1. Factor VIII Inhibitors: After Seven Decades Still a Nightmare and a Mystery Georges E Rivard Centre Hospitalier Universitaire Sainte-Justine Montréal November 2009

2. For this presentation, I declare no conflict of interest with any pharmaceutical industry November 2009 My Disclosure

3. Lozner EI, Jolliffe LS, Taylor FHL. Hemorrhagic diathesis with prolonged coagulation time associated with a circulating anticoagulant. Am J Med Sc 1940;199:318-27 A 61 Y. old man with acquired hemophilia. Died of bleeding after removal of a lymph node which showed tuberculosis. Whole blood transfusion did not stop bleeding. Normal plasma did not shorten coagulation of patient’s plasma in vitro Munro FL, Jones MD. The detrimental effect of frequent transfusions in the treatment of a patient with hemophilia. Am J Med Sc 1943;206:710-3 A 36 Y. old man with severe hemophilia from a family with many severe hemophiliacs.Treated successfully for hematuria "with 3 successive days of 50 cc. of plasma". Further to this success, as need arose, he was successfully treated with 25 to 50 cc. of lyophilized plasma. After about 6 months this treatment failled to control his bleeds and his plasma inhibited clotting of normal plasma in vitro. “We feel that in view of these observations, care should be taken in treating hemophiliacs by transfusions, and that they probably should not be given them as prophylactic measure…" Coagulation Inhibitors: Not a New Story

4. Coagulation Inhibitors: Still a Very Serious Problem FS. 8 y boy. Severe FVIII def. High titre inhibitor since age of 12 m. Severe allergic reaction to any product that has even trace of FVIII. Multiple severe haemophilic arthropathies. Wheel-chair bound…for life! FR. 15 y boy. Severe FIX def. High titre inhibitor since age of 6 m. Severe allergic reaction to any product that has even trace of FIX. Has had 3 IC bleeds. Last one 6 m ago…and the next one… CO. 15 y girl. Severe FXIII def. High titre inhibitor since age of 2 y. IC bleed at age 2 y. Died of IC bleed last summer…poor parents! YM. 42 y man. Severe FVII def. Father of two. High titre inhibitor for one month. No response to any treatment. Died of IC last year on Christmas eve. His 6 y old boy…when is daddy coming back?

5. A Crippling Problem in Congenital Hemophilia A

7. A Major Problem in the Frail Old Subject with Acquired hemophilia A

8. An Expensive Problem in Congenital Hemophilia A Cost for Products: $1,285,892 Right Leg Amputation in a 42 y Man with Hemophilia A

9. An Expensive Problem in Acquired Hemophilia A Cost for Products: $1,518,768 Surgery for Colon Cancer in a 71 y Woman with Acquired Hemophilia A

10. Cumulative Incidence of Factor VIII Inhibitors HEMOPHILIACS :10% to 30% NON-HEMOPHILIACS :1/10 6 per year A Relatively Rare Condition

11. Plan of my Presentation Acquired hemophilia A in a nut shell Hemophilia A with inhibitors…in an egg shell

12. Plan of my Presentation Acquired hemophilia A in a nut shell Hemophilia A with inhibitors…in an egg shell

13. Acquired Haemophilia A UK National Surveillance Study From May 2001 to May 2003 with follow up until May 2004 Data from 255/256 centers: 172 patients Clinical information for 156/172 patients PW Collins et al. Blood 2007

14. Presenting Characteristics Incidence: 1.48/million/y Median age: 78 (range 2-98) Sex: 43% men, 4/4 women in age group 21-40 Associated with pregnancy 4.3%, or 1 case per 350 000 births Underlying diagnosis: 5/5 under age 40 55% between 40 and 59 42% between 60 and 79 23% over 80 PW Collins et al. Blood 2007

15. Underlying Diagnosis None 95 (63.3%) Autoimmune disease 25 (16.7%) Malignancy 22 (14.7%) Dermatologic 5 (3.3%) Pregnancy 3 (2%) PW Collins et al. Blood 2007

16. Sites of Bleeding PW Collins et al. Blood 2007

17. More on Presenting Characteristics No hemostatic therapy required for 34% Bleeding as cause of death in 9% (at 1-146 d) Factor VIII level and inhibitor titer did not predict severity of bleeding Older patients more likely to have died during follow up (P < 0.001) but achieved remission more quickly (P< 0.042) PW Collins et al. Blood 2007

18. International Recommendations on the Diagnosis and Treatment of Patients with Acqquired Hemophilia A Anti-hemorrhagic treatment - rFVIIa 90 µg/kg every 2-3 h until hemostasis is achieved OR - aPCC 50-100 IU/kg every 8-12 h until hemostasis is achieved Inhibitor eradication - Corticosteroids 1 mg/kg/day for 4-6 weeks alone or with: - Cyclophosphamide 1.5 – 2 mg/kg/day for up to 6 weeks A Huth-Kühne et al. Haematologica 2009;94:566-75

19. Long-term Prognosis: Overall Survival Quebec Reference Center for Inhibitors Years At 5 years: 69% (95% CI: 55–80%) St-Louis J, et al. Haemophilia 2008;14(suppl 2):1 (abstract)

20. Plan of my Presentation Acquired hemophilia in a nut shell Hemophilia A with inhibitors…in an egg shell

22. Laboratory Investigation APTT: 78/34 APTT ( 1P + 1N): 65/34 Factor VIII inhibitor: 10 BU

23. Age and Number of Exposure Days at Inhibitor Development Median age at inhibitor development: 16 m Cumulative Incidence of Inhibitors: 23.8% (n=87/366) HM van den Berg et al. 2006 Median number of exposure days at inhibitor development: 12

24. Facteur VIII Inhibitors Nature: Antibodies directed against one or more epitopes of factor VIII and leading to partial or complete inhibition of its procoagulant function

25. Factor VIII Inhibitors : Nature 622Kappa + Lambda 55Only Lambda 2615Only Kappa ALLOANTIBODIESAUTOANTIBODIESLIGHT CHAINS 02IgA 12IgM 00Only IgG 1 00Only IgG 2 31Only IgG 3 127Only IgG 4 128IgG + Other subtypes ALLOANTIBODIESAUTOANTIBODIESHEAVY CHAINS Hoyer 1982

26. The Bethesda Unit Kasper, 1975 One Bethesda Unit: The amount of antibody that inhibits half of the factor VIII activity in a 1 to 1 mixture of patient plasma and normal plasma incubated at 37°C for 2 hours

27. Type I AlloantibodiesType II Autoantibodies Inactivation of Factor VIII by Inhibitors

28. Facteur VIII Inhibitors Nature: Antibodies directed against one or more epitopes of factor VIII and leading to partial or complete inhibition of its procoagulant function

29. A2 Activated protein A1A3C1C2 Me 2+ Mature protein NH 2 COOH A1A2 B C1C2A3 2332 aa 300 kDa 26 exons 186 kb FVIII- gene Exon 26 14 22 1 cDNA 7 kb Factor VIII: From Gene to Protein Slide courtesy of Dr. Johannes Oldenburg

30. Astermark J. et al. Haemophilia 2008; 14 (Suppl.3):36-42 Binding of Factor VIII to von Willebrand Factor

31. vWF/PL Factor VIII Inhibitory Antibodies FIXavWFFIXaFX COOH 2332217320251648719336 NH 2 Inhibitory antibodies measured by the Bethesda assay interfere with: –Xase function –Binding to phospholipids –Binding to von Willebrand factor –Behave as serine esterase and hydrolyse FVIII –The Bethesda assay does not see antibodies against the B domain Modified from Oldenburg et al. Haemophilia. 2002;8(suppl 2):23-29.

32. Neutralization Assay of Plasmas from 21 Autoantibody Inhibitors Inhibitor % Neutralization by FVIII Region Epitopes of Major inhibitor(s ) A2C2 Domain(s) EM≥ 95-A2 JM≥ 95 ≤ 5A2 NS≥ 95 ≤ 5A2 MR ≤ 5≥ 95C2 MS ≤ 5≥ 95C2 D ≤ 5 81C2 SLC ≤ 5 70C2 HR ≤10≥ 95C2 LK ≤ 5≥ 95C2 AA ≤10 79C2 UJ ≤10 74C2 DP ≤ 5≥ 95C2 D. Scandella et al.Blood 1997;89; 3663-71

33. Neutralization Assay of Plasmas from 21 Autoantibody Inhibitors (suite) Inhibitor % Neutralization by FVIII Region Epitopes of Major inhibitor(s ) A2C2 Domain(s) PF ≤ 591C2 FM 8119A2, C2 WC 5932A2, C2, AR3-A3-C1 F ≤ 565C2 EH ≤ 582C2, AR3-A3-C1 SL≤1057C2, AR3-A3-C1 WT ≤ 548C2, AR3-A3-C1 SC 5125A2, C2, AR3-A3-CA D. Scandella et al. Blood 1997

34. Neutralization Assay of Plasmas from 23 Hemophilic Inhibitor Patients Treated with Plasma-Derived FVIII Inhibitor % Neutralization by FVIII RegionMajor Inhibitor Epitope(s) Domain(s) A2C2 CHA≥ 95-A2 RC≥ 95-A2 RM 86-A2 L≤ 10≥ 95C2 WD 56≤10A2, PEP 341-63 RJ≥ 95 14A2, C2 RDU 70 28A2, C2 CC 67 37A2, C2 RMA 62 39A2, C2 KB ≤ 5 33C2, AR3-A3-C1 YA≤ 10 62C2, AR3-A3-C1 MP ≤ 5 20C2, AR3-A3-C1 D. Scandella et al.Blood 1997; 89; 3663-71

35. Neutralization Assay of Plasmas from 23 Hemophilic Inhibitor Patients Treated with Plasma-Derived FVIII (suite) Inhibitor % Neutralization by FVIII RegionMajor Inhibitor Epitope(s) Domain(s) A2C2 GK ≤ 531C2, AR3-A3-C1 MU ≤ 515C2, AR3-A3-C1 SCN ≤ 533C2, AR3-A3-C1 GK ≤ 540C2, AR3-A3-C1 RI≤ 1026C2, AR3-A3-C1 JR 3129A2, C2, AR3-A3-C1 WG 4226A2, C2, AR3-A3-C1 HG 5025A2, C2, AR3-A3-C1 GK 1228A2, C2, AR3-A3-C1 MS 2730A2, C2, AR3-A3-C1 GK 4019A2, C2, AR3-A3-C1 D. Scandella et al.Blood 1997; 89; 3663-71

36. Neutralization Assay of Plasmas from 11 Hemophilic Inhibitors Treated only with Recombinant Factor VIII Inhibitor No. % Neutralization by FVIII Region Epitopes of Major inhibitor(s ) A2C2 Domain(s) R7611 74 27A2, C2 R1911 37 74A2, C2 R1113 85 26A2, C2 R3511 49 41A2, C2 R7717 ≤ 5≤ 10AR3-A3-C1 R2113 27 54A2, C2, AR3-A3-C1 K66 ≥ 95 ≤ 5A2 K126 68 51A2, C2 K129 24 26A2, C2, AR3-A3-C1 K147 37 43A2, C2, AR3-A3-C1 K184 28 51A2, C2, AR3-A3-C1 Scandella et al. Blood 1997

37. vWF/PL Factor VIII Inhibitory Antibodies FIXavWFFIXaFX COOH 2332217320251648719336 NH 2 The Bethesda assay does not see antibodies against the B domain Modified from Oldenburg et al. Haemophilia. 2002;8(suppl 2):23-29.

38. Vincent AM, Lillicrap D, Boulanger A, Meilleur C, Amesse C, St-Louis J, Rivard GE. Non-neutralizing anti-FVIII antibodies: different binding specificity to different recombinant FVIII concentrates. Haemophilia 2009;15:374-6 ELISA with 3 coating antigens: Kogenate FS, Advate, ReFacto Bethesda + : ≥ 0.6 BU ELISA + : AU ≥ 3 AU: SD of the mean OD of 6 N plasma Number of AU for a given specimen = OD of the specimen – (OD of blank + mean OD of 6 N plasma) SD of the mean OD of 6 N plasma

39. Subjects with Acquired Hemophilia A 25105< 0.6*Lymphoma 76M 46861Pancreatitis 36F 91851057Idiopathic 87F 62605210Idiopathic 63M 81 8430Idiopathic 83M 90100134160 Autoimmune vasculitis 49M 941411331984Colon cancer 71 M AU BUAssociated condition Age Gender ReFacto Advate Kogenate FS ELISA BETHESDA *Subject known to have been Bethesda positive in the past

40. *Subject known to have been Bethesda positive in the past. Subjects with Congenital Hemophilia A and Inhibitor

41. 0.223 < 0.60,03 0.124< 0.6< 0,01 0310< 0.6< 0,01 2321 < 0.6< 0,01 -0.54436 < 0.6< 0,01 0.26366< 0.6 0,05 AU BUBaseline FVIII ReFactoAdvateKogenate FS ELISA BETHESDA Subjects with Congenital Hemophilia A without Inhibitor

42. Contributing Factors to Development of FVIII Inhibitors Genetic Related to factor VIII Unrelated to factor VIII Environmental Conditions of treatment Therapeutic product

43. Contributing Factors to Development of FVIII Inhibitors Genetic Related to factor VIII Unrelated to factor VIII Environmental Conditions of treatment Therapeutic product

44. Family Incidence Malmo International Brother Study 388 hemophilia A Overall inhibitor incidence 32% Risk if inhibitor in family 48% Risk if inhibitor in brother 78% Astermark J, et al. Haemophilia 2001;7:267-72

45. Race and Inhibitor Incidence in Severe Hemophilia A Meta-Analysis on PUP Studies(Scharrer et al. 1999) Kogenate (Lusher et al. 1997) Recombinate (Gruppo et al. 1998) U.S. retrospective study (Addiego et al. 1994) Caucasians inhibitors: 51/198 25.8% African origin inhibitors: 14/27 51.9% MIBS (Astermark et al. 2001) Caucasians inhibitors 27.4% African origin inhibitors 55.6%

46. Contributing Factors to Development of FVIII Inhibitors Genetic Related to factor VIII Unrelated to factor VIII Environmental Conditions of treatment Therapeutic product

47. 364 1 7.4%Small deletion or insertion 35.7%Large deletion or insertion 4.3%Missense 38.4%Nonsense 34.4% Intron 22 inversion Inhibitor Prevalence Type of Mutation 1 Schwaab R et al. Thromb Haemost. 1995; 74: 1402-1406 2 Oldenburg J and Pavlova A. Haemophilia 2006;12 (Suppl.6)15-22 Factor VIII Mutations and Inhibitor Development 753 2 21% 31% 5% 41% 16%

48. Nonsynonymous Single Nucleotide Polymorphisms of Factor VIII Gene Viel KR et al. NEJM 2009;360: 1618-27

49. Inhibitor of Factor VIII in Black Patients with Hemophilia Kevin R. Viel et al. NEJM 2009; 360: 1618-27 Nonsynonymous Single Nucleotide Polymorphisms of Factor VIII Gene and Risk of Inhibitor Development

50. Contributing Factors to Development of FVIII Inhibitors Genetic Related to factor VIII Unrelated to factor VIII Environmental Conditions of treatment Therapeutic product

51. Non-Factor VIII Genetics and Inhibitor Development OR 19.2 (95% Cl, 2.4 – 156.5; P < 0.001) for presence of TNFA – 308 A/A among patients with severe hemophilia A and inhibitors. Polymorphisms in the TNFA gene and the risk of inhibitor development in patients with hemophilia A. Astermark J. et al. Blood 2006;108:3739-45 OR 5.4 (95% Cl 2.1-13.7 : P < 0.001) for the presence of allele 134 among patients with severe hemophilia A and inhibitors. Polymorphisms in the IL10 but not in the IL1Beta and IL4 genes are associated with inhibitor development in patients with hemophilia A. Astermark J. et al. Blood 2006;107:3167-72 OR 0.3 (95% CI 0.1-0.8; P = 0.012) for the presence of the T allele among all patients with severe hemophilia A and inhibitors. Polymorphisms in the CTLA-4 gene and inhibitor development in patients with severe hemophilia A. Astermark J. et al. J Thromb Haemost 2007;5:263-5

52. Contributing Factors to Development of FVIII Inhibitors Genetic Related to factor VIII Unrelated to factor VIII Environmental Conditions of treatment Therapeutic product

53. –Associated inflammatory reactions –Continuous infusion 1 –Age at first treatment 2 –Episodes of intensive treatment 1 Sharathkumar 2003; 2 van der Bom 2003 Contributing Factors to Development of FVIII Inhibitors: Conditions of Treatment

54. Contributing Factors to Development of FVIII Inhibitors Genetic Related to factor VIII Unrelated to factor VIII Environmental Conditions of treatment Therapeutic product

55. Contributing Factors to Development of FVIII Inhibitors:Therapeutic Product In vivo human data In vivo animal data In vitro data

56. Influence of the type of factor VIII concentrate on the incidence of factor VIII inhibitors in previously untreated patients with severe hemophilia A. Goudemand J. et al. Blood 2006; 107: 46-51 Cumulative incidence of inhibitors at 50 ED rFVIII 32.3% VWF-FVIII 10.3% Univariate analysis Multivariate analysis p < 0.05 p < 0.009* *adjusted for intron 22, ethnic origin and age at first exposure n = 62 on plasma VWF-FVIII n = 86 on rFVIII (62 Recombinate ® ; 24 Kogenate ® ) Median ED 120 86

57. Early factor VIII exposure and subsequent inhibitor development in children with severe haemophilia A. Chalmers EA.et al. Haemophilia 2007; 13: 149-155 N = 172 on rFVIII N = 132 on plasma vWF- FVIII Cumulative incidence of inhibitors at 50 ED rFVIII = 27 % vWF-FVIII = 14 % P=0.009

58. Kreuz Courter Wight J, Paisley S. The epidemiology of inhibitors in haemophilia A: a systematic review. Haemophilia 2003; 9: 318-35 Cumulative risk of inhibitor The Proof of the Poutine is in the Poutine * * * Addition to the original figure

59. Treatment of Subjects with Hemophilia A and FVIII Inhibitors: General Comments Control of hemostasis: Human FVIII, not effective except in low titres Porcine FVIII, no longer available Activated Prothrombin Concentrates (FEIBA) Recombinant Activated FVII (NiaStase) Elimination of inhibitors: Immune Tolerance Induction Immunosuppression/Immunomodulation

60. Summary Acquired hemophilia A is a rare but serious condition with a peak of incidence in elderly subjects; it could be life threatening but usually responds well to appropriate treatment Factor VIII inhibitor development is a common and severe complication of hemophilia A treatment There are many genetic and environment factors that contribute to development of inhibitors in hemophilia A There are some reasonably effective treatment for the control of hemostasis and the eradication of inhibitors: both aspects of treatment are extremely expensive Better strategies for prevention and treatment of this condition are badly needed; more research is needed…

61. An Interesting Bed Time Story…

62. Interesting???