Virologic breakthrough in a patient with chronic hepatitis B by combination treatment with tenofovir disoproxil fumarate and entecavir

Abstract

Tenofovir disoproxil fumarate (TDF) is widely used to treat hepatitis B virus (HBV) patients in the USA and Europe. No confirmed report of resistance selection during treatment with TDF in treatment-naïve and nucleoside/nucleotide analog-treated chronic hepatitis B patients has yet been reported. Here, we report for the first time a patient with chronic hepatitis B and cirrhosis who emerged with virologic breakthrough during combination therapy with TDF and entecavir (ETV), against ETV-resistant virus. A 51-year-old Japanese woman with hepatitis B e-antigen (HBeAg), whose genotype was C, received ETV monotherapy continuously followed by TDF and ETV combination therapy, because her HBV DNA levels had been >3.5 log copies/mL. At the start of combination therapy, amino acid substitutions of the reverse transcriptase (rt) gene, rtL180M, rtT184I/M, and rtM204V, were detected. After this, serum HBV DNA decreased to less than 2.1 log copies/mL and remained at this level until 31 months of combination therapy, when it again began to increase. Amino acid substitutions of rtL180M, rtS202G, and rtM204V emerged and were associated with an increase in serum HBV DNA at virologic breakthrough. Long-term therapy with TDF against the ETV-resistant virus has the potential to induce virologic breakthrough and resistance, and careful follow-up should be carried out.

