hépatite c pourquoi le génotype 3 est-il si particulier final hcv... · liver steatosis in...
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Hépatite C Pourquoi le génotype 3
est-il si particulier ?
Francesco Negro University Hospitals of Geneva, Switzerland
Lyon – 9 Juin 2015
• Steatosis
• Insulin resistance (?)
• Accelerated fibrosis progression rate
• Increased risk of hepatocellular carcinoma
• Reduced response to IFN-free therapy
HCV-3a associated features
• Steatosis
• Insulin resistance (?)
• Accelerated fibrosis progression rate
• Increased risk of hepatocellular carcinoma
• Reduced response to IFN-free therapy
HCV-3a associated features
"Steatosis is frequent in post-transfusion hepatitis"
HCV genotype and liver steatosis n=1383 without risk factors (BMI <25 kg/m2, no alcohol, no T2D)
RR=4.81 p=2.33x10-27
LEANDRO et al, Gastroenterology 2006;130:1636–1642
Pre
vale
nce o
f liver
ste
ato
sis
(%
)
Pre
vale
nce o
f liver
ste
ato
sis
(%
)
Severity of steatosis correlates with serum (+) and liver (-) HCV RNA in HCV-3
r P
All 0.49 0.01
HCV-1 0.32 NS
HCV-3 0.64 0.004
RUBBIA-BRANDT et al, J Hepatol 2000;33:106-15 FARTOUX et al, Gut 2005;54:1003-8
Serum, HCV-3
Steatosis in HCV non-3 genotypes mostly correlates with body weight
ADINOLFI et al, Hepatology 2001;33:1358-64
Steatosis improves after SVR (more often in HCV genotype 3 infected patients)
POYNARD et al, Hepatology 2003;38:75-85
17%
Liver steatosis in post-LT hepatitis C and response to a-interferon
Liver steatosis in a patient with recurrent hepatitis C after liver transplantation: 1a - before a-IFN therapy, 1b - at the time of on-treatment virological response 1c – when the patient relapsed after the end of treatment
RUBBIA-BRANDT et al, J Hepatol 2001; 35: 307
• Steatosis
• Insulin resistance (?)
• Accelerated fibrosis progression rate
• Increased risk of hepatocellular carcinoma
• Reduced response to IFN-free therapy
HCV-3a associated features
HOMA-IR as a function of viral load and genotype
MOUCARI et al, Gastroenterology 2008;134:416-23
HOMA-IR >2 in 153/381 HCV-1/4 (40.1%) vs 22/108 HCV-2/3 (20.3%) (p<.001)
HCV-3a : more or less insulin resistance??
HCV-1 (n=186) HCV-3 (n=160) p
Glucose (mmol/L) 5.1 (4.7, 5.4) 5.0 (4.7, 5.4) 0.8
C-Peptide (nmol/L) 0.77 (0.50, 1.21) 0.76 (0.51, 1.31) 0.6
Insulin (μU/mL) 10.0 (7.0, 15.0) 9.3 (6.0, 13.0) 0.1
HOMA-IR 2.31 (1.43, 3.48) 2.14 (1.27, 2.89) 0.2
CUA et al, Hepatology 2008;48:723-31
HCV RNA was not associated with HOMA-IR in the whole cohort (r = 0.02, P = 0.7) Genotype-specific subgroup analysis did not alter this finding.
(Sydney Westmead Cohort, n=346; median, interquartile values)
Insulin resistance (HOMA-IR >3) in low metabolic risk patients
A multicenter study from Greece
n HCV non-3 HCV-3 p
BMI <25 kg/m2 133 17% 23% NS
BMI <25 kg/m2, F1-F2 92 16% 24% NS
TSOCHATZIS et al, Aliment Pharmacol Ther 2009;30:947-54
Viral steatosis does not induce IR
p<0.05
FARTOUX et al, Gut 2005;54:1003-8
HCV
Hepatic Insulin Resistance
(post-insulin receptor interactions)
Extrahepatic Insulin Resistance
(mediated by soluble factors)
Whole body insulin resistance
20-34% 66-80%
VANNI E et al, Hepatology 2009 MILNER KL et al, Gastroenterology 2010
Endogenous glucose production (EGP) and glucose disposal in hepatitis C without MS (euglycemic hyperinsulinemic clamp + tracers infusion + indirect calorimetry, n=14)
VANNI et al, Hepatology 2009;50:697-706
3
non-3
(under clamp conditions)
• Steatosis
• Insulin resistance (?)
