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EMOGLOBINURIA PAROSSISTICA NOTTURNA

Lucio Luzzatto, Scientific Director,

Istituto Toscano TumoriProfessor of Haematology,

University of Firenze.Firenze, ITALY

SIE - Corso di Ematologia CllinicaRoma, 29 maggio 2007

Haemolytic anaemia with characteristic clinical triad:

1. Intravascular haemolysis2. Thrombosis3. Cytopenias

PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA:definition

•Malaria•Auto-immune•Drug-induced•Micro-angiopathic•Other

ParoxysmalNocturnal

Haemoglobinuria(PNH)

Acquired

Familial HUS•Haemoglobinopaties•Enzimopathies•Membranopathies•Other

Hereditary

Extracorpuscolarcauses

Intracorpuscolarcauses

CLASSIFICATION OF HAEMOLYTIC ANAEMIAS

1. Hemolytic2. Hemolytic/hypoplastic3. Sub-clinical

PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA:classification

HAEMOGLOBINURIA

Indicates intravascular haemolysis

WORLDWIDE PNH PATIENTSWORLDWIDE PNH PATIENTS

22

88

77

3838 132132

33

Patient RBCPatient RBCControlControlRBCRBC

dAcdAc dSdS dAcdAc dHidHi pHipHi pAcpAc HH22

00

Ham Test in a PNH Patient

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CD59

M.B.c o

u n

t s

c o

u n

t s

Flow-cytometry Analysis of Red Cells from Patients with PNH

PNH III

normal

C.J

Control

normal

PNH II

Control

R.K

PNH IIInormal

PNH II

Control

CD59, CD90, CD109

CD55CD58*CD59CD48CDw52PrPcCD16*

CD24 CD55CD58* CD59 CD48 PrPCCD73 CDw108

CD55CD58*CD59CD109PrPCGP500Gova/b

CD55CD58*CD59PrPCAChEJMH AgDombrochHG Ag

CD55 CD58*CD59 CD14CD16 CD24CD48 CD66bCD66c CD87CD109 CD157LAPNB1 PrPCp50-80 GPI-80ADP-RT NA1/NA2

CD14 CD55 CD58*CD59 CD48 CDw52CD87 CD109 CD157Group 8 PrPC GPI-80CD16*

CD55 CD58*CD59 CD48CDw52 CD87 CDw108 PrPcADP-RT CD73CD90 CD109CD16*

PROTEINS DEFICIENT ON PNH BLOOD CELLS

Hematopoietic Stem Cell

Platelets

RBC

PMN

B cells

Monocytes

T cells

NK cells

QuickTime™ and aGIF decompressor

are needed to see this picture.All these proteins are GPIAll these proteins are GPI--linkedlinked

ERLumen

Cytoplasm

GPI BIOSYNTHESIS IN MAMMALIAN CELLS

Dol-P-Man

Dol-P + GDP-Man

P

NAc

P

P

EtNP

P EtN

P

P EtN

P

P EtN

P

P

P

To proteins

P

EtNP

P EtN

P EtN

P P

MannoseGlucosamineDPM3

PIG-A

PIG-H PIG-BPIG-FPIG-C

PIG-LGAA1

GPI1/PIG-Q

DPM1/PIG-EDPM2 SL15

GPI8/PIG-K

PIG-X PIG-N PIG-OPIG-P

PIG-SPIG-T

GPI7PIG-F

DPM2

PIG-U

PIG-W PIG-VPIG-M

PIG-Y

SOME GENES OF MEDICAL IMPORTANCE ON THE X-CHROMOSOME

PIG-AParoxysmalNocturnal Haemoglobinuria

G6PD

..

4

982 11898497161

1

1452Null mutations

3

AvaIpolymorphism

del 735 bp

Large deletions

***** *

****

*****

50 bp

**

652

del exons 3-4-5

Non-null mutations

Mutations in the PIG-A Gene

COMPLEXITY OF THECOMPLEXITY OF THE COMPLEMENT COMPLEMENT CCASCAASCADEDE

Cell ActivationCell ActivationLysisLysis

C6 C7 C8 C9C6 C7 C8 C9

Potent AnaphylatoxinPotent AnaphylatoxinChemotaxisChemotaxis

Cell ActivationCell Activation

C5bC5b--99

C3 ConvertaseC3 Convertase

C3C3

C1qC1q Activated C1Activated C1

C4+C2C4+C2

C5 ConvertaseC5 Convertase

C3bC3b

Classical PathwayClassical PathwayActivationActivation

Antibody/Antigen ComplexesAntibody/Antigen Complexes

C3aC3aWeakWeak

AnaphylatoxinAnaphylatoxin

C4b2aC4b2a C4b2a3bC4b2a3b

C5bC5b

C5aC5a

C5C5Immune ComplexImmune Complexand Microbialand MicrobialOpsonizationOpsonization

