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
s Tr
ansf
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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
gn
itiv
e*
Dys
pn
ea†
Pai
n*
Inso
mn
ia*
Co
nst
ipat
ion
Nau
sea
Dia
rrh
ea
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
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