paroxysmal nocturnal hemoglobinuria (pnh) - enerca. notaro.pdf · paroxysmal nocturnal...

6th EUROPEAN SYMPOSIUM ON RARE ANAEMIAS 1st Dutch-Belgian meeting for patients and health professionals

21st - 22nd November 2015

Amsterdam - The Netherlands

Paroxysmal Nocturnal Hemoglobinuria (PNH)

Introduction to PNH Rosario Notaro

Cancer Genetics and Gene Transfer

Core Research Laboratory

Istituto Toscano Tumori

Florence, Italy

6th European Symposium on Rare Anaemias - 1st Dutch-Belgian meeting for patients and health professionals

Disclosures Company name Research

support Employee

Consultant Stockholder Speakers bureau Advisory board Other

No conflicts to declare


This talk is applicable for:

Definite Probable

Thalassemia’s

Sickle cell disease

Membrane disorders (e.g. sferocytosis)

Enzym defects (e.g. PKD, G6PD)

PNH Yes

Other forms of hemolytic disease Yes


Paroxysmal Nocturnal Hemoglobinuria

Acquired disease (it is not transmitted to offspring)

Prevalence: 5-10 cases per million

Geographic distribution: worldwide

Age: any

Gender: same frequency in men and in women


Chronic intravascular hemolysis:

with crisis

Thrombosis: Often Multiple, Venous, Abdominal

PNH is a rare clonal acquired blood disorder

characterized by the clinical triads:

Cytopenias (Bone marrow failure): Common, sometimes severe

WBC /µl Plts /µl



Transmembrane Proteins and GPI-linked Proteins

Cell Membrane

Cytoplasm

Proteins

glycophosphatidylinositol- GPI Anchor

CD59, CD90, CD109

CD55

CD58*

CD59

CD48

CDw52

PrPc

CD16*

CD24 CD55

CD58* CD59

CD48 PrPC

CD73 CDw108

CD55

CD58*

CD59

CD109

PrPC

GP500

Gova/b

CD55

CD58*

CD59

PrPC

AChE

JMH Ag

Dombroch

HG Ag

CD55 CD58*

CD59 CD14

CD16 CD24

CD48 CD66b

CD66c CD87

CD109 CD157

LAPNB1 PrPC

p50-80 GPI-80

ADP-RT

NA1/N

A2

CD14 CD55

CD58* CD59

CD48 CDw52

CD87 CD109

CD157 Group 8

PrPC GPI-80

CD16*

CD55 CD58*

CD59 CD48

CDw52 CD87

CDw108 PrPc

ADP-RT CD73

CD90 CD109

CD16*

GPI-linked Proteins Deficient on PNH Blood Cells

Hematopoietic

Stem Cell

Platelets

RBC

PMN

B cells

Monocytes

T cells

NK cells

nucleus

ER

X chr.

PIGA

+ GPI protein

Etn-P GlcN

Inositol-P

glycan

PIGA

GPI anchor

nucleus

ER

X chr.

PIGA

+ GPI protein

PIGA

GPI anchor

Etn-P GlcN

Inositol-P

glycan

Hematopoietic Stem Cell

Normal cell Normal cell PNH cell

Paroxysmal Nocturnal Hemoglobinuria (PNH)

Pathogenesis


• Intravascular hemolysis

• Thrombosis

• Bone marrow failure


C5

C3

CD55

CD59

Factor H

CD46

CR1

Factor I

C5

C3

C4bp

Complement Inhibitors

Membrane Soluble

Complement System Microorganisms Antigen-Antibody

Constitutive/

Microorganisms

Classical pathway

Lectin pathway

Alternative pathway

modified from Brown et al, Nat Clin Pract Nephrol 2007

Clusterin

Protein S

C1 Inh

Factor I

Membrane

Attack Complex

CD55

CD59

in PNH


Effects of intravascular hemolysis

Hemosiderinuria

Iron loss

ANEMIA (low Hgb)

Nitric oxide

depletion

smooth muscle

dystonia

• Abdominal pain

• Fatigue

• Dysphagia

• Erectile disfunction

• (Renal failure)

• (Pulmonar hypertension)

Intravascular hemolysis

Hemoglobinemia

Hemoglobinuria

High LDH

High Retics



• Thrombosis




• 30-40% of PNH patients suffer from at least a thrombotic event

• 40–67% of deaths in PNH results from thromboembolisms

• More frequent in hemolytic than in aplastic PNH patients

• There is a rough correlation with the size of PNH population - but thrombosis can occur also in patients with small PNH population

• More frequent in patients with congenital and acquired thrombophilic conditions

• Substantial increased risk after the first thrombotic event

PNH is a severe acquired thrombophilic state - thrombosis in PNH -


Hillmen et al, Blood 2007

Ictus/transient ischaemic attack (TIA)

Myocardial infarction/unstable angina

Arterial Thrombosis 15.3%

Deep vein thrombosis

- lower extremity

- other

Mesenteric/splenic vein thrombosis

Hepatic/portal vein thrombosis

Pulmonary embolus

Cerebral/internal jugular thrombosis

Superficial vein thrombosis

Venous Thrombosis 84.7%

% of Total

33.1

18.5

14.5

18.5

16.9

6.5

5.6

4.0

13.7

1.6

Sites of thrombosis IN PNH


Coagulation Complement

Mo

difie

d fro

m M

ark

iew

ski e

t a

l, T

ren

ds Im

mu

no

l 2

00

7

Coagulation, Complement and PNH

Partners in Crime?

