PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

Dr.Fariha Saleem

PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

Dr.Fariha Saleem

Outline:

Introduction Pathogenesis Clinical features and complications Investigations Treatment

Introduction:

PNH is a clonal non-malignant hematological disease characterized by the expansion of hematopoietic stem cells and progeny mature cells, whose surface lack all the proteins linked through the glycosyl-phosphatidyl inositol anchor.

Acquired somatic mutation in the X- linked phosphatidylinositol glycan class A gene.

PNH

Hemolytic anemia

Thromboembolis

m

Bone marrow failure

History:

Investigator Year Contribution Gull 1866 Described nocturnal and paroxysmal

nature of “intermittent haematinuria” in a young man.

Strubing 1882 Distinguished PNH from paroxysmal cold haemoglobinuria and

march haemoglobinuria. Attributed the problem to the red cells.

van den Burgh 1911 Red cells lysed in acidified serum. Suggested a role for complement.

Enneking 1928 Coined the name “paroxysmal nocturnal haemoglobinuria”.

Marchiafava 1928- Described perpetual hemosiderinemia. and Micheli 1931 Their names became eponymous for PNH

in Europe. Ham 1937- Identified the role of complement in lysis of

PNH red 1939 cells. Developed the acidified serum test, also

called the Ham test, which is still used to diagnose PNH. Demonstrated that only a portion of PNH red cells are

abnormally sensitive to complement. Davitz 1986 Suggests defect in membrane protein

anchoring system responsible Hall & Rosse 1996 Flow cytometry for the diagnosis of PNH

Epidemiology:

Rare disease.

Incidence of PNH in UK = 1.3 newly diagnosed patients per million per year.

Observed in all ages but most common in young adults.

No evidence of family clustering.

Pathophysiology

Two kinds of membrane proteins: transmembrane and glycosyl phosphatidyl inositol (GPI)-linked. The latter are anchored to cell membranes through a covalent attachment to a glycosyl phospatidyl inositol moiety. In PNH, GPI cannot be synthesized, leading to a global deficiency of GPI-linked membrane proteins

GPI – anchor:

GPI (Glycosylphosphatidylinositol)-anchor is a glycolipid consisting of phosphatidylinositol (PI), glucosamine (GlcN), mannose (Man) and ethanolaminephosphate (EtNP).

Acts as a lipid anchor for various plasma-membrane proteins.

Synthesis of Glycosylphosphatidylinositol (GPI) Anchor

Synthesized in the endoplasmic reticulum.

Transferred en bloc to the carboxyl terminus of a protein that has a GPI-attachment signal peptide.

Involves at least 10 reactions and more than 20 different genes.

Synthesis of GPI Anchor: (cont.)

The first step in GPI anchor biosynthesis is the transfer of N-acetylglucosamine (GlcNAc) from uridine 5′ diphospho-N-acetylglucosamine (UDPGlcNAc) to phosphatidylinositol (PI) to yield GlcNAc-PI.

This step is catalyzed by GlcNAc:PI α1-6 GlcNAc transferase, an enzyme whose subunits are encoded by 7 different genes: PIG-A, PIG-C, PIG-H, GPI1, PIG-Y, PIG-P and DPM2.

The core is modified with side groups during or after synthesis.

The GPI anchored proteins then transit the secretory pathway to reach their final destination at the plasma membrane where they reside in 50 to 350-nm microdomains known as lipid rafts.

Biosynthesis of GPI-anchored proteins GPI-anchored biosynthesis takes place in the endoplasmic reticulum. PIG-A is one of 7 genes

required for the first step, the transfer of N-acetylglucosamine (GlcNAc) from uridine 5 -′diphospho-N-acetylglucosamide (UDP-GlcNAc:PI ) to phosphatidylinositol (PI) to yield GlcNAc-PI. After synthesis of the mature GPI precursor, the cognate protein is attached and then transported to the plasma membrane where the GPI-anchored protein resides in membrane rafts. PIG-A mutations lead to a defect in the first step in GPI anchor biosynthesis resulting in intracellular degradation of the cognate protein and a lack of cell surface GPI anchored proteins.

Failure to synthesize a mature GPI anchor causes the cognate protein to be degraded intracellularly and results in an absence of all GPI anchored proteins from the cell surface.

To date, all PNH patients have been shown to have genetic mutations in PIG-A gene located on short arm of Ch X. (band Xp22)

More than 180 mutations identified, majority of which are small insertions/deletions producing frameshifts, nonsense mutations. Only 2 large deletions identified.

The remaining mutations are missense or small in-frame deletions.

