Detection and significance of PD-1.3 SNP (rs11568821) and IL28B SNP (rs12979860) in patients with current or past hepatitis B virus (HBV) infection Asterios Saitis 1, Nikolaos K. Gatselis 1, Kalliopi Azariadi 1, Kalliopi Zachou 1, George K. Koukoulis 2, George N. Dalekos 1 1 Department of Medicine and Research Laboratory of Internal Medicine, School of Medicine, Thessaly University, Larissa, Greece 2 Department of Pathology, School of Medicine, Thessaly University, Larissa, Greece
Thomas et al, Nature 2009 Ge et al, Nature 2009 Chronic Hepatitis C IL28B polymorphism (rs 12979860) Response to Peg-IFN +RIBA Spontaneous HCV clearance
Chronic Hepatitis C PD1.3 polymorphism (rs11568821) Asociation analysis of PD-1 polymorphism with response to antiviral treatment Genotypes SVR (n=210) NR (n=197) p value PD-1.3 <0.05 G/G 150 (71.4%) 164 (83.2%) G/A 50 (23.8%) 29 (14.7%) A/A 10 (4.8%) 4 (2.1%) 70 60 50 OR (95% CI) 47.7 64.5 53.3 40 30 20 35.3 G/G G/A or AA 10 0 Response (n=210) Non-Response (n=197) Vidal-Castiñeira et al. Journal of Hepatology 2012
Chronic Hepatitis B IL28B polymorphism PD1.3 polymorphism Susceptibility to HBV infection? Determinants of interferon response?
Chronic Hepatitis B Chronic infection 4-5% Chronic hepatitis 0.8-1% Past infection 95% Determinants Age of infection Genetic? Genetic? EASL Clinical Practice Guidelines, Journal of Hepatology 2017
Chronic Hepatitis B Inteferons HBsAg clearance 48-week duration or Nucleoside/ Nucleotide analogues High efficacy High safety Per os therapy Genetic determinants of IFN response? EASL Clinical Practice Guidelines, Journal of Hepatology 2017
Study Design 401 HBV patients chronic hepatitis N=208 chronic infection N=100 past infection N=93 IFN-treated N=78 IL28B & PD1.3 polymorphism Clinical- Laboratory features Treatment Response
Fluorescence (533-580) Fluorescence (533-580) Methods Allelic Discrimination Endpoint C T Allele specific fluorescent DNA Probes 20.0 16.0 12.0 20.000 16.000 12.000 Allele y samples Heterozygous samples 8.0 8.000 4.0 0.0 4.000 2.000 0.500 Allele x samples 1 3 6 9 13 18 23 28 33 38 Cycles 2.000 4.000 6.000 8.000 10.000 12.000 Fluorescence (465-510) Gatselis et al, J Hepatol 2013; 58: S187
Methods IL28B genotyping In house allelic end point discrimination assay Residual DNA of serum samples Direct sequencing Gatselis et al, J Hepatol 2013; 58: S187
Methods PD1.3 genotyping In house allelic end point discrimination assay Genomic DNA of total blood samples In house PCR restriction fragment length polymorphism (RFLP)
Results IL28B & Susceptibility to HBV infection N=403 Chronic Hepatitis B Chronic HBV infection Past Infection TT 18% CC 41% TT 10% CC 40% TT 11% CC 46% CT 41% CT 50% CT 43%
Results PD1.3 & Susceptibility to HBV infection N=120 Chronic hepatitis B Chronic infection Past infection GA 19% GA 21% GA 20% AA 3% GG 81% GG 79% GG 77%
Study Design 401 HBV patients chronic hepatitis N=208 chronic infection N=100 past infection N=93 IFN-treated N=78 IL28B & PD1.3 polymorphism Clinical- Laboratory features Liver Disease Progression
