Cacoub hcvmehdu12

Extrahepatic Manifestations

of Hepatitis C Virus Infection

Service de Médecine Interne, et CNRS UMR 7087 Université Pierre et Marie Curie

Centre National de Référence Maladies Autoimmunes

Hôpital La Pitié-Salpêtrière, Paris, FRANCE

Pr. Patrice CACOUB

Manifestation Prevalence

certainly associated with HCV %-------------------------------------------------------• Vasculitis (PAN, cryoglobulinemia) 5-40 • Fatigue 35-54• Arthralgia-myalgia 25-35• Sicca syndrome 10-25• Autoantibodies 10-40• Thrombocytopenia 20-40• Lymphoma ?

3

• Hepatitis • Cirrhosis• Hepatocarcinoma

• Cryoglobulinemia• Auto-Ab• B-NHL

HepatocyteChoo. Science 1989

Lymphocyte

Zignego. J Hepatol 1992

Ferri. Blood 1993

Hepatitis C Virus Chronic Infection: Two Main Target Cells

Cryoglobulinémies mixtes

Saadoun, Arch Intern Med, 2006

Infection VHC +++

5

Endothelial cells

Cryoprecipitation

6

Pathogenesis of

cryoglobulinae

mic vasculitis

Roccatello, D. et al. Nephrol. Dial. Transplant. 2004

7

Skin Purpura

Membrano-proliferative Glomerulonephritis CNS Vasculitis

Neuropathy

Cryoglobulinemia-Systemic Vasculitis

10

HCV Mixed Cryoglobulinemia & Digestive Tract

Mesenteric artery stenosis

Intestinal wall thickening

Terrier B et al, GUT 2011

Distal Polyneuropathy 80%

Cacoub P et al, AIDS 2005

Mixed Cryoglobulin and Neuropathy

• Chronic progressive course,

• Distal, symetric, axonal PN, mainly

sensory and painful

• Few extra neurological signs :

purpura

• Severe liver involvement

• Moderate inflammatory syndrome

- important peri-vascular infiltrate of lymphocyte- around small vessels i.e. venules, capillaries- no PMN, no destruction of the vascular wall

Mixed Cryoglobulin and Distal Polyneuropathy

Peripheral Nerve Biopsy

Cryoglobulinemic Membrano-Proliferative Glomerulonephritis

Central Nervous System Involvement in HCV-

Cryoglobulinemia Vasculitis

HCV-vasculitis HCVControls

(n=40) (n=11) (n=36)--------------------------------------------------------------------------------------Gender (F/M) 23/17 6/5 20/16Age (yrs) 59 ± 13 56 ± 10

58 ± 12WMHS 7.0 ± 9.9 0.9 ± 1.8 *2.0 ± 3.1

PVHS 2.5 ± 3.1 0.4 ± 0.5 * 0.8 ±

1.4

NCFD 2.2 ± 1.8 0.9 ± 0.8 * -

--------------------------------------------------------------------------------------* P<0.01WMHS: White Matter Hypersignals PVHS: Periventricular HypersignalsNCFD: Number of Cognitive Function Deficiency

Casato M et al, J Hepatol 2004

17

Age at disease onset 54 ± 13 (29-72) Female/Male ratio 3 Purpura 98% Weakness 98% Arthralgias 91% Arthritis (non-erosive) 8% Raynaud's phenomenon 32% Sicca syndrome 51% Peripheral neuropathy 81% Renal involvement 31% B-cell non-Hodgkin's lymphoma 11% Hepatocellular carcinoma 3%

Ferri C, Mascia MT, Saadoun D, Cacoub P. 2009

Demographic & Clinical Features of 250 Mixed Cryoglobulinemic Patients

18

HCV Core Protein in Skin Vascular Structures

Who’s the culprit ?

