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/gpi-anchored-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 PNH arises through a block in the biosynthesis of the GPI anchor. (Left) Normal erythrocytes and the biosynthesis of GPI. (A) With phosphatidylinositol (PI) on the cytoplasmic side of the ER, the first biosynthetic step, the addition of N-acetylglucosamine to PI is catalyzed by an enzyme complex that comprises a subunit encoded by the X-linked gene PIG-A. (B-C) The biosynthetic pathway proceeds through several enzymatic steps (broken arrow) that add further sugar moieties (green symbols), until the complete GPI molecule is on the luminal side of the ER. Preformed protein (ochre) becomes covalently linked to the GPI anchor through a transamidation reaction. This step is catalyzed by an enzyme complex that comprises a subunit encoded by the gene PIG-T on chromosome 20. (D) Among the red cell proteins that are tethered to the membrane through GPI anchors, ≥2 are important in protecting red cells from activated complement: CD55 that regulates the C3 convertase and CD59 that impedes the lytic action of the membrane attack complex (MAC). (Right) Abnormal PNH erythrocytes: the tethering to the membrane of proteins that need the GPI anchor can be compromised by failure of any of the enzyme reactions above. (A) In the majority of patients with PNH, a somatically acquired inactivating mutation11 of PIG-A (of which in each cell there is only 1 active allele on the X-chromosome) blocks the very take-off of the GPI biosynthesis. (B) Krawitz et al report for the first time a patient in whom the block is instead at the very last step: the GPI anchor is ready, but the protein does not become linked to it because of mutations in both alleles of the PIG-T gene. (C) PNH erythrocytes, lacking CD55, bind C3 convertase; moreover, because they lack CD59, they are vulnerable to MAC attack, and when this takes place, they are lysed. The elements of this figure pertaining to the GPI biosynthetic pathway (top two and middle two panels) were redrawn from the work of Kinoshita et al.,12 and the elements pertaining to the action of complement on normal and on PNH red cells (bottom two panels) were redrawn from Luzzatto et al.13 Professional illustration by Xavier Studio. Lucio Luzzatto Blood 2013;122:1099-1100 ©2013 by American Society of Hematology

CD55 and CD 59 CD 55 CD 59 Decay Activating Factor (DAF) MAC Inhibitory Protein (MAC-IP) Membrane Inhibitor of Reactive Lysis (MIRL)

Nat. Rev. Nephrol. doi:10.1038/nrneph.2012.195 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 Pathogenesis and therapeutic targets in hemolysis-associated pulmonary hypertension and vasculopathy. Intravascular hemolysis releases hemoglobin into plasma which reacts with and destroys endothelial derived nitric oxide (NO). Arginase is also released from the red cell into plasma during hemolysis and degrades arginine, further reducing NO formation from arginine. Xanthine oxidase bound to endothelium produces superoxide which also inhibits NO. Reduced NO bioavailability promotes vasoconstriction, activation of adhesion molecules (VCAM), activation of endothelin-1, a potent vasoconstrictor, and activation of platelets and thrombosis (tissue factor). Splenectomy is associated with pulmonary hypertension and may increase thrombotic risk as well as intravascular hemolysis. A number of therapies that target these pathways are shown on the outside of the blood vessel. Abbreviations: Hb, hemoglobin; PDE5, phosphodiesterase 5; VCAM-1, vascular cell adhesion molecule-1.Figure reproduced with permission from Lin et al.63 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 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  Significant increases in the percentage of PNH‐type cells among the patients at the diagnosis of bone marrow failure. (A) PNH‐type granulocytes in patients with various diseases. The numbers above the diagnosis represent the prevalence of increased PNH‐type granulocytes (%) and the number of patients studied. The solid line denotes a threshold for the significant increase in the PNH‐type cell percentage. (B) Correlation between PNH‐type granulocytes and erythrocytes. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. 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%  Pattern diagram of the PNH‐type cell transition. Phase 1 refers to the first phase which occurs before the diagnosis of BM failure while phase 2 refers to change in the PNH‐type cell percentage after the diagnosis. 5Y, 10Y; 5 and 10 years, respectively. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. 24% IST did not appear to influence this fate among the 35 AA patients treated compared with the untreated patients 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

