1. Extrahepatic manifestations of HCV infection 1) Association defined on the basis of high prevalence and pathogenesis:  Mixed cryoglobulinemia and cryoglobulinemic nephropathies  Mixed cryoglobulinemia and cryoglobulinemic nephropathies 1) Association defined on the basis of high prevalence and pathogenesis:  Mixed cryoglobulinemia and cryoglobulinemic nephropathies  Mixed cryoglobulinemia and cryoglobulinemic nephropathies 2) Association defined on the basis of higher prevalences than in controls:  B-cell non-Hodgkin’s lymphoma  Monoclonal gammopathies  Porphyria cutanea tarda  Lichen planus 3) Less close associations :  Autoimmune thyroiditis  Thyroid cancer  Sicca syndrome  Lung fibrosis  Non-cryoglobulinemic nephropathies

2. Among extrahepatic manifestations of HCV infection, the most common is MIXED CRYOGLOBULINEMIA MC is a systemic disease characterized by vasculitis of the skin and multiple organs involvement secondary of immunocomplexes deposition (cryoglobuylins plus complement). By tradition MC is classified as a systemic vasculitis involving medium and small vessels. Given the presenc of a monoclonal B-cell population in PBMC and in bone marrow, MC can be considered as a lymphoproliferative disease, with a “benign” behaviour, but with the possibility to evolve in a non-Hodgkin’s lymphoma (NHL), at least in a small fraction of cases.

6. Cryoglobulinemia classification (according to Brouet) Type I (Single Cryoglobulin): Monoclonal IgM, rarely IgG, these cases are generally affected by Waldenstrom’s macroglobulinemia or by Mieloma. Type II (Mixed Cryoglobulinemia) Monoclonal IgM and polyclonal IgG Monoclonal IgM are endowed with anti-IgG activity (RF) Type III (Mixed Cryoglobulinemia) Polyclonal IgG and IgM without anti-IgG activity

7. MIXED CRYOGLOBULINEMIA Anti-HCV antibodies and HCV-RNA are present in 80 -90 % of the patients with MC, and viral RNA can be amplificated in the 98% of the cryoprecipitates. Viral RNA is present in PBMC and in bone marrow of MC patients even in absence of a liver disease.

8. HOW MANY HCV PATIENTS HAVE MC? (I)Adinolfi et al.: 46% ; 20% type II It J Gastroenterol 1996 (D) Hartmann et al.: 51% ; 33% type II Z Gastroenterol 1995 (E) Horcojda et al. : 43% ; 13% typo II Ann Int Med 1999

9. Prevalence of Cryoglobulins and of Cryoglobulinemic Syndrome in 500 HCV+ Patients Cryoglobulins190/500(38%) Type II 61/190 (32%) Type III129/190 (68%) -Cryoglobulinemic syndrome50/190 (26%) Type II 45/50(90%) Type III 6/50(10%) Lenzi M,2004

10. HOW MANY HCV PATIENTS HAVE MC? (Korea) Lee et al.: 60% but 0 % type II Ann Rheum Dis 1998 (UK) Wong et al.: 20% but 2 (9%) type II Clin Exp Rheumatol 1998 (USA) Kerr et al.: 40% but 1 case (2%) type II J Rheumatol 1997

11. These data suggest a different prevalence of MC between the HCV patients from Mediterranean basin and the HCV patients from far-east and USA/UK Since a monoclonality of B-lymphocytes is detectable, in PBMC, of each MC patient and in at least 25% of patients with HCV infection without cryoglobulins, we compared: A group of Japanese HCV patients without MC and without dosable levels of cryoglobulins A group of Italian HCV patients without MC and without dosable levels of cryoglobulins Positive controls (MC) Negative controls (HBV, Alcoholic cirrhosis, Normal subjects)

12. Ethnic difference in the prevalence of B-cell monoclonality in HCV positives Among 50 Italian HCV+ without CM, 24 % showed B-cell monoclonality in PBMC Among 44 Japanese HCV+ without CM, 0% showed B-cell monoclonality in PBMC All the patients affected by alcoholic liver disease, HBV+ and normal subjects were negatives for B-cell monoclonality All the cases affected by MC showed B-cell monoclonality in PBMC

13. Ethnic difference in the prevalence of B-cell monoclonality in HCV positives These data indicate that HCV is able to induce a monoclonal B-cell proliferation only in a fraction of infected patients. The different distribution of MC in the world is not due to differences in the clinical or epidemiological approach to the problem, but to some (at present not fully understood) ethnic difference in immune response to HCV infection.

14. How HCV is able to interact with the immune system?

15. HCV INFECTION OF HAEMATOPOIETIC AND PERIPHERAL BLOOD CELLS Infection of PBMC by HCV in MC (Ferri et al. 1993) HCV involves CD34+ haematopoietic cells (Sansonno et al. 1998) Neutrophils are site of replication of HCV (Pozzato et al. 2000)

16. From these studies HCV seems able to infect and to replicate in: Haematopoietic cells B lymphocytes Monocytes PMN HCV INFECTION OF HAEMATOPOIETIC AND PERIPHERAL BLOOD CELLS

17. Through CD81 receptor (tetraspanin) interacting with E2 viral region (Pileri et al. 1998) Through LDL receptor (Monazahian et al. 1999) Through Scavenger Receptor Class B Type 1 (SR-B1) (Scarselli et al. 2002) HOW HCV INFECT HUMAN CELLS ?

