International Society Laboratory Hematology Milan, May Current and Emerging Approaches for Assessing von Willebrand disease (VWD) in 2016

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1 International Society Laboratory Hematology Milan, May 2016 Current and Emerging Approaches for Assessing von Willebrand disease (VWD) in 2016 Augusto B. FEDERICI Hematology and Transfusion Medicine Luigi Sacco University Hospital, University of Milan

2 Disclosures A.B. Federici Employment Research support Scientific advisory board Consultancy Speakers bureau Major stockholder Patents Honoraria Travel support Other NONE NONE BAXALTA, CSL-BEHRING, GRIFOLS, KEDRION- LFB, OCTAPHARMA, WERFEN-IL NONE BAXALTA, CSL-BEHRING, GRIFOLS, KEDRION- LFB, OCTAPHARMA, WERFEN-IL NONE NONE BAXALTA, CSL-BEHRING, GRIFOLS, KEDRION- LFB, OCTAPHARMA, WERFEN-IL NONE NONE

3 History of VWD 1959 Nilsson IM et al, Acta Med Scand; 164:26378 February 1926 E.A. von WILLEBRAND 1971 Zimmerman TS et al, JCI; 50:24454

4 VWD: Clinical and Lab Diagnosis Background 2016 (1) VWD is the most common inherited bleeding disorder and is due to quantitative (VWD3 & VWD1) and/or qualitative (VWD2A, VWD2B, VWD2M, VWD2N) defects of VWF: in severe forms of VWD3, VWD1 & VWD2N FVIII is also reduced Despite the complex and heterogeneous nature of the VWF defects, nowadays all VWD types can be managed efficiently in most patients.

5 How Does VWF Work A Intact vessel wall B Damaged vessel wall C Platelet plug formation collagen fibrils endothelial cell torque matrix VWF plasma VWF platelet initial platelet tethering GpIba platelet rolling extracellular matrix Non activated aiibb3 activated aiibb3 platelet activation and adhesion

6 VWD: Clinical and Lab Diagnosis Background 2016 (2) Correct VWD diagnosis and classification cannot be always available in several Centers to provide the best therapeutic approach. Differently from HA easily classified (severe, moderate, mild) by baseline FVIII levels, clinical severity of different VWD forms is not well defined within types so far.

7 List of Clinical and Laboratory Tools Used for VWD Diagnosis More Than One Test Always Needed Basic Tests Patient & Family History Bleeding Score Bleeding Time PFA 100 PTT FVIII:C Specific tests VWF:Ag VWF:RCo VWF:CB VWF:RCo/Ag VWF:CB/Ag VIII:C/VWF:Ag Additional tests RIPA test VWF:FVIIIB Multimeric analysis Molecular genetics

8 Classification of VWD Types Based on Several Assays NIH US Guidelines

9 Heterogeneity of VWD Patients Based on Cohort Studies Bleeders versus Non Bleeders Heterogeneous VWD Cohort: Italian Registries (RENAWI) VWF:RCo <10 U/dL + FVIII:C < 20 U/dL VWD Severe Forms: VWD1, VWD2A, VWD3 VWD Moderate Forms VWD1, VWD2B, VWD2M, VWD2N VWF:RCo U/dL + FVIII:C U/dL Bleeders: VWD1 Mild Forms Non bleeders: Low Levels of VWF VWF:RCo U/dL + FVIII:C U/dL Diagnosed VWD: the tip of the iceberg? Federici AB et al, Blood 2014; 123:

10 Criteria for Correct Diagnosis (Bleeding History, Low VWF Activity, Inheritance) Platelet GPIba A1 Collagen VI Heparin Sulphatide VWF:RCo C C A2 ADAMTS 13 C C A3 VWF:CB SubEndothelium Collagen I and III I I:1 W Tosetto et al JTH 2006 II W III W III:3 W

11 Clinical and Lab Diagnosis of VWD Outlines Definitions and classification of VWD Clinical parameters for VWD First-level laboratory tests Second-level laboratory tests Additional and automatic assays Severe or mild VWD types: outcomes

12 VWF:RCo (Platelet Aggregometric Method) Normal Fixed Platelets + Patient Plasma Dilutions + Ristocetin [1 mg/ml] Parameters That Influence the VWF:RCo VWF:Ag Multimeric Pattern (2A & 2B) Mutations in the A1 Domain (2M)

13 VWF:RIPA (Ristocetin Induced Platelets Agglutination) Normal VWD 2A VWD 2B Transmission Ristocetin mg/ml 1 min. 1 min. 1 min. Platelet Rich Plasma from Patients + RISTOCETIN [ mg/ml] Ruggeri ZM et al, JCI 1978