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 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. High-sensitivity flow cytometric analysis of erythrocytes. By careful gating and by using dual-color flow cytometry, GPI-deficient cells that comprise less than 1% of the total erythrocyte population can be reliably and reproducibly demonstrated (B-C).2 Using this technique, GPI-AP–deficient cells are not identified in the peripheral blood of the controls (volunteer donors; A). A combination of FITC-labeled anti-CD55 and anti-CD59 was used along with phycoerythrin (PE)–labeled anti–glycophorin A for the dual staining. These data were kindly provided by Dr Shinji Nakao and Dr Chiharu Sugimori, Kanazawa University, Japan, and are used with their permission. Illustration enhanced by A. Y. Chen. Charles Parker et al. Blood 2005;106:3699-3709 ©2005 by American Society of Hematology

Flow Cytometry Endo et al. Blood 1996;87:2546-2557 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 Phenotypic mosaicism in PNH. Hypothetical histograms of erythrocytes from patients with PNH stained with anti-CD59 are illustrated. The proportion and type of abnormal erythrocytes varies greatly among patients with PNH and these characteristics are important determinants of clinical manifestations. In general, patients with a high percentage of type III erythrocytes have clinically apparent hemolysis (A). If the erythrocytes are partially deficient in GPI-AP, hemolysis may be modest even if the percentage of the affected cells is high (B). A patient may have a diagnosis of PNH, but if the proportion of type III cells is low, only biochemical evidence of hemolysis may be observed (C). Illustration enhanced by A. Y. Chen. 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 Analysis of PMNs is more informative than analysis of RBCs due to selective destruction GPI-AP deficient RBCs

Classification of PNH Classic Hemolysis Marrow Flow 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. Complement regulation and eculizumab. The lectin, classical, and alternative pathways converge at the point of C3 activation. In PNH, hemolysis is usually chronic because the alternative pathway is always in a low-level activation state through a process known as tick-over. Terminal complement begins with cleavage of C5 to C5a and C5b. C5b oligomerizes with C6, C7, C8, and multiple C9 molecules to form the MAC. CD55 inhibits proximal complement activation by blocking the formation of C3 convertases; CD59 inhibits terminal complement activation by preventing the incorporation of C9 into the MAC. The absence of CD55 and CD59 on PNH cells leads to hemolysis, inflammation, platelet activation, and thrombosis. Eculizumab inhibits terminal complement activation by binding to C5 and preventing generation of C5a and C5b. Robert A. Brodsky Blood 2014;124:2804-2811 ©2014 by American Society of Hematology

What is the Clinical Effect of Eculizumab Therapy? On symptoms of fatigue and QOL? On renal function? On thrombosis? On hemolysis? On marrow failure? On transfusion needs? Overall,’ Improved symptoms and QOL Improved renal function Decreased thrombosis Improved intravascular hemolysis Does not help the marrow failure May decrease of eliminate need for transfusions depending on to degree to which the anemia persists either due to (1) marrow failure and (2) ongoing hemolysis? Why might the hemolysis not been eliminated?

Overall survival of eculizumab treatment compared with an age- and sex-matched normal population. Overall survival of 79 patients from initiation 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. 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 ? CR1 polymorphisms have been noted with some being associated with higher (others lesser) biding affinity for C3 and C4. Increased CR1 binding affinity is associated with increased decay of the C3 and C5 convertases. This leads to decreased complement C3 binging to the RBC membrane  less opsonization --> less macrophage-induced extracellular hemolysis in these patients during eculizumab therapy. Thus, less residual hemolysis, higher hemoglobin levels, and decreased need for transfusions. C5 mutation c.2654G→A has been described in ~3.5% of Japanese individuals. It blocks Eculizumab biding rendering it ineffective. It does not effect the cellular binding of C5 not its complement function in activating the MAC complex.

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

Prognosis after IST compared between patients with PNH+ and with PNH-. Prognosis after IST compared between patients with PNH+ and with PNH-. (A) Overall survival; (B) failure-free survival; (C) incidence of clonal hematologic disorders, including PNH, myelodysplastic syndrome, and acute myelogenous leukemia; and (D) incidence of relapse. Chiharu Sugimori et al. Blood 2006;107:1308-1314 ©2006 by American Society of Hematology

Treatment algorithm based on disease classification Treatment algorithm based on disease classification. Disease classification is based on the recommendations of the International PNH Interest Group.13 Charles J. Parker Hematology 2011;2011:21-29 ©2011 by American Society of Hematology