• Accelerated fibrosis progression rate
• Increased risk of hepatocellular carcinoma
• Reduced response to IFN-free therapy
HCV-3a associated features
Liver fibrosis progression varies according to HCV genotype (n=957 with assessable date of infection; Swiss Hepatitis C Cohort Study)
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
0 5 10 15 20 25 30 35 40
HCV1_4, N=552, ref
HCV2, N=89, P=0.301
HCV3, N=315, P=0.000
BOCHUD et al, J Hepatol 2009;51:655-666
Fibrosis progression rates estimated from one biopsy [HCV genotype 3 (n=730) vs other genotypes (n=1619)]
PROBST et al, J Viral Hep 2011;18:745-59
Attributable fraction of risk for liver fibrosis progression in chronic hepatitis C
(The Swiss Hepatitis C Cohort Study; n=590)
RUEGER et al, Gut 2014 Sep 11 [Epub ahead of print]
Most liver fibrosis progression in chronic hepatitis C is attributable to non modifiable factors
The severity of steatosis at baseline predicts liver fibrosis progression in chronic hepatitis C
Retrospective study on serial liver biopsies (n=135, 60% HCV-1, 16% HCV-3, median FU 61 mo [18-158])
FARTOUX et al, Hepatology 2005;41:82-87
Steatosis (presumably viral) is not an independent factor of stage-constant fibrosis progression in HCV-3a
(Swiss Hepatitis C Cohort Study; n = 315 with assessable date of infection)
BOCHUD et al, J Hepatol 2009;51:655-66
OR 95% CI p
Male sex 2.012 1.007 – 4.019 0.05
Age at infection 1.081 1.029 – 1.135 0.002
ALT 1.004 1.000 – 1.007 0.05
Metavir activity 2.128 1.099 – 4.121 0.03
LEANDRO et al, Gastroenterology 2006;130:1636-42
Activity vs HCV genotype (HCV MAID database, all patients, n=3068)
• Steatosis
• Insulin resistance (?)
• Accelerated fibrosis progression rate
• Increased risk of hepatocellular carcinoma
• Reduced response to IFN-free therapy
HCV-3a associated features
HCV genotype 3 is associated with increased risk of HCC development
NKONTCHOU et al, J Viral Hepat 2011;18:e516-22
HR (95% CI) p
SVR 0.19 (0.08 – 0.44) <.001
Age, per y 1.09 (1.06 – 1.12) <.001
Male sex 2.00 (1.07 – 3.76) .03
HCV 3 2.07 (1.06 – 4.05) .03
T2D 2.01 (1.07 – 3.80) .03
Alcohol 2.20 (1.23 – 3.94) .008
Ishak 5 2.93 (1.23 – 6.95) .02
Ishak 6 2.62 (1.20 – 5.70) .02
VAN DER MEER et al, JAMA 2012;308:2584-93
HCV-3 increases the risk of developing HCC (Adjusted HR = 1.80, 95% CI = 1.61-2.03 in HCV-3 vs HCV-1)
KANWAL et al, Hepatology 2014;60:98-105
• Steatosis
• Insulin resistance (?)
