbiopsy at time of enrolment is compulsory and is
advisable at end of treatment (week 48).

Study population: The study population will consist of 150 patients chronically infected with
hepatitis B virus with low viral load and HBeAg negativity.

Main study parameters/endpoints:

The aim of this study is to investigate what proportion of HBeAg negative, inactive carriers of
the hepatitis B virus with low (< 20,000 IU/mL) load will lose HBsAg when treated with
nucleot(s)ide analogue/Peg-IFN combination therapy.

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 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

Nature and extent of the burden and risks associated with participation, benefit and
group relatedness:
During a period of 1.5 year blood samples will be collected and tested at 21 defined
timepoints, the number of site visits will be 21 (see appendix table 1).
Questionnaires about compliance and quality of life will be asked to be filled in. Potential
physical and physiological discomfort associated with participation are the burden of
undergoing a liver biopsy, taking medication, extensive site visits, extra blood samples that
will be taken, adverse reactions and serious adverse reactions.

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 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

1. INTRODUCTION AND RATIONALE

Approximately 400 million individuals worldwide, 6% of the world population, are chronically
infected with hepatitis B virus (HBV), and 2 billion have serological evidence of past or
present HBV infection.(7)
Worldwide HBV is transmitted mostly perinatally or horizontally early during childhood,
whereas in western countries infection is mainly transmitted via sexual intercourse.
Despite the availability of a safe and effective vaccine for more than two decades, HBV
infection is still a major global health problem. People with chronic HBV infection are at
increased risk of developing hepatic decompensation, liver cirrhosis, and hepatocellular
carcinoma (HCC). It is estimated that worldwide 30% of liver cirrhosis and 25% of HCC is
due to HBV infection.(8) Mortality estimation models estimated 563,000-1.000,000 deaths
due to HBV infection each year, thereby making chronic hepatitis B the seventh leading
cause of death in the world. (8-10) Prevalence of chronic HBV infection however is low (0.1-
2.0%) in Western Europe and the USA, intermediate in Mediterranean countries and Japan
(2.0-8.0%), and high in Southeast Asia and sub-Saharan Africa (8.0-20.0%) (11). The annual
incidence of spontaneous HBsAg loss, signifying clearance of the virus, is only 0.1%-0.8% in
patients from Southeast Asia, and 1-2% in patients from Western Europe and the USA.
Progression from acute to chronic hepatitis B is influenced by the patient’s age at acquisition
of the virus. When the virus is acquired perinatally, infection becomes chronic in more than
90% of new borns. In contrast when the infection is acquired during adulthood, most acute
individuals clear the infection and only approximately 5% of persons become chronically
infected (12).
Figure 1 represents the natural course of chronic hepatitis B, when infection is acquired
perinatally. Chronic hepatitis B can present as two distinct forms: HBeAg-positive and
HBeAg-negative infection. Patients are rendered for treatment when the patient develops
active disease regardless of HBeAg positivity or negativity.
In the last decade therapeutic options for chronic hepatitis B have dramatically improved,
which resulted in more patients achieving a state of inactive disease. Unfortunately treatment
is not yet optimal, and a cure (i.e. loss of HBsAg) is still far away.
Two types of therapies are currently available for the treatment of chronic hepatitis B:
1) nucleos(t)ide analogues, which are antiviral agents inhibiting the activity of reverse
transcriptase, a viral DNA polymerase. Several nucleoside/nucleotide analogues are licensed
for the treatment of HBV in the Netherlands; lamivudine, entecavir, telbivudine, which are
nucleoside analogues and adefovir dipivoxil (ADF) and tenofovir disoproxil fumarate (TDF),

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 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

which are a nucleotide analogues. The mode of action of nucleotide and nucleoside
analogues is the same, by inhibiting the HBV reverse transcriptase.(13-16)
2) Conventional interferon and pegylated interferon (13, 17, 18).
Interferon alpha has a dual mode of action, with both antiviral and immunomodulatory effects
(19-21) and has been recommended as one of the first-line therapies for both HBeAg-
positive and HBeAg-negative chronic hepatitis B patients according to the EASL guidelines
2009 (22). Pegylated interferon (Peginterferon alpha2a (Peg-IFN)) is registered for the
treatment of HBeAg positive as well as HBeAg negative chronic hepatitis B. Peg-IFN can be
administered once weekly due to attachment of interferon alfa-2a to 40 KD branched-chain
polyethylene glycol (Peg).
Dutch guidelines advise treatment with anti-virals of all chronic hepatits B patients with HBV-
DNA > 2*104 IU/mL with persistent or intermittent elevation of ALT (alanine
aminotransferase) above the upper limit of normal (ULN) and with moderate/severe hepatitis
on biopsy using a standardized scoring system (e.g. at least grade A2 or stage F2 by
METAVIR scoring), regardless if they are HBeAg positive or negative (34).
These types of therapies have suboptimal efficacy. Peg-IFN is administered once weekly
subcutaneous and is associated with dose-limiting adverse events. Nucleos(t)ide analogues
are better tolerated but require life long administration and therefore nucleoside analogue
treatment has been associated with development of drug resistance (23).
For example, the long-term use of lamivudine promotes viral resistance at an estimated rate
of 14 to 32% per year. The 5 year resistance for lamivudine is 70%, for adefovir 30%, for
entecavir 1.2%, for tenofovir unknown but 0% after 2 years (24). Emergence of resistant
mutants or discontinuation of antiviral therapy has been associated with acute exacerbations
of liver disease with viral rebound and ALT elevations(13). Although ADF is active against
lamivudine resistant mutants, long-term ADF therapy has been found to be associated with
development of a, ADF-resistant, rtN236T HBV viral mutation in a small number of patients
(25).
In HBeAg-positive patients, HBeAg loss/seroconversion has long been selected as the
primary goal of treatment. This is because studies on the natural history of HBV indicate that,
in the majority of patients with HBeAg-positive chronic hepatitis B, HBeAg
loss/seroconversion (either spontaneous or induced by antiviral therapy) is closely correlated
with suppression of HBV DNA, ALT normalization and disease remission (26-28).
Eradication of HBV infection is rendered difficult because stable, long enduring covalently
closed circular DNA (cccDNA) and HBV DNA becomes integrated in the hepatocyte nuclei.
As the course for chronic hepatitis B into developing cirrhosis/HCC is slow, the major goals
of therapy are the long term prevention of progression to irreversible liver damage.

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Relatively short-term follow up studies use different surrogate outcomes, which have been
proven to be durable and predictive for further progression of the disease. As HBV DNA
levels are associated with the progression to irreversible liver damage, the goal of treatment
at present has been effective suppression of HBV replication, rather than curing the disease.
Recently however, increasing attention is paid to the loss of HBsAg as the primary goal for
treatment. HBsAg loss is the most reliable efficacy parameter for both HBeAg-positive and
HBeAg-negative chronic hepatitis B. Serum HBsAg is a marker of HBV infection, and
antibodies against HBsAg (anti-HBs) signify recovery.
When in current strategies loss of HBsAg and anti-HBs conversion has been achieved, it is
considered as the closest outcome to clinical cure of chronic hepatitis B infection. Especially
in patients who clear HBsAg before they have developed cirrosis, the prognosis is excellent
compared to patients who remain HBsAg positive. Treatment with nucleoside analogues for
one year does not increase the loss of HBsAg for both HBeAg positive and negative patients
and remains approximately 1%. One year treatment with Peg-IFN results in HBsAg loss of
approximately 3% in the HBeAg positive patients and 4% in the HBeAg negative patients.
(29). In a study with Peg-IFN and lamuvidine therapy for one year with patients with HBeAg
positivity, in 11% of patients HBsAg surface loss was seen after 3 years of follow up.
Another long-term follow up study in HBeAg negative patients, treated with Peg-IFN with or
without lamivudine for one year showed a 12% loss (both with and without lamivudine) of
HBsAg 5 years post-treatment (30).
In our currently ongoing study in patients with a high viral load (HBV DNA ≥ 20,000 IU/mL)
combination therapy of Peg-IFN and ADF, a rate of 23% HBsAg loss was reported after one
year of therapy, which is much higher than currently is observed with other treatment
regimens (6). Moreover in this study a positive predictive value of HBsAg loss of 85% was
seen when baseline HBsAg levels were less than 675 IU/mL, suggesting that patients with
lower baseline HBsAg levels are more susceptible to HBsAg clearance. Treatment in these
patients was well tolerated, 4% developed thyroid disease which is comparable to other
studies (31). Recent studies have shown that tenofovir is a more potent viral suppressor
than adefovir (32). Moreover it has recently been reported that the rate of HBsAg loss in
HBeAg positive patients after 2 years of tenofovir (TDF) therapy was 6% (33).
Chronic hepatitis B patients with low viral load (HBV DNA < 20,000 IU/mL) most often have
inactive disease en don’t require viral suppression according to Dutch guidelines (34).
However in the light of the currently highly successful rate of HBsAg loss seen in our ongoing
study (6) we hypothesize that a similar rate of HBsAg clearance can be achieved in HBeAg
negative patients with low HBV DNA viral load, thereby focusing on cure of the disease
rather than viral suppression.

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In the light of the suboptimal therapies currently available e.g. the dose limiting adverse
reactions of Peg-INF treatment and the development of drug resistance, it is of great
practical value to establish predictors of response (or non-response) before or early in the
course of anti-viral treatment to assess the success rate of antiviral agents and to guide
therapeutic interventions in hepatitis B.
In this study we hypothesize that both treatment with peg-interferon and ADF or Peg-IFN and
TDF in HBeAg negative chronic hepatitis B patients with low HBV DNA viral load will induce
a high rate of HBsAg loss, comparable to that in patients with high viral load after treatment
with ADF and Peg-IFN.

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2. OBJECTIVES

2.1 Primary Objective:

The primary objective is to demonstrate the efficacy of combination therapy (Peg-IFN and
adefovir or Peg-IFN and tenofovir) for inducing loss of HBsAg compared to no-treatment in
HBeAg negative chronic hepatitis B patients with low viral load. HBsAg loss is defined as
HBsAg level < 0.05 IU/mL.

