Paroxysmal Nocturnal Hemoglobinuria (PNH): A Chronic, Systemic, and Life-Threatening Disease. Actuarial Survival From the Time of

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1 PNH Current Thinking on the Disease, Diagnosis, and Treatment Bart Scott, MD Associate Professor of Medicine, Division of Oncology, University of Washington Associate Member, Fred Hutchinson Cancer Research Center What is PNH? PNH is a disease of chronic complement-mediated hemolysis characterized by a somatic (acquired) mutation of the PIG-A gene in which blood cells lack key, naturally occurring terminal complement inhibitors (e.g. CD55 and CD59) on cell surfaces 1-4 This mutation results in a deletion of GPI anchors, rendering proteins unable to attach to the surface of the cell PNH is a progressive and destructive disease that leads to: 5 End-organ damage 1,6 Increased mortality 1,7 PNH: What it s Not It is not paroxysmal 1 Even in the absence of symptoms, destructive progression of hemolysis is ongoing It is not nocturnal 1 Hemolysis in PNH is subtle and constant, 24 hours a day Hemoglobinuria is a less commonly seen complication ¾ patients present without hemoglobinuria Brodsky R. Paroxysmal nocturnal hemoglobinuria. In: R Hoffmanet al., eds. Hematology - Basic Principles and Practices. 4th ed. Philadelphia, PA: Elsevier Churchill Livingstone 25; ;2. Rosse WFet al. Hematology (Am Soc Hematol Educ Program)24:48 62;3.Wiedmer T et al. Blood 1993;82: ;4. Rother RP et al. JAMA 25;293: ; 5. Hillmen P et al. Blood 27;11: ; 6. Hillmen P et al. Am J Hematol 21;85: ;7. Hillmen P et al. N Engl J Med 1995;333: Rother R et al. Nature Biotechnology 27;25,11: ;2. International PNH Interest Group.Blood. 25;16: PNH Classification Classic PNH Intravascular hemolysis Reticulocytosis Increased LDH Increased indirect bilirubin Low haptoglobin PNH + Bone Marrow Disorder Intravascular hemolysis + AA/MDS/Myelofibrosis Subclinical PNH No clinical/lab evidence of hemolysis Detected by very sensitive flow cytometric analysis In association with AA/MDS Paroxysmal Nocturnal Hemoglobinuria (PNH): A Chronic, Systemic, and Life-Threatening Disease Prevalence: 15.9 / million 1 Diagnosed at all ages Median age early 3s 3,4 Progressive disease 2 4 Uncontrolled complement activation underlies the morbiditiesand mortality Despite best supportive care 5 year mortality: 35% 2 Patients Surviving (%) Actuarial Survival From the Time of Diagnosis in 8 Patients With PNH Years After Diagnosis Age- and Gender- Matched Controls Patients With PNH The expected survival of an age- and gender-matched control group is shown for comparison (Hillmen et al. 1995) The Defect in PNH PNH clones are defined as PNH cells with a deficiency of proteins that require a GPI anchor for attachment to the cell membrane 1 CD59 (MIRL) Forms a defensive shield for red blood cells (RBCs) from complement-mediated lysis Inhibits the assembly of the membrane attack complex CD59 GPI-anchor CD55 (DAF) Prevents formation and augments instability of the C3 convertases, attenuating the complement cascade CD55 Parker et al. Blood 25;16: de Latour et al. Blood 28;112: Hill A et al. Blood 26;18:29a. Abstract 985; 2. Hillmen P et al. N Engl J Med 1995;333: ; 3. Nishimura JI et al. Medicine 24;83:193 27;4. Socié G et al. Lancet 1996;348: GPI = glycerophosphatidylinositol. 1. Borowitz MJ et al. Cytometry B Clin Cytom 21;78: Adapted from:johnsonrj et al. J Clin Pathol:Mol Pathol 22;55: ;Brodsky R. Paroxysmal nocturnal hemoglobinuria.in: R Hoffmanet al, eds. Hematology - Basic Principles and Practices. 4thed. Philadelphia, PA: ElsevierChurchill Livingstone;25;

2 Bone Marrow Failure Syndromes PNH MDS AML AA STAT3-Mutations Indicate the Presence of Subclinical Self-Reactive Cytotoxic T Cell Clones in Aplastic Anemia and Myelodysplastic Syndromes RESULTS Distribution of the clinicaland sublinical D661Y and Y64F mutated clones according to the disease type Aplastic Anemia Large Granular Lymphocyte Leukemias Myelodysplastic syndromes PRCA LGL Young NS. Ann Intern Med. 22 Apr 2;136(7): MPN Jerez et al. Blood 213;122: STAT3+ Sub-clinical clones of LGL in AA patients detected by Deep Sequencing, n=1 Clinical STAT3+ clones of LGL in AA patients, n=6 9% 6% 3% % % of patients Aplastic Anemia (n=14) Myelodysplastic syndromes (n=367) STAT3+ Sub-clinical clones of LGL in AA patients detected by Deep Sequencing, n=9 Clinical STAT3+ clones of LGL in MDS patients, n=9 7 8 Jerez et al. Blood 213;122: Autoreactive T Cells from Patients with PNH Specifically Recognize Glycosyl-Phosphatidyl-Inositol (GPI) X chr. PIG-A nucleus Paroxysmal Nocturnal Hemoglobinuria Pathogenesis Etn-P glycan GlcN PIG-A Inositol-P GPI anchor X chr. PIG-A nucleus PIG-A GPI anchor Intravascular Hemolysis PNH Triad Absence of complement regulators ER + protein GPI ER protein Absence of complement regulators Bone Marrow Failure and cytopenia Mechanism? Gargiulo et al. Blood 213 April 4; 121 (14): Normal cell Hematopoietic Stem Cells PNH cell

3 Evidence for a role of autoimmunity in PNH PNH is closely related to aplastic anemia a T-cell-mediated autoimmune disorder: T cells are the likely agents suppressing normal hematopoiesis in PNH. The target of T cells could be either a GPI-linked protein or GPI itself. GPI has been found within the presentation groove of CD1d (Joyce et al. Science 1998; De Silva et al. J Immunol. 22) Identical or quasi-identical TCRβ CDR3 sequences found in CD8+CD57+ T cells in a group of HLA- disparate PNH patients suggesting antigen not HLA - restricted (Gargiulo et al. Blood 25) CD1d is expressed on human and murine hematopoietic stem and progenitor cells (Kotsianidis et al. Blood 26; Broxmeyer et al. Blood 212) Increased frequency of novel invariant TCRVa21 mrna chain within the whole TCRVa21 family repertoire of PNH patients CD1d-dependent GPI-specific T cells in PNH Normal Controls PNH Patients Gargiulo et al. Blood 213 April 4; 121 (14): PNH Patients *Wilcokson Rank Test 13 Gargiulo et al. Blood 213 April 4; 121 (14): X chr. PIG-A nucleus Paroxysmal Nocturnal Hemoglobinuria Pathogenesis Etn-P + protein GPI ER glycan GlcN PIG-A Inositol-P GPI anchor X chr. PIG-A nucleus ER protein PIG-A GPI anchor Parodoxical Expansion of PNH Clone (Escape Theory) Hypothesis: GPI-specific, CD1d-restricted T cells responsible for selection of PNH cells T cell receptor GPI T cell Killing T cell No Killing CD1d/ 2 m Normal cell Hematopoietic Stem Cells PNH cell 16 PIG-A+ cells PIG-A null cells Time crippled PIG-A+ cells Noxious agent Rotoli & Luzzatto, GPI+ ER GPI-anchored protein GPI- ER Karadimitris and Luzzatto, Leukemia

