- Email: [email protected]
731 CHANGES OF SERUM APOPTOTIC CASPASE ACTIVITY IN HBEAG-NEGATIVE CHRONIC HEPATITIS B (CHB) PATIENTS UNDER ORAL ANTIVIRAL THERAPY
POSTERS prevalence and the patterns of HBV infection in a large cohort of immigrants in Rome. Methods: Between August 2008 and July 2009, 1203 consecutive patients were visited at our Department (68.2% female, 32.8% male, mean age 30 years). 58.78% were from Africa, 22.04% from Asia, 13.06% from Eastern-Europe and 6.12% from South-America. A screening form with demographic, clinical and laboratory data was collected for each HBsAg-positive patient, in order to identify the patterns of HBV infection, to optimize their management and treatment and to observe their evolution over time. Results: The prevalence of HBsAg-positive patients was 16.5% (199/1203). 71.44% of these subjects were from Africa, 16.66% from Eastern-Europe, 9.52% from Asia and 2.38% from South-America. Mean age was 27 years (61.9% male, 38.1% female). HBeAg was positive in 373 patients (30.9%). Respectively 8.7% and 15% of HBV infected immigrants were co-infected with HCV and HDV. The diagnostic categories assigned were: inactive carrier state 58.7%, chronic hepatitis B (CHB) 31.6% and HBV-related cirrhosis 9.7%. Conclusions: Chronic infection with HBV is an important health issue in immigrated patients, partly as a result of absence of vaccine prophylaxis in many countries. Although universal vaccination has dramatically reduced the incidence of HBV in Italy, there is a risk of a rise in HBV infections in our country, as result of the increase of imported cases through immigration from highly endemic countries. Another alarming finding of this study is the high prevalence of anti-HDV antibodies in immigrants. This is a particularly serious issue because co-infection with HDV has been shown to accelerate the progression to cirrhosis, and rates of response to anti-viral treatment are lower than in mono-infected patients. 730 HUMAN GENETIC VARIANTS LINKED TO CHRONIC HEPATITIS B ARE ASSOCIATED WITH EXPRESSION OF HLA-DPA1 AND HLA-DPB1 T. O’Brien1 , I. Kohaar1 , R. Pfeiffer1 , D. Maeder1 , M. Yeager1 , L. Prokunina-Olsson1 , E. Schadt2,3 . 1 Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD, 2 Sage Bionetworks, Seattle, WA, 3 Pacific Biosciences, Menlo Park, CA, USA E-mail: [email protected] Background and Aims: Genetic regulation of HLA may play a role in viral control. A recent genome-wide association study (GWAS; Kamatani, Nature Genetics, 2009) strongly linked two single nucleotide polymorphisms (SNPs) to chronic hepatitis B: rs3077, which lies in the 3’ untranslated region (3 UTR) of HLA-DPA1, and rs9277535, which lies in the 3 UTR of HLA-DPB1. We examined whether these SNPs are associated with expression of these genes. Methods: The Human Liver Cohort (HLC) database identifies gene expression associated SNPs (eSNPs) by integrating genotype (Illumina Sentrix HumanHap650Y Genotyping BeadChip) and gene expression (custom Agilent array) data. The current analysis is based on 651 liver samples without disease. The Kruskal-Wallis test was used to calculate p-values for associations between expression and genotype. To confirm findings, we measured allelic expression imbalance (AEI) of mRNA compared to DNA in an independent set of liver specimens from subjects who were heterozygous for rs3077 and rs9277535. Results: Among ~650,000 SNPs in the HLC, rs3077 was the variant most strongly associated with expression of HLA-DPA1 (p = 10−48 ); the allele associated with HBV clearance (rs3077-A) was associated with higher expression of HLA-DPA1 (Figure 1). AEI testing verified the association of rs3077-A with increased expression of HLA-DPA1 (p = 3.0×10−7 ). Similarly, rs9277535-A was associated with both HBV clearance and greater HLA-DPB1 expression (p = 10−15 ), and this association was confirmed by AEI (p = 0.001). S284
Conclusions: We found that the SNPs previously associated with chronic hepatitis B are also strongly associated with expression of HLA-DPA1 and HLA-DPB1, suggesting that expression of these genes is important in HBV infection. Therapies to increase expression of HLA-DPA1 or HLA-DPB1 might improve treatment of chronic hepatitis B.
Figure 1. (a) Odds ratio (OR) for chronic HBV (adapted from Kamatani, 2009) and difference in expression of HLA-DPA1 in liver tissue, by rs3077 genotype. (b) OR for chronic HBV and difference in expression of HLA-DPB1 in liver tissue, by rs9277535 genotype. Gene expression values represent the arithmetic difference in mean-log gene expression from the referent genotype (rs3077GG or rs9277535GG).