Schematic representation of the structure and mutations in the PIGA gene:

GPI Linked Proteins:

Rosti, Haematologica, 2000

The Role of Complement in Intravascular Hemolysis:

PNH red cells are more vulnerable to complement-mediated lysis due to a reduction, or complete absence, of two important GPI-anchored, complement regulatory proteins. (CD55 and CD59)

CD59 is a 19,000 molecular weight glycoprotein that directly interacts with the membrane attack complex (MAC) to prevent lytic pore formation by blocking the aggregation of C9.

CD55, a 68,000 molecular weight glycoprotein, controls early complement activation by inhibiting C3 and C5 convertases.

Of the two, CD59 is more important in protecting cells from complement.

All three cell lines are effected by the mutation but only RBCs are the one to suffer hemolysis.

The Role of Complement in Intravascular Hemolysis:

Consequences of Chronic Hemolysis and Free Hemoglobin:

1. International PNH Interest Group. Blood. 2005;106:3699-3709. 2. Brodsky R Paroxysmal Nocturnal Hemoglobinuria. In: Hematology - Basic Principles and Practices. 4th ed. R Hoffman; EJ Benz; S Shattil et al, eds. Philadelphia, PA: Elsevier Churchill Livingstone; 2005;419-427. 3. Rother RP et al. JAMA. 2005;293:1653-1662. 4. Socie G et al. Lancet. 1996;348:573-577. 5. Hill A et al. Br J Haematol. 2007;137:181-92. 6. Lee JW et al. Hematologica 2010;95 (s2):Abstract #505 and 506. 7. Hill A et al. Br J Haematol. 2010; May;149(3):414-25. 8. Hillmen P et al. Am. J. Hematol. 2010;85:553-559.

Thrombosis

Fatigue

Renal Failure

Abdominal Pain

Dyspnea

Dysphagia

Hemoglobinuria

Erectile Dysfunction

Normal red blood cells are protected from complement attack by a shield of terminal complement inhibitors

Without this protective complement inhibitor shield, PNH red blood cells are destroyed

Intact RBC

ComplementActivation

Significant Impact on Survival

Significant Impact on Morbidity

Free Hgb/Anemia

Pulmonary Hypertension

NO↓

Nitric oxide scavenging in PNH: Nitric oxide is a major regulator of vascular physiology

and many clinical manifestations of PNH are readily explained by depletion of nitric oxide at the tissue level.

Free hemoglobin in the plasma has enormous affinity for nitric oxide and serves as a potent nitric oxide scavenger.

Haptoglobin is one compensatory mechanism for free hemoglobin removal, but the concentration of plasma hemoglobin in PNH exceeds the capacity of haptoglobin to remove the hemoglobin from the plasma.

Manifests clinically as fatigue, abdominal pain, esophageal spasm, male erectile dysfunction, and possibly thrombosis.

Nitric oxide scavenging in PNH Under normal conditions (A) Nitric oxide synthase (NOS) combines with oxygen (O2) and arginine

to form nitric oxide (NO) and citrulline. (B) Intravascular hemolysis releases free hemoglobin into the plasma. Oxygen bound Fe2+ from free hemoglobin enters the plasma and converts NO to inert nitrite and oxidizes hemoglobin to methemoglobin. In addition, intravascular hemolysis releases erythrocyte arginase, which depletes arginine, the substrate for NOS. Depletion of NO at the tissue level leads to many of the symptoms of PNH including smooth muscle dystonias.

Thrombosis:

Thrombosis is an ominous complication of PNH and leading cause of death from the disease.

Occurs in about 40% of PNH patients and predominantly involves the venous system.

Patients with PNH granulocyte clones of greater than 60% appear to be at greatest risk for thrombosis.

Mechanism of thrombosis in PNH :

Nitric oxide depletion has been associated with increased platelet aggregation, increased platelet adhesion and accelerated clot formation.

In an attempt to repair complement-mediated damage, PNH platelets undergo exocytosis of the complement attack complex. This results in the formation of microvesicles with

phosphatidylserine externalization, a potent in vitro procoagulant. These prothrombotic microvesicles have been detected in the blood of PNH patients.

Mechanism of thrombosis in PNH : (cont.)

Fibrinolysis may also be perturbed in PNH given that PNH blood cells lack the GPI anchored urokinase receptor.

Tissue factor pathway inhibitor (TFPI), a major inhibitor of tissue factor, has been shown to require a GPI anchored chaperone protein for trafficking to the endothelial cell surface.