2.2. Secondary Objective:

The secondary objectives are to evaluate:
a. the rate of HBsAg loss and anti-HBs serconversion,
b. To establish predictive markers at baseline and during the first 12 weeks of treatment for
response of primary and secondary endpoints:

serologic markers
HBsAg levels
immunologic markers
T cell responses, cytokine production of T cells.
histologic markers
HAI (histology activity index)
Fibrosis grade
HBsAg level
cccDNA level (liver)
HBV-DNA level (liver)
biochemical markers (livertransaminases)
ALT
AST
LDH
AF
yGT
virologic markers
HBsAg level
cccDNA (plasma)
HBV-DNA level
HDV co-infections

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Hostgenomics
DNA and RNA expression levels in PBMC’s, liver
Proteomics
Possible protein candidates include: SR-B1(CD36), CD81, certain Claudins, STAT-1 and
2, ApoE and more
Inflammatory markers
CRP, neoptorin
Neoplastic markers
Rate of development of hepatocellulair carcinoma

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3. STUDY DESIGN

This is a three arm open-label prospective randomized controlled trial. (n=150) The trial is
designed to compare the efficacy of treatment with Peg-IFN and ADF or Peg-IFN and TDF
versus no treatment for clearance of the HBsAg in chronic hepatitis B patients with low viral
load.

Patients will be enrolled into the study after assessment of elegibility. Group 1 will consist of
patients treated with Peg-IFN 180µg once weekly and ADF 10mg once daily (standard
doses), group 2 will consist of patients treated with Peg-IFN 180µg once weekly and TDF
300mg (equivalent to 245 mg tenofovir disoproxil) once daily (standard doses) and group 3
will consist of untreated patients as negative controls.

The trial consists of a screening period of approximately 4 weeks, a 48 week treatment

period, a 24 week follow-up period and a long term follow up period of 5 year. A schematic
overview of the study design is presented in table 1 and 2 (see appendix).
150 patients will be randomized in a 50:50:50 ratio to one of the treatment groups.
Randomization will be stratified to genotype A to optimize balance between treatment
groups. All patients will receive medication for the period of 48 weeks. For enrolment into the
study a liver biopsy at time of enrolment is compulsory for patients selected in the treatment
groups. For enrolment of patients that are selected in the non-treatment group a liverbiopsy
is optional but not compulsory for participation. In patients selected in the treatment group a
second liver biopsy at week 48 is recommended for evaluation of therapeutic responses and
to guide any further possible therapy. Due to the burden of undergoing a liver biopsy, the
second liver biopsy is not compulsory to take part into the study. Patients that are selected in
the non treatment group will not undergo a biopsy at week 48. Patients will be clinically
monitored intensively according to the assessment schedule (table 1). In the long term follow
up period patients will be clinically monitored each year.

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4 STUDY POPULATION

4.1 Population (base)

Patients will be enrolled into the study after assessment of eligibility. Target population will
consist of adult patients (≥ 18 years) with chronic hepatitis B ( HBsAg positivity ≥ 6 months),
HBeAg negativity,HBV DNA < 20,000 IU/ml and ALT < 5* upper limit of normal.

4.2 Inclusion criteria

1. Male and female patients > 18 and ≤ 70 years of age
2. Positive HBsAg for more than 6 months.
3. Negative for HBeAg for more than 6 months.

4. HBV DNA < 20,000 IU/ml

5. Patients with chronic hepatitis B who are either naive to antiviral treatment, or have
received either interferon (IFN) or nucleoside/nucleotide analogues in the past but are
still positive for HBsAg.
6. Serum ALT ≤ 5 * ULN as determined by two values taken >14 days apart during the six
months before the first dose of study drug with at least one of the determinations
obtained during the screening period.
7. Negative urine or serum pregnancy test (for women of childbearing potential)
documented within the 24-hour period prior to the first dose of test drug.

4.3. Exclusion criteria

1. Patients co-infected with HCV, HIV or who have decompensated liver disease, hepato-
cellular carcinoma, significant cardiac disease, significant renal disease, seizure
disorders or severe retinopathy.
2. Patients who have received nucleos(t)ide analogues for their chronic hepatitis B within 6
weeks before enrollment or have received Peg-IFN within 3 months before enrollment.
3. Patients must not have received any other systemic anti-viral, anti-neoplastic or immuno-
modulatory treatment (including supraphysiologic doses of steroids or radiation) ≤3
months prior to the first dose of study drug or the expectation that such treatment will be
needed at any time during the study.
4. Positive test at screening for anti-HAV IgM, anti-HIV, HCV RNA. (Patients that have cleared
the hepatitis C virus can be included in the study)

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5. Patients who are expected to need systemic antiviral therapy other than that provided by
the study at any time during their participation in the study are also excluded. Exception:
patients who have had a limited (≤7 day) course of acyclovir for herpetic lesions more
than 1 month prior to the first administration of test drug are not excluded.
6. Evidence of decompensated liver disease (Child pugh B-C)
7. Serum total bilirubin > twice the upper limit of normal at screening
8. History or other evidence of bleeding from esophageal varices or other conditions
consistent with decompensated liver disease.
9. History or other evidence of a medical condition associated with chronic liver disease
other than HBV (e.g., hemochromatosis, autoimmune hepatitis, metabolic liver diseases
including Wilson’s disease and alfa1-antitrypsin deficiency, alcoholic liver disease, toxin
exposures, thalassemia).
10. Women with ongoing pregnancy or who are breast feeding.
11. Neutrophil count <1500 cells/mm3 or platelet count <80,000 cells/mm3 at screening.
12. Hemoglobin < 7.1 mmol/L (< 11.5 g/dL) for females and < 7.8 mmol/L (< 12.5 g/dL) for
men at screening.
13. Serum creatinine level >1.5 times the upper limit of normal at screening.
14. Unstable ongoing severe psychiatric disease, especially depression (stable patients can
be included).
15. History of immunologically mediated disease (e.g., inflammatory bowel disease,
idiopathic thrombocytopenic purpura, lupus erythematosus, autoimmune hemolytic
anemia, scleroderma, severe psoriasis, rheumatoid arthritis).
16. History or other evidence of chronic pulmonary and cardiac disease associated with
functional limitation. Severe cardiac disease (e.g., NYHA Functional Class III or IV,
myocardial infarction within 6 months, ventricular tachyarrhythmias requiring ongoing
treatment, unstable angina or other significant cardiovascular diseases).
17. History of a severe seizure disorder or current anticonvulsant use and clinically unstable
disease.
18. Evidence of an active or suspected cancer or a history of malignancy where the risk of
recurrence is ≥20% within 5 years. Patients with a lesion suspicious of hepatic
malignancy on a screening imaging study will only be eligible if the likelihood of
carcinoma is ≤10% following an appropriate evaluation.
19. Major organ transplantation. (patients with skin, cornea or bone transplantation are
allowed to be included into the study)

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20. Thyroid disease with thyroid function poorly controlled on prescribed medications.
Patients with elevated thyroid stimulating hormone or T4 concentrations, with elevation of
antibodies to thyroid peroxidase and any clinical manifestations of thyroid disease that
are not stable on prescribed medication are excluded. Stable patients can be included.
21. History or other evidence of severe retinopathy (e.g. CMV retinitis, macula degeneration)
or clinically relevant ophthalmological disorder due to diabetes mellitus or hypertension.
22. Inability or unwillingness to provide informed consent or abide by the requirements of the
study.
23. History or other evidence of severe illness or any other conditions which would make the
patient, in the opinion of the investigator, unsuitable for the study.
24. Patients with a value of alfa-fetoprotein >100 ng/mL are excluded, unless stability (less
than 10% increase) has been documented over at least the previous 3 months.
25. Evidence of current hard drug(s) (i.e. cannabis products are allowed) and/or alcohol
abuse (20g/day for women and 30g/day for men).
26. Patients included in another trial or having been given investigational drugs within 12
weeks prior to screening.

4.4 Sample size calculation

Per treatment group 44 per group (88 for both groups) is needed to achieve 81% power to
detect a difference between the two treatment groups and the control group. The proportion
in the Peg-IFN and adefovir group is assumed to be 0.01 under the null hypothesis and 0.20
under the alternative hypothesis. The proportion in the control group (no treatment) is 0.01.
The test statistic used is the two-sided Fisher's Exact test . The significance level of the test
was targeted at 0.05. A group sample of 44 persons in the control group is needed to
achieve a 81% power to detect a difference between the Peg-IFN adefovir or Peg-IFN
tenofovir.
Regarding a 10% drop-out due to Peg-IFN side-effects or other reasons a total of 50 patients
are needed in each group.

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5. TREATMENT OF SUBJECT

5.1 Investigational product/treatment

Peginterferon alfa-2a (40KD) (PEGASYS®) + Adefovir (ADF) dipivoxil (Hepsera®)
Peginterferon alfa-2a (40KD) (PEGASYS®) + Tenofovir (TDF) disoproxil fumarate (Viread®)

5.2 Use of co-intervention

All medication (prescription and over- the- counter) administered from signing of informed
consent onwards until the safety follow-up visit should be recorded in the CRF.
When having heterosexual intercourse, female subjects of childbearing potential and non-
vasectomized male subjects who have a female partner of childbearing potential must use a
combination of 2 effective birth control methods during dosing with Peg-IFN and TDF or ADF
and for 6 months after discontinuation of therapy.
Use of co-intervention (e.g. painkillers) will be to the discretion of the investigator

5.3 Escape medication

Use of escape medication (e.g. switch of antiviral therapy due to side effects) will be to the
discretion of the investigator.