Results Chronic HBV Hepatitis Clinical & laboratory features N IL28B genotype CC CT or TT P value AST, IU/l, mean ±SD 77 72.4± 67.3 73.8± 94.5 0.755 ALT, IU/l, mean ±SD 190 131.1± 143.5 143.4± 203.7 0.210 γgt, IU/l, mean ±SD 75 36.6±23.3 34.5± 31.3 0.781 Bilirubin, mg/dl, mean ±SD 75 1.2±1.8 0.9±0.44 0.061 Albumin, g/dl, mean ±SD 74 4.3±0.6 4.4± 0.4 0.085 Prothrombin time, sec, mean ±SD 74 13.3±3.12 13± 1.2 0.069 Platelets, 10 9 /μl, mean ±SD 64 180478±52816 203731± 53677 0.355 Baseline HBV DNA, IU/ml, mean ±SD 174 254647± 5280185 3137776 ± 7517539 0.292 Fibrosis at Baseline: (frequencies, %) Baseline Cirrhosis: (frequencies, %) Progression to Cirrhosis: (frequencies, %) F0-F2 F3-F4 yes no yes no 180 124 138 27(69.2%) 12(31.8%) 11 (14%) 58 (86%) 3(5%) 56(95%) 31 (44.9%) 31 (77.5%) 33(30%) 78(70%) 6 (7.6%) 73 (92.4%) 0.120 0.049 0.732 Progression to Decompensation: yes (frequencies, %) no 179 1 (1.4%) 67(98.6%) 8 (28.6%) 103 (64.3%) 0.156 HCC development : (frequencies, %) yes no 180 8 (11.8%) 61 (88.4%) 14 (14.4%) 97(85.6%) 1.000
Results Chronic HBV Hepatitis IL28B & Baseline Cirrhosis Genotypes IL28B Non- Cirrhotic (n=135) Cirrhotic (n=44) p value CC 58 (84.5%) 11(15.5%) <0.05 CT or TT 78 70%) 33 (30%) CC Cirrhosis 16% P<0.05 Cirrhosis 30% CT or TT Non-Cirrhosis 84% Non-Cirrhosis 70%
Results Chronic HBV Hepatitis Clinical & laboratory features N PD1.3 genotype GA or AA GG P value AST, IU/l, mean ±SD 16 63.3± 30.6 57.5± 44.6 0.836 ALT, IU/l, mean ±SD 34 80± 50.2 157.9± 231.5 0.428 γgt, IU/l, mean ±SD 16 41.6± 40.5 29± 22 0.451 Bilirubin, mg/dl, mean ±SD 16 0.56±0.29 0.77±0.36 0.386 Albumin, g/dl, mean ±SD 15 4.3±0.36 4.25± 0.61 0.906 Prothrombin time, sec, mean ±SD 15 12.7±3.1.06 13.3± 1.1 0.462 Platelets, 10 9 /μl, mean ±SD 5 221600± 50361 Baseline HBV DNA, IU/ml, mean ±SD 32 729708± 916225 3137776 ± 7517539 0.014 Fibrosis at Baseline: (frequencies, %) Baseline Cirrhosis: (frequencies, %) Progression to Cirrhosis: (frequencies, %) F0-F2 F3-F4 yes no yes no 21 33 24 4 (80.0%) 1 (20.0%) 11 (14%) 58 (86%) 0 (0%) 5 (100%) 10 (62.5%) 6 (37.5%) 33(30%) 78(70%) 2 (10.5%) 17 (89.5%) 0.624 0.637 0.449 Progression to Decompensation: yes (frequencies, %) no 33 0 (0 %) 6(100%) 4 (14.8%) 23 (85.2%) 1.000 HCC development : (frequencies, %) yes no 33 0 (0 %) 6(100%) 4 (14.8%) 23 (85.2%) 1.000
Results Chronic HBV Hepatitis PD1.3 & Baseline HBV DNA Genotypes PD1.3 HBV-DNA (n=132) p value GA or AA 729708 ± 916225 <0.05 GG 4514630 ± 7128748 10000000 1000000 Baseline HBV DNA p<0.05 100000 10000 1000 100 10 1 GA or AA GG
Study Design 401 HBV patients chronic hepatitis N=208 chronic infection N=100 past infection N=93 IFN-treated N=78 IL28B & PD1.3 polymorphism Treatment Response
Results IFN-treated patients IL28B & Response to IFN treatment N=78 80 75.5 70 60 50 48.3 CC CT or TT 51.7 40 30 24.5 20 10 0 Response Non-Response
Results IFN-treated patients PD1.3 & Response to IFN treatment N=13 12 10 10 8 6 GG 4 2 2 1 1 GA or GA 0 Response Non-Response
Conclusions PD-1.3 (rs11568821) and IL28B (rs12979860) SNPs were successfully genotyped by in-house assays No influence of IL28B & PD1.3 polymorphisms on genetic susceptibility to HBV infection
Conclusions Association of IL28B polymorphism with baseline advanced liver fibrosis Prognostic Significance of IL28B rs12979860 for response to interferon treatment
Conclusions-Perspective Association of PD1.3 polymorphism with baseline HBV DNA levels Larger studies to determine the prognostic significance of PD1.3 polymorphism in interferon treatment
Thank you for your attention This study has been supported by Asklepios GILEAD Grants (Greece)