4 Summary Expansion of the PNH Clone Is Necessary to Result in Clinical PNH CD1d-restricted GPI-specific CD8+ T cells are present and expanded in PNH patients. T cells with a novel invariant TCR chain have been discovered in some PNH patients. Step 1 Somatic Mutation of PIG-A Step 2 Immunologic Attack Selective Damage Step 3 Growth Advantage GPI-Deficient Cell Selected Cells Expanded Cells The Role of Complement These T cells could be the noxious agent responsible for the pathogenesis of PNH. Normal Hematopoietic Stem Cells GPI-Anchor Deficiency Immunologic Selection Benign Tumor- Like Expansion Expansion may be due to another somatic mutation The need for both selectionand expansionmay explainthe rarityof PNH 19 Adapted from: Inoue N et al. Int J Hematol 23;77: The Complement System: Always on, Strongly Amplified, Dependent on Natural Regulators The complement system is a vital component of the natural (innate) protective immune system 1 Complement is activated by three mechanisms (classical, alternative, and lectin) which allow the system to respond to inflammatory, infectious, ischemic, necrotic, as well as foreign and self antigens Always on to allow rapid immune response 1 Rapid amplification leads to powerful and destructive immune reactions 2 Natural inhibitors of complement keep amplification in check and prevent uncontrolled complement activation 2 Factors That Amplify Complement Activation Infection Surgery Autoimmune Pregnancy Proximal Terminal Chronic Uncontrolled Complement Activation Leads to Devastating Consequences in PNH Anaphylaxis Inflammation Consequences Lectin pathway Classical pathway Alternative pathway Immune complex clearance Microbial Opsonization Weak anaphylatoxin C3a C5a Potent anaphylatoxin Chemotaxis Pro-inflammatory Leucocyte activation Endothelial activation Pro-thrombotic C3 C3b C5-convertase C5 C5b C6 C7C8 C9 C3 + H 2O: always active (chronic) Amplification ic3b - C5b-9 Membrane attack complex Cell lysis Pro-inflammatory Platelet activation Leucocyte activation Endothelial activation Pro-thrombotic Natural inhibitors: CD55 - Natural inhibitor: CD59 Cell destruction Consequences Inflammation 1. Holers VM et al. Immunol Rev 28;223:3 316;2. Zipfel PF et al. Curr Opin Nephrol Hypertens 21;4: Zipfel PF et al. Vaccine.28;26(Suppl 8):I67-74; 2. Figueroa JE, Densen P. Clin Microbiol Rev. 1991;4:359-95; 3. Walport MJ. N Engl J Med. 21;344:158-66; Glovsky MM et al. Ann Allergy Asthma Immunol 24;93: ; Rubio MT et al. Bone Marrow Transplant 28;41(Suppl. 1):S22. Abstract P766; 4. Rother RP et al. Nat Biotechnol.27;25: ; 5. Meyers G et al. Blood. 27;11:abs 3683; 6. Hill A et al. Br J Haematol.21;149:414-25; 7. Hillmen P et al. Am J Hematol.21;85:553-9; 8. Parker C et al. Blood. 25;16: ; 9. Hillmen P et al. N Engl J Med. 1995;333:1253-8; 1. Nishimura J et al. Medicine(Baltimore). 24;83:193-27; 11. Caprioli J et al. Blood. 26;18: ; Mastellos D et al. Immunologic Res 23;3: ; Mergenhagen STE et al. J Infect Dis 1973;128:S86; Chenoweth DE et al. N Engl J Med 12. Noris M et al. ClinJ Am Soc Nephrol. 21;5: ; 13. George JN. Blood.21;116:46-9; 14. Loirat C et al. Pediatr Nephrol.28;23: ; 1981;34:497 53;Giradi G. Am J Reprod Immunol 28;59: Ståhl AL et al. Blood.28;111:537-15;16. Hosler GA et al. Arch Pathol Lab Med. 23;127;834-9;17. Ariceta G et al. Pediatr Nephrol.29;24:

5 Absence of CD59 Allows Terminal Complement Complex Formation Complement Cascade Regulation and Erythrocytes Complement Cascade Regulation and Erythrocytes C9 C9 x C5 C5 convertase C5a C5b C5 convertase C6 C7 C6 C7 C5b C8 X C6 C5b CD59 C7 C6 C5b C7 CD59 X C6 C5b C7 C8 C8 C9 C8 C5b-9 C5b,6,7 C5b-8 Adapted from: Cellular and Molecular Immunology AK Abbas, AH Litchman and JS Pober, 3rd Edition WB Saunders; Philadelphia Lucio Luzzatto et al. Haematologica 21;95: Lucio Luzzatto et al. Haematologica 21;95: Complement Cascade Regulation and Erythrocytes Chronic Uncontrolled Complement Activation Leads to Tissue and End Organ Damage Strict regulation is needed to avoid unnecessary damage to self due to overt or mistargeted activation 1,2 Tight control needed especially for the alternative pathway which is continuously turned on Historically Viewed as a Hemolytic Anemia Normal red blood cells (RBCs) are protected from complement attack by a shield of terminal complement inhibitors Complement Activation Without this protective complement inhibitor shield, PNH RBCs are destroyed Lack of Bound CD55, CD59 Leads to Uncontrolled Complement Activation Intact RBC Reduced Red Cell Mass Lucio Luzzatto et al. Haematologica 21;95: Membrane Attack Complex (MAC) on PNH Erythrocyte Photo: W Rosse. Reprinted with Permission. 1. Holers VM et al. Immunol Rev 28;223:3 316;2. Zipfel PF et al. Curr Opin Nephrol Hypertens 21;4: Endothelial Cells Damaged by Complement Attack Photo: S. Meri, Univ. of Helsinki. Reprinted with Permission. 29 Free AHneemmoigalobin 1. International PNH Interest Group.Blood 25;16: BrodskyR 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;25; Rother RP et al. JAMA 25;293: Socie G et al. Lancet 1996;348: Hill A et al. Br J Haematol 27;137:

6 Hemolysis Leads to NO Consumption in PNH Patients Consequences of Nitric Oxide (NO) Depletion Chronic Uncontrolled Complement Activation Leads to Devastating Consequences Plasma Free Hb (µmol/l) R=.594, P< LDH (u/l) Plasma Free Hb (µmol/l) R=.9529, P<.1 NO consumption assay: NO chemiluminescence NO Consumption (µmol/l) LDH significantly correlates with free hemoglobin (Hgb) 1 Confirms LDH as a biomarker for hemolysis LDH 1.5x at diagnosis had a 4.8-fold greater mortality 2 Free Hgb significantly correlates with NO consumption Hgb is in reduced state and reactive with NO Reduced Nitric Oxide Can Cause Smooth muscle dystonias 1 Vascular constriction pulmonary and systemic hypertension, erectile dysfunction 2 Gastrointestinal contractions dysphagia, abdominalpain Platelet activation and aggregation 1 4 Platelet hyperreactivity Hypercoagulability Complement Activation Elevated LDH Free Hemoglobin Decreased NO Renal Failure Pulmonary Hypertension Abdominal Pain Chest Pain Dyspnea Dysphagia Fatigue Hemoglobinuria Erectile Dysfunction Significant Impact on Survival Significant Impact on Morbidity 1. Hill A et al. Br J Haematol 21;149: ; 2. Lee JW et al. ASH 211. Abstract Rother R et al. JAMA 25;293: ;2.Hill A et al. Br J Haematol 21;149: ;3.WeitzI. Res 21;125:S16 S17; 4. Helley D et al. Haematologica 21;95: LDH = lactate dehydrogenase. 1. International PNH Interest Group. Blood 25;16: ; 2. Brodsky R. Paroxysmal nocturnal hemoglobinuria. In: R Hoffmanet al, eds. Hematology - Basic Principles and Practices. 4th ed. Philadelphia, PA: Elsevier Churchill Livingstone; 25; ; 3. Rother RP et al. JAMA. 25;293: ; 4. Socie G et al. Lancet 1996;348: ; 5. Hill A et al. Br J Haematol 27;137: ; 6. Lee JW et al. Hematologica 21;95(s2): Abstracts 55 and 56; 7. Hill A et al. Br J Haematol 21;149: ; 8. Hillmen P et al. Am J Hematol 21;85: Historical Management of PNH Is the Leading Cause of Death in PNH 1 Supportivecare optionsdo not impactprogression andrisk for severe morbidities and mortality 1 Transfusions 1 risk of iron overload Anticoagulants 1 ineffective in many patients Red cell supplements 1 may expand clone and elevate hemolysis Steroids/androgen hormones 1 adverse events Morbidities and Mortality in PNH Accounts for 4 67% of deaths 2 First thrombotic event (TE) can be fatal 2,3 First TE increases risk for death 5- to 1-fold 2 Up to 44% of patients experience clinical thrombotic events 2 Occurs in typical and atypical sites 4 AlthoughBMT is the onlypotentiallycurative therapyfor PNH, BMT is associated with significant morbidities and mortality 2,3 Is not adequately managed with anticoagulation 2 All patients with PNH are at risk for thrombosis 2 In a study examining PNH patients (n=23) 2 5% chronic GVHD; 42% acute GVHD 3 Transplant-related mortality was 42% BMT has a significant impact on quality of life (QoL) post-transplant 4,5 1. International PNH Interest Group. Blood 25;16: ; 2. Santaraone S et al. Haematologica 21;95: ; 3. de Latour PF et al. EBMT 29:Abstract 316; 4. Bieri S et al. Bone Marow Transplant 28;42: ; 5. Fraser CJ et al. Blood 26;18: International PNH Group et al. Blood 25;16: ;2. Hillmen et al. Blood 27;11: ; 3. Audebert HJ et al. J Neurol 25;252: ;4. Lee JW et al. Hematologica 21;95(s2):Abstract

