1. Rare Bleeding Disorders (RBDs) Flora Peyvandi Hemophilia and Thrombosis Center, University of Milan ISTH Educational Course: Advanced Course in Haemostasis and Thrombosis Moscow, September 17-19, 2014

2. Bleeding disorders Hemophilia A and B (prevalence 1:5,000 and 1:30,000) von Willebrand disease (VWD) (prevalence 1:100) Platelet disorders (prevalence 1:1,000,000) Rare bleeding disorders (deficiency of fibrinogen, FII, FV, FV+FVIII, FVII, FX, FXI and FXIII) (prevalence ranging from 1:500,000 for FVII deficiency to 1:2 million for FII and FXIII deficiencies) Erhardtsen http://www.springerreference.com/index/chapterdbid/33723 2012 Poon M-C et al. Vasc Health Risk Manag 2007;3:655–64 Peyvandi F et al. Semin Thromb Hemost 2009;35:349–355 Bolton Maggs P Pediatr Blood Cancer 2013;60(Suppl 1):S37-40 Inherited bleeding deficiencies affect 65,000 people in Europe RBDs represent 3-5% of all inherited coagulation deficiencies Usually transmitted as autosomal recessive traits In some countries, prevalence is higher due to consanguineous marriage

3. Epidemiology Data collection: registers Clinical manifestations Phenotype diagnosis Association between laboratory phenotype and clinical severity Genotype characterization Treatments and available products Outlines of discussion on RBDs

4. Epidemiology WFHWORLDEU N° of countries 10928 N° of people identified with HA and B 172,37344,494 N° of people with VWD 66,14434,518 N° of people with rare coagulation disorders 17,5198,959 N° of people with platelet disorders 11,4302,716 N° of people with unknown type of hemophilia or coagulation disorder 4,1921,763 Total271,65892,450 Funded by the European Community - action programme for public health (DG SANCO) Survey updated to December 2013 (http://www1.wfh.org/publications/files/pdf-1574.pdf)

5. World map of bleeding disorders other than hemophilia and VWD

6. Bleeding disorders in Russia Uncertain diagnosis

7. Data collection: registers Improving knowledge on prevalence Observing course of disease Understanding variations in symptoms Associating the laboratory phenotype and clinical picture Monitoring treatments Assessing effectiveness of existing treatments F. Peyvandi, M. Spreafico. Blood Trand 2008; 6(Suppl. 3):s34-8. Soucie JM et al. Am J Prev Med 2010; 38:S475-81. Orphanet Report Series – Rare Disease collection. Rare Disease Registries in Europe. Jan 2014

8. National registries in EU Belgian patient registry for rare bleeding disorders FranceCoag: French prospective cohort of patients affected with haemophilia or severe form of other hereditary hemorrhagic diseases except platelet disorders Austrian Haemophilia Registry Serbian registry of patients with rare bleeding disorders Slovenian registry of patients with inherited bleeding disorder EMOWEB: Italian registry of coagulopathies Registry of inherited bleeding disorders in Emilia Romagna region UK - NHD: national haemophilia database DHR: German Haemophilia Registry European network of Rare Bleeding Disorders (EN-RBD) PRO-RBDD : Prospective data collection on patients with coagulation fibrinogen and factor XIII deficiencies

9. Clinical manifestations Highly variable bleeding tendency The most typical symptoms are -mucosal tract bleedings -excessive bleeding at the time of invasive procedures, -Bleeding at labour and delivery in women, bleeding after circumcision in affected boys CNS bleeding, umbilical cord bleeding, hemarthroses and soft tissue haematomas are frequent in fibrinogen, FVII, FX and FXIII deficiencies GI bleeding occurs mainly in FX deficiency Peyvandi F et al. Semin Thromb Hemost 2013;39:579-84; Peyvandi F et al. Hematology Education Book 2010;4(1):63- 68; Karimi M et al. Haematologica 2008;93:934-938; Mariani G et al. Thromb Haemost 2005;93:481-7 Heterozygous patients (coagulant activity level >30%): – 20% have mucocutaneous bleedings and bleeding during surgery – Post-traumatic hemarthrosis and hematomas are rarely reported in FVII and FX deficiencies – Menorrhagia, spontaneous abortion and bleeding during vaginal delivery were reported in roughly 20% of women with all type of deficiencies

