1. Sindrome HCV dieci anni dopo:
Nuovi algoritmi terapeutici
nella crioglobulinemia mista
Dario Roccatello
Centro di Ricerche di Immunopatologia (CMID),
Dipartimento di Malattie Rare, Immunologiche, Ematologiche,
Immunoematologiche,
ASL 2 Torino Nord e Università di Torino
Coordinamento Interregionale delle Malattie Rare
del Piemonte e della Valle d’Aosta

2. MIXED CRYOGLOBULINEMIA 7.8
WEGENER’S GRANULOMATOSIS 2.2
GIANT CELL ARTERITIS 1.6
POLYARTERITIS NODOSA 1.5
BEHÇET’S DISEASE 1.3
MICROSCOPIC POLYANGIITIS 1.3
KAWASAKI DISEASE 0.7
TROMBOTIC MICROANGIOPATHY 0.7
CHURG-
CHURG-STRAUSS VASCULITIS 0.4
TAKAYASU’S ARTERITIS 0.3
SISTEMIC VASCULITIS IN PIEDMONT:
#/100000
Prevalenza anti - HCV
>10%
5-10%
1-5%
< 1%
Sconosciuta
Rapporto OMS 2000

3. PATHOGENESIS of TISSUE INJURY in MIXED CRYOGLOBULINEMIA
ROCCATELLO D, EXPERT. REV. CLIN. IMMUNOL. 2008 ALPERS CE, CURR OP NEPHR HYPERT 2008

4. Kidney survival in patients with cryoglobulinemic nephritis
female male
1,0
survival probability
0,9 GIIPR’ study
0,8 AJKD, 2007
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0,0
0 50 100 150 200 250
months
Tarantino, KI,1995 , AJKD, 2007
GIIPR

5. Sustained virological response in chronic Hepatitis C and MC:
cumulative data from Mazzaro (2005), Rossi (2003) and Kayali (2006)
Romero-Gòmez et al, Rev Esp de Enfermedades Digestiva, 2008

6. Drug Pros Cons
Steroids - Inhibition of the inflammatory cascade through - Direct or indirect ↑ of HCV
NFkB replication in vivo and in vitro
- Effects on mineral metab and endocrine system
Leflunomide -Inhibition of T lymphocyte clonal expansion - Leflunomide induced necrotizing
- Anti-inflammatory properties vasculitis
- Inhibition of selected tyrosine kinases - Liver toxicity
- L-analogue FK778 vasculoprotective in exp
models
Cyclos A - Inhibition of IL-2 and proinflammatory cytokines - Vasospastic effect on macro and
- Inhibition of HCV replication in vitro and in vivo microcirculation
↑
-↑ Blood viscosity
- ↑ PLT aggregattion worsening
of vascular manifestations
-Nephrotoxicity, hypertension,
neurotoxicity
Mycophenola -Inhibition of T and B lymp clonal expansion -GI adverse events (liver toxicity)
te mofetil / -Ribavirin-like action in vitro
Azathioprine -Prevention of arterial smooth muscle cell
proliferation and proliferative arteriopathy in
animal model
Anti-TNF Inhibition of TNF-α -↑ B cell number and activity in
Anti-inflammatory activity peripheral blood
TNF-α implicated in refractoriness to IFN - Autoantibodies (ANA, anti nDNA)
Anti-CD20 Inhibition of B cells -↑ HCV RNA (?)
Effective targeting of autoreactive clones - progression of HCV infection (??)

7. Fornasieri e Roccatello
Manuale di terapia delle glomerulonefriti
Peg IFN a 2a (40 kD) 180 ug week
kD)
Mycophenolate mofetil 2g/day
2g/day
Peg IFN a 2b (12 kD)1.5 ug /kg week
kD)1.5
plus
Ribavirin 800-1200 mg/die
800- mg/die

8. Drug Pros Cons
Steroids - Inhibition of the inflammatory cascade through NFkB - Direct or indirect ↑ of HCV
- Effects on mineral metab and endocrine system replication in vivo and in vitro
Leflunomide -Inhibition of T lymphocyte clonal expansion - Leflunomide induced necrotizing
- Anti-inflammatory properties vasculitis
- Inhibition of selected tyrosine kinases - Liver toxicity
- L-analogue FK778 vasculoprotective in exp models
Cyclos A - Inhibition of IL-2 and proinflammatory cytokines - Vasospastic effect on macro and
- Inhibition of HCV replication in vitro and in vivo microcirculation
-↑ Blood viscosity
- ↑ PLT aggregattion worsening of
vascular manifestations
-Nephrotoxicity, hypertension,
neurotoxicity
Mycophenolat -Inhibition of T and B lymp clonal expansion -GI adverse events (liver toxicity)
e mofetil / -Ribavirin-like action in vitro
Azathioprine -Prevention of arterial smooth muscle cell proliferation
and proliferative arteriopathy in animal model
Anti-TNF α
Inhibition of TNF-α ↑
-↑ B cell number and activity in
Anti-inflammatory activity peripheral blood
α
TNF-α implicated in refractoriness to IFN - Autoantibodies (ANA, anti nDNA)
Anti-CD20 Inhibition of B cells -↑ HCV RNA (?)
Effective targeting of autoreactive clones - progression of HCV infection (??)