7 Multifactorial Pathogenesis of in PNH Chronic Uncontrolled Complement Activation Leads to Vasoconstriction and The Incidence of TE is Increased in Patients with Elevated LDH at Diagnosis Pathogenesis of thrombosis in PNH is a result of uncontrolled complement activation 1 Activation of complement C5b 9 Hemolysis leads to reduced nitric oxide levels and vasoconstriction 2 4 Platelets undergo morphological changes, release microparticles, and aggregate 2,4 Activation of complement C5a Leukocytes release tissue factor and inflammatory cytokines (IL-6) to initiate coagulation 2,3,5 Leukocytes decrease expression of plasminogen activator receptor (PAR) leading to impaired fibrinolysis 4 Impaired regulation of smooth muscles Local vasoconstriction Chronic Pro-inflammatory effect Chronic Hemolysis on endothelial cells [NO] Uncontrolled Complement C5b-9 Activation C5a C5b-9 Platelet Inflammation Activation Leukocyte Platelet Activation Aggregation Chronic hemolysis Platelet activation Local vasoconstriction CLOT Inflammation Endothelial cell injury Systemic thrombosis Patients With TE (%) LDH 1.5x ULN 3.8 P<.1 LDH <1.5x ULN Univariate analysis showed that the incidence of TE was significantly increased in patients with LDH 1.5x ULN at diagnosis (43/171; 25.1%) compared with patients with LDH <1.5x ULN (2/53; 3.8%; OR 8.57) 1. McKeage K. Drugs 211;71: ;2.Hill A et al. Br J Haematol 27;137: ;3. WeitzI. Res 21;125:S16 S17; 4. Helley D et al. Hematologica.21;95: ; 5. Markiewski MM et al. Trends Immunol 27;28: Adapted from: Gladwin MT et al. Free Rad Biol & Med 24;36:77 717; Rother RP et al. JAMA 25;293: Data from South Korean National Registry. Lee JW et al. Presented at the 54th Annual Meeting of the AmericanSociety of Hematology;December 8 11,212;Atlanta, GA. Abstract Risk of First Ever Ischemic Stroke (FEIS) Elevated in PNH is Associated With Risk of Early Mortality Occurs in Both Typical and Atypical Sites* First Ever Ischemic Stroke (per 1 patient years) Age of FEIS in PNH patientsis markedly less than in the general population PNH Population 2,3 General Population 1 PNH Population 2,3 <54 Years Old.3 General Population Years Old 1 1. Gostynski M et al. J Neurol 26;253:86 91; 2.Hillmen P et al. Blood 27;11: ; 3. Data on file. Alexion Pharmaceuticals, Inc. 4 TE increases risk of death 15-fold over patients with no TE Hazard Ratio French PNH Patients (n=415) TE was an independent prognostic factor related to poor survival (HR 15.4; 95% CI ; P<.1) in a large cohort of French PNH patients de Latour RP et al. Blood 28;112: Superficial vein thrombosis 13.7 Cerebral/internal jugular thrombosis 5.6 Pulmonary embolus 6.5 Hepatic/portal vein thrombosis VENOUS *124 events, 63 patients 16.9 Myocardial Infarction/ 1.6 unstable angina 13.7 Cerebrovascular accident/ transient ischemic attack 33.1 Deep vein thrombosis* 18.5 Mesenteric/splenic vein thrombosis *Includes 18.5% lower extremity and 14.5% other (inferior vena cava, bilateral lower extremity, pelvic, ureter, axillary, subclavian, and brachiocephalic veins). Hillmen P et al. Blood 27;11: ARTERIAL 42 7

8 PNH Patients are at Risk of Despite Anticoagulation or Minimal Transfusion Requirements Can Occur Regardless of Clone Size in PNH Conclusions Rate (TE per 1 patient years) Patients Treated With Anticoagulants (n=91) 4.87 Patients With 1 Transfusions Per Year (n=22) TE (%) <2 2-5 >5 PNH Granulocyte Clone Size (%) (n=43) 4 67% mortality in PNH results from thrombosis 1 is the leading cause of death in PNH 2 First TE increases risk for death 5- to 1-fold 1 LDH 1.5 ULN at diagnosis is associated with TE and mortality 3 DVT or PE most common clinical presentation 1 Arterial thromboses are also common 1 Anticoagulant therapy may not be adequate to control thrombosis in PNH 1 Clinicalthrombosis evident in PNH patients: No transfusion history 1 Smaller clone size 4 Hillmen P et al. Blood 27;11: Data from South Korean National Registry. Lee JW et al. Hematologica 21;95(s2): Abstract Hillmen P et al. Blood 27;11:12: ; 2. International PNH Group et al. Blood 25;16: ; 3. Lee JW et al. Blood (ASH Annual Meeting Abstracts)Nov 211;118:3166;4.Lee JW et al. Hematologica 21;95(s2):Abstract Kidney Pathology in PNH Chronic Kidney Disease Chronic Kidney Disease Chronic Kidney Disease: Morbidity and Mortality in PNH Chronic Kidney Disease 64% of PNH Patients Exhibit Clinical Chronic Kidney Disease (CKD) Complement-mediated hemolysis and cell-free plasma hemoglobin lead to chronic kidney disease in PNH 1 5 Repetitive exposure of tissue to cell-free hemoglobin may lead to renal damage in PNH 3,4 Interstitial scarring on the left Micrograph of a Renal Biopsy from a PNH Patient, Indicative of Vascular Damage Normal tissue on the right Kidney failure is the cause of 8 18% of PNH-related deaths 1 8% of PNH patients (median age of 31.5 years) had MRI evidence of significant renal hemosiderosis 2,3 Marked hemosiderin deposits in the proximal renal tubule are a common feature in all autopsy and biopsy reports dealing with PNH Demonstrable by MRI even when no overt hemoglobinuria is seen Proportion of Patients (%) Stage 3-5 CKD (n=4) Stage 1-2 CKD (n=84) No CKD (n=69) Clark DA, et al. Blood. 1981;57: Reprinted with Permission. 1. Brodsky R. Hematology: Paroxysmal nocturnal hemoglobinuria. In: R Hoffmanet al, eds. Hematology - Basic Principles and Practices. 4th ed. Philadelphia, PA: Elsevier Churchill Livingstone 25; ;2. Rother R et al. JAMA 25;293: ;3. Clark DA et al. Blood 1981;57:83 89; 4. Hillmen P et al. Am J Hematol 21;85: ; 5. McKeage K. Drugs 211;71: Hillmen P, et al. Am J Hematol 21;85: ;2. Brodsky R. Hematology:Paroxysmal nocturnal hemoglobinuria. In: R Hoffmanet al, eds. Hematology - Basic Principles and Practices. 4thed. Philadelphia, PA: Elsevier Churchill Livingstone 25; Hill A. et al. Blood 26;18: Abstract Hillmen P et al. Am J Hematol 21;85:

9 Chronic Kidney Disease Common Symptoms of PNH Common Symptoms of PNH Kidney Disease in PNH: Conclusions Clone Size Does Not Correlate to Symptom Severity Common PNH Symptoms are Associated With TE Kidney failure is the cause of 8 18% of PNH-related deaths 1 Kidney disease in PNH is caused by complement-mediated hemolysis 2,3 64% of patients with PNH exhibit chronic kidney disease at any one time 4 Kidney disease is underappreciated in PNH 4 Patients (%) 1% 8% 6% 4% 2% % Abdominal Pain International PNH Registry Gran Clone <1% Gran Clone 1 49% Gran Clone 5% Shortness Chest Pain of Breath 33 Clinical Symptoms 6 74 Discolored Urine Fatigue TE Odds Ratio (1 = no effect on risk) Abdominal Pain P=.4 Elevated LDH ( 1.5 ULN) in combination with abdominal pain, chest pain, and dyspnea are associated witha higher risk of TE 2.6 Chest Pain P= Dyspnea P= Nishimura JI et al. Medicine 24;83:193 27;2. Clark DA et al. Blood 1981;57:83 89;3. McKeage K. Drugs 211;71: ; 4. Hillmen P et al. Am J Hematol 21;85: ; 5. Kim JS et al. Hematologica 211;96(s2): Abstract Urbano-Ispizua A et al. Hematologica 21;95(s2): Abstract Study description: a retrospective analysis of the medical charts of 286 PNH patients in a South Korean National Registry. Lee JW et al. Hematologica 21;95(s2): Abstracts55 and Chronic Complement-Mediated Hemolysis in Combination With Clinical Symptoms Increase the Risk of LDH 1.5 ULN LDH and Abdominal Pain LDH and Chest Pain LDH and Dyspnea LDH and Hemoglobinuria Odds Ratio for TE (MultivariateAnalysis) The risk of TE in patients with LDH 1.5 ULN was 7.1 times greater than in patients withldh <1.5 ULN (P=.13). Common Symptoms of PNH Common Symptoms of PNH: Conclusions Common symptoms in PNH associated with TE should be considered as part of a comprehensive clinical assessment Abdominal pain, chest pain, dyspnea, hemoglobinura Abdominal pain and dyspnea are linked by underlying hemolysis and the threat of thrombosis 2 66% of patients report shortness of breath 3 59% of patients report abdominal pain 4 97% of patients report fatigue 1 Fatigue and severe dyspnea are prominent clinical features that can be associated with pulmonary hypertension and cardiac dysfunction 76% of patients with PNH have disruptions in daily activities 1 Clone size does not correlate to symptom severity 5 Common Symptoms of PNH Early Identification of Patients at High Risk for PNH Data from South Korean National Registry. Lee JW et al. Presented at the 54th Annual Meeting of the American Society of Hematology;December 8 11,212;Atlanta, GA. Abstract Weitz I et al. Intern Med J. 212;2. Lee JW et al. Hematologica 21;95(s2):Abstract Meyers G et al. Blood 27;11:Abstract 3683; 4. Lee JW et al. Hematologica 21;95(s2): Abstract 55; 5. Urbano-Ispizua A et al. Hematologica 21;95(s2): Abstract

10 Advancements in Treatment Options Warrant Early Diagnosis and Intervention Two Independent International Groups Recommend Testing High-Risk Patientsfor PNH Suggestions for PNH Testing by ICCS PNH Guidelines Early diagnosis is essential for improved patient prognosis: PNH Bone marrow failure 1 International PNH Registry Provides evidence to inform clinical decision making Over 21 patients from 26 countries are currently enrolled in the PNH registry Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry Michael J. Borowitz *, Fiona E. Craig, Joseph A. DiGiuseppe, Andrea J. Illingworth, Wendell Rosse, D. Robert Sutherland, Carl T. Wittwer, Stephen J. Richards,On behalf of the Clinical Cytometry Society Clinical Indications for PNH Testing Intravascular hemolysis as evidenced by hemoglobinuria or elevated plasma hemoglobin Evidence of unexplained hemolysis with accompanying: - Iron deficiency, or - Abdominal pain or esophageal spasm, or - (see below), or - Granulocytopenia and/or thrombocytopenia Other acquired Coombs-negative, non-schistocytic, non-infectious hemolytic anemia with unusual features Unusual sites: - Hepatic veins (Budd Chiari syndrome) - Other intra-abdominal veins (portal, splenic, splanchnic) - Cerebral sinuses - Dermal veins With signs of accompanying hemolytic anemia (see below) With unexplained cytopenia Evidence of bone marrow failure Suspected or proven aplastic or hypoplastic anemia Refractory cytopenia with unilineage dysplasia Other cytopenias of unknown etiology after adequate workup 1. Sugimori C et al. Blood 26;17: BorowitzMJ et al. International Clinical Cytometry Society. Part B Clin Cytometry 21;78B:211 23; International PNH Interest Group. Blood 25;16: BorowitzMJ et al; Clinical Cytometry Society. Cytom B Clin Cytom 21;78B: Incidence of PNH Clones by Diagnostic Code in the Clinical Setting Utilizing High-Sensitivity Flow Cytometry (HSFC) Objective: To use multi-parameter HSFC to analyze the incidence of PNH clones in patientsrecommended for testing in the clinicalsetting Key Results: 6987 individualswere screenedfor PNH clones utilizinghsfc 421 of 6987 patients (6.1%) across allhigh-riskpopulations recommendedfor testing were found to be PNH positive PNH clinicalclone (>1%) detection rate was 3.8% Subclinicalclone (<1%) detection rate was 2.3% Incidence of PNH Clones in High-Risk Patient Populations ICD-9 General Description Incidence of PNH Clone Patients (%) with PNH Clone Hemolytic anemia 147/ * Hemoglobinuria 14/ AA 94/ MDS 32/ Unexplained cytopenia 13/ Pancytopenia 63/ / Anemia unspecified 4/ Hemolysis 18/ Unspecified iron deficiency 7/ PNH Clonal Expansion in an AA Representative Population n=75 n=114 At the Start of Follow Up PNH+ Patients PNH- Patients At the Last of Follow Up Transitional pattern (Classic PNH) Expansion n (%) (8) (11%) 13 (17%) Persistent 44 (59%) Newly developed 5 (4%) Disappearance 18 (24%) 19 (96%) Movalia MK et al. Presented at the American Society of Hematology 53rd Annual Meeting; December 1 13, 211; San Diego, CA. Abstract ICD-9 = International Classification of Diseases, 9th revision. *Includes patients already diagnosed as having PNH. Patients with PNH clone.1% High-risk patients may be included in more than 1 ICD-9 category. Movalia MK et al. Presented at the American Society of Hematology 53rd Annual Meeting; December 1 13, 211; San Diego, CA. Abstract Sugimori C et al. BJH. 29;147:

11 Immunosuppressive Therapy (IST) Has Increased Efficacy in AA Patients With PNH Cells Patients With Response 1% 8% 6% 4% 2% * 82% 46% * 36% PNH+ PNH- AA Considerations for Managing the PNH/AA Patient PNH with hemolysis PNH Intermediate + hemolysis PNH AA/PNH Evidence of PNH Cells in RA-MDS EXPLORE TRIAL Patient Population Description RA-MDS (n=1293) RA-MDS PNH cells/clone (Grans + RBC type III) >.1% 17.16% (222 / 1293) WBC PNH clone 1% 1.54% (2 / 1293) Clone 1% and LDH > ULN 4.% (8 / 2) WBC PNH Clone size 1% % Overall Response Complete Response The presence of PNH cells was the only significant predictor of response to IST in 14 AA patients (P<.1) in multivariate analysis 2 Complete response (CR) was defined as hemoglobin normal for age, neutrophil count more than /L, and platelet count more than 15 19/L; Partial response (PR) was defined as transfusion independent and no longer meeting criteria for severe disease in patients with severe AA. 1 Overall response = CR + PR *P value = complete response, P=.3; Overall response, P< Sugimori C et al. Blood 26;17: ;2.Sugimori C et al. Exp Hematol 27;35: % 61 Eculizumab Moderate AA with hemolysis De Latour RP, Amoura Z, and Socie G. La revue de medecine interne. 21; 2-27.? Moderate AA without hemolysis Prophylactic Anticoagulaion Severe AA without hemolysis IST 62 BMT 62 Mean clone 32.19% (n=2) Interim Results from EXPLORE, a Multi-centerPrevalenceStudy of PNH Clone Size in Patientswith AA, MDS, and other BMF Galili N et al. Poster presentation at: American Society of Clinical Oncology 45th Annual Meeting; May 29 June 2, 29; Orlando, FL.. 63 PatientsWith Unexplained Cytopenias are at High Risk for PNH 1 Unexplained Cytopenias 93% of PatientsWith PNHHave Peripheral Blood Abnormalities Unexplained Cytopenias 66 Test the Following Cytopenic Patients for PNH 5,6 Other combinations* No concomitant cytopenias Unexplained Cytopenia After thorough work-up Cytopenia and Evidence of Hemolysis LDH Haptoglobin Reticulocyte count (with or without anemia) Cytopenia With Any of These Coexisting Findings Anemia Coombs-negative hemolytic anemia Bone marrow failure disorder Hemoglobinuria (dark colored urine) Anemia and neutropenia Anemia and thrombocytopenia 17% 7% 4% 7% Anemia 32% 33% Pancytopenia *.1% PNH cell threshold. 1. Movalia MK et al. Blood 211;118: Abstract 133; 2. Barzi A, Sekeres MA. Clev Clin J Med 21;77:37 44; 3. Jordan MB et al. Blood 211;118: ;4. Moreno C et al. Blood 21;116: ; 5. Borowitz MJ et al; Clinical Cytometry Society.Cytom B Clin Cytom 21;78B:211 23;6. BrodskyRA. Blood 29;113: Anemia: hemoglobin <12. g/l. Neutropenia: ANC < /L. Thrombocytopenia: platelet count < /L. *Other combinations: thrombocytopenia alone, neutropenia alone, both thrombocytopenia and neutropenia. Socie et al. Lancet 1996;348:

12 Standard Diagnostic Test for PNH Testing for PNH in RBCs How Do You Test forpnh? 67 Flow cytometry performed on peripheral blood Granulocytes and at least one additional cell line should be evaluated RBCs Monocytes Quantitative results Optimal high sensitivity analysis:.1% Routine analysis: 1% Easy to understand PNH reports Use more than one reagent against GPI-anchored proteins BorowitzMJ et al.; Clinical Cytometry Society. Cytom B Clin Cytom 21;78B: Gating on GPA+ RBCs GPA = glycophorin A. Data Source: Dahl-Chase Diagnostic Services.. Patient 1: Normal RBCs with normal CD59 expression (Type I cells) Patient 2: PNH clone with complete CD59 deficiency (Type III cells) Patient 3: PNH clone with complete CD59 deficiency (Type III cells) and partial CD59 deficiency (Type II cells) 69 Why Look Beyond RBCs for PNH? PNH Patient With an 8% WBC Clone Size and 31% RBC Clone Size Indicating Hemolysis ICCS Recommendations for Follow-Up Testing of PatientsWith an Identified PNH Clone Granulocytes provide more accurate representation of PNH clone size 1 Percentages of PNH RBCs may be affected by: Hemolysis Blood transfusion PNH reports should provide quantitative results expressing clone size on both granulocytes and RBCs CD24- Granulocytes WBC FLAER- GPI Anchor Binding Marker RBC CD59 GPI Anchored Protein 8.1 % of Granulocytes lack GPI proteins 31.4% RBCs are Type III PNH cells Annual monitoring 1 Stable patients Patients with aplastic anemia and small PNH clone Patients with refractory cytopenia with unilineage dysplasia (RCUD) and small PNH clone More frequent monitoring to evaluate for expanding clones Patients with changing symptoms or lab values Patients in early stages of treatment 1. Borowitz MJ et al; Clinical Cytometry Society. Cytom B Clin Cytom 21;78B: Data Source: Dahl-Chase Diagnostic Services Borowitz MJ et al.; Clinical Cytometry Society.Cytom B Clin Cytom 21;78B:

13 Importance of Monitoring Granulocytes and RBCs Over Time CD24-Granulocytes September 28 December 28 March 29 FLAER-GPI Anchor Marker Gran clone: 3.8% RBC clone:.8% CD24-Granulocytes s te y o c ul ran G CD FLAER-GPI Anchor Marker Gran clone: 7.6% RBC clone: 1.6% CD24-Granulocytes FLAER-GPI Anchor Marker Gran clone: 14.2% RBC clone: 1.8% CD24-Granulocytes May 29 FLAER-GPI Anchor Marker Gran clone: 23.3% RBC clone: 2.4% Conclusions Chronic complement-mediatedhemolysis is the underlying cause of progressive morbidities and mortality in PNH is the leading cause of death in PNH 1 Renal failure has been identified as the cause of death in approximately 8 18% of PNH patients 2,3 PNH may be more common than you think Treatment of PNH with Complement Inhibitor Eculizumab CD59 GPI Anchor Protein 3 Months CD59 GPI Anchor Protein 6 Months 9 Months CD59 GPI Anchor Protein CD59 GPI Anchor Protein Delays in diagnosis range from 1 to more than 1 years 4 High sensitivity flow cytometry, performed on peripheral blood, is the gold standard test Advancements in treatment options warrant early diagnosis and intervention Data Source: Dahl-Chase Diagnostic Services. PNH Clone Expanded in <1 Year Hillmen P et al. Blood 27;11: ; 2. Nishimura JI et al. Medicine 24;83:193 27; 3. Hillmen P et al. Am J Hematol 21;85: ; 4. Hillmen P et al. N Engl J Med 1995;333: Eculizumab (soliris) Humanized First in Class Anti - C5 Antibody Human IgG 2 Heavy Chain Constant Region 1 and Hinge (Eliminates Fc receptor binding) Human Framework Regions No mutations Germline Hinge CH2 CH3 Complementarity Determining Regions (murine origin) Human IgG 4 Heavy Chain Constant Regions 2 and 3 (Eliminates complement activation) Eculizumab Blocks Terminal Complement 1,2 Proximal Terminal Complement Cascade 2,3 C3 C3b C5 C5b C3a C5a C5b-9 Soliris Eculizumabbinds with high affinity to C5 1,2 Terminalcomplement - C5a and C5b-9 formation blocked 1,2 Proximal functions of complement remain intact 1,2 Weak anaphylatoxin 2,4 Immune complex clearance 2 Microbial opsonization 2 Therapeutic Indications and Usage Eculizumabis a Complement Inhibitor Indicated for: The treatment of patients with paroxysmal nocturnal hemoglobinuria (PNH) to reduce hemolysis The treatment of patients with atypicalhemolytic uremic syndrome (ahus) to inhibit complement-mediatedthrombotic microangiopathy The effectivenessof Eculizumabin ahus is basedon the effects on thromboticmicroangiopathy (TMA) and renal function Prospectiveclinicaltrials in additional patientsare ongoingto confirm the benefitof Eculizumab in patients with ahus Rother R et al. Nat Biotech 27;25: Please see full prescribing informationfor Soliris (eculizumab). 1. Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; 212; 2. Rother RP et al. Nature Biotech 27;25: ; 3. Walport MJ.N Engl J Med 21;344: ;4. Figueroa JE, Densen P. Clin Microbiol Rev 1991;4: Please see full prescribing informationfor Soliris (eculizumab). Soliris (eculizumab) Summary of Product Characteristics. AlexionEurope SAS;