• Accelerated fibrosis progression rate
• Increased risk of hepatocellular carcinoma
• Reduced response to IFN-free therapy
HCV-3a associated features
68
SOF / R 24
naive
SPARE (HCV-1) (Osinusi et al, JAMA 2013)
EVERSON et al, EASL 2014
SOFOSBUVIR 400 mg + GS-5816 25 or 100 mg, 12 weeks HCV-1 to -6, treatment-naive, NO cirrhosis
100
80
60
40
20
0
SVR
24
(%
)
96%
GS-5816
100% 100% 93%
86%*
100%
93% 91%
25 25 100 100 25 100 25 100
100%
100
100% 100%
25 100
HCV-1 HCV-2 HCV-3 HCV-4 HCV-5 HCV-6
*Only 1 out of 7 relapsed
PIANKO et al, AASLD 2014
SOFOSBUVIR 400 mg + GS-5816 25 or 100 mg ± RBV, 12 weeks HCV-1 or 3, treatment-experienced, with/without cirrhosis
100
80
60
40
20
0
SVR
24
(%
)
85%
GS-5816 RBV
58%
F4
84%
F4
88%
F4
96%
F4
100% 100% 96%
25 -
25 +
100 +
100 -
25 +
96%*
25 -
100%
100 +
96%*
100 -
100%
HCV-3 HCV-1
*Two relapses only (1 cirrhotic, 1 non-cirrhotic)
1b (Con1) 1a (H77) 2a (JFH-1) 1b/2a NS5B 1b/2b NS5B 1b/3a NS5B
48 ± 13 44 ± 4.7 37 ± 3.6 4.7 ± 1.7 20 ± 4.4 16 ± 3.4
CHENG et al, EASL 2013
LAM et al, Antimicrob Agent Chemother 2012;56:3359-68
Steatosis (>5%) predicts relapse in HCV-3 with RVR (ACHIEVE-2/3, n=402 with HCV-3 and RVR; Albuferon and pegIFN pooled data)
SHAH et al, Clin Gastroenterol Hepatol 2011;9:688-93
Predictors of response in HCV-2 and -3 treated with sofosbuvir and ribavirin
• 127 patients from FUSION, POSITRON, FISSION trials (HCV-2, n=50; HCV-3, n=77)
• Serum levels of ApoE, ApoB, cholesterol and 11 post-squalene metabolites of cholesterol synthetic pathway (by GC/MS)
• By MV logistic regression analysis of SVR, after adjustment for gender and HCV-2, the only independent predictor of SVR was lathosterol (OR 2.08, 95% CI 1.03 – 4.18; p=0.041)
• No other factor predicted SVR, including ApoB
YOUNOSSI et al, AASLD 2014
Le génotype 3 du VHC est particulier, mais pourquoi?
JE NE SAIS PAS !!!!!!
The HCV-3a core is sufficient to induce large LD in Huh-7
1b 2a
3a 4a
(courtesy of Sophie Clément)
Huh-7 Cells Expressing the HCV-3a Core Are Enriched in Cholesterol Esters
LOIZIDES-MANGOLD, CLEMENT et al, PLoS One 2014;9:e115309
SOAT: sterol O-acyl-transferase TMP-153: SOAT inhibitor
Lipid Droplets of Huh-7 Expressing the HCV-3a Core Are Enriched in Cholesterol Esters
LOIZIDES-MANGOLD, CLEMENT et al, PLoS One 2014;9:e115309
10 mM lovastatin
TMP-153
HCV-3a core induces CE and increased LD size via a sphingolipid biosynthesis-dependent mechanism
LOIZIDES-MANGOLD, CLEMENT et al, PLoS One 2014;9:e115309
3a + scrambled 3a + siSPT
3a CONT 3a + myriocin
SPT: serine palmitoyl transferase
+ HCV
Ceramide (Cer), glycosylceramide (GlcCer) and sphingomyelin (SM) profiles in Huh-7 expressing HCV 2a or 3a core
The increased level of ceramides, observed in whole cell extracts, is not due to
an enrichment of ceramides within the LD core or LD monolayer but to an overall
increase in ceramides in different membrane compartments
LOIZIDES-MANGOLD, CLEMENT et al, PLoS One 2014;9:e115309
KAPADIA & CHISARI, Proc Natl Acad Sci USA 2005;102:2561-6 WANG et al, Mol Cell 2005;18:425-34; IKEDA et al, Liver Int 2011;31:871-80
LAMBERT et al, Hepatology 2013;57:1697-1704; PEYROU, CLEMENT et al, J Hepatol 2013;59:420-6 LOIZIDES-MANGOLD, CLEMENT et al, PLoS One 2014;9:e115309
geranylgeranyl PP
acetylCoA + acetoacetylCoA
mevalonate
HMGCoA
steroid hormones
vitamin D
bile acids
squalene
mevalonate PP
farnesyl PP
cholesterol
lanosterol
HMG CoA synthase
HMG CoA reductase
statins -
FBL2
CE
+
HCV +
ceramides
+
Second messenger?
Membranes?
