1. Extrahepatic Manifestations of
Hepatitis C Virus Infection
Pr. Patrice CACOUB, MD, PhD
Service de Médecine Interne, et CNRS UMR 7087
Université Pierre et Marie Curie
Hôpital La Pitié-Salpêtrière, Paris, FRANCE

2. Manifestation Prevalences
certainly associated with HCV %
--------------------------------------------------
• Vasculitis (PAN, cryoglobulinemia) 4-40
• Fatigue 35-54
• Arthralgia-myalgia 25-35
• Sicca syndrome 10-25
• Autoantibodies 10-40
• Thrombocytopenia 20-40
• Lymphoma (SLVL) ?

3. Cryoprecipitation
Endothelial cells

4. Pathogenesis of
cryoglobulinaemic
nephritis
Roccatello, D. et al. Nephrol. Dial.
Transplant. 2004

5. Distal Polyneuropathy 80%
Peripheral Nerve Biopsy
- important peri-vascular infiltrate of lymphocyte
- around small vessels i.e. venules, capillaries
- no PMN, no destruction of the vascular wall

6. Skin Purpura Neuropathy
Cryoglobulinemia-Systemic Vasculitis
Membrano-proliferative CNS Vasculitis
Glomerulonephritis

7. Prevalence of HCV infection in patients with
essential cryoglobulinemia
100
90
80
70
60
50
40
30
20
10
0
Ferri Disdier Casato Pechere Misiani Agnello Cacoub Dupin Monti

8. Hepatitis C Virus Chronic Infection :
two main target cells
Hepatocyte Lymphocyte
Choo. Science 1989 Zignego. J Hepatol 1992
Ferri. Blood 1993
• Hepatitis • Cryoglobulinemia
• Cirrhosis • B-NHL
• Hepatocarcinoma

9. FREQUENCY OF HBV-RELATED PAN: 1972-1999
60%
53%
50% 48%
40% 41%
30% 28%
20%
10% 12%
0%
1972-79 1980-84 1985-89 1990-94 1995-99
Guillevin L

10. Clinical features of 231 MC Patients
end beginning
follow-up follow-up p°
Purpura 89% 81% .05
Weakness 91% 80% .001
Arthralgias 90% 72% .001
Arthritis 6% 8% ns
Raynaud's phen. 44% 36% ns
Sicca syndrome 48% 29% .001
Skin ulcers 20% 11% .02
Periph. neuropathy 73% 58% .001
Liver involvement 70% 58% .02
Renal involvement 27% 20% ns
B-cell lymphoma 9% 0.4% .001
Hepat. carcinoma 3% 0% .05
Ferri C, Sem Arthr Rheum 2004

11. MC and Skin

12. Cutaneous Manifestations of HCV

13. HCV Core Protein in Skin Vascular Structures

14. Systemic Vasculitis and Hepatitis C virus
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
insufficiency
polyneurop.
Severe HTA
Livedo-ulcers-
Purpura
eight loss
ultifoc.
Sensory
Neurop.
Ery nod.
Renal
SM m
W
Cacoub P et al. Arthritis Rheum 2002

15. MC and Neuropathy
Distal Polyneuropathy 80%
• First symptoms : 61 years
• Chronic course, progressive
• Distal, symetric, axonal
polyneuropathy, mainly sensory and
painful
• Few extra neurological signs :
purpura, Raynaud, kidney ...
• Severe liver involvement
• Moderate inflammatory syndrome
Cacoub P et al, AIDS 2005

16. MC and Neuropathy
Mononeuropathy
Multiplex 20%
Cacoub P et al, AIDS 2005

17. Mononeuropathy multiplex (20%)
• Younger age at first symptoms (< 60 years)
• Acute or subacute involvement
• Severe, sensory-motor, mononeuritis multiplex
• Weight loss, inflammatory syndrome
• Extra-neurological manifestations
• Moderate liver involvement

18. Pathological data in HCV related vasculitis
Cacoub P et al. Arthritis Rheum 2002
N %
Membranoproliferative GN 5 -
Leucocytoclastic vasculitis 6 -
PAN-type vasculitis 7/23 30 %
Mixed cryo-type vasculitis 14/23 61 %
Both 2/23 9%
Knodell score 6.5 (1-12) -
Cirrhosis 3/26 12 %

19. Central Nervous System Involvement
in HCV-Infected Patients
Stroke (ischemic or haemorraghic)
- usually associated with numerous extra-
neurologic manifestations, i.e. renal, PNS,
skin, digestive tract
- 4 cases with isolated CNS involvement
Encephalopathy with coma or convulsions
- multiple ischemic strokes,
- in two cases, brain biopsy showed small vessel
vasculitis
-> Possible improvement under steroids,
immunosuppressive and anti-viral treatment.