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6. INVESTIGATIONAL MEDICINAL PRODUCT

6.1. Name and description of investigational medicinal product

6.2. Product Information

Peginterferon alfa-2a (40KD) (PEGASYS®)

Summary of findings from non-clinical and clinical studies

Peginterferon alfa-2a (40KD) (PEGASYS®) was developed through the process of
pegylation, whereby a large, 40-kDa, branched-chain polyethylene glycol (PEG) molecule is
attached to the base interferon alfa-2a molecule to produce a drug with a prolonged half-life.
The size of the PEG molecule and its structure are factors affecting the pharmacokinetic
properties of pegylated interferons. The attachment of a 40-KD, branched PEG moiety to
interferon alfa-2a results in a drug with a once weekly frequency of administration which
maintains effective concentrations of peginterferon alfa-2a (40KD) throughout the dosing
interval while substantially reducing the peak-to-trough ratio. Hence, sharp increases to peak
serum concentrations can be avoided, which are linked to a number of adverse events
associated with thrice-weekly IFNα dosing.(35)
PEGASYS®, as monotherapy and in combination with ribavirin has been approved for use in
patients with chronic hepatitis C (36).
PEGASYS® was recently approved in the EU for treatment of HBeAg positive as well as
HBeAg-negative chronic hepatitis B. The clinical development of PEGASYS for chronic
hepatitis B consists of a phase II study in patients with HBeAg-positive HBV (37) and two
pivotal phase III trials in HBeAg-positive and HBeAg-negative disease (38). The HBeAg-
positive study results were presented in 2004.
The primary aim of the phase II study was to compare the efficacy and safety of PEGASYS
administered once weekly for 24 weeks at 90 μg, 180 μg and 270 μg with conventional IFNα-
2a 4.5 MIU thrice weekly in patients with HBeAg-positive chronic hepatitis B.
A total of 194 adult patients were enrolled in this randomized, open-label, multicenter study.
Included patients were IFNα naïve and had received less than 6 months treatment with
nucleoside/nucleotide analogues and/or had not received nucleoside/nucleotide therapy in
the 6 months prior to the study. Patients with decompensated cirrhosis were excluded. All
patients were serum positive for HBeAg, HBsAg and had viral loads >500,000 HBV DNA
cop/mL. Chronic hepatitis B status was confirmed by liver biopsy and, at the time of
screening, all patients had baseline ALAT levels over 2-fold greater but <10 x ULN. Patients

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were randomized to four treatments arms and treated for 24 weeks with either conventional
IFNα-2a or with one of three doses of PEGASYS®. All patients were monitored for an
additional treatment-free 24-week follow-up period.
The percentages of patients achieving a combined response (HBeAg loss, HBV DNA
<500,000 cop/mL, ALAT normalization) at end-of-follow-up (week 48) were 12%, 27%, 28%
and 19% for conventional IFNα-2a, PEGASYS 90 µg, 180 µg and 270 µg once weekly,
respectively. When the data from all PEGASYS doses were pooled, the combined
response rate was 24% and this was statistically significantly different compared with
conventional IFNα-2a (P=0.036). In terms of on-treatment HBV DNA suppression, a
PEGASYS dose of 180 µg gave a greater response at the end of week 24. At week 24,
HBV DNA levels had not yet reached a plateau but continued to decline (37). Of the patients
treated with 180 µg PEGASYS 33% had HBeAg seroconversion and 35% had HBeAg loss
after 24 weeks of treatment and 24 weeks follow-up.
Advantages of PEGASYS over interferon alfa-2a were noted also in the subgroups of
patients with difficult-to-treat disease such as those with liver cirrhosis, low ALT, and/or high
HBV DNA at baseline as well as patients with HBV genotype C (37).
Overall, the data from this phase II study demonstrates that PEGASYS has substantially
higher efficacy than conventional IFNα-2a in patients with HBeAg-positive CHB .
Two phase III, randomized clinical trials with PEGASYS® in chronic hepatitis B have been
performed. These trials investigated the potential of PEGASYS® (180 mcg once weekly)
alone and in combination with lamivudine (100 mg daily) in the treatment of HBeAg-positive
and HBeAg-negative CHB. A treatment duration of 48 weeks was proposed for the phase III
trials as a result of the phase II data, which demonstrated a continuing decline in HBV DNA
levels at week 24.
The principal objective of the first phase III study was to compare the efficacy and safety of
PEGASYS® 180 μg once weekly with and without lamivudine 100 mg daily, to that of
lamivudine 100 mg daily in the treatment of HBeAg-positive patients with CHB. A total of 814
adult patients have been enrolled and they have been randomized to one of the three
treatment arms receiving 48 weeks of anti-viral therapy and monitored for a further 24 weeks
post-treatment. At end of follow-up HbeAg seroconversion and HBV-DNA < 100,000 cop/mL
was observed in 32 % of patients treated with Pegasys® with placebo (p<0.001), 27 %
(p<0.023) with Pegasys® and Lamivudine and 19 % with Lamivudine alone. Also HbeAg loss
and ALT normalization was significantly higher in 2 groups treated with Pegasys® than in the
group with Lamivudine alone (38).

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The aim of the second phase III study was to compare the efficacy and safety of PEGASYS®
180 μg once weekly with and without lamivudine 100 mg daily, to that of lamivudine 100 mg
daily in the treatment of HBeAg-negative patients with CHB. The 537 adult patients in this
trial have been randomized to one of the three treatment arms. All patients have received 48
weeks of anti-viral treatment and have been monitored for a further 24 weeks post-treatment
period.
After 24 weeks of follow-up, the percentage of patents with normalization of alanine
aminotransferase levels or hepatitis B virus (HBV) DNA levels below 20,000 IU/mL was
significantly higher with peginterferon alfa-2a monotherapy (59 % and 43 % respectively) and
peginterferon alfa-2a plus lamivudine (60 % and 44 %) than with lamivudine monotherapy
(44 %, P=0.004 and P=0.003 respectively; and 29 %, P=0.007 and P=0.003 respectively). At
the end of follow-up rates of sustained suppression of HBV DNA to below 400 copies per
milliliter were 19 % with peginterferon alfa-2a monotherapy, 20 % with combination therapy
and 7 % with lamivudine alone (P<0.001 for both comparisons with lamivudine alone). Loss
of hepatitis B surface antigen occurred in 12 patients in the peginterferon groups, as
compared with 0 patients in the group given lamivudine alone.
At end of follow-up (week 72), using the intent to treat analysis, the percentage with
normalised ALAT or HBV-DNA (< 20,000 cop/mL) was significantly higher in the PEGASYS®
plus placebo and PEGASYS® plus lamivudine groups than in the lamivudine group (38) .

In multivariate analyses, high ALT, low HBV-DNA and low HBeAg levels at baseline were
significant predictors of HBeAg seroconversion (P< 0.001).In HBeAg-positive CHB, marked
ALT flares during INF-based treatment have been associated with HBeAg seroconversion
and viral suppression. Marked on-treatment ALT elevations were generally more frequent
with peginterferon alfa-2a monotherapy and were more common in patients with HBeAg
seroconversion 24 weeks after the end of treatment (37, 39).

Summary of known and potential risks and benefits

PEGASYS can cause a broad variety of serious adverse reactions . The most common life-
threatening or fatal events induced or aggravated by PEGASYS were depression, suicide,
relapse of drug abuse/overdose, and bacterial infections, each occurring at a frequency of <
1%. Hepatic decompensation occurred in 2% (10/574) of CHC/HIV patients .

In clinical trials of 48 week treatment duration, the adverse event profile of PEGASYS® in
chronic hepatitis B was similar to that seen in chronic hepatitis C PEGASYS® monotherapy

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use, except for exacerbations of hepatitis. Six percent of PEGASYS® treated patients in the
hepatitis B studies experienced one or more serious adverse events.

The most common or important serious adverse events in the hepatitis B studies were
infections (sepsis, appendicitis, tuberculosis, influenza), hepatitis B flares, anaphylactic
shock, thrombotic thrombocytopenic purpura.

The most commonly observed adverse reactions were pyrexia (54% vs. 4%), headache
(27% vs. 9%), fatigue (24% vs. 10%), myalgia (26% vs. 4%), alopecia (18% vs. 2%), and
anorexia (16% vs. 3%) in the PEGASYS and lamivudine groups respectively.

Overall 5% of hepatitis B patients discontinued PEGASYS therapy and 40% of patients

required modification of PEGASYS dose. The most common reason for dose modification in
patients receiving PEGASYS therapy was for laboratory abnormalities including neutropenia
(20%), thrombocytopenia (13%), and ALT disorders (11%).

Description and justification of route of administration and dosage

The recommended dose of PEGASYS monotherapy for hepatitis B is 180 μg (1.0 Ml vial or
0.5 mL prefilled syringe) once weekly for 48 weeks by subcutaneous administration in the
abdomen or thigh.

When dose modification is required for moderate to severe adverse reactions (clinical and/or
laboratory), initial dose reduction to 135 μg (which is 0.75 mL for the vials or adjustment to
the corresponding graduation mark for the syringes) is generally adequate. However, in
some cases, dose reduction to 90 μg (which is 0.5 mL for the vials or adjustment to the
corresponding graduation mark for the syringes) may be needed. Following improvement of
the adverse reaction, re-escalation of the dose may be considered

Dosages, dosage modifications and method of administration

Hematological:
Dose reduction from 180μg to 135μg is required if absolute neutrophil counts are < 750/mm³.
If ANC < 500/mm³, treatment should be suspended until ANC values return to more than
1000/mm³. Treatment must be reinstituted at 90 μg and monitor ANC needs to be monitored.
When platelet counts are < 50,000/mm³, dosage must be reduced to 90 μg. Treatment must
be discontinued if platelet count < 25,000/mm³.
Psychiatrical
Dose reduction from 180μg to 135μg is required in cases of moderate depression. Treatment
should be discontinued in severe depression.

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Renal Function

In patients with end-stage renal disease requiring hemodialysis, dose reduction to 135 μg
PEGASYS is recommended. Signs and symptoms of interferon toxicity should be closely
monitored.

Liver Function

If ALT increases are progressive despite dose reduction or accompanied by increased

bilirubin or evidence of hepatic decompensation, therapy should be immediately
discontinued.

In chronic hepatitis C patients with progressive ALT increases above baseline values, the
dose of PEGASYS should be reduced to 135 μg and more frequent monitoring of liver
function should be performed. After PEGASYS dose reduction or withholding, therapy can be
resumed after ALT flares subside.

In chronic hepatitis B patients with elevations in ALT ( > 5 x ULN), more frequent monitoring
of liver function should be performed and consideration should be given to either reducing
the dose of PEGASYS to 135 μg or temporarily discontinuing treatment. After PEGASYS
dose reduction or withholding, therapy can be resumed after ALT flares subside.

In patients with persistent, severe (ALT > 10 times above the upper limit of normal) hepatitis
B flares, consideration should be given to discontinuation of treatment. (35).

