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Virological breakthrough and resistance in patients with chronic hepatitis B
receiving nucleos(t)ide analogs in clinical practice
Short title: Virological breakthrough during HBV treatment
Chanunta Hongthanakorn, Watcharasak Chotiyaputta, Kelly Oberhelman,
Robert J. Fontana, Jorge A. Marrero, Tracy Licari, and Anna S.F. Lok
Division of Gastroenterology, Department of Internal Medicine, University of Michigan Health
System, Ann Arbor, MI, USA
Key words: Entecavir, Tenofovir, HBV treatment, HBV DNA, antiviral resistance mutations
Address for correspondence
Anna SF Lok, MD
Division of Gastroenterology
University of Michigan Health System
3912 Taubman Center, SPC 5362
1500 East Medical Center Drive
Ann Arbor, MI 48109
Fax: 734-936-7392
Email: aslok@med.umich.edu
Total word count: 4,983 words
Total 3 tables, 4 Figures and 1 supplementary table
Abbreviations
NUCs nucleos(t)ide analogs
LAM lamivudine
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ADV adefovir
ETV entecavir
TBV telbivudine
TDF tenofovir
CHB chronic hepatitis B
HBV hepatitis B virus
HIV human immunodeficiency virus
VBT virological breakthrough
GR genotypic resistance
Anti-HBe hepatitis B e antibody
HBeAg hepatitis B e antigen
ALT alanine aminotransferase
E-mail and Financial Disclosures:
Chanunta Hongthanakorn Email: chanunta@med.umich.edu
No financial disclosure
Watcharasak Chotiyaputta Email: watchara@med.umich.edu
No financial disclosure
Kelly Oberhelman Email: kellmo@med.umich.edu
No financial disclosure
Robert J. Fontana Email: rfontana@med.umich.edu
Consulting/ research: Bristol-Myers Squibb, GlaxoSmithKline
Speaker’s Bureau: Gilead Sciences, Genetech
Jorge A. Marrero Email: jmarrero@med.umich.edu
No financial disclosure
Tracy Licari Email: bsiegel@med.umich.edu
No financial disclosure
Anna SF Lok Email: aslok@med.umich.edu
Consulting: Gilead Sciences, Roche, and Bristol-Myers Squibb,
GlaxoSmithKline; Grant/Research Support: Bristol-Myers Squibb,
Gilead Sciences, GlaxoSmithKline, Schering-Plough, and Roche.
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ABSTRACT
Virological breakthrough (VBT) is the first manifestation of antiviral drug resistance during
nucleos(t)ide analog (NUC) treatment of chronic hepatitis B (CHB) but not all VBTs are due to
drug resistance. The aims of this study were to determine the incidence of virological
breakthrough (VBT) and genotypic resistance (GR) in CHB patients receiving NUCs in clinical
practice. Records of CHB patients receiving NUCs were reviewed. All patients with VBT were
tested for drug resistance mutations. Of 148 patients included, 73% were men, mean age was
44.9 years. During a mean follow-up of 37.5±20.1 months, 39 (26%) patients had at least 1 VBT.
Of these 39 patients, 15 (38%) were not confirmed to have VBT on retesting and 10 of these 15
had no evidence of GR. The cumulative probability of VBT, confirmed VBT, and GR at 5 years
was 46.1%, 29.7%, and 33.9%, respectively. In multivariate analysis, failure to achieve
undetectable HBV DNA was the only factor significantly associated with VBT. Among the 10
patients who had VBT but no confirmed VBT or GR and who were maintained on the same
medications, serum HBV DNA decreased in all 10 and 9 had undetectable HBV DNA a mean of
6.8 months after the VBT. Four patients had persistently undetectable HBV DNA while six had
transient increase in HBV DNA during follow-up but none had GR. Conclusion: VBT was
common in CHB patients receiving NUCs in clinical practice, but nearly 40% of the VBTs were
not related to antiviral drug resistance. Counseling of CHB patients on medication adherence
and confirmation of VBT and/or GR can avoid unnecessary changes in antiviral medications.
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INTRODUCTION
Five nucleos(t)ide analogs (NUCs): Lamivudine (LAM), Adefovir (ADV), Entecavir (ETV),
Telbivudine (TBV), and Tenofovir (TDF) have been approved for the treatment of chronic
hepatitis B (CHB). Nucleos(t)ide analogs are administered orally and have very few side effects;
however, these medications suppress but do not eradicate hepatitis B virus (HBV). Therefore,
most patients with CHB will require long-term treatment to derive clinical benefit. However, long-
term NUC treatment is associated with increasing risk of drug resistance particularly when NUCs
with low genetic barrier to resistance are used as monotherapies.