Cellular Infiltrate in HCV-Vasculitis

19

Detection of Genomic Viral RNA in Nerve and Muscle of Patients with HCV

Neuropathy

Inflammatory vascular lesions in 26/30 (87%) patients

Positive-strand genomic HCV RNA detected in 10/30 patients (muscle 9, nerve 3)

Negative-strand replicative HCV RNA never detected

--> HCV neuropathy probably results from virus-triggered immune-mediated mechanisms rather than direct nerve infection and in situ replication

Authier JF et al, Neurology, 2003

20

A Major Role for T Cell Immunity in HCV-Vasculitis

Abnormal T lymphocytes distribution

Predominant T lymphocytes infiltration in vasculitis lesions

MHC-II polymorphism (DR11)

Th1 cytokines profile in vasculitis lesions

Deficit in Treg lymphocytes

21

Quantitative Deficit in Treg Lymphocytes (CD4+CD25+) in HCV-Systemic Vasculitis

Boyer O, Saadoun D et al, Blood 2004

23

Before treatmentOn treatmentEarly F/u Late F/U3

4

5

6

CD

25

hig

h (

% o

f C

D4

+)

4 4

5

6

Before

treat.

On Treat.

Early F/U

Late F/U.

**†

**†

-CR

-NR/PR

0

10

20

30

40

CD25h

igh

(ce

lls/μl)

†*

BeforeTreat.

CR NR/PRAfter Treat.

C

After Treat.

A

Complete clinical response of HCV-vasculitis to anti-viral treatment is

associated with an increase in CD4+CD25high levels

24

0 20 40 60 80 1000.0

0.2

0.4

CD25high (cells /μl)C

4 (

g/l

)

R²-0.16, p<0.005

0 20 40 60 80 1000

1

2

3

CD25high (cells /μl)

Cry

og

lob

uli

ns

(g

/l)

R²-0.1, p<0.005

Correlation between Immune Response and Treg Lymphocytes in HCV MC Vasculitis

25

Chronic HCV infection

Poly- oligoclonal

B-cell expansion

AutoantibodiesRF - IC

Mixed cryoglobulins

Cryoglobulinemic vasculitis

Monoclonal B-cellproliferation

Overt lymphoma

HCV eradication

Immunosuppressors

Chemotherapy

Plasma exchange

Steroids

26

HCV Treatment Efficacy in HCV-Vasculitis%

im

pro

vem

en

t

Zuckerman, J Rheumatol 2000. Naarendorp, J Rheumatol 2001. Cacoub, Arthritis Rheum 2002, Zaja F, Blood 2003. Sansonno D, Blood 2003 , Cacoub, Arthritis Rheum 2005, Saadoun, Arthritis Rheum 2007

27

Predictive Factors of Clinical Response to HCV Therapy in Mixed Cryoglobulinemia

VasculitisMultivariate Analysis

Odds ratio [95%CI]

p

• Renal involvement 0.27 [0.08-0.87]

0.02

• Renal insufficiency (GFR<70) 0.18 [0.05-0.67]

0.01

• Daily proteinuria > 1g 0.32 [0.09-1.11]

0.05

• Early virological response 3.53 [1.18-10.59]

0.02

28

Rationale for Rituximab

treatment in cryoglobulinemic

vasculitis

Rocatello D, Nephrol Dial Transplant, 2004Roccatello, D. et al. Nephrol. Dial. Transplant. 2004

29

Treatment of Mixed Cryoglobulinemia Resistant

to Interferon with Rituximab*

Sansonno D et al, Zaja F et al, Blood 2003

31

10

20

30

40

50

60

70

80

90

MONTHS

100

6 12

15 (93.7)

13 (81.2)

12 (75)

1 2 3 4 5 7 8 9 1011 24 36 48

10 (62.5)

6 (37.5)

Cryoglobulinemia Vasculitis: Response Maintenance after Discontinuation of

Rituximab

Sansonno D et al, 2007

32

HCV Vasculitis: a Two-Faces Disease

…Needs a Two Faces Treatment Strategy

Rituximab

PegIFN plus Ribavirin

34

RITUXIMAB (375 mg/m²)

Time (months)0 1

RIBAVIRIN (600-1200 mg/d)

PEGYLATED INTERFERON 2b (1.5 μg/Kg/wk)

