Advances in Paroxysmal Nocturnal Hemoglobinuria (PNH)

1. Advances in Paroxysmal Nocturnal Hemoglobinuria (PNH) Bart Scott, MD Assistant Professor of Medicine, Division of Oncology, University of Washington Director of Hematology and Hematologic Malignancies, Seattle Cancer Care AllianceSeattle, WA

2. Actuarial Survival From the Time ofDiagnosis in 80 Patients With PNH2 100 80 Age- and Gender-Matched Controls 60 Patients Surviving (%) 40 Patients With PNH 20 0 0 5 10 15 20 25 Years After Diagnosis Paroxysmal Nocturnal Hemoglobinuria (PNH):A Chronic, Systemic, and Life-Threatening Disease • Prevalence: 15.9 / million1 • Diagnosed at all ages • Median age early 30s3,4 • Progressive disease2–4 • Uncontrolled complement activation underlies the morbidities and mortality • Despite best supportive care • 5 year mortality: 35%2 The expected survival of an age- and gender-matched control group is shown for comparison (Hillmen et al. 1995) 1. Hill A et al. Blood 2006;108:290a. Abstract 985;2. Hillmen P et al.N Engl J Med 1995;333:1253–1258;3. Nishimura JI et al.Medicine 2004;83:193–207; 4. Socié G et al. Lancet 1996;348:573–577.

3. 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 surfaces1-4 • 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: • Thrombosis5 • End-organ damage1,6 • Increased mortality1,7 1. Brodsky R. Paroxysmal nocturnal hemoglobinuria. In: R Hoffman et al., eds. Hematology - Basic Principles and Practices. 4th ed. Philadelphia, PA: Elsevier Churchill Livingstone 2005;419–427; 2. Rosse WF et al.Hematology (Am SocHematolEduc Program) 2004:48–62; 3. Wiedmer T et al.Blood 1993;82:1192–1196; 4. Rother RP et al.JAMA 2005;293:1653–1662; 5. Hillmen P et al. Blood 2007;110:4123–4128; 6. Hillmen P et al.Am J Hematol 2010;85:553–559; 7. Hillmen P et al.N Engl J Med 1995;333:1253–1258.

4. PNH: What it’s Not • It is not paroxysmal1 • Even in the absence of symptoms, destructive progression of hemolysis is ongoing • It is not nocturnal1 • Hemolysis in PNH is subtle and constant, 24 hours a day • Hemoglobinuria is a less commonly seen complication • ¾ patients present without hemoglobinuria2 1. RotherR et al. Nature Biotechnology 2007;25,11:1256–1264; 2. International PNH Interest Group. Blood. 2005;106:3699–3709.

5. 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 membrane1 • CD59 (MIRL) • Forms a defensive shield for red blood cells (RBCs) from complement-mediated lysis • Inhibits the assembly of the membrane attack complex • CD55 (DAF) • Prevents formation and augments instability of the C3 convertases, attenuating the complement cascade CD59 CD55 GPI-anchor GPI = glycerophosphatidylinositol. 1. Borowitz MJ et al.Cytometry B ClinCytom 2010;78:211–230. Adapted from: Johnson RJ et al. J ClinPathol: Mol Pathol 2002;55:145–152;Brodsky R. Paroxysmal nocturnal hemoglobinuria. In: R Hoffman et al, eds. Hematology - Basic Principles and Practices. 4th ed. Philadelphia, PA: Elsevier Churchill Livingstone; 2005;419–427.

6. Autoreactive T Cells from Patients with PNH Specifically RecognizeGlycosyl-Phosphatidyl-Inositol (GPI) Abstract 647 Gargiulo et al

7. PIG-A Etn-P GlcN glycan nucleus PIG-A Inositol-P X chr. GPI anchor + GPI protein ER Paroxysmal Nocturnal Hemoglobinuria Pathogenesis PIG-A PIG-A nucleus GPI anchor X chr. protein ER Normal cell PNH cell Hematopoietic Stem Cells

8. Intravascular Hemolysis Absence of complement regulators Thrombosis Absence of complement regulators Bone Marrow Failure and cytopenia Mechanism? PNH Triad

9. crippled PIG-A+ cells Noxious agent Parodoxical Expansion of PNH Clone(Escape Theory) Time PIG-A+ cells PIG-A null cells Rotoli & Luzzatto, 1989

