Li 2017
Li 2017
Li 2017
Available online at
ScienceDirect
www.sciencedirect.com
ORIGINAL ARTICLE
a
Department of Infectious Disease, Shandong Provincial Hospital Affiliated to Shandong University,
324 Jingwu Road, Ji’nan, Shandong 250021, China
b
Department of Oncology, Shandong Provincial Hospital Affiliated to Shandong University, 324 Jingwu
Road, Ji’nan, Shandong 250021, China
c
Division of Gastroenterology and Hepatology, Stanford University Medical Center, Palo Alto, CA,
United States
Summary
Background: This study aimed to investigate the relationship between intrahepatic cccDNA and
serum HBsAg in chronic Hepatitis B (CHB) patients with undetectable serum HBV DNA during
antiviral therapy.
Methods: We investigated HBsAg serum levels and their relationship to intrahepatic total
cccDNA and HBV DNA in CHB patients with undetectable serum HBV DNA during oral antivi-
ral therapy. Intrahepatic cccDNA and HBV DNA quantitation were performed in the same needle
biopsy material, while serum HBsAg, HBeAg and HBV DNA levels were measured in samples
drawn on the day of the liver biopsy.
Results: A total of 90 patients who had a liver biopsy were enrolled, including 80 patients
with CHB and 10 patients with liver cirrhosis (LC). All the CHB patients were divided into
HBeAg-positive and HBeAg-negative group. By using real-time PCR detection, we found that
intrahepatic cccDNA and HBV DNA levels were higher in CHB patients than those in LC patients
(Intrahepatic cccDNA: 6.15 ± 1.19 vs. 6.12 ± 0.36, HBV DNA: 7.26 ± 0.49 vs. 5.59 ± 0.45, both
∗
Corresponding author.
E-mail addresses: lijier@sina.com (J. Li), mw50881212@sohu.com (X. Sun), sdshij@sohu.com (J. Fang), chuanxiwang@yeah.net
(C. Wang), gqinghan@126.com (G. Han), whr631212@sina.com (W. Ren).
http://dx.doi.org/10.1016/j.clinre.2017.03.004
2210-7401/© 2017 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Li J, et al. Analysis of intrahepatic total HBV DNA, cccDNA and serum HBsAg level in
Chronic Hepatitis B patients with undetectable serum HBV DNA during oral antiviral therapy. Clin Res Hepatol Gastroenterol
(2017), http://dx.doi.org/10.1016/j.clinre.2017.03.004
+Model
CLINRE-994; No. of Pages 9 ARTICLE IN PRESS
2 J. Li et al.
P < 0.05). Intrahepatic cccDNA level was positively correlated with serum HBsAg in HBeAg-
negative (r = 0.66, P = 0.02) and lower serum HBeAg (≤50S/CO) CHB patients (r = 0.47, P = 0.03),
but not in higher serum HBeAg (>50S/CO) CHB patients (both P > 0.05). In HBeAg negative
patients, serum HBsAg level was correlated with intrahepatic total HBV DNA level (r = 0.52,
P = 0.006). However, no relationship between HBsAg level and intrahepatic total HBV DNA level
was found in HBeAg positive patients (both P > 0.05).
Conclusions: Serum HBsAg can be used to predict intrahepatic cccDNA and HBV DNA level in
CHB patients with low serum HBeAg statues, especially in HBeAg negative patients.