10. Menstruation Pregnancy Delivery Bleeding during pregnancy Miscarriage Post-partum hemorrhage Menorrhagia Age Women with RBDs: clinical problems Kadir R and James A WFH Reproductive health in women with bleeding disorders 2008

11. Bleeding disorder MenorrhagiaAbortionPost partum haemorrhage Rare bleeding disorders 35-78% (Lak 1999, Siboni 2009, James 2010, Khair 2013, Mariani 2013, Shetty 2014, Napolitano 2014) Afibrinogenemia 30-50% Hypofibrinogenemia 29% Dysfibrinogenemia 38% FXI 10% FXIII 50-63% (Goodwin 1989, Haverkate 1995, Burrows 2000, Lak 2003, Myers 2007, Mensah 2011) Hypofibrinogenemia 45% FV 76% FVII,FX,FXI,FXIII high rates (Goodwin 1989, Noia 1997, Kadir 1998, Burrows 2000, Kulkarni 2006, Teixeira 2012, Baumann Kreuziger 2013) Haemophilia A and B carriers 10-67% (Siboni 2009, James 2010, Khair 2013) 14-20% (Chi 2008, Knol 2010) the most significant cases in women with FVIII <50% and no hemostatic coverage during labor and postpartum (Kadir et 1997) VWD 32-100% (Lak 2000, Siboni 2009, James 2010 and 2011, Khair 2013) 15-25% (Kadir 1998, Kirtava 2003) 16-29% primary 20-29% secondary (Ramsahoye 1995, Kadir 1998) Glanzmann’s thrombasthenia 50-98% (James 2010, Khair 2013) 11% (Siddiq 2011) 34% primary 24% secondary (Siddiq 2011) Prevalence of obstetric-gynecological symptoms Full references in slide notes

12. Anemia due to iron deficiency Dysmenorrhea Hysterectomy: effective and definitive treatment - However, it is a major surgical procedure with significant physical and emotional complications and social and economic costs Maternal death (post-partum bleeding) Complications in women 12 Peyvandi F Expert Opinion 2014

13. Geographical variation in causes of maternal death Khan K et al. Lancet 2006;367:1066–74 *Represents HIV/AIDS. †Represents embolism. ‡Represents ectopic pregnancy. §Represents anaemia.

14. Phenotype diagnosis aPTTPT TT TR D-Dimer Fibrinolysis VII Tissue factor + calcium Extrinsic pathway Trombin Fibrinogen Fibrin Reptilase (XII) XI IX VIII Kallikrein + Kininogen + calcium Intrinsic pathway X II V XIII

15. Screening tests aPTT and PT tests: Usually sensitive enough to detect abnormal coagulant factor actiivity: fibrinogen, FII, FV, FV+FVIII, FVII, FX and FXI May be normal in mild deficiencies, especially fibrinogen and FII Normal in FXIII deficiency Mixing tests: Correction of the abnormality indicates that a factor deficiency No correction indicates presence of an inhibitor TT test: Required for the diagnosis of fibrinogen deficiency Interpretation in relation to the personal and family history Kitchen S, McCraw A, Echenagucia A. Diagnosis of Hemophilia and Other Bleeding Disorders. A LABORATORY MANUAL. Second Edition. WFH 2010

16. Factor activity level One-stage coagulation assays: - aPTT based – FXI deficiency - PT based – FII, FV, FVII, FX deficiencies Fibrinogen assays FXIII assays Factor antigen level Immunochemical methods Specific factor assays Not mandatory except for fibrinogen and FII deficiencies. Kitchen S, McCraw A, Echenagucia A. Diagnosis of Hemophilia and Other Bleeding Disorders. A LABORATORY MANUAL. Second Edition. WFH 2010 Peyvandi F et al. Semin Thromb Hemost 2009; 35(4): 349-55.

