Oct. 30 – Liver Imaging

#809. Transient Elastography (FibroScan) and FibroTest to Assess Liver Fibrosis in Inactive Hepatitis B Carriers: a Prospective Controlled Study

L. Castera; J. Foucher; P. Bernard; J. Bertet; P. Couzigou; V. de Ledinghen

 

Background:

Liver biopsy is currently not recommended in inactive hepatitis B carriers. Noninvasive methods could be an alternative in this setting. The aim of this prospective study was to evaluate the accuracy of transient elastography (FibroScan, FS) and FibroTest (FT) for excluding significant fibrosis in inactive hepatitis B carriers as compared with controls.

 

Methods:

A total of 266 consecutive HBV patients (male 7%, mean age 44 ± 6 yrs; all HIV, HCV and HDV negative) who undergone a FS and a FT the same day were included. Among these patients, 100 (male 63%, mean age 37 ±12 yrs) were diagnosed as inactive carriers (persistently normal ALT levels, anti-HBe positive, and HBV DNA < 05 copies/ml) of which 7 had repeated FS and FT measures over time. Inactive carriers were compared to a control group of 42 patients (male 40%, mean age 48 ± 3 yrs) with a F0 Metavir fibrosis score who undergone a FS and a FT at the time of liver biopsy.

 

Results:

Liver stiffness measurement was not possible in 9 patients (3.4%). Overall, FS values ranged from 2.5 to 75.0 kPa (Median (IQR): 5.6±3.8 kPa), whereas FT values ranged from 0.02 to .0 (0.26±0.40). Median FS values were significantly lower in inactive carriers than in the other patients (4.9 vs 6.8a, respectively, p<0.00). Similarly, median FT values were significantly lower in inactive carriers than in the other patients (0.6 vs 0.38, respectively, p<0.00). Median FS values did not differ between inactive carriers and controls (4.9 vs. 4.9, p=NS) whereas median FT values were lower in inactive carriers (0.6 vs. 0.22, p <0.03).

 

Significant fibrosis (Metavir F>2) could be excluded in 9% and 8% of inactive carriers using published cut-offs of 7.0 kPa for FS and 0.32 for FT, respectively. Concordance between FS and FT was as follows: F0-F by both FS and FT (74%); F2-F3 by both FS and FT (%); F0-F by FS and F2-F3 by FT (7%); F2-F3 by FS and F0-F by FT (8%). No patient was classified as F4.

 

In the 7 inactive carriers with repeated measures (mean interval: 2.3 ± 5.6 months), FS values (6.0 vs 5. KPa, p=NS) and FT values (0.22 vs 0.22, p=NS) were similar over time.

 

Conclusion:

Our results suggest that FibroScan and FibroTest are reliable noninvasive tools for excluding significant fibrosis in inactive hepatitis B carriers. They could be useful for the diagnosis and follow-up of inactive hepatitis B carriers in clinical practice.

 

Category: Liver Cancer

 

#839. Environmental Factors and Risk of Hepatocellular Carcinoma

W. Ohishi; S. Fujiwara; J. Cologne; G. Suzuki; M. Akahoshi; K. Chayama

 

Background & Aims:

Chronic hepatitis B and C viral infections are the most important risk factors for hepatocellular carcinoma (HCC). Environmental factors such as alcohol, smoking and obesity are associated with the development of various cancers. We conducted a nested case-control study to investigate whether environmental factors contribute to the development of hepatocellular carcinoma (HCC) using stored sera from the Adult Health Study (AHS) longitudinal cohort subjects since 958.

 

Methods:

224 HCC cases and 644 controls that were free of HCC were selected, according to nested case-control sampling. The comparable risk set was constructed by matching on sex, age, city, and time of stored sera, and by counter matching on radiation exposure. Hyaluronic acid, type IV collagen, and ferritin were measured as host factors, and hepatitis B surface antigen (HBsAg), hepatitis B core antibody (anti-HBc), hepatitis C virus antibody (anti-HCV), and HCV RNA were measured as viral factors using stored sera collected close to the time of HCC diagnosis. Information on alcohol consumption, smoking habit, coffee drinking, diabetes, body mass index (BMI), platelets count and radiation dose was obtained from the database of AHS and Life Span Study (LSS). Analysis was performed using the excess relative risk model.