12

Rituximab plus Peg-IFNα2b-Ribavirin in Refractory HCV-Related Systemic

Vasculitis

2

Saadoun D et al, Ann Rheum Dis 2008

38

Outcome of HCV-MC pts according to treatment

Parameters All PegIFN-ribavirin RTX-PegIFN-

ribavirinn=93 n=55 n=38 P

Time clinical response, months

6.8 ± 4.7

8.4 ± 4.75.4 ± 4.0

0.004

Clinical response

CR68

(73.1) 40 (72.7) 28 (73.7) 0.98PR 22 (23.6) 13 (23.6) 9 (23.7)NR 3 (3.2) 2 (3.6) 1 (2.6)Relapse 17 (18.3) 10 (18.1) 7 (18.4)

Immunological response

CR49

(52.7) 24 (43.6) 26 (68.4) 0.001PR 35 (37.6) 25 (45.4) 10 (26.3)NR 8 (8.6) 6 (10.9) 2 (5.2)Relapse 17 (18.3) 10 (18.1) 7 (18.4)

Virological response

SVR55

(59.1) 33 (60) 22 (57.9) 0.94Death 5 (5.4) 2 (3.6) 3 (7.9) 0.70

39

Course of kidney parameters in HCV-MC patients according to the type of treatment

PegIFN-ribavirin RTX-PegIFN-

ribavirinn=10 p n=21 p

CR kidney involvement 4 (40) 17 (80.9) 0.04Creatininemia (µmol/l)Baseline 150 ± 30 217 ± 47EOF 169 ± 44 0.28 136 ± 27 0.03GFR (ml/min)Baseline 58 ± 7 42 ± 5EOF 59 ± 9 0.41 57 ± 4 0.01Daily Proteinuria (gr/d)Baseline 3.1 ± 0.9 3 ± 1EOF 1.2 ± 0.5 0.046 0.4 ± 0.1 <0.001Hematuria (n,%)Baseline 10 (100) 19 (90.5)EOF 2 (20) 2 (10.5) <0.001

40

Antiviral therapy alone decreases the

memory B cells

n=38 n=55

Saadoun D et al, Blood 2010

41

Antiviral therapy alone decreases the memory B

cells

Antiviral therapy plus Rituximab

decrease naive B-cells

Saadoun D et al, Blood 2010

44

Time Course of HCV Viral Load

Terrier B et al. Arthritis Rheum 2009

• If failure or CI of PegINF/riba: RTX alone• Place to be defined for PegIFN/Riba/Previr

48

Years

Overa

ll su

rviv

all

Overall Survival of 151 HCV-Vasculitis Patients


49

Years

Overa

ll su

rviv

all

Overall Survival of 151 HCV-Vasculitis Patients


32 deaths after a median follow-up of 54

months (IQR 26-89)

Causes of death:- Infection (n=10)

- Cirrhosis (n=10; 4 HCC)- Non-HCC neoplasia (n=4)

- Cardiovascular (n=4)- Renal failure (n=2)

- Vasculitis (n=2)- Unknown (n=2)

50

Baseline Prognostic Factors of HCV-Vasculitis Patients

51

• Metavir fibrosis score:HR = 10.8 (3.63-32.14),

P<0.0001

• Five Factor Score:HR = 2.49 (1.29-4.8),

P=0.007

Liver Fibrosis and Five Factor Scores are Associated with a Poor Prognosis in HCV

vasculitis Patients Multivariate Analysis

52

Multivariate analysis

- Metavir fibrosis score:HR 10.8 (3.63-32.14), P<0.0001-FFS:HR 2.49 (1.29-4.8), P=0.007

Metavir Fibrosis

FFS F0-F2 F3-F4

0 1.0

1 2.49

> 1 6.2

FFS is a good predictorof outcome

Interaction Between Liver Fibrosis and Five Factor Score in HCV-Vasculitis

Patients

53

Multivariate analysis

- Metavir fibrosis score:HR 10.8 (3.63-32.14), P<0.0001-FFS:HR 2.49 (1.29-4.8), P=0.007

