1. IL RITUXIMAB NEL TRATTAMENTO DELLA
PORPORA TROMBOTICA TROMBOCITOPENICA
Erica Daina
Centro Centro di Ricerche Cliniche
per le Malattie Rare
Aldo e Cele Daccò
Istituto Mario Negri
22 Gennaio 2010
Torino

2. THROMBOTIC MICROANGIOPATHY
Hemolytic Uremic Syndrome - Thrombotic Thrombocytopenic Purpura
Definition
A multisystem disease with predominant renal
involvement in HUS and neurological signs in
TTP, characterized by a triad of symptoms:
- microangiopathic hemolytic anemia
- thrombocytopenia
- formation of platelet-rich thrombi in the
microcirculation

3. THROMBOTIC THROMBOCYTOPENIC PURPURA
Incidence of TTP estimated at 4 cases per 1 million/year
TTP is more frequent among women
(female/male ratio 3:2)
The most commom form of TTP is acquired ( “sporadic”)
Relapses following an initial episode of acquired TTP are
described, with about one third of cases becoming recurrent
Familial TTP usually manifests in the postnatal period or
during infancy, although in some cases the onset is later at
20-30 years
Patients with familial TTP tipically exhibit a relapsing course

4. • Deficiency of the von Willebrand Factor (VWF)-
cleaving protease, ADAMTS13, has been
reported in the majority of patients with TTP

5. VWF-CLEAVING PROTEASE
Member of ADAMTS family (A Disintegrin-like And
Metalloprotease, with ThromboSpondin type 1 motif)
Named ADAMTS 13
1427 aa residues
Genomic DNA mapped to human chromosome 9q34
mRNA detected in liver
Zheng et al., J Biol Chem, 2001
Gerritsen et al., Blood, 2001
Fujikawa et al., Blood, 2001
Soejima et al., J Biochem, 2001

6. REDUCED ADAMTS13 ACTIVITY IN HEALTH AND DISEASE
- elderly
- newborns
- third trimester of pregnancy
- uremia
- after major surgery
- inflammatory states
- liver cirrhosis Mannucci et al., Blood, 2001

7. ADAMTS13 MUTATIONS IN TTP CAUSE SEVERELY REDUCED
PLASMA ADAMTS13 CONCENTRATION
Family A
ADAMTS13 antigen (ng/mL) 77 1580 79 731 82 786
ADAMTS13 activity (%) < 6% 108% < 6% 62% < 6% 67%
5 10 1 6 5 10 6 10 5 10 5 1
Heterozygous 3 5 5 5 3 5 5 5 3 5 3 5
2 4 3 5 2 4 / 4 2 4 2 /
Val88Met(metalloprotease) 7 7 7 7 7 7
8
7 7 7 7 7 7
7
5 8 7 6 5 6 8 5 8 5
7 7
Gly1239Val (CUB1) 3 7 7 / 3 7 7 3 7 3
Family B Family C
ADAMTS13 antigen (ng/mL) < 62.5 < 62.5 < 62.5
ADAMTS13 activity (%) < 6% < 6% < 6%
6 6 6 6 6 6
Homozygous 5 5 Homozygous 5
1
5
1
5
1
5
1
3 3
6 6 5 5 5 5
Arg1123Cys (TSP8) 5 5 Arg1219Trp (CUB1) 6 6 6 6
4 4 6 6 6 6

8. ACQUIRED IDIOPATHIC TTP
Undetectable plasma levels of ADAMTS13
Furlan et al., Blood, 1998
The activity is normal after recovery
Antibodies that inhibit enzyme activity in plasma are
found in 48 to 80 % of these patients
Tsai et al., N Engl J Med, 1998
Veyradier et al., Blood, 2001

9. • Deficiency of the von Willebrand Factor (VWF)-
cleaving protease, ADAMTS13, has been
reported in the majority of patients with TTP
• ADAMTS13 deficiency may be either
constitutive, due to mutations in the ADAMTS13
gene, or acquired due to the presence of
circulating anti-ADAMTS13 autoantibodies

10. OLD DAYS OF TREATMENT OF HUS/TTP
Plasma manipulation
100
Aspirin
80
Survival (%)
60
40
20
0
1925 1964 1980 1990
Bell et al NEJM, 1991
Rock et al NEJM,1991
Goldenfarb et al, JAMA 1973

11. • Plasma exchange is the treatment of choice for
patients with acquired TTP
• Congenital TTP responds to plasma infusion
Author: Deborrah Symonette
http://emedicine.medscape.com/article/779969-overview
Updated: Sep 16, 2009

12. DIFFERENTIAL DIAGNOSIS BETWEEN TTP ASSOCIATED WITH
GENETIC OR IMMUNE-MEDIATED ADAMTS13 DEFICIENCY IS
IMPORTANT TO GUIDE SPECIFIC TREATMENTS
Genetic ADAMTS13 deficiency:
- replacement with plasma of the defective activity
Immuno-mediated ADAMTS13 deficiency:
- plasmapheresis to remove anti-ADAMTS13 autoantibodies
and to replace the metalloprotease
- inhibition of the autoantibodies production through treatment
with glucocorticoids, immunosuppressive agents, or
rituximab

13. INTERNATIONAL REGISTRY OF
RECURRENT AND FAMILIAL HUS/TTP
Participating Centers 180
UK Switzerland Denmark Patients 600
Germany HUS/TTP 473/127
Familial 86/19
Trento Sporadic 387/108
Belgium
VareseBergamo
BresciaTreviso
Monza
MilanoVicenza
Torino Padova
Genova Pavia Parma
Canada
Firenze
Poland Czech R. Estonia
USA Roma
Portugal
Sassari Foggia
Salerno Bari
Spain Greece Iran
Serbia
Cagliari
Reggio Calabria
Palermo
Israel Turkey
Chile Argentina
South Africa
UAE Saudi Arabia
http://villacamozzi.marionegri.it Malaysia Japan Australia

14. INTERNATIONAL REGISTRY OF
RECURRENT AND FAMILIAL HUS/TTP
• A congenital deficiency of ADAMTS13 was found in 15 patients
• Undetectable ADAMTS13 activity (<6%) due to the presence of anti-
ADAMTS13 inhibitors was found in 28 of 32 patients, in the acute
phase, and in 14 of 53 patients, during remission
• 10 patients with a severe and recurrent form of TTP showed
persistence of high titers of autoantibodies in remission

15. RITUXIMAB, A HUMANIZED CHIMERIC ANTIBODY
DIRECTED AGAINST THE CD20 ANTIGEN IN B
CELLS, HAS BEEN PROVEN EFFECTIVE IN
INDUCING REMISSION IN PATIENTS
REFRACTORY TO ANY OTHER TREATMENT
Chemnitz et al., Am J Hematol, 2002
Gutterman et al., Blood Cells Mol Dis, 2002
Tsai et al., Eur J Haematol, 2003
Yomtovian et al., Br J Haematol, 2004
Fakhouri et al., Ann Int Med, 2004
Sallah et al., J Thromb Haemost, 2004

16. http://www.clinicaltrials.gov/ct2/home
• Found 17 studies with search of: Thrombotic Thrombocytopenic Purpura
Use of Rituximab Treatment in Addition to Standard Care for Newly Presenting TTP
Rituximab in Patients With Relapsed or Refractory TTP-HUS
Evaluating the Effectiveness of Adding Rituximab to Standard Treatment for TTP
(The STAR Study)
The Use of Rituximab in Acute Thrombotic Thrombocytopenic Purpura (TTP)

17. REGISTRY ANALYSES - Aims of the evaluation
• To verify if Rituximab, in patients with anti-ADAMTS13 antibodies,
can induce remission of acute refractory TTP and can prevent
relapses of recurrent forms
• To verify if Rituximab can restore a significant ADAMTS13 plasma
activity (>10%) with no detectable inhibitors
• To evaluate whether the reapparance of high titer of ADAMTS13
inhibitors may be an indicator for retreatment

18. Patients
• Adult patients with at least one episode of TTP
• Reduced ADAMTS13 activity (<6%) and high titers of anti-
ADAMTS13 antibodies at onset in patients with refractory TTP
• Reduced ADAMTS13 activity (<6%) and high titers of anti-
ADAMTS13 antibodies in at least two assays performed at least 3
months apart, and at the time of prophylactic treatment

19. CHARACTERISTICS OF TTP PATIENTS BEFORE TREATMENT
WITH RITUXIMAB
Patient Age at Age at 1st Total number Brain/Kidney Previous
Sex diagnosis treatment with of TTP involvement treatments
(yr) Rituximab (yr) episodes
Plasma, steroids, vincristine,
01/F 23 23 1 ?/No
defibrotide
Plasma, steroids, vincristine,
02/F 57 57 1 Yes/Yes cyclophosphamide,
cyclosporine
Plasma, steroids, intravenous
03/M 48 58 >10 Yes/Yes
Ig, cyclosporine, splenectomy
Plasma, steroids,
04/M 37 42 3 Yes/Yes
intravenous Ig
Plasma, steroids,
05/F 32 49 10 No/Yes
intravenous Ig, azathioprine
Plasma, steroids, intravenous
06/M 27 31 5 No/Yes
Ig, vincristine, vinblastine
Data from the International Registry of Familial and Recurrent HUS/TTP

20. RITUXIMAB TREATMENT IN ACUTE
REFRACTORY TTP
FOLLOW-UP
Patient Treatment
no no Pre 2 mo 6 mo 10 mo 12 mo 15 mo 27 mo
ADAMTS13#
-activity <6 52 54 55 70 67
01 1 -inhibitors 2.2 Neg Neg Neg Neg Neg
CD20% 11 1 0 1 nd nd
ADAMTS13#
-activity <6 62 69 <6 68 76
02 1 -inhibitors 2.3 Neg Neg Pos Neg Neg
Prophylactic
treatment
CD20% 8 1 1 nd nd
# ADAMTS13 activity is expressed as % (normal range: 50-150%)
Inhibitors are expressed as Bethesda units
Data from the International Registry of Familial and Recurrent HUS/TTP

21. PROPHYLAXIS WITH RITUXIMAB IN RECURRENT TTP
A patient with recurrent TTP due to high titers of ADAMTS13 inhibitors who
used to have two relapses of TTP a year
+ATG
+CHP
+CHP
+ATG
+CHP
+ATG
+ATG
700
600
+CyA
Vin
Vin
Vin
Vin
+Vin
+Vin
Vin
+Vin
+Vin
+Ig
+Ig
+Ig
500
Vin ATG
400 ##
300
+FFP
200
100
0
0 10 20 30 40 50 60 70 80 90 100 110 120
months
: Plasma exchange;
Vin: vincristine; ATG: antiplatelet agents;
CyA: cyclosporin A; CHP: cyclophosphamide;
#: plasma exchange, vincristine, antiplatelet agents and corticosteroids;
Ig: immunoglobulins; FFP: fresh frozen plasma;
: corticosteroids;
: splenectomy;
: transient ischemic attack;
: ADAMTS13 activity <6%, anti-ADAMTS13 inhibitors present. Galbusera et al., Blood 2005

22. PROPHYLAXIS WITH RITUXIMAB IN RECURRENT TTP
- Rituximab caused progressive disappearance of inhibitors and increase of protease activity
that lasted 14 months
- A second course of rituximab induced a prompt recovery of ADAMTS13 activity and
disappearance of inhibitor
- Plasmapheresis had a small transient effect on ADAMTS13 activity and on inhibitor titer
ADAMTS-13 activity (%)
35 18
Inhibitor titer (BU)
30 15
25 12
9
20
13
6
15
3
10
0
6
-25 -20 -15 -10 -5 0 0 100 200 300 400 500 600 700 800
days
3 courses of plasmapheresis 4 rituximab doses
: anti ADAMTS13 IgG titer: 1:1600
: anti ADAMTS13 IgG titer: negative
Galbusera et al., Blood 2005

23. PROPHYLAXIS WITH RITUXIMAB IN RECURRENT TTP
Patient Treatment FOLLOW-UP
no no
Day 0 3 mo 6 mo 12 mo 18 mo 24 mo 30 mo 40 mo
03 1 ADAMTS13#
-activity <6 15 21 14 <6 32 10 <6
-inhibitors 2.3 Neg Neg Neg 6.5 Neg Neg 4.4
Prophylactic Relapse
CD20% 9 1 3 6 retreatment 1 4 10 mo later
04 1 ADAMTS13#
-activity <6 55 74 91 70 84 56 45
-inhibitors 1.9 Neg Neg Neg Neg Neg Neg Neg
CD20% 7 0 0.2 3 3.2 7.5 nd nd
05 1 ADAMTS13#
-activity <6 59 64 50 24 23 <6 25
-inhibitors 3.4 Neg Neg Neg Neg Neg Pos Neg
Prophylactic
CD20% nd 1 1 nd nd nd retreatment 4.1
1 infusion
# ADAMTS13 activity is expressed as % (normal range: 50-150%)
Inhibitors are expressed as Bethesda units
Data from the International Registry of Familial and Recurrent HUS/TTP

24. TTP PATIENT TREATED WITH RITUXIMAB BOTH
IN ACUTE AND REMISSION PHASES
FOLLOW-UP
Patient Treatment
no no
Day 0 2 mo 3 mo 6 mo 9 mo 12 mo
ADAMTS13#
06 1 -activity <6 33 56 46 <6
acute -inhibitors + Neg Neg Neg 3.6
Prophylactic
treatment
ADAMTS13#
-activity <6 75 78 <6 <6
06 2
-inhibitors 3.6 Neg Neg +/- 2.5
remission
Mild disease
relapse
ADAMTS13#
3
06 -activity <6 60 53 16
acute -inhibitors 2.5 Neg Neg Neg
# ADAMTS13 activity is expressed as % (normal range: 50-150%)
Inhibitors are expressed as Bethesda units
Data from the International Registry of Familial and Recurrent HUS/TTP

25. CONCLUSIONS
Our observations confirmed that Rituximab as adjunctive
therapy in patients with TTP not responding to
conventional therapies induced remission
• Rituximab can be used as preventive therapy in patients
at high risk of relapses
• ADAMTS13 activity and inhibitors should be monitored
during follow-up
• Trials are needed to evalute:
- parameters that should be used to predict
relapse and indicate retreatment
- response rate and long term side effects

26. RITUXIMAB FOR TREATMENT OF aHUS WITH ANTI-CFH ANTIBODIES
In a 10-year-old girl with high levels of anti-CFH autoantibodies, plasma
exchange combined with prednisone and azathioprine only transiently
decreased the antibody titer.
Rituximab (375 mg/m2/week x 4 weeks) led to a complete B cell
depletion and maintained anti-CFH antibody at low levels during the
following 4 months.
Kwon et al, Nephrol Dial Transplant , 2008
In a 7 year old boy, combined treatment with FFP and Rituximab
resulted in reduction in CFH autoantibody levels and induced clinical
remission.
Wigger et al, Pediatric Nephrol 2008

27. Laboratory Clinical
Marina Noris Giuseppe Remuzzi
Roberta Donadelli Piero Ruggenenti
Chiara Mossali Erica Daina
Chiara Fenili Elena Bresin
Annalisa Sorosina Sara Gamba
Jessica Caprioli
Collaborations
Rossella Piras
Caterina Mele
Peter Zipfel
Erica Rurali
Matthew Pickering
Veronique Fremeaux-Bacchi
Grants Tim Goodship
John Atkinson
Santiago Rodriguez DeCordoba