10. 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. • GPIhas been found within the presentation groove of CD1d(Joyce et al. Science 1998; De Silva et al.J Immunol. 2002) • Identical or quasi-identical TCRβ CDR3 sequences found in CD8+CD57+ T cells in a group of HLA- disparate PNH patientssuggesting antigen not HLA -restricted(Gargiulo et al. Blood 2005) • CD1d is expressed on human and murine hematopoietic stem and progenitor cells(Kotsianidis et al. Blood 2006; Broxmeyer et al. Blood 2012)

11. Hypothesis: GPI-specific, CD1d-restricted T cells responsible for selection of PNH cells T cell T cell T cell receptor No Killing Killing GPI CD1d/b2m GPI-anchored protein GPI+ GPI- Karadimitris and Luzzatto, Leukemia 2001

12. *p=0,0355 C1R C1R- CD1d with GPI addition 6 PNH Patients CD1d-dependent GPI-specific T cells in PNH *Wilcokson Rank Test

13. Normal Controls PNH Patients Increased frequency of novel invariant TCRVa21 mRNA chain within the whole TCRVa21 family repertoire of PNH patients

14. Summary CD1d-restricted GPI-specific CD8+ T cells are present and expanded in PNH patients. T cells with a novel invariant TCRa chain have been discovered in some PNH patients. These T cells could be the “noxious agent” responsible for the pathogenesis of PNH.

15. GPI-specific, CD1d-restricted T cells are present in PNH patients and could be the responsible of the suppression of GPI+ HSC. T cell T cell No Killing T cell receptor Killing GPI CD1d/b2m GPI-anchored protein GPI+ GPI- Future work: further structural and functional characterization

16. Expansion of the PNH Clone Is Necessary to Result in Clinical PNH Step 1 Somatic Mutation of PIG-A Step 2 Immunologic Attack Selective Damage Step 3 Growth Advantage • Expansion may be due to another somatic mutation • The need for both selection and expansion may explain the rarity of PNH Selected Cells Expanded Cells GPI-Deficient Cell Normal HematopoieticStem Cells GPI-Anchor Deficiency Immunologic Selection Benign Tumor- Like Expansion Adapted from: Inoue N et al.Int J Hematol2003;77:107.

17. The Role of Complement

18. The Complement System: Always on, Strongly Amplified, Dependent on Natural Regulators The complement system is a vital component of the natural (innate) protective immune system1 • 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 response1 • Rapid amplification leads to powerful and destructive immune reactions2 • Natural inhibitors of complement keep amplification in check and prevent uncontrolled complement activation2 1. Holers VM et al. ImmunolRev 2008;223:300–316; 2. Zipfel PF et al.CurrOpinNephrolHypertens2010;4:372–378.

19. Factors That Amplify Complement Activation Glovsky MM et al.Ann Allergy Asthma Immunol2004;93:513–523; Rubio MT et al.Bone Marrow Transplant 2008;41(Suppl. 1):S220. Abstract P766; Mastellos D et al. Immunologic Res 2003;3:367–385; Mergenhagen STE et al. J Infect Dis 1973;128:S86; Chenoweth DE et al.N Engl J Med 1981;304:497–503; Giradi G. Am J ReprodImmunol2008;59:183–192.

20. Chronic Uncontrolled Complement ActivationLeads to Devastating Consequences in PNH Lectin pathway Classical pathway Alternative pathway Immune complex clearanceMicrobial Opsonization C3 C3 + H2O: always active (chronic) Proximal Amplification Weak anaphylatoxin C3a Natural inhibitor: CD59 - Natural inhibitors: CD55 - C3b iC3b C5-convertase C5 Terminal C5b C6 C7 C8 C9 • C5a • Potent anaphylatoxin • Chemotaxis • Pro-inflammatory • Leucocyte activation • Endothelial activation • Pro-thrombotic • C5b-9Membrane attack complex • Cell lysis • Pro-inflammatory • Platelet activation • Leucocyte activation • Endothelial activation • Pro-thrombotic Anaphylaxis Inflammation Thrombosis Cell destruction Inflammation Thrombosis Consequences Consequences 1. Zipfel PF et al. Vaccine. 2008;26(Suppl 8):I67-74; 2. Figueroa JE, Densen P. ClinMicrobiolRev. 1991;4:359-95; 3. Walport MJ. N Engl J Med. 2001;344:1058-66; 4. Rother RP et al. Nat Biotechnol. 2007;25:1256-64; 5. Meyers G et al. Blood. 2007;110:abs 3683; 6. Hill A et al. Br J Haematol. 2010;149:414-25; 7. Hillmen P et al. Am J Hematol. 2010;85:553-9; 8. Parker C et al. Blood.2005;106:3699-709; 9. Hillmen P et al. N Engl J Med. 1995;333:1253-8; 10. Nishimura J et al. Medicine (Baltimore). 2004;83:193-207; 11. Caprioli J et al. Blood. 2006;108:1267-79; 12. Noris M et al. Clin J Am Soc Nephrol. 2010;5:1844-59; 13. George JN. Blood. 2010;116:4060-9; 14. Loirat C et al. PediatrNephrol. 2008;23:1957-72; 15. Ståhl AL et al. Blood. 2008;111:5307-15; 16. Hosler GA et al. Arch Pathol Lab Med. 2003;127;834-9; 17. Ariceta G et al. PediatrNephrol.2009;24:687-96.