© 2017 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Li J, et al. Analysis of intrahepatic total HBV DNA, cccDNA and serum HBsAg level in
Chronic Hepatitis B patients with undetectable serum HBV DNA during oral antiviral therapy. Clin Res Hepatol Gastroenterol
(2017), http://dx.doi.org/10.1016/j.clinre.2017.03.004
+Model
CLINRE-994; No. of Pages 9 ARTICLE IN PRESS
Analysis of intrahepatic HBV DNA, cccDNA and serum HBsAg level in Chronic Hepatitis B patients 3
the upper limit of detection (250 IU/ml), the samples were instructions of the manufacturer. PSAD (Epicentre, Madison,
diluted (1:500) and reassessed. HBV DNA was extracted from WI, USA) was used to digest HBV rcDNA, replicative dsDNA
serum samples and quantified by using a commercial poly- and ssDNA. Afterwards, rolling Circle Amplification (RCA)
merase chain reaction (PCR) diagnostic kit with a detect was conducted to selectively amplify circle DNA. Using the
limit of 500 copies/ml (PG Biotech, Shenzhen, China). RCA products as template, HBV cccDNA was further ampli-
fied and quantified with TaqMan real-time PCR mediated
by a pair of cccDNA-selective primers. Intrahepatic HBV
Quantitation of intrahepatic HBV cccDNA and HBV cccDNA detection range is between 2.50 × 102 copies/ml and
DNA 2.50 × 109 copies/ml. The detect limit of intrahepatic HBV
DNA is 500 copies/ml [19].
HBV DNA and cccDNA were amplified from genomic DNA
extracted from liver specimens obtained from all patients.
Briefly, about 30 mm formalin fixed paraffin-embedded Statistical interpretation
(FFPE) liver biopsy tissue was sectioned to 6 mm each
for DNA extraction. To prevent contamination, disposable Data was presented as M+ and inter-quartile range. Serum
tweezers, brush and interleaver were used and sectioning HBV DNA and HBsAg levels were expressed in logarithm. Dif-
blades were carefully cleaned with 70% ethanol after every ferences between variables were tested by Student’s t-test
sampling. The DNA was extracted using QIAamp FFPE DNA or one-way ANOVA test where it is appropriate. Regression
Mini Kit (QIAGEN, GmbH, Hilden, Germany) according to the and Pearson’s correlation analysis were applied to compare
Please cite this article in press as: Li J, et al. Analysis of intrahepatic total HBV DNA, cccDNA and serum HBsAg level in
Chronic Hepatitis B patients with undetectable serum HBV DNA during oral antiviral therapy. Clin Res Hepatol Gastroenterol
(2017), http://dx.doi.org/10.1016/j.clinre.2017.03.004
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CLINRE-994; No. of Pages 9 ARTICLE IN PRESS
4 J. Li et al.
Figure 2 Intrahepatic HBV cccDNA, HBV DNA and serum HBsAg levels in chronic Hepatitis B and liver cirrhosis patients. Comparison
of intrahepatic HBV cccDNA (A), HBV DNA (B) and serum HBsAg (C) levels between 80 CHB and 10 LC patients. The data were analyzed
by the Mann—Whitney U test. LC, liver cirrhosis; CHB, chronic Hepatitis B.
Please cite this article in press as: Li J, et al. Analysis of intrahepatic total HBV DNA, cccDNA and serum HBsAg level in
Chronic Hepatitis B patients with undetectable serum HBV DNA during oral antiviral therapy. Clin Res Hepatol Gastroenterol
(2017), http://dx.doi.org/10.1016/j.clinre.2017.03.004
+Model
CLINRE-994; No. of Pages 9 ARTICLE IN PRESS
Analysis of intrahepatic HBV DNA, cccDNA and serum HBsAg level in Chronic Hepatitis B patients 5
Figure 3 Intrahepatic HBV cccDNA, HBV DNA and serum HBsAg levels in different groups of Chronic Hepatitis B patients. Com-
parison of intrahepatic HBV cccDNA (A), HBV DNA (B) and serum HBsAg (C) levels between different groups of Chronic Hepatitis
B patients divided according to the serum HBeAg level. Compared with * group, both P < 0.05. The data were analyzed by the
Mann—Whitney U test.
significantly higher intrahepatic HBV DNA than those in LC HBeAg-negative to HBeAg-positive A—C group, respectively)
patients (7.26 ± 0.49 vs. 5.59 ± 0.45, P < 0.05). In addition, (Fig. 3B and C).
intrahepatic HBV cccDNA level was higher in CHB patients
than those in LC patients (6.15 ± 1.19 vs. 6.12 ± 0.36, Correlation of intrahepatic HBV cccDNA with serum
P < 0.05) (Fig. 2B). Additionally, we found no significant HBsAg in CHB patients with different serum HBeAg
difference in the serum HBsAg level between CHB and LC
level
patients (3.33 ± 0.67 vs. 3.20 ± 0.34, P = 0.36) (Fig. 2C).