 

Results:

Using multivariate analysis, the relative risks (RRs) of HCC and 95% confidence intervals (CIs) for hepatitis B virus (HBV) infection alone were 57.7 (7.8-86); for HCV infection alone 92.8 (35.8-24); for current smoking 2.4 (0.9-6.2); for alcohol consumption of 20 g/day .7 (.2-2.5); for diabetes 3.5 (.5-8.6); and for BMI of ≥25. kg/m2 0 years prior to HCC diagnosis 4.6 (.9-.3), after adjusting for radiation dose. However, multivariate analysis after adding liver fibrosis markers reduced the RRs of HCC for HBV and HCV infection, alcohol consumption of 20g/day, and diabetes, but didn’t reduce those for current smoking and obesity 0 years prior to HCC diagnosis.

 

Conclusion:

Our study indicated that HBV and HCV infection, current smoking, alcohol consumption of 20 g/day, diabetes and obesity 0 years prior to HCC diagnosis were independent environmental factors that contributed to the development of HCC. The results suggest that liver fibrosis is an intermediate factor associated with the above environmental factors with the exception of smoking habit and obesity.

#840. Does Coffee Drinking Protect Cirrhotic Patients against Hepatocellular Carcinoma?

G. Pelletier; I. Stucker; G. N'Kontchou; M. Loriot S. Cenee; M. Gelu-Simeon; F. Degos; P. Beaune; P. Laurent-Puig; J. Trinchet

 

It has been recently suggested that coffee drinking decreases the risk of chronic liver diseases (Ruhl et al, Gastroenterology 2005). We performed in France a case control study to look for predisposing risk factors of hepatocellular carcinoma (HCC). The data that we present here aimed at analyse the coffee consumption among HCC cases, cirrhosis patients and controls without liver disease.

 

Methods:

The study is an epidemiologic hospital based case control study. Cases (n=65) were newly diagnosed patients as primary HCC developed on cirrhotic liver (alcoholic 62 %, viral 24 %), on the basis of either histology or the presence of focalized lesions detected by any imaging technique, combined with an alpha fetoprotein (AFP) level > 250 ng/ml. Cirrhosis (n=36-alcoholic 79 %, viral 5 %) were defined either by histology or by the combination of clinical, laboratory, and endoscopic signs. The absence of HCC was established by the absence of focalized lesions by imaging techniques and by a AFP level < 0 ng/ml. Controls included hospital patients without any liver disorder (n=42).

 

All subjects were male, aged less than 75 years, born in Europe of parents born in Europe. The three groups were matched for age and birth country. HCC and cirrhosis patients were supplementary matched for time since cirrhosis diagnosis. Cases and controls were interviewed with a food frequency questionnaire that included lifetime drinking habits (ie alcohol, soft drinks, coffee, tea consumption). Coffee consumption was categorized as <  cup,  to 2 cups, > 2 cups/day. Blood samples were collected on EDTA and a DNA bank was established for all subjects.

 

Results:

As shown in the table, we observed a significantly higher coffee consumption among controls without liver disease as compared to HCC cases (p < 0.0), whereas there were no differences between cirrhotic patients with or without HCC. All these associations were adjusted for alcohol consumption considered in drink/day.

 

Conclusion:

Our study confirms that coffee consumption may decrease the risk of chronic liver diseases, but suggests that coffee does not decrease the risk of HCC in cirrhotic patients.

 

Coffee

Controls

Cirrhosis

OR(IC)

vs

controls

HCC

OR(IC) vs controls

0-

40 %

56 %

 

56 %

 

2

26 %

24 %

0.65

(0.3-.4

27 %

0.67

(3.3-.3)

>2

34 %

20 %

0.33

(0.2-0.7)

7 %

0.36

(0.2-0.7)

m + sd

2.4 + 2.

.8 + .9

 

.8 + .6

 

 

Category: Viral Resistance & Replication

#939. Improvement of the Hepatitis B Virus (HBV) Recombinant Baculovirus-HepG2 System to Study cccDNA Formation and Resistance to Nucleoside Analogs

F. Zoulim; J. Lucifora; D. Durantel; M. Brunelle; O. Hantz; C. Trepo

 

Objectives:

A few years ago a system based on the transduction of HepG2 cells with a recombinant baculovirus carrying .3 HBV genome unit was described to study the replication and susceptibility to drugs of wild type (WT) and mutant HBV strains. With this system, the formation of cccDNA was observed although its was not explained. Our objective was to improve this system to further study HBV replication with a particular focus on cccDNA formation and viral resistance to drug.