731 CHANGES OF SERUM APOPTOTIC CASPASE ACTIVITY IN HBEAG-NEGATIVE CHRONIC HEPATITIS B (CHB) PATIENTS UNDER ORAL ANTIVIRAL THERAPY G.V. Papatheodoridis, J. Giannousis, S. Manolakopoulos, E. Hadziyannis, A. Georgiou, A.J. Archimandritis. 2nd Dept. of Internal Medicine, Athens University Medical School, Hippokration General Hospital, Athens, Greece E-mail: [email protected] Background and Aim: The effects of CHB treatment on hepatocyte apoptosis have not been evaluated. We investigated the changes in serum levels of the apoptotic caspase-generated fragments of keratin-18 (K-18), a major intermediate filament protein in hepatocytes, in HBeAg-negative CHB patients under nucleos(t)ide analogue(s). Methods: We included 46 patients (age:52±14 years, M/F:38/8) with histologically documented HBeAg-negative CHB who had received nucleos(t)ide analogue(s) (lamivudine:42, adefovir:4) for ≥12 months. Of the 42 lamivudine treated patients, 23 developed
Journal of Hepatology 2010 vol. 52 | S183–S317
POSTERS virological with or without biochemical breakthroughs and received add-on adefovir therapy for ≥12 months. Sera drawn at baseline, at 6 and 12 months of initial therapy as well as at the diagnosis of breakthroughs and at 6 and 12 months after adefovir addition were tested blindly using the M30-Apoptosense ELISA Kit (PEVIVA) that measures the apoptosis-associated neoepitope in the C-terminal domain of K-18 (aa 387–396). Results: During initial therapy, K-18 fragments (U/L) levels significantly reduced from baseline [mean±SD (range): 446±459 (149–2461)] to 6 months [180±74 (110–454) U/L, P < 0.001] and from 6 to 12 months [162±47 (113–280), P = 0.031]. K-18 fragments levels increased at breakthroughs [250±198 (124–1063)] compared to last previous visits [197±146 (118–809), P = 0.042] but remained lower than baseline (P = 0.016). Changes of K-18 fragments levels correlated positively with changes of ALT and HBV-DNA from baseline to 6 (r = 0.869, P < 0.001 and r = 0.396, P = 0.014) or 12 months of initial therapy (r = 0.873, P < 0.001 and r = 0.544, P < 0.001). In contrast to initial therapy, in patients with breakthroughs, K-18 fragments levels had only a trend for decline from adefovir addition [303±290 (124–1219)] to 6 [274±377 (122– 1576), P = 0.109] and mainly to 12 months [223±201 (122–878), P = 0.055]. In the latter subgroup, changes of K-18 fragments levels correlated positively with changes of ALT at 6 (r = 0.624, P = 0.017) or 12 months (r = 0.765, P < 0.001) and with changes of HBV-DNA only at 12 months (r = 0.535, P = 0.010) after adefovir addition. Conclusions: Apoptotic caspase activity decreases significantly during oral antiviral therapy in HBeAg-negative CHB in parallel with the improvements in ALT and HBV-DNA levels. It increases again during breakthroughs and may improve after an effective rescue treatment but at a slower rate compared to the initial therapy. 732 PROPORTION OF DEFECTIVE HBV DNA ISSUED FROM A SINGLY SPLICED RNA AND ENCODING FOR HBSP IS RELATED TO THE COURSE OF VIRAL INFECTION F. Redelsperger1 , B. Lekbaby1 , N. Desir ´ e´ 2 , A.M. Roque-Afonso3 , 4 5 S. Brichler , G. Perlemuter , P. Dubreuil6 , N. Bacon2 , F. Zatla2 , C. Le Pendeven2 , D. Kremsdorf1 , P. Soussan1,2 . 1 INSERM U845, 2 Virology Unit, CHU Tenon, 3 Virology Unit, CHU P. Brousse, 4 Virology Unit, CHU Avicenne, 5 Hepatology Unit, 6 Virology Unit, CHU A Beclere, Paris, France E-mail: [email protected] Background: Defective hepatitis B virus particles (dHBV), generated from a singly spliced HBV RNA are detected during HBV infection. We and others have observed that proportion of circulating dHBV particles varied in chronic carriers and could be related to severe liver disease. This relationship is controversial, probably due to criteria of included patients and/or quantification methods. The present study was designed to quantify defective and wild-type HBV (wtHBV) genomes through the course of viral infection using two previously described methods and subsequently to search for viral factors involved in dHBV formation. Patients and Methods: HBV patients with 1. anti-HBc IgM positive acute (developing or not chronic infection and fulminant; n = 40) or reactivation (n = 5) hepatitis; 2. moderate or severe chronic liver disease untreated (n = 72, HBV >105 copies/ml), and 3. a longitudinal follow-up during 9 to 23 years (n = 5), were studied. Wild-type HBV and dHBV DNA were quantified by in-house qPCR (using two previous published methods for dHBV DNA). HBV Posttranscriptional Regulatory Element (PRE) region was studied by direct sequence analysis. Mutations identified were inserted by mutagenesis in HBV plasmid and impact on splicing modulation was analyzed in HuH7 cells.