14 WARNING: SERIOUS MENINGOCOCCAL INFECTIONS Eculizumab increases the patient s susceptibility to meningococcal infection due to its mechanism of action To reduce the risk of infection, a tetravalent vaccine against serotypes A, C, Y and W135 (preferably conjugate), is strongly recommended to vaccinate patients at least 14 days prior to receiving the first dose of eculizumab Patients less than 2 years of age or not vaccinated at least 14 days before starting treatment with eculizumab must receive treatment with appropriate prophylactic antibiotics until 14 days after vaccination. Revaccinate according to current medical guidelines for vaccine use Vaccination may not be sufficient to prevent meningococcal infection and the use of antibacterial agents may need to be considered Monitor patients for early signs of meningococcal infection; evaluate immediately if infection is suspected and treat with antibiotics if necessary Please see full prescribing informationfor Soliris (eculizumab). Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; Clinical Trials With Eculizumab Pilot Study NEJM 24 N=11 Primary endpoint: reduction of hemolysis TRIUMPH NEJM 26 Pivotal Phase III, Double-Blind, Placebo-Controlled Trial, N=87 SHEPHERD Blood 28 Broader patient population, including those receiving minimal transfusions or with thrombocytopenia, N = 97 Long-Term Extension Trial Hillmen Blood 27 Evaluated long-term safety, efficacy and effect on thrombosis; Placebo patients switched to eculizumab N=187 Please see full prescribing informationfor Soliris (eculizumab). Soliris (eculizumab) Summary of Product Characteristics. AlexionEurope SAS; Eculizumab is a Chronic Treatment for a Chronic Disease Dose within ±2 days. Soliris PNH Dosing Schedule Pretreatment Induction Phase Maintenance Phase 2 weeks Week before induction X 9 X 9 q14d Neisseria meningitidis vaccination Soliris dose, mg In clinical trials all patients were vaccinated against Neisseria meningitidis¹ Concomitant medications allowed: Steroids, immunosuppressant drugs, anti-clotting agents and hematinics² Eculizumab should be administered via IV infusionwithin minutes every 7 days during induction and every 14 days during maintenance¹ Eculizumab dose adjustment to every 12 days may be necessary for some patients to maintain LDH reduction¹ Please see full prescribing informationfor Soliris (eculizumab). 1. Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; Hillmen P et al. N Engl J Med 24;35: LDH (U/L) % Reduction in LDH: TRIUMPH and SHEPHERD 1% response after the first dose Time, Weeks TRIUMPH placebo patients switched to Eculizumab after Week 26 All TRIUMPH patients entered the long-term extension study TRIUMPH Placebo/Extension TRIUMPH Soliris/Extension SHEPHERD Eculizumab P<.1 at all measured time points. Please see full prescribing informationfor Soliris (eculizumab). Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; 212. Hillmen P et al. Blood 27;11: % Reduction in Thrombotic Events Thrombotic Events (#) Pre-Eculizumab Treatment 63% of patients received concomitant anticoagulants 1 Eculizumab Treatment The effect of anticoagulant withdrawal was not studied 2 Events observed in both venous and arterial sites 3 There were fewer thromboticevents with Eculizumab treatment than during the same period of time prior to treatment 2 Please see full prescribing informationfor Soliris (eculizumab). 1. Brodsky R et al. Blood 28;111: ; 2. Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; 212; 3. Hillmen P et al. Blood 27;11: N=195 P=.1 Eculizumab Reduced in Patients Treated with Anticoagulants 1 Event Rate (TE per 1 patient-years) 12% 1% 8% 6% 4% 2% % Pre-Soliris Treatment.72 Soliris Treatment 94% reduction in event rate with Eculizumab P<.1 N=91 *Excludes patients on antiplatelet agents. Please see full prescribing informationfor Soliris (eculizumab). Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; Hillmen P et al. Blood 27;11:

15 64% of Patients Exhibit Chronic Kidney Disease (CKD) 1 Renal Function With Eculizuamb in Different Baseline PNH Populations 6 Months 1 Effect of Eculizumab on Chronic Kidney Disease in PNH Proportion of Patients (%) 5% 4% 3% 2% 1% % 2.5 Stage 3-5 CKD (n=4) 43.1 Stage 1-2 CKD (n=84) 35.4 No CKD (n=69) Proportion of Patients (%) P< Overall (n=189) Stage 1 2 (n=81) Segment of PNH Population 75. P= Stage 3 5 (n=4) 59% of patients with minimal ( 1) transfusion history had CKD (n=22) No Change Improvement Worsening Hillmen P et al. Am J Hematol 21;85: Please see full prescribing informationfor Soliris (eculizumab). Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; Hillmen P et al. Am J Hematol 21;85: Renal Function With Eculizumab in Different Baseline PNH Populations 18 Months 1 Chronic Kidney Disease in PNH: Summary Proportion of Patients (%) P< P< P= Early intervention with eculizumab has shown a timedependent improvement in renal function in protecting against progression of renal damage 1 Is the Primary Cause of Fatigue in PNH Anemia or Hemolysis? Overall Stage 1 2 Stage 3 5 (n=189) (n=81) (n=4) Segment of PNH Population No Change Improvement Worsening Please see full prescribing informationfor Soliris (eculizumab). Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; Hillmen P et al. Am J Hematol 21;85: Please see full prescribing informationfor Soliris (eculizumab). Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; Hillmen P et al. Am J Hematol 21;85:

16 TRIUMPH Demonstrated That Improvement in Fatigue Occurred Independent ofhemoglobin Response 73% Reduction in Mean Units Transfused Across All Subgroups: TRIUMPH Eculizumab Treatment Results in Large and Clinically Meaningful Improvements in Patient-Reported Outcomes Hemoglobin, g/dl 12. P< or more points denotes a clinically significant improvement Time, Weeks FACIT-Fatigue Score Hgb Level FACIT-Fatigue Score Hgb Placebo Ecu (n=43) Patients not on Ecu (n=44) Ecu Hgb Placebo Hgb Change From Baseline FACIT-Fatigue Score Median Units Transfused Patients not on Ecu (n=44) 14 Ecu (n=43) * 2 * * * (n=87) (n=3) (n=35) (n=22) Overall >25 Pre-treatment Transfusion Strata 51% of Ecu patients achieved transfusion independence vs % of patients not on Ecu 1 Standard Effect Size (SES) FACIT-Fatigue* EORTC Fatigue* (P<.1) (P<.1).69 Dyspnea* (P=.2).65 Pain (P<.1) Large Clinical Impact Moderate Clinical Impact Small Clinical Impact In SHEPHERD, 78% of patients reported a significant improvement in fatigue 1 Patients with concomitant bone marrow dysfunction may continue to require transfusions 2 FACIT = Functional Assessment of Chronic Illness Therapy. Please see full prescribing informationfor Soliris (eculizumab). Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; 212. Adapted from: Hillmen P et al. N Engl J Med 26;355: ; Brodsky R et al. Blood Rev 28;22:65 74; Hill A et al. Haematologica 28;93(Suppl.1):359. Abstract 94;Brodsky R et al. Blood 28;111: ;Schubert J, et al. Br J Haematol 28;142(2): *P<.1. Transfusiondata obtained during 12 months beforetreatment; values were normalized for a 6-month period. Please see full prescribing informationfor Soliris (eculizumab). Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; Hillmen P et al. N Engl J Med 26;355; ;2.Schubert J. Br J Haematol 28;142: Please see full prescribing informationfor Soliris (eculizumab). Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; Brodsky R et al. Blood 26;18: Abstract 377; 2. Data on file. Alexion Pharmaceuticals. 3. Weitz I, et al. Internal Medicine Journal 212. Accepted Article; doi: /j x 93 Eculizumab Reduces Hemolysis and Improves Fatigue in IST-Treated Patients Mean LDH (U/IL) IST Mean FACIT Fatigue 1,45 1, LDH N= FACIT-Fatigue N=1 MMonotnhsthosfoEfcEucliuzulimzuamb ab * * * Months of Eculizumab IST: Severe hemolysis Ecu: Significant and sustained reduction in hemolysis IST: Severe fatigue Ecu: Significant and sustained reduction in fatigue Demonstrates effectiveness of treatment with Eculizumab to reduce hemolysis and fatigue in AA patients despite concomitantist treatment How Does Eculizumab Impact Pulmonary Hypertension and Dyspneain PNH? Reduction of Pulmonary Hypertension With Eculizumab as Measured by NT-ProBNP Proportion of Patients With Evidence of Pulmonary Hypertension Soliris vs Placebo (P<.1) % Reduction 52.5 Baseline Week 26 Baseline Week 26 Placebo Eculizumab Treatment Group - TRIUMPH (n=73) Pulmonary Hypertension Defined as NT-proBNP 16 pg/ml 2 P<.37 * P<.1 when prior and post months compared.. P<.5 when prior and post months compared. Schrezenmieier et al Blood Please see full prescribing informationfor Soliris (eculizumab). Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; Hill A et al. Br J Haematol 212;158: Machada et al. JAMA