Adefovir (ADF) dipivoxil (Hepsera®)

Summary of findings from non-clinical and clinical studies

Adefovir (ADF) dipivoxil (Hepsera®) is a synthetic adenine nucleotide analogue that has
recently been licensed for the treatment of CHB in the United States and Europe.
In vitro, the concentration of adefovir required to inhibit 50% of HBV DNA synthesis ranged
from 0.2-2.5 µmol/L in a variety of HBV DNA-producing human hepatoma cell lines. Additive
antiviral effects were observed when adefovir was combined with lamivudine, or with one of
the experimental drugs entecavir or telbivudine.
Large, pacebo-controlled clinical trials have shown potent activity against HBV in both
HBeAg-positive and HBeAg-negative patients that is associated with significant biochemical,
virologic, and histologic improvement. Moreover, resistance to this agent has not been
observed in up to 48 weeks of therapy. The drug has been shown to be effective both in wild-
type and lamivudine-resistant forms of HBV both in vitro and in vivo. Clinical trials to date

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show that virologic and biochemical improvement are observed after adefovir dipivoxil is
added to ongoing lamivudine therapy in lamivudine-resistant patients or when adefovir
dipivoxil is administered as monotherapy (15, 16, 40). In HBeAg positive chronic hepatits B
patients (n=515) ADF given in dosages of 10 mg, 30 mg or placebo per day for 48 weeks
showed that after 48 weeks of treatment, significantly more patients who received 10 mg or
30 mg of ADF dipivoxil per day than who received placebo had histologic improvement (53
percent [P<0.001], 59 percent [P<0.001], and 25 percent, respectively), a reduction in serum
HBV DNA levels (by a median of 3.52 [P<0.001], 4.76 [P<0.001]. and 0.55 log cop/mL,
respectively), undetectable levels (fewer than 400 cop/mL) of serum HBV DNA (21 percent
[P<0.001], 39 percent [P<0.001], and 0 percent, respectively), normalization of ALAT levels
(48 percent [P<0.001], 55 percent [P<0.001], and 16 percent, respectively), and HBeAg
seroconversion (12 percent [P=0.0491], 14 percent [P=0.01], and 6 percent, respectively).
No ADF-associated resistance mutations were identified in the HBV DNA polymerase gene.
The safety profile of the 10-mg dose of ADF dipivoxil was similar to that of placebo; however,
there was a higher frequency of adverse events and renal laboratory abnormalities in the
group given 30 mg of ADF dipivoxil per day (15).
In HBeAg negative CHB patients (n=185) ADF given in a dosage of 10 mg or placebo once
daily for 48 weeks shows that at week 48, 64 percent of patients who had base-line liver-
biopsy specimens available in the ADF dipivoxil group had improvement in histologic liver
abnormalities (77 of 121), as compared with 33 percent of patients in the placebo group (19
of 57, p<0.001). Serum hepatitis B virus (HBV) DNA levels were reduced to fewer than 400
cop/mL in 51 percent of patients in the ADF dipivoxil group (63 of 123) and in 0 percent of
those in the placebo group 0 of 61, p<0.001). The median decrease in log-transformed HBV
DNA levels was greater with ADF dipivoxil treatment than with placebo (3.91 vs. 1.35 log
cop/mL, P<0.001). ALAT levels had normalized at week 48 in 72 percent of patients
receiving ADF dipivoxil (84 of 116), as compared with 29 percent of those receiving placebo
(17 of 59, P<0.001). No HBV polymerase mutations associated with resistance to ADF were
identified. The safety profile of ADF dipivoxil was similar to that of placebo (16).
Follow-up results after 48 weeks of treatment with 10 mg AFD were presented at the ISVHLD
2003 meeting in Sydney [45]. At the end of 24 weeks follow-up without treatment, 5 % of the
ADF group had undetectable HBV-DNA levels (HBV-DNA < 1000 cop/mL). After 48 weeks of
follow-up 8% had undetectable HBV-DNA levels (HBV-DNA < 1000 cop/mL).

Two studies (40, 41) showed that in chronic hepatitis B with compensated and
decompensated liver disease due to YMDD related Lamivudine resistance, ADF 10 mg once
daily given for 48-52 weeks resulted in a significant virological and biochemical improvement.

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Summary of known and potential risks and benefits

Adverse reactions to HEPSERA identified from placebo-controlled and open label studies
include the following: asthenia, headache, abdominal pain, diarrhea, nausea, dyspepsia,
flatulence, increased creatinine, and hypophosphatemia.

Adefovir dipivoxil 10 mg/day is generally well tolerated in patients with chronic HBV infection;
there were no marked increases in adverse events or laboratory abnormalities compared
with placebo in a pooled analysis of 48-week data from two double-blind trials. Asthenia and
diarrhoea occurred more frequently than with placebo in one of the placebo-controlled
studies, while headache and abdominal pain occurred more frequently in the other study.
Within 48 weeks 2% of patients from one study and no patients in the other study
discontinued treatment because of adverse events.

Nephrotoxicity characterized by a delayed onset of gradual increases in serum creatinine

and decreases in serum phosphorus was historically shown to be the treatment-limiting
toxicity of adefovir dipivoxil therapy at substantially higher doses in HIV-infected patients (60
and 120 mg daily) and in chronic hepatitis B patients (30 mg daily). Chronic administration of
HEPSERA (10 mg once daily) may result in delayed nephrotoxicity. The overall risk of
nephrotoxicity in patients with adequate renal function is low. However, this is of special
importance in patients at risk of or having underlying renal dysfunction and patients taking
concomitant nephrotoxic agents such as cyclosporine, tacrolimus, aminoglycosides,
vancomycin and non-steroidal anti-inflammatory drugs It is important to monitor renal
function for all patients during treatment with HEPSERA, particularly for those with pre-
existing or other risks for renal impairment. Patients with renal insufficiency at baseline or
during treatment may require dose adjustment. The risks and benefits of HEPSERA
treatment should be carefully evaluated prior to discontinuing HEPSERA in a patient with
treatment-emergent nephrotoxicity.

Severe acute exacerbation of hepatitis has been reported in patients who have discontinued
anti-hepatitis B therapy, including therapy with HEPSERA. Hepatic function should be
monitored at repeated intervals with both clinical and laboratory follow-up for at least several
months in patients who discontinue HEPSERA. If appropriate, resumption of anti-hepatitis B
therapy may be warranted.

In clinical trials of HEPSERA, exacerbations of hepatitis (ALT elevations 10 times the upper
limit of normal or greater) occurred in up to 25% of patients after discontinuation of

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HEPSERA. These events were identified in studies GS-98-437 and GS-98-438 (N=492).
Most of these events occurred within 12 weeks of drug discontinuation. These exacerbations
generally occurred in the absence of HBeAg seroconversion, and presented as serum ALT
elevations in addition to re-emergence of viral replication. In the HBeAg-positive and HBeAg-
negative studies in patients with compensated liver function, the exacerbations were not
generally accompanied by hepatic decompensation. However, patients with advanced liver
disease or cirrhosis may be at higher risk for hepatic decompensation. Although most events
appear to have been self-limited or resolved with re-initiation of treatment, severe hepatitis
exacerbations, including fatalities, have been reported. Therefore, patients should be closely
monitored after stopping treatment.

Prior to initiating HEPSERA therapy, HIV antibody testing should be offered to all patients.
Treatment with anti-hepatitis B therapies, such as HEPSERA, that have activity against HIV
in a chronic hepatitis B patient with unrecognized or untreated HIV infection may result in
emergence of HIV resistance. HEPSERA has not been shown to suppress HIV RNA in
patients; however, there are limited data on the use of HEPSERA to treat patients with
chronic hepatitis B co-infected with HIV.

Lactic acidosis and severe hepatomegaly with steatosis, including fatal cases, have been
reported with the use of nucleoside analogs alone or in combination with antiretrovirals.

A majority of these cases have been in women. Obesity and prolonged nucleoside exposure
may be risk factors. Particular caution should be exercised when administering nucleoside
analogs to any patient with known risk factors for liver disease; however, cases have also
been reported in patients with no known risk factors. Treatment with HEPSERA should be
suspended in any patient who develops clinical or laboratory findings suggestive of lactic
acidosis or pronounced hepatotoxicity (which may include hepatomegaly and steatosis even
in the absence of marked transaminase elevations).

Description and justification of route of administration, dosages, dosage modifications and

method of administration

The recommended dose of HEPSERA in chronic hepatitis B patients for patients ≥12 years
of age with adequate renal function is 10 mg, once daily, taken orally, without regard to food.
The optimal duration of treatment is unknown (42).

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Adefovir dipivoxil is absorbed rapidly following oral administration. The drug is cleaved to
adefovir which, in turn, is phosphorylated intracellularly to adefovir diphosphate (the active
moiety). Following a single dose of adefovir dipivoxil, the bioavailability of adefovir was 59%.

Adefovir is excreted renally as unchanged drug (steady-state renal clearance [CLR] 154
mL/h/kg). The median steady-state terminal elimination half-life was ≊7 hours in 14 patients
with chronic hepatitis B. In patients with moderate to severe renal dysfunction, the systemic
exposure of adefovir increased and the CLR decreased.

In healthy volunteers, there were no clinically relevant drug interactions when lamivudine,
paracetamol (acetaminophen), ibuprofen or cotrimoxazole (trimethoprim/sulfamethoxazole)
were coadministered with adefovir dipivoxil.

Significantly increased drug exposures were seen when HEPSERA was administered to
adult patients with renal impairment. Therefore, the dosing interval of HEPSERA should be
adjusted in adult patients with baseline creatinine clearance <50 mL/min using the following
suggested guidelines (see Table 4). The safety and effectiveness of these dosing interval
adjustment guidelines have not been clinically evaluated. Renal function should be monitored
in all patients; patients receiving concomitant drugs that are excreted renally or known to
affect renal function should be closely monitored (43). Additionally, it is important to note that
these guidelines were derived from data in patients with pre-existing renal impairment at
baseline. They may not be appropriate for patients in whom renal insufficiency evolves
during treatment with HEPSERA. Therefore, clinical response to treatment and renal function
should be closely monitored in these patients. (42). Patients who discontinue adefovir
dipivoxil should be closely monitored for exacerbation of hepatitis (43).

Creatinine Clearance (mL/min)Creatinine clearance calculated by Cockcroft-Gault

method using lean or ideal body weight.

≥50 30–49 10–29 Hemodialysis Patients

Recommended
dose 10 mg every 10 mg every 10 mg every 10 mg every 7 days following
and dosing 24 hours 48 hours 72 hours dialysis
interval

Table 4.

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The pharmacokinetics of adefovir have not been evaluated in non-hemodialysis patients with
creatinine clearance <10 mL/min; therefore, no dosing recommendation is available for these
patients.