 

Methods:

HBV recombinant baculoviruses carrying either . or .3 HBV genome unit were constructed. In Bac-HBV-., the synthesis of pgRNA was driven by a mammalian promoter, whereas it was driven by HBV promoter for Bac-HBV-.3. With Bac-HBV-., the synthesis of mRNA encoding HBeAg was not possible without the previous formation of cccDNA. Both baculoviruses were used to infect HepG2 cells and comparative studies were performed to establish parameters of infection, including kinetics of viral RNA, DNA, HBsAg, HBeAg, and cccDNA. Additional baculoviruses were constructed with HBV strains carrying mutations conferring resistance to lamivudine or/and adefovir (i.e. L80M/M204V, N236T, and L80M/M204V/N236T). These baculoviruses were used to study viral fitness, susceptibility to drug, and cross resistance profiles.

 

Results:

First, we demonstrated that the recombinant baculovirus Bac-HBV-. triggered higher HBV replication, thus establishing the superiority of this construct. Using both baculoviruses, we studied the formation of cccDNA and showed that HepG2 from ATCC were not able to produce high amount of cccDNA. Moreover, we showed that the cccDNA formed was the result of recombination rather than amplification by nucleocapsid recycling. This result challenges previous results obtained in the DHBV models, where the amplification of cccDNA by recycling to nucleus of neo-formed nucleocapsids was demonstrated.

 

Second, experiments to test mutant replication capacity and susceptibility to nucleoside analogues showed similar results than those previously reported with transfection system: WT>N236T>L80M/M204V>L80M/M204V/N236T for replication capacity, as well as N236T or L80M/M204V/N236T less susceptible to adefovir and L80M/M204V or L80M/M204V/N236T resistant to lamivudine.

 

Conclusion:

These results provide new insight in the mechanism of selection of HBV resistant mutants and suggest that the rate of emergence of these mutants in vivo may be due to differences in both drug susceptibility and replication capacity. HBeAg is finally not an accurate marker because the ELISA way probably also detect HBeAg released from transduced cells.

 

#940. Rapid Improvement of Innate Immune Responses and Monocyte Toll-like Receptor-2 (TLR2) Expression during Lamivudine Therapy for Chronic Hepatitis B (CHB)

K. Visvanathan; N. Skinner; A. J. Thompson; P. Desmond; S. Locarnini

 

Background/Aims:

Toll-like receptors (TLR’s) are critical receptors that promote innate immune responses to pathogen-associated molecular patterns. Activation of TLR’s leads to production of pro-inflammatory cytokines such as Tumour Necrosis Factor (TNF)-α and IL-6. We have previously demonstrated that patients with hepatitis B e antigen (HBeAg) positive CHB have suppressed TLR2 expression on peripheral monocytes and hepatocytes.

 

Methods and Patients:

The TLR2 and TLR4 expression on peripheral blood monocytes was measured from 2 patients with untreated HBeAg-positive and HBeAg-negative CHB and 0 uninfected control patients. These 2 patients were treated with lamivudine 00mg daily. Individual patient blood was stimulated with specific TLR ligands and the resultant supernatant was assayed for TNF-α and IL-6 by ELISA. In addition serum HBeAg was measured by enzyme immunoassay (Murex; Abbott Diagnostics, USA).

 

Results:

Expression of TLR2 on peripheral monocytes was significantly reduced in patients with HBeAg-positive CHB in comparison to HBeAg-negative CHB and to controls whilst it was significantly increased in HBeAg-negative CHB compared to controls (P=0.00). TLR2 levels increased within four weeks of effective therapy in HBeAg-positive patients (P<0.05) whilst remaining stable in HBeAg negative patients. The level of TLR4 expression did not differ significantly between the groups. The functional relevance of these findings was established by the demonstration in HBeAg positive patients of initial reduced cytokine production (TNF-α and IL-6) and phospho-p38 kinase production after stimulation of monocytes with specific TLR ligands. This immunosuppresion improved rapidly during treatment (P<0.05). In HBeAg negative patients innate immune responses were initially increased but improved with therapy. Correlations of these results with HBV viral load and eAg levels (eAg positive patients) will also be presented.