18. SR-B1 and LDL receptors are not expressed in B or T lymphocytes, but in the hepatocytes and other organs (ovary, adrenal glands). They are important for virus internalization and, therefore, for cell infection, but are not involved in the immune response. CD81 is expressed mainly in B-lymphocytes, where it is complexed with CD19 and CD21, two receptors involved in the B-cell activation.

21. On these basis we investigated whether HCV was able to modify the immunological status of B cells through the interaction with CD81

22. “IN VITRO” EFFECTS OF NORMAL B-CELLS STIMULATION WITH anti-CD81 ON THREE MARKERS OF LYMPHOID ACTIVATION % of B-cells expressing markers

23. ACTIVATION OF THE B-LYMPHOCYTES IN PATIENTS WITH HCV INFECTION AND IN CONTROLS % of B-cells expressing markers * ** ** ** * ** **

24. % of B-cells expressing receptors * ** ** “IN VITRO” EFFECTS OF NORMAL B-CELLS STIMULATION WITH anti-CD81 ON CHEMOKINE RECEPTORS

25. CHEMOCKINE RECEPTORS IN B-CELLS OF PATIENTS WITH HCV INFECTION AND IN CONTROLS % of B-cells expressing receptors * * ** ** *

26. RESPONDER NON-RESP. EFFECTS OF THE ANTIVIRAL THERAPY ON CD71 EXPRESSION IN B-CELLS

27. RELAPSER NON-RESP.

28. These data indicate: 1) In HCV-positive patients, B-cells show an activation pattern (over-expression of CD69/CD71/CD86) and express a chemokine receptor pattern similar to what obtained in vitro through the CD81 engagement 2) B “naïve” lymphocytes are much more activated than “memory” B-cells

29. The results obtained in the patients undergoing antiviral therapy indicate that B-cells activation depends from HCV replication, as demonstrated by the disappearance of this condition after HCV-RNA elimination. This distinctive activation pattern of B-cells can explain the well-known clinical association between HCV and several autoimmune or lymphoproliferative disorders.

30. HCV genotypes differences ? N°1a1b2a2b2a/c4Co ND Williams, ‘9411-101----- Sinico, ‘9549-2721----1 Crovatto, ‘9565-461054-1 Zehender, ‘9522- 714--1-- Zignego, ‘966313512-13-2- WHY SOME HCV PATIENTS DEVELOP MC ?

31. Haplotype HLA-DR3 confers susceptibility to hepatitis C virus-related Mixed cryoglobulinaemia. Lenzi M et al. Blood 1998 Different HLA haplotype distribution ?

32. The susceptibility to develop cryoglobulins in HCV patients is not associated with HLA-DR or DQ. The HLA-DRB1*11 positive individuals are protected from serious chronic liver disease. Amoroso et al. J. Hepatol. 1998; 29(1): 36-44 WHY SOME HCV PATIENTS DEVELOP MC ? Different HLA haplotype distribution ?

33. HCV Receptors polymorphisms ? No polymorphisms of CD81 ! No polymorphisms of SR-B1 ! WHY SOME HCV PATIENTS DEVELOP MC ?

34. At present, no explanations ! WHY SOME HCV PATIENTS DEVELOP MC ? Possible relationship with Fibronectin gene polymorphisms

35. From MC to NHL HCV infection in NHL complicating MC (Ferri et al. 1994) HCV infection in patients with NHL (Ferri et al. 1995)

36. From MC to NHL Cases 108 patients affected by NHL Control Group 1 75 patients affected by other haematological B-cell monoclonal disorders HL, CLL, MM Control Group 2 General population (6.917 subjects)

37. From MC to NHL Patients with NHLHCV positivity: 28,0 %* Control group 1 HCV positivity: 3,1 % Control group 2 (Gen.Pop.)HCV positivity: 2,9 % * p<0.000001

38. From MC to NHL Low-grade NHL :42 % Intermediate NHL :27 % High grade NHL :20 %

39. From MC to NHL Prevalence of HCV in lymphoproliferative disorders (Silvestri et al. Blood 1996) HCV in subsets of neoplastic lymphoproliferative... (Luppi et al. Leukemia 1996) HCV in cryogl. and non-cryoglobulinemic NHL (Silvestri et al. Haematologica 1997)

40. 9% 28% 22% 16% 42% 11% 32% 33%

41. PREVALENCE OF HCV INFECTION IN ITALY 2.9% (6,917 cases) 26.7% (2,472 cases) 8.4% (681 cases) 14.4% (1,352 cases)

42. PREVALENCE HCV+ OVER 60 YEARS OF AGE PREVALENCE HCV+ OVER 60 YEARS OF AGE 18.2% 41.7% 33.0% 10.2%

43. From MC to NHL The prevalence of HCV positivity in NHL should be compared with the prevalence of HCV infection in the general population of the same age. The association HCV-LNH is still significant in the North and Centre Italy but not in the South (were the prevalence of HCV infection is very high)

44. HCV and NHL in Europe 0% (65 cases) 0% (72 cases) 1.4% (136 cases) 2.5% (1485 cases) 9.4% (180 cases) 19.8% (2,668)

45. THE ASSOCIATION BETWEEN HCV AND NHL HAS BEEN RECENTLY CONFIRMED BY SEVERAL STUDIES. From Italy: Mele et al (Blood 2003): prevalence in NHL: 17.5% vs 5.6% in the general population Talamini et al (Int. J. Cancer 2004) prevalence in NHL 19.6% vs 8.9% in the controls. From France: Seve et al. (Eur. J. Gastroenterol. 2004) prevalence in NHL 2.8% vs 2% in the controls. From Spain: Gisbert et al. (Eur. J. Gastroenterol. 2004) prevalence of NHL 7% vs 0.93% un the controls.

46. HCV and NHL in the world Data are available only from USA, Canada and Japan

47. 22% (120 cases) NB: 78% hispanic 1.8% (78 cases) 25% (32 cases) 3.9% (813 cases)

48. Toronto0% (100 cases) British Columbia 2.3% (88 cases)

49. 17% (100 cases ) 16% (150 cases ) 16% (180 cases) In Japan the prevalence of HCV infection in NHL ranges from 5.7 to 22.2% (mean 11.3%)

50. From other countries, few studies are available: In Egypt 42% In Turkey 8% In Romania 29.5% In Hungaria 14.3% In these studies a control group is generally lacking and the prevalence of HCV infection in the general population is high

51. From MC to NHL The distribution of HCV-associated NHL is the mirror of the prevalence of MC in HCV-positive patients. This indicates that the same pathogenetic mechanism underlies the MC as well as the HCV- NHL

52. Which NHL subtypes are associated with HCV infection ? Plasmacytoid immunocytomas in the 75% of cases (cryo+) The remaining 25% includes marginal zone lymphomas (cryo+/-) follicle center lymphomas (cryo+/-) diffuse large cell lymphomas (cryo-) splenic lymphoma with villous lymphocytes (cryo+)

53. Immunophenotype CD19 + CD20 + CD22 + CD24 + DBA44 + negative for CD5 negative for CD10 negative for CD25 Morphology SPLENIC LYMPHOMA WITH VILLOUS LYMPHOCYTES

54. THERAPY OF MIXED CRYOGLOBULINEMIA To eliminate the etiologic agent (IFN, Ribavirin) To inhibit the cryoglobulin synthesis by B-cells (IFN, steroids, ciclophosphamide,other immunosuppressive agents, anti-CD20 antibodies) To reduce the immunocomplexes formation (diet) To remove the circulating immunocomplexes circolanti (plasmapheresis) To reduce inflammation (immunosuppressive agents, colchicine)

55. Rational: Inhibition of Ig production by cytolitic effects on B-cell Drugs: Chlorambucil and Cyclophosphamide (EDX) In the past, EDX was the drug of choice in NHL with CM, but its use has been reconsidered for the nearly constant presence of HCV. Cytostatic agents

56. ANTIVIRAL THERAPY IN MC The antiviral treatment is the therapy of choice for MC. Combination therapy is able to remove the etiologic agent of MC, therefore, after the virus elimination, the disease recovers. Caution in presence of moderate or severe vasculitis.

57. THE RESPONSE RATE EVOLUTION IN CHRONIC HEPATITIS C AND MIXED CRYOGLOBULINEMIA

58. Mabthera (anti-CD20) in MC Sansonno et al. Blood 2002 (20 cases) Zaja et al. Blood (15 cases) Very good clinical response (90%), but high relapse rate in (30 – 50%) after 12 – 18 months. The treatment can be repeated without problems. HCV replication increases, but no cases of liver failure or worsening of liver function tests have been observed. The costs are very high (€ 12.000 each therapy)

59. Which therapy of HCV-NHL? Indolent course (no B symptoms; no Bulky disease, no double time less than 1 year) Normal – Aggressive course Cryoglobulin syndrome No cryglobulin syndrome

60. Indolent course ± cryglobulin syndrome ANTIVIRAL THERAPY Normal/Aggressive course ± cryglobulin syndrome CONVENTIONAL CHEMOTHERAPY

61. Which is the impact of HCV+ CLD on the outcome of therapy? Few studies are available. The most relevant has been recently (2005) published on JCO. Among more than 5000 cases of DLCL only 26 (0.5%) were infected by HCV. However, this small population shows some peculiar characteristics: 1) High proportion of low-grade transformed NHL 2) More frequent spleen involvement 3) Worse survival due to increased toxicity of chemotherapy (dose reduction and/or treatment discontinuation) 4) High frequency and severity of hepatic toxicity

62. This study indicates: The HCV replication determines a more frequent evolution of NHL from a low- to a high- grade NHL. The liver disease reduces the possibility of a favourable outcome of the therapy As consequence …. antiviral therapy should be raccomended in any HCV-positive patient with evidence of viral replication.