Gene Expression and HCV InfectionGene Expression and HCV Infection

44th Annual Meeting of the 44th Annual Meeting of the

Italian Association for the Study of the LiverItalian Association for the Study of the Liver

Roma, 24 February 2011Roma, 24 February 2011

Tarik AsselahTarik Asselah

Service dService d’’HHéépatologie & INSERM U773, CRB3 patologie & INSERM U773, CRB3 Hôpital Beaujon, ClichyHôpital Beaujon, Clichy

tarik.asselah@bjn.aphp.frtarik.asselah@bjn.aphp.fr

•• IntroductionIntroduction•• VirologyVirology

•• Genetic (GWAS)Genetic (GWAS)

•• Gene expression (transcriptomic)Gene expression (transcriptomic)

•• Clinical ImplicationsClinical Implications•• Fibrosis ProgressionFibrosis Progression

•• Response to treatmentResponse to treatment

•• ConclusionConclusion

Gene Expression and HCV InfectionGene Expression and HCV Infection

USA & USA & CanadaCanada

4 M4 M

SOUTHSOUTHAMERICAAMERICA

10 M10 M

AFRICA AFRICA 32 M32 M

EASTERNEASTERNMEDITERRANEANMEDITERRANEAN

21.3M21.3M SOUTH EAST SOUTH EAST ASIAASIA

32.3 M32.3 M

AUSTRALIAAUSTRALIA0.2 M0.2 M

WHO, 1999WHO, 1999

EUROPE EUROPE 9 M9 M

FAR EASTFAR EAST/ASIA/ASIA60 M60 M

170 170 Millions Hepatitis C virus (HCV) carriers Millions Hepatitis C virus (HCV) carriers

HCV infection worldwideHCV infection worldwideHCV infection worldwide

JapanJapan

2M2M

Asselah T et al. Liver International 2009Asselah T et al. Liver International 2009

Asselah T et al. Liver International 2011Asselah T et al. Liver International 2011

The International HapMap Consortium. The International HapThe International HapMap Consortium. The International Hap-- Map Project. Nature 2003.Map Project. Nature 2003.Evans WE, McLeod HL. PharmacogenomicsEvans WE, McLeod HL. Pharmacogenomics----drug disposition, drug targets, and side effects. drug disposition, drug targets, and side effects. NEJM 2003.NEJM 2003.

«« The good drug, to the good The good drug, to the good patient, at the good timepatient, at the good time »»

«« Sequence of Sequence of Human GenomeHuman Genome »»

1. Transcription1. Transcription

2. Translation2. Translation

3. Maturation3. Maturation

DNADNA

mRNAmRNA

ExportationExportation

CytoplasmCytoplasm

CoreCore

4. Assembly and4. Assembly andreleaserelease

mRNAmRNA

Endoplasmic Endoplasmic ReticulumReticulum

RNAsmRNA (Transcriptomic)

sRNA non codants

Proteins(Proteomic)

Extraction

Tissue

DNA(Genomic)

PatientsN=500

ControlsN=500

1 22~3 000 000 SNPs in the Genome

P-va

lue

1 22

3 domains significant association

Chromosomal Location

Genome Wide Association Studies (GWAS)

Human GenomeHuman Genome(2001)(2001)

CtCt

Real time RTReal time RT--PCRPCR Microarrays Microarrays

11--500 selected genes500 selected genes ~~ 25 000 g25 000 gèènesnes

• Introduction• Virology

• Genetic (GWAS)

• Gene expression (transcriptomic)

• Clinical Implications• Fibrosis Progression

• Response to treatment

• Conclusion

Gene Expression and HCV Infection

Bedossa et al. Hepatology 1996

• Initially mild fibrosis with progression• 271 untreated chronic hepatitis C patients

• Paired liver biopsies (FU 108 ± 71 months)

• No progression 24.4%, 1 Stage 45.0%, 2 stages 10.3%

• Cirrhosis risk score (CRS)• 7 SNPs : AZIN1 (ch 8); TLR4 (ch 9); TRPM5 (ch 11);

AQP2 (ch 9) and 3 other SPNs (chr 1, 3, and 15)

• Increased CRS value associated with increased

fibrosis progression.

Genetic and Fibrosis Progression

Marcolongo et al. Hepatology 2009

Friedman SL. Physiol Rev. 2008

240 genes

Extracellular matrix (14) Matrix proteases and Inhibitors (13)

Cell adhesion and cell junction (9) Hepatic stellate cells (12)

Cytokines (12) Chemokines (10)

Oxydatif stress (11) Angiogenesis (17)

Apoptosis (8) Cell cycle (10)

Growth factors & receptors (12) Various (25)

IFN inducible genes (13) Serum markers of fibrosis (7)

Discovery of Fibrosis Biomarkers

F1 (18 samples)

F2 (21 samples)

F3(17 samples)

F4 (6 samples)

COL1A1

0

5

10

15

20

1.0(0.34-3.10)

1.65 (0.81-14.25)

2.00(0.08-12.84)

4.06(1.01-13.64)

Stan

dard

i zed

mR

NA

l eve

l

1.0(0.41-2.93)

2.95(0.87-9.40)

4.0(1.41-27.73)

8.8(2.4-12.7)

0

5

10

15

20

Stan

dard

i zed

mR

NA

l eve

l

KRT19

AsselahAsselah T et al. Gastroenterology 2006T et al. Gastroenterology 2006

F1 F2

F1 F2

Cytokeratin 19Cytokeratin 19

IL 8IL 8

F2F2F2

F2

F2F2F2

F2F2F2F1

F2

F2F2F2F2

F2F2F2F2

F2

F2

F1

F1F1

F1

F1F1

F1

F1F1

F1F1F1

F1F1

F1F1F1

•

••

•

19/22 (86 %) F2

15/17 (88 %) F1

Molecular Signature 11 genes : KRT19, COL1A1, STMN2/SCG10, CXCL6, CCR2, TIMP1, IL8, IL1A, ITGA2, CLDN4, and IL2.

