Paroxysmal%20Nocturnal%20Hemoglobinuria

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ParoxysmalNocturnalHemoglobinuria

• New ideas about an old disease
• Ahmad Shihada Silmi Msc, FIBMS
• Staff Specialist in Hematology
• Medical Technology Department
• Islamic University of Gaza

2
Objectives

• 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

3
What is PNH?

• 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.
• There is no ethnic preference for the disorder.
• It may present early or late in life.
• The manifestations may be classic or obscure.

4
PNH Prognosis

• 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
• 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
• Likely incident of leukemia lower than 5

5
What is PNH Mutation?

• 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.
• gt100 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

6
What is PNH?

• 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

7
What is PNH?

• 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

8
Stem Cells
Egg
Sperm
Egg
Embryonic Stem Cell
Egg or Sperm
Blood
Muscle
Nerve
Etc.
Somatic Stem Cells
The Cells of Each Specific Organ
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Stem Cells
Egg
Sperm
Egg
Embryonic Stem Cell
Egg or Sperm
Blood
Muscle
Nerve
Etc.
Somatic Stem Cells
The Cells of Each Specific Organ
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Stem Cells
Egg
Sperm
Egg
Embryonic Stem Cell
Egg or Sperm
Blood
Muscle
Nerve
Etc.
Somatic Stem Cells
The Cells of Each Specific Organ
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• Stem Cells

T LYMPHOCYTES
STEM CELL
B LYMPHOCYTES
ERYTHROCYTES
GRANULOCYTES
MONOCYTES
PLATELETS
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• Stem Cells

T LYMPHOCYTES
STEM CELL
B LYMPHOCYTES
ERYTHROCYTES
GRANULOCYTES
MONOCYTES
ALTERED GENE
PLATELETS
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• Stem Cells

NORMAL
CLONES
ABNORMAL
CLONE
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GPI-AP BiosynthesisInvolves 10 Steps and gt20
Genes
Plasma Membrane
Raft
N
N
PIG-A
PI
GlcNAc-PI
Endoplasmic Reticulum
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The Beginning of PNH

• The change that occurs in PNH stops the
  production of an anchor that ties protein
  molecules to the cell
• Sometimes the stop is only partial and PNH II
  cells occur

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• The GPI Anchor Defect in PNH

PNH
O
O-P-O
O
CH
CH
CH
2
2
O
O
CO
(180,1)
(224,5)
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Pathogenesis - The Defect
GPI Anchor

• 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
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What is PNH?

• 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
  cant attach to the cells.
• The most important proteins for PNH are CD 59,
  CD55.

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Proteins anchored by GPI Anchorand
Surface Proteins Missing on PNH Blood
Cells Antigen
Expression
Pattern Enzymes Acetylcholinesterase (AchE)
Red blood cells Ecto-5'-nucleotidase
(CD73) Some B- and T-lymphocytes Neutrophi
l alkaline phosphatase(NAP) Neutrophils ADP-rybo
syl transferase Some T-lymphs,
Neutrophils Adhesion molecules Blast-I/CD48
Lymphocytes Lymphocyte
function- associated antigen-3(LFA-3 or CD58)
All blood cells CD66b Neutrophils Complem
ent regulating surface proteins Decay
accelerating factor (DAF or CD55) All
blood cells Homologous restriction
factor, Membrance inhibitor of reactive lysis
All blood cells (MIRL or CD59)
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Surface Proteins Missing on PNH Blood
Cells Antigen
Expression
Pattern Receptors Fc-? receptor III (Fc ? Rlll
or CD16) Neutrophils, NK-cells,
macrophages, some T-lymphocytes Monocyte
differentiation antigen Monocytes,
macrophages (CD14) Urokinase-type Plasminogen
Monocytes, granulocytes Activator Receptor
(u-PAR, CD87) Blood group antigens Comer
antigens (DAF) Red
blood cells Yt antigens (AchE)
Red blood cells Holley Gregory antigen
Red blood cells John Milton
Hagen antigen (JMH) Red blood cells,
lymphocytes Dombrock reside
Red blood cells Neutrophil antigens NB1/NB2

Neutrophils
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Surface Proteins Missing on PNH Blood
Cells Antigen
Expression
Pattern Other surface proteins of unknown
functions CAMPATH-1 antigen (CDw52)
Lymphocytes, monocytes CD24
B-lymphocytes, Neutrophils,
eosinophils p5O-80 Neutrophils GP500

Platelets GPI75
Platelets
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PathogenesisCD 55

• 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

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PathogenesisCD59

• 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

24
Pathogenesis of CD55 CD59

• Inherited absences of both proteins in humans
  have been described
• 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

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Pathogenesis

• 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
  marrow-WHY?

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Pathogenesis - Clonal evolution and cellular
selection

• Expansion of abnormal hematopoietic stem cell
  required for PNH disease expression
• Theories for expansion
• 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
• 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???)

