Transcript What Is PNH?

Paroxysmal
Nocturnal
Hemoglobinuria
New ideas about an old disease
Ahmad Shihada Silmi
Msc, FIBMS
Staff Specialist in Hematology
Medical Technology Department
Islamic University of Gaza
Objectives
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Try to answer some of the frequently
asked questions about:
The cause of the PNH
The clinical presentation of PNH
Diagnosing PNH
The complications of PNH
New treatments for PNH
What is PNH?
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A disorder of blood affecting all the cells which
come the bone marrow.
Prevalence 1-4 per million
The disease is quite rare, only 10, 000 patients
in the US and Europe.
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There is no ethnic preference for the disorder.
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It may present early or late in life.
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The manifestations may be “classic” or obscure.
PNH Prognosis
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Significant morbidity and mortality
Patients have lived for extended periods of time and have
seen spontaneous recovery
Report of 80 patients showed that 60% of deaths due to
venous thrombosis or bleeding
Retrospective study of 220 patients
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Kaplan-Meier survival 78, 65, and 48% at 5, 10, and 15 years
8 year rates of major complications of PNH (pancytopenia, thrombosis,
MDS) 15, 28, and 5%
Up to 5% of patients can develop acute leukemia; onset about 5 years
after Dx of PNH
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Likely incident of leukemia lower than 5%
What is PNH Mutation?
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PNH is due to a mutation in a gene in a blood stem
cell.
The gene is called the PIG-A gene
(phosphatidylinositol glycan complementation group A)
and is located on the X chromosome.
 >100 mutations in PIG - A gene known in PNH
 The mutations (mostly deletions or insertions)
generally result in stop codons - yielding
truncated proteins which may be non or partially
functional - explains heterogeneity seen in PNH
What is PNH?
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In most cases of PNH, the change in the
gene (mutation) is acquired, not
something you are born with. When and
why is unknown.
The gene contains the genetic information
for the GPI anchors which link proteins to
the cell membrane
What is PNH?
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A mutation is a “mistake” or a “change” in
the gene that arises during copying and is
not corrected
When the cell divides, the mutation is
transmitted to daughter cells
The effect of a mutation:
None
 An altered protein (sickle hemoglobin)
 No protein is produced as in PNH, hemophilia
etc