In order to further comparison, the total 80 CHB
After analyzing the different groups of chronic HBV infected
patients were divided into HBeAg-positive and HBeAg-
patients separately, we found that the significant positive
negative group. HBeAg-positive CHB patients were
correlation between intrahepatic HBV cccDNA level and
divided into 3 groups according to the serum HBeAg
serum HBsAg level was only observed in HBeAg-negative
level (group A: 1S/CO ≤ HBeAg < 50S/CO, group B:
(r = 0.66, P = 0.00), lower HBeAg level (<50S/CO) patients
50S/CO ≤ HBeAg < 100S/CO, group C: HBeAg ≥ 100S/CO).
(r = 0.47, P = 0.03) rather than in other patients (both
The intrahepatic cccDNA levels were 6.03 ± 0.71,
P > 0.05) (Fig. 4).
6.07 ± 0.85, 6.01 ± 0.60, 6.70 ± 1.00 log10 IU/ml in
these groups, respectively (from HBeAg-negative to
HBeAg-positive A—C group). HBV cccDNA level was Correlation of intrahepatic HBV DNA with serum
significantly higher in HBeAg ≥ 100S/CO patient than HBsAg in CHB patients with different serum HBeAg
those in HBeAg < 100S/CO patients (Fig. 3A). Intrahep- level
atic HBV DNA and serum HBsAg levels were significantly
higher in HBeAg-negative patients than those in HBeAg- Next, we analyzed the correlation between intrahepatic
positive patients (intrahepatic HBV DNA: 6.90 ± 0.49, HBV DNA and serum HBsAg in different CHB patients. Our
7.22 ± 0.36, 7.06 ± 0.35, 7.31 ± 0.88; HBsAg level: date indicated that intrahepatic HBV DNA and serum HBsAg
2.83 ± 0.83, 3.53 ± 0.58, 3.42 ± 0.36, 3.80 ± 0.42, from level were significantly higher in HBeAg-negative patients
Please cite this article in press as: Li J, et al. Analysis of intrahepatic total HBV DNA, cccDNA and serum HBsAg level in
Chronic Hepatitis B patients with undetectable serum HBV DNA during oral antiviral therapy. Clin Res Hepatol Gastroenterol
(2017), http://dx.doi.org/10.1016/j.clinre.2017.03.004
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CLINRE-994; No. of Pages 9 ARTICLE IN PRESS
6 J. Li et al.
Figure 4 Correlation of intrahepatic HBV cccDNA with serum HBsAg in patients with different serum HBeAg level. Significant
positive correlation was found between the intrahepatic HBV cccDNA and serum HBsAg in HBeAg-negative (r = 0.66, P = 0.00) and
lower HBeAg level (<50S/CO) (r = 0.47, P = 0.03) patients (A and B), but not in 50S/CO ≤ HBeAg < 100S/CO (r = 0.10, P = 0.64) and
HBeAg ≥ 100S/CO (r = 0.37, P = 0.12) patients (C and D). Dates were shown as X—Y scatter plots. P values were calculated using the
Spearman rank correlation. Solid line: linear growth trend, r: correlation coefficient.
Please cite this article in press as: Li J, et al. Analysis of intrahepatic total HBV DNA, cccDNA and serum HBsAg level in
Chronic Hepatitis B patients with undetectable serum HBV DNA during oral antiviral therapy. Clin Res Hepatol Gastroenterol
(2017), http://dx.doi.org/10.1016/j.clinre.2017.03.004
+Model
CLINRE-994; No. of Pages 9 ARTICLE IN PRESS
Analysis of intrahepatic HBV DNA, cccDNA and serum HBsAg level in Chronic Hepatitis B patients 7
Figure 5 Correlation of intrahepatic HBV DNA with serum HBsAg in CHB patients with different serum HBeAg level. Significant
positive correlation was found between the intrahepatic HBV DNA and serum HBsAg in HBeAg-negative (r = 0.52, P = 0.006) patients
(A), but not in HBeAg-positive patients (group A: r = −1.21, P = 0.59; group B: r = 0.16, P = 0.45; group C: r = 0.43, P = 0.07) (B—D).
