EMOGLOBINURIA PAROSSISTICA NOTTURNA Lucio Luzzatto, Scientific Director, Istituto Toscano Tumori Professor of Haematology, University of Firenze. Firenze, ITALY SIE - Corso di Ematologia Cllinica Roma, 29 maggio 2007 PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA: definition Haemolytic anaemia with characteristic clinical triad: 1. Intravascular haemolysis 2. Thrombosis 3. Cytopenias CLASSIFICATION OF HAEMOLYTIC ANAEMIAS Intracorpuscolar Extracorpuscolar causes causes Hereditary •Haemoglobinopaties •Enzimopathies Familial HUS •Membranopathies •Other Acquired •Malaria Paroxysmal •Auto-immune Nocturnal Haemoglobinuria •Drug-induced (PNH) •Micro-angiopathic •Other PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA: classification 1. Hemolytic 2. Hemolytic/hypoplastic 3. Sub-clinical HAEMOGLOBINURIA Indicates intravascular haemolysis WORLDWIDEWORLDWIDE PNHPNH PATIENTSPATIENTS 132132 88 3838 77 3 3 22 Ham Test in a PNH Patient dAcdAc dSdS dAcdAc dHidHi pHipHi pAcpAc HH2 00 Control Patient RBC RBC QuickTime™ and a GIF decompressor are needed to see this picture. Flow-cytometry Analysis of Red Cells from Patients with PNH Control Control Control c o u n t s c o u n t s C.J M.B. R.K PNH III PNH II PNH III normal normal PNH II normal CD59 PROTEINS DEFICIENT ON PNH BLOOD CELLS CD55 B cells CD24 CD55 CD58* CD58* CD59 CD59 CD48 PrPC PrPC CD73 CDw108 AChE JMH Ag Dombroch Hematopoietic HG Ag RBC Stem Cell T cells CD55 CD58* CD59 CD48 CDw52 CD87 CD55 CDw108 PrPc CD58* ADP-RT CD73 CD59 CD90 CD109 CD109 CD59, CD90, CD109 CD16* PrPC GP500 Platelets Gova/b NK cells CD55 CD58* CD55 CD59 CD14 CD58* CD16 CD24 Monocytes CD59 CD48 CD66b PMN CD48 CD66c CD87 CDw52 CD109 CD157 CD14 CD55 CD58* CD59 CD48 CDw52 PrPc LAPNB1 PrPC CD16* p50-80 GPI-80 CD87 CD109 CD157 ADP-RT NA1/NA2 Group 8 PrPC GPI-80 CD16* QuickTime™ and a GIF decompressor All these proteins are GPI-linkedare needed to see this picture. GPI BIOSYNTHESIS IN MAMMALIAN CELLS PIG-A GPI8/PIG-K PIG-C PIG-M PIG-F PIG-F GAA1 PIG-H PIG-L PIG-W PIG-X PIG-V PIG-N PIG-B PIG-O GPI7 PIG-S PIG-P PIG-T GPI1/PIG-Q PIG-U PIG-Y DPM2 To proteins EtN EtN P P PEtN PEtN PEtN PEtN PEtN Lumen P P P P P P P P ER P P P Dol-P-Man NAc DPM1/PIG-E Cytoplasm Dol-P + GDP-Man DPM2 SL15 Mannose DPM3 Glucosamine SOME GENES OF MEDICAL IMPORTANCE ON THE X-CHROMOSOME PIG-A Paroxysmal Nocturnal Haemoglobinuria G6PD . Mutations in the PIG-A Gene Large deletions del 735 bp del exons 3-4-5 * * ** * * Null mutations * * * * * * 1 716 * 849 982 * *1189 1452 * * 1 2 3 4 5 6 Non-null mutations AvaI polymorphism 50 bp COMPLEXITYCOMPLEXITY OFOF THETHE COMPLEMENTCOMPLEMENT CCASCAASCADEDE Classical Pathway Activation Antibody/Antigen Complexes Lectin Pathway Potent Anaphylatoxin Chemotaxis C1q Activated C1 Activation C1q Cell Activation MBL C3 Convertase C5 Convertase C4+C2 C4b2a C4b2a3b Weak C5a Anaphylatoxin C3a Immune Complex C3 C3b C5 C5b C5b-9 and Microbial Opsonization C6 C7 C8 C9 C3b C3, C3H2O C3bBb C3bBb3b Cell Activation C3 Convertase C5 Convertase Factor B+D Lysis Alternative Pathway Activation Microbiological Membranes Bacterial LPS Immune Complexes Mammalian Cell Membranes MechanismMechanism ofof ActionAction ofof CD59CD59 C5b-8 C5b-8 C9 C9 CD59 with CD59 4ºC 37ºC