Metavir Fibrosis

FFS F0-F2 F3-F4

0 1.0 10,8

1 2.49 10,25

> 1 6.2 9,74

FFS is a good predictorof outcome

Interaction Between Liver Fibrosis and Five Factor Score in HCV-Vasculitis

Patients

No more prognostic

value of FFS

54

Use of Peg-IFN/riba had a positive prognostic impact

HR = 0.34 (0.16-0.67)

Prognostic Factors

During follow-up

After adjustment on vasculitis severity

• Negative impact of immunosuppressantsHR = 4.05 (1.75-9.36), P=0.001

•… but not of corticosteroidsHR = 1.79 (0.77-4.16), P=0.17

55

Reversible Quantitative Deficit in Treg Lymphocytes (CD4+CD25+) in HCV-Systemic

Vasculitis

Before treatmentOn treatmentEarly F/u Late F/U3

4

5

6

CD

25

hig

h (

% o

f C

D4

+)

4 4

5

6

Before

treat.

On Treat.

Early F/U

Late F/U.

**†

**†

-CR

-NR/PR

After Treat.

A

0 20 40 60 80 1000

1

2

3

CD25high (cells /μl)

Cry

og

lob

uli

ns

(g

/l)

R²-0.1, p<0.005

0 20 40 60 80 1000.0

0.2

0.4

CD25high (cells/μl)

C4

(g/l )

R²-0.16, p<0.005

57

Effects of Low-Dose Interleukin-2 on Levels of CD4-Treg (c) and CD8-Treg (sq) in Patients with HCV-Vasculitis, According to

Treatment Course.

58

Effects of Low-Dose Interleukin-2 on Levels on the Ratio of Treg Cells to the sum of Effector T Cells CD4 + CD8 in Patients with

HCV-Vasculitis, According to Treatment Course.

60

Effects of Low-Dose Interleukin-2 on Levels on CD19+ total B Cells (c) and Marginal-Zone B Cells (sq) in Patients with HCV-Vasculitis,

According to Treatment Course.

Baseline C1 C2 C3 C4 Post IL-2 Baseline C1 C2 C3 C4 Post IL-2 Baseline C1 C2 C3 C4 Post IL-2 Baseline C1 C2 C3 C4 Post IL-2

Baseline C1 C2 C3 C4 Post IL-20

10

20

30


10

20

30


10

20

30


10

20

30

PurpuraNeuropathy

ArthralgiaFatigueKidney Involvement

CD

4+Tr

eg

(%

)C

LIN

ICA

LR

ES

PO

NS

ETemporal Effects of Low-Dose Interleukin-2 on Clinical

Features, Levels of Regulatory T Cells, and Cryoglobulin for Each Study Patient

62

BEFORE IL-2 AFTER IL-2CCL3CCL3L1CCL3L3

IL1ACCL20

IL6CLECL1CD79A

BLKCCL4L2

EBF1CCL4L1CXCR5

IER3

CXCR7OLR1PDE48PTGS2IL1B

BAFFR

4-1BBL

PLAURNLRP3RIPK2ATF3

NAMPT-PBEF1

TNFRSF21-DR6ETS2

MAPK3K8-COT

GOS2

CD83

Up Down Khi2 test

Inflammation 0 251 1,30E-40

Immune Response 16 684 3,40E-94

Lymphocyte 77 555 7,00E-49

Cell Cycle 1701 208 1,50E-138

Control 226 343 2,50E-01

Autoimmune & transplantation pathologies

0 46 7,60E-09

Inflammatory infectious diseases 6 242 7,60E-36

Other diseases 190 211 4,15E-02

Saadoun D et al. NEJM 2011

Anti-inflammatory Effects of Low-Dose Interleukin-2 Revealed through Unsupervised Transcriptome Analyses of PBMCs.

63

Regulatory T Cell Recovery in HCV-Vasculitis through Low-Dose IL-2

Treatment

We provide the first evidence of Treg recovery through low-dose IL-2 therapy in a human autoimmune disease.