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CASE REPORT
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Open Access Full Text Article
http://dx.doi.org/10.2147/DDDT.S65349
Virologic breakthrough in a patient with chronic
hepatitis B by combination treatment with
tenofovir disoproxil fumarate and entecavir
Fumitaka Suzuki1,2
Hitomi Sezaki1
Norio Akuta1
Yoshiyuki Suzuki1
Yusuke Kawamura1
Tetsuya Hosaka1
Masahiro Kobayashi1
Satoshi Saitoh1
Yasuji Arase1
Kenji Ikeda1
Mariko Kobayashi3
Sachiyo Watahiki3
Rie Mineta3
Yukiko Suzuki3
Hiromitsu Kumada1
1Department of Hepatology,
Toranomon Hospital, Tokyo, Japan;
2Okinaka Memorial Institute for
Medical Research, Tokyo, Japan;
3Research Institute for Hepatology,
Toranomon Branch Hospital,
Kawasaki, Japan
Correspondence: Fumitaka Suzuki
Toranomon Hospital, Department
of Hepatology, 2-2-2 Toranomon,
Minato-ku, Tokyo 105-8470, Japan
Tel +81 44 877 5111
Fax +81 44 860 1623
Email fumitakas@toranomon.gr.jp
Abstract: Tenofovir disoproxil fumarate (TDF) is widely used to treat hepatitis B virus (HBV)
patients in the USA and Europe. No confirmed report of resistance selection during treatment
with TDF in treatment-naïve and nucleoside/nucleotide analog-treated chronic hepatitis B patients
has yet been reported. Here, we report for the first time a patient with chronic hepatitis B and
cirrhosis who emerged with virologic breakthrough during combination therapy with TDF and
entecavir (ETV), against ETV-resistant virus. A 51-year-old Japanese woman with hepatitis B
e-antigen (HBeAg), whose genotype was C, received ETV monotherapy continuously followed
by TDF and ETV combination therapy, because her HBV DNA levels had been .3.5 log copies/
mL. At the start of combination therapy, amino acid substitutions of the reverse transcriptase (rt)
gene, rtL180M, rtT184I/M, and rtM204V, were detected. After this, serum HBV DNA decreased
to less than 2 .1 log copies/mL and remained at this level until 3 1 months of combination therapy,
when it again began to increase. Amino acid substitutions of rtL180M, rtS202G, and rtM204V
emerged and were associated with an increase in serum HBV DNA at virologic breakthrough.
Long-term therapy with TDF against the ETV-resistant virus has the potential to induce virologic
breakthrough and resistance, and careful follow-up should be carried out.
Keywords: hepatitis B virus, resistant
Introduction
Hepatitis B virus (HBV) infection is a common disease that can induce a chronic
carrier state and is associated with the risk of progressive disease and hepatocellular
carcinoma.1 Interferon (IFN) and several nucleoside/nucleotide analogs (NAs), such
as lamivudine (LAM), adefovir dipivoxil (ADV), entecavir (ETV), and tenofovir
disoproxil fumarate (TDF), are currently approved for the treatment of chronic
hepatitis B (CHB) in most countries.2–5 Because NA analogs inhibit reverse transcription
of the HBV polymerase but do not directly interfere with the formation of covalently
closed circular DNA (cccDNA), they require long-term administration, which is usu-
ally accompanied by the emergence and selection of drug-resistant mutations in the
viral polymerase.6
TDF is widely used to treat HBV patients in the USA and Europe. This agent is
equally effective against multiple HBV genotypes (A–H) as well as against LAM-
resistant isolates.7 No confirmed report of resistance selection during treatment with
TDF in treatment-naïve CHB patients has yet been reported.8–10 In a recent study,
long-term TDF monotherapy provided durable antiviral efficacy for 240 or up to
288 weeks (6 years) of treatment, and comprehensive genotypic and phenotypic analy-
ses detected no evidence of TDF resistance.11,12 Additionally, longer treatment duration
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did not increase the incidence of virologic breakthrough.12
Moreover, TDF monotherapy has demonstrated the long-term
(median 23 months) efficacy of this agent in NA-experienced
patients with treatment failure, and virologic breakthrough
was not observed in any patient during the entire observa-
tion period.13
Here, we report for the first time a patient with CHB
and cirrhosis who emerged with virologic breakthrough
during TDF and ETV combination therapy against ETV-
resistant virus.
Case report
A 51-year-old Japanese woman with CHB underwent a
checkup in February 1999 and was found to be seropositive for
hepatitis B surface antigen (HBsAg), with mild alanine amin-
otransferase (ALT) elevation. Hepatitis B e-antigen (HBeAg)
was positive, and serum HBV DNA was .7.6 log copies/mL
(Amplicor HBV Monitor assay; F Hoffman-La Roche Ltd,
Basel, Switzerland). The HBV genotype was C, and human
immunodeficiency virus (HIV) status was negative. She
was diagnosed with cirrhosis by peritoneoscopy and liver
biopsy (moderate hepatitis [A2] and severe fibrosis [F4])
in February 2000. She received LAM (100 mg/day) mono-
therapy from September 2006. The nadir of HBV DNA
was 2.5 log copies/mL in January 2007. HBV DNA levels
gradually increased, and LAM-resistant virus emerged
(reverse transcriptase [rt] M204I). Treatment was switched
from LAM to ETV (0.5 mg/day) in October 2007 (HBV
DNA 3.9 log copies/mL) following the emergence of ETV-
resistant virus (rtL180M, rtS202G, and rtM204V) and
higher elevation in HBV DNA. However, she discontinued
therapy of her own volition from February 2009 to May
2010. She returned to our hospital in May 2010 because of
general fatigue and ascites, at which time serum HBV DNA
was .7.6 log copies/mL, ALT was 687 IU/L, and bilirubin was
3.8 mg/dL. Treatment with ETV (0.5 mg/day) was restarted
immediately, and ALT and serum HBV DNA levels gradually
decreased. However, because HBV DNA levels remained
at .3.5 log copies/mL until September 2010, she was started
on TDF (300 mg/day) and ETV combination therapy (HBV
DNA 3.9 log copies/mL). Serum HBV DNA then decreased
to less than 2.1 log copies/mL (COBAS® TaqMan® HBV Test,
v2.0; F Hoffman-La Roche Ltd) at November 2011 (month 14
of TDF and ETV treatment) and remained at this level until
April 2013 (month 31 of TDF and ETV treatment), when
it again began to increase (HBV DNA 3.9 log copies/mL).
Moreover, ALT was elevated in September 2013 (Figure 1).
Compliance with TDF and ETV was good throughout the
course of treatment.
During treatment, nucleotide sequences of the polymerase
gene were determined by polymerase chain reaction (PCR)
direct sequencing, as previously described.14 The viral poly-
merase reverse transcriptase (rt) gene at the baseline of LAM
treatment (September 2006) showed the wild type sequence
00
1
2
3
4
5
HBV DNA (log copies/mL)
6
7
8
9
8.6
9.0
687
307
Sept 2006
Sept 2010
Feb 2009 May 2010 Nov 2011 Oct 2013
Oct 2007 Apr 2013
100
200
300
400
ALT (IU/L)
500
600
700
800 HBV DNA
HBV DNA
HBV DNA
ALT
ALT ALT
LAM ETV
ETV
TDF
Figure 1 Clinical course of lamivudine or entecavir and tenofovir disoproxil fumarate therapy.
Abbreviations: ALT, alanine aminotransferase; ETV, entecavir; HBV, hepatitis B virus; LAM, lamivudine; TDF, tenofovir disoproxil fumarate.