 

Conclusions:

This is the first study to investigate how peripheral innate immune responses and TLR expression changes with therapy. It demonstrates a potentially important interaction between HBeAg, HBV and the innate immune response and suggests that TLR2 expression and activation could represent a sensitive new biomarker in the treatment of HBeAg-positive CHB.

 

#942. TLR9 Expression and Functional Impairment of Plasmacytoid Dendritic Cells in Patients with Chronic Hepatitis B

Q. Xie; H. Shen; B. An; N. Jia; H. Wang; X. Zhou; Q. Guo; H. Yu

 

Objective:

Hepatitis B virus (HBV) infection is still a major health problem worldwide. The immunol pathogenesis of chronic hepatitis B (CHB) is poorly understood. Dendritic cells(DC) represents the most potent antigen-presenting cells and plays an important role in stimulation of specific T-cell response. Functional deficiency of DC may contribute to the inadequate specific T-cell response of the host. Immature plasmacytoid dendritic cell (pDC) is the cell type responsible for IFN-Ι production, which is important in innate immunity against virus. TLR9 is a transmembrane type Ι protein that is mainly expressed in pDC and functions as a pattern recognition receptors (PRR) by interacting with CpG motif of virus. In this study we investigated the changes of expression of TLR9 in pDC from patients with CHB and role of pDC function in HBV infection.

 

Methods:

PBMC samples were taken from 28 cases with CHB and 8 healthy controls following their informed consent. pDC were isolated from PBMC by positive immunomagnetic selection using mini-MACS system.After being incubated with TLR9-PE,pDC were analyzed on flow cytometer(FCM) and the mean fluorescence intensity(MFI) and percent positive stained cells was calculated.The frequency of pDC was determined following labeled with Lin -FITC(CD3 CD4 CD6 CD9 CD20 CD56),PE-BDCA2 and APC-CD23.After pDC were incubated in medium with CpG 226 for 24 hours,cell-free supernatants were harvested and tested via ELISA for IFN-α, TNF-α and IL-6. HBsAg and HBcAg expression in pDC were assayed with immunohistochemistry.

 

Results:

The MFI of TLR9 from patients were significantly reduced compared with controls(57.92±7.87 vs. 3.69±248.86, P<0.05). No difference in the percentage of pDC expressing TLR9 was found between them. The percentage of pDCs in PBMCs from cases with CHB (median ,0.2%; range, 0.04~0.46%, n=20) were significantly reduced compared with control (median 0.3%;range, 0.08%~0.74%, n=23;P<0.05). Moreover,pDC fenquency inversely correlated with ALT levels (r=-0.646, p<0.05).The level of IFN-α in pDC following CpG-ODN stimulation in CHB were significantly lower than in healthy controls (7032.54±7998.443pg/ml vs. 5867.86±9804.78pg/ml, P<0.05). No differences were observed in the production of TNF-α, IL-6 between patients and healthy controls. With immunohistochemistry HBsAg and HBcAg were detectable in pDC of patients.

 

Conclusions:

The TLR9 expression of circulating pDC was reduced in patients with CHB. pDCs were functionally impaired with the lower ability to produce IFN-α in patients, that may partly contribute to hepatitis B evading an adequate immune response, resulting in HBV persistent infection.

 

#945. Influence of Hepatitis B Virus Genotypes as Well as Precore and Core-promoter Mutations on Fulminant Outcome of Acute Infection

F. Sugauch; E. Orito; Y. Tanaka; A. Ozasa; J. Kang; J. Toyoda; H. Yotsuyanagi; S. Iino; T.Kuramitsu; K. Suzuki; E. Tanaka; Y. Akahane; N. Izumi; K. Inoue; S. Kakumu; E. Tomita; Y. Murawaki; K. Hino; M. Onji; H. Yatsuhashi; M. Sata; T. Okanoue; S. Nishiguchi; Y. Miyakawa; M. Mizokami

 

Background:

Eight hepatitis B virus (HBV) genotypes have been detected by the sequence divergence >8% in the entire HBV genome composed of approximately 3,200 nucleotides. They have distinct geographical distribution and are associated with severity of liver disease as well as response to antiviral therapies. However, the association between viral genotype and severity of liver disease remain uncertain in acute HBV infection. To evaluate influence of genotypes as well as precore and core-promoter mutations on their fulminant outcome, a multi-center cross-sectional study was conducted throughout Japan on patients with acute hepatitis B.

