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