Low-dose IL-2 dramatically increases CD4+CD25highCD127– Foxp3+ Treg cells that are functional

Treg expansion persists after IL-2 therapy.

IL-2 therapy was well tolerated with no flare of vasculitis.

Saadoun D et al. NEJM 2011

64

The Yin and Yang of IL-2-Mediated Immunotherapy

Balance of Pathogenic Effector T Cells and Regulatory T Cells

Bluestone JA, NEJM 2011

Hepatitis C virus : extrahepatic manifestations, an update 2007Hepatitis C virus : extrahepatic manifestations, an update 2007

% of patients

n = 1614

% of controls

n = 412

Fatigue without depression

Fatigue with depression

Depression without fatigue

No fatigue and no depression

Total

48

5

2

45

100

0.7

0

0

99.3

100

Fatigue without EM

Fatigue with EM

EM without fatigue

No fatigue and no EM

Total

19

35

21

25

100

0.5

0.2

3.4

96

100

Association between fatigue, depression and clinical extrahepatic manifestations (EM)

Poynard T et al. J Viral Hep, 2002

Hepatitis C virus : extrahepatic manifestations, an update 2007Hepatitis C virus : extrahepatic manifestations, an update 2007Multivariate analysisMultivariate analysis

Fatigue (moderate or severe) in comparison to absence of fatigue was associated with:

• female gender,

• age > 50 years,

• cirrhosis or many septa,

• purpura. Independently of these associations, fatigue

(moderate-severe) was associated with : arthralgia, myalgia, paresthesia, sicca sd & pruritus.

Poynard T et al. J Viral Hep, 2002Poynard T et al. J Viral Hep, 2002

Hepatitis C virus : extrahepatic manifestations, an update 2007Hepatitis C virus : extrahepatic manifestations, an update 2007Prevalence of fatigue at baseline and at 18 months follow-up in treated

and untreated patients

Baseline 18 months 18 months vsbaseline

Non treated (n=72) No fatigue Moderate Severe

39 %35 %26 %

42 %39 %19 %

P = 0.74

Sustained responders(n=82) No fatigue Moderate Severe

41 %37 %22 %

69 %24 %7 %

P < 0.001

Relapsers (n= 47) No fatigue Moderate Severe

45 %43 %13 %

40 %45 %15 %

P = 0.68

Non responders (n= 224) No fatigue Moderate Severe

40 %42 %18 %

46 %40 %14 %

P = 0.18

Poynard T et al. J Viral Hep, 2002


certainly associated with HCV %-------------------------------------------------------• Vasculitis (PAN, cryoglobulinemia) 5-40 • Fatigue 35-54• Arthralgia-myalgia 25-

35• Sicca syndrome 10-25• Autoantibodies 10-40• Thrombocytopenia 20-40• Lymphoma ?


0%5%

10%

15%20%25%30%

35%40%

Sustained responders (n = 83)

Impact of Treatment on Extra hepatic Manifestations in HCVpatients.

At Baseline and 18 months Follow-up in Responders.

Cacoub P et al. J Hepatol 2002


0%5%

10%15%20%25%30%35%40%

Sustained responders (n = 83) Non responders - RNA + (n = 348)

Cacoub P et al. J Hepatol 2002

Impact of Treatment on Extra hepatic Manifestations in HCVpatients.

At Baseline and 18 months Follow-up in Responders.

Auto-antibody production in chronic HCV infection.

0

10

20

30

40

50

60

70

%

A-nuclearA-phospholipidA-thyroglobulinA-smooth muscle≥ one auto-Ab≥ three auto-Ab

Pawlotsky JM, Hepatology 1994. Pawlotsky JM, Ann Intern Med 1994.Prieto J, Hepatology 1996. Cacoub P, J Rheumatol 1997. Cacoub P, Medicine 2000.