17 Extension Trial Experience With Eculizumab? Pilot Study NEJM 24 N=11 Primary endpoint: reduction of hemolysis TRIUMPH NEJM 26 Pivotal Phase III, Double-Blind, Placebo-Controlled Trial, N=87 SHEPHERD Blood 28 Broader patient population, including those receiving minimal transfusions or with thrombocytopenia, N = 97 Long-Term Extension Trial Hillmen Blood 27 Evaluated long-term safety, efficacy and effect on thrombosis; Placebo patients switched to Eculizumab N=187 86% Reduction in LDH Sustained Over Entire Course of 36 Month Treatment Period Mean LDH (U/L) * * * * *P<.1 * Dashed line represents the upper limit of the normal range ( U/L) Study Months * * * Treatment Expectations: What is the Benefit ofeculizumab Therapy in PNH Patients? 6 12 Study Month Baseline Patients (n) Please see full prescribing informationfor Soliris (eculizumab). Soliris (eculizumab) Summary of Product Characteristics. AlexionEurope SAS; Please see full prescribing informationfor Soliris (eculizumab). Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; 212. Brodsky R et al. Blood 21;116: Abstract Eculizumab Treatment Expectations PNH Patients in ClinicalTrialsShoweda Benefit from EculizumabTherapy Treatment with Eculizumab should not alter anticoagulant management. 1 In 1 Week Reduction in hemolysis (as measured by LDH) 3 Reduction in fatigue 3 Between 2 and 3 Weeks Improvement in quality of life 1 Improvement in dyspnea 4 Between 2 and 6 Months Reduction in frequency of transfusions 5 Stabilization of hemoglobin levels 5 >6 Months Continued improvement in quality of life 3,6 LDH levels maintained at upper limit of normal 3,6 Continued improvement in fatigue 3,6 Continued reduction in frequency of transfusions 3,6 Continued stabilization of hemoglobin levels 5 At 36 Months and Up to 1 Years Reduction in hemolysis (as measured by LDH) was sustained 1,7,8 Improvement in TE was maintained 1,7,8 Most commonly reported adverse events were mild or moderate in severity 1 Please see full prescribing information for Soliris (eculizumab). 1. Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; Hillmen P et al. Blood 27;11: Brodsky R et al. Blood 28;111: Hill A et al. Br J Haematol 212;158: Hillmen P et al. N Engl J Med 26;355: Socié G et al. Blood (ASH Annual Meeting Abstracts) 27;11: Abstract Brodsky RA et al. In: Abstracts of the 52 nd ASH Annual Meeting and Exposition; December 4 7, 21; Orlando, FL. Abstract Hill A et al. In: Abstracts of the 54 th Annual Meeting of the American Society of Hematology (ASH). December 8 11, 212, Atlanta, GA. Abstract #3472. Appears in Blood 212;12(21). 9. Szer J et al. In: Abstracts of the 54 th Annual Meeting of the American Society of Hematology (ASH). December 8 11, 212, Atlanta, GA. Abstract #126. Appears in Blood 212;12(21). 1 Reduction in Thrombotic Event Rate 2 Adverse Reactions Reported in 5% of Eculizumab Treated Patients in TRIUMPH Reaction Soliris (n=43) Patients, n (%) Placebo (n=44) Headache 19 (44) 12 (27) Nasopharyngitis 1 (23) 8 (18) Back pain 8 (19) 4 (9) Nausea 7 (16) 5 (11) Fatigue 5 (12) 1 (2) Cough 5 (12) 4 (9) Herpes simplex virus infections 3 (7) Sinusitis 3 (7) Respiratory tract infection 3 (7) 1 (2) Constipation 3 (7) 2 (5) Myalgia 3 (7) 1 (2) Pain in extremity 3 (7) 1 (2) Influenza-like illness 2 (5) 1 (2) Hillmen P, et al. NEJM. 26;355: Please see full prescribing informationfor Soliris (eculizumab). Soliris SmPC: Soliris (eculizumab) summary of product characteristics. Alexion Europe SAS Recommendations for Monitoring PNH Patients on Eculizumab Monthly Complete Blood Count Reticulocyte count Serum LDH Yearly PNH Flow Cytometry If Evidence of Extravascular Hemolysis (anemia and increased retic) Direct Antiglobulin Test Brodsky et al.. Blood 214;124:

18 Summary of Clinical Efficacy % sustained reduction in hemolysis as measured by LDH Maintained over a 36 month treatment period % reduction in thrombotic events 73% reduction in transfusion requirements across all patient populations 78% clinicallymeaningful improvement in fatigue Sustained improvementin overall quality of life Patients treated with eculizumabexperienced improvement in CKD and pulmonary hypertension Eculizumab provideda rapidand durableeffect on dyspnea, a key marker of hemolysis-induced PHT Please see full prescribing informationfor Soliris (eculizumab). 1. Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; Hillmen P et al. N Engl J Med 26;355: ; 3. Brodsky R et al. Blood 21;116: Abstract 4237; 4. Hillmen P et al. Blood 27;11: ; 5. Socie G et al. Blood 27;11: Abstract Szer J et al. In: Abstracts of the 54th Annual Meeting of the American Society of Hematology (ASH). December 8 11, 212, Atlanta, GA; Abstract #126. Appears in Blood 212;12(21). 13 Summary of Clinical Efficacy and Safety 1 5 In a multicenter analysis eculizumab showed a major impact on survivalin PNH; survival is comparable to age- and gendermatched controls Eculizumab significantly reduced hemolysis, the underlying cause of morbidity and mortality in PNH Significant reductions in AEs were observed suggesting good tolerability and a favorable risk/benefit ratio over the long term Please see full prescribing informationfor Soliris (eculizumab). 1. Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; Hillmen P et al. N Engl J Med 26;355: ; 3. Brodsky R et al. Blood 21;116: Abstract 4237; 4. Hillmen P et al. Blood 27;11: ; 5. Socie G et al. Blood 27;11: Abstract Szer J et al. In: Abstracts of the 54th Annual Meeting of the American Society of Hematology (ASH). December 8 11, 212, Atlanta, GA; Abstract #126. Appears in Blood 212;12(21). 14 New Directions in PNH: What s on the Horizon Bart Scott, MD Associate Professor of Medicine, Division of Oncology, University of Washington Associate Member, Fred Hutchinson Cancer Research Center 15 What is the Clinical Data on the Long-term Outcome of Treatment with Eculizumab in Patients with PNH? What is the Impact of Long-term Soliris Treatment on Clinical Outcomes and Survival? Uncontrolled Complement Activation Hemolysis Complications Associated With Elevated Hemolysis (LDH) End Organ Damage TE Renal Gastrointestinal Pulmonary Cardiac Hepatic DInecreased Mortality? Long-term Treatment With Soliris in PNH: Sustained Efficacy and Improved Survival 153 consecutive patients with PNH treated with Soliris May 22 April 212 Duration mean of treatment 3.5 years (range < years) Mortality and disease symptoms were evaluated Hillmen et al. Br Jnl Haematol. 213;162:

19 Patient Characteristics (n=153) Presenting Features Number (%) Male 76 (49.7%) Age at diagnosis (median) 34 yrs (12-8) Age at initiation of Soliris Concomitant immunosuppression Concomitant anticoagulation 42 yrs (14 84) 25 (16.3%) 93 (6.7%) LDH level (normal <43IU/L) 232 (151 1,3) Eculizumab in PNH: 1-Year Multicenter Experience Patient Characteristics at Baseline Characteristic N=153 Female, n (%) 77 (5.3) Median age at diagnosis, years (range) 34 (12-8) Median age at commencement of Eculizumab, years (range) *Normal <43 IU/L. IU=international units. 42 (14-84) Concomitant immunosuppression, n (%) 25 (16.3) Concomitant anticoagulation, n (%) 93 (6.8) LDH level, IU/L (range) 232 (151 1,3)* Eculizumab in PNH: 1-Year Multicenter Experience Reduction in TE with Long-term Eculizumab Therapy Number of TE Months Prior to Eculizumab P<.5 3 Most Recent 12 Months on Eculizum*ab Hillmen et al. Br Jnl Haematol. 213;162: Hillmen et al. Br Jnl Haematol. 213;162:62-73 Please see full prescribing informationfor Soliris (eculizumab). Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; 212. Hill A et al. Presented at the 54th Annual Meeting of the American Society of Hematology (ASH). December 8 11, 212, Atlanta, GA. Abstract #3472. Appears in Blood 212;12(21). 11 Hillmen et al. Br Jnl Haematol. 213;162:62-73 *Patients received eculizumab treatment from May 22 to April 212 Please see full prescribing informationfor Soliris (eculizumab). Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; 212. Hill A et al. Presented at the 54th Annual Meeting of the American Society of Hematology (ASH). December 8 11, 212, Atlanta, GA. Abstract #3472. Appears in Blood 212;12(21) Thrombotic Events in 5 Patients Prior to Soliris Treatment Thrombotic Events in Equivalent Time Periods Discontinuing Anticoagulation 12 months prior to initiating Soliris 36 thrombotic episodes in 22 (14.3%) patients In the most recent 12 months on therapy 3 thromboses in 3 (2.%) patients 1 Budd-Chiari during complement blockade breakthrough caused by infection 1 CVA during reversal of warfarin overanticoagulation 1TIA/lacunarinfarctthought to be due to diabetic smallvessel disease Primary prophylaxis with warfarin has been safely stopped in 43/5 (86%) patients Secondary prophylaxis discontinued in 4 (<1%) due to haemorrhages, risk of bleeding from varices and/ or thrombocytopenia With no thrombotic sequelae 28 thrombotic episodes in 15 patients anticoagulated prior to initiation of Soliris therapy Hill A. ASH 212. Hill A. ASH 212. Hill A. ASH