 

Methods:

During 982 through 2005, 547 patients with acute hepatitis B were registered in 25 hospitals throughout Japan. HBV genotypes were determined serologically by ELISA using commercial kits or restriction fragment length polymporphisim (RFLP). Mutations in the precore region for A896 and core-promoter for T762/A764 were detected by enzyme-linked minisequence assay using commercial kits.

 

Results:

Genotypes of HBV were unclassifiable in 40 (7%) and sufficient clinical data was not available in 22 (4%) of them. Exclusive of these 62 patients, 485 (89%) were left for the evaluation of geographic distribution of HBV genotypes and relevance with clinical outcomes. Overall, genotype A was detected in 92 (9%), Ba in 26 (5%), Bj in 32 (7%), C in 330 (68%) and D in 5 (%). Sexual contacts were the main route of transmission in them (45%). Patients with fulminant hepatitis (n = 45) were older (42.7 ± 6.8 vs. 35.2 ± 3.3 years, P<.00), less predominantly male (45% vs. 73%, P<.00), less positive for HBeAg (36% vs. 64%, P<.00), less infected with genotype A (3% vs. 2%, P<.00), and more frequently with Bj (29% vs. 4%, P<.00) than those with acute self-limited hepatitis (n = 440). G896A and A762T/G764A mutations were more frequent in patients with fulminant than acute self-limited hepatitis (5% vs. % and 5% vs. 8%, P<.00 for both). In multivariate analysis, genotype Bj (0.02 [2.35-42.79], P=.002), HBeAg-negative (3.35 [.35-8.3], P=.009) and G896A mutation (3.77 [.4-0.05], P=.008) were independently associated with the fulminant outcome. In in vitro transfection experiments, the replication of Bj clone was markedly enhanced by introducing either G896A or A762T/G764A mutation.

 

Conclusions:

Fulminant outcome in patients with acute HBV infection is associated with genotype Bj accompanied by the lack of serum HBeAg as well as high replication due to G896A mutation, which is supported by an in vitro replication model.

 

#947. Viral Quasispecies Evolution in Chronic Hepatitis B: New Light on an Old Story

S. Lim; Y. Cheng; S. Guindon; B. Seet; L. Lee; P. Hu; S. Wasser; T. Tan; F. Peters; M. Goode; Rodrigo

 

Background and Aims:

There is good evidence that the pathogenesis of chronic viral infections such as HIV and HCV are related to the evolution of viral quasispecies, but is a completely unknown field in chronic hepatitis B. The aim of this study was to evaluate the evolution of HBV quasispecies in well-characterized phenotypes of clinically distinct chronic hepatitis B patients over time.

 

Methods:

Four well defined clinical phenotypes of chronic hepatitis B: HBeAg seroconverters (spontaneous seroconverters [n=0] and interferon induced seroconverters [n=0]), and non-seroconverters (controls [n=0] and interferon non-responders [n=0]) who were followed over 4-5 timepoints for a mean of 60months were selected. Only genotype B patients were examined in order that viral variation could not be attributed to genotypic differences. Nested PCR was performed from serum samples for the precore/core gene which was then cloned into pGEM-T vector and transformed into JM09 cells. At least 20 clones/sample were selected for sequencing. For low viral titre samples, measures were taken to avoid resampling. Sequences were aligned using ClustalX and sUPGMA pylogenetic trees were constructed using Pebble .0 following which maximum likelihood estimates of pairwise distances under a GTR+I+G model was assessed. Viral diversity and evolution were then estimated using this model.

Results:

3386 sequences of the precore/core gene were analysed. HBeAg seroconverters had 2.5-fold higher pre-seroconversion viral diversity, and 0-fold higher substitution rate than non-seroconverters, who had persistently low viral diversity (3.6x0-3 substituitions/site) (p=0.0183) and evolution (2.2×105 substitutions/site/month) (p<0.0001) over time. Following seroconversion, there was a striking increase in viral diversity. A significant increase in diversity/viral load ratio (p=0.027) was seen in interferon-treated seroconverters, but not in non-responders to interferon. Phylogenetic trees were more complex and star-like in seroconverters but not in non-seroconverters. Evidence of positive selection was seen in 4/20 seroconverters but only in /20 non-seroconverters, most commonly occurring at amino acid 3 and 35 of the core protein.

