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 Saitis1, Nikolaos K. Gatselis1, Kalliopi Azariadi1, Kalliopi Zachou1, George K. Koukoulis2, George N. Dalekos1

1Department of Medicine and Research Laboratory of Internal Medicine, School of Medicine, Thessaly University, Larissa, Greece

2Department of Pathology, School of Medicine, Thessaly University, Larissa, Greece

Chronic Hepatitis C

Thomas et al, Nature 2009

Ge et al, Nature 2009

Response to Peg-IFN +RIBA Spontaneous HCV clearance

IL28B polymorphism (rs 12979860)

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 OR(95% CI)

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%)47.7

53.3

64.5

35.3

0

10

20

30

40

50

60

70

Response (n=210) Non-Response (n=197)

G/G

G/A or AA

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

EASL Clinical Practice Guidelines, Journal of Hepatology 2017

Past infection95%

Chronic infection4-5%

Chronic hepatitis0.8-1%

Determinants

• Age of infection

• Genetic?• Genetic?

Chronic Hepatitis B

or

HBsAg clearance

48-week duration

High efficacy

High safety

Per os therapy

Inteferons Nucleoside/ Nucleotideanalogues

Genetic determinants of IFN response?

EASL Clinical Practice Guidelines, Journal of Hepatology 2017

Study Design

401 HBV patients

chronic hepatitisN=208

chronic infectionN=100

past infectionN=93

IL28B & PD1.3 polymorphism

Clinical- Laboratory featuresTreatment Response

IFN-treatedN=78

Methods

Gatselis et al, J Hepatol 2013; 58: S187

Allelic Discrimination Endpoint

Flu

ore

sce

nce

(53

3-5

80

)

1

Cycles

3 6 9 13 18 23 28 33 38

0.0

4.0

8.0

12.0

16.0

20.0

Flu

ore

sce

nce

(53

3-5

80

)

0.500

4.000

8.000

12.000

16.000

20.000

2.000

Fluorescence (465-510)2.000 4.000 6.000 8.000 10.000 12.000

Allele ysamples

Allele x samples

Heterozygoussamples

T

CAllele specific fluorescent

DNA Probes

IL28B genotyping

Methods

‘Residual DNA’ of serum samples

Gatselis et al, J Hepatol 2013; 58: S187

‘In house’ allelic end point discrimination assay

Direct sequencing

‘In house’ allelic end point discrimination assay

Genomic DNA of total blood samples

Methods

PD1.3 genotyping

‘In house’ PCR restriction fragment length polymorphism (RFLP)

IL28B & Susceptibility to HBV infection

Results

CC41%

CT41%

TT18%

Chronic Hepatitis B

CC40%

CT50%

TT10%

Chronic HBV infection

CC46%

CT43%

TT11%

Past Infection

N=403

PD1.3 & Susceptibility to HBV infection

Results

GG81%

GA19%

Chronic hepatitis B

GG79%

GA21%

Chronic infection

GG77%

GA20%

AA3%

Past infection

N=120

Study Design

chronic hepatitisN=208

IL28B & PD1.3 polymorphism

Clinical- Laboratory featuresLiver Disease Progression

401 HBV patients

chronic infectionN=100

past infectionN=93

IFN-treatedN=78

Chronic HBV Hepatitis Results

Clinical & laboratory features NIL28B genotype P

valueCC CT or TT

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, 109/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: F0-F2(frequencies, %) F3-F4

18027(69.2%)12(31.8%)

31 (44.9%)31 (77.5%)

0.120

Baseline Cirrhosis: yes(frequencies, %) no

12411 (14%)58 (86%)

33(30%)78(70%)

0.049

Progression to Cirrhosis: yes(frequencies, %) no

1383(5%)

56(95%)6 (7.6%)

73 (92.4%)0.732

Progression to Decompensation: yes (frequencies, %) no

1791 (1.4%)

67(98.6%)8 (28.6%)

103 (64.3%)0.156

HCC development : yes(frequencies, %) no

1808 (11.8%)61 (88.4%)

14 (14.4%)97(85.6%)

1.000

IL28B & Baseline Cirrhosis

Results

GenotypesIL28B

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%)

Non-Cirrhosis84%

Cirrhosis16%

Non-Cirrhosis70%

Cirrhosis30%

CC CT or TT

P<0.05

Chronic HBV Hepatitis

Chronic HBV Hepatitis Results

Clinical & laboratory features NPD1.3 genotype P

valueGA or AA GG

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, 109/μl, mean ±SD 5 221600± 50361

Baseline HBV DNA, IU/ml, mean ±SD 32 729708± 916225 3137776 ± 7517539 0.014

Fibrosis at Baseline: F0-F2(frequencies, %) F3-F4

214 (80.0%)1 (20.0%)

10 (62.5%)6 (37.5%)

0.624

Baseline Cirrhosis: yes(frequencies, %) no

3311 (14%)58 (86%)

33(30%)78(70%)

0.637

Progression to Cirrhosis: yes(frequencies, %) no

240 (0%)

5 (100%)2 (10.5%)

17 (89.5%)0.449

Progression to Decompensation: yes (frequencies, %) no

330 (0 %)6(100%)

4 (14.8%)23 (85.2%)

1.000

HCC development : yes(frequencies, %) no

330 (0 %)6(100%)

4 (14.8%)23 (85.2%)

1.000

PD1.3 & Baseline HBV DNA

Results

GenotypesPD1.3

HBV-DNA(n=132)

p value

GA or AA 729708 ± 916225 <0.05

GG 4514630 ± 7128748

1

10

100

1000

10000

100000

1000000

10000000

GA or AA GG

Baseline HBV DNA

p<0.05

Chronic HBV Hepatitis

Study Design

IL28B & PD1.3 polymorphism

Treatment Response

IFN-treatedN=78

401 HBV patients

chronic hepatitisN=208

chronic infectionN=100

past infectionN=93

IL28B & Response to IFN treatment

Results

48.351.7

24.5

75.5

0

10

20

30

40

50

60

70

80

Response Non-Response

CC

CT or TT

N=78

IFN-treated patients

PD1.3 & Response to IFN treatment

Results

2

10

1 1

0

2

4

6

8

10

12

Response Non-Response

GG

GA or GA

N=13

IFN-treated patients

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

• Prognostic Significance of IL28B rs12979860 for response to interferon treatment

• Association of IL28B polymorphism with baseline advanced liver fibrosis

Conclusions

• Larger studies to determine the prognostic significance of PD1.3polymorphism in interferon treatment

Conclusions-Perspective

• Association of PD1.3 polymorphism with baseline HBV DNA levels

Thank you for your attention

This study has been supported by Asklepios GILEAD Grants (Greece)