Stem Cells
Egg
Egg or
Sperm
Blood
Sperm
E
g
g
Embryonic Stem Cell
Muscle
Nerve
Etc.
Somatic
Stem Cells
The Cells of Each Specific Organ
Stem Cells
Egg
Egg or
Sperm
Blood
Sperm
E
g
g
Embryonic Stem Cell
Muscle
Nerve
Etc.
Somatic
Stem Cells
The Cells of Each Specific Organ
Stem Cells
Egg
Egg or
Sperm
Blood
Sperm
E
g
g
Embryonic Stem Cell
Muscle
Nerve
Etc.
Somatic
Stem Cells
The Cells of Each Specific Organ
Stem Cells
T LYMPHOCYTES
STEM CELL
B LYMPHOCYTES
ERYTHROCYTES
GRANULOCYTES
MONOCYTES
PLATELETS
Stem Cells
T LYMPHOCYTES
STEM CELL
B LYMPHOCYTES
ERYTHROCYTES
GRANULOCYTES
MONOCYTES
ALTERED GENE
PLATELETS
Stem Cells
NORMAL CLONES
ABNORMAL CLONE
GPI-AP Biosynthesis:
Involves 10 Steps and >20
Genes
Plasma Membrane
Raft
N
N
N
PIG-A
PI
GlcNAc-PI
Endoplasmic Reticulum
The Beginning of PNH
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The change that occurs in PNH stops the
production of an anchor that ties protein
molecules to the cell
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Sometimes the stop is only partial and PNH II
cells occur
The GPI Anchor Defect in
PNH
PROTEIN
Asp
NORMAL
PNH
C=O
NH
CH
2
CH
2
O
O=P=O
O
( 1-2)
( 1-6)
( 1-4)
O
N
O
O-P=O
O CH
2
CH
2
NH
2
O
O-P-O
CH
2
O
CH
CH
O
2
O-P-O
O
CH
C=O
2
O
(22:4,5
CH
O
C=O
C=O
(18:0,1
)
CH
(16:0)
(18:0,1
)
(22:4,5)
2
O
Pathogenesis - The Defect
GPI Anchor
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PIG - A gene codes for 60 kDa protein
glycosyltransferase which effects the first
step in the synthesis of the glycolipid GPI
anchor (glycosylphosphatidylinositol).
Results in clones lacking GPI anchor - in
turn, attached proteins
PIG - A protein
What is PNH?
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As a result of the PIG-A mutation, there is
little of no GPI anchor produced.
PNH II cells- mild reduction
 PNH III cells- severely reduced.
 When the anchor is reduced, certain proteins
can’t attach to the cells.
 The most important proteins for PNH are
CD 59, CD55.
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Proteins anchored by GPI Anchor
and
Surface Proteins Missing on PNH Blood Cells
Antigen
Enzymes
Acetylcholinesterase (AchE)
Ecto-5'-nucleotidase (CD73)
Neutrophil alkaline phosphatase(NAP)
ADP-rybosyl transferase
Adhesion molecules
Blast-I/CD48
Lymphocyte functionassociated antigen-3(LFA-3 or CD58)
CD66b
Complement regulating surface proteins
Decay accelerating factor (DAF or CD55)
Homologous restriction factor,
Membrance inhibitor of reactive lysis
(MIRL or CD59)
Expression Pattern
Red blood cells
Some B- and T-lymphocytes
Neutrophils
Some T-lymphs, Neutrophils
Lymphocytes
All blood cells
Neutrophils
All blood cells
All blood cells
Surface Proteins Missing on PNH Blood Cells
Antigen
Receptors
Fc- receptor III (Fc  Rlll or CD16)
Monocyte differentiation antigen
(CD14)
Urokinase-type Plasminogen
Activator Receptor (u-PAR, CD87)
Expression Pattern
Neutrophils, NK-cells, macrophages,
some T-lymphocytes
Monocytes, macrophages
Monocytes, granulocytes
Blood group antigens
Comer antigens (DAF)
Yt antigens (AchE)
Holley Gregory antigen
John Milton Hagen antigen (JMH)
Dombrock reside
Red blood cells
Red blood cells
Red blood cells
Red blood cells, lymphocytes
Red blood cells
Neutrophil antigens
NB1/NB2
Neutrophils
Surface Proteins Missing on PNH Blood Cells
Antigen
Other surface proteins
of unknown functions
CAMPATH-1 antigen (CDw52)
CD24
p5O-80
GP500
GPI75
Expression Pattern
Lymphocytes, monocytes
B-lymphocytes, Neutrophils,
eosinophils
Neutrophils
Platelets
Platelets
Pathogenesis
CD 55
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Also known as decay accelerating factor
Accelerates decay of enzyme that destroys
red cell membranes
CD55 inhibits the formation or destabilizes
complement C3 convertase (C4bC2a)
When GPI anchor absent, CD55 not
available and RBC membranes hemolyze
Pathogenesis
CD59
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Also known as membrane inhibitor of reactive
lysis, protectin, homologous restriction factor,
and membrane attack complex inhibitory factor
CD59 Protects the membrane from attack by the
C5-C9 complex
Absence or reduction of CD59 leads to increased
hemolysis and perhaps thrombosis
Pathogenesis of CD55 & CD59
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Inherited absences of both proteins in
humans have been described
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Most inherited deficiencies of CD55 - no distinct
clinical hemolytic syndrome
Inherited absence of CD59 - produces a clinical
disease similar to PNH with hemolysis and
recurrent thrombotic events
Pathogenesis
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Many normal people have very small numbers
(perhaps 6 per 1,000,000 bone marrow cells)
In PNH, the abnormal cells have an advantage and
become a major population in the marrow and blood
(anywhere from 1% to over 90%)