Virological breakthrough (VBT) is the first clinical manifestation of antiviral drug
resistance and may precede biochemical breakthrough (1-3). Phase III clinical trials of
nucleos(t)ide analogs in NUC-naïve patients revealed that 0 to 87.5% of patients with VBT had
confirmed genotypic resistance (GR) (4-9). In the phase III trial of telbivudine vs. lamivudine, 32
of 680 (4.7%) and 99 of 687 (14.4%) patients who received telbivudine and lamivudine,
respectively, experienced VBT after one year of treatment but only 28 (87.5%) and 75 (75.8%)
patients with VBT were confirmed to have GR (9). In the phase III trial of entecavir vs.
lamivudine, 11 of 679 (1.6%) and 88 of 668 (13.2%) patients who received entecavir and
lamivudine, respectively, experienced VBT after one year of treatment but 0 (0%) and 65
(73.9%) of the patients with VBT, respectively were confirmed to have GR (7, 8). In the phase III
trial of tenofovir, 10 of 426 (2.3%) patients who received tenofovir experienced VBT after one
year of treatment but none of these patients were confirmed to have GR (4). These data indicate
that not all VBTs are related to antiviral drug resistance.
Possible explanations for the discrepancy between the rates of VBT and GR include poor
adherence to medications, failure to detect drug-resistance mutations due to insensitive assays,
and failure to recognize new mutations associated with antiviral drug resistance. VBT during the
first year of treatment was attributed to medication non-adherence in patients who received
entecavir or tenofovir in the phase III trials (4, 7). Medication adherence is likely to be lower in
clinical practice than in phase III clinical trials where highly motivated patients are recruited and
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closely monitored. Differentiating between VBT due to medication non-adherence and VBT due
to drug resistance is important as virological response can be restored by reinforcement of
adherence in the former case while rescue therapy is needed in the latter situation.
The aims of this study were (1) to determine the incidence of VBT and GR in CHB
patients treated with nucleos(t)ide analogs in clinical practice, (2) to determine the factors
associated with VBT in CHB patients receiving nucleos(t)ide analogs, and (3) to determine the
outcomes of patients with VBTs that were not confirmed to be associated with antiviral drug
resistance mutations.
PATIENTS AND METHODS
Consecutive adult patients with CHB seen at the liver clinic of the University of Michigan Health
System between January 2000 and July 2010, who had received nucleos(t)ide analog treatment
for at least one year with serum HBV DNA <10,000 IU/mL after one year of treatment were
included. Patients who were receiving combination therapy of nucleos(t)ide analog and
interferon, patients receiving nucleos(t)ide analogs to prevent reactivation of hepatitis B during
immunosuppressive or cancer treatment or to prevent recurrent hepatitis B after liver
transplantation, patients with human immunodeficiency virus (HIV), hepatitis C virus or hepatitis
D virus co-infection, and patients with impaired renal function requiring dose adjustment of
nucleos(t)ide analogs were excluded. The study was approved by the Institutional Review Board
of the University of Michigan.
Medical records were reviewed and information on patient demographics (age, gender,
race) body weight, HBV markers (hepatitis B e antigen [HBeAg], hepatitis B e antibody [anti-
HBe], HBV DNA); hepatic panel (albumin, aspartate aminotransferase [AST], alanine
aminotransferase [ALT], total bilirubin [TB], and alkaline phosphatase [alk phos]); complete blood
count (CBC); prothrombin time (PT)/international normalized ratio (INR); and liver histology were
recorded. HBV treatment history was reviewed. Index treatment was defined as the first course
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of nucleos(t)ide analog therapy initiated at our liver clinic. Start and stop dates of index and prior
HBV treatment, medications used during each course of treatment, and serial results of HBeAg,
anti-HBe, HBV DNA, AST, and ALT during treatment were recorded.
During treatment, serum HBV DNA and hepatic panel was tested every 3 months and
HBeAg/anti-HBe every 6-12 months. Serum HBV DNA was quantified by commercial
polymerase chain reaction (PCR) assays – Amplicor HBV monitor test (Roche Molecular
Diagnostics, Indianapolis, IN) with a lower limit of detection of 200-1,000 copies/mL (40-200
IU/mL) between 2000 and 2005, and real-time polymerase chain reaction assays, COBAS
TaqMan HBV (Roche Molecular Diagnostics) with a lower limit of detection of 29 IU/mL from July
2005 onwards.
Tests for antiviral drug resistance mutations
HBV DNA from serum samples of patients with VBT was amplified and sequenced as described
previously (10). The DNA sequences were aligned with SeqmanTM II and EditSeq
TM software
(DNASTAR Inc, Madison, WI) and compared to consensus sequences of the respective HBV
genotype. All samples were also tested for antiviral drug resistance mutations by a line probe
assay, INNO-Lipa HBV DR v.2 and v.3 (Innogenetics NV, Gent, Belgium) according to the
manufacturer’s instructions (10, 11).