No clinical data are available to make dosing recommendations in adolescent patients with
renal insufficiency

Tenofovir disoproxil fumarate (Viread®)

Summary of findings from non-clinical and clinical studies

Tenofovir disoproxil fumarate (VIREAD®), the oral prodrug of tenofovir, is a nucleotide
analog reverse transcriptase inhibitor with activity against HIV and HBV. (G. alvarez-uria,
british hiv association 2009) For this reason it has been widely used in HIV-infected patients
with HBV coinfection.
The pharmacological action is exerted through inhibiting viral polymerase- reverse
transcriptase by direct binding, and after incorporation into DNA, by termination of the DNA
chain due to the absence of a requisite 3’hydroxyl on the tenofovir molecule.
Functioning of the enzyme polymerase (reverse transcriptase) is necessary for the
replication of HIV and HBV by producing viral DNA. It remains active against lamivudine-
resistant HBV, and it has known activity against HBV both in patients with HBV
monoinfection and in patients with HIV-1 and HBV coinfection.

Tenofovir is structurally similar to adefovir. In vitro studies showed that tenofovir and
adefovir are equipotent. Because tenofovir appears to be less nephrotoxic, the approved
dose is much higher than that of adefovir, 300mg vs 10mg daily. This may explain why
tenofovir has more potent antiviral activity in clinical studies. Tenofovir is generally well
tolerated but it has been rarely reported to cause Fanconi syndrome and renal insufficiency
(44).

Tenofovir DF, is currently approved in the United States and more than 50 other countries for
the treatment of humon-immunodeficiency virus type 1 (HIV-1), and it was recently approved
for the treatment of chronic HBV infection in the United States, Canada, Europe, Australia
and Turkey.
Recently two fase 3 studies were performed to compare the safety and efficacy of tenofovir
DF with adefovir dipivoxil in HBeAg-negative or HBeAg positive patients (32). In these
studies among patients with chronic HBV infection, tenofovir DF at a daily dose of 300mg

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had superior antiviral efficacy with a similar safety profile as compared with adefovir dipivoxil
at a daily dose of 10mg through week 48. The primairy endpoint of both HBV DNA level of
less than 400 copies/ml and histologic improvement was reached in 71% vs. 49% among
HBeAg negative patients and 67% vs. 12% among HBeAg positive patients.
Another recent long-term follow-up study of 34 months showed that tenofovir DF was able to
controle HBV replication in most HIV-coinfected patients, regardless of previous lamivudine
treatment. A sustained virologic respons rate of HBV DNA was achieved in 83%.

Summary of known and potential risks and benefits

Lactic acidosis and severe hepatomegaly with steatosis, including fatal cases, have
been reported with the use of nucleoside analogs, including VIREAD, in combination
with other antiretrovirals. A majority of these cases have been in women. Obesity and
prolonged nucleoside exposure may be risk factors. Particular caution should be
exercised when administering nucleoside analogs to any patient with known risk factors
for liver disease; however, cases have also been reported in patients with no known risk
factors. Treatment with VIREAD should be suspended in any patient who develops
clinical or laboratory findings suggestive of lactic acidosis or pronounced hepatotoxicity
(which may include hepatomegaly and steatosis even in the absence of marked
transaminase elevations).

Discontinuation of anti-HBV therapy, including VIREAD, may be associated with severe

acute exacerbations of hepatitis. Patients infected with HBV who discontinue VIREAD
should be closely monitored with both clinical and laboratory follow-up for at least
several months after stopping treatment. If appropriate, resumption of anti-hepatitis B
therapy may be warranted.

Tenofovir is principally eliminated by the kidney. Renal impairment, including cases of

acute renal failure and Fanconi syndrome (renal tubular injury with severe
hypophosphatemia), has been reported with the use of VIREAD .
It is recommended that creatinine clearance be calculated in all patients prior to
initiating therapy and as clinically appropriate during therapy with VIREAD. Routine
monitoring of calculated creatinine clearance and serum phosphorus should be
performed in patients at risk for renal impairment.
Dosing interval adjustment of VIREAD and close monitoring of renal function are
recommended in all patients with creatinine clearance <50 mL/min.
No safety or efficacy data are available in patients with renal

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impairment who received VIREAD using these dosing guidelines, so the potential
benefit of VIREAD therapy should be assessed against the potential risk of renal
toxicity. VIREAD should be avoided with concurrent or recent use of a nephrotoxic agent.

Description and justification of route of administration and dosage

Dosages, dosage modifications and method of administration

For the treatment of HIV-1 or chronic hepatitis B: The dose of VIREAD is 300 mg once
daily taken orally, without regard to food.
In the treatment of chronic hepatitis B, the optimal duration of treatment is unknown.

Significantly increased drug exposures occurred when VIREAD was administered to

patients with moderate to severe renal impairment.
Therefore, the dosing interval of VIREAD should be adjusted in patients with baseline
creatinine clearance <50 mL/min. Dosing interval needs to be adjusted to every 48 hours or
every 72 to 96 hours in patients with a creatinine clearance of 30-49 or 10-29 ml/min
respectively. In hemodialysis patients dose should be given every 7 days or after a total of
approximately 12 hours of dialysis. (Generally once weekly assuming three hemodialysis
sessions a week of approximately 4 hours duration).
These dosing interval recommendations are based on modeling of single-dose
pharmacokinetic data in non-HIV and non-HBV infected subjects with varying degrees of
renal impairment, including end-stage renal disease requiring hemodialysis. The safety and
effectiveness of these dosing interval adjustment recommendations have not been clinically
evaluated in patients with moderate or severe renal impairment, therefore clinical response
to treatment and renal function should be closely monitored in these patients
No dose adjustment is necessary for patients with mild renal impairment (creatinine
clearance 50–80 mL/min). Routine monitoring of calculated creatinine clearance and
serum phosphorus should be performed in patients with mild renal impairment

The pharmacokinetics of tenofovir have not been evaluated in non-hemodialysis

patients with creatinine clearance <10 mL/min; therefore, no dosing recommendation is
available for these patients.

VIREAD is available as tablets. Each tablet contains 300 mg of tenofovir disoproxil

fumarate, which is equivalent to 245 mg of tenofovir disoproxil. The tablets are
almondshaped, light blue, film-coated, and debossed with “GILEAD” and “4331” on one side

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and with “300” on the other side (45).

No preparation and labelling of Investigational Medicinal Products is applicable to all of the

investigational products.

Drug accountability
The investigator or the hospital pharmacist must maintain an adequate record of the receipt
of all trial supplies. Dispensation and return, or if applicable, destruction, of the study
medication must be documented by using the appropriate forms. All these records must be
available for inspection at any time.
During the trial compliance will be assessed by counting returned dosage units. Subject will
also be asked to record their drug intake in a diary.

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7. METHODS

7.1 Study parameters/endpoints

7.1.1. Main study parameter/endpoint

The primary objective is to demonstrate the superior efficacy of combination therapy (Peg-
IFN and adefovir or Peg-IFN and tenofovir) in loss of HBsAg compared to no treatment in
subjects with chronic HBV and low viral loads.
HBsAg loss is defined as HBsAg level < 0.05 IU/mL.

7.1.2. Secondary study parameters/endpoints

The secondary objectives are to evaluate:
a. the rate of HBsAg loss and anti-HBs serconversion,
b. To establish predictive markers for response at baseline and during the first 12 weeks of
treatment. ( see paragraph 2. objectives)

7.1.3. Other study parameters

Baseline characteristics will be documented of all patients. Age, weight, sex, medical history,
cause of infection, interferon naïve, previous treatment, histology activity index, ISHAK
fibrosis score, prescence of cirrhosis, ethnicity will be documented.

7.2. Randomisation, blinding and treatment allocation

Patients will be randomized unblinded to one of the 3 groups. A stratification for hepatitis B
genotype A will be performed.

7.3. Study procedures

Study procedures are listed in table 2.

7.4. Withdrawal of individual subjects

Subjects can leave the study at any time for any reason if they wish to do so without any
consequences. The investigator can decide to withdraw a subject from the study for urgent
medical reasons.

7.4.1.Specific criteria for withdrawal

Subjects may be withdrawn from the treatment if:
1. a serious side effect occurs.

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2. they fail to comply with the protocol requirements or to cooperate with the
investigator.
Subjects must discontinue all study medication or have their treatment modified for the
following reasons:
1. all study medication must be discontinued and a subject must be withdrawn from the
trial if he/she withdraws informed consent.
2. all study medication must be discontinued if a subject has a positive pregnancy
test,or if the subject/partner is non-compliant with contraception requirements.

7.5. Replacement of individual subjects after withdrawal

After withdrawal of an individual subjects no replacement will take place, 10% drop out is
calculated in the power of this study.

7.6.Follow-up of subjects withdrawn from treatment

Follow up of subject withdrawn from treatment will be done according the EASL HBV
guidelines.

7.7.Premature termination of the study

The study will be terminated prematurely in case of serious adverse events.

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8.0 SAFETY REPORTING

8.1 Safety Assessments

Safety assessments will be performed during screening, at baseline, at weeks 1, 2, 4, 6, 8,

12 and then every 6 weeks throughout the 48 weeks treatment period. Safety assessments
will continue at weeks 50, 52, 56, 60 and 72 during the follow up period, as outlined in
Section 4.1. Patients prematurely discontinued from test drug therapy will have a safety
assessment 4 and 12 weeks after their last dose of study medication. Clinically significant
laboratory abnormalities should prompt repeat measures no less frequently than every 4
weeks or in shorter intervals as clinically indicated, with appropriate clinical management,
until values return to normal or baseline levels.
Measures of safety will include assessment of AEs, vital signs (systolic and diastolic blood
pressure and pulse rate), and laboratory tests in addition to those carried out for efficacy as
well as documentation of dose adjustments and premature withdrawals from treatment for
safety or tolerability reasons. Patients with preexisting ophthalmologic disorders (e.g. diabetic
or hypertensive retinopathy) should receive periodic ophthalmologic exams during therapy.

8.2 Section 10 WMO event

In accordance to section 10, subsection 1, of the WMO, the investigator will inform the
subjects and the reviewing accredited METC if anything occurs, on the basis of which it
appears that the disadvantages of participation may be significantly greater than was
foreseen in the research proposal. The study will be suspended pending further review by
the accredited METC, except insofar as suspension would jeopardise the subjects’ health.
The investigator will take care that all subjects are kept informed.

8.3. Adverse and serious adverse events

Adverse events are defined as any undesirable experience occurring to a subject during a
clinical trial, whether or not considered related to the investigational drug.