Coagulation Complement



• Thrombosis




Bone Marrow Failure and PNH Aplastic Anemia and PNH relationship

Bone Marrow Failure and PNH

Dual Pathogenesis of PNH

Hemolysis

Cytopenia

PNH

AA

PNH/AA

Bone Marrow Failure targeting GPI+ cells (?)

Complement Susceptibility targeting GPI– cells

Rotoli & Luzzatto, 1989

PIG-A+

cells

PIG-A null

cells

crippled

PIG-A+ cells

Noxious agent

Time

Paradoxical Expansion of PNH Clone (Escape Theory)

Y

Y

Y

Y

Y

T Lymphocytes autoreactive T Lymphocytes Hematopoietic Stem cell

?? IFN

IFN

IFN

IFN

GPI protein

GPI protein

ID of Lymphocytes that

damage normal cells and spare PNH cells Which is the target?

Skewed TCR repertoire (Karadimitris et al, Blood 2000)

Similar TCR sequences in oligoclonal T cells (Gargiulo et al, Blood 2007)

GPI specific and CD1d restricted T cells (Gargiulo et al, Blood 2013)

Normal Stem Cell (GPI+) PNH Stem cell (GPI-)

A Unified Pathogenetic Model for PNH and AA CD1d restricted autoreactive T cells target the GPI anchor

anti-GPI TCR

CD1d


Hematopoietic stem cells GPI-targeted

noxious agent

Healthy

Hematopoietic System

Damaged

Hematopoietic stem cells

PNH clone(s)

(GPI-

deficient)

No clinical

consequences

PIG-A

mutation

Selective

expansion

of PNH clone(s)

PNH Intravascular

haemolysis Thrombosis

CD59(-)

red cells

Abnormal

platelets

Pancytopenia

Aplastic

Anaemia

A Unified Pathogenetic Model for PNH and AA


Clinical Management



Support treatments • Folic Acid • Iron supply • Red blood cell transfusions • Anti-coagulant Profilaxis • (Thrombolysis)

Phatogenetic treatments • Allogeneic HSC Transplantation • Immuno-suppression (ALG/CSA) • (Androgens)

Paroxysmal Nocturnal Hemoglobinuria therapeutic options in pre-eculizumab era


Complement Blockade

- eculizumab -



CH

3

CH

2

Rother et al, Nature Biotech 2007

Eculizumab

Anti-C5 humanized monoclonal antibody

Variable

heavy chain

Variable light

chain

Human constant

light chain Ck

Human constant

heavy chain

IgG4 CH2 and CH3

Human constant

heavy chain

IgG2 CH1 and hinge

Human framework

regions

Murine

complementarity

determining regions


in Healthy Subjects in PNH Patients

C5 C3

CD

55

CD

59

CD

46

CR

1


C5 C3

Regulation of Complement Cascade

CD

55

CD

59

C5b-C9

Facto

r H

Facto

r I

C4b

p

Classical

pathway

Lectin

pathway

Alternative

pathway


in PNH Patients Treated with Eculizumab

C5 C3

CD

55

CD

59

CD

46

CR

1


C5 C3

Regulation of Complement Cascade

CD

55

CD

59

Eculizumab Facto

r H

Facto

r I

C4b

p

Classical

pathway

Lectin

pathway

Alternative

pathway

Effect of eculizumab on intravascular haemolysis LDH levels

2500

LD

H

2000

1500

1000

500

TRIUMPH – placebo

0

weeks

10 20 30 40 50

Pilot

TRIUMPH – eculizumab

SHEPHERD

Eculizumab

Hillmen, N Eng J Med 2004; Hillmen, N Eng J Med 2006; Brodsky, Blood 2008

Eculizumab

Placebo

Eculizumab

Hillmen et al, Brit J Haematol 2013

Eculizumab abrogates the need of blood transfusion in most PNH patients

Patients

without

blo

od tra

nsfu

sio

n

du

rin

g th

e p

revio

us 6

mo

nth

s

35%

18%

92%

PRE

Eculizumab


(P<0,001)

Patients = 103 10.61

0.62

0

4

8

12

Pre eculizumab On eculizumab

TE

/ 1

00

pa

tie

nt

ye

ars

Hillmen et al, Blood 2007

Eculizumab and Thrombosis thromboembolism in patients on prophylaxis


100

80

60

40

20

0

0 10 20

Years since diagnosis

Su

rviv

al

(%

)

PNH (n 80; Hillmen 1995)

30 40

PNH (n 424; De Latour 2008)

Overall Survival of PNH Patients in pre-eculizumab era

Hillmen et al, Brit J Haematol 2013

in eculizumab era

Mortality by: Thrombosis

Bone marrow failure

(MDS/leukemia?)