 This may be a result of change in the immune
system –inability to recognize something foreign.
 Or it may be related to aplastic anemia, a disease
of poor production of blood from the marrowWHY?
Pathogenesis - Clonal evolution and
cellular selection
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Expansion of abnormal hematopoietic stem cell required
for PNH disease expression
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Theories for expansion
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Blood cells lacking GPI-linked proteins have intrinsic ability to grow
abnormally fast
 In vitro growth studies demonstrate that there are no differences
in growth between normal progenitors and PNH phenotype
progenitors
 In vivo - mice deficient for PIG -A gene also demonstrates no
growth advantage to repopulation of BM.
Additional environmental factors exert selective pressure in
favor of expansion of GPI anchor deficient blood cells
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Close association with AA - PNH hematopoitic cells cells may be
more resistant to the Immune System 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???)
PNH Clinical Features
Aplastic Anemia
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Some PNH patients have aplastic anemia or a
history of aplastic anemia
Many PNH patients have evidence of a bone
marrow that doesn’t work well or well enough to
maintain normal blood counts
Therefore, whatever causes aplastic anemia
(immune suppression or dysregulation or
damage to the stem cells) may allow PNH to
develop
What is PNH?
Complement
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Complement is a group of blood proteins that
act together to help the body get rid of
microbiological invaders
 One of the ways it does this is by
penetrating the membrane (outside
surface) of the invading bacteria or
viruses.
 When this happens to PNH blood cells, the
cells are destroyed.
What is PNH?
Complement circulates in an inactive form
 It is activated spontaneously and by a
variety of events
It is normally activated more at night
 It is more active with infections,
trauma, vaccinations, surgery, immune
complexes, autoimmune diseases
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What is PNH?
Complement
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Complement activity is regulated by proteins in
the blood and on the membranes of the cell.
Proteins on the cell surface interfere with
complement to prevent breakdown (lysis)
of the cell membrane
The most important of these is CD59, which
is missing on the abnormal cells of PNH
 For this reason, PNH red cells are extremely
sensitive to very small amounts of activated
complement
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Absence of CD59 Allows
Terminal Complement Complex Formation
C5a
C5
C6
C3
C3b
C5
convertase
Bb
+
C5
convertase
C5b
C7
C8
C6
C7
C5b
C6
C7
C8
C9
C9
X
X
C5b
CD59
C6
C7
C8
C5b
CD59
C9
x 12 - 15
C6
C5b
C7
C8
C5b-9
C5b,6,7
C5b-8
Adapted from Cellular and Molecular Immunology AK Abbas, AH Litchman and JS Pober,
3rd Edition. 1991 WB Saunders; Philadelphia.
What is PNH?
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Complement successfully attacks the red
cells and they break up (hemolysis)
This releases hemoglobin (the red pigment in
red cells) into the plasma
 Causes anemia
 Pieces of the membrane come off.
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The white cells release granule contents
and change to express other proteins.
The platelets form vesicles (membrane
blisters) and activate.
What is PNH?
Normal red blood
cells are protected
from complement
attack by a shield of
terminal
complement
inhibitors
Complement
activation
Without this
protective
complement
inhibitor shield, PNH
red blood cells are
destroyed
What is PNH?
Clinical Features
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Some of the hemoglobin passes through
the kidneys and into the urine, causing red
to dark brown urine (hemoglobinuria)
 This causes a loss of iron from the body
 In the long run, this may damage the
kidney
Free hemoglobin binds nitric oxide causing
vascular and smooth muscle spasm
Causes inflammation
Action of Nitric Oxide (NO)
NO
Smooth Muscle
Relaxation
Smooth Muscle
Contraction
NO
Free Hemoglobin Binds NO
NO
NO
Smooth Muscle
Relaxation
NO
Free Hemoglobin
Smooth Muscle
Contraction
What Are The Effects of Nitric
Oxide Trapping by Hemoglobin
Spasm of the esophagus
 Abdominal pain
 Erectile dysfunction
 Other symptoms such as “fatigue”
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What is PNH
Clinical aspects
Vascular (arterial constriction, HBP)
 Pulmonary artery pressure increase
(PHTN)
 Spasm of the esophagus
 Abdominal pain
 Erectile dysfunction
 Other symptoms such as “fatigue”
 Platelets are more “reactive”