Effect of GPI- AP deficiency on blood cell populations:

Sites of thrombotic events in haemolytic PNH:

Blood 2007;110:4123– 8. © American Society of Hematology

Clonal evolution and cellular selection:

‘ESCAPE THEORY’ of PNH: Expansion of abnormal hematopoietic stem cell required

for PNH disease expression. In vitro growth studies, demonstrate that there are no

differences in growth between normal progenitors and PNH phenotype progenitors.

Close association with AA - PNH hematopoitic cells cells may be more resistant to the immune attack than normal hematopoitic cells.

Evidence in AA is that the decrease in hematopoitic cells is due to increased apoptosis via cytotoxic T cells by direct cell contact or cytokines (escape via deficiency in GPI linked protein???)

The dual pathophysiology theory:

Bone Marrow Failure Occurrence of PNH blood cells

Anemia

Infections

Gallstones

Bleeding

Inappropriate complement activation

Extravascular Hemolysis

Low blood cell counts

Blood Clot

Abdominal painBloatingBack painHeadacheErectile dysfunctionEsophagospasmFatigue

Hemoglobinuria

Kidney failure

Intravascular Hemolysis

The Mechanisms of Disease in PNH

Classification of PNH:

Classification of PNH

A Classic PNH

B PNH in the presence of another specified bone marrow disorder (e.g. PNH/AA or PNH/refractory anemia-MDS)

C PNH sub clinical (PNH-sc) in the setting of another specified bone marrow disorder (e.g., PNH-sc ⁄ AA)

Classification of PNH:

Category Hemolysis GPI - clone Bone marrow

Classical +++ large Erythroid hyperplasia and normal or near normal morphology

PNH in the presence of another specified bone marrow disorder

+/++ variable Defined underline marrow abnormality

sub clinical - Small population

Defined underline marrow abnormality

Clinical features:

Fatigue due to anemia. (mild to severe)

Passing of (very) dark colored urine. Attacks of abdominal pain associated

with either diarrhea or constipation. Recurrent dysphagia. Erectile dysfunction. May present as a case of Aplastic

Anemia (AA)

Common Symptoms in Patients With PNH:

1. Meyers G et al. Blood. 2007;110(11):Abstract 3683.3. 2. Hill A et al. Br. J. Hematol. 2010;149(3):414-425. 3. Hillmen P et al. Am. J. Hematol. 2010; 85:553-559. 4. International PNH Interest Group. Blood. 2005;106(12):3699-3709. 5. Hillmen P et al. N Engl J Med. 1995;333:1253-8. 6. Nishimura J et al. Medicine. 2004;83(3):193-207.

41% Dysphagia1

47% Pulmonary Hypertension2

66% Dyspnea1

57% Abdominal Pain1

64% Chronic Renal Insufficiency3

47% Erectile dysfunction1

26% Hemoglobinuria4

40% Thrombosis5

89% Anemia6

96% Fatigue, Impaired QoL1

PNH Symptom Incidence Rate (%)

Examination findings:

Pallor. Mild jaundice. Deep palpation abdominal

tenderness. Hepatosplenomegaly (rare) : usually

occur when there is thrombosis in the hepatic vein (Budd-Chiari syndrome) splenic or in the portal vein.

Complications of PNH:

Chronic Kidney Disease Renal insufficiency Dialysis Hypertension

End Organ Damage Brain Liver GI

Anemia Transfusions Hemosiderosis

Fatigue / ImpairedQuality of Life

Abdominal pain Dysphagia Poor physical functioning Erectile dysfunction

Pulmonary Hypertension Dyspnea Cardiac Dysfunction

ThrombosisVenous PE/DVT Cerebral Dermal Hepatic/Portal Abdominal ischemia

Arterial Stroke/TIA MI

1. International PNH Interest Group. Blood. 2005;106:3699-3709. 2. Brodsky R. Paroxysmal Nocturnal Hemoglobinuria. In: Hematology - Basic Principles and Practices. 4th ed. R Hoffman; EJ Benz;S Shattil et al, eds. Philadelphia, PA: Elsevier Churchill Livingstone; 2005; p. 419-427. 3. Hillmen P et al. N Engl J Med. 1995;333:1253-1258. 4. Rosse W et al. Hematology (Am Soc Hematol Educ Program). 2004:48-62. 5. Rother R et al. JAMA. 2005;293:1653-1662. 6. Socie G et al. Lancet. 1996;348:573-577. 7. Hill A et al. Br J Haematol. 2007;137:181-92. 8. Lee JW et al. Hematologica 2010. 95 (s2): Abstract #505 and 506. 9. Hill A et al. Br J Haematol. 2010; May;149(3):414-25. 10. Hillmen P et al. Am. J. Hematol. 2010; 85:553–559.