20. Central Nervous System Involvement in
HCV-Cryoglobulinemia Vasculitis
HCV-
HCV-vasculitis HCV Controls
(n=40) (n=11) (n=36)
---------------------------------------------------------
-----------------------------
Sex ratio F/M 23/17 6/5 20/16
Age (yrs) 59 ± 13 56 ± 10 58 ± 12
WMHS 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
* P<0.01 2.2 ± 1.8 0.9 ± 0.8 * -
---------------------------------------------------------
-----------------------------
WMHS: White Matter Hypersignals
Casato M et al, J Hepatol 2004
PVHS: Periventricular Hypersignals

21. HCV and membranoproliferative
glomerulonephritis
• Proteinuria (g/d) 3.1 ± 2.2
• Albumin (g/L) 29 ± 5
• Creatinine (µmol/L) 118 ± 41
• Cryoglobulin (II/III) 16 / 2
• Cryoglobulin level (g/L) 1.4 ± 1.8
• ALT (IU x N/ml) 1.5 ± 1
• Genotype 1/ 2/ 3/ 4 11/ 3/ 2/ 2
• Treatment of nephrotic sd
plasmapheresis 132 (66%)
steroids 8 (44%)
furosemide 18 (100%)
ACE 12 (66%)
Alric L. Am J K Dis, 2004

22. Therapeutic strategy in HCV+ Mixed Cryoglob.
Chronic HCV infection HCV eradication
Poly- oligoclonal Immunosuppressors
B-cell expansion
Autoantibodies Monoclonal B-cell
RF - IC proliferation Chemotherapy
Mixed cryoglobulins Overt lymphoma
Plasma exchange
Cryoglobulinemic vasculitis Steroids

23. Treatment Efficacy in HCV-Related Systemic Vasculitis
100
90
80
% improvement
70
60
IFN + RBV
50
40
30
20
10
0
Skin Renal Nerve
Zuckerman, J Rheumatol 2000. Naarendorp, J Rheumatol 2001. Cacoub, Arthritis Rheum 2002,

24. Treatment Efficacy in HCV-Related Systemic Vasculitis
100
90
% improvement
80
70
60
50 IFN + RBV
40 PegIFN + RBV
30
20
10
0
Skin Renal Nerve
Zuckerman, J Rheumatol 2000. Naarendorp, J Rheumatol 2001. Cacoub, Arthritis Rheum 2002, Zaja F, Blood 2003.
Sansonno D, Blood 2003 , Cacoub, Arthritis Rheum 2005

25. All MC patients α2b-Riba
α2
IFNα2 α2b-Riba
α2
PegIFNα2
Parameter (n=72) (n=32) (n=40)
Treatment
Duration of anti-HCV therapy (months) 16.63 ± 7.8 18.35 ± 10.0 13.25 ± 4.4
Ribavirin dosage (mg/day) 915.9 ± 182.8 875.9 ± 195.7 945 ± 169.3
Previous antiviral therapy (n,%) 20 (27.8) 7 (21.9) 13 (32.5)
Corticosteroids use (n,%) 29 (40.3) 15 (46.9) 14 (35)
Plasmapheresis (n,%) 9 (12.5) 8 (25) 1 (2.5)
Immunosuppressors (n,%) 4 (5.6) 4 (12.5) 0 (0)
All adverse events (n,%) 39 (54.2) 17 (53.1) 22 (55)
Outcome
Lower use of associated treatments in patients
Deaths (n,%) 8 (11.1) 6 (18.8) 2 (5)
Clinical CRreceived a combination of PegIFN + Ribavirin
who † (n,%) 40 (55.5) 12 (37.5) 28 (70)*
Virological CR † (n,%) 49 (68.0) 19 (59.3) 30 (75)
Immunological CR † (n,%) 33 (45.8) 9 (28.1) 24 (60)*

26. Predictive Factors of Clinical Response to HCV
Therapy in Mixed Cryoglobulinemia Vasculitis
Multivariate 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
• Renal insufficiency (GFR<70) 0.19 [0.04-0.69] 0.01
• Daily proteinuria > 1g 0.32 [0.09-1.11] 0.05
Early virological resp. (M3) 3.53 [1.18-10.59] 0.02
• Early virological response (M3) 2.86 [0.97-8.78] 0.05