Definition of virological breakthrough, genotypic resistance, and biochemical
breakthrough
Virological breakthrough (VBT) was defined as any increase in serum HBV DNA by >1 log10 from
nadir or redetection of serum HBV DNA at levels 10-fold the lower limit of detection of the HBV
DNA assay after having an undetectable result. Thus, a patient who previously had undetectable
serum HBV DNA by an assay with a lower limit of detection of 29 IU/mL would be considered to
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have a VBT if serum HBV DNA is subsequently detected at levels 290 IU/mL. A cutoff 10-fold
the lower limit of detection was chosen because the consensus definition of VBT required a 10-
fold increase in HBV DNA(2, 12). In addition, HBV DNA levels slightly above the limit of
detection may not be reproducible on retesting of the same serum sample. Confirmed virological
breakthrough (confirmed VBT) was defined as persistence of VBT on repeat test (fulfilling same
criteria) 1-3 months later (with or without further increase in serum HBV DNA). Genotypic
resistance (GR) was defined as detection of signature resistance mutations by direct
sequencing. Signature resistance mutations included alanine to threonine or valine at codon 181
(rtA181T/V), threonine to alanine, cysteine, glycine, isoleucine, leucine, methionine, or serine at
codon 184 (rtT184A/C/G/I/L/M/S), alanine to threonine substitution at codon 194 (rtA194T),
serine to cysteine, glycine, or isoleucine at codon 202 (rtS202C/G/I), methionine to valine or
isoleucine substitution at codon 204 (rtM204V/I), asparagine to threonine at codon 236
(rtN236T), and methionine to isoleucine or valine at codon 250 (rtM250I/V). Compensatory
mutations such as leucine to methionine substitution at codon 180 (rtL180M) were not included
(13).
Biochemical breakthrough (BBT) was defined as ALT above the upper limit of normal
(ULN) (35 IU/L) in patients who had normalized ALT and ALT >2 times nadir in those who never
had normal ALT. ALT flare was defined as ALT >5 times ULN in patients who had normalized
ALT and ALT >5 times nadir in those who never had normal ALT.
Statistical analyses
Continuous variables were expressed as mean ± SD, or median and range. Serum HBV DNA
was expressed as log10 IU/mL. Categorical variables were expressed as number and percent.
Continuous variables were compared with two-tailed student t-test or Mann Whitney test
depending on the distribution, and categorical variables were compared with chi-squared test or
Fisher’s exact test. The Kaplan-Meier method was used to estimate the cumulative probability of
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VBT, confirmed VBT, and GR. For these analyses, patients were censored when index
treatment was changed. The Log-rank method was used to compare the cumulative probability
of VBT, confirmed VBT, and GR of different treatment groups. Cox regression analysis was used
to identify factors associated with VBT. The following variables were included in the analysis:
HBV markers (HBeAg, HBV DNA) at the start of treatment, HBV DNA levels after 1 year of
treatment, nadir virological response, history of HBV treatment (NUC-naïve or NUC-
experienced), and medication used in the index regimen (lamivudine vs. others for NUC-naïve
patients and combination therapy or tenofovir vs. others for NUC-experienced patients).
Variables with a p value <0.1 on univariate analysis were further analyzed by multivariate Cox
regression to determine the independent factors associated with VBT. Data analyses were
performed using SPSS software version 15 (SPSS Chicago, IL).
RESULT
Characteristics of patients at the start of the index regimen
A total of 148 CHB patients were included. Five patients who met other criteria but whose serum
HBV DNA exceeded 10,000 IU/mL after 1 year of treatment were excluded, all five patients had
>1 log10 decrease in HBV DNA after 6 months of treatment. Characteristics of the patients at the
start of the index regimen are shown in table 1. Seventy-three percent of the patients were men
and the mean age was 44.9 ± 12.3 years. Approximately half (48.7%) of the patients were Asian,
41.2% were Caucasian, and the remainder were of other races. Roughly half (52.7%) of the
patients were HBeAg positive and the mean HBV DNA was 6.2 ± 1.8 log10 IU/mL. Most (87.2%)
patients had an elevated serum ALT based on our hospital laboratory reference range and 42
(28.4%) had ALT level >5 times the upper limit of normal.
Index regimen and initial response
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Table 2 lists the medications used in the index regimen. Among the 81 NUC-naïve patients,
entecavir alone (n=43) and lamivudine alone (n=26) were the most common medications. Of the
67 NUC-experienced patients; 19 received combination therapy, 16 received adefovir, 15
received tenofovir, 13 received entecavir, and 4 received lamivudine. The mean duration of
follow-up was 37.5 ± 20.1 months (median 31.5 (range 12-102) months). Forty-two (28.4%)
patients had been receiving the index treatment regimen for more than 48 months.
