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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
[email protected]@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