Factor B+DFactor B+DC3 ConvertaseC3 Convertase C5 ConvertaseC5 Convertase

C3, C3HC3, C3H22OO C3bC3b

Alternative PathwayAlternative PathwayActivationActivation

Microbiological MembranesMicrobiological MembranesBacterial LPSBacterial LPS

Immune ComplexesImmune ComplexesMammalian Cell MembranesMammalian Cell Membranes

C3bBb3bC3bBb3bC3bBbC3bBb

LectinLectin PathwayPathwayActivationActivation

MBLMBL

adapted fromadapted from: Meri: Meri et et al, al, 19901990

C5bC5b--8844ººCCC9 loosely boundC9 loosely bound(displaceable)(displaceable)

C9C9

C5bC5b--883737ººCCC9 tightly boundC9 tightly bound(not displaceable)(not displaceable)

C9C9

C5bC5b--88

PolyPoly--C9C9

C5bC5b--88

C9C9

C5bC5b--88

CD59CD59C9C9

Mechanism of Action of CD59Mechanism of Action of CD59

with CD59with CD59

without CD59without CD59QuickTime™ and aGIF decompressor

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• Why acquired?• Why the thrombosis?

PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA: features

Thrombosis: Thrombosis: Often Multiple, Venous, AbdominalOften Multiple, Venous, Abdominal

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LIFELIFE--THREATENING BUDDTHREATENING BUDD--CHIARI IN PNHCHIARI IN PNH

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TISSUE PLASMINOGEN ACTIVATOR IS A POTENT THROMBOLYTIC AGENT

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OTHER SITES OF THROMBOSIS IN PNH

• Splenic vein • Inferior vena cava• Superior mesenteric vein• Inferior mesenteric vein• Renal veins• Subclavian vein

(LL, personal observations)

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Cavernous Transformation of Portal VeinCavernous Transformation of Portal Vein

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ANGIOGRAPHIC PICTURE OF THE SPLEEN

pre-Embolization post-Embolization

THE RISK OF THROMBOSIS IN PNHIS 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

55 1515 2525YEARSYEARSAraten et Araten et al., al.,

Thromb HaemostThromb Haemost 9393:88,2005.:88,2005.

• 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?)

PNH is a severe acquired thrombophilic state.

POSSIBLE MECHANISMS:

A COMPROMISE PROTOCOLre Anticoagulant Prophylaxis

1. Haemolytic versus aplastic PNH2. Thrombophilia screen (Prot S, Prot C,

Factor V Leiden, ATIII, PT20010, MTHFR)3. Large size of granulocyte PNH population4. Previous episode of thrombosis

A COMPROMISE PROTOCOLre Anticoagulant Prophylaxis1. Haemolytic versus aplastic PNH2. Thrombophilia screen (Prot S, Prot C,

Factor V Leiden, ATIII, PT20010, MTHFR)3. Large size of granulocyte PNH population4. Previous episode of thrombosis

With 4, must start anti-coagulants

Depending on 1-3, considerstarting anti-coagulants

nucleus

ER

GPI anchorprotein

PIG-A

Chr X

p

q

P

glycan

inositol-P

GlcNEtn-P

proteinPIG-A

GPI anchor

Normal cellNormal cell

Pinositol-P

PIG-Aprotein

P

Normal cellNormal cell

nucleus

ER

protein

PIG-A

Chr X

p

q

Pinositol-P

PIG-Aprotein

PNH cellPNH cell

P

nucleus

ER

GPI anchorprotein

PIG-A

Chr X

p

q

P

glycan

inositol-P

GlcNEtn-P

proteinPIG-A

GPI anchor

Normal cellNormal cell

P

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• Why acquired?• Why the thrombosis?• Why the cytopenias?

PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA: features

Erythroid Erythroid hyperplasiahyperplasia AplasiaAplasia QuickTime™ and aGIF decompressor

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PIGPIG--AA plusplusblood cellblood cell

PIGPIG--AA plusplusdamaged blood celldamaged blood cell

PIGPIG--AA minusminusblood cellblood cell

Auto immune Auto immune attackattack

TimeTime

Pathogenesis of PNHPathogenesis of PNH((facts and speculationsfacts and speculations))

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CAN PNH CURE APLASTIC ANEMIA?

SHARED TCR-β SEQUENCES IN PATIENTS WITH PNH

2.5CATS RTAGETQ YFGP152S5b

2.5CATS RIGGETQ YFGP159S5a

2.5CATS RVAGETQ YFGP155, 7, 9S5

2.1CATS GIAGETQ FFGP155; 7; 9S4

1.1CA WEQVIA FFGQ308; 19S3

1.2CATS RGRTQGLDYG YTFG156; 16S2

2.7CASS LVGGPEQ YFGP7.97; 9S1

TRBJVβ nDn JβTRBVPatientsSequence ID

TCR-β CDR3

PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA:Issues in management. I.