Chronic Kidney Disease:

Renal failure: cause of death in 8% to 18% of patients with PNH2

Renal insufficiency prevalence in PNH is 6.6x higher than reported for the general population1,3

80% of patients with PNH had renal hemosiderosis (median age 32)4

1. Hillmen P, Elebute MO, Kelly R, et al. [ASH abstract]. Blood. 2007;110: Abstract 3678.2. Nishimura J-I, Kanakura Y, Ware RE, et al. Medicine. 2004;83:193-207. 3. Stevens LA, Coresh J, Greene T, Levey AS. N Engl J Med. 2006;354:2473-2483. 4. Hill A, Reid SA, Rother RP, et al. [ASH abstract]. Blood. 2006;108: Abstract 979.

64% of Patients With PNH Have Chronic Kidney Disease (CKD)1

47

Chronic Kidney Disease

Acute Renal Failure

Pulmonary Hypertension

Cardiac Dysfunction

Stroke / TIAIschemic Bowel

DVT

Hepatic Failure

Signal the Underlying Threat of Catastrophic ConsequencesCommon Symptoms of Hemolysis

FatigueImpaired QoL

Anemia

HemoglobinuriaDyspneaDysphagia

Abdominal Pain

Erectile Dysfunction

Diagnosis of PNH

Who should be screened for PNH?

Patients with hemoglobinuria. Patients with Coombs-negative intravascular hemolysis ,

especially patients with concurrent iron deficiency. Patients with venous thrombosis involving unusual

sites: Budd-Chiari syndrome Other intra-abdominal sites (eg, mesenteric or portal

veins) Cerebral veins Dermal veins

Patients with aplastic anemia (screen at diagnosis and once yearly even in the absence of evidence of intravascular hemolysis)

Patients with refractory anemia-MDS. Patients with episodic dysphagia or abdominal pain with

evidence of intravascular hemolysis.

Laboratory findings:

Urine : hemoglobinuria. Anemia : may be normocytic or macrocytic on the

account of reticulocytosis. If MCV is normal rather high, there probably is

superimposed iron deficiency. Neutrophills : range from normal to below 1 x

109/L. Platelets : range from normal to below 20 x 109/L. Lymphocytes : normal count, lymphopenia or

increase in large granular lymphocytes. Bilirubin : moderately increased.

LDH : markedly increased. Haptoglobin : markedly decreased. Serum iron and transferrin

saturation index : may be decreased. Serum ferritin : might be normal. Coomb’s test : negative. Bone marrow aspirate and trephine:

may be cellular with erythroid hyperplasia or hypoplastic or MDS-like changes in one or more cell lineages.

Diagnostic Tests:

Complement based tests Acidified-serum lysis test (Ham test) Sucrose lysis test

Flow cytometry

Thomas Hale Ham (1905-1987)7th President of the

American Society of Hematology

Acidified-Serum Lysis test (Ham Test)

Principle:The patient’s red cells are

exposed at 37*C to the action of normal or patient’s own serum suitably acidified to the optimum pH for lysis (pH 6.5 – 7.0)

First described in 1937Patient’s red cells can be

obtained from defibrinated, heprainized, oxalated, citrated or EDTA blood but the best is by defibrination.

The Acidified-serum lysis test with added magnesiumTest (ml) Controls (ml)

Reagent 1 2 3 4 5 6

Fresh Normal serum 0.5 0.5 0 0.5 0.5 0

Heat-inactivated normal serum 0 0 0.5 0 0 0.5

0.2 mol/L HCl 0 0.05 0.05 0 0.05 0.05

50 % patient’s red cells 0.05 0.05 0.05 0 0 0

50 % normal red cells 0 0 0 0.05 0.05 0.05

MgCl (250 mmol/L; 23.7 g/L) 0.01 0.01 0.01 0.01 0.01 0.01

Lysis (in a positive modified test) Trace (2%)

+++ (30%)

- - - -

PNH Control

S HS S HSPNH patient

Diagnosis of PNH by the Ham Test:

Ham Test : (cont.)

Three populations of red cells are demonstrated

Type III cells: 10 – 15 times more sensitive than normal cells.

Type II cells : Medium sensitivity, 3 – 5 times more sensitive

than normal cells. Type I cells :

Normal sensitivity.

Sensitivity of Ham test:

Reasonably good at estimating the proportion of PNH red cells, if they are PNH type III cells and comprise less than 20% of the total.

