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Paroxysmal Nocturnal
Hemoglobinuria
J. Christian Barrett, MD
PATHOPHYSIOLOGY OF PNH
Paroxysmal Nocturnal Hemoglobinura
• A disease of the hematopoietic stem cell
– Non-malignant clonal disease
• Defective post-translational processing of
glycosylphosphotidylinositol biosythesis
– Involves >26 different gene products
– Mutations in any of them could potentially lead to
PNH phenotype.
PIG-A Gene
• Acquired somatic mutation of the PIG-A gene
– Located on X-chromosome
– Involved in the first step of the GPI anchor
biosynthesis
• The addition of n-acetylglucosamine to the
phosphatidylinositol on the cytoplasmic side of the
endoplasmic reticulum membrane
Glycosylphosphatidylinositol Anchor
Glycosylphosphatidyinositol Anchor
http://www.sigmaaldrich.com/technical-documents/articles/biology/glycobiology/gpianchored-glycoproteins.html
Transport to the Cellular Surface
http://www.fbs.osaka-u.ac.jp/organelle-network/eng/research/22/
PNH arises through a block in the biosynthesis of the GPI anchor.
PIG-T mutations
also described in
a patient with a
double hit on
the PIG-T gene
located on
chromosome
20—involves the
last step of
attaching the
surface protein
to the formed
GPI-anchor
Lucio Luzzatto Blood 2013;122:1099-1100
©2013 by American Society of Hematology
CD55 and CD 59
• CD 55
– Decay Activating Factor (DAF)
• CD 59
– MAC Inhibitory Protein (MAC-IP)
– Membrane Inhibitor of Reactive Lysis (MIRL)
Noris, M. et al. (2012) STEC-HUS, atypical HUS and TTP are all diseases of complement
activation
Nat. Rev. Nephrol. doi:10.1038/nrneph.2012.195
Normal State: Protection Against
Complement Deposition
Lucio Luzzatto, Antonio Maria Risitano, Rosario Notaro
Haematologica April 2010 95: 523-526; Doi:10.3324/haematol.2009.017848
Complement-Medicated Hemolysis
Lucio Luzzatto, Antonio Maria Risitano, Rosario Notaro
Haematologica April 2010 95: 523-526; Doi:10.3324/haematol.2009.017848
Hemolysis-Induced Nitric Oxide Depletion
Smooth muscle contraction
Vascular constriction
systemic HTN
pulmonary HTN
GI dysmotility
Erectile dysfunction
Platelet activation
Thrombosis formation
American Society of Hematology Hematology
2005;2005:544-552
©2005 by American Society of Hematology
PNH and Thrombosis Risk
• Most common cause of PNH-related mortality
• ~44% of clinical PNH patients have a thrombus
• Venous thrombosis most common
– Often in unusual sites
• Arterial events possible and also increased
• Etiology is multifactorial
PNH and Thrombosis Risk
• Nitric oxide depletion by free HGB
– Vasoconstriction
– Platelet activation
• Complement deposition on platelets
– Platelets microparticle release
• Increased C5a due to complement activation
– Leads to inflammatory cell release of IL-6, IL-8 and TNF-alpha
• Decreased GPI-anchored tissue factor pathway inhibitor
receptor
– Leads to increased coagulation cascade activation
• Decreased GPI-anchored plasminogen activator receptor
– Leads to impaired fibrinolysis
More than Just Complement Disease
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Prion Protein (PrP)
FcgIII receptor (CD16) -- APCs
Tissue factor pathway inhibitor receptor
Urokinase-type plasminogen activator
receptor
Clonal Evolution and Expansion
Hypothesis of Clonal Selection and
Expansion
Bone Marrow Failure States
• Association with Aplastic Anemia and MDS
– Long appreciated
– ? Mechanism
– ? Causal relationship
PNH Clone in Patients with Bone Marrow Failure
British Journal of Haematology
Volume 147, Issue 1, pages 102-112, 28 JUL 2009 DOI: 10.1111/j.1365-2141.2009.07822.x
http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2141.2009.07822.x/full#f1
Fate of PNH Clone in Patients with Bone Marrow
Failure
17%
59%
24%
British Journal of Haematology
Volume 147, Issue 1, pages 102-112, 28 JUL 2009 DOI: 10.1111/j.1365-2141.2009.07822.x
http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2141.2009.07822.x/full#f3
IST did not appear to influence this fate
among the 35 AA patients treated
compared with the untreated patients
DIAGNOSIS OF PNH
Clinical Presentation
• Intravascular hemolysis
– Fatigu,. lethargy, and asthemia
– Dyspnea with exertion
– Chest pain
– Jaundice
– Hemoglobinuria
Clinical Presentation
• Vascular thrombosis
– Leg pain and/or edema
– Chest pain and/or Dyspnea
– Liver failure
– Abdominal pain
– Headaches
Laboratory Investigation
• Routine labs
– CBC
– Hemolysis labs
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Reticulocyte count
LDH and fractionated bilirubin
Haptoglobin
Urine hemosiderin
Direct Antigen Test (DAT)
– Iron studies
• Flow cytometry looking at CD55 and CD59
– RBC and Granulocytes
Flow Cytometry Results
• Percentage of PNH cells is highly variable
High-sensitivity flow cytometric analysis of erythrocytes.