Potential role of ceramide in the development and progression of nonalcoholic fatty liver disease and related complications
PAGADALA et al, Trends Endocrinol Metab 2012;23:365-71
Serum sphingolipid levels associate with liver fibrosis progression and poor response to IFNa in chronic hepatitis C
(INDIV2 study cohort, n=203, all HCV-1)
Variable OR (95% CI) p
Age 1.126 (1.065 ± 1.191) <.001
Sex, female vs male 6.444 (2.150 ± 19.312) .001
AST 1.056 (1.029 ± 1.084) <.001
Ferritin 0.996 (0.993 ± 0.999) .015
Sphingosin, ng/mL 1.111 (1.028 ± 1.202) .007
Sphinganine, ng/mL 0.634 (0.435 ± 0.925) .01
GRAMMATIKOS et al, Hepatology 2015;61:812-22
F0-F2 vs
F3-F4
Variable OR (95% CI) p
Alkaline phosphatase 1.020 (1.001 ± 1.039) .03
Ferritin 1.006 (1.003 ± 1.010) <.001
IL28b rs12979860 9.483 (3.139 ± 28.643) <.001
C24Cer, ng/mL 0.998 (0.997 ± 0.999) .001
C18:1Cer, ng/mL 0.973 (0.947 ± 0.999) .048
C24:1Cer, ng/mL 1.001 (1.000 ± 1.039) .059
SVR
Ceramide profile* of Huh-7 expressing the HCV core
*Ceramide concentrations are expressed relative to internal standard and normalized to phosphate, according to fatty acid chain length
LOIZIDES-MANGOLD, CLEMENT et al, PLoS One 2014;9:e115309
Ether phospholipids are increased in Huh-7 expressing HCV-3a core
LOIZIDES-MANGOLD, CLEMENT et al, PLoS One 2014;9:e115309
What are ether lipids (aka plasmalogens)?
• Ether lipids are lipids in which one or more of the carbon atoms on glycerol is bound to an alkyl chain via an ether linkage, as opposed to the usual ester linkage (like in TG)
• They may function as alkyl donors during glycosylphosphatidylinositol (GPI)
anchor biosynthesis (allowing anchoring of cell surface proteins), or as (precursors of) second messenger lipid forms, or as antioxidants
ester
ether bonds
PTEN (Phosphatase and TENsin homolog, phosphatidylinositol-3,4,5-
triphosphate 3-phosphatase, MMAC1)
• Ubiquitously expressed phosphatase
• As lipid phosphatase, it downregulates the PI3K signaling pathway via dephosphorylation of PIP3, the major product of PI3K in response to growth factor stimulation
• As protein phosphatase, it modulates the activity of factors involved in cell adhesion, migration and invasion
• In the nucleus, it is required for chromosome stability
• Potent tumor suppressor, mutated, deleted or downregulated in numerous human cancers (endometrial, prostate, glioma, melanoma, small cell lung cancer and HCC)
Binds to pleckstrin homology (PH) domain (PDK1, Akt/PKB, PKCm, IRS-1, ARF...)
Phosphatidylinositol-3,4,5-triphosphate (PIP3)
PTEN SHIP2
HCV-3a downregulates the tumor suppressor PTEN, thereby increasing the LD size
Core 3a
Core 3a + PTEN
Control
PTEN
CLEMENT et al, Hepatology 2011;54:38-49
IHC PTEN expression in HCV-related HCC is associated with survival
RAHMAN et al, Int J Cancer 2002;100:152–157
Conclusions
• HCV-3a is associated with: – severe steatosis – accelerated fibrosis progression rate – increased risk of HCC development – suboptimal response to DAA therapy
• HCV-3a increases the production of C16 and C24 ceramides, and ether lipids
• HCV-3a downregulates the tumor suppressor PTEN • The role of these alterations in the pathogenesis of
the above features is to be determined
Acknowledgments University Hospitals of Geneva
Laura Rubbia-Brandt Gilles Mentha Emiliano Giostra Laurent Spahr Sophie Clément Vincent Kaddai Gaia Trincucci Claudio de Vito Rafael Quadri Emilie Branche Stéphanie Conzelmann Nicolas Goossens
University of Geneva, Faculty of Sciences Howard Riezman Ursula Loizides-Mangold
INGM, Milan Annalisa Bianco Raffaele De Francesco
University of Geneva, Faculty of Medicine Michelangelo Foti Marion Peyrou Karim Bouzakri Philippe Halban
University of Lausanne / CHUV Pierre-Yves Bochud Zoltan Kutalik
Swiss Hepatitis C Cohort Study Group DITTO-HCV Study Group MAID Study Group HepaCute Consortium VIRGIL Network University of Sydney
Jacob George University of Heidelberg
Ralf Bartenschlager University of California, San Diego
Aleem Siddiqui