After one year on the index regimen, 70.9% of the patients had undetectable HBV DNA
(Table 2). With continued treatment, 86.5% (87.7% NUC-naïve and 85.1% NUC-experienced) of
patients achieved undetectable HBV DNA. The mean interval from the start of the index
treatment to nadir virological response was 9 ± 6.3 months. Of the 136 patients who had
baseline elevated ALT, 105 (77.2%) achieved ALT normalization after a mean of 8.4 ± 8.3
months.
Virological breakthrough during treatment
Thirty-nine (26.4%) patients experienced at least one VBT after the first year of
treatment, 24 (62%) of these patients had confirmed VBT, 24 (62%) had GR by direct
sequencing, 13 (33%) had biochemical breakthrough and only one had ALT flare (Figure 1). Of
the 36 patients who had repeat HBV DNA after VBT, the mean interval from VBT to confirmed
VBT was 2.4 ± 1.6 months. Line probe assay revealed two additional patients had GR. Both
were receiving adefovir and found to have rtN236T mutation (Figure 1). The first patient had
confirmed VBT and was switched to combination of tenofovir and emtricitabine with undetectable
HBV DNA 6 months after start of rescue therapy (supplementary table1, patient T). The second
patient (patient 8) was not confirmed to have VBT on retesting and continued to receive adefovir
monotherapy.
The baseline characteristics of the patients who did or did not experience VBT were
similar except for lower serum albumin among the patients who subsequently experienced VBT
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(Table 1). The cumulative probability of VBT at 3 and 5 years was 21.5% and 46.1%, confirmed
VBT was 13.7% and 29.7%, and GR was 10.7% and 33.9%, respectively (Figure 2). If the
traditional definition of VBT (increase in HBV DNA by >1 log from nadir or redetection of any
level of HBV DNA after becoming undetectable) was used, 45 (30%) patients would be
considered to have experienced at least 1 VBT and 26 (58%) of these patients would have
confirmed VBT.
Among the NUC-naïve patients, the cumulative probability of VBT, confirmed VBT, and
GR at 3 and 5 years was 18% and 54%, 12% and 31%, and 13% and 39%, respectively. NUC-
naïve patients who experienced VBT were more likely to be receiving lamivudine than other
nucleos(t)ide analogs (Table 2). Fourteen of 26 (53.8%) patients receiving lamivudine
monotherapy experienced at least one VBT compared to 3 of 43 (7%) patients receiving
entecavir monotherapy. Antiviral drug resistance mutations were detected in 12 of the 14
patients receiving lamivudine and 1 of the 3 patients receiving entecavir who experienced VBT.
The cumulative probability of VBT, confirmed VBT, and GR at 3 years was 37%, 30%, and 33%,
respectively among the patients receiving lamivudine monotherapy (Figure 3A); and 13%, 3.1%,
and 3.1%, respectively among the patients receiving entecavir monotherapy (Figure 3B) (P =
0.001, 0.001, and <0.001, respectively).
Among the NUC-experienced patients, the cumulative probability of VBT, confirmed VBT,
and GR at 3 and 5 years was 22% and 33%, 14% and 28%, and 8% and 29%, respectively.
Sixteen of 33 (48.5%) patients who were receiving lamivudine, adefovir or entecavir
monotherapy but only 2 of 34 (5.9%) patients receiving combination therapy or tenofovir
monotherapy experienced VBT. The cumulative probability of VBT, confirmed VBT, and GR at 3
years was 32.3%, 23.6%, and 13.8%, respectively among the patients receiving lamivudine,
adefovir or entecavir monotherapy; and 8.7%, 3.8%, and 0%, respectively among the patients
receiving combination therapy or tenofovir monotherapy (P = 0.005, 0.01, and 0.04,
respectively).
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Univariate analyses found that failure to achieve undetectable HBV DNA after 1 year of
treatment (HR 2.6, 95% CI 1.39-5.0, P = 0.003) and at nadir response (HR 6.92, 95% CI 3.4-
14.1, P = <0.001) significantly increased the risk of VBT. Multivariate analyses showed that
failure to achieve undetectable HBV DNA at nadir response was the only factor significantly
associated with VBT in the overall population (HR 5.5, 95% CI 2.49-12.28, P=<0.001). For sub-
group analyses, the predictors of VBT among NUC-naïve patients were failure to achieve
undetectable HBV DNA at 1 year (HR 2.79, 95% CI 1.11-7.01, P = 0.03) and at nadir response
(HR 3.95, 95% CI 1.46-10.71, P = 0.007), and receipt of lamivudine monotherapy (HR 3.19, 95%
CI 1.22-8.33, P = 0.02). The predictors of VBT among the NUC-experienced patients were
failure to achieve undetectable HBV DNA at nadir response (HR 9.59, 95% CI 1.64-56.07, P =
0.01), and monotherapy with drugs other than tenofovir (HR 8.15, 95% CI 1.61-41.29, P = 0.01)
(Table 3).