17. Fibrinogen activity assays Clauss fibrinogen assay PT-derived Fg assay Recommendations Clauss fibrinogen assay remains the reference method PT-Fg assays are not recommended for routine clinical use Mackie IJ et al. Guidelines on fibrinogen assays. Br J Haematol 2003; 121: 396-404. Levels measured by different assays may vary significantly Uncertainty in assigning the severity of the hypofibrinogenemia

18. FXIII activity assays Clot solubility assay Functional FXIII activity assays: - Photometric assays - Fluorimetric assays - Putrescine incorporation assays Note Association in patients with FXIII < 5% is difficult to interpret and may not be accurate Clot solubility assay is no longer recommended since it is qualitative, poorly standardized, and detects only severe FXIII deficiency Schroeder V et al. FXIII deficiency: An Update. Semin Thromb Hemost 2013; 39: 632-41 Recommendations by the FXIII and Fibrinogen SSC subcommittee of the ISTH 1. First-line screening test Quantitative functional FXIII activity assay If FXIII activity is decreased 2. Establish subtype of FXIII deficiency Measurement of antigen concentration (FXIII-A2B2, FXIII-A, FXIII-B) Only in case of suspected acquired FXIII deficiency: 3. Detection of autoantibodies Mixing study (detection of neutralizing antibodies against FXIII-A) Binding assay (detection of nonneutralizing antibodies) For research purpose only: Characterization of molecular genetic defect

19. Phenotype diagnosis: a summary

20. Problems with clotting assays Despite the existence of recommendations, a number of problems still remain, particularly for some deficiencies (Fibrinogen and FXIII): ─ Assay accuracy at low factor levels ─ Lack of standardization – high inter-laboratory variability

21. Laboratory phenotype and clinical severity 3 years of data collection 13 European centers from 11 countries 489 patients registered in the database

22. Assigned categories of clinical bleeding severity Peyvandi F et al J Thromb Haemost2012;10:615-621

23. Linear regression analysis: adjusted for age, gender, and country where diagnosis was made Excellent correlationGood correlationNo correlation Factor activity (Y) = Beta* Bleeding severity (X) + Constant Results

24. SSC – ISTH recommendations on RBDS Coagulant factor deficiency Laboratory phenotype Severe (coagulant activity associated with spontaneous major bleeding) Moderate (coagulant activity associated with minor spontaneous or triggered bleeding) Mild (coagulant activity associated with a mostly asymptomatic disease course) Coagulant activity Fibrinogenundetectable clot≤0,1 g/L>0,1 g/L FIIundetectable activity≤10%>10% FVundetectable activity10%≥10% FV+FVIII<20%20-40%>40% FVII<10%10-20%>20% FX<10%10-40%>40% FXIIIundetectable activity30%≥30%

25. Do clotting factor levels predict severity? The relationship between coagulation factor activity level and clinical bleeding severity is heterogeneous A strong association exists for fibrinogen, FX and FXIII deficiencies No association for FXI deficiency The minimum factor levels to prevent spontaneous major bleeding is different in various deficiencies It is not appropriate to use a single criterion of classification for all types of RBDs

26. Global hemostasis assays Tests investigating the global hemostatic capacity could help: -to predict clinical phenotype -to determine the effectiveness of therapies -to monitor treatment particularly FXI deficiency where standard assays fail to correlate with bleeding risk TGT TEG

27. Recently, these tests have been used to evaluate hemostasis in patients with RBDs, specifically FV and FXI deficiency However test standardization to reproduce reliable measurements facilitated by standardized pre-analytical and analytical procedures is required before widespread clinical use. In global hemostasis assays, a number of barriers may prevent implementation and quality assurance. Quality assurance studies have observed that regular proficiency testing is needed to ensure accuracy of such methods Al Dieri R et al, Thromb Haemost. 2002; Strey RF et al, Pathophysiol Haemost Thromb. 2005; Schols SE et al, Thromb Haemost 2008; Rugeri L et al, Haemophilia 2010; Guegen P et al, Br J Haematol 2011; Riddell A et al, Thromb Haemost 2011: Van Geffen M et al, Haemophilia. 2012; Spiezia L et al, Haemophilia. 2012; Livnat T et al. Blood Coag Fibrynol 2012