27. Is there a place for other treatments in HCV-
systemic vasculitis ?
• Steroids
– at the initial phase, multivisceral lifethreatening
disease, i.e. kidney, CNS, digestive tract involvement.
– in combination with anti-HCV treatments.
– prednisone 0.5-1 mg/kg/d, rapidly tapered to 10 mg/d
• Immunosuppressive
– cyclophosphamide: if no response with CT + IFN +
ribavirin
– azathioprine, methotrexate: cautious with liver disease
• Plasmapheresis
– if multivisceral involvement, particularly kidney.
– if no response with CT + IFN + ribavirin

28. Pathogenesis of
cryoglobulinaemic
nephritis
and
rationale for
Rituximab treatment
Roccatello, D. et al. Nephrol. Dial.
Transplant. 2004

29. Treatment of Mixed Cryoglobulinemia Resistant to
Interferon-alfa with an Anti-CD 20 Monoclonal
Antibody (Rituximab*)
Sansonno D et al, Zaja F et al, Blood 2003

30. HCV-Vasculitis Treatment : PegIFN-Ribavirin vs. Rituximab
90
80
% improvement
70
60
50
40
30
20
10
0
ia e s
ura alg er v ney ryo pse
P urp rth
r N Kid C
Re
la
A
PegIFN-RBV (n=40) Rituximab (n=43)

31. Cryoglobulinemia Vasculitis : Response
Maintenance after Discontinuation of Rituximab
100 15 (93.7) RESPONSE MAINTENANCE (%)
90
13 (81.2)
80
12 (75)
70
60
10 (62.5)
50
40
6 (37.5)
30
20
10
1 2 3 4 5 6 7 8 9 10 11 12 24 36 48
MONTHS
Sansonno D et al, 2007

32. Rituximab plus Peg-IFNα2b-Ribavirin in Refractory
Peg-IFNα2b-
HCV-
HCV-Related Systemic Vasculitis
PEGYLATED INTERFERON α2b (1.5 μg/Kg/wk)
RIBAVIRIN (600-1200 mg/d)
RITUXIMAB (375 mg/m²)
0 1 2 12
Time (months)
Cacoub P, 2007

33. Figure 1
Response rate of HCV-cryoglobulinemia vasculitis
during Rituximab & Peg-IFNα2b + Ribavirin.
Rituximab Peg-Interferon-ribavirin
70
% of complete responders
62.5
50 50
37.5
30
20
18.7
10
1 2 3 4 5 6 7 8 9 10 11 12 Months

34. Immunologic parameters in HCV-MC patients during
treatment with Rituximab & Peg-IFNα2b-ribavirin.
Figure 4
A B
Cryoglobulin C4
g/l g/l
2 0,18
1,6 0,15
0,12
1,2
0,09
0,8
0,06
0,4
0,03
0
0
0 3 6 9 12 EOF
0 3 6 9 12 EOF
Months Months
C D
g/l
IgM RF
IU/l
2,8 240
2,4 200
2
160
1,6
120
1,2
80
0,8
0,4 40
0 0
0 3 6 9 12 EOF 0 3 6 9 12 EOF
Months Months

35. HCV RNA viral load during treatment with Rituximab &
Peg-IFNα2b + Ribavirin in HCV-cryoglobulinemia vasculitis.
7
6
Log copies/ml
5
4
3
2
1
0 Months
0 3 6 9 12 EOF
Rituximab Peg-Interferon-ribavirin

36. HCV-
Outcome of 93 HCV-MC patients according to the
type of treatment
Parameters All α
PegIFNα-ribavirin RTX-PegIFNα-α
ribavirin
n=93 n=55 n=38 p
Time of clinical response (months) 6.8 ± 4.7 8.4 ± 4.7 5.4 ± 4.0 0.004
Clinical response
CR 68 (73.1) 40 (72.7) 28 (73.7) 0.98
PR 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
CR 49 (52.7) 24 (43.6) 26 (68.4) 0.001
PR 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
SVR 55 (59.1) 33 (60) 22 (57.9) 0.94
NR 38 (40.8) 22 (40) 16 (42.1)
Death 5 (5.4) 2 (3.6) 3 (7.9) 0.70
Cirrhosis 1 (1.1) _ 1 (2.6)
Liver carcinoma 3 (3.2) 2 (3.6) 1 (2.6)
Unknown 1 (1.1) _ 1 (2.6)