C5b-8 C9 loosely bound C5b-8 C9 tightly bound C5b-8 (displaceable) (not displaceable) C9 C9 Poly-C9 without CD59 QuickTime™ and a GIF decompressor adapted from: Meri et al, 1990are needed to see this picture. PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA: features • Why acquired? • Why the thrombosis? Thrombosis:Thrombosis: Often Multiple, Venous, Abdominal QuickTime™ and a GIF decompressor are needed to see this picture. LIFE-THREATENING BUDD-CHIARI IN PNH QuickTime™ and a GIF decompressor are needed to see this picture. QuickTime™ and a GIF decompressor are needed to see this picture. TISSUE PLASMINOGEN ACTIVATOR IS A POTENT THROMBOLYTIC AGENT QuickTime™ and a GIF decompressor are needed to see this picture. QuickTime™ and a GIF decompressor are needed to see this picture. OTHER SITES OF THROMBOSIS IN PNH •Splenic vein • Inferior vena cava • Superior mesenteric vein • Inferior mesenteric vein • Renal veins • Subclavian vein (LL, personal observations) Cavernous Transformation of Portal Vein QuickTime™ and a GIF decompressor are needed to see this picture. ANGIOGRAPHIC PICTURE OF THE SPLEEN pre-Embolization post-Embolization QuickTime™ and a GIF decompressor are needed to see this picture. THE RISK OF THROMBOSIS IN PNH IS RELATED TO THE SIZE OF THE PNH CELL POPULATION (Hall, Richards & Hillmen, Blood, 102: 3587-3591, 2003) THE INCIDENCE OF THROMBOSIS IN PNH IS INFLUENCED BY GENETIC BACKGROUND 5 15 25 Araten et al., YEARS Thromb Haemost 93:88,2005. PNH is a severe acquired thrombophilic state. POSSIBLE MECHANISMS: • Impaired fibrinolysis (Urokinase receptor is GPI-linked) • Hyper-coaugulability (release of micro-particles with “thromboplastin activity”?) • Hyper-activity of platelets (hyper-sensitivity to activated complement?) • Increased release of TF by monocytes (hyper-sensitivity to activated complement?) A COMPROMISE PROTOCOL re Anticoagulant Prophylaxis 1. Haemolytic versus aplastic PNH 2. Thrombophilia screen (Prot S, Prot C, Factor V Leiden, ATIII, PT20010, MTHFR) 3. Large size of granulocyte PNH population 4. Previous episode of thrombosis A COMPROMISE PROTOCOL re Anticoagulant Prophylaxis 1. Haemolytic versus aplastic PNH 2. Thrombophilia screen (Prot S, Prot C, Factor V Leiden, ATIII, PT20010, MTHFR) 3. Large size of granulocyte PNH population 4. Previous episode of thrombosis With 4, must start anti-coagulants Depending on 1-3, consider starting anti-coagulants Etn-P Gl c N Etn-P Gl c N glycan glycan protein p proteinprotein p PIG-A p PIG-APIG-A PIG-A PIG-APIG-A inositol-PP inositol-Pinositol-Pinositol-PP P P nucleus nucleus q GPI anchor q GPI anchor Chr X Chr X protein protein GPI anchor GPI anchor ER ER NormalNormal cellcell NormalNormalPNHPNH cellcellcellcell QuickTime™ and a GIF decompressor are needed to see this picture. PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA: features • Why acquired? • Why the thrombosis? • Why the cytopenias? QuickTime™ and a GIF decompressor Erythroid hyperplasia Aplasia are needed to see this picture. PathogenesisPathogenesis ofof PNHPNH ((factsfacts andand speculationsspeculations)) Time PIG-A plus PIG-A minus PIG-A plus Auto immune blood cell blood cell damaged blood cell attack CAN PNH CURE APLASTIC