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Virologic breakthrough with TDF and ETV treatment for HBV
(with no LAM, ADV, ETV, or TDF resistance substitutions). In
October 2007 (after switching from LAM to ETV), an amino
acid substitution of the rt gene, rtM204I (LAM resistance
substitution), was detected. Moreover, amino acid substitu-
tions of rtL180M, rtS202G, and rtM204V (ETV resistance
substitutions) emerged during ETV treatment in February
2009. The rt gene analysis at the baseline of ETV retreat-
ment (May 2010) returned the wild type sequence (with no
LAM or ETV resistance substitutions). In June 2010 (week 3
of ETV retreatment), amino acid substitutions of rtL180M,
rtT184I, and rtM204V (ETV resistance substitutions) were
simultaneously detected. Moreover, amino acid substitu-
tions of rtL180M, rtT184M, and rtM204V coexisted with
the above mutants at the end of June 2010 (week 6 of ETV
retreatment). In October 2010 (week 4 of TDF and ETV
treatment), these amino acid substitutions were replaced
by wild type virus (no ETV resistance substitutions). Since
April 2013 (month 31 of TDF and ETV treatment), amino
acid substitutions of rtL180M, rtS202G, and rtM204V
have emerged and have been found to be associated with an
increase in serum HBV DNA (Figure 2). In comparison with
those at the start of TDF therapy, the amino acid substitu-
tions changed from rtL180M, rtT184M, and rtM204V to
rtL180M, rtS202G, and rtM204V, and no other amino acid
substitutions apart from these in the rt region were observed.
Further, there were no substitutions that could be associated
with reduced TDF susceptibility (rtA181V/T, rtN236T, or
rtA194T) in April 2013.
Discussion
Genotypic resistance to TDF has been detected in several
patients with HIV-HBV coinfection. The substitution
rtA194T (plus rtL180M + rtM204V) has been associated
with TDF resistance,15 albeit that a second report failed to
confirm this.16 It has been shown that rtA181V + rtN236T
double mutants are resistant to TDF in vitro, but clinical
data suggest that patients with rtA181 or rtN236T remain
susceptible to TDF.17 The substitution rtP177G and rtF249A
reduced susceptibility to TDF in an in vitro study, but no
clinical findings have yet been reported.18 Moreover, rescue
therapy with ETV and TDF in CHB patients harboring viral
resistance patterns (for LAM, ADV, or ETV) or showing
only partial antiviral responses to preceding therapies was
efficient in patients both with and without advanced liver
disease.19 To date, there have been no confirmed reports of
resistance selection during treatment with TDF for CHB.9–12
Moreover, virologic breakthrough occurs infrequently and
has been associated with nonadherence to medication in the
majority of cases.12
To our knowledge, this is the first report of a patient with
virologic breakthrough during TDF therapy. In our case,
compliance with TDF and ETV was good throughout the
Figure 2 Evolution of the viral polymerase reverse transcriptase protein sequence (amino acids 1–344) during lamivudine, entecavir, and tenofovir disoproxil fumarate
therapy.
Notes: The AB033550 strain was reported by Okamoto et al.23 In June 2010, two kinds of strain were identied, June 2010–1 and –2.
Abbreviation: rt, reverse transcriptase.

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Suzuki et al
course of treatment. Virologic breakthrough in compliant
patients is generally related to viral resistance.6 Amino acid
substitutions of rtL180M, rtS202G, and rtM204V have
emerged in cases in which serum HBV DNA increased dur-
ing TDF and ETV therapy. Moreover, these amino acid sub-
stitutions changed from rtL180M, rtT184M, and rtM204V
to rtL180M, rtS202G, and rtM204V. This clinical course
suggests that these amino acid substitutions are resistant
to TDF and ETV therapy, although in vitro confirmation
is necessary. Kim et al reported that among 18 patients
who failed multiple NA treatments, including LAM, ADV,
and ETV, 17 patients achieved virologic response and one
patient showed a viral reduction of 3.9 log IU/mL, nearly
reaching virologic response within 24 months.20 These
findings indicate that genotypic resistance to ETV does not
affect the probability of an initial virologic response to TDF
therapy.20 Petersen et al reported that four patients harboring
ETV-resistant virus achieved a virologic response within
9 months.19 Recently, Seto et al reported 142 Asian CHB
patients with at least 6 months exposure to other NAs (includ-
ing ETV) who received TDF with or without LAM. With a
median 2.25 years of follow-up, 45 patients had detectable
viremia in at least one time point.21 For these 45 patients,
which included ten with virologic breakthrough, both line
probe assay and direct sequencing revealed no new amino
acid substitutions, including substitutions that could be asso-
ciated with reduced TDF susceptibility (rtA181V/T, rtN236T,
or rtA194T). Moreover, Karatayli et al reported that HBV
DNA, in seven of eight patients with ETV resistance muta-
tions (T184F/A/L/I, S202G, and M250V), became unde-
tectable with TDF and LAM after 6 months of treatment.22
In vitro drug susceptibility showed that TDF displayed
one- to twofold resistance to ETV-resistant viral strains
(N123D + H124Y + L180M + S202G + M204V + Y257H,
I163V + L164M + L180M + S202G + M204V + C256S, and
H124Y + L180M + S202G + M204V + Y257H). However, in
other cases, the treatment period was relatively shorter. In our
case, virologic breakthrough occurred at month 31 of TDF
and ETV therapy, and the ETV-resistant strain (L180M +
S202G + M204V) of our case was not identical with that in
the in vitro drug susceptibility study above. Clarification of
virologic breakthrough and resistance of TDF against patients
with NA-resistant virus, especially ETV, will likely require
further studies with a longer time frame.
In conclusion, this study shows that long-term treatment
of ETV-resistant virus with TDF has the potential to induce
virologic breakthrough and resistance, and careful follow-up
should be done.
Acknowledgments
This study was supported in part by a Grant-in-Aid for Scientific
Research (C) (grant number 24590999) from the Japan Society
for the Promotion of Science and by a Grant-in-Aid from the
Ministry of Health, Labor and Welfare of Japan.
Disclosure
Dr Kumada reports having received investigator, lecture,
and consulting fees from Bristol-Myers KK, Tokyo, Japan
and GlaxoSmithKline KK, Tokyo, Japan. The other authors
report no conflicts of interest in this work.
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