Results: Whatever dHBV DNA quantification method used, higher amount of dHBV DNA was associated in severe compared to moderate chronic disease (8.5±2.6% versus 1.7±0.5%, respectively; p = 0.02). In acute infection, dHBV DNA proportion was independent to disease progression. A significant lower proportion of dHBV DNA was observed in acute compared to chronic carriers, suggesting regulation of the dHBV DNA proportion during infection (dHBV DNA amount >5% for 2/40 versus 16/72 patients, respectively, p = 0.02). Investigation of follow-up of HBV chronic carriers showed that despite an overall constant proportion of dHBV DNA, highest levels (>50%) were observed at 2 timepoints for 2 patients and seemed associated to liver disease progression. Mutations in PRE region, involved in splice regulation, were detected by sequence comparison of higher or lower proportion of dHBV DNA. Transfection of HBV with PRE mutations or wild-type showed an impact in dHBV DNA expression. Conclusion: Our data demonstrate, independently to quantification methods, an increase proportion of dHBV DNA during the progression of viral disease, probably through PRE mutations. 733 REACTIVATION OF OVERT AND OCCULT HBV INFECTION IN IMMUNOSUPPRESSIVE SETTINGS N. Coppola1 , G. Tonziello1 , M. Pisaturo1 , M. Fiore1 , V. Iodice1 , S. Guastrafierro1 , C. Sagnelli1 , N. Capoluongo1 , V. Messina2 , G. Pasquale1 , E. Sagnelli1,2 . 1 Second University of Naples, Naples, 2 Sant’Anna Hospital of Caserta, Caserta, Italy E-mail: [email protected] Aim: To evaluate differences in clinical and virological presentation between patients with overt and occult HBV infection during reactivation of HBV infection (r-HBV). Methods: 23 consecutive patients with symptomatic r-HBV (HBsAg/HBV-DNA-positivity, ALT serum level at least 5 times higher the mean value in the preceding 6-months) during or after immunosuppressive therapy were enrolled: 10 with reactivation of overt HBV infection (HBsAg positive from 6 months or more before r-HBV; Overt Group) and 13 of occult HBV infection (HBsAgnegative/anti-HBc-positive from at least 6 months at r-HBV; Occult Group). No patient had received prophylaxis for r-HBV. Anti-HBV Nucleot(s)ide analogue treatment was given at first observation after r-HBV in 21 patients. Results: All patients in Occult Group and 6 in Overt Group (p = 0.05) had onco-hematological diseases. r-HBV was observed both during and after immunosuppressive therapy. Regimens including rituximab or fludarabine were frequently administered in Occult Group (53% and 31%, respectively). No significant difference was observed between Overt and Occult groups as far age, sex, clinical and biochemical presentation. r-HBV was frequently severe both in Overt (40%) and in Occult groups (38.4%). Patients in Overt Group showed higher HBVDNA titres than those in Occult Group (1.1×108 ±1.4×108 vs 5.1×105 ±6.8×105 IU; p < 0.005). All patients had HBV genotype D. Seven patients died during r-HBV, 2 in Overt and 5 in Occult group, two for hepatic failure (one in each group) and 5 for progression of ongoing diseases after mandatory discontinuation of immunosuppressive treatment (1 in Overt and 4 in Occult group). Of patients who died, 2 were untreated and 5 received Lamivudine; of the 16 with remission of r-HBV, 4 received Lamivudine and 12 other recently developed analouges or an analogues combination. Conclusions: r-HBV is a severe complication in patients undergoing immunosuppressive therapy that may be prevented or treated with appropriate anti-HBV analogues treatment.
Journal of Hepatology 2010 vol. 52 | S183–S317
S285
Conclusions: We found that the SNPs previously associated with chronic hepatitis B are also strongly associated with expression of HLA-DPA1 and HLA-DPB1, suggesting that expression of these genes is important in HBV infection. Therapies to increase expression of HLA-DPA1 or HLA-DPB1 might improve treatment of chronic hepatitis B.