9. Anti TNF therapy and C virus hepatitis
Parke, Arhtr & Rheum 2004

10. Treatment with Abatacept of a relapsing case of MC
C4 mg/dl
14
12
10
8
6
4
2
0
Proteinuria mg/24h
1200 Serum creatinine mg/dl
3
1000
2,5
800
2
600
1,5
400
1
200 0,5
0 0

11. Roccatello, NDT, 2004
Rituximab is expected
# to improve the subclinical lymphoproliferation
and interfere with IgM monoclonal production
and cryoglobulin synthesis

12. Roccatello, Expert Reviews in Clinical Immunology, 2008

13. Rituximab in patients with HCV-related cryoglobulimia
HCV-
Study Year Patients Rtx dose Other Side effects HCV viral Relapse
(# of nephritis) treatment load (#)
Sansonno 2003 20 (1) 375 mg/m2 S (low Septic fever (1) Responder 4 / 16
weekly x 4 weeks doses) = (> 7
nonrespon months)
der
Zaja 2003 15 (2) 375 mg/m2 S (< 0.5 Retinal 2/8 6 (3-6
weekly x 4 weeks mg/kg/day) thrombosis (1) 1/8 months)
=5/8
Roccatello 2004 6 (5) 375 mg/m2 Transient 14 2
weekly x 4 weeks bradycardia (2) unchanged (> 12
+ 375 mg/m2 months)
monthly x 2
months
Quartuccio 2008 5 (5) 375 mg/m2 S (one Transient NR 3 (>5, > 7
weekly x 4 weeks case) neutropenia (1) and > 12
months)
Basse 2006 7 (7) (post 375 mg/m2 CNI, MM Lethal infection NR NR
kidney weekly x 2-4 (2, fungal and
transplant) weeks HSV)
Visentini 2007 5 (1) (available 250 mg/m2 S, IF, CYC, 2 /5 NR
for analysis) weekly x 2 weeks PE =3/5
Terrier 2009 12 (4) 375 mg/m2 S, PE in Serum sickness Unchanged 4/12 (18±7
weekly or 1000 nephritic pts neutropenia months)
mg twice
Ibidem 2009 20 (10) 375 mg/m2 Combined Serum sickness decreased 3/20(23±12
weekly or 1000 PEG-IFN hematologic months)
mg twice and toxicity
Ribavirin flare of psoriasis
hepatocarcinoma
poor compliance

14. ANTI-
ANTI-CD20 MONOCLONAL ANTIBODY THERAPY FOR TYPE II MC SYNDROME:
SYNDROME:
A CONTROLLED STUDY vs. BEST AVAILABLE TREATMENT
vs.
• Randomized, controlled multicentre study
1.Udine – S. De Vita (Co-Ordinating Centre)
• Study phase III
2. Pordenone – C. Mazzaro
• No sponsor – no profit study 3. Brescia – P. Scaini
4. Salerno – S. Scarpato
• Duration 24 months (end of recruitment 31/12/2006) 5. Bologna – M. Lenzi
6. Novara – M. Campanini
7. Bergamo – M. Pietrogrande
• Target population:
population: 8. Pisa – A. Tavoni, S. Bombardieri
9. Modena – M.T. Mascia, C. Ferri
patients with HCV-related or –unrelated mixed cryoglobulinemia
HCV- 10. Torino – D. Roccatello
with skin ulcers, active glomerulonephritis or peripheral neuropathy 11. Saronno – G. Monti, Saccardo
12. Napoli – S. Migliaresi
13. Napoli Cotugno – C. De Pascale
Primary end point 14. Milano Niguarda – B. Canesi, Filippini
To evaluate the efficacy of rituximab in comparison with the conventional, best currently 15. Ancona – A. Gabrielli
available therapeutical approaches (as chosen by expert clinicians) 16. Firenze – A.L. Zignego, M. Matucci
17. Monza – P. Pioltelli
Additional end points
• To determine the steroid-sparing effects of rituximab, if any
• To determine the duration of clinical and biologic efficacy of rituximab treatment in type II
MC, to better plan retreatment and/or maintenance regimens in future studies
• To determine the effects on the quality of life and disease
• To determine whether rituximab may be useful to rescue patients where best available
treatment has failed
• Pharmacoeconomic assessments of rituximab therapy vs. best available treatment
GROUP A: Best conventional treatment GROUP B: Rituximab (rescue from group A allowed)
1) corticosteroids (maximal initial doses 1 mg/kg/day of Rituximab 1000 mg IV on day +1 and +15
prednisone/eqivalents) with or without preceding 6- with or without concomitant corticosteroids.
methylprednisolone pulses (500 to 1000 mg/day for 3
consecutive days Only corticosteroids are allowed as concomitant treatment
2) azathioprine or cyclophosphamide 1- 2 mg/kg/day, with or - if already administered
without corticosteroids - if introduced concomitantly with rituximab treatment, only
3) Plasmapheresis with or without corticosteroids low doses (≤ 0.1 mg/kg/day) allowed.
Randomization: GROUP A: 30, GROUP B: 29 Rescue therapy” to rituximab: 22/30 (73.3%)