21. AdBoard Master_Sept 14, 2010 Absence of CD59 Allows Terminal Complement Complex Formation C5a C8 C6 C6 C6 C6 C6 C5 CD59 CD59 C7 C7 C7 C7 C7 C5b C5b C5b C5b C5b C9 C9 x 12 - 15 C8 X X C5 convertase C5 convertase 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.

22. 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 activation1,2 • Tight control needed especially for the alternative pathway which is continuously turned on Membrane Attack Complex (MAC) on PNH Erythrocyte Photo: W Rosse. Reprinted with Permission. Endothelial Cells Damaged by Complement Attack Photo: S. Meri, Univ. of Helsinki. Reprinted with Permission. 1. HolersVM et al. ImmunolRev 2008;223:300–316; 2. ZipfelPF et al. CurrOpinNephrolHypertens 2010;4:372–378.

23. Historically Viewed as a Hemolytic Anemia Normal red blood cells (RBCs) are protected from complement attack by a shield of terminal complement inhibitors Without this protective complement inhibitor shield, PNH RBCs are destroyed ComplementActivation Lack of Bound CD55, CD59 Leads to Uncontrolled Complement Activation Intact RBC Reduced Red Cell Mass Anemia Free Hemoglobin 1. International PNH Interest Group. Blood 2005;106:3699–3709. 2. Brodsky R Paroxysmal Nocturnal Hemoglobinuria. In: Hematology - Basic Principles and Practices. 4th ed. R Hoffman; EJ Benz; S Shattilet al.eds. Philadelphia, PA: Elsevier Churchill Livingstone;2005;419–427. 3.Rother RP et al.JAMA 2005;293:1653–1662. 4. Socie G et al. Lancet 1996;348:573–577. 5. Hill A et al.Br J Haematol 2007;137:181–192.

24. Hemolysis Leads to NO Consumption in PNH Patients R=0.5094, P<0.001 R=0.9529, P<0.0001 NO consumption assay: NO chemiluminescence 100 100 Plasma Free Hb (µmol/l) Plasma Free Hb (µmol/l) 10 10 1 1 1 100 10 1000 10000 LDH (u/l) 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 mortality2 • Free Hgb significantly correlates with NO consumption • Hgb is in reduced state and reactive with NO 1. Hill A et al. Br J Haematol 2010;149:414–425; 2. Lee JW et al. ASH 2011. Abstract 3166.

25. Consequences of Nitric Oxide (NO) Depletion Reduced Nitric Oxide Can Cause • Smooth muscle dystonias1 • Vascular constriction – pulmonary and systemic hypertension, erectile dysfunction2 • Gastrointestinal contractions – dysphagia, abdominal pain • Platelet activation and aggregation1–4 • Platelet hyperreactivity • Hypercoagulability 1. Rother R et al. JAMA 2005;293:1653–1662; 2. Hill A et al.Br J Haematol 2010;149:414–425; 3. Weitz I. Thrombosis Res 2010;125:S106–S107; 4. Helley D et al.Haematologica2010;95:574–581.

26. Chronic Uncontrolled Complement Activation Leads to Devastating Consequences Thrombosis Renal Failure Significant Impact on Survival Pulmonary Hypertension Abdominal Pain ComplementActivation Chest Pain Dyspnea Elevated LDH Dysphagia Free Hemoglobin Significant Impact on Morbidity Fatigue Decreased NO Hemoglobinuria Erectile Dysfunction LDH = lactate dehydrogenase. 1. International PNH Interest Group. Blood 2005;106:3699–3709; 2. Brodsky R.Paroxysmal nocturnal hemoglobinuria. In: R Hoffman et al, eds. Hematology - Basic Principles and Practices. 4th ed. Philadelphia, PA: Elsevier Churchill Livingstone; 2005;419–427; 3. Rother RP et al.JAMA. 2005;293:1653–1662; 4. Socie G et al.Lancet 1996;348:573–577; 5. Hill A et al. Br J Haematol 2007;137:181–192; 6. Lee JW et al.Hematologica2010;95(s2): Abstracts 505 and 506; 7. Hill A et al.Br J Haematol 2010;149:414–425;8. Hillmen P et al.Am J Hematol 2010;85:553–559.