Figure 1. (a) Odds ratio (OR) for chronic HBV (adapted from Kamatani, 2009) and difference in expression of HLA-DPA1 in liver tissue, by rs3077 genotype. (b) OR for chronic HBV and difference in expression of HLA-DPB1 in liver tissue, by rs9277535 genotype. Gene expression values represent the arithmetic difference in mean-log gene expression from the referent genotype (rs3077GG or rs9277535GG).
731 CHANGES OF SERUM APOPTOTIC CASPASE ACTIVITY IN HBEAG-NEGATIVE CHRONIC HEPATITIS B (CHB) PATIENTS UNDER ORAL ANTIVIRAL THERAPY G.V. Papatheodoridis, J. Giannousis, S. Manolakopoulos, E. Hadziyannis, A. Georgiou, A.J. Archimandritis. 2nd Dept. of Internal Medicine, Athens University Medical School, Hippokration General Hospital, Athens, Greece E-mail: [email protected] Background and Aim: The effects of CHB treatment on hepatocyte apoptosis have not been evaluated. We investigated the changes in serum levels of the apoptotic caspase-generated fragments of keratin-18 (K-18), a major intermediate filament protein in hepatocytes, in HBeAg-negative CHB patients under nucleos(t)ide analogue(s). Methods: We included 46 patients (age:52±14 years, M/F:38/8) with histologically documented HBeAg-negative CHB who had received nucleos(t)ide analogue(s) (lamivudine:42, adefovir:4) for ≥12 months. Of the 42 lamivudine treated patients, 23 developed
Journal of Hepatology 2010 vol. 52 | S183–S317
POSTERS virological with or without biochemical breakthroughs and received add-on adefovir therapy for ≥12 months. Sera drawn at baseline, at 6 and 12 months of initial therapy as well as at the diagnosis of breakthroughs and at 6 and 12 months after adefovir addition were tested blindly using the M30-Apoptosense ELISA Kit (PEVIVA) that measures the apoptosis-associated neoepitope in the C-terminal domain of K-18 (aa 387–396). Results: During initial therapy, K-18 fragments (U/L) levels significantly reduced from baseline [mean±SD (range): 446±459 (149–2461)] to 6 months [180±74 (110–454) U/L, P < 0.001] and from 6 to 12 months [162±47 (113–280), P = 0.031]. K-18 fragments levels increased at breakthroughs [250±198 (124–1063)] compared to last previous visits [197±146 (118–809), P = 0.042] but remained lower than baseline (P = 0.016). Changes of K-18 fragments levels correlated positively with changes of ALT and HBV-DNA from baseline to 6 (r = 0.869, P < 0.001 and r = 0.396, P = 0.014) or 12 months of initial therapy (r = 0.873, P < 0.001 and r = 0.544, P < 0.001). In contrast to initial therapy, in patients with breakthroughs, K-18 fragments levels had only a trend for decline from adefovir addition [303±290 (124–1219)] to 6 [274±377 (122– 1576), P = 0.109] and mainly to 12 months [223±201 (122–878), P = 0.055]. In the latter subgroup, changes of K-18 fragments levels correlated positively with changes of ALT at 6 (r = 0.624, P = 0.017) or 12 months (r = 0.765, P < 0.001) and with changes of HBV-DNA only at 12 months (r = 0.535, P = 0.010) after adefovir addition. Conclusions: Apoptotic caspase activity decreases significantly during oral antiviral therapy in HBeAg-negative CHB in parallel with the improvements in ALT and HBV-DNA levels. It increases again during breakthroughs and may improve after an effective rescue treatment but at a slower rate compared to the initial therapy. 732 PROPORTION OF DEFECTIVE HBV DNA ISSUED FROM A SINGLY SPLICED RNA AND ENCODING FOR HBSP IS RELATED TO THE COURSE OF VIRAL INFECTION F. Redelsperger1 , B. Lekbaby1 , N. Desir ´ e´ 2 , A.M. Roque-Afonso3 , 4 5 S. Brichler , G. Perlemuter , P. Dubreuil6 , N. Bacon2 , F. Zatla2 , C. Le Pendeven2 , D. Kremsdorf1 , P. Soussan1,2 . 1 INSERM U845, 2 Virology Unit, CHU Tenon, 3 Virology Unit, CHU P. Brousse, 4 Virology Unit, CHU Avicenne, 5 Hepatology Unit, 6 Virology Unit, CHU A Beclere, Paris, France E-mail: [email protected] Background: Defective hepatitis B virus particles (dHBV), generated from a singly spliced HBV RNA are detected during HBV infection. We and others have observed that proportion of circulating dHBV particles varied in chronic carriers and could be related to severe liver disease. This relationship is controversial, probably due to criteria of included patients and/or quantification methods. The present study was designed to quantify defective and wild-type HBV (wtHBV) genomes through the course of viral infection using two previously described methods and subsequently to search for viral factors involved in dHBV formation. Patients and Methods: HBV patients with 1. anti-HBc IgM positive acute (developing or not chronic infection and fulminant; n = 40) or reactivation (n = 5) hepatitis; 2. moderate or severe chronic liver disease untreated (n = 72, HBV >105 copies/ml), and 3. a longitudinal follow-up during 9 to 23 years (n = 5), were studied. Wild-type HBV and dHBV DNA were quantified by in-house qPCR (using two previous published methods for dHBV DNA). HBV Posttranscriptional Regulatory Element (PRE) region was studied by direct sequence analysis. Mutations identified were inserted by mutagenesis in HBV plasmid and impact on splicing modulation was analyzed in HuH7 cells.