In cases in which the PNH cells are type II and more than 20% are present, the standard Ham test significantly underestimates the proportion of PNH red cells.

The standard Ham test can be negative when there are less than 5% PNH type III cells or less than 20% PNH type II cells.

With supplementation of Ham tset with magnesium, the percentage lysis gives a more accurate estimation of the proportion of PNH cells.

Significance of the Acidified-Serum Lysis Test:

Positive Ham test: PNH.

False-positive acidified-serum test: congenital dyserythopoietic anaemia

type II. Positive test in inactivated serum:

Markedly spherocytic red cells.

Sucrose Lysis Test:

Principle: Red cells absorb complement components from

serum at low ionic concentrations. PNH cells, because of their greater sensitivity, undergo lysis but normal red cells do not.

An iso-osmotic solution of sucrose (92.4g/l) is required.

In PNH, lysis usually varies from 10% - 80%. Sucrose lysis test can be positive in other

conditions like megaloblastic anemia, autoimmune haemolytic anemia, myelofibrosis, leukaemia.

Flow Cytometric analysis of GPI-linked Proteins:

Flow cytometry is a rapid, sensitive and reproducible diagnostic tool for the detection of PNH clones in different peripheral blood cell populations.

It was first described in 1985

‘Gold Standard’ for diagnosis of PNH

Analysis of RBCs by flow cytometry:

Quantitation of at least 2 GPI-APs is recommended to exclude the possibility that the clinical process is a consequence of an inherited, isolated deficiency of a single GPI-AP.

CD59 expression is stronger on RBCs than CD55 and hence CD59 gives much better separation of different types of cells.

Analysis ideally should be performed prior to transfusion or during a period of transfusion abstinence.

IIIIIIIII III

Co

un

ts

CD59

I

Flow Cytometric Analysis of Red Blood Cells

Normal PNH

PNH I PNH II PNH I + III PNH I + II + III

PNH PNH

Testing for PNH in Red Blood Cells:

GPA = glycophorin A.Data Source - Dahl-Chase Diagnostic Services.

RBC’s with normal CD59 expression

(Type I cells)

clone with complete CD59 deficiency (Type III cells) and partial CD59 deficiency (Type II cells)

clone with complete CD59 deficiency (Type III cells)

Gating on GPA+ RBC’s

Analysis of Granulocytes:

In contrast to GPI-AP-deficient red cells, the life span of PNH granulocytes is normal. So, the proportion of abnormal granulocytes more accurately reflects the PNH clone size and is unaffected by red cell transfusion.

CD55 is better than CD59 on granulocytes as against RBCs.

Other proteins include CD16, CD24, CD55, CD59 and CD67.

Flow cytometric analysis of granulocytes in PNH using a combination of anti-CD15 FITC, anti-CD24 PE, and anti-CD16 PE:

Diagnosis and management of paroxysmal nocturnal hemoglobinuria, blood-2005-04-1717

Recommendations for flow cytometric analysis in diagnosis and management of PNH:

For patients with clinical evidence of hemolysis (classic PNH and PNH/aplastic anemia) At diagnosis, flow cytometric analysis of both

erythrocytes and granulocytes is recommended. After establishment of the diagnosis, flow cytometric

analysis is recommended every 6 months for 2 years and yearly thereafter if the parameters are stable.

If there is evidence of clinical progression (or amelioration), more immediate analysis should be performed.

Diagnosis and management of paroxysmal nocturnal hemoglobinuria, blood-2005-04-1717

Recommendations for flow cytometric analysis: (cont.)

For patients with aplastic anemia or refractory anemia-MDS without clinical evidence of hemolysis

At diagnosis, analysis of erythrocytes and granulocytes using high-sensitivity flow cytometry.

Every year, even in the absence of clinical evidence of hemolysis (including patients treated with immunosuppressive therapy).

FLAER:

An alternative flowcytometric approach.

This assay utilizes Aerolysin, the toxin of the bacterium Aeromonas hydrophila, which binds directly to the GPI anchor. It is secreted as an inactive protoxin, proaerolysin, that is converted to the active form, through proteolytic removal of a C-terminal peptide. Aerolysin, thus generated binds to cell surface structures and oligomerizes, forming channels that result in cell lysis.

Initially, this reagent was used to demonstrate the resistance of PNH erythrocytes to aerolysin and also to enrich GPI-negative PNH cells

Two point mutations were introduced to obtain a protein that still binds GPI upon activation but lacks lytic activity.