Outcome of patients who had virological breakthrough
Of the 39 patients who had at least 1 episode of VBT, 24 (62%) had confirmed VBT, 12 were not
confirmed to have VBT on retesting, and three were not available for retesting because they
received rescue therapy when VBT was first detected (Figure 1). Two of the latter three patients
had GR. BBT was observed in 9 (37.5%) of 24 patients with confirmed VBT, 3 (25%) of 12 not
confirmed to have VBT and 1 of the 3 patients who received rescue therapy when VBT was first
detected (Figure 1). Only one patient had an ALT flare. This patient had confirmed VBT, nadir
ALT was 30 IU/L, and ALT at the time of VBT was 34 IU/L. This patient was non-compliant and
did not have repeat HBV DNA testing until 8 months after the initial VBT. At the time of
confirmed VBT, ALT was 258 IU/L.
Among the 24 patients who had confirmed VBT, 19 patients had GR, and all 19 received
rescue therapy. The index treatment, mutations detected at the time of VBT, rescue therapy
administered, and response to rescue therapy in these 19 patients are shown in supplementary
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table 1 (patients A-S). Seventeen of these 19 patients had undetectable HBV DNA within 6
months of initiating rescue therapy.
Of the 5 patients who did not have GR by direct sequencing, two (patients 1 and 2)
continued on the same treatment (Figure 4) and the other three received rescue therapy
(supplementary table 1, patients T-V). Patient 1 had further decrease in HBV DNA level during
continued treatment with the same medication but HBV DNA remained detectable. Serum HBV
DNA at the last visit was 2.2 log10 IU/mL, 12 months after the VBT. Patient 2 had a slow decline
in serum HBV DNA level, retesting for GR 10 months later did not reveal any signature antiviral
resistance mutations and HBV DNA became undetectable 19 months after the VBT. This patient
subsequently developed two more episodes of VBT but testing for GR did not reveal any
signature antiviral resistance mutations and HBV DNA became undetectable again after each
episode of VBT. No BBT was observed during subsequent episodes of VBT. The three patients
who received rescue therapy had undetectable HBV DNA within 6 months.
Among the 12 patients who had VBT that was not confirmed on retesting, 3 were found
to have signature antiviral resistance mutations. All three patients were receiving lamivudine
monotherapy and were found to have M204I mutation. Two patients (supplementary table 1,
patients W and X) had undetectable HBV DNA within 6 months after rescue therapy while the
third was lost to follow-up. The remaining 9 patients who did not have confirmed VBT had no
evidence of GR, 2 had BBT with ALT of 56 IU/L and 65 IU/L. Eight of these nine patients
(patients 3-10) continued on the same treatment while one patient received rescue therapy. HBV
DNA levels in four patients (patients 3-6) who continued on the same treatment became
consistently undetectable during follow-up. Three (patients 7-9) patients’ HBV DNA levels
became undetectable during continued treatment with the same medication but HBV DNA
subsequently became detectable at levels below our VBT criteria. Retesting did not reveal any
signature antiviral resistance mutations and HBV DNA levels of these 3 patients became
undetectable again with continued treatment on the same medication. The last patient (patient
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10) had an initial decline in HBV DNA during continued treatment with the same medication but
developed a second VBT 33 months after the first episode of VBT. This second episode of VBT
was not confirmed on retesting and no signature antiviral resistance mutation was detected. HBV
DNA level subsequently became undetectable (Figure 5).
In total, 10 patients who experienced VBT continued on the same treatment and had
been followed for a mean of 29.3 ± 20.5 months (median 24.5 (range 9-75) months) after the
initial episode of VBT. All 10 patients had further decrease in serum HBV DNA and nine had
undetectable HBV DNA a mean of 6.8 ± 6.3 months after the VBT. Six patients had one or more
episodes of transient increase in serum HBV DNA; of these, two met our criteria for VBT but
none had GR. Two of these six patients had a mild increase in ALT with peak values of 56 and
65 IU/L during subsequent episodes of HBV DNA increase.
Of the remaining 29 patients with VBT, 28 received rescue therapy and all but three had
undetectable HBV DNA within 6 months of initiating rescue therapy while one was lost to follow-
up.
DISCUSSION
This study examined the rates of VBT, confirmed VBT and GR in 148 CHB patients treated with
nucleos(t)ide analogs in clinical practice. We found a high rate of VBT, 39 (26%) patients
experienced at least 1 episode of VBT with a cumulative probability of 46% at 5 years. Twenty-
four (16%) patients had confirmed VBT; of these, 19 (79%) had GR by direct sequencing. An
additional 5 patients had GR but were not confirmed to have VBT on retesting or received
rescue therapy without retesting. Thus, in total 24 (16%) patients had GR. The finding that 38%
of patients who experienced VBT were not confirmed to have VBT on retesting and 38% did not
have antiviral resistance mutations on direct sequencing suggests that medication non-
adherence may be the cause of the VBT in these patients.
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We acknowledge that direct sequencing is insensitive and will not detect viral variants
that comprise <20% of the viral population. In this study, all patients were also tested by a line
probe assay which is more sensitive and can detect viral variants that comprise >5% of the viral
population. Two additional patients were found to have N236T mutation by the line probe assay.
Thus, signature antiviral resistance mutations were not detected in 13 (33%) patients with VBT
by both direct sequencing and line probe assay. None of these 13 patients was noted to have
other substitutions in the reverse transcriptase region of the HBV polymerase gene. Only one
had BBT. In phase III clinical trials of nucleos(t)ide analogs for CHB, 65.7% to 87.5% of patients
receiving lamivudine or telbivudine and 0% of patients receiving entecavir or tenofovir, who
experienced VBT during the first year had GR (4, 7-9).
Although antiviral resistance mutations may be detected if we had used more sensitive
methods such as single genome sequencing or pyrosequencing, follow-up data suggest that
most of the patients who experienced VBT but did not have confirmed VBT or GR were not
adherent to their antiviral medication(s). Because this was a retrospective study, data on
medication adherence was not available; however, follow-up data suggest that non adherence
was an important cause of these unconfirmed VBTs. All 10 patients who continued treatment
with the same medication(s) had further decrease in serum HBV DNA levels and all but one had
undetectable HBV DNA after the VBT. Six of these 10 patients experienced 1 episode of
transient increase in serum HBV DNA level during follow-up despite counseling on the
importance of medication adherence. None of the five retested for GR was found to have
antiviral resistance mutations and all had subsequent decline in serum HBV DNA during
continued treatment with the same medication(s). We acknowledge that our attribution that
nonadherence was an important cause of VBT is speculative. In a separate prospective study of
105 patients in whom adherence was evaluated using a self-administered questionnaire, 26%
admitted to missing their medication at least once in the past 30 days and patients who had
<100% adherence on serial assessments had a trend towards a higher rate of VBT (14).
Medication adherence has been shown to be important in maintaining response in patients
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receiving treatment for other conditions. Published studies showed that patients who were
adherent to anti-hypertensive medications were more likely to have adequately controlled blood
pressure (15-17). Several studies of antiretroviral medications in patients with HIV infection also
revealed that failure to adhere to HIV treatment regimens and repeated drug holidays (defined
as stopping treatment entirely for 48 hours) were associated with a higher rate of virological
failure (18, 19). In one study of HIV treatment that included a protease inhibitor, 80% of patients
with <80% adherence had virological failure, compared to 22% of those with 95% adherence
(20).
There are very little data on adherence to nucleos(t)ide analog treatment for hepatitis B.
In a previous study of pharmacy claims database including 11,100 patients receiving
nucleos(t)ide analogs for CHB, we found that mean adherence 1 year after enrollment (defined
as percent of days in which the patient had medications during that year) was 87.8% (21). In that
study, we were not able to correlate medication adherence with virological response or
occurrence of VBT. In the current study, we found that nadir response and type of medication
used were significantly associated with the occurrence of VBT. Thus, patients who had rapid
response with undetectable serum HBV DNA within 1 year of treatment and those who had
undetectable serum HBV DNA at some point during the course of treatment were less likely to
experience VBT. These data are in accord with previous studies showing that undetectable
serum HBV DNA after 24 weeks of treatment is associated with significantly lower rates of
antiviral resistance (22, 23). As expected, among NUC-naïve patients, those receiving entecavir
monotherapy were less likely to experience VBT than those receiving lamivudine monotherapy.
Among NUC-experienced patients, those receiving combination therapy (lamivudine + adefovir
or emtricitabine + tenofovir) or tenofovir monotherapy were less likely to experience VBT than
those receiving lamivudine, adefovir or entecavir monotherapy. This is not surprising because
these patients had previous nonresponse or resistance to lamivudine or adefovir and it is now
known that switching from lamivudine to adefovir or entecavir monotherapy in patients with prior
lamivudine resistance is associated with a high rate of subsequent resistance to adefovir or
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entecavir (24-26). In this study, all the patients who received entecavir or tenofovir alone or in
combination with another nucleos(t)ide analog had undetectable HBV DNA within 6 months of
rescue therapy while three patients with lamivudine resistance still had detectable HBV DNA 6
months after rescue therapy with adefovir alone or in combination with lamivudine.
In conclusion, this study revealed that VBT was common in clinical practice. However,
VBT was not always related to antiviral drug resistance. CHB patients receiving nucleos(t)ide
analog therapy who experienced VBT should be counseled on medication adherence and for
patients who are immunocompetent and have compensated liver disease, confirmation of VBT
and/or determination of GR is prudent before the initiation of rescue therapy to avoid
unnecessary changes in antiviral medications. We acknowledge that this study is limited by the
small number of patients, the heterogeneity in treatment regimens, and the lack of data on
medication adherence. However, the results of this study highlight the importance of HBV DNA
monitoring and counseling on medication adherence throughout the course of nucleos(t)ide
analog treatment and the fine balance between prompt initiation of rescue therapy versus
avoidance of unnecessary changes to the treatment regimen.
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Reference
1. Lok AS, Lai CL, Leung N, Yao GB, Cui ZY, Schiff ER, et al. Long-term safety of
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26. Lampertico P, Vigano M, Manenti E, Iavarone M, Sablon E, Colombo M. Low resistance
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Figure legends
Figure 1: Outcome of patients with virological breakthrough (VBT)
Abbreviations; VBT, virological breakthrough; GR, genotypic resistance; BBT, biochemical
breakthrough
*This patient was lost to follow up.
**1 patient had ALT flare.
# 1 patient was found to have N236T mutation by line probe assay but did not receive rescue
therapy (patient 8).
## 1 patient was found to have N236T mutation by line probe assay and received rescue therapy.
Figure 2. Cumulative probability of virological breakthrough (VBT), confirmed virological
breakthrough, and genotypic resistance (GR) (n=148)
Virological breakthrough
Confirmed virological breakthrough
Genotypic resistance
Figure3. Cumulative probability of virological breakthrough, confirmed virological breakthrough,
and genotypic resistance in NUC-naïve patients receiving (A) lamivudine monotherapy (n=26)
and (B) entecavir monotherapy (n=43)
Virological breakthrough
Confirmed virological breakthrough
Genotypic resistance
Figure4. Serum HBV levels in 10 patients who had virological breakthrough but no confirmed
virological breakthrough or genotypic resistance during continued treatment with the same
medication
LLOD, lower limit of detection
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Table 1: Baseline Characteristics of Patients
Virological
Breakthrough
No Virological
Breakthrough
All Patients P value
No. of Patients 39 109 148
Age (year) 48.1 ± 12.4 43.7 ± 12.1 44.9 ± 12.3 0.06
Gender (male) 31 (79.5) 77 (70.6) 108 (73) 0.40
Race
Caucasian 15 (38.5) 46 (42.2) 61 (41.2) 0.36
Asian 22 (56.4) 50 (45.9) 72 (48.7)
Other 2 (5.1) 13 (11.9) 15 (10.1)
Weight (pound) 159 ± 40 169 ± 46 167 ± 45 0.35
Previous HBV Treatment
None 21 (53.8) 61 (56) 82 (55.4) 0.95
1 Regimen 11 (28.2) 28 (25.7) 39 (26.4)
> 1 Regimen 7 (18) 20 (18.3) 27 (18.2)
Liver Histology 24 64 88
Cirrhosis 12 (30.8) 29 (26.6) 41 (27.7) 0.68
HBV DNA (log IU/ml) 6.6 1.7 6.0 ± 1.9 6.2 ± 1.8 0.11
<5 log 7 (17.9) 34 (31.2) 41 (27.7) 0.28
5-7 log 15 (38.5) 35 (32.1) 50 (33.8)
>7 log 17 (43.6) 40 (36.7) 57 (38.5)
HBeAg-positive 21 (55.8) 57 (52.3) 78 (52.7) 0.99
Laboratory values
Total bilirubin (mg/dL) 1.7 4.4 1.0 ± 1.3 1.2 ± 2.5 0.11
AST (IU/L)
76 (23-920) 62 (15-2854) 66 (15-2854) 0.24
ALT (IU/L)
97 (22-1720) 97(13-4324) 97 (13-4324) 0.19
Albumin (g/dL) 3.9 0.6 4.2 ± 0.5 4.1 ± 0.5 0.03
Alk phos (IU/L) 102 53 91 ± 46 94 ± 48 0.16
Platelets (K/mm3) 177 84 194 ± 75 189 ± 78 0.08
Results expressed as number (%), mean ± SD or median (range)
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Table 2: Index Treatment Regimen and Response to Treatment
Virological
breakthrough(N=39)
No Virological
breakthrough (N=109)
All Patients
(N=148)
P value
Regimen
NUC-naïve (N=81)
Lamivudine 14 (35.9) 12 (11) 26 (17.6) <0.001
Adefovir 3 (7.7) 1 (0.9) 4 (2.7)
Entecavir 3 (7.7) 40 (36.7) 43 (29.1)
Tenofovir 0 6 (5.5) 6 (4.1)
Telbivudine 1 (2.6) 1 (0.9) 2 (1.3)
NUC-experienced (N=67)
Lamivudine 2 (5.1) 2 (1.8) 4 (2.7) 0.002
Adefovir 9 (23) 7 (6.4) 16 (10.8)
Entecavir 5 (12.8) 8 (7.3) 13 (8.8)
Tenofovir 1 (2.6) 14 (12.8) 15 (10.1)
Combination
1 (2.6) 18 (16.5) 19 (12.8)
Duration of index treatment (month
)
Mean
SD
45.5 18.4
34.6 20
37.5 20.1
0.001
< 24 3 (7.7) 47 (43.1) 50 (33.8) <0.001
25-48 20 (51.3) 37 (33) 56 (37.8)
>49 16 (41) 26 (23.9) 42 (28.4)
Response at 1 year
HBV DNA undetectable
All patients 21/39 (53.8) 84/109 (77.1) 105 (70.9) 0.008
NUC-naïve 12/21 (57.1) 50/60 (83.3) 62/81 (76.5) 0.03
NUC-experienced 9/18 (50) 34/49 (69.4) 43/67 (64.2) 0.16
HBV DNA* (log IU/mL) 2.4 0.7 2.4 0.6 2.4 0.7 0.91
ALT <ULN# 25/37 (67.6) 54/99 (54.5) 79/136 (58.1) 0.24
Nadir Response
HBV DNA undetectable
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All patients 26/39 (66.7) 102/109 (93.6) 128 (86.5) <0.001
NUC-naïve 15/21 (71.4) 56/60 (93.3) 71 (87.7) 0.02
NUC-experienced 11/18 (61.1) 46/49 (93.9) 57 (85.1) 0.003
HBV DNA* (log IU/mL) 2.3 0.7 1.7 0.7 2.1 0.8 0.09
ALT <ULN# 31/37 (83.8) 74/99 (74.7) 105/136 (77.2) 0.36
Interval from Nadir to 1st VBT
(month) Mean SD
20.5 17
0
20.5 17
Duration of on-treatment F/U after
nadir (month)
Mean SD
35.6 19.5
25.8 20.7
28.4 23
0.003
Results expressed as number (%), mean SD
*Among patients who had detectable HBV DNA
#For patients who had elevated baseline ALT
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Table 3: Multivariate Analyses of Factors Associated with Virological Breakthrough
All Patients NUC- naï ve patients NUC-exper ienced patients
Variables HR (95% CI) P-value HR(95%CI) P-value HR(95%CI) P-value
Undetectable HBV
DNA after 1 year
1.57 (0.75-3.26)
0.23
2.79 (1.11-7.01)
0.03
1.92 (0.36-10.34)
0.45
Undetectable HBV
DNA at nadir
response
5.53 (2.49-12.28)
<0.001
3.95 (1.46-10.71)
0.007
9.59 (1.64-56.07)
0.01
Index regimen
LAM monotherapy
vs. Others
-
-
3.19 (1.22-8.33)
0.02
-
-
Other monotherapy
vs. TDF monotherapy
or combination
therapy
-
-
-
-
8.15 (1.61-41.29)
0.01
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Patient Index
treatment
Mutation(s) at time of VBT Rescue therapy HBV DNA 6 months after start of
rescue therapy
A LAM M204I, L180M ADV <2 log10 decrease
B LAM M204I, L180M LAM + ADV >2 log10 decrease
C LAM M204I TDF + FTC UD
D LAM M204I TDF + FTC UD
E LAM M204I TDF + FTC UD
F LAM M204I TDF UD
G LAM M204V, L180M LAM + ADV UD
H LAM M204I, L180M TDF + FTC UD
I LAM M204I/V, L180M ADV UD
J ADV N236T, A181V TDF + FTC UD
K ADV N236T, A181V TDF + FTC UD
L ADV N236T, A181V TDF UD
M ADV A181V TDF + FTC UD
N ADV A181V, M204V, L180M TDF + FTC UD
O ADV N236T, M204V, L180M TDF + FTC UD
P ETV M204V, L180M, T184L TDF UD
Q ETV M204V, T184A ETV + TDF UD
R ETV M204V, L180M, S202G ETV + TDF UD
S ETV M204V, L180M, A181V, T184S,
V173L
TDF + FTC UD
T ADV N236T* TDF + FTC UD
U ADV No mutation TDF UD
V TDF No mutation TDF + FTC UD
W LAM M204I, L180M LAM + TDF UD
X LAM M204I TDF + FTC UD
Y LAM M204I LAM + ADV <2 log10 decrease
Z ETV M204V, L180M, T184S TDF + FTC UD
AA LAM No mutation LAM + ADV UD
AB TBV No mutation ETV UD
LAM, lamivudine; ADV, adefovir; ETV, entecavir; TBV, telbivudine; TDF, tenofovir; FTC, emtricitabine; UD, undetectable
*mutation detected by line probe assay but not by direct sequencing
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