A serious adverse event is any untoward medical occurrence or effect that at any dose
results in death;
- is life threatening (at the time of the event);
- requires hospitalisation or prolongation of existing inpatients’ hospitalisation;
- results in persistent or significant disability or incapacity;
- is a congenital anomaly or birth defect;

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- is a new event of the trial likely to affect the safety of the subjects, such as an
unexpected outcome of an adverse reaction, lack of efficacy of an IMP used for the
treatment of a life threatening disease, major safety finding from a newly completed
animal study, etc.

Medical and scientific judgment should be exercised in deciding whether expedited reporting
is appropriate in other situations, such as important medical events that may not be
immediately life-threatening or result in death or hospitalization but may jeopardize the
patient or may require intervention to prevent one of the outcomes listed in the definitions
above. These situations should also usually be considered serious.

The term severe is a measure of intensity, thus a severe adverse event is not necessarily
serious. For example, nausea of several hours’ duration may be rated as severe, but may not
be clinically serious.

A death occurring during the study or which comes to the attention of the investigator within 4
weeks after stopping the treatment or during the 24 weeks of protocol-defined follow-up
period, whether considered treatment-related or not, must be reported.

Any pregnancy which occurs during a clinical study with an investigational drug must be
reported as an SAE for tracking purposes. All pregnancies which are identified during this
study need to be followed to conclusion and outcome reported.

Female patients should immediately inform the Investigator of any pregnancies and should
be instructed by the Investigator to stop taking study medication. (Pregnancies occurring up
to 3 months after the completion of the study must also be reported to the Investigator). The
Investigator should counsel the patient, discuss the risks of continuing with the pregnancy
and the possible effects on the fetus. Monitoring of the patient should continue until the
conclusion of the pregnancy.

Such preliminary reports will be followed by detailed descriptions later which will include
copies of hospital case reports, autopsy reports and other documents when requested and
applicable.

For serious and all other AEs, the following must be assessed and recorded on the AE page
of the CRF: intensity, relationship to test substance, action taken regarding test substance,
and outcome to date.

On the SAE form a drug/event relationship must be provided for each study medication (Yes
or No) and each event. “Yes” must be entered for a causality of remote, possible, probable,

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i.e., there is a reasonable suspected causal relationship, or causality is unknown. No is to be

entered for a causality of not related.

The investigator will notify the Ethics Committee (METC) of such an event in writing in
accordance with local laws and regulations. The investigator will notify the Health Authorities.
The definitions for and procedures for reporting SAEs to Health Authorities will be taken from
the ICH-GCP and the Dutch law ‘Wet medisch-wetenschappelijk onderzoek met mensen’
(WMO).

8.3.1. uspected unexpected serious adverse reactions (SUSAR)

Adverse reactions are all untoward and unintended responses to an investigational product
related to any dose administered.

Unexpected adverse reactions are adverse reactions, of which the nature, or severity, is not
consistent with the applicable product information (e.g. Investigator’s Brochure for an
unapproved IMP or Summary of Product Characteristics (SPC) for an authorised medicinal
product).

The investigator will report expedited the following SUSARs to the METC:
− SUSARs that have arisen in the clinical trial that was assessed by the METC;
− SUSARs that have arisen in other clinical trial of the same sponsor and with the
same medicinal product, and that could have consequences for the safety of the
subjects involved in the clinical trial that was assessed by the METC.
The remaining SUSARs are recorded in an overview list (line-listing) that will be submitted
once every half year to the METC. This line-listing provides an overview of all SUSARs from
the study medicine, accompanied by a brief report highlighting the main points of concern.
The sponsor will report expedited all SUSARs to the competent authority, the Medicine
Evaluation Board and the competent authorities in other Member States.

The expedited reporting will occur not later than 15 days after the investigator has first
knowledge of the adverse reactions. For fatal or life threatening cases the term will be
maximal 7 days for a preliminary report with another 8 days for completion of the report.

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8.3.2 Annual safety report

In addition to the expedited reporting of SUSARs, the sponsor will submit, once a year
throughout the clinical trial, a safety report to the accredited METC, competent authority,
Medicine Evaluation Board and competent authorities of the concerned Member States.
This safety report consists of:
− a list of all suspected (unexpected or expected) serious adverse reactions, along
with an aggregated summary table of all reported serious adverse reactions,
ordered by organ system, per study;
− a report concerning the safety of the subjects, consisting of a complete safety
analysis and an evaluation of the balance between the efficacy and the
harmfulness of the medicine under investigation.

8.3.3. Pregnancy

Pregnancy is to be strictly avoided during the course of this trial. However, if a female subject
becomes pregnant during the study she must be instructed to stop taking the trial medication
and immediately inform the investigator. Pregnancies occurring up to 12 weeks after the
completion of the trial medication must also be reported to the investigator. The investigator
should counsel the subject, discuss the risks of continuing with the pregnancy and the
possible effects on the fetus. Monitoring of the patient should continue until conclusion of the
pregnancy.

8.4 Follow-up of adverse events

All adverse events will be followed until they have abated, or until a stable situation has been
reached. Depending on the event, follow up may require additional tests or medical
procedures as indicated, and/or referral to the general physician or a medical specialist.

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9 STATISTICAL ANALYSIS

9.1 Descriptive statistics

The main efficacy variable in the comparison of treatments A and B to treatment C ( no

treatment) is the proportion of subjects in each treatment group achieving loss of HBsAg.
The primary objective is to demonstrate superior efficacy of treatment A and/or B to
treatment C.

The sample sizes were calculated as follows:

Per treatment group 44 per group (88 for both groups) is needed to achieve 81% power to
detect a difference between the two treatment groups and the control group. The proportion
in the PEG/IFN and adefovir group is assumed to be 0.01 under the null hypothesis and 0.20
under the alternative hypothesis. The proportion in the control group (no treatment) is 0.01.
The statistical test used is the two-sided Fisher's Exact test . The significance level of the test
was targeted at 0.05. A group sample of 44 persons in the control group is needed to
achieve a 81% power to detect a difference between the PEG/IFN adefovir or PEG/IFN
tenofovir.
Regarding a 10% drop-out due to peginterferon side-effects or other reasons a total of 50
patients are needed in each group.

Univariate logistic regression analyses will be performed to identify predictors of response to

the combination therapy of PEGASYS and ADF dipivoxil (Hepsera) and PEGASYS and
tenofovir. Significant predictors from the univariate analysis will be combined into a
multivariate logistic regression analysis. As a rule of thumb, the number of potential
predictors to be included in a multivariate analysis should not exceed the number of
responders (or non-responders, whichever is the lowest) divided by 10. With 100 patients
included, the maximum number of predictors in a multivariate analysis is five, assuming a
response rate of 0.50. Initially, the number of significant predictors from univariate analysis to
be included in the multivariate analysis will be restricted following multi colinearity
diagnostics. If the number of potential predictors remains too large however, a bootstrap
procedure will be followed drawing 200 samples of the same size as the original (n=100) with
replacement, each time repeating the multivariate logistic regression analysis. Descriptives of
this bootstrap procedure will be reported to identify a stable predictor set. The level of
significance during all these analyses is set at 0.05.

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 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

9.2 .Interim analysis

After inclusion of 50,100 and 150 patients and yearly during longterm follow-up an interim
analysis will be performed by the investigator. The statistical test is the two-sided Fisher's
exact test. Stopping rules for the study are the occurrence of serious adverse events.

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 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

10 ETHICAL CONSIDERATIONS

10.1 Regulation statement

This trial will be conducted in accordance with the current ICH-GCP Guidelines.
Good clinical practice (GCP) is an international ethical and scientific quality standard for
designing, conducting, recording, and reporting trials that involve the participation of human
subjects. Compliance with these standard provides public assurance that the rights, safety
and well being of trial subjects are protected, consistent with the principles that have their
origin in the Declaration of Helsinki, and that the clinical trial data are credible.

10.2 Recruitment and consent

Prior to entry in the study, the investigator or a person designated by the investigator must
explain to potential subjects or their legally acceptable representative the study and the
implications of participation. Subjects will be informed that their participation is voluntary and
that they may withdraw from the trial at any time. They will be informed that choosing not to
participate or to withdraw from the trial will not have an impact on the care the subject will
receive for the treatment of his/her disease. Finally, they will be told that their records may be
accessed by the IEC/IRB, regulatory authorities and authorized representatives of the
sponsor without violating the confidentiality of the subject, to the extent permitted by the
applicable laws and or regulations. By signing the ICF, the subject is authorizing such
access.
In case the subject is unable to read and write, an impartial witness must confirm the
informed consent.
The subject will be given sufficient time to read the ICF and to ask additional questions. After
this explanation and before entry in the trial, consent should be appropriately recorded by
means of the subject’s personally dated signature or by the signature of an independent
witness who certifies the subject’s consent in writing. After having obtained the consent, a
copy or the signed and dated informed consent must be given to the subject.
Any information relevant to the subject’s willingness to participate in the study will be
provided to the subject in a timely manner by means of updated ICF. This amended ICF will
be signed and dated by the subject and the investigator to document the willingness of the
subject to continue with the trial.
This signed and dated amended version will be filled together with initial signed and dated
ICF.

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 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

10.3 Compensation for injury

The sponsor/investigator has a liability insurance which is in accordance with article 7,
subsection 6 of the WMO.

The sponsor (AMR) has an insurance which is in accordance with the legal requirements in
the Netherlands (Article 7 WMO and the Measure regarding Compulsory Insurance for
Clinical Research in Humans of 23th June 2003). This insurance provides cover for damage
to research subjects through injury or death caused by the study.
1. € 450.000,-- (i.e. four hundred and fifty thousand Euro) for death or injury for each
subject who participates in the Research;
2. € 3.500.000,-- (i.e. three million five hundred thousand Euro) for death or injury for
all subjects who participate in the Research;
3. € 5.000.000,-- (i.e. five million Euro) for the total damage incurred by the
organisation for all damage disclosed by scientific research for the Sponsor as
‘verrichter’ in the meaning of said Act in each year of insurance coverage.

The insurance applies to the damage that becomes apparent during the study or within 4
years after the end of the study.

For subject’s participating in this study insurance is covered by the AMR (Academisch
Medisch Centrum Medical Research)

10.4 Incentives

Except for travelling expenses no special compensation or incentives will be applicable to

participating subjects.

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11. ADMINISTRATIVE ASPECTS AND PUBLICATION

11.1 Handling and storage of data and documents

Storage of al study information will be carried out by a database. The outline for this
database needs to be specially suited and designed for HBV patients. A person designated
by the investigator concerning the development of this database will be applied and will take
care of processing this information. Human materials (PBMC’s) will be kept in a -800C freezer
until ready for processing. Information concerning this study will only be available by the
study investigator and the designated people by the use of passwords.