Extrahepatic manifestations associated with HCV infection.(Prospective study in 321 HCV patients)

Autoantibody Number %

----------------------------------------------------- Antinuclear 124 41

• A-nucleosome 6 2

• A-DNA 8 3

• A-histone 9 3

• A-ENA 10 3

Cacoub P et al. Medicine 2000; 79: 47-56


B-cell-Non Hodgin’s LymphomaB-cell-Non Hodgin’s Lymphoma

Hepatitis C virusHepatitis C virus

2462 tested2462 tested

13.5 % positive • vs 0-5 % in controlsvs 0-5 % in controls

• vs 5 % in other malignant vs 5 % in other malignant hemopathyhemopathy

469 tested469 tested

0 - 39 %

Hepatitis C virus : extrahepatic manifestations, an update 2007Hepatitis C virus : extrahepatic manifestations, an update 2007Effects of alpha-interferon on HCV+/SLVL course

After 6 months of IFN alpha treatment in SLVL/HCV+: Complete clinical hematologic response (spleen size < 12

cm, lymphocytosis <4500/mm3, No cytopenia ):

---> 7/9 HCV RNA negative Partial clinical hematologic response

(spleen size or lymphocytosis decrease >50%) :

---> 2/9 HCV RNA +

Hermine O. et al, N Engl J Med 2002; 347: 89-94

HCV antibodies : B-NHL (< 3%) vs SLVL (15%)HCV antibodies : B-NHL (< 3%) vs SLVL (15%)

----> Splenic lymphoma with villous lymphocytes may be associated with HCV infection


Median Follow-up of 3 years (2-5)

6 Complete Responses ---> HCV RNA still negative6 Complete Responses ---> HCV RNA still negative

1 relapse off therapy at 1 year,1 relapse off therapy at 1 year,

• associated with positivity of HCV RNA. associated with positivity of HCV RNA.

• second CR following IFN & negativity HCV RNAsecond CR following IFN & negativity HCV RNA

2 Partial Responses 2 Partial Responses

• CR after Combination of Interferon and Ribavirin CR after Combination of Interferon and Ribavirin

• PR after Interferon and Ribavirin PR after Interferon and Ribavirin


Effects of alpha-interferon on HCV+/SLVL course

Hepatitis C virus : extrahepatic manifestations, an update 2007Hepatitis C virus : extrahepatic manifestations, an update 2007HCV negative / SLVL Patients Treated with Alpha-Interferon

Median age 65 (54-72)Median age 65 (54-72)

Prior therapy (2/6), chemotherapy (1), splenectomy(1)Prior therapy (2/6), chemotherapy (1), splenectomy(1)

Splenomegaly (4/6)Splenomegaly (4/6)

Hyperlymphocytosis Median 25,000 (500-100.000)Hyperlymphocytosis Median 25,000 (500-100.000)

Cytopenia (2/6)Cytopenia (2/6)

Cryoglobulinemia or rheumatoid factor (0/6)Cryoglobulinemia or rheumatoid factor (0/6)

Alpha-Interferon 3 M IU x 3/W during 6 monthsNo response



Conclusion

Extrahepatic manifestations of HCV

infection are frequent, and may be cured

by HCV treatment :

• Systemic vasculitis (cryoglobulinemia,

PAN)

• Fatigue

• Arthralgia - myalgia - arthritis (±)

• Auto-antibodies (?)

• Splenic lymphoma with villous

lymphocytes

• Thrombocytopenia

82

S. Caillat-Zucman, Paris

P. Ghillani, Paris D. Klatzmann, Paris L. Musset, Paris M. Rosenzwajg, Paris

D. Saadoun, Paris D. Sene, Paris B. Terrier, Paris G. Géri, Paris P. Hausfater, Paris O. Lidove, Paris A. Gatel, St Brieuc J-M. Léger, Paris N. Limal, Paris T. Maisonobe, Paris JC Piette, Paris

Thanks

L. Alric, Toulouse M. Bourlière, Marseille P. Halfon, Marseille S. Pol, Paris T. Poynard, Paris V. Thibault, Paris Les membres du

GERMIVIC

L. Calabrese, Cleveland

M. Casato, Roma C. Ferri, Modena G. Kerr, Washington E. Sasso, Seattle JA. Schifferli, Basel V. Soriano, Madrid

Cacoub hcvmehdu12

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