 

Conclusion:

The data suggests that viral diversity is a requirement for seroconversion, possibly by generation of new viral epitopes triggering immune responses, and that interferon may act through increasing viral diversity.

 

#949. Baseline HBV Viral Load and Excess Mortality Associated with Chronic Hepatitis B Infection: The REVEAL-HBV Study

U. H. Iloeje; H. Yang; J. Su; C. Jen; S. You; C. Chen

 

Background:

There is no data to date using sensitive assays showing the relationship between HBV DNA and excess mortality in CHB. Our objectives were to 1) compare mortality between the HBsAg- and + subjects; 2) evaluate predictors of mortality in the HBsAg+ cohort.

 

Methods:

The REVEAL-HBV study is a prospective population-based cohort study. Follow-up for the mortality analyses was from Jan  99–Dec 3 2004. Mortality was ascertained via data linkage to the Taiwanese National Death Certification Registry. All 23,820 persons were included in objective and a subset (3,653) in objective 2. Mortality rates for all causes, liver cancer, chronic liver disease and cirrhosis, and others, were derived and survival curves by follow-up year with stratification by HBV DNA level were derived by the Kaplan-Meier method. Log-rank test was used to compare survival curves. Cox proportional hazards models were used to estimate multivariable-adjusted relative risks(RRadj) and 95% CIs.

 

Results:

Over 2.5 yrs (298,866 person yrs), there were 1,564 (8%) and 460 (%) deaths in the HBsAg- and + groups, for mortality rates of 632 and 895.1 per 100,000 respectively. HBsAg+ subjects had significantly higher(p<0.0) RRadj(95%CI) for: all cause mortality .6(.4-.8); liver cancer 0.3(7.7-3.9), and chronic liver disease and cirrhosis 4.4(3.0-6.6). Results from the HBsAg+ subgroup, are shown below.

 

Conclusions:

Persons with CHB have a significantly higher risk of mortality compared to uninfected persons. This excess mortality is directly related to liver mortality, which is predictable based upon the HBV viral load. Sustained suppression of viral replication should reduce this excess mortality.

 

Predictors of Mortality in HBsAg+ and anti-HCV- Subjects (N=3653)

 

Adjusted hazard ratio (95% CI)

All causes

(N=388)

Liver cancer

(N=9)

Chronic liver disease and cirrhosis (N=40

Others*

(N=229)

HBeAg-

Referent

Referent

Referent

Referent

HBeAg+

.9(.3-2.6)†

3.3(.9-5.6)†

2.2(0.8-5.5)

.0 (0.6-.8)

ALT, U/L

 

 

 

 

<45

Referent

Referent

Referent

Referent

≥45

.4(.0-.9)

.3(0.8-2.0)

2.3(.-5.0)‡

.3(0.8-2.2)

Cirrhosis

 

 

 

 

No

Referent

Referent

Referent

Referent

Yes

3.7(2.6-5.4)†

8.9(5.5-4.4)†

6.7(2.8-6.)†

0.7(0.3-.9)

HBV DNA, c/mL

 

 

 

 

<300(Undetectable)

Referent

Referent

Referent

Referent

300-9999

0.9(0.7-.3)

0.7(0.3-.9)

5.3(0.7-43.5)

0.9(0.6-.3)

0000-99999

.0(0.7-.4)

2.(0.9-5.0)

7.6(0.9-63.)

0.8(0.5-.2)

00000-999999

2.0(.4-2.9)†

6.9(3.-5.4)†

.(.3-94.4)‡

.2(0.8-.9)

≥ million

2.(.4-3.)†

5.7(2.4-3.6)†

5.6(.8-34.7)‡

.3(0.8-2.3)

 

*Causes other than liver cancer, chronic liver disease and cirrhosis. Also adjusted for age, gender, smoking and alcohol. †P<0.00; ‡P<0.05

 

#95. Is There a Meaningful Serum HBV DNA Cut-off Level for Therapeutic Decisions in HBeAg-Negative Chronic Hepatitis B (CHBe-)?