28. Genotype characterization Currently based on the mutation search in the genes that encode each corresponding coagulation factor Exceptions: -combined deficiency of coagulation FV and FVIII (mutations in genes for FV and FVIII intracellular transport – MCFD2 and LMAN1) -combined deficiency of the vitamin-K-dependent proteins (mutations in genes for post-translational modifications and vitamin K metabolism (GGCX and VKORC1) Pattern of inheritance is autosomal recessive for all RBDs, except for some cases of FXI, where some missense mutations were shown to exert a dominant negative effect, and of dysfibrinogenemia

29. 5-10% of patients remain with no identified genetic defect The use of next generation sequencing (NGS) might help to identify novel pathways in coagulation disorders While the potential of these genome-wide strategies is indisputable, these approaches have yet to be utilized in the analysis of RBDs for which causative mutations remain elusive Type of deficiencyGeneIndels (%)Missense (%)Nonsense (%)Splicing (%)3'-5' UTR (%) Fibrinogen FGA FGB FGG 20,957,311,57,52,8 FIIF21377,85,53,70 FVF527,348,512,911,30 FV+VIII LMAN1508,820,6 0 MCFD227,3504,518,20 FVIIF712,362,27,811,56,2 FXF1010,5801,97,60 FXIF1110,47010,58,20,9 FXIIIF132947,19,1140,8 Vit-K factors GGCX10600300 VKORC10100000 Peyvandi F et al. Blood 2013;122:3423-31. Mutations causing RBDs

30. Treatment of RBDs Non-transfusional treatment: antifibrinolytic amino acids fibrin glue oestrogen-progesterone preparations desmopressin (DDAVP) Replacement therapy: FFP Cryoprecipitate (also virus-inactivated) prothrombin complex concentrates (PCC) single factor concentrates (plasma-derived or recombinant) Flora Peyvandi

31. Registered RBDs concentrates Registry of Clotting Factor Concentrate, Ninth Edition Mark Brooker, WFH

32. Novel PD concentrates: ongoing studies Fibrinogen ─ NCT01575756 – Octapharma (safety/efficacy, phase 2) ─ NCT02065882 – Biotest (PK/safety/efficacy, phase 1-2) ─ NCT00916656 – CSL (Phase 3b non inferiority safety/efficacy - withdrawn prior to enrollment) ─ NCT02094430 – LFB (Phase 2-3 - clinical pharmacology/efficacy/safety in paediatric patients) ─ FIBGrifols FX (high purity) ─ NCT01721681 – BPL (prophylaxis in <12 years, phase 3) FV ─ Only recently a concentrate has been developed. Preclinical studies are currently being performed for the orphan drug designation application (EMA and FDA)

33. Deficient factor Plasma half-life Recommended trough levels On demand dosages Recommended trough levels to maintain asymptomatic state after publication of the EN-RBD results Fibrinogen2–4 days0.5-1g/L Cryoprecipitate (15-20 mL/kg) SD-treated plasma (15–30 mL/kg) Fibrinogen concentrate (50–100 mg/kg) 1 g/L Prothrombin3–4 days20–30% SD-treated plasma (15–25 mL/kg) FIX concentrate and PCC (20–40 units/kg) >10 % Factor V36 hours10–20%SD-treated plasma (15–25 mL/kg)10% Factor V and factor VIII FV 36 hours FVIII 10–14 hours 10–15%As for FV40% Factor VII4–6 hours10–15% FVII concentrate (30–40 mL/kg) PCC (20–30 units/kg) rFVIIa (15–30 μg/kg every 4–6 hours) >20% Factor X40–60 hours10–20% SD-treated plasma (10–20 mL/kg) PCC (20–30 units/kg) FX/FIX concentrate (10–20 units/kg) >40% Factor XI50 hours15–20% SD-treated plasma (15–20 mL/kg) FXI concentrate (15–20 units/kg) 15-20% Factor XIII9–12 days2–5% Cryoprecipitate (2–3 bags) SD-treated plasma (3 mL/kg) FXIII concentrate (till 50 units/kg for high hemorrhagic events) rFXIII-A (35 units/kg) 30% Vitamin K dependent Vitamin K (10 mg) IV. or SC. for minor bleeding PCC (20–30 units/kg) with vitamin K (5–20 mg) for severe bleeding or major surgery FFP 15–25 ml/kg is an alternative to PCC No data available On-demand treatment Peyvandi F. Haemophilia 2002; Mannucci PM. Blood. 2004; Peyvandi F et al J Thromb Haemost2012;10:615-621; Mumford AD, Br J Haematol 2014