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What is PNH?
Clinical Features
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WBC:
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Granulocytes - release content stimulating
inflammation
Monocytes - activate expressing TF which leads to
blood clots. TF-Microvesicles
Platelets become “activated”
They stick together and form clumps
 The membrane changes, allowing them to bind
to monocytes
 Pieces of the membrane come off
(microvesicles)
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What is PNH?
Clinical Features
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Hemolytic anemia due to complement
activation
Hemoglobinuria and, eventually, kidney
damage
 Anemia to a variable degree
 Effects of NO depletion- HBP, smooth muscle
dystonia, reduced blood flow to the kidney
and lungs
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Impaired bone marrow function
What is PNH?
Clinical Features
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Thrombosis (Blood clots)
 Often in unusual places (liver veins,
abdominal veins, cerebral veins, dermal
veins)
Fatigue – overwhelming, poor correlation to
level of hemoglobin
 inflammation
 anemia
 Portal hypertension (PHTN).
Thrombosis in PNH Pathophysiology
COMPLEMENT INJURY
NO
PS
C5b-9
HEMOLYSIS
COMPLEMENT
MONOCYTES
PLATELET
Endothelial cells
THROMBIN
GENERATION
C5a
TF
TF = Tissue factor.
CYTOKINES IL-6
INFLAMMATION
Laboratory Evaluation of PNH
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Acidified Serum Test (Ham Test 1939)
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Acidified serum activates alternative complement
pathway resulting in lysis of patient’s rbcs
May be positive in congenitial dyserythropoietic
anemia
Still in use today
Sucrose Hemolysis Test (1970)
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10% sucrose provides low ionic strength which
promotes complement binding resulting in lysis of
patient’s rbcs
May be positive in megaloblastic anemia,
autoimmune hemolytic anemia, others
Less specific than Ham test
Laboratory Evaluation of PNH
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PNH Diagnosis by Flow Cytometry (1986)
 Considered method of choice for diagnosis
of PNH (1996)
 Detects actual PNH clones lacking GPI
anchored proteins
 More sensitive and specific than Ham and
sucrose hemolysis test
PNH Diagnosis by Flow Cytometry
Of the long list of GPI anchored protein, monoclonal antibodies
to the following antigens have been used in the diagnosis of PNH
The most useful Abs are to CD14, 16, 55, 59, and 66. Are all
required? Probably not - more studies needed
Antigen
Cell Lineage
Function
CD14
monocytes
LPS receptor, MDF
CD16
neutrophils
FcIII receptor
CD24
neutrophils
B-cell differentiation marker
CD55
all lineages
DAF
CD58
all lineages
possible adhesion
CD59
all lineages
MIRL, HRF, protectin
CD66b
neutrophils
CEA-related glycoprotein
PNH Diagnosis by Flow Cytometry
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Antigen expression is generally categorized into three
antigen density groups
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type I Normal Ag expression
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type II Intermediate Ag expression
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type III No Ag expression
Patient samples that demonstrate cell populations with
diminished or absent GPI-linked proteins (Type II or III
cells) with multiple antibodies are considered to be
consistent with PNH.
Should examine multiple lineages (ie granulocytes &
monocytes)
PNH Diagnosis by Flow Cytometry
Examples of variable GPI linked CD59 expression on
granulocytes on four PNH patients
PNH Diagnosis by Flow Cytometry
Example of variable
expression of several
GPI linked Ags on
several lineages
From Purdue Cytometry CD-ROM vol3 97
PNH Diagnosis by Flow Cytometry
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Flow Cytometry is method of choice but only
supportive for/against diagnosis
More studies are needed to better define whether
the type (I, II, or III), cell lineage, and size of the
circulating clone can provide additional prognostic
information.
Theoretically - should be very valuable
Historical Management Options for
PNH
Generally conservative, supportive, and dependent on
symptom severity1,2
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Transfusions
Anticoagulants
Supplements
 Folic acid
 Iron
 Erythropoiesis stimulating agents
Steroids/androgen hormones
Allogeneic bone marrow transplant
(limited eligibility)
What is
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®
Soliris ?
Monoclonal antibody (protein) that blocks
complement at C5 preventing the
formation of the terminal complement
complex
Quickly and markedly reduces hemolysis
Stops hemoglobinuria
 Increases hemoglobin level