By coupling this mutant proaerolysin to a fluorescent marker (Alexa Fluor 488), a reagent (FLAER) was produced that stains cells containing GPI proteins but not PNH cells lacking GPI. As this reagent detects the GPI anchor itself, it can be used to investigate all peripheral blood cell types except erythrocytes, which do not express the necessary activating proteases

Display of FLAER vs CD24 in three PNH patients:

A multiparameter gating strategy for granulocytes and monocytes:

Multiparameter Flow Cytometry analysis of peripheral blood in PNH. (A-D) Aplastic anemia patient with small (2%) PNH clone; (E-H) classic PNH patient. (A,E) Forward scatter (FSC)/side scatter (SSC) display showing initial gate to exclude lymphocytes and debris. (B,F) Granulocytes (green) are identified as bright CD15 and low CD33, whereas monocytes (blue) are bright CD33 and low CD15. (C,G) Population of GPI anchor protein–deficient granulocytes showing lack of staining with both anti-CD24 and FLAER. (D,H) Population of GPI anchor protein–deficient monocytes showing lack of staining with both anti-CD14 and FLAER.

PNH Patient With an 80% WBC Clone Size and 31% RBC Clone Size Indicating Hemolysis:

Data Source - Dahl-Chase Diagnostic Services.

CD

24

- G

ranulo

cyte

s

FLAER- GPI Anchor Binding Marker CD59 – GPI Anchored Protein

80.1 % of Granulocytes lack GPI proteins 31.4% RBCs are Type III PNH cells

WBC RBC

Antibodies Useful in PNH Testing:

Comparison between FLAER and immunophenotyping for the diagnosis of PNH

FLAER Immunophenotyping using monoclonal antibodies against GPI-AP

Sensitive as a single agent and hence moreeconomical as screening test

At least two antibodies required

Detection of PNH clone only on leukocytes

Detection of PNH clone on all peripheral blood cells

Better separation of Type I, II, and III cells on granulocytes

Separation of Type I, II, and III cells on granulocytes is not always clear

Better estimation of clone size on granulocytes and monocytes and hence useful for estimation of small clone of granulocytes in AA and MDS using multiparametric assay

Essential for estimation of clone size on RBCs and monitoring of RBC clone size in patients on Eculizumab therapy

More robust assay for detection of clone on granulocytes, can be performed on samples stored up to 48 h

Analysis on granulocyte needs to be performed within 8 h of collection, but analysis on RBCs can be done in samples stored up to 21–30 d

MANAGEMENT OF PNH:

Supportive: Management of haemolysis and anemia Management of thrombosis Management of marrow failure

Curative:

Supportive management:

Management of haemolysis and anemia Management of thrombosis Management of marrow failure

Management of hemolysis and anemia:

Corticosteroids Androgens – in the cases with marrow

impairment RBC transfusions Iron and folate supplementation

Management of thrombosis: Propensity toward thrombosis appears roughly

proportional to the size of the PNH clone. The risk of thromboembolic disease appears higher in

white and African-American patients than in patients of Asian/Pacific Island or Hispanic ancestry even when adjusted for clone size.

White and African-American patients with greater than 50% GPI-AP-deficient granulocytes who have no contraindications are candidates for prophylactic anticoagulation with warfarin.

Patients with PNH who have experienced a thromboembolic event should remain anticoagulated indefinitely.

Management of thrombosis: (cont.)

LWMH Warfarin Anti-platelet agents Fibrinolytic agents

Management of marrow failure:

Immunosuppressive therapy Antithymocyte/antilymphocyte globulin High dose prednisone Cyclosporin A Alemtuzumab

Curative strategies:

Stem cell transplantation

Inhibition of complement activation - Eculizumab

ALLOGENIC BONE MARROW TRANSPLANTATION: Indications:

Bone marrow failureDecision on transplantation is based on underlying

marrow abnormality (eg aplastic anemia) Major complication of PNH

Recurrent, life-threatening thromboembolic disease

Refractory, transfusion-dependent hemolytic anemia

PNH-specific transplant-related issues: The conditioning regimen of

cyclophosphamide/ATG is recommended for patients with PNH/aplastic anemia.

For patients with classic PNH, a more myeloablative regimen is indicated.

Additional investigation is required to define the role of nonmyeloablative regimens.

For syngeneic twin transplants, a myeloablative conditioning regimen is recommended to prevent recurrence of PNH.

PNH-specific transplant-related issues: (cont.) There are no PNH-specific adverse events

associated with transplantation; severe, acute graft-versus-host disease (GVHD) occurs in more than a third of the patients and the incidence of chronic GVHD is roughly 35%.