34. Prophylaxis The choice of prophylaxis is related to: – the frequency of bleeding – the risk of severe spontaneous bleeding – the risk of long-term disabilities associated with the occurrence of bleeding in a particular region of the body despite on-demand treatment (e.g., CNS, GI and joint bleedings)

35. Deficient factor Recommended trough levels Reported dose schedule for successful long-term prophylaxis Notes ProductsDoseFrequency Fibrinogen0.5-1g/L Cryoprecipitate 1 unit3 times/week Afibrinogenemic patients with recurrent life-threatening bleedings or undergoing surgeries 3 unitsEvery 7–10 days Fibrinogen concentrate 30–100 mg/kgEvery week Prothrombin20–30%PCC20–40 units/kg1/ week— Factor V10–20%SD-treated plasma20-30 mL/kg2/week Only in patients with life- threatening bleedings, as CNS Factor V and Factor VIII 10–15%No data— Factor VII10–15% SD-treated plasma10–15 mL/kg2/week Prevention of bleeding during surgery or in children with recurrent hemarthrosis or CNS pdFVII10–40 units/kg3 times/week rFVIIa20–40 µg/kg2-3 times/week Factor X10–20% PCC20–40 units/kg2-3 times/week Patients with recurrent life- threatening bleedings or undergoing surgeries FX/FIX concentrate 20–40 units/kg 1-2 times/week Factor XI15–20%No data— Factor XIII2–5% Cryoprecipitate2 unitsEvery 3 weeks Highly recommended in severe patients SD-treated plasma15–20 mL/kgEvery 4–6 weeks FXIII concentrate10-40 units/kgEvery 4–6 weeks rFXIII-A35 units/kgEvery 4 Vitamin K dependent Vitamin K5-20 mg1/week orally — Mannucci PM. Blood. 2004; Castaman G. Blood Transfusion 2008; Mumford AD, Br J Haematol 2014 Prophylactic treatment

36. Due to the RBDs rarity, little information is available on the optimal management of patients with these disorders, particularly on long- term prophylaxis In addition, the technical limitations of laboratory testing, particularly in patients with factor activity <5%, make difficult to adopt the appropriate prophylactic treatment To overcome these limitations, new strategies are required: – creation of global partnerships – networking between treatment centers – increasing the support provided by public health organizations – Prospective data collection – Better and more sensitive assays Conclusion

37. The needs SPECIALISED CARE IN HOSPITALS Institutions FUNDING AND COORDINATION Patients association TRAINING AND SUPPORT Companies NEW PRODUCTS CLINICAL AND SCIENTIFIC RESEARCH Prevention Early diagnosis Assays development Data collection Clinical trials Guidelines DATA COLLECTION

38. “The PRO-RBDD project” Write to info@rbdd.eu Visit our website eu.rbdd.org

39. ACKNOWLEDGMENTS European Commission Executive Agency for Health and Consumers (EAHC) European Commission Executive Agency for Health and Consumers (EAHC) Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico University of Milan Luigi Villa Foundation Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico University of Milan Luigi Villa Foundation The European Hemophilia Network project (EUHANET) Novo Nordisk Health Care AG (for Extra-EU support All patients and clinicians who are collaborating with us