11.2 Amendments
Amendments are changes made to the research after a favourable opinion by the accredited
METC has been given. All amendments will be notified to the METC that gave a favourable
opinion.

11.3 Annual progress report

The investigator will submit a summary of the progress of the trial to the accredited METC
once a year. Information will be provided on the date of inclusion of the first subject, numbers
of subjects included and numbers of subjects that have completed the trial, serious adverse
events/ serious adverse reactions, other problems, and amendments.

11.4 End of study report

The investigator will notify the accredited METC of the end of the study within a period of 8
weeks. The end of the study is defined as the last patient’s last visit.
In case the study is ended prematurely, the investigator will notify the accredited METC,
including the reasons for the premature termination.
Within one year after the end of the study (end of short term follow-up after 72 weeks), the
investigator/sponsor will submit a final study report with the results of the study, including any
publications/abstracts of the study, to the accredited METC.

11.5 Public disclosure and publication policy

After interim analysis and yearly during long term follow up (5 years) the results of this study
will be presented and published.
In accordance with generally recognized principles of scientific collaboration, co-authorship
with any company personnel wil be discussed and mutually agreed upon before submission
of a manuscript to a publisher.

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 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

12. REFERENCES
Reference List

1. World Health Organization. Hepatitis B. World Health Organization Fact Sheet 204
(Revised August 2008). World Health Organization 2007.

2. Lee WM. Hepatitis B virus infection. N Engl J Med 1997 Dec 11;337(24):1733-1745.

3. Yang HI, Lu SN, Liaw YF, You SL, Sun CA, Wang LY, et al. Hepatitis B e antigen and
the risk of hepatocellular carcinoma. N Engl J Med 2002 Jul 18;347(3):168-174.

4. Chen YC, Sheen IS, Chu CM, Liaw YF. Prognosis following spontaneous HBsAg
seroclearance in chronic hepatitis B patients with or without concurrent infection.
Gastroenterology 2002 Oct;123(4):1084-1089.

5. Yuen MF, Wong DK, Sablon E, Tse E, Ng IO, Yuan HJ, et al. HBsAg seroclearance in
chronic hepatitis B in the Chinese: virological, histological, and clinical aspects.
Hepatology 2004 Jun;39(6):1694-1701.

6. Takkenberg RB, Zaaijer HL, Weegink C.J., Terpstra V, Dijkgraaf MG, Jansen P.L.M., et
al. Baseline HBsAg level predict HBsAg loss in HBeAg negative chronic hepatitis B
patients treated with a combination of Peginterferon alfa-2a and Adefovir: An interim
analysis. J Hepatol 2009 Apr;50(Supp 1):S9-S10.

7. World Health Organization. The World Health Repotrt 1997. 1997.

8. Perz JF, Armstrong GL, Farrington LA, Hutin YJ, Bell BP. The contributions of hepatitis
B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. J
Hepatol 2006 Oct;45(4):529-538.

9. Goldstein ST, Zhou F, Hadler SC, Bell BP, Mast EE, Margolis HS. A mathematical
model to estimate global hepatitis B disease burden and vaccination impact. Int J
Epidemiol 2005 Dec;34(6):1329-1339.

10.World Health Organization. Hepatitis B. World Health Organization Fact Sheet 204
(Revised October 2000). 2007.

11.Lavanchy D. Hepatitis B virus epidemiology, disease burden, treatment, and current

and emerging prevention and control measures. J Viral Hepat 2004 Mar;11(2):97-107.

12.Dienstag JL. Hepatitis B virus infection. N Engl J Med 2008 Oct 2;359(14):1486-1500.

13.Lok AS, McMahon BJ. Chronic hepatitis B. Hepatology 2001 Dec;34(6):1225-1241.

14.Dienstag JL, Schiff ER, Wright TL, Perrillo RP, Hann HW, Goodman Z, et al.
Lamivudine as initial treatment for chronic hepatitis B in the United States. N Engl J
Med 1999 Oct 21;341(17):1256-1263.

15.Marcellin P, Chang TT, Lim SG, Tong MJ, Sievert W, Shiffman ML, et al. Adefovir
dipivoxil for the treatment of hepatitis B e antigen-positive chronic hepatitis B. N Engl J
Med 2003 Feb 27;348(9):808-816.

16.Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, Chang TT, Kitis G, Rizzetto M, et al.
Adefovir dipivoxil for the treatment of hepatitis B e antigen-negative chronic hepatitis B.
N Engl J Med 2003 Feb 27;348(9):800-807.

Version number: 4, date 16-07-2013 46 of 54

 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

17.Janssen HL, Gerken G, Carreno V, Marcellin P, Naoumov NV, Craxi A, et al. Interferon
alfa for chronic hepatitis B infection: increased efficacy of prolonged treatment. The
European Concerted Action on Viral Hepatitis (EUROHEP). Hepatology 1999
Jul;30(1):238-243.

18. Wong DK, Cheung AM, O'Rourke K, Naylor CD, Detsky AS, Heathcote J. Effect of
alpha-interferon treatment in patients with hepatitis B e antigen-positive chronic
hepatitis B. A meta-analysis. Ann Intern Med 1993 Aug 15;119(4):312-323.

19.Tompkins WA. Immunomodulation and therapeutic effects of the oral use of interferon-
alpha: mechanism of action. J Interferon Cytokine Res 1999 Aug;19(8):817-828.

20. Kuhen KL, Samuel CE. Mechanism of interferon action: functional characterization of
positive and negative regulatory domains that modulate transcriptional activation of the
human RNA-dependent protein kinase Pkr promoter. Virology 1999 Feb 1;254(1):182-
195.

21.Kuhen KL, Vessey JW, Samuel CE. Mechanism of interferon action: identification of
essential positions within the novel 15-base-pair KCS element required for
transcriptional activation of the RNA-dependent protein kinase pkr gene. J Virol 1998
Dec;72(12):9934-9939.

22. Franchis de R., Hadengue A, Lau G, Lavanchy D, Lok A, McIntyre N, et al. EASL
International Consensus Conference on Hepatitis B. 13-14 September, 2002 Geneva,
Switzerland. Consensus statement (long version). J Hepatol 2003;39 Suppl 1:S3-25.

23.Lok AS, Zoulim F, Locarnini S, Bartholomeusz A, Ghany MG, Pawlotsky JM, et al.
Antiviral drug-resistant HBV: standardization of nomenclature and assays and
recommendations for management. Hepatology 2007 Jul;46(1):254-265.

24.Dienstag JL. Benefits and risks of nucleoside analog therapy for hepatitis B.
Hepatology 2009 May;49(5 Suppl):S112-S121.

25.Westland CE, Yang H, Delaney WE, Gibbs CS, Miller MD, Wulfsohn M, et al. Week 48
resistance surveillance in two phase 3 clinical studies of adefovir dipivoxil for chronic
hepatitis B. Hepatology 2003 Jul;38(1):96-103.

26.Chu CJ, Hussain M, Lok AS. Quantitative serum HBV DNA levels during different
stages of chronic hepatitis B infection. Hepatology 2002 Dec;36(6):1408-1415.

27.Fattovich G, Rugge M, Brollo L, Pontisso P, Noventa F, Guido M, et al. Clinical,

virologic and histologic outcome following seroconversion from HBeAg to anti-HBe in
chronic hepatitis type B. Hepatology 1986 Mar;6(2):167-172.

28.Hoofnagle JH, Dusheiko GM, Seeff LB, Jones EA, Waggoner JG, Bales ZB.
Seroconversion from hepatitis B e antigen to antibody in chronic type B hepatitis. Ann
Intern Med 1981 Jun;94(6):744-748.

29.Hoofnagle JH, Doo E, Liang TJ, Fleischer R, Lok AS. Management of hepatitis B:
summary of a clinical research workshop. Hepatology 2007 Apr;45(4):1056-1075.

30.Moucari R, Mackiewicz V, Lada O, Ripault MP, Castelnau C, Martinot-Peignoux M, et

al. Early serum HBsAg drop: a strong predictor of sustained virological response to
pegylated interferon alfa-2a in HBeAg-negative patients. Hepatology 2009
Apr;49(4):1151-1157.

31.Fernandez-Soto L, Gonzalez A, Escobar-Jimenez F, Vazquez R, Ocete E, Olea N, et

al. Increased risk of autoimmune thyroid disease in hepatitis C vs hepatitis B before,

Version number: 4, date 16-07-2013 47 of 54

 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

during, and after discontinuing interferon therapy. Arch Intern Med 1998 Jul
13;158(13):1445-1448.

32.Marcellin P, Heathcote EJ, Buti M, Gane E, de Man RA, Krastev Z, et al. Tenofovir
Disoproxil Fumarate versus Adefovir Dipivoxil for Chronic Hepatitis B. N Engl J Med
2008 Dec 4;359(23):2442-2455.

33.Heathcote EJ, Gane E, de Man RA, Chan S, Sievert W, Mauss S, et al. Two year
tenofovir disiproxil fumarate (TDF) treatment and adefovir dipivoxil (ADV) switch data in
HBeAg positive patients with chronic hepatitis B (study 103), preliminary results.
Hepatology 2008 Oct 1;48(S4):376A.

34.Buster EH, van Erpecum KJ, Schalm SW, Zaaijer HL, Brouwer JT, Gelderblom HC, et
al. Treatment of chronic hepatitis B virus infection - Dutch national guidelines. Neth J
Med 2008 Jul;66(7):292-306.

35. EMEA summary of product characteristics PEGASYS; 2009.

36. Perry CM, Jarvis B. Spotlight on peginterferon-alpha-2a (40KD) in chronic hepatitis C.

BioDrugs 2002;16(3):213-217.

37.Cooksley WG, Piratvisuth T, Lee SD, Mahachai V, Chao YC, Tanwandee T, et al.
Peginterferon alpha-2a (40 kDa): an advance in the treatment of hepatitis B e antigen-
positive chronic hepatitis B. J Viral Hepat 2003 Jul;10(4):298-305.

38.Marcellin P, Lau GK, Bonino F, Farci P, Hadziyannis S, Jin R, et al. Peginterferon alfa-
2a alone, lamivudine alone, and the two in combination in patients with HBeAg-
negative chronic hepatitis B. N Engl J Med 2004 Sep 16;351(12):1206-1217.