• Supportive treatment• BMT• Immunosuppression• Anti-C5• Anticoagulant prophylaxis• Thrombolytic therapy• PNH and pregnancy

55 1010 1515 2020

2525

5050

7575

100100

2525

yearsyears

Pat

ient

s su

rviv

ing

(%)

Pat

ient

s su

rviv

ing

(%)

AllogeneicAllogeneic BMT BMT (IBMTR)(IBMTR)

Natural HistoryNatural History

The Fate of PNH PatientsThe Fate of PNH Patients

AllogeneicAllogeneic BMT BMT ((GenovaGenova))

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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 Hillmen et al., et al., New Eng J MedNew Eng J Med 355355:1233,2006):1233,2006)

TARGETING THE COMPLEMENT TARGETING THE COMPLEMENT CCASCAASCADEDE

Cell ActivationCell ActivationLysisLysis

C6 C7 C8 C9C6 C7 C8 C9

Potent AnaphylatoxinPotent AnaphylatoxinChemotaxisChemotaxis

Cell ActivationCell Activation

C5bC5b--99

C3 ConvertaseC3 Convertase

C3C3

C1qC1q Activated C1Activated C1

C4+C2C4+C2

C5 ConvertaseC5 Convertase

C3bC3b

Classical PathwayClassical PathwayActivationActivation

Antibody/Antigen ComplexesAntibody/Antigen Complexes

C3aC3aWeakWeak

AnaphylatoxinAnaphylatoxin

C4b2aC4b2a C4b2a3bC4b2a3b

C5bC5b

C5aC5a

C5C5Immune ComplexImmune Complexand Microbialand MicrobialOpsonizationOpsonization

Factor B+DFactor B+DC3 ConvertaseC3 Convertase C5 ConvertaseC5 Convertase

C3, C3HC3, C3H22OO C3bC3b

Alternative PathwayAlternative PathwayActivationActivation

Microbiological MembranesMicrobiological MembranesBacterial LPSBacterial LPS

Immune ComplexesImmune ComplexesMammalian Cell MembranesMammalian Cell Membranes

C3bBb3bC3bBb3bC3bBbC3bBb

LectinLectin PathwayPathwayActivationActivation

MBLMBL

XX

AntiAnti--C5 C5 (ECULIZUMAB)(ECULIZUMAB)

Baseline Characteristics of the Patients

Hillmen P et al. N Engl J Med 2006;355:1233-1243

(Hillmen et al., New Eng J Med355:1233,2006)

Levels of Lactate Dehydrogenase and PNH Type III Erythrocytes during Treatment with Eculizumab

Hillmen P et al. N Engl J Med 2006;355:1233-1243

ECULIZUMAB ARRESTS THE SELECTIVE INTRAVASCULAR

LYSIS OF PNH III RED CELLS

(Hillmen et al., New Eng J Med355:1233,2006)

Kaplan-Meier Curves for the Time to the First Transfusion during Treatment

Hillmen P et al. N Engl J Med 2006;355:1233-1243

ECULIZUMAB CAN ABROGATE THE NEED FOR BLOOD TRANSFUSION

(Hillmen et al., New Eng J Med 355:1233,2006)

Change in Fatigue Scores from Baseline to Week 26

Hillmen P et al. N Engl J Med 2006;355:1233-1243

ECULIZUMAB HAS AN IMPACT ON THE QUALITY OF LIFE

(Hillmen et al., New Eng J Med 355:1233,2006)

Eculizumab appears to reduce the risk of thrombosis in PNH

TE event rate (A/B x 100)

Patient Years

Thrombo-embolic (TE) events

1.17.4C

2811683B

3124A

On anti-C5

Pre-treatment

n = 195

3 BONE MARROW DISORDERS; 2 PATHOGENETIC FACTORS;

1 PATHOGENETIC MECHANISM

None?

Trisomy 8, trisomy 6,

other

PIG-Amutation

Somatic genetic change

Universal+++AA

?+/+++MDS

GPI or GPI-linked protein

++PNH

Target of auto-immune attack

Failure of ‘normal’stem cells

PIG-A mutation

GPI-targeted noxious agent

PNH clone (GPI-proteins

deficient)

Selective expansion

of PNH clone(s)

CD59(-) red cells

Abnormal platelets

No clinical consequences

Intravascular haemolysis

Thrombosis

Pancytopenia

Damage toHematopoietic

stem cells

PNH

APLASTIC ANAEMIA

A SYNOPSIS OF THE PATHOGENESIS OF PNH

IbadanS B ONIB O OSUNKOYAG J F ESAN

NapoliBRUNO ROTOLIFIORELLA ALFINITO

LondonPETER HILLMENMONICA BESSLERDAVID SWIRSKYINDERJEET DOKALPHILIP MASONMARY F MCMULLIN

New YorkMONICA BESSLERANASTASIOS KARADIMITRISDAVID ARATENROSARIO NOTAROKHEDOUJA NAFAVITTORIO ROSTIPIERPAOLO PANDOLFI

GenovaROSARIO NOTAROLUCIA GARGIULOALESSANDRO POGGIGIANFRANCO GAETANICARLO FERROANDREA BACIGALUPO

FirenzeGIANGIACOMO GIANFALDONIALBERTO BOSIELISABETTA ANTONIOLI

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ITTIstituto Toscano Tumori

Missione: CAPIRE, CURARE, PREVENIRE I TUMORIAL MEGLIO, PER TUTTI