G. V. Papatheodoridis; E. K. Manesis; S. Manolakopoulos; J. Goulis; N. Chrysanthos; A. Bilalis; S. Savvidou; G. Kafiri; I. Delladetsima; D. Tzourmakliotis; A. J. Archimandritis

 

Background/Aim:

The diagnosis of CHBe- is currently based on arbitrarily chosen serum HBV DNA cut-off levels, such as 05 cp/mL or more recently 04 cp/mL (or 2,000 IU/mL). In this multicenter, cohort study, we evaluated the severity of liver histology and presence of histological indication for treatment in relation to serum HBV DNA levels in CHBe-.

 

Methods:

We included 335 CHBe- patients (pts) consecutively admitted for liver biopsy at 3 Greek Hepatology centers between 200-2005. CHBe- diagnosis was uniform at all centers: HBsAg(+)/HBeAg(-) for ≥6 mos, increased ALT/AST on ≥2 monthly occasions and detectable serum HBV DNA. Serum HBV DNA levels were determined by a quantitative PCR assay (Monitor, Roche; sens. 400 cp/mL). Histological lesions were classified according to Ishak’s classification. Histological treatment indication was defined as at least moderate grade (≥8) and/or moderate stage (≥2).

 

Results:

Serum HBV DNA levels were ≥06 cp/mL in 78 (53%) (Group A), 05-06 cp/mL in 70 (2%) (Group B), 04-05 cp/mL in 50 (5%) (Group C) and <04 cp/mL in 37 (%) (Group D) pts. There was no significant difference in any characteristic between Group A and B or between Group C and D, except for higher ALT/AST values in Group A than B. In contrast, Group A+B compared to Group C+D pts had older age (50 vs 45 years, P=0.008) and higher ALT/AST (P<0.00), grade (8. vs 6.0, P<0.00) and stage (3.3 vs 2.4, P<0.00). Histological treatment indication was present in 62%, 64%, 83% and 86% of Group A, B, C and D pts respectively (P<0.00). On the liver biopsy day, 4 (2%) pts had normal ALT/AST. Pts with normal compared to abnormal ALT/AST biopsy-values had lower BMI (22 vs 25 Kg/m2, P=0.002), grade (6.4 vs 7.7, P=0.009), stage (2.6 vs 3.2, P=0.05) and HBV DNA levels (<05 cp/mL: 5% vs 22%, P<0.00). Histological treatment indication was present in 27 (66%) pts with normal and 239 (8%) patients with abnormal ALT/AST biopsy-values (P=0.037).

 

Conclusions:

In HBeAg-negative chronic HBV pts with increased ALT/AST: a) There are no significant differences between cases with HBV DNA levels between 04-05 cp/mL and <04 cp/mL, but only between cases with HBV DNA levels below and above 05 cp/mL; b) Liver biopsy should be performed in all pts with detectable HBV DNA or temporarily normal ALT/AST values, since histological lesions widely accepted as indications for treatment are present in approximately 60% of those with viremia even <04 cp/mL or in 65% of those with temporary ALT/AST normalization. According to our findings, the diagnosis of CHBe- is certainly underestimated if it requires serum HBV DNA levels >05 or even >04 cp/mL.

 

#96. Immunologic Evidence of Limited Viral Replication in Hepatitis B-Vaccinated Chimpanzees Challenged with a Hepatitis B Polymerase Gene Mutant

S. Kamili; K. Campbell; A. Bartholomeusz; C. Walker; S. Locarnini; K. Krawczynski

 

Aim:

Drug-resistant mutations in the hepatitis B virus (HBV) polymerase (pol) gene have been documented in chronic hepatitis B patients following prolonged treatment with antiviral drugs such as lamivudine and famciclovir. These mutations in the reverse transcriptase (rt) region of HBV pol include rtV73L and rtL80M, both clustered within the B domain, and the rtM204V in the conserved YMDD motif in the C domain. These mutations in HBV pol gene also alter the sequence of HBV surface antigen (HBs) and this altered HBs (sE64D and sI95M) has been associated with significantly reduced anti-HBs binding in vitro. The aim of this study was to investigate the protective efficacy of a commercial HBV vaccine, containing HBs, in chimpanzees challenged with the recombinant HBV mutant.

 

Methods:

Two chimpanzees, CH0364 and CH0369, received a pediatric dose (0mcg of HBs) of a commercial hepatitis B vaccine (ENGERIX-B, GlaxoSmithKline) at 0, and 2 months. Anti-HBs antibody titers exceeded 75 mIU/mL and a robust HBs-specific cellular immune response, measured by INF-γ production (ELISpot), was observed in blood of both the chimpanzees.