Reduces transfusions
 Hematocrit may not be quite normal

Microorganisms
SOLIRIS Blocks Terminal
Complement
Proximal
Classical
C3
Terminal
Lectin
Antigen-Antibody
Complexes
C5
Constitutive/
Microorganisms
Alternative
C3a
• SOLIRIS binds with high
affinity to C5
• Terminal complement
activity is blocked
C3b
C5b
SOLIRIS
C5a
C5b-9
Cause of Hemolysis
in PNH
• Proximal functions of
complement remain intact
•Weak anaphylatoxin
•Immune complex and apoptotic
body clearance
Figueroa, et al. Clin Microbiol Rev. 1991;4:359-395.
•Microbial
Walport. N Engl J Med. 2001;344:1058.
SOLIRIS™ (eculizumab) [package insert]. Alexion Pharmaceuticals; 2007.
opsonization
Reduction in LDH During Soliris® Treatment
in TRIUMPH and SHEPHERD
Lactate Dehydrogenase (U/L)
3000
TRIUMPH – Placebo/extension
TRIUMPH – SOLIRIS/extension
SHEPHERD – SOLIRIS
2500
2000
1500
1000
500
0
0
10
20
30
40
50
Time, Weeks
PI: All patients sustained a reduction in intravascular hemolysis over a total SOLIRIS exposure time
ranging from 10 to 54 months.
TRIUMPH placebo patients switched to SOLIRIS after week 26. All TRIUMPH patients entered the long term extension study.
Urine score 2 weeks
before & after Eculizumab
4 4 7 7 5 5 8 8 5 84 1 1 8
Pre-eculizumab
1 1 1 1 1 1 1 1 1 1 1 1 1 1
Post-eculizumab
Hemoglobin Stabilization (%)
Effect of Soliris® on Ability to
Maintain a Good Hemoglobin
50
40
P<0.000000001
30
20
10
0
Placebo
SOLIRIS
Effect of Soliris® on
Transfusion in PNH
Transfused
Units/Patient (median)
10
8
P<0.0000001
6
4
2
0
Placebo
Soliris®
What is the effect of Soliris®
in PNH

Stops the symptoms associated with
hemolysis
“Fatigue”
 Esophageal and abdominal spasm
 Erectile dysfunction
 Improves sense of well being
 Reduced the need for transfusion


Appears to reduce thrombosis (blood
clots)
Effect of Eculizumab on the blood
clots in PNH
45
40
35
30
25
20
15
10
5
0
39
3 P=0.0001
Patients on Antithrombotics n=103
(P < 0.000000001
12.00
10.61
Thrombosis Event Rate
(TE per 100 Pt-Years)
Thrombotic Events (#)
Equalized Patient Years (195)
10.00
8.00
6.00
4.00
2.00
0.62
0.00
Pre-Eculizumab
Pre-Soliris
Treatment
Soliris
Treatment
Post-Eculizumab
92% reduction in TE events with
Eculizumab
Pre-Soliris Treatment
Pre-Eculizumab
Soliris
Treatment
Post-Eculizumab
91% reduction in TE event rate with
Eculizumab
What is the Effect of
Soliris®?
Improves kidney function
reduced hemoglobinuria and iron deposition
 Reduced thrombosis

Improves hypertension
May in part be due to availability of nitric oxide
Side Effects of Soliris®
Treatment

Susceptibility to sepsis by meningococcal
organism






All patients must be vaccinated at least 2 weeks
before starting Soliris
All patients must know to seek medical help at once
when fever happens
All patients must carry cards describing this
complication
Headache – first week or 2
Cost
Inconvenience

Must be given every 12-14 days by vein
What

®
Soliris
Cannot Do
Does not appear to improve impaired
bone marrow function
Low white count or low platelet count may
persist in some patients, especially if it is due
to aplastic anemia
 Other treatments may be indicated

Bone marrow transplantation
 immunosuppressives

When is Soliris Ineffective
or Less Effective




Patient has been incorrectly diagnosed
with PNH.
Patient has a very small PNH clone (less
than 10%)
bone marrow failure- AA
Breakthrough – infections, increased
clearance, delayed dosing
Extravascular hemolysis
Which PNH Patients are
Candidates for Soliris?








Patients with hemolysis (LDH )
Patients with large CD 59 deficient clones- RBC ,
WBC
Patients with hemolysis with evidence of renal
impairement GRF<90, proteinuria etc
Patients with history of thrombosis
Patients with hemolysis and elevated Ddimers
Patients with hemolysis with fatigue
Patients with hemolysis and transfusion
dependence
Patients prior to ?BMT, surgery, ???pregnancy
Who Should Get
®
Soliris ?
In all cases, the decision should be made by
a physician that understand PNH, its
complications and its treatment in
conjunction with the patient, whose life is
affected by the disorder.