Dates were shown as X—Y scatter plots. P values were calculated using the Spearman rank correlation. Solid line: linear growth
trend, r: correlation coefficient.
than those in HBeAg-positive patients. Further analyzing incoming viruses from the blood. Consistent with previ-
showed a significant positive correlation between intrahep- ous studies [21,22], our data showed that high levels of
atic HBV DNA and serum HBsAg level was only observed HBV DNA and cccDNA were still been detected in the liver
in HBeAg-negative (r = 0.52, P = 0.006), but not in HBeAg- tissues although in those patients who have undetectable
positive patients (both P > 0.05) (Fig. 5). serum HBV DNA during antiviral therapy (Fig. 1). In contrast,
persistent intrahepatic HBV DNA and cccDNA is the main
cause of viral relapse after NA therapy is stopped. There-
Conclusions fore, the measurement of intrahepatic cccDNA and/or HBV
DNA has additional value when evaluating anti-HBV thera-
The goal of current treatment of CHB is to suppress viral peutic efficacy and estimating treatment endpoint to CHB
replication to the lowest possible level thus halting disease patients.
progression. NA have a suppressive effect on the transcrip- The major obstacle for quantitation of intrahepatic HBV
tion of pregenomic RNA, and the administration of these DNA and cccDNA in clinic is the requirement for invasive liver
agents can induce a rapid and dramatic decrease in serum biopsy. Thus there has been increasing research to identify
HBV DNA [20]. However, currently NA therapy is unable to surrogate, non-invasive markers of the amount of intrahep-
induce a complete elimination of intrahepatic HBV DNA and atic HBV DNA and cccDNA in the liver. Both HBeAg and HBsAg
cccDNA in CHB patients. NA treatment does not directly titer have been proposed as surrogates for infected liver
affect the cccDNA content of the liver. Decreases in cccDNA cell mass. If the hepatocyte were considered in isolation,
levels are derived from the lack of sufficient re-entry of HBsAg and HBeAg would be expected to directly corre-
viral nucleocapsids to the nucleus, due to strong inhibi- late with liver cccDNA, as all are translated from separate
tion of viral DNA synthesis in the cytoplasm, and fewer transcripts (Pre-S1, Pre-S2/S, precore/core, and pregenomic
Please cite this article in press as: Li J, et al. Analysis of intrahepatic total HBV DNA, cccDNA and serum HBsAg level in
Chronic Hepatitis B patients with undetectable serum HBV DNA during oral antiviral therapy. Clin Res Hepatol Gastroenterol
(2017), http://dx.doi.org/10.1016/j.clinre.2017.03.004
+Model
CLINRE-994; No. of Pages 9 ARTICLE IN PRESS
8 J. Li et al.
Please cite this article in press as: Li J, et al. Analysis of intrahepatic total HBV DNA, cccDNA and serum HBsAg level in
Chronic Hepatitis B patients with undetectable serum HBV DNA during oral antiviral therapy. Clin Res Hepatol Gastroenterol
(2017), http://dx.doi.org/10.1016/j.clinre.2017.03.004
+Model
CLINRE-994; No. of Pages 9 ARTICLE IN PRESS
Analysis of intrahepatic HBV DNA, cccDNA and serum HBsAg level in Chronic Hepatitis B patients 9
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Please cite this article in press as: Li J, et al. Analysis of intrahepatic total HBV DNA, cccDNA and serum HBsAg level in
Chronic Hepatitis B patients with undetectable serum HBV DNA during oral antiviral therapy. Clin Res Hepatol Gastroenterol
(2017), http://dx.doi.org/10.1016/j.clinre.2017.03.004