Overall survival for unselected PNH patients who undergo transplantation using an HLA-matched sibling donor is 50% to 60%.

Inhibition of terminal complement activation:

Eculizumab : Eculizumab is a humanized monoclonal antibody

against C5 that inhibits terminal complement activation.

Prevention of C5 cleavage blocks the generation of the potent proinflammatory and cell lytic molecules C5a and C5b-9.

C5 blockade preserves the critical immunoprotective and immunoregulatory functions of upstream components that culminate in C3b-mediated opsonization and immune complex clearance.

Most effective in Classical PNH.

Eculizumab was engineered to reduce immunogenicity and eliminate effecter function. Human IgG2 and IgG4 heavy-chain sequences were combined to form a hybrid constant region that is unable to bind Fc receptors or to activate the complement cascade. Eculizumab exhibits high affinity for human C5, effectively blocking its cleavage and downstream proinflammatory and cell lytic properties.

The complement cascade and C5 blockade by Eculizumab:

Rationale for Eculizumab use in PNH:

Extravascular hemolysis with eculizumab therapy:

Eculizumab: (cont.)

Treatment with eculizumab decreases or eliminates the need for blood transfusions, improves quality of life and reduces the risk of thrombosis

Two weeks before starting therapy, all patients should be vaccinated against Neisseria meningitides because inhibition of complement at C5 increases the risk for developing infections with encapsulated organisms, particularly N meningitides and N gonorrhoeae

Eculizumab: (cont.)

Dosage :

I/V, 600 mg weekly for the first 4 weeks, then 900 mg biweekly starting on week 5

Must be continued indefinitely because it does not treat the underlying cause of the disease

Dosing Schedule of Eculizumab:

Pretreatment Induction Phase Maintenance Phase

2 weeks before induction

Week→ 1 2 3 4 5 6 7 8

9 and every

2 weeks thereafter

Neisseria meningitidis vaccination

SOLIRIS® dose, mg

→600 600 600 600 900 X 900 X 900

Eculizumab: (cont.)

Indications for Therapy: No widely accepted evidence-based

indications for treatment. Eculizumab is usually for patients with

disabling fatigue, thromboses, transfusion dependence, frequent pain paroxysms, renal insufficiency, or other end-organ complications from disease.

Watchful waiting is appropriate for asymptomatic patients or those with mild symptoms.

Eculizumab: (cont.)

Adverse Affects: Most common side effect is headache and it

occurs in approximately 50% of patients, after the first dose or two, but rarely occurs thereafter.

Neisserial sepsis is the most serious complication of eculizumab therapy.

0.5% yearly risk of acquiring Neisserial sepsis even after vaccination.

Patients should be revaccinated against N meningitidis every 3 to 5 years after starting treatment.

Eculizumab: (cont.)

Monitoring patients on eculizumab: Symptomatic improvement within hours to days

after the first dose of eculizumab. Complete blood count, reticulocyte count, LDH,

and biochemical profile weekly for the first 4 weeks and then at least monthly thereafter.

LDH usually returns to normal or near normal within days to weeks after starting eculizumab.

Reticulocyte count usually remains elevated because extravascular hemolysis persists and the hemoglobin response is highly variable.

In patients who are transfusion-dependent, a marked decrease in red cell transfusions is observed in virtually all patients, with more than 50% achieving transfusion independence.

Breakthrough intravascular hemolysis and a return of PNH symptoms occur in less than 2% of PNH patients treated with eculizumab.

Infections might be a cause.

86% Reduction in LDH:TRIUMPH and SHEPHERD

P<0.001 at all measured time points.Hillmen P et al. Blood. 2007;110(12):4123-8.

TRIUMPH placebo patients switched to SOLIRIS® after week 26.All TRIUMPH patients entered the long-term extension study.

TRIUMPH – Placebo/Extension

TRIUMPH – SOLIRIS®/Extension

SHEPHERD – SOLIRIS®

Lac

tate

Deh

ydro

gen

ase

(U/L

)

0

500

1000

1500

2000

2500

3000

Time, Weeks

0 4 8 12 16 20 24 28 32 36 40 44 48 52

100% response after the first dose

73% Reduction in Mean Units Transfused Across all Subgroups: TRIUMPH

*P<0.001. †Transfusion data obtained during 12 months before treatment; values were normalized for a 6-month period.1. Hillmen P et al. N Engl J Med. 2006;355;1233-1243. 2. Schubert J. Br. J Haematol. 2008;142(2):263-72.