20 Reduction in Intravascular Haemolysis All patients had a rapid reduction in LDH P <.1 Median Transfusion Requirementsin the 12 Months Prior to Soliris and Most Recent 12 Months on Soliris 66% of transfused patients become transfusion independent Reasons for Transfusion in the 25 Patients in Leeds Not Transfusion Independent Aplasia Breakthrough haemolysis (required higher dosing) Myelodysplasia Breakthrough haemolysis (minimally transfused, maximum of 3 units in 12 months) Cause unclear Extravascular haemolysis 25 patients still requiring transfusions: The meannumberof transfusionsfell significantly from24.6units (range 4-44) to 11.4units (range 2-45), P=.2 Hill A. ASH 212. Hill A. ASH Outcomes (n=153 patients) 137 patients on treatment as of April discontinued eculizumab therapy 1 predominant AA 2 spontaneous remissions of PNH clone 3 treated for indication of pregnancy alone 1 successfully transplanted for VSAA 9 patients died Outcomes 9 patientsdied 3 due to progression of their underlying bone marrow failure to MDS/AML One died immediately after BMT (veno-occlusive disease) 27 yrs old 2 AA 29 PNH Mar 211 AML May 211 Ecu Oct 211 transplant; VOD Remaining 5 not directly related to PNH Causes of Death Hill A. ASH 212. Hill A. ASH

21 Paroxysmal Nocturnal Hemoglobinuria: Compelling Long Term ClinicalBenefits in PNH Patients Patients Surviving, % 1 Pre-Soliris from Time of Diagnosis in 8 Patients With PNH 1, Age- and gendermatched controls Patients with PNH 5 year mortality of 35% recently confirmed Years After Diagnosis N= 8 Despite best supportive care - 5 year mortality: 35% Survival of PNH Patients Treated With Soliris Compared With the Normal UK Population 3 Age- and gender-matched normal population Soliris Treated PNH Population Numbers at Risk: Time (years) 1. Hillmen P et al. N Engl J Med. 1995;333: Kelly RJ et al. Blood. 211;117: Hillmen et al. Br Jnl Haematol.213;162:62-73 N= Sustained Complement Inhibition Leads to Reduced Hemolysis, and Improvements in Survival Uncontrolled Complement Activation Soliris Hemolysis Complications Associated With Elevated Hemolysis (LDH) Reduction in LDH (P<.1) 1% response rate in pivotal clinical trial programs (As measured by reduction in LDH) 92% (P<.1) reduction in TE End Organ Damage Renal Gastrointestinal Pulmonary Cardiac Hepatic Significant reduction in abdominal pain Improvement in dyspnea, dysphagia, fatigue, hemoglobinuria 4 fold improvement in CKD over placebo (P<.4) DInecreased Mortality Soliris appears to normalize survival in patients with PNH 1. Hillmen P et al. Blood. 27;11(12): Brodsky R et al. Blood. 21;116(21): Abstract # Hill A et al. Blood. 24;14:772: Abstract # Data on file. Alexion Pharmaceuticals, Inc. 5. Hillmen et al. N Engl J Med. 24; Brodsky et al. Blood.28;111: Hillmen et al. Blood.27;11: Hill A et al. Br J Haematol.21; May;149(3): Hillmen P et al. Am J Hematol.21;85: Hill et al. Blood. 28;112:Abstract A Long-Term Experience with Eculizumab in PNH Up to 1 year multicenter experience of eculizumab shows: Improved survival with a well-tolerated safety profile Persistent and significant improvement in symptoms and quality of life Significant reduction in the number of thrombotic events Significant reduction in transfusion requirements Long-term use of eculizumab demonstrates the impact on quality of life and reduction in complications, thereby improving long-term outcomes for patients with PNH Please see full prescribing informationfor Soliris (eculizumab). Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; 212. Hill A et al. Presented at the 54th Annual Meeting of the American Society of Hematology (ASH). December 8 11, 212, Atlanta, GA. Abstract #3472. Appears in Blood 212;12(21). 123 Complement Cascade Regulation and Erythrocytes DAT in Patients with PNH Pre and Post-Eculizumab Role of Complement 3 in Continued Extravascular Hemolysis 124 Lucio Luzzatto et al. Haematologica 21;95: Hill et al. Haematologica 21;95:

22 Flow Cytometry on C3b Fragment Deposition on Red Blood Cells C3b Fragments Are Only Found on PNH + RBCs Transfusion requirement and Hg in DAT-positive or -negative PNH patients treated with eculizumab Eculizumab Hill et al. Haematologica 21;95: Hill et al. Haematologica 21;95: Hill et al. Haematologica 21;95: PNH: Hemolysis Before and After Eculizumab Hemoglobin Normalization after Splenectomy Role ofsplenectomy RisitanoAM et al. N Engl J Med 21;363: Risitano et al. Blood 28;112:

23 Complete Attack in PNH Effect of C3 inhibitors on hemolysis and C3 fragment deposition on PNH erythrocytes. Novel Complement Inhibitors 133 Joshua M. Thurman Blood 214;123: Risitano et al. Blood 214;123: HCT for PNH Long Term Results HCT for PNH Transplantation for PNH N=48 MRD 56% MA 15 RIC N=22 MRD 65% N=26 overall 57% BM 2 PBPC Related 136 Unrelated 75 BM 135 PBSC 71 CBT Sašo et al Br J Haematol. 1999:14; Santarone et al. Haematologica 21:95; De Latour Haematologica 212;97:

24 RIC HCT for PNH Meningitis Vaccines Meningitis Vaccine in PNH Meningitis A, C, Y, W-135 (Quadrivalent Vaccines) MenHibrix (Hib-Men CY-TT) BIVALENT children 6 weeks-18 mos Menveo (Men ACWY-CRM) 2 months-55 years of age Menactra (Men ACWWY-D) 9 months-55 years of age Menomune (MPSV4) polysaccharide allergic reactions Older than 55 No mucosal immunity Duration of immunity less than 3 years no memory T cells Farah et al. ASH 211 abstract Current ACIP Recommendations for Complement Deficiencies Distribution of Meningitis Serotype in Norway Meningitis Vaccines Meningitis A, C, Y, W-135 (Quadrivalent Vaccines) MenHibrix (Hib-Men CY-TT) BIVALENT children 6 weeks-18 mos Menveo (Men ACWY-CRM) 2 months-55 years of age Menactra (Men ACWWY-D) 9 months-55 years of age Menomune (MPSV4) polysaccharide allergic reactions Older than 55 No mucosal immunity Duration of immunity less than 3 years no memory T cells Meningitis B Bexsero (Novartis) 1-25 years of age 2 dose series ( and 1-6 months) Trumenba (Pfizer) 1-25 years of age 3 dose series (,2, and 6 months)

25 Meningitis Vaccines Meningitis X North America, Europe, Australia and West Africa No commercially available vaccine 145 Global, observational, non-interventional study to collect real-world safety, effectiveness, and QoL data Open to all physicians treating patients with PNH regardless of therapy Objectives Database for publications to enhance understanding of disease and improve outcomes Promote evidence-based medicine Current enrollment Over 2 patients enrolled Participation in 24 countries, including Argentina, Australia, Belgium, Canada, Denmark, Finland, France, Germany, The Netherlands, New Zealand, Russia, South Korea, Spain, Sweden, Switzerland, Taiwan, United Kingdom, United States Enrollment information: