Hepatitis B Virus Surface Antigen Levels: A Guide to

Sustained Response to peginterferon alfa-2a in

HBeAg-Negative Chronic Hepatitis B
Maurizia Rossana Brunetto,1 Francesco Moriconi,1 Ferruccio Bonino,2 George K. K. Lau,3 Patrizia Farci,4
Cihan Yurdaydin,5 Teerha Piratvisuth,6 Kangxian Luo,7 Yuming Wang,8 Stephanos Hadziyannis,9 Eva Wolf,10
Philip McCloud,11 Richard Batrla,12 and Patrick Marcellin13

We investigated the relationship between hepatitis B virus surface antigen (HBsAg) serum level
decline and posttreatment response in patients with hepatitis B e antigen (HBeAg)-negative
chronic hepatitis B from a large multinational study of pegylated interferon alfa-2a (peginter-
feron alfa-2a), with or without lamivudine, versus lamivudine alone. Serum HBsAg was quanti-
fied using the Architect assay (Abbott Diagnostics) at pretreatment, end of treatment (week 48),
and 6 months after the end of treatment (week 72) in sera from 386 of the 537 patients who
participated in the multinational study (peginterferon alfa-2a, 127; peginterferon alfa-2a plus
lamivudine, 137; lamivudine monotherapy, 122). Pretreatment HBsAg levels varied according to
genotype, with the highest levels present in patients infected with genotypes A (median, 4.11
log10 IU/mL) and D (median, 3.85 log10 IU/mL). Significant on-treatment decline in HBsAg was
observed during treatment with peginterferon alfa-2a (alone or combined with lamivudine; mean
decline at week 48, ⴚ0.71 and ⴚ0.67 log10 IU/mL, respectively, P < 0.001), but not during
treatment with lamivudine alone (ⴚ0.02 log10 IU/mL). Significantly more patients treated with
peginterferon alfa-2a (21%) or peginterferon alfa-2a plus lamivudine (17%) achieved HBsAg
levels <100 IU/mL at the end of treatment compared with lamivudine (1%) (both P < 0.001
versus lamivudine). End-of-treatment HBsAg level correlated strongly with HBV DNA suppres-
sion to <400 copies/mL 6 months posttreatment. An HBsAg level <10 IU/mL at week 48 and
on-treatment decline >1 log10 IU/mL were significantly associated with sustained HBsAg clear-
ance 3 years after treatment (both P < 0.0001). Conclusion: On-treatment quantification of
HBsAg in patients with HBeAg-negative chronic hepatitis B treated with peginterferon alfa-2a
may help identify those likely to be cured by this therapy and optimize treatment strategies.
(HEPATOLOGY 2009;49:1141-1150.)

treatment for patients with chronic hepatitis B (CHB) is

See Editorial on Page 1063
to decrease progression of liver disease to cirrhosis and

C
hronic hepatitis B is a global health problem ac-
counting for 1 million deaths each year.1 Age-
adjusted death rates are 3 to 3.6 times higher in
carriers of hepatitis B virus (HBV) surface antigen (HB-
sAg) than in persons without HBV infection.2 The aim of
hepatocellular carcinoma, with the ultimate aim of im-
proving survival. This can be pursued by maintaining
constant inhibition of viral replication through a long-
term treatment with nucleos(t)ide analogs or by inducing,
through the combined antiviral and immunomodulatory

Abbreviations: ALT, alanine aminotransferase; CART, classification and regression tree; CHB, chronic hepatitis B; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B
virus surface antigen; HBV, hepatitis B virus; peginterferon alfa-2a, pegylated interferon alfa-2a.
From 1UO Epatologia, Azienda Ospedaliero Universitaria Pisana, Pisa, Italy; 2Scientific Direction, Foundation IRCCS Policlinico of Milan and University of Pisa,
Italy; the 3Department of Medicine, Queen Mary Hospital, Hong Kong, China; 4Dipartimento di Scienze Mediche, Università di Cagliari, Monserrato, Italy; the
5Department of Gastroenterology, University of Ankara, Turkey; the 6Department of Internal Medicine, Songklanagarind Hospital, Prince of Songkla University, Hat Yai,

Thailand; the 7Department of Infectious Diseases, Nanfang Hospital, Guangzhou, China; the 8Infectious Disease Department, Xinan Hospital, Chongqing, China; the
9Department of Medicine and Hepatology, Henry Dunant Hospital, Athens, Greece; 10MUC Research, Munich, Germany; 11Roche, Dee Why, Australia; 12Roche, Basel,

Switzerland; and 13Hôpital Beaujon, University of Paris, Clichy, France.

Received June 25, 2008; accepted November 23, 2008.
Editorial assistance supported by Roche.

1141
1142 BRUNETTO ET AL.
effects of interferon, a sustained immune response. Main-
tenance of the inactive carrier state (HBV DNA ⬍2,000
IU/mL and normal alanine aminotransferase [ALT]) after
treatment discontinuation has been associated with good
prognosis in both HBeAg-positive and HBeAg-negative
CHB.3-7 Nevertheless, HBsAg seroconversion, character-
ized by the loss of serum HBsAg and development of
anti-HBs antibodies, is the hallmark of a successful im-
munological response to HBV infection and the closest
outcome to clinical cure. It is associated with favorable
long-term clinical outcomes, including reduced incidence
of cirrhosis and hepatocellular carcinoma and longer sur-
vival.8-12 HBsAg seroconversion can be achieved after in-
terferon-based therapy as demonstrated by conventional
interferon5-7 and, more recently, by peginterferon alfa-2a
and peginterferon alfa-2b in patients with HBeAg-posi-
tive disease13,14 and by peginterferon alfa-2a in patients
with HBeAg-negative disease.15
Interferon-based therapy leads to an off-therapy re-
sponse in approximately one third of patients, and these
patients have an increasing likelihood of achieving HBsAg
clearance during long-term follow up. In contrast, HBsAg
clearance is rarely achieved in patients treated with nu-
cleos(t)ide analogs, especially in those with HBeAg-nega-
tive disease15,16 where rates are typically 0% with 1 year of
therapy. Nucleos(t)ide analogs need to be given long
term, because they suppress HBV DNA only during ther-
apy. Although quantification of HBV DNA during treat-
ment is appropriate for determining response to
nucleos(t)ide analogs, it is not as suitable for monitoring
the mechanisms responsible for the achievement of sus-
tained antiviral response to interferon-based therapy in
chronic hepatitis B patients.
A recent retrospective analysis suggested that quantita-
tive determination of HBsAg level in patients treated with
interferon may provide an insight into the likelihood of
eventual HBsAg seroconversion.17 We hypothesized that
the analysis of response to interferon-based therapy using
the additional marker of HBsAg quantification might im-
prove our understanding of which patients are most likely
to achieve a complete posttreatment response. We per-
formed a comparative analysis of HBsAg levels in patients
Address reprint requests to: Maurizia Brunetto, UO Epatologia, Azienda Ospedaliero Universitaria Pisana, Pisa, Italy. E-mail: brunetto@med-club.com; fax:
(39)-050-995457.
Copyright © 2008 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/hep.22760
Potential conflict of interest: Dr. Brunetto is on the speakers’ bureau of Roche. Dr. Bonino is a consultant for, advises, and is on the speakers’ bureau of Abbott,
Bristol-Myers Squibb, Gilead, and Novartis. He is also in the speakers’ bureau of Schering-Plough. Dr. Lau is on the speakers’ bureau of and received grants from Roche
and Novartis. Dr. Piratvisuth advises and is on the speakers’ bureau of Roche. He advises Novartis and Schering-Plough and is on the speaker’s bureau of GlaxoSmith Kline.
Dr. Hadziyannis is on the speakers’ bureau of and received grants from Roche. He advises and received grants from Gilead. He advises Novartis and Bristol-Myers Squibb.
Dr. McCloud owns stock in Roche.
HEPATOLOGY, April 2009
with HBeAg-negative chronic hepatitis B who were
treated as part of a large randomized multinational study
of peginterferon alfa-2a, with or without lamivudine, and
lamivudine alone. The main objectives of this study were
(1) to investigate pretreatment levels of HBsAg and their
possible correlation with other baseline factors including
HBV genotype, (2) to quantify changes in HBsAg level
during different treatment regimens, and (3) to determine
if on-treatment HBsAg decline is correlated with response
6 months posttreatment and subsequent HBsAg clear-
ance up to 3 years posttreatment.
Patients and Methods
Patients. This was a retrospective analysis of HBsAg
levels in sera from a total of 386 of 537 patients with
HBeAg-negative CHB who participated in a randomized,
placebo-controlled study conducted at 54 sites in 13
countries.15 The selection criterion for inclusion in the
present study was availability of stored sera for quantifi-
cation of HBsAg level pretreatment, at end of treatment
(week 48), and 6 months after end of treatment (week
72). Sera for the remaining patients were not available.
The study was conducted according to the guidelines of
the Declaration of Helsinki and with the principles of
Good Clinical Practice and was approved by local ethics
committees. All patients gave written informed consent.
All patients were positive for HBsAg and negative for
anti-HBs antibody pretreatment. Treatment comprised
180 ␮g peginterferon alfa-2a once weekly plus oral pla-
cebo once daily (n ⫽ 127), 180 ␮g peginterferon alfa-2a
once weekly plus 100 mg lamivudine once daily (n ⫽
137), or 100 mg lamivudine once daily (n ⫽ 122) for 48
weeks. Results 6 months posttreatment have been pub-
lished previously.15 For the purpose of this analysis, re-
sponse 6 months posttreatment was defined as HBV
DNA suppression to ⱕ400 copies/mL or biochemical
(ALT ⱕ30 U/L). Response parameters assessed 3 years
after the end of therapy were HBV DNA ⱕ400 cop-
ies/mL and HBsAg clearance.
Analyses. Pretreatment HBsAg levels for all 386 pa-
tients were analyzed according to the baseline factors
HEPATOLOGY, Vol. 49, No. 4, 2009
HBV DNA level, ALT, HBV genotype, and host factors
(age, sex). Decline in HBsAg and HBV DNA over time
(pretreatment, end of treatment, and 6 months posttreat-
ment) was determined for all 386 patients according to
treatment group. Stratification of decline in HBsAg levels
over time according to HBV genotype was performed for
the 264 patients treated with peginterferon alfa-2a ⫾
lamivudine.
HBsAg level at end of treatment was analyzed with
respect to HBV DNA and ALT response 6 months post-
treatment for all 386 patients stratified according to treat-
ment group. The relationship between end-of-treatment
HBsAg level and HBsAg clearance 3 years posttreatment
was investigated in a total of 198 patients treated with
peginterferon alfa-2a ⫾ lamivudine who entered a long-
term observational rollover study. Patients with missing
data for HBsAg clearance at 3 years posttreatment were
considered to be nonresponders.
End-of-treatment HBsAg levels associated with a
3-year posttreatment response were identified using clas-
sification and regression tree (CART) analyses. Multiple
logistic regression analyses were performed to identify po-
tential baseline or on-treatment parameters associated
with sustained HBsAg clearance.
Assays. HBsAg was quantified using the Architect
HBsAg assay (Abbott Laboratories, Abbott Park, IL; dy-
namic range, 0.05-250.0 IU/mL) after 1:100 dilution.
Samples with HBsAg levels ⬎250.0 IU/mL at 1:100 di-
lution were retested at a final dilution of 1:1,000. Samples
with HBsAg levels ⬍0.05 IU/mL at 1:100 dilution were
retested undiluted. HBV DNA was measured using the
Amplicor HBV test (Roche Diagnostics, Basel, Switzer-
land; range, 400-200,000 copies/mL); samples with vire-
mia ⬎200,000 copies/mL were retested after 1:100
dilution according to the manufacturer’s recommenda-
tions. Serum ALT was measured at the time of sampling.
Normal ALT was considered to be ⱕ1⫻ the upper limit
of normal (30 IU/mL).
Statistical Analyses. Multiple logistic regression
analysis was used to investigate baseline factors other than
treatment with peginterferon alfa-2a that might be asso-
ciated with HBsAg reduction (as a continuous variable)
from baseline to end of treatment (week 48) and from
baseline to 6 months posttreatment (week 72) for the 198
patients treated with peginterferon alfa-2a ⫾ lamivudine.
Factors included in the analyses were: treatment with
peginterferon alfa-2a versus peginterferon alfa-2a plus
lamivudine, sex, genotype, and—as continuous vari-
ables—age, body mass index, baseline ALT, baseline
HBV DNA, and baseline HBsAg level.
For the subset of 65 patients (treated with peginterferon
alfa-2a ⫾ lamivudine) with HBsAg quantification and avail-
BRUNETTO ET AL. 1143
able 3-year follow-up data, multiple logistic regression anal-
ysis was used to investigate factors that might be associated
with HBsAg clearance 3 years after end of treatment. Vari-
ables included in the analyses were baseline factors (treat-
ment with peginterferon alfa-2a versus peginterferon alfa-2a
plus lamivudine, age, baseline ALT, baseline HBV DNA)
and week 48 variables (change in HBsAg level from baseline,
absolute HBsAg level, HBV DNA level).
CART analyses18 were performed using CART soft-
ware19 to investigate negative and positive predictive val-
ues as well as the sensitivity and specificity of end-of-
treatment HBsAg levels for predicting sustained response
to peginterferon alfa-2a 3 years after end of treatment.
Test data were generated using 10-fold cross-validation;
the tree models were then fitted to all the data excluding
each subsample in turn. The misclassification cost was
calculated for each of the 10 trees by validating each in
their respective subsamples.
Results
Pretreatment HBsAg Level and Correlation with
Other Baseline Factors. Patient demographic and labo-
ratory characteristics were comparable across the three
treatment arms (Table 1). The mean HBsAg level was 3.4
log10 IU/mL (2,500 IU/mL; range, 0.78-4.89 IU/mL);
21.5% of patients had an HBsAg level ⬍3 log10 IU/mL
(1,000 IU/mL), 63.2% had levels ⱖ3 and ⬍4 log10
IU/mL (1,000-10,000 IU/mL), and 15.3% had levels ⱖ4
log10 IU/mL (10,000 IU/mL).
Pretreatment HBsAg showed a positive correlation
with baseline HBV DNA level (r ⫽ 0.38), but there was
no apparent correlation with baseline ALT level (r ⫽
0.09) or age (r ⫽ ⫺0.14). Median HBsAg levels were
similar in males (3.39 log10 IU/mL, n ⫽ 326) and females
(3.42 log10 IU/mL, n ⫽ 60). Variation in pretreatment
HBsAg levels was observed between genotypes (Fig. 1),
with the highest median levels in patients infected with
HBV genotypes A (median, 4.11 log10 IU/mL) and D
(median, 3.85 log10 IU/mL).
HBsAg and HBV DNA Decline According to Treat-
ment Regimen. The magnitude of HBsAg decline from
pretreatment to week 48 was approximately 30 times higher
with peginterferon alfa-2a (alone or combination with lami-
vudine) versus lamivudine alone (mean declines of ⫺0.71
and ⫺0.67 log10 IU/mL, respectively, versus ⫺0.02 log10
IU/mL in patients treated with lamivudine monotherapy; P
⬍0.01) (Fig. 2A). The pattern of decline in HBsAg and
HBV DNA levels from pretreatment to weeks 48 and 72
according to treatment group is provided in Fig. 2B. HBV
DNA level declined considerably in all three treatment arms,
1144 BRUNETTO ET AL. HEPATOLOGY, April 2009

Table 1. Baseline Demographics and Laboratory Characteristics

Peginterferon Peginterferon
Characteristic alfa-2a ⴙ Placebo alfa-2a ⴙ Lamivudine Lamivudine

Total number of patients 127 137 122

Male/female, n (%) 107/20 (84/16) 115/22 (84/16) 104/18 (85/15)
Region, n (%)
Asia-Pacific 31 (24) 29 (21) 28 (23)
China 67 (53) 70 (51) 66 (54)
Europe 29 (23) 38 (28) 28 (23)
Mean age, years ⫾ SD 38.76 ⫾ 11.13 38.94 ⫾ 10.28 38.80 ⫾ 10.58
Mean height, cm ⫾ SD 168.97 ⫾ 7.90 169.07 ⫾ 8.10 170.31 ⫾ 7.48
Mean weight, kg ⫾ SD 68.94 ⫾ 11.24 68.40 ⫾ 12.75 69.80 ⫾ 12.20
BMI ⫾ SD 24.06 ⫾ 3.4 23.82 ⫾ 3.84 23.93 ⫾ 3.33
Genotype, n (%)
A 8 (6) 9 (7) 5 (4)
B 41 (32) 37 (27) 43 (35)
C 56 (44) 61 (45) 50 (41)
D 17 (13) 26 (19) 22 (18)
Other 5 (4) 4 (3) 2 (2)
Mean pretreatment HBV DNA, log10
copies/mL ⫾ SD 7.20 ⫾ 1.94 7.34 ⫾ 2.08 7.31 ⫾ 1.79
Pretreatment HBsAg, log10 IU/mL
Mean ⫾ SD 3.32 ⫾ 0.61 3.46 ⫾ 0.58 3.43 ⫾ 0.57
Median 3.30 3.50 3.39
Range 0.78–4.70 1.73–4.78 1.58–4.89

Abbreviation: BMI, body mass index; SD, standard deviation.

with the greatest decline at the end of treatment in the pegin-
terferon alfa-2a plus lamivudine arm.
Baseline Factors Associated with Decline in HBsAg
to End of Treatment (Week 48) and 6 Months Post-
treatment (Week 72) in Patients Treated with Pegin-
terferon alfa-2a ⴞ Lamivudine. We analyzed whether
baseline factors might influence the degree of decline of
HBsAg observed in patients treated with a peginterferon
alfa-2a– containing regimen. Patients aged ⬍25 years
showed a greater on-treatment decline in HBsAg level
Fig. 1. Pretreatment HBsAg level distribution according to genotype.
(⬎⫺0.8 log10 IU/mL mean decline) than patients aged
⬎55 years (⬍⫺0.4 log10 IU/mL mean decline), whereas
there was no apparent pattern in on-treatment decline in
HBsAg level according to baseline ALT level.
Analysis of on-treatment HBsAg decline according to ge-
notype showed that the mean decline was most pronounced
in patients infected with genotypes A and B (approximately
⫺0.8 log10 IU/mL; 95% confidence intervals, ⫺1.58 to
⫺0.02 and ⫺1.03 to ⫺0.50, respectively) and least pro-
nounced in patients with genotype D (⫺0.3 log10 IU/mL;
95% confidence interval, ⫺0.58 to ⫺0.06). The distribu-
tion of HBsAg levels over time in patients infected with
HBV genotypes A to D is shown in Fig. 3. Median levels of
HBsAg decreased from pretreatment to end of treatment in
all genotypes, with less rebound evident between end of
treatment and 6 months posttreatment in patients infected
with HBV genotypes A and B.
The only baseline factors identified by multivariate
analysis that were significantly associated with decline in
HBsAg to end of treatment were baseline HBV DNA
level (P ⫽ 0.003) and age (P ⫽ 0.04). Baseline HBV
DNA level was significantly associated with reduction in
HBsAg level from baseline to 6 months posttreatment
(P ⫽ 0.005). No significant association was found be-
tween decline in HBsAg from end of treatment or 6
months posttreatment and treatment regimen (peginter-
feron alfa-2a versus peginterferon alfa-2a plus lamivu-
dine), sex, HBV genotype, body mass index, baseline
ALT, and baseline HBsAg level.
HEPATOLOGY, Vol. 49, No. 4, 2009
Fig. 2. (A) Decline in HBsAg levels from pretreatment to end of
treatment (week 48) and 6 months after treatment (week 72) according
to treatment group. (B) Pattern of HBsAg and HBV DNA decline according
to treatment group.
BRUNETTO ET AL. 1145
Association Between Serum HBsAg Level at End of
Treatment and Response to Therapy 6 Months Post-
treatment. Overall, the mean decline in HBsAg from
pretreatment levels to week 48 was greater for those who
achieved a virological response than for those who did not
(Fig. 4). The mean HBsAg decline was ⫺1.077 log10 for
the HBV DNA ⱕ400 copies/mL endpoint responders
versus ⫺0.263 for nonresponders (P ⬍ 0.001). This as-
sociation held true only for the subgroup of patients
treated with peginterferon alfa-2a– based therapy. Among
patients treated with lamivudine who achieved an HBV
DNA level ⱕ400 copies/mL at week 72, the mean HBsAg
decline was negligible (⫺0.003 log10 IU/mL) and lower
than that seen in patients who did not achieve suppression
of HBV DNA to ⱕ400 copies/mL (⫺0.022 log10 IU/
mL).
Significantly more patients treated with peginterferon
alfa-2a ⫾ lamivudine achieved a substantial on-treatment
decline in HBsAg compared with those treated with lami-
vudine only (Fig. 5A); HBsAg levels ⱕ100 IU/mL at the
end of treatment were observed in 21% and 17% of pa-
tients treated with peginterferon alfa-2a and peginter-
feron alfa-2a plus lamivudine compared with only 1% of
those treated with lamivudine alone (P ⬍ 0.001 for pegin-
terferon alfa-2a ⫾ lamivudine versus lamivudine).
We performed a detailed analysis of end-of-treatment
HBsAg levels and response 6 months posttreatment ac-
cording to treatment group. There was a strong associa-
tion between level of HBsAg at end of treatment and
suppression of HBV DNA to ⱕ400 copies/mL, in partic-
ular for patients treated with a peginterferon alfa-2a– con-
taining regimen. The association was less apparent for
ALT response (Fig. 5B). A very high proportion of pegin-
terferon alfa-2a–treated patients with end-of-treatment
HBsAg levels ⱕ10 IU/mL had HBV DNA suppressed to
ⱕ400 copies/mL 6 months posttreatment (88% and 73%
for peginterferon alfa-2a and peginterferon alfa-2a plus
lamivudine, respectively).
Association Between Serum HBsAg Level at End of
Treatment and Response to Therapy 3 Years After
Treatment with Peginterferon alfa-2a. In total, 198
peginterferon alfa-2a–treated patients with available
data on quantitative HBsAg entered the long-term ob-
servational follow-up study, and we analyzed the rela-
tionship between end-of-treatment HBsAg level at
subsequent HBsAg clearance 3 years after treatment.
Of the 198 patients, 16 (8%) had cleared HBsAg by the
3-year posttreatment analysis. Of the 23 patients with
an HBsAg level ⱕ10 IU/mL, 12 (52%) had cleared
HBsAg 3 years after treatment compared with only
four (2.3%) of the 171 patients with an HBsAg level
⬎10 IU/mL at the end of treatment (P ⬍ 0.0001;
1146 BRUNETTO ET AL.
relative risk, 22.8; 95% confidence interval, 8-649)
(Table 2). Similarly, an on-treatment decline of HBsAg
of ⬎1.1 log10 IU/mL was significantly associated with
HBsAg clearance (P ⬍ 0.0001; relative risk, 14.6; 95%
confidence interval, 5.5-38.5).
Multivariate logistic regression analysis using selected
baseline and on-treatment factors identified a decline in
HBsAg levels from pretreatment to end of treatment as
the only factor significantly associated with HBsAg clear-
ance 3 years after treatment. Baseline parameters (age,
Fig. 4. Decline in HBsAg from pretreatment to end of treatment
according to virological response and treatment group.
HEPATOLOGY, April 2009
Fig. 3. HBsAg level over time ac-
cording to HBV genotype.
HBsAg level, HBV DNA level, ALT level) or treatment
with peginterferon alfa-2a versus peginterferon alfa-2a
plus lamivudine were not associated with HBsAg clear-
ance 3 years after treatment.
Given the association between HBsAg level at week
48 and HBsAg clearance 3 years after treatment, we
aimed to identify cutoff levels of HBsAg at week 48
that could be used to provide the best predictive value
of response. CART analyses were performed for 3-year
posttreatment response including HBsAg clearance or
HBV DNA suppression to ⱕ400 copies/mL. For re-
sponse defined as HBsAg clearance 3 years after treat-
ment, the best predictive values were an absolute
HBsAg level of ⱕ380 IU/mL at week 48 or a change in
HBsAg level from pretreatment to week 48 of ⬎1.87
log10 IU/mL (Table 3). Of the 64 patients with an
end-of-treatment HBsAg level ⱕ380 IU/mL, 16
(25%) had HBsAg clearance by 3 years after treatment
compared with none of the 134 patients who had an
end-of-treatment HBsAg level ⬎380 IU/mL. Of the
27 patients with a change in HBsAg level from pre-
treatment to week 48 of ⬎1.87 log10 IU/mL, 12 pa-
tients (44%) had sustained HBsAg clearance 3 years
after treatment. Of the 171 patients with a decline in
HBsAg level from pretreatment to week 48 of ⬍1.87
log10 IU/mL, only four (2%) had HBsAg clearance 3
years after treatment.
The cutoff level associated with reduction in HBV
DNA level to ⱕ400 copies/mL 3 years after treatment
HEPATOLOGY, Vol. 49, No. 4, 2009
Fig. 5. (A) Distribution of HBsAg level at end of treatment according
to treatment group. (B) Response 6 months after treatment according to
end-of-treatment HBsAg level and treatment group.
BRUNETTO ET AL. 1147
was HBsAg ⱕ19 IU/mL at week 48 and an on-treat-
ment reduction in HBsAg of ⬎0.46 log10 IU/mL. Of
27 patients with HBsAg ⱕ19 IU/mL at week 48, 17
(63%) had HBV DNA ⱕ400 copies/mL 3 years after
treatment, whereas only 14 of 171 patients (8%) with
HBsAg ⬎19 IU/mL at week 48 reached this endpoint.
Eighty-two patients had a reduction in HBsAg of
⬎0.46 log10 from baseline to week 48; of these, 25
(30%) had HBV DNA ⱕ400 copies/mL 3 years after
treatment.
Discussion
Our analyses provide an insight into levels of HBsAg in
patients with HBeAg-negative chronic hepatitis B, a di-
rect comparison of the effects of different treatment regi-
mens upon them and how end-of-treatment HBsAg levels
relate to sustained posttreatment response. Considerable
variation in HBsAg level is observed within the patient
population, and levels appear to be affected by HBV ge-
notype, with the highest levels in those patients infected
with HBV genotypes A and B. There was some degree of
correlation of pretreatment HBsAg with HBV DNA
level, lower HBsAg levels being associated with lower lev-
els of HBV DNA.
Serum HBsAg levels declined significantly during 48
weeks of treatment with peginterferon alfa-2a ⫾ lamivu-
dine, but not with lamivudine alone (P ⬍ 0.001). The
decline in HBsAg was similar in the two peginterferon
alfa-2a– containing regimens with approximately 10% of
patients achieving HBsAg levels ⬍10 IU/mL at end of
treatment. These patients had the highest rates of HBV
DNA suppression to below 400 copies/mL 6 months after
treatment. This suggests that the HBsAg level reached at
the end of treatment could be used as an indicator of
anticipated sustained response in peginterferon alfa-2a–
treated patients. Previous studies have shown that patients
who achieve sustained virological posttreatment response
to interferon therapy have an increasing chance of achiev-
ing subsequent clearance of HBsAg,20 which is considered
the closest outcome to clinical cure in patients with
chronic hepatitis B.
Of the 23 patients in the long-term follow-up study
who had achieved an end-of-treatment HBsAg level ⬍10
IU/mL, 52% had cleared HBsAg by 3 years after treat-
ment. In contrast, there was no significant association
between suppression of HBV DNA to ⬍400 copies/mL
at end of treatment and sustained HBsAg clearance 3
years after treatment. HBsAg level may therefore be a
more appropriate marker of response to peginterferon
than HBV DNA level.
Despite lowering HBV DNA levels through its an-
tiviral effects, lamivudine had little effect on HBsAg
1148 BRUNETTO ET AL. HEPATOLOGY, April 2009

Table 2. Association Between End– of-Treatment Levels of HBsAg or HBV DNA and HBsAg Clearance 3 Years After
Treatment
Patients with HBsAg Loss
3 Years After Treatment,
Parameter Value No. of Patients n (%) Relative Risk P Value

HBsAg level at week 48, IU/mL (n ⫽ 194) ⱕ10 23 12 (52) 22.8 (8–649) ⬍0.0001
⬎10 171 4 (2.3)
Decline in HBsAg from baseline to week
48, log10 IU/mL (n ⫽ 198) ⬎2.0 26 11 (42.3) 14.6 (5.5–38.5) ⬍0.0001
ⱕ2.0 172 5 (2.9)
⬎1.0 43 13 (30) 10.8 (3.7–31.8) ⬍0.0001
ⱕ1.0 155 4 (2.6)
HBV DNA level at week 48, copies/mL
(n ⫽ 194) ⱕ400 161 15 (9) 3.1 (0.4–22.5) NS
⬎400 33 1 (3)

Abbreviation: NS, not significant.

levels, as shown by Manesis et al.17 Thus, antiviral in the absence of the high degree of HBV DNA inhi-
suppression alone is clearly not sufficient to reduce bition induced by the nucleos(t)ide analogs.21 Other
HBsAg levels. Interferon has not only antiviral activity oral antivirals, including entecavir— despite being
that suppresses viral replication, but also immuno- more potent inhibitors of HBV replication— have not
modulatory effects which can prevent infection of pre- so far induced HBsAg clearance rates comparable to
viously uninfected hepatocytes and also result in those achieved by peginterferon alfa-2a in patients with
clearance of infected hepatocytes. It is the combined HBeAg-negative disease, despite treatment durations
effect of HBV DNA suppression and immunological of 2 to 5 years.22-24
enhancement that may help tip the balance to allow the The potential value of quantifying serum HBsAg to
host to gain immunological control and thereby a sus- predict long-term response to conventional interferon
tained off-therapy response and eventual HBsAg clear- was suggested previously in patients with HBeAg-pos-
ance. Therefore, the reduction of HBsAg serum levels itive CHB. A significant reduction in serum HBsAg
during peginterferon alfa-2a therapy may be consid- levels was seen in patients who responded to interferon
ered the hallmark for achievement of the immune com- (as determined by HBeAg seroconversion), but not in
petence that is needed for persistent control of chronic patients without HBeAg seroconversion (P⬍ 0.001).25
HBV infection. Accordingly, the modeling of HBV The on-treatment reduction in HBsAg afforded by
dynamics during therapy showed a similar or even peginterferon alfa-2a and its association with sustained
greater impact on the clearance of the infected cell by response suggests that its monitoring could be benefi-
peginterferon alfa-2a compared with lamivudine, even cial in several ways. It may be possible, for example, to
identify early during the course of therapy which pa-
Table 3. HBsAg Cutoff Levels at Week 48 and Predictive
tients are achieving HBsAg decline and thus most likely
Values for Response 3 Years After Treatment to respond. For example, patients treated with pegin-
Response Parameter 3 Years Post
terferon alfa-2a who achieved an end-of-treatment
Treatment HBsAg level ⬍10 IU/mL had a very high chance (88%)
HBV DNA <400 of achieving suppression of HBV DNA to ⱕ400 cop-
Copies/mL HBsAg Clearance ies/mL 6 months after treatment. Conversely, given the
HBsAg level at week 48 ⬍19 IU/mL ⬍380 IU/mL fact that not all patients are responsive to peginter-
Sensitivity (%) 92 74 feron, lack of HBsAg decline could be used as a poten-
Specificity (%) 75 100
tial indicator of nonresponse and thus be used to
NPV (%) 98 100
PPV (%) 44 25 consider switching a patient to an alternative treatment
Reduction in HBsAg approach.
from pretreatment to week 48 ⬎0.46 log10 IU/mL ⬎1.87 log10 IU/mL
We found a significant association between on-
Sensitivity (%) 66 92
Specificity (%) 81 75 treatment decline in serum HBsAg of ⬎1 or ⬎2 log10
NPV (%) 95 98 IU/mL and HBsAg clearance 3 years after treatment.
PPV (%) 30 44 On-treatment decline of HBsAg ⬍1.87 log10 IU/mL or
Abbreviations: NPV, negative preditive value; PPV, positive predictive value. an absolute HBsAg level of ⬎380 IU/mL were negative
HEPATOLOGY, Vol. 49, No. 4, 2009
predictors of sustained HBsAg clearance 3 years after
treatment. Similarly on-treatment decline of HBsAg
⬍0.46 log10 IU/mL or an absolute HBsAg level of ⬎19
IU/mL could be used as a negative predictor for HBV
DNA suppression to ⬍400 copies/mL 3 years after
treatment, with negative predictive values of 95% and
92%, respectively. Such a negative prediction could be
taken into account when deciding whether a patient
with a large decline in HBsAg levels after 12 months of
treatment might stop treatment or whether a patient
without significant HBsAg decline might benefit from
switching to an alternative treatment strategy. Con-
versely, prolongation of treatment might be indicated
in patients with a slow decline in HBsAg levels. In fact,
extending interferon treatment duration was shown to
benefit some patients with HBeAg-negative CHB,7 and
a recent pilot study has investigated extending treat-
ment duration with peginterferon alfa-2a.26 This small
study of 13 patients suggests that prolonging treatment
with peginterferon alfa-2a to 60 weeks may be a viable
way to increase response rates; quantification of HBsAg
demonstrated a ⬎90% decrease in HBsAg levels in five
of 13 patients after 60 weeks of treatment. The authors
concluded that further clinical studies in which pegin-
terferon alfa-2a is given for even longer than 60 weeks
are warranted.27 Although the optimal duration of in-
terferon-based treatment in patients with HBeAg-neg-
ative CHB remains unresolved, it is generally regarded
as being longer than that for the HBeAg-positive form
of the disease.8,9,26,27 An on-treatment marker predict-
ing sustained off-treatment response may be used to
determine the optimal treatment duration of peginter-
feron in patients with HBeAg-negative CHB and,
therefore, enable the individualization of therapy and
optimization of response.
In conclusion, HBsAg clearance represents the best
possible and closest to cure outcome of antiviral ther-
apy in patients with CHB, but is realistic almost exclu-
sively among patients receiving interferon-based
regimens, which are recommended as a first-line ther-
apeutic approach. Quantitative on-therapy measure-
ment of serum HBsAg may be useful to identify
patients who are most likely to benefit from interferon-
based therapy and may help tailor and optimize treat-
ment duration. Further studies to determine the
optimal time points to measure HBsAg level are there-
fore warranted.
Acknowledgment: We thank Giovanni Pellegrini and
Giovanna Moscato for their technical assistance.
BRUNETTO ET AL. 1149
References
1. Hui CK, Lau GK. Current issues and future directions in treatment. Semin
Liver Dis 2006;26:192-197.
2. Chen G, Lin W, Shen F, Iloeje UH, London WT. Chronic hepatitis B
infection and mortality from non-liver causes: results from the Haimen
cohort study. Int J Epidemiol 2005;34:132-137.
3. Hsu YS, Chien RN, Yeh CT, Sheen IS, Chiou HY, Chu CM, et al.
Long-term outcome after spontaneous HBeAg seroconversion in patients
with chronic hepatitis B. HEPATOLOGY 2002,35:1522-1527.
4. Niederau C, Heintges T, Lange T, Oldmann G, Niederau CM, Mohr
L, et al. Long-term follow-up of HBeAg-positive patients treated with
interferon alfa for chronic hepatitis B. N Engl J Med 1996;334:1422-
1427.
5. Hadziyannis SJ, Vassilopoulos D. Hepatitis B e antigen-negative chronic
hepatitis B. HEPATOLOGY 2001;34:617-624.
6. Brunetto MR, Oliveri F, Coco B, Leandro G, Colombatto P, Gorin JM, et
al. Outcome of anti-HBe positive chronic hepatitis B in alpha-interferon
treated and untreated patients: a long term cohort study. J Hepatol 2002;
36:263-270.
7. Lampertico P, Del Ninno E, Vigano M, Romeo R, Donato MF, Sablon E,
et al. Long-term suppression of hepatitis B e antigen-negative chronic
hepatitis B by 24-month interferon therapy. HEPATOLOGY 2003;37:756-
763.
8. Lok AS, McMahon BJ. Chronic hepatitis B: update of recommendations.
HEPATOLOGY 2004;39:857-861.
9. The EASL Jury. EASL International Consensus Conference on Hepatitis
B, 13–14 September, 2002, Geneva, Switzerland: consensus statement
(long version). J Hepatol 2003;39:S3-S25.
10. Lok AS, McMahon BJ. Chronic hepatitis B. HEPATOLOGY 2007;45:507-
539.
11. Fattovich G, Giustina G, Sanchez-Tapias J, Quero C, Mas A, Olivotto PG,
et al. Delayed clearance of serum HBsAg in compensated cirrhosis B:
relation to interferon alpha therapy and disease prognosis. Am J Gastroen-
terol 1998;93:896-900.
12. Lin SM, Yu ML, Lee CM, Chien RN, Sheen IS, Chu CM, et al. Interferon
therapy in HBeAg positive chronic hepatitis reduces progression to cirrho-
sis and hepatocellular carcinoma. J Hepatol 2007;46:45-52.
13. Lau GK, Piratvisuth T, Luo KX, Marcellin P, Thongsawat S, Cooksley
G, et al. Peginterferon alfa-2a, lamivudine and the combination for
HBeAg-positive chronic hepatitis B. N Engl J Med 2005;352:2682-
2695.
14. Janssen HL, van Zonneveld M, Senturk H, Zeuzem S, Akarca US, Cakalo-
glu Y, et al. HBV 99-01 Study Group; Rotterdam Foundation for Liver
Research. Pegylated interferon alfa-2b alone or in combination with lami-
vudine for HBeAg-positive chronic hepatitis B: a randomised trial. Lancet
2005;365:123-129.
15. 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 for HBeAg-negative chronic hepatitis B. N Engl J Med
2004;351:1206-1217.
16. Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, Chang TT, Kitis G,
Rizzetto M, et al; Adefovir Dipivoxil 438 Study Group. Long-term therapy
with adefovir dipivoxil for HBeAg-negative chronic hepatitis B. N Engl
J Med 2005;352:2673-2681.
17. Manesis EK, Hadziyannis ES, Angelopoulou OP, Hadziyannis SJ. Predic-
tion of treatment-related HBsAg loss in HBeAg-negative chronic hepatitis
B: a clue from serum HBsAg levels. Antivir Ther 2007;12:73-82.
18. Breiman L, Friedman JH, Olshen RA, Stone CJ. Classification and Re-
gression Trees. Monterey: Wadsworth and Brooks/Cole, 1984.
19. Steinberg D, Colla P. CART—Classification and Regression Trees. San
Diego, CA: Salford Systems, 1997.
20. Korevaar A, Moucari R, Asselah T, Lada O, Boyer N, Martinot-Peignoux
M, et al. High rates of HBsAg seroconversion ion chronic hepatitis B
patients responding to interferon therapy: a long term follow-up study
[Abstract]. HEPATOLOGY 2006:46:679A.
1150 BRUNETTO ET AL.
21. Colombatto P, Civitano L, Bizzarri R, Oliveri F, Choudhury S, Gi-
eschke R, et al; Peginterferon Alfa-2a HBeAg-Negative Chronic Hep-
atitis B Study Group. A multiphase model of the dynamics of HBV
infection in HBeAg-negative patients during pegylated interferon-
alpha2a, lamivudine and combination therapy. Antivir Ther 2006;11:
197-212.
22. Lai CL, Shouval D, Lok AS, Chang TT, Cheinquer H, Goodman Z, et al.;
BEHoLD AI463027 Study Group. Entecavir versus lamivudine for pa-
tients with HBeAg-negative chronic hepatitis B. N Engl J Med 2006;354:
1011-1020.
23. Shouval D, Akarca US, Hatzis G, Kitis G, Lai CL, Cheinquer H, et al.
Continued virologic and biochemical improvement through 96 weeks of
entecavir treatment in HBeAg(-) chronic hepatitis B patients (study ETV-
027). J Hepatol 2006;Suppl:45A.
HEPATOLOGY, April 2009
24. Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, Chang TT, Kitis G,
Rizzetto M, et al.; Adefovir Dipivoxil 438 Study Group. Long-term ther-
apy with adefovir dipivoxil for HBeAg-negative chronic hepatitis B for up
to 5 years. Gastroenterology 2006;131:1743-1751.
25. Janssen HL, Kerhof-Los CJ, Heijtink RA, Schalm SW. Measurement of
HBsAg to monitor hepatitis B viral replication in patients on alpha-inter-
feron therapy. Antiviral Res 1994;23:251-257.
26. Keeffe EB, Dieterich DT, Han S-H, Jacobson IM, Martin P, Schiff ER, et
al. A treatment algorithm for the management of chronic hepatitis B virus
in the United States: an update. Clin Gastroenterol Hepatol 2006;4:936-
962.
27. Gish RG, Lau DT, Schmid P, Perrillo R. A pilot study of extended dura-
tion peginterferon alfa-2a for patients with hepatitis B e antigen-negative
chronic hepatitis B. Am J Gastroenterol 2007;102:2718-2723.