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PNH Clinical Features Aplastic Anemia

• Some PNH patients have aplastic anemia or a
  history of aplastic anemia
• Many PNH patients have evidence of a bone marrow
  that doesnt 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

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What is PNH? Complement

• 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.

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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

• 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
C9
C9
x 12 - 15
C8
X
X
C5 convertase
C5 convertase
Bb

C3b
C3
C9
C8
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.
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What is PNH?

• 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.
• The white cells release granule contents and
  change to express other proteins.
• The platelets form vesicles (membrane blisters)
  and activate.

35
What is PNH?
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
Complement activation
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What is PNH?Clinical Features

• 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

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Action of Nitric Oxide (NO)
NO
NO
Smooth Muscle Relaxation
Smooth Muscle Contraction
38
Free Hemoglobin Binds NO
NO
NO
Smooth Muscle Relaxation
NO
Free Hemoglobin
Smooth Muscle Contraction
39
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 PNHClinical 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

41
What is PNH?Clinical Features

• WBC
• 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

• 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
• Impaired bone marrow function

44
What is PNH?Clinical Features

• 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).

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Thrombosis in PNH Pathophysiology
COMPLEMENT INJURY
NO PS
C5b-9 HEMOLYSIS
MONOCYTES PLATELET Endothelial cells
THROMBIN GENERATION
COMPLEMENT
C5a
TF
CYTOKINES IL-6 INFLAMMATION
TF Tissue factor.
46
Laboratory Evaluation of PNH

• Acidified Serum Test (Ham Test 1939)
• Acidified serum activates alternative complement
  pathway resulting in lysis of patients rbcs
• May be positive in congenitial dyserythropoietic
  anemia
• Still in use today
• Sucrose Hemolysis Test (1970)
• 10 sucrose provides low ionic strength which
  promotes complement binding resulting in lysis of
  patients rbcs
• May be positive in megaloblastic anemia,
  autoimmune hemolytic anemia, others
• Less specific than Ham test

47
Laboratory Evaluation of PNH

• 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

48
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 LP
S 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
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PNH Diagnosis by Flow Cytometry

• Antigen expression is generally categorized into
  three antigen density groups
• type I Normal Ag expression
• type II Intermediate Ag expression
• 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)

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PNH Diagnosis by Flow Cytometry
Examples of variable GPI linked CD59 expression
on granulocytes on four PNH patients
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PNH Diagnosis by Flow Cytometry
Example of variable expression of several GPI
linked Ags on several lineages
From Purdue Cytometry CD-ROM vol3 97
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PNH Diagnosis by Flow Cytometry

• 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 thecirculating clone can provide additional
  prognosticinformation.
• Theoretically - should be very valuable

53
Historical Management Options for PNH
Generally conservative, supportive, and dependent
on symptom severity1,2

• Transfusions
• Anticoagulants
• Supplements
• Folic acid
• Iron
• Erythropoiesis stimulating agents
• Steroids/androgen hormones
• Allogeneic bone marrow transplant(limited
  eligibility)

54
What is 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

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SOLIRIS Blocks Terminal Complement
Antigen-Antibody Complexes
Constitutive/Microorganisms
Microorganisms
Lectin
Classical
Alternative
SOLIRIS

• SOLIRIS binds with high affinity to C5

C3
C3a
Proximal

• Terminal complement activity is blocked

C3b

• Proximal functions of complement remain intact
• Weak anaphylatoxin
• Immune complex and apoptotic body clearance
• Microbial opsonization

C5
C5a
Terminal
C5b-9 Cause of Hemolysis in PNH
C5b
Figueroa, et al. Clin Microbiol Rev.
19914359-395. Walport. N Engl J Med.
20013441058. SOLIRIS (eculizumab) package
insert. Alexion Pharmaceuticals 2007.
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Reduction in LDH During Soliris Treatment in
TRIUMPH and SHEPHERD
3000
TRIUMPH Placebo/extension
TRIUMPH SOLIRIS/extension
2500
SHEPHERD SOLIRIS
2000
Lactate Dehydrogenase (U/L)
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.
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Urine score 2 weeks before after Eculizumab
4
7
7
5
5
8
8
5
8
4
1
1
8
1
1
1
1
1
1
1
1
1
1
1
1
1
1
4
Pre-eculizumab
Post-eculizumab
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Effect of Soliris on Ability to Maintain a Good
Hemoglobin
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Effect of Soliris on Transfusion in PNH
10
8
Plt0.0000001
Transfused Units/Patient (median)
6
4
2
0
Soliris
Placebo
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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)

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Effect of Eculizumab on the blood clots in PNH

• Equalized Patient Years (195)

• Patients on Antithrombotics n103
• (P lt
  0.000000001

Pre-Eculizumab
Post-Eculizumab
Pre-Eculizumab
Post-Eculizumab
92 reduction in TE events with Eculizumab
91 reduction in TE event rate with Eculizumab
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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

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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

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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

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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

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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 GRFlt90, 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

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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.