Overall 4-14 15-25 >25

Pre-treatment Transfusion Strata†

Patients not on SOLIRIS® (n=44)

SOLIRIS (n=43)

**

*

*

(n=87) (n=30) (n=35) (n=22)0

2

4

6

8

10

12

14

16M

edia

n U

nit

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18

• 51% of SOLIRIS patients achieved transfusion independence vs 0% of patients not on SOLIRIS1• Patients with concomitant bone marrow dysfunction may continue to require minimal transfusions

Patients Report Rapid and Sustained Improvement Across Broad Range of Measures

*P<0.05.†P<0.001.1. Brodsky R et al. Blood. 2006;108(11): Abstract 3770. 2. Data on file. Alexion Pharmaceuticals.

Moderate Impact

Small Impact

Large Impact

Sta

nd

ard

Eff

ect

Siz

e (S

ES

)

EORTC Functioning

EORTC Symptoms

FAC

IT-F

atig

ue†

EO

RT

C F

atig

ue†

Glo

bal

Hea

lth†

Ph

ysic

al†

Ro

le†

Co

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e*

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ea†

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ia*

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ion

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sea

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0

0.2

0.4

0.6

0.8

1

1.2

92% Reduction in Thrombotic Events:

63% of patients received concomitant anticoagulants1 The effect of anticoagulant withdrawal was not studied2

Events observed in both venous and arterial sites3

PI: There were fewer thrombotic events with SOLIRIS treatment than during the same period of time prior to treatment.1. Brodsky R et al. Blood. 2008;111(4):1840-47. 2. SOLIRIS® (eculizumab) [package insert]. Alexion Pharmaceuticals; 2009. 3. Hillmen P, et al. Blood. 2007;110:4123-4128.

39

3

05

101520253035

4045

Pre-SOLIRIS® Treatment SOLIRIS Treatment

Th

rom

bo

tic

Eve

nts

(#)

P=0.0001

N=195

Eculizumab-Pro’s and Con’s

Pro’s Very effective at

reducing hemolysis Well tolerated Improvements in

QOL, reduction in transfusions proven

Reduction in burden of disease

Probable reduction in clots

Con’s $$$$ Infusion weekly

X5, then every 2 weeks

Infection risk: meningococcal meningitis

Burden of treatment

Plan for lifetime therapy

Does not improve other blood counts

Correction of CD59 deficiency: An alternative approach to the prevention of

hemolysis in PNH is to restore CD59 (membrane inhibitor of reactive lysis) expression to the surface of the PNH red cells and thus reestablish membrane complement inhibitory activity

In a recent study, a novel synthetically modified recombinant human CD59 (rhCD59-P), a soluble protein that attaches to cell membranes was assessed for its ability to correct CD59 deficiency on PNH red cells both in vitro (human red cells) and in vivo

In vitro treatment of PNH erythrocytes with rhCD59-P resulted in levels of CD59 equivalent to normal erythrocytes and effectively protected erythrocytes from complement-mediated hemolysis

Clinical management of PNH:

Impact of PNH on Quality of Life

59% patients were transfusion-free for at least 12 mo or had never been transfused

76% were forced to modify their daily activities to manage their PNH

17% were unemployed due to PNH

*Moderate to severe; N=29.Meyers G et al. Blood. 2007;110 (11): Abstract 3683.

~75% of Patients Reported Symptoms as Moderate to Very Severe

Paroxysmal Nocturnal Hemoglobinuria:A Chronic Disabling and Life-Threatening Disease

5 year mortality: 35%1

Quality of life diminished2

Progressive disease.

The expected survival of an age- and sex-matched control group is shown for comparison (Hillmen et al 1995). In a patient population where ½ the patients have <30% clone, 1 in 7 patients died by 5 years.de Latour et al. Blood. 2008; 112: 3099-3106.

Years After Diagnosis

Pati

en

ts S

urv

ivin

g (

%)

Actuarial Survival From the Time ofDiagnosis in 80 Patients With PNH1

100

80

60

40

20

0

0 5 10 15 20 25

Age- and Gender-Matched Controls

Patients with PNH

1. Hillmen P et al. N Engl J Med. 1995;333:1253-1258. 2.Hill A et al. Br J Haematol. 2007;137:181-92.

Poor prognostic factors:

Development of thrombosis Progression to pancytopenia MDS or acute leukemia Age ≥ 55 years Thrombocytopenia at diagnosis Aplastic anemia antedating PNH

Future Research Topics:

Many research questions still to be answered: Why do PNH cells survive immune

mediated insults better? Why clotting? Why does the PNH clone expand?

Better treatments Improvement in supportive care and

transplantation