Extravascular Hemolysis

- a pattern change in PNH -



C5 blockade is associated with C3-binding on GPI- RBC

CD

59

-PE

CD

59-P

E

C3-FITC

CD

59-P

E

CD

59-P

E

Untreated PNH patient

PNH patients during eculizumab treatment

Hgb≥12 8.5≤ Hgb <10 Transfused

C3-FITC C3-FITC

2% of

PNH RBC

45% of

PNH RBC 67% of

PNH RBC

Risitano AM, Notaro R et al, Blood 2009

C3-FITC


Eculizumab treatment is associated

with C3-binding on PNH red cells

CD

59-P

E

C3-FITC

CD

59-P

E

Patient not on eculizumab

Patient on eculizumab

45% of

PNH RBC

C3-FITC

Risitano, Notaro et al, Blood 2009

C5 C3 Alternative

pathway C5 C3

C3

Eculizumab

C5 C3 Alternative

pathway C3

C3


A clinical case


years

0

1

2

3

4

5

6

7

8

9

10

11

12

pre-ecu

0 1 2 3 4 5 6 7 8 9

Hb (g/dl)

Neutrophils *103/µl

LDH (*UNL)

Retics *105/µl

Plts *105/µl

13 Packed RBC

2 U/mo

years from starting eculizumab

Eculizumab (900 mg/14 days)

Heavily transfused PNH patients suffering from recurrent thrombosis

no more blood transfusions and no more thrombosis

(despite the discontinuation of anti-coagulant prophylaxis)

-2 -1

recurrent

thrombosis

(Budd Chiari)

Hb (g/dl)

LDH (*UNL)

Retics *105/µl

Plts *105/µl

eculizumab starts and the story changes ……………

Blood

Transfusions

Eculizumab stops intravascular hemolysis and related symptoms

abrogates or reduce transfusion need

reduces thrombotic risk

Hemoglobinuria

Anemia

Hemoglobinemia

• Fatigue

• Dysphagia

• Erectil Dysfunction

•Abdominal Pain

is safe (also during the pregnancy)

However, it is not curative

extravascular hemolysis may result in transfusional need

it is not available to all patients (even in EU)

Thrombosis

Future Developments



C5

C3

CD55

CD59

Factor H

CD46

CR1

Factor I

C5

C3

C4bp

CD55

CD59

Complement Inhibitors Membrane Soluble

New complement inhibitors

Eculizumab

MoAb AntiC3

Compstatin

TT30 (FH-CR2 hybrid)

CR1-like (no EPN)

Fattore H-like

MoAb AntiCFD (no EPN)

MoAb AntiCFB

siRNA anti CFB

Small molecules

Aptamers

Coversin

MoAb AntiC5 (no EPN)

Cyclic Peptids

siRNA

CRL- ITT, Florence Genetics and Gene Transfer

Rosario Notaro

Michela Sica, Lucia Gargiulo, Tommaso Rondelli, Margherita Berardi, Giulia Talini, Annamaria Pellecchia,

Emanuele Di Lorenzo, Benedetta Peruzzi, Chiara Pescucci,

Maria De Angioletti

Imperial College - London (UK) Anastassios Karadimitris, Lucia Gargiulo

Federico II University - Naples (Italy) Antonio M. Risitano, Giuseppina Ruggiero, Fiorella Alfinito,

New York University - New York (USA) David Araten

Hopital Saint-Louis, Paris (France) Gerard Sociè, Regis Peffault de Latour

Kanazawa University, Kanazawa (Japan) Shinji Nakao, Yoshitaka Zaimoku

Sapienza University - Rome (Italy) Anna Paola Iori

Ospedale Maggiore - Milan (Italy) Carla Boschetti, Wilma Barcellini

AOU Careggi - Florence (Italy) Roberto Caporale, Luigi Rigacci, Renato Alterini, Valentina

Carrai, Giacomo Gianfaldoni,

Barbara Scappini, Giovanni Longo, Grazia Gelli,

Francesco Attanasio,

University of Florence (Italy) Alberto Bosi, Valeria Santini, Francesco Mannelli,

Ilaria Cutini, Cristina Nativi, Barbara Richichi

Nurse Staff Careggi Hospital

Istituto Toscano Tumori

Lucio Luzzatto

Core Research

Laboratory


Dank aan de Patiënten en hun Families

Merci aux patients et à leurs familles

Thanks to Patients and their Families

Grazie ai Pazienti ed alle loro Famiglie


There is a soul of goodness in things evil, Would men observingly distil it out.

William Shakespeare, King Henry V, IV, i

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