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 Damage to GPI-targeted PNH clone noxious Hematopoietic (GPI-proteins agent stem cells deficient) PIG-A mutation
Selective expansion of PNH clone(s)
CD59(-) Abnormal red cells platelets APLASTIC ANAEMIA Pancytopenia
Intravascular Thrombosis PNH haemolysis THANK YOU!
Ibadan S B ONI Firenze B O OSUNKOYA GIANGIACOMO GIANFALDONI G J F ESAN ALBERTO BOSI ELISABETTA ANTONIOLI Genova ROSARIO NOTARO LUCIA GARGIULO Napoli ALESSANDRO POGGI BRUNO ROTOLI GIANFRANCO GAETANI FIORELLA ALFINITO CARLO FERRO ANDREA BACIGALUPO New York London MONICA BESSLER PETER HILLMEN ANASTASIOS KARADIMITRIS MONICA BESSLER DAVID ARATEN DAVID SWIRSKY ROSARIO NOTARO INDERJEET DOKAL KHEDOUJA NAFA PHILIP MASON VITTORIO ROSTI MARY F MCMULLIN PIERPAOLO PANDOLFI ITT
Istituto Toscano Tumori
Missione: CAPIRE, CURARE, PREVENIRE I TUMORI AL MEGLIO, PER TUTTI