37. HCV-
Course of kidney parameters in HCV-MC patients
according to the type of treatment
α
PegIFNα-ribavirin RTX-PegIFNα-α
ribavirin
n=10 p n=21 p
- CR of kidney involv. 4 (40) 17 (80.9) 0.04
- Creatininemia (µmol/l)
Baseline 150.0 ± 30.6 217.5 ± 47.4
EOF 169.2 ± 44.2 0.28 136.9 ± 27.1 0.03
- GFR (ml/min)
Baseline 58.0 ± 7.4 42.8 ± 5.8
EOF 59.5 ± 9.9 0.41 57.6 ± 4.5 0.01
- Daily Proteinuria (g)
Baseline 3.1 ± 0.9 3.5 ± 0.9
EOF 1.2 ± 0.5 0.046 0.35 ± 0.1 <0.001
- Hematuria (n,%)
Baseline 10 (100) 19 (90.5)
EOF 2 (20) 2 (10.5) <0.001

38. HCV-
Course of B lymphocytes in HCV-MC patients according
to the type of treatment
n=38 n=55

39. Antiviral therapy alone decreases the memory B cells
HCV-
in HCV-MC patients
n=38
n=55

40. Antiviral therapy in association with Rituximab
B- HCV-
decreases naive B-cells in HCV-MC patients :
n=38 n=55

41. Time Course of HCV Viral Load

44. Therapeutic Strategies in
HCV-related Cryoglobulinemic Vasculitis
Mild to Moderate Severe disease Life threatening
disease (Progressive renal disease, (Rapidly progressive nephritis,
(Purpura, arthralgia, mononeuritis multiplex, skin CNS, digestive and/or pulmonary
polyneuropathy)
ulcer) involvement)
Peg IFN-α + Ribavirin Rituximab Steroids, plasma exchange,
Peg IFN-α + Ribavirin cyclophosphamide and/or
rituximab.
Peg IFN-α + Ribavirin
(differed)

45. Hepatitis C virus : extrahepatic manifestations, an update 2007
Manifestation Prevalences
certainly associated with HCV %
---------------------------------------------------------------
Vasculitis (PAN, cryoglobulinemia) 4-40
Fatigue 35-54
Arthralgia-myalgia-arthritis 25-35
Sicca syndrome 10-25
Autoantibodies 10-40
Thrombocytopenia 20-40
Lymphoma (SLVL) -

46. Association between fatigue, extrahepatic manifestations, an update 2007
Hepatitis C virus : depression and clinical
extrahepatic manifestations (EM)
% of patients % of controls
n = 1614 n = 412
Fatigue without depression 48 0.7
Fatigue with depression
5 0
Depression without fatigue
2 0
No fatigue and no depression
45 99.3
Total
100 100
Fatigue without EM 19 0.5
Fatigue with EM 35 0.2
EM without fatigue 21 3.4
No fatigue and no EM 25 96
Total 100 100
Poynard T et al. J Viral Hep, 2002

47. Multivariate analysis Hepatitis C virus : extrahepatic manifestations, an update 2007
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, 2002

48. Prevalence of fatigue at Hepatitis C virus :at 18 months follow-up in treated
baseline and extrahepatic manifestations, an update 2007
and untreated patients
Baseline 18 months 18 months vs
baseline
Non treated (n=72)
No fatigue 39 % 42 % P = 0.74
Moderate 35 % 39 %
Severe 26 % 19 %
Sustained responders
(n=82) P < 0.001
No fatigue 41 % 69 %
Moderate 37 % 24 %
Severe 22 % 7%
Relapsers (n= 47)
No fatigue 45 % 40 % P = 0.68
Moderate 43 % 45 %
Severe 13 % 15 %
Non responders (n= 224)
No fatigue 40 % 46 % P = 0.18
Moderate 42 % 40 %
Severe 18 % 14 %
Poynard T et al. J Viral Hep, 2002

49. Hepatitis C virus : extrahepatic manifestations, an update 2007
Manifestation Prevalences
certainly associated with HCV %
---------------------------------------------------------------
Vasculitis (PAN, cryoglobulinemia) 4-40
Fatigue 35-54
Arthralgia-myalgia-arthritis 25-35
Sicca syndrome 10-25
Autoantibodies 10-40
Thrombocytopenia 20-40
Lymphoma (SLVL) -

50. Impact of Treatment on Extra hepatic Manifestations in
HCVpatients.
Hepatitis C virus : extrahepatic manifestations, an update 2007
At Baseline and 18 months Follow-up in Responders.
40%
35%
30%
25%
20%
15%
10%
5%
0%
0
0
0
0
18
18
18
18
M
M
M
M
M
M
M
M
ia
a
ia
sd
si
lg
lg
he
a
ya
ra
cc
st
th
M
Si
re
Ar
Pa
Sustained responders (n = 83)
Cacoub P et al. J Hepatol
2002

51. Impact of Treatment on Extra hepatic Manifestations in
HCVpatients.
Hepatitis C virus : extrahepatic manifestations, an update 2007
At Baseline and 18 months Follow-up in Responders.
40%
35%
30%
25%
20%
15%
10%
5%
0%
18
18
18
18
0
0
0
0
M
M
M
M
M
M
M
M
ia
sd
a
ia
lgi
lg
es
a
ya
ra
cc
sth
th
M
Si
re
Ar
Pa
Sustained responders (n = 83) Non responders - RNA + (n = 348)
Cacoub P et al. J Hepatol
2002

52. Hepatitis C virus : extrahepatic manifestations, an update 2007
Manifestation Prevalences
certainly associated with HCV %
---------------------------------------------------------------
Vasculitis (PAN, cryoglobulinemia) 4-40
Fatigue 35-54
Arthralgia-myalgia-arthritis 25-35
Sicca syndrome 10-25
Autoantibodies 10-40
Thrombocytopenia 20-40
Lymphoma (SLVL) -

53. Auto-antibody production in chronic HCV infection.
70
60
50 A-nuclear
A-phospholipid
40
A-thyroglobulin
30 A-smooth muscle
20 ≥ one auto-Ab
≥ three auto-Ab
10
0
%
Pawlotsky JM, Hepatology 1994. Pawlotsky JM, Ann Intern Med 1994.
Prieto J, Hepatology 1996. Cacoub P, J Rheumatol 1997. Cacoub P, Medicine 2000.

54. Auto-antibody production in chronic HCV infection.
Most patients were negative for all other autoAbs :
• neutrophil cytoplasmic, β2 GP1
• Langherans islet, insulin, GAD
• liver-kidney microsome, mitochondria
There was no correlation between :
• Clinical or immunological abnormalities
• α-IFN & clinical/immunological abnormalities

55. 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

56. Hepatitis C virus : extrahepatic manifestations, an update 2007
Manifestation Prevalences
certainly associated with HCV %
---------------------------------------------------------------
Vasculitis (PAN, cryoglobulinemia) 4-40
Fatigue 35-54
Arthralgia-myalgia-arthritis 25-35
Sicca syndrome 10-25
Autoantibodies 10-40
Thrombocytopenia 20-40
Lymphoma (SLVL) -

57. Hepatitis C Virus Chronic Infection :
two main target cells
Hepatocyte Lymphocyte
Choo. Science 1989 Zignego. J Hepatol 1992
Ferri. Blood 1993
• Hepatitis • Cryoglobulinemia
• Cirrhosis • B-NHL
• Hepatocarcinoma

59. Hepatitis C virus : extrahepatic manifestations, an update 2007
B-cell-Non Hodgin’s Lymphoma
2462 tested
13.5 % positive 469 tested
• vs 0-5 % in controls
• vs 5 % in other malignant
0 - 39 %
hemopathy
Hepatitis C virus

60. Effects of alpha-interferon on HCV+/SLVL course update 2007
Hepatitis C virus : extrahepatic manifestations, an
HCV antibodies : B-NHL (< 3%) vs SLVL (15%)
----> Splenic lymphoma with villous lymphocytes may be
associated with HCV infection
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

61. Effects of alpha-interferon on HCV+/SLVL course update 2007
Hepatitis C virus : extrahepatic manifestations, an
Median Follow-up of 3 years (2-5)
6 Complete Responses ---> HCV RNA still negative
1 relapse off therapy at 1 year,
• associated with positivity of HCV RNA.
• second CR following IFN & negativity HCV RNA
2 Partial Responses
• CR after Combination of Interferon and Ribavirin
• PR after Interferon and Ribavirin
Hermine O. et al, N Engl J Med 2002; 347: 89-94

62. HCV negative / SLVL Patients Treated with Alpha-Interferon 2007
Hepatitis C virus : extrahepatic manifestations, an update
Median age 65 (54-72)
Prior therapy (2/6), chemotherapy (1), splenectomy(1)
Splenomegaly (4/6)
Hyperlymphocytosis Median 25,000 (500-100.000)
Cytopenia (2/6)
Cryoglobulinemia or rheumatoid factor (0/6)
Alpha-Interferon 3 M IU x 3/W during 6 months
No response
Hermine O. et al, N Engl J Med 2002; 347: 89-94

63. Conclusion
Hepatitis C virus : extrahepatic manifestations, an update 2007
Extra hepatic manifestations of HCV infection are
frequent, & may be curred by HCV treatment :
• Systemic vasculitis (cryoglobulinemia, PAN)
• Fatigue
• Arthralgia - myalgia - arthritis (±)
• Auto-antibodies (?)
• Splenic lymphoma with villous lymphocytes
• Thrombocytopenia