39.Janssen HL, van ZM, Senturk H, Zeuzem S, Akarca US, Cakaloglu Y, et al. Pegylated
interferon alfa-2b alone or in combination with lamivudine for HBeAg-positive chronic
hepatitis B: a randomised trial. Lancet 2005 Jan 8;365(9454):123-129.

40.Perrillo R, Hann HW, Mutimer D, Willems B, Leung N, Lee WM, et al. Adefovir dipivoxil
added to ongoing lamivudine in chronic hepatitis B with YMDD mutant hepatitis B virus.
Gastroenterology 2004 Jan;126(1):81-90.

41.Peters MG, Hann HH, Martin P, Heathcote EJ, Buggisch P, Rubin R, et al. Adefovir
dipivoxil alone or in combination with lamivudine in patients with lamivudine-resistant
chronic hepatitis B. Gastroenterology 2004 Jan;126(1):91-101.

42.EMEA product information HEPSERA; 2009.

43.Dando T, Plosker G. Adefovir dipivoxil: a review of its use in chronic hepatitis B. Drugs
2003;63(20):2215-2234.

44.Lok AS, McMahon BJ. Chronic hepatitis B. Hepatology 2007 Jan 26;45(2):507-539.

45.EMEA product information VIREAD; 2009.

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 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

13. APPENDIX

Figure 1. Natural history of chronic HBV infection

HBeAg positive HBeAg negative; anti-HBe positive

11 10

10 HBV DNA
9
ALT
9 8
log HBV DNA (cop/mL)

8
7
7

ALT (xULN)
6
6
5
5
4
4
3
3

2 2

1 1

0 0
years
Immuno tolerant phase Immuneactive Immune controle phase Chronic active hepatitis B,
phase HBeAg negative

Mild Moderate/ None/ Mild Moderate/ severe

Severe

Histology Activity Index

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 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

Schedule of Assessments

Table 1. Schedule of Assesments

Assessment/ Procedure Screening Study treatment Period Peg-INF and ADF, Peg-IFN and tenofovir or no treatment Follow-up

(weeks (weeks) (weeks)

Study weeks -4 to 0 B Day 3 1 2 4 6 8 12 18 24 30 36 42 48 2 4 8 12 16 20 24

Informed consent, medical history X

Physical examination X X X X X X X X X X X X X

Liver biopsy (preceding 12 months) X X

Urine or serum pregnancy test a X x X X X X X X X X X X X X X X

Chest X-ray, selected patients b X

Ultrasound of the liver X __

Fibroscan X X

CT or MRI, selected patients c

Electrocardiogram, selected patients d X

Anti-HAV IgM, anti-HIV, anti-HCV, X

HCV-RNA, anti-HDV

Ophthalmoloic examination e (on indication) X

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 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

Assessment/ Procedure Long term Follow-up

(years)

Years 1 2 3 4 5

Physical examination X X X X X

Complete hematology with differential WBC, and platelets g

(on indication)

Chemistry 1 g X X X X X

g
Chemistry 2 (on indication)

Chemistry 3 (on indication)

Fibroscan f X X X

HBeAg, anti-HBe (frozen for central processing)HBV-DNA X X X X X

i
(frozen for central processing) HBsAg, anti-HBs (frozen for

central processing)

Ultrasound, CT or MRI (on indication) c

PBMC’s (frozen for central processing) X X X X X

Paxgene TM RNA (k) X X X X X

Plasmatube (proteomics profiling) (l) X X X X X

Quality of life (m) X X X X X

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 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

Table 2 (cont.) Schedule of Assessments

Assessment / Procedure Screening Study treatment Period Follow-up

(weeks) (weeks) (week)

a a a a a a
Study weeks -4 to 0 B day 3 1 2 4 6 8 12 18 24 30 36 42 48 50 52 56 60 64 68 72

Complete hematology with differential WBC, and platelets g X X X X X X X X X X X X X X X X X X X X

g
Chemistry 1 X X X X X X X X X X X X X X X X X X

Chemistry 2 g X X X X X X X X X

Chemistry 3 X X X

g
Urinalysis, dipstick (on indication) X X X X X X X X X X X

Ceruloplasmin, alfa-1-antitrypsin X

Alfa-fetoprotein X X X X

AMA, ANA, ASMA, thyroid peroxidase antibodies, X X

h
selected patients

HBeAg, anti-HBe (frozen for central processing) X X X

HBsAg, anti-HBs (frozen for central processing) X X X X X X X X X X X X X X X X X X X X X X

HBV-DNA (frozen for central processing) i X X X X X X X X X X X X X X X X X X X X X X

Alpha-2 macro globulin, Haptoglobin, Apolipoprotein A1 X

Viral sequencing X X X X X X X X X X X X X X X X X X X X

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 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

PBMC’s (frozen for central processing) X X X X X X X X X X X X

TM
PAXgene DNA (j) (human genomics) X

TM (k)
PAXgene RNA (human genomics) X X X X X X

Plasma tube (proteomics profiling ) (l) X X X X X X

Adverse events X X X X X X X X X X X X X X X X X X X X X

Quality of life (m) X X X X X

Chemistry 1 : ASAT, ALAT,

Chemistry 2: sodium, chloride, potassium, bilirubin, creatinine, urea, prothrombin time, APTT, antitrombine III, alkaline phosphatase, calcium, phosphorus, total protein, albumin, uric acid, TSH, free
T4 and glucose.
Chemistry 3: cholesterol, triglycerides, sober glucose

B: baseline
(a) For females of child bearing potential only, a pregnancy test will be performed within 24 hours prior to first dose. A pregnancy test must be done at any time a secondary amenorrhea of more
than 1 week occurs.
(b) Only for patients with pre-existing pulmonary disease (Not necessary if (1) a chest X-ray available to the investigator has been obtained within the past 12 months and (2) the patient’s
pulmonary disease has been clinically stable)
(c) Patients with cirrhosis or marked fibrosis on liver biopsy or raised AFP need to have a liver imaging study during the screening period to rule out hepatic neoplasia.
(d) For anyone with preexisting cardiac disease.
(e) Eye examination will be done on indication. Any patient complaining of decrease or loss of vision must have a prompt and complete eye examination. Patients with preexisting
ophthalmologic disorders (eg, diabetic or hypertensive retinopathy) should receive periodic ophthalmologic exams during Peg-IFN therapy. Peg-IFN treatment should be discontinued in
patients who develop new or worsening ophthalmologic disorders.
(f) non-invasive measurent of the liver to detect fibrosis and cirrhosis
(g) Only for patients with decreased kidney function. If there are clinically significant laboratory abnormalities, repeat no less frequently than every 2 weeks or as clinically indicated, with
appropriate toxicity management, until they return to normal or baseline values. Urinalysis to be performed via dipstick, with subsequent microscopic evaluation if positive for hemoglobin at
the discretion of the investigator.
(h) Only for certain patients at risk, don’t repeat if data from previous assessments are available
(i) Roche TaqMan®; when HBV DNA is more then the maximal detection limit, ten or hundred time dilution is proceded.
(j) Paxgene TM DNA sample (tube) will be collected. The DNA samples will be used exclusively for exploratory DNA research (genotyping of hostgenomics) to evaluate drug disposition
genes.(e.g. metabolic enzymes and drug transporters), to expore possible underlying genetic variants involved in therapy response in HBV chronically infected.Samples will only be
processed in a number of subjects. No other testing will be performed on these samples.
(k) Paxgene TM RNA sample (tube) will be collected to assess the expression of RNA in peripheral blood using whole genome microarray technology. These samples ares collected as part of
an effort to better understand the effects of medication. Samples will only be processed in a number of subjects. No other testing will be performed on these samples.
(l) plasma tube will be collected for proteomics profiling. These samples are collected as part of a study to better understand the effects of medication on liver activity. Samples will only be
processed in a number of subjects. No other testing will be performed on these samples. Possible protein candidates include: SR-B1(CD36), CD81, certain Claudins, STAT-1 and 2, ApoE
and more.
(m) During the study a quality of life index according to SF36 standardized questionnaires will be administered

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 HBsAg loss during treatment with Peg-IFN combined with ADV or TDF

Screening Assessments
Table 3. Screening Assessments
Medical History And Includes body weight, vital signs
Physical Examination
Eye examination Eye examination by an ophthalmologist at screening will be done on
indication.Patients with preexisting ophthalmologic disorders (eg,
diabetic or hypertensive retinopathy) should receive periodic
ophthalmologic exams during Peg-IFN therapy. Peg-IFN treatment
should be discontinued in patients who develop new or worsening
ophthalmologic disorders.
Clinical Chemistry ASAT, ALAT, bilirubin, creatinine, urea, sodium, chloride, potassium,
calcium, phosphorus, prothrombin time, alkaline phosphatase, total
protein, albumin, BUN, uric acid, cholesterol, triglycerides, glucose
Hematology Leukocyte count, differential WBC, red blood count, platelets
Immunology and Special HBeAg, anti-HBe, HBsAg, anti-HBs
Chemistry anti-HIV, anti-HCV, anti-HDV, Alfa-fetoprotein, ceruloplasmin,
alfa1-antitrypsin, ferritin
Only for patients at risk without previous data being available: AMA,
ANA, ASMA, thyroid peroxidase antibodies.
Virology Quantitative HBV-DNA measurement
Thyroid Function Tests TSH, free T4
Urinalysis Dipstick with subsequent microscopic evaluation if positive for
hemoglobin
Chest X-Ray Will be done during screening (Not necessary if (1) a chest X-ray
available to the investigator has been obtained within the past 12
months and (2) the patient’s pulmonary disease has remained
clinically stable.)
Electrocardiogram Only for anyone with a history of pre-existing cardiac disease.
(a)
Liver Biopsy Performed at start of the study and repeated after 48 weeks.
(b)
Fibroscan Performed at start of the study and repeated after 48 weeks.
Liver Imaging (ultrasound) Performed at screening or start of the study, repeated after 48 weeks
and on indication.
Liver Imaging (CT or Only for patients suspected to have hepatic neoplasia.
MRI)
HCG Pregnancy Test For women of childbearing potential a negative urine (or serum) HCG
test needs to be documented within 24 hours prior to the first dose.

(a) When a participant has objections against a second liver biopsy at week 48 ,he or she can
refuse it. The participant will than not be excluded from the study.
(b) A fibroscan measures the liver stiffness, expressed in kilopascals (kPA)

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