Charles Parker et al. Blood 2005;106:3699-3709
©2005 by American Society of Hematology
Flow Cytometry
Type II
cells
Type III
cells
Type I
cells
Type
I
cells
Endo et al. Blood 1996;87:2546-2557
Phenotypic Mosaicism:
Hypothetical histograms of RBC stained with anti-CD59
Charles Parker et al. Blood 2005;106:3699-3709
©2005 by American Society of Hematology
Flow Cytometry: PMNs vs. RBCs
PMNs are more reliably
informative
Classification of PNH
• Classic
– Hemolysis
• Florid (markedly abnormal LDH often with episodic
macroscopic hemoglobinuria)
– Marrow
• Cellular marrow due to erythroid hyperplasia and normal or
near-normal morphology††
– Flow
• Large population (>50%) of GPI-AP deficient PMNs
– Effectiveness of Eculizumab
• Yes
Based on recommendations of the International PNH Interest
Group(Blood2005;106:3699-3709)
Classification of PNH
• PNH in the setting of another bone marrow failure syndrome§
– Hemolysis:
• Mild (often with minimal abnormalities of biochemical markers of hemolysis)
– Marrow
• Evidence of a concomitant bone marrow failure syndrome§
– Flow
• Although variable, the percentage of GPI-AP deficient PMNs is usually
relatively small (<50%)
– Effectiveness of Eculizumab
• Typically no, but some patients have relatively large clones and clinically
significant hemolysis and may benefit from treatment
§ Aplastic anemia or low risk myelodysplastic syndrome
Based on recommendations of the International PNH Interest
Group(Blood2005;106:3699-3709)
Classification of PNH
• Subclinical
– Hemolysis
• No clinical or biochemical evidence of intravascular hemolysis
– Marrow
• Evidence of a concomitant bone marrow failure syndrome
– Flow
• Small (<1%) population of GPI-AP deficient PMNs detected by
high-resolution flow cytometry
– Effectiveness of Eculizumab
• No
§ Aplastic anemia or low risk myelodysplastic syndrome
Based on recommendations of the International PNH Interest
Group(Blood2005;106:3699-3709)
MANAGEMENT OF PNH
Management of Classic PNH
• Anticoagulation therapy
– NOT ALWAYS effective
– Duration is controversial
• Complement blockade
• Hematopoietic stem cell transplantation
Management of Classic PNH
• Complement blockade
– Eculizumab
• Humanized monoclonal antibody
• Binds C5
– Administration
• IV every 7 days x 5 doses  every 14 days
Eculizumab Toxicities
• Headache
– Due to sudden increase in nitric oxide
– ~50% with the first dose
• Improves with repeated dosing
• Life-threatening Neissserial infections
– ~0.5%/year (5% after 10 years)
– Should vaccinate
• Ciprofloxacin prophylaxis x 2 weeks if must urgently
start therapy concomitantly with vaccination
Complement regulation and eculizumab.
Robert A. Brodsky Blood 2014;124:2804-2811
©2014 by American Society of Hematology
What is the Clinical Effect of
Eculizumab Therapy?
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On symptoms of fatigue and QOL?
On renal function?
On thrombosis?
On hemolysis?
On marrow failure?
On transfusion needs?
Overall survival of eculizumab treatment compared with an age- and sex-matched normal
population.
Richard J. Kelly et al. Blood 2011;117:6786-6792
©2011 by American Society of Hematology
Overall Survival of Patients before and after Eculizumab.
Richard J. Kelly et al. Blood 2011;117:6786-6792
©2011 by American Society of Hematology
Blood transfusion requirements in the 12 months before eculizumab therapy and the most
recent 12 months on eculizumab treatment in 64 patients.
Richard J. Kelly et al. Blood 2011;117:6786-6792
©2011 by American Society of Hematology
PNH Treated with Eculizumab
Now often DAT C3+
Lucio Luzzatto, Antonio Maria Risitano, Rosario Notaro
Haematologica April 2010 95: 523-526; Doi:10.3324/haematol.2009.017848
What is the Clinical Effect of
Eculizumab Therapy?
• Effect of Complement Receptor 1 (CR1)
polymorphisms?
• Effect of C5 mutation c.2654G→A ?
Hematopoietic Stem Cell
Transplantation
• Failure to respond to Eculizumab
Bone Marrow Failure States
• Treat as you otherwise would
– Aplastic anemia
• Allogeneic transplantation
• Immunosuppressive therapy
– Myelodysplasia
• Hypomethylating agent
• Allogeneic transplantation
Prognosis after IST compared between patients with PNH+ and with PNH-.
Chiharu Sugimori et al. Blood 2006;107:1308-1314
©2006 by American Society of Hematology
Charles J. Parker Hematology 2011;2011:21-29
©2011 by American Society of Hematology