14 VWF Multimeric Analyses (Kindly Provided by U. Budde)

15 VWF:FVIIIB (Binding Assay - ELISA) 1500 Normal Bound FVIII:C 750 Heterozygous R854Q Homozygous R854Q VWF Immobilized

16 VWF Pro-Peptide Usefulness of this assay The assay for VWF pro-peptide measures in circulation the amount of protein cleaved from PRE-PRO-VWF synthesized in Endothelial Cells Increased VWF:pp/VWF:Ag ratio identifies those patients with shortened VWF survival Shortened VWF survival can also be observed during the infusion trial with DDAVP

17 DDAVP Challenge Test: An important assessment at diagnosis Ruggeri et al, Blood 1982 Federici AB et al, Blood 2004; 103:

18 Clinical and Lab Diagnosis of VWD Outlines Definitions and classification of VWD Clinical parameters for VWD First-level laboratory tests Second-level laboratory tests Additional and automatic assays Severe or mild VWD types: outcomes

19 VWF:CB (Collagen Binding Activity) Evaluates VWF capability to bind to collagen - Mimics VWF interaction with sub-endothelial collagen matrix at site of vascular injury Dependent on VWF multimeric size - Collagen binds more readily with HMWM - Studies show VWF:CB can serve as a surrogate measure for presence of HMWM When tested with VWF:Ag and VWF:RCo can improve differentiation between VWD types 1, 2A, 2B and 2M

20 VWF:CB (ELISA) Anti-VWF- HPR Conjugated Polyclonal Antibody 95% type I and 5% type III Collagens (Nycomed-Horm) Collagen Diluted Plasma VWF A3 anti VWF VWF A3 Add substrate Read OD 492 nm anti VWF VWF A3 Methods not standardized Type I collagen is more sensitive to discriminate VWD types and is more sensitive to HMWM but it has poor reproducibility A B C D Federici et al, Haematologica 2004 Parameters That Influence the VWF:CB Multimeric Pattern Mutations in the A1 (2B) A3 (2M)

21 Platelet Dependent-VWF Activity (Nomenclature and Methodology) Bodó et al on behalf of ISTH-SSC-SC on VWF JTH 2014

22 VWF:RCo (Recent Automated Methods) Immunoturbidimetric Chemiluminescence HemosIL VWF:RCo Assay HemosILAcuStar VWF:RCo Assay Reaction Mechanism Latex particles coated with a recombinant fragment of platelet gp1b, through a monoclonal antibody, which binds VWF in the presence of ristocetin resulting in agglutination. Reaction Mechanism Latex particles coated with a recombinant fragment of platelet gp1b, through a monoclonal antibody, which binds VWF in the presence of ristocetin resulting in agglutination.

23 Gain-of-Function Mutant GPIb-Binding (VWF:GPIbM) Assays *Not available for sale in the US. Bodó et al on behalf of ISTH-SSC-SC on VWF JTH 2014

24 Flow chart for VWD Diagnosis Used in Italian Registry

25 Clinical and Lab Diagnosis of VWD Outlines Definitions and classification of VWD Clinical parameters for VWD First-level laboratory tests Second-level laboratory tests Additional and automatic assays Severe or mild VWD types: outcomes

26 Aims of the RENAWI-2 To evaluate the incidence, types and severity of spontaneous bleeding episodes requiring DDAVP and/or VWF concentrates in a large cohort of VWD patients To characterize bleeding phenotype in different VWD types and to predict clinical outcome in these patients.

27 Bleeding Phenotype in VWD Evidence-Based Methods Restricted Cubic Spline Curve Cox s Proportional Hazard Model Federici AB et al, Blood 2014; 123:

28 Bleeding Phenotype in VWD types Type of Bleeding Symptoms

29 DDAVP Treatment Biological Response to Predict Effective Therapy Biological Response in VWD1 No Biological Response in VWD2A (Ruggeri et al Blood 1982)

30 Clinical Use of DDAVP According to Clinical Phenotype (BS) of VWD

31 VWF/FVIII Concentrates In VWD3 patients VWF:RCo = VWF:Ag = FVIII:C = < 3 < <

32 Clinical Use of VWF/FVIII Concentrates According to Clinical Phenotype (BS) of VWD

33 Conclusions of RENAWI-2 The bleeding score (BS) correlates with VWF levels in VWD and helps to predict clinical outcomes in adult patients with VWD.

34 Clinical and Lab Diagnosis of VWD Current Perspectives in 2016 BS and of specific tests for VWF activities should be always used together in Adults to identify VWD patients with bleeding phenotype More specific and automatic lab tests should be available in most laboratory world-wide for a rapid VWD diagnosis of bleeding individuals

35 VWD: Clinical and Lab Diagnosis ACKNOWLEDGEMENTS AB BONOMI Hemophilia Thrombosis Center (Milan): P. BUCCIARELLI, P.M. MANNUCCI, F. PEYVANDI R. BADER, L. BARONCIANI, M.T. CANCIANI and VWD LAB Italian Association of Hemophilia Centers (AICE): G. CASTAMAN, M.G. MAZZUCCONI, M. MORFINI, A. ROCINO, F. RODEGHIERO, M. SCHIAVONI