15. CMID’s open study
22 pts, mean age years 61.7 (range 36-78 years), with HCV infection in 21
cases, genotype 2a/2c (7 cases), 3 (2 cases) , 1b (10 cases)
– intolerance to standard therapy 6
– resistance to standard therapy 6
– severe BM lymphocyte infiltration 5
– front-line therapy 5
12 with severe renal involvement, 10 with MPGN, 1 with renal vasculitis
13 with multiple mononeuritis (extremely severe in 5)
9 large skin ulcers (necrotizing in 8)
Rituximab was administered intravenously according to the 4 plus 2
protocol (Roccatello, NDT 2004): 375 mg/m2 on days 1, 8, 15 and 22
with two more doses administered 1 and 2 months later
13 pts were given further infusions:
8 received a re-induction (2 doses in 2 weeks plus 1 monthly infusion for 2 months) after 12-51 months
5 were allocated in a maintenance protocol (1 infusion at 3 months interval)
3 out of 18 pts developed low levels of anti-Rituximab abs (14-22 AU/ml, normal<12)

16. Clinical profiles of 22 patients with severe crioglobulinemia
Arthralgia 15/15 improved VAS >50%
undergoing the 4 plus 2 infusion protocol of anti-CD20 MoAb
BM ab reversal or improvement :
5/5 re- examined pts
re-

17. Laboratory profiles of 12 patients with crioglobulinemic nephritis
undergoing the 4 plus 2 infusion protocol of anti-CD20 MoAb
P<0.001
• Mean follow-up 55.4 months (range 8-87 months)
• Re-inductions in 4 cases at 12,13,17 and 45 months, respectively
• Two cases allocated in a maintenance protocol (1 infusion at 3 months interval for 1 year)
• Dose prednisone at the last observation:
8 pts without maintenance treatment
2 pts with 2.5 mg/d
2 pts with 5 mg/d

18. 59 yr-old man with hemophilia with NS and severe renal failure due to MC
yr-
Pre 2 m 6 m 12 24 36 48
1997: nephrotic syndrome
sCr 6.8 4.6 4.9 5.0 3.5 3.5 3.7
full clinical picture of MC
HCV infection (genotype 1b) Pu g/d 3.5 0.9 0.6 0.6 0.4 0.3 0.8
Given CS, cyclo and PE TP 6.5 6.5 6.5 7.5 7.6 7.6 7.1
2003: scheduled for artero-
artero- ESR 54 30 19 19 19 20 28
venous fistula to start dialysis RF 298 24 30 249 105 95 102
Consultation in our Unit (CMID)
CMID)
Severe renal insufficiency IgM 397 80 60 142 69 75 96
with nephrotic syndrome
4-drug resistant hypertension C3 65 80 78 79 78 82 81
4-limb sensitive motor C4 4 11 28 16 28 24 18
polyneuritis
Arthralgia,
Arthralgia, weakness Cryo 4% 2% 0.5 0 1 0.5 0.5
Iatrogenic diabetes, purpura
VL 1.3 nd 1.0 0.7 0.3 0.4 0.4
ALT 26 27 16 44 43 36 38

20. “4 plus 2” Rituximab protocol: effects of therapy

21. Symptom changes in 13 patients with polyneuropathy
Previous treatments:
CS (11), PE (3), CYC or MMF(4), IFN (4)
15 - 45% - 66% - 83%
10
5
0
paresthesia burning foot RLS weakness
pre 11 4 1 12
post 6 2 1 2
Cavallo and Roccatello J Neurol, 2009

22. Cryoglobulinemic polyneuropathy in 13 pts:
pts:
EMG changes after anti-CD 20 MoAb
anti-
PRE POST p
SPE ampl mV 1.08±0.95 1.50±1.32 0.04
SPE MCV m/s 41.29±8.0 41.77±7.35 n.s.
SPE Lat ms 4.16±1.05 4.38±1.32 n.s.
Sural ampl µV 0.39 7.13 0.085
Sural SCV m/s 33.85±0.77 47.48±5.72 0.018
Cavallo and Roccatello J Neurol, 2009
CMAP changes
5
4,5
4
3,5
3
mV
2,5
2
1,5
1
0,5
0
1.08±0.95 pre pos t 1.50±1.32
p<0.04

23. Dario Roccatello
Research Center of Immunopathology
University of Turin, Italy
Inter-regional Coordinating Center for Rare Diseases of Piedmont and Aosta Valley
NorthTurin Emergency Hospital San Giovanni Bosco,Turin, Italy
effective therapeutic option
severe worsening of renal
function, mononeuritis
multiplex, extensive skin
ulcers, and distal necrosis
persistence of effects is
often finite, but long lasting
response (48 mths) is
observed in about a half of
the patients
Roccatello, Expert Reviews in Clinical Immunology, 2008