Group B Group B SurgicalSurgicalcontrolcontrol

Group A Group A NonNon--surgicalsurgicalcontrolcontrol

Asselah et al, Hepatology, 2008

Caveat: The Importance of an Adequate Normal Caveat: The Importance of an Adequate Normal Tissue Control in Gene Expression StudiesTissue Control in Gene Expression Studies

IL8 gene97.9

Group A(n=14)

10

53

01.0 1.1 1.8 2.2 2.8

8.6

[0.6–2.1] [0.4–2.1] [0.7–6.9] [0.5–5.2] [0.6–5.5] [1.2–30.7] [3.8–434.7]

A1F1(n=11)

A2F1(n=9)

A1F2(n=10)

A2F2(n=10)

A2F3(n=15)

Group B(n=14)

vs. vs.HCV

• Introduction• Virology

• Genetic (GWAS)

• Gene expression (transcriptomic)

• Clinical Implications• Fibrosis Progression

• Response to treatment

• Conclusion

Gene Expression and HCV Infection

Progress in HCV Treatment

PEG-IFN IFN+Riba

6 16%18 23%

47% 63%

35 43%

PEG-IFN+Riba

1989 2011IFN

Manns et al. Lancet 2001Manns et al. Lancet 2001Fried et al. NEJM 2002 Fried et al. NEJM 2002 Hadziyannis et al. Hadziyannis et al. Annals of Internal Medicine 2004Annals of Internal Medicine 2004

PEGPEG--IFNIFN--αα + Ribavirin+ RibavirinRates of FailureRates of Failure

PEGPEG--IFNIFN--αα 2a2a+ ribavirin + ribavirin (Fried et al., 2002)(Fried et al., 2002)

PEGPEG--IFNIFN--αα 2b2b+ ribavirin+ ribavirin(Manns et al., 2001)(Manns et al., 2001)

Manns et al., Lancet 2001; Manns et al., Lancet 2001; Fried et al., N Engl J Med 2002; Fried et al., N Engl J Med 2002; Hadziyannis et al., Ann Intern Med 2004.Hadziyannis et al., Ann Intern Med 2004.

54%54%

24%24%

Genotype 1Genotype 1 Genotypes 2/3Genotypes 2/3

58%58%

48%48%

18%18%16%16%

PEGPEG--IFNIFN--αα 2a2a+ ribavirin + ribavirin (Hadziyannis et al., 2004)(Hadziyannis et al., 2004)

RVPRVP

Chronic Hepatitis C

NRPEG-IFN + RBV

Prediction of Non response

50 %50 %

50 %

Manns et al, Nature review in drug discovery 2007

Side Effects, Costs

Genome Wide Association Studies

25%

80%

0%

20%

40%

60%

80%

100%

SVR

(%)

T/T C/C

40 %

T/C

rs12979860 Predict Response to Treatment

Ge et al, Nature 2009

SNPs identified in chromosome 19 for genotype 1 patients with an SVR

Genome wide association results for SVR in genotype 1 patients

EASL 2010 – Asselah T., A 1180

rs12979860 localized within IL28B promotor

EASL 2010 – Asselah T., A 1180

IL28B genotype C/C associated with SVR in genotype 2/3 with no RVR

Mangia et al, Gastroenterology 2010

IL28B polymorphism (rs 8099917) associated with SVR : Telaprevir + PEG-IFN + RBV

Akuta et al, Hepatology 2010

Asselah T. Journal of Hepatology, 2010Asselah T. Journal of Hepatology, 2010

Chen et al. Gastroenterology 2005

Cytokines (12)Chemokines (10)ISG stimulated genes (26)Others (10)

Discovery of biomarkers to Discovery of biomarkers to predict response (58) predict response (58)

Signature: IFI27, CXCL9

Groupe A Patients correctly classified : 31/40 (78%)

Patients correctly classified : : 23/29 ((79%)Validation (B)

69 patients

Group A40 patients

(14 NR, 26 SVR)

Group B29 patients

(9 NR, 20 SVR)

ConstructionSignature

Validation Signature

Asselah et al, Gut 2008

Prediction of treatment response

Gene NR/SVR P-value IFI6 3.5 0.002IFI27 4.2 0.002ISG15 3.7 0.002MIX1 2.7 0.006

HERC5 2.2 0.006TGFB2 2.7 0.006OAS2 1.8 0.016

VEGFD 2.4 0.020IL8 3.2 0.020

Asselah et al, Gut 2008.

Genes differentially expressed betweenNR and SVR

IFI27

CXCL9

Interleukin 8Interleukin 8

Serum Markers

In SituIn Situ SerumSerum

Activated Stellate CellsDeposition of Scar Matrix

Kupffer Cell Activation

Endothelial Cell

Hepatocytes

3

–– GenotypeGenotype–– Viral loadViral load–– AgeAge–– Fibrosis stageFibrosis stage–– EtcEtc……....

Prediction of treatment response

20 markers

2010 2011

25 000 Genes100 000 RNA

1 000 000 Proteins

1

2

• Fibrosis progression is related to genetic factors

• IL28B SNP associated with response

• Validated in different ethnic groups

• Exact mechanism unknown, no relation with expression

• Might be a marker of immune response

• Interteron stimulated genes (ISG) up-regulated in NR

• Role in clinical practice not established

• IL28B and triple therapy ?

• Other markers ?

• Other populations ?

Conclusion