Results: Whatever dHBV DNA quantification method used, higher amount of dHBV DNA was associated in severe compared to moderate chronic disease (8.5±2.6% versus 1.7±0.5%, respectively; p = 0.02). In acute infection, dHBV DNA proportion was independent to disease progression. A significant lower proportion of dHBV DNA was observed in acute compared to chronic carriers, suggesting regulation of the dHBV DNA proportion during infection (dHBV DNA amount >5% for 2/40 versus 16/72 patients, respectively, p = 0.02). Investigation of follow-up of HBV chronic carriers showed that despite an overall constant proportion of dHBV DNA, highest levels (>50%) were observed at 2 timepoints for 2 patients and seemed associated to liver disease progression. Mutations in PRE region, involved in splice regulation, were detected by sequence comparison of higher or lower proportion of dHBV DNA. Transfection of HBV with PRE mutations or wild-type showed an impact in dHBV DNA expression. Conclusion: Our data demonstrate, independently to quantification methods, an increase proportion of dHBV DNA during the progression of viral disease, probably through PRE mutations. 733 REACTIVATION OF OVERT AND OCCULT HBV INFECTION IN IMMUNOSUPPRESSIVE SETTINGS N. Coppola1 , G. Tonziello1 , M. Pisaturo1 , M. Fiore1 , V. Iodice1 , S. Guastrafierro1 , C. Sagnelli1 , N. Capoluongo1 , V. Messina2 , G. Pasquale1 , E. Sagnelli1,2 . 1 Second University of Naples, Naples, 2 Sant’Anna Hospital of Caserta, Caserta, Italy E-mail: [email protected] Aim: To evaluate differences in clinical and virological presentation between patients with overt and occult HBV infection during reactivation of HBV infection (r-HBV). Methods: 23 consecutive patients with symptomatic r-HBV (HBsAg/HBV-DNA-positivity, ALT serum level at least 5 times higher the mean value in the preceding 6-months) during or after immunosuppressive therapy were enrolled: 10 with reactivation of overt HBV infection (HBsAg positive from 6 months or more before r-HBV; Overt Group) and 13 of occult HBV infection (HBsAgnegative/anti-HBc-positive from at least 6 months at r-HBV; Occult Group). No patient had received prophylaxis for r-HBV. Anti-HBV Nucleot(s)ide analogue treatment was given at first observation after r-HBV in 21 patients. Results: All patients in Occult Group and 6 in Overt Group (p = 0.05) had onco-hematological diseases. r-HBV was observed both during and after immunosuppressive therapy. Regimens including rituximab or fludarabine were frequently administered in Occult Group (53% and 31%, respectively). No significant difference was observed between Overt and Occult groups as far age, sex, clinical and biochemical presentation. r-HBV was frequently severe both in Overt (40%) and in Occult groups (38.4%). Patients in Overt Group showed higher HBVDNA titres than those in Occult Group (1.1×108 ±1.4×108 vs 5.1×105 ±6.8×105 IU; p < 0.005). All patients had HBV genotype D. Seven patients died during r-HBV, 2 in Overt and 5 in Occult group, two for hepatic failure (one in each group) and 5 for progression of ongoing diseases after mandatory discontinuation of immunosuppressive treatment (1 in Overt and 4 in Occult group). Of patients who died, 2 were untreated and 5 received Lamivudine; of the 16 with remission of r-HBV, 4 received Lamivudine and 12 other recently developed analouges or an analogues combination. Conclusions: r-HBV is a severe complication in patients undergoing immunosuppressive therapy that may be prevented or treated with appropriate anti-HBV analogues treatment.
Journal of Hepatology 2010 vol. 52 | S183–S317
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