ANEMIA? QuickTime™ and a GIF decompressor are needed to see this picture. SHARED TCR-β SEQUENCES IN PATIENTS WITH PNH TCR-β CDR3 Sequence ID Patients TRBV Vβ nDn Jβ TRBJ S1 7; 9 7.9 CASS LVGGPEQ YFGP 2.7 S2 6; 16 15 CATS RGRTQGLDYG YTFG 1.2 S3 8; 19 30 CA WEQVIA FFGQ 1.1 S4 5; 7; 9 15 CATS GIAGETQ FFGP 2.1 S5 5, 7, 9 15 CATS RVAGETQ YFGP 2.5 S5a 9 15 CATS RIGGETQ YFGP 2.5 S5b 2 15 CATS RTAGETQ YFGP 2.5 PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA: Issues in management. I. • Supportive treatment •BMT • Immunosuppression •Anti-C5 • Anticoagulant prophylaxis • Thrombolytic therapy • PNH and pregnancy TheThe FateFate ofof PNHPNH PatientsPatients 100 Allogeneic BMT (Genova) 75 Allogeneic BMT (IBMTR) 50 Natural History 25 Patients surviving (%) Patients surviving (%) 5 10 15 20 25 years QuickTime™ and a GIF decompressor are needed to see this picture. MULTIPLE ACTIONS OF ALLOGENEIC BMT IN PNH • Elimination of PNH clone(s) • Supply of normal hematopoietic stem cells (HSC) • Ablation of immune cells causing damage to non-PNH HSC THE ECULIZUMAB TRIAL, 2005- 2006 • In this randomized trial, eculizumab, a humanized monoclonal antibody against C5 that inhibits terminal complement activation, was compared with placebo as a treatment for PNH (Hillmen et al., New Eng J Med 355:1233,2006) TARGETINGTARGETING THETHE COMPLEMENTCOMPLEMENT CCASCAASCADEDE Classical Pathway Lectin Pathway Activation Activation Antibody/Antigen Complexes Potent Anaphylatoxin MBL Chemotaxis C1q Activated C1 C1q Cell Activation C3 Convertase C5 Convertase C4+C2 C4b2a C4b2a3b Weak C5a Anaphylatoxin C3a Immune Complex C3 C3b C5 C5b C5b-9 and Microbial XX Opsonization C6 C7 C8 C9 C3b C3, C3H2O C3bBb C3bBb3b Cell Activation C3 Convertase C5 Convertase Factor B+D Lysis Alternative Pathway Activation Microbiological Membranes Anti-C5 Bacterial LPS (ECULIZUMAB) Immune Complexes Mammalian Cell Membranes Baseline Characteristics of the Patients (Hillmen et al., New Eng J Med 355:1233,2006) Hillmen P et al. N Engl J Med 2006;355:1233-1243 ECULIZUMAB ARRESTS Levels of Lactate Dehydrogenase and PNH Type III Erythrocytes during Treatment with THE SELECTIVEEculizumab INTRAVASCULAR LYSIS OF PNH III RED CELLS (Hillmen et al., New Eng J Med 355:1233,2006) Hillmen P et al. N Engl J Med 2006;355:1233-1243 ECULIZUMAB CAN ABROGATE Kaplan-Meier Curves for the Time to the First Transfusion during Treatment THE NEED FOR BLOOD TRANSFUSION Hillmen P et al. N Engl J Med 2006;355:1233-1243 (Hillmen et al., New Eng J Med 355:1233,2006) ECULIZUMABChange in Fatigue Scores HAS from Baseline AN IMPACTto Week 26 ON THE QUALITY OF LIFE Hillmen P et al. N Engl J Med 2006;355:1233-1243 (Hillmen et al., New Eng J Med 355:1233,2006) Eculizumab appears to reduce the risk of thrombosis in PNH Pre- On treatment anti-C5 A Thrombo-embolic (TE) 124 3 events B Patient Years 1683 281 C TE event rate (A/B x 100) 7.4 1.1 n = 195 3 BONE MARROW DISORDERS; 2 PATHOGENETIC FACTORS; 1 PATHOGENETIC MECHANISM Failure of Somatic Target of auto- ‘normal’ genetic immune attack stem cells change PNH ++ PIG-A GPI or GPI- mutation linked protein MDS +/+++ Trisomy 8, ? trisomy 6, other AA +++ None? Universal A SYNOPSIS OF THE PATHOGENESIS OF PNH No clinical consequences