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 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 OF MEDICAL IMPORTANCE ON THE X-

PIG-A Paroxysmal Nocturnal Haemoglobinuria

G6PD . Mutations in the PIG-A

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 ( 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 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