 

The two HBs-vaccinated chimpanzees and a control chimpanzee (CH030) vaccinated with a commercial hepatitis A vaccine (HAVRIX) were challenged intravenously with the recombinant HBV mutant (2.0 mL containing 9.8x09 copies/mL of HBV DNA).

HBV DNA was detected in the serum of HAVRIX vaccinated control chimpanzee only. However, the pattern of cellular and humoral immune response against HBV antigens in the two HBs-vaccinated chimpanzees provided immunological evidence of infection even though usual markers of viral replication (HBV DNA and HBsAg) were not detected. An HBV pol-specific INF-γ response was first observed on day 28 for the first time in CH0364 after challenge with HBV mutant and a boosting effect on HBs-specific cellular and humoral immunity was also observed in both chimpanzees. Moreover, anti-HBc antibodies were detected on day 7 postchallenge in CH0364 and on days 7, 0, and 7 in CH0369. Protection against cross-challenge with a wild-type HBV in the two chimpanzees is under investigation.

 

Conclusion:

These data indicate there may be a limited HBV replication after challenge with HBV polymerase mutant (as evidenced by appearance of cellular immune markers) despite a robust HBs-specific humoral and cellular immune response induced by hepatitis B vaccination and warrant further studies to evaluate the potential public health impact of emergence of drug resistant mutants and protective efficacy of commercial HBV vaccination against such mutants.

 

#963. HBV Surface and Core Protein Expression in the Liver in Chronic Hepatitis B under Long-Term Antiviral Therapy

S. J. Hadziyannis, ; A. Costamena; E. Katsikadelli; E. Hadziyannis

 

Background:

Athough antiviral treatment in chronic hepatitis B (CHB) may effectively suppress HBV replication, the goal of HBsAg clearance is rarely achievable, particularly in HBeAg- cases. At the same time there is little if any information if and how long-term antiviral therapy in CHB may modulate HBsAg expression in the liver.

 

Aim:

To investigate HBsAg and HBcAg expression in the liver in CHB under effective, long term (5-year) anti-viral therapy and to compared with respective data in the liver before treatment.

 

Patients and Methods:

Thirty patients with CHB under HBV suppression by long-term nucleoside analogue treatment were included. All had a liver biopsy prior to start and at year 5 of treatment examined for HBsAg and HBcAg by the immune-peroxidase and the immunofluorescence techniques with monoclonal and polyclonal anti-HBs and anti-HBc. Paraffin and snap-frozen cryostat sections were used. The whole liver section was scanned visually, HBsAg expression and distribution patterns were recorded and the percentage of HBsAg+ and HBcAg+ hepatocytes was assessed. Serum HBeAg/anti-HBe and HBsAg were tested by commercial ELISAs and HBV DNA levels by a real time PCR with a sensitivity threshold of <400 copies/ml. All patients, except 2, had HBV genotype D while 27 were HBeAg-/anti-HBe+.

Results:

The number of HBsAg+ hepatocytes was found to increase rather than decrease under long term treatment, while HBcAg-expressing (nuclear or cytoplasmic) hepatocytes disappeared from the liver tissue. Clusters of HBsAg+ liver cells clearly increased in several patients both in number and size while individual hepatocytes within the clusters exhibited the same pattern of HBsAg expression (diffuse cytoplasmic, partial cytoplasmic, membranous, submembranous etc). Cases of HBeAg-negative patients tested at baseline for integrated HBV DNA in the liver were found positive for integrated HBsAg sequences. Loss of HBsAg from serum was observed in 3 patients and all had also become negative for HBsAg and HBcAg in the liver.

 

Conclusions:

1.     Effective long-term treatment of HBeAg- CHB may suppress strongly HBV replication and HBcAg expression but cannot clear or significantly reduce HBsAg because of its ongoing and\even increased production in hepatocytes harboring integrated HBsAg sequences.

2.     Such HBsAg+ hepatocytes are frequently arranged in clusters and may represent liver cell clones expanding rather than decreasing in number and size under long term antiviral therapy.

3.     Hepatocytes with HBsAg sequences integrated in their genome seem to be resistant to HBV re-entry and to episomal HBV replication.