27. Historical Management of PNH Supportive care options do not impact progression and risk for severe morbidities and mortality1 • Transfusions1 – risk of iron overload • Anticoagulants1 – ineffective in many patients • Red cell supplements1 – may expand clone and elevate hemolysis • Steroids/androgen hormones1 – adverse events Although BMT is the only potentially curative therapy for PNH, BMT is associated with significant morbidities and mortality2,3 • In a study examining PNH patients (n=23)2 • 50% chronic GVHD; 42% acute GVHD3 • Transplant-related mortality was 42% • BMT has a significant impact on quality of life (QoL) post-transplant4,5 1. International PNH Interest Group. Blood 2005;106:3699–3709; 2. Santaraone S et al. Haematologica2010;95:983–988; 3. de Latour PF et al.EBMT 2009:Abstract 316; 4. Bieri S et al.Bone Marow Transplant 2008;42:819–827; 5. Fraser CJ et al. Blood 2006;108:2867–2873.

28. Morbidities and Mortality in PNH

29. Thrombosis Thrombosis Is the Leading Cause of Death in PNH1 • Accounts for 40–67% of deaths2 • First thrombotic event (TE) can be fatal2,3 • First TE increases risk for death 5- to 10-fold2 • Up to 44% of patients experience clinical thrombotic events2 • Occurs in typical and atypical sites4 • Is not adequately managed with anticoagulation2 • All patients with PNH are at risk for thrombosis2 1. International PNH Group et al.Blood 2005;106:3699–3709; 2. Hillmenet al. Blood 2007;110:4123–4128; 3. AudebertHJ et al.J Neurol 2005;252:1379–1386; 4. Lee JW et al.Hematologica2010;95(s2): Abstract 506.

30. Thrombosis Multifactorial Pathogenesis of Thrombosis in PNH • Pathogenesis of thrombosis in PNH is a result of uncontrolled complement activation1 • Activation of complement C5b–9 • Hemolysis leads to reduced nitric oxide levels and vasoconstriction2–4 • Platelets undergo morphological changes, release microparticles, and aggregate2,4 • Activation of complement C5a • Leukocytes release tissue factor and inflammatory cytokines (IL-6) to initiate coagulation2,3,5 • Leukocytes decrease expression of plasminogen activator receptor (PAR) leading to impaired fibrinolysis4 1. McKeage K. Drugs 2011;71:2327–2345; 2. Hill A et al.Br J Haematol 2007;137:181–192; 3. Weitz I. Thrombosis Res 2010;125:S106–S107; 4. Helley D et al.Hematologica.2010;95:574–581; 5. Markiewski MM et al.Trends Immunol 2007;28:184–192.

31. Thrombosis Chronic Uncontrolled Complement Activation Leads to Vasoconstriction and Thrombosis Impaired regulation of smooth muscles Local vasoconstriction Pro-inflammatory effect on endothelial cells Chronic hemolysis Platelet activation Local vasoconstriction Chronic Hemolysis Chronic UncontrolledComplementActivation [NO] C5b-9 C5b-9 C5a CLOT Inflammation Endothelial cell injury Systemic thrombosis Platelet Activation Inflammation Platelet Aggregation Leukocyte Activation Adapted from: Gladwin MT et al.Free Rad Biol & Med 2004;36:707–717; Rother RP et al. JAMA 2005;293:1653–1662.

32. Thrombosis The Incidence of TE is Increased in Patients with Elevated LDH at Diagnosis P<0.001 • 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) 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, 2012; Atlanta, GA. Abstract 1273.

33. Thrombosis Risk of First Ever Ischemic Stroke (FEIS) Elevated in PNH Age of FEIS in PNH patients is markedly less than in the general population1 2,3 1 2,3 1 1. Gostynski M et al.J Neurol 2006;253:86–91; 2.Hillmen P et al. Blood 2007;110:4123–4281; 3. Data on file. Alexion Pharmaceuticals, Inc.

34. Thrombosis Thrombosis is Associated With Risk of Early Mortality TE increases risk of death 15-fold over patients with no TE • TE was an independent prognostic factor related to poor survival (HR 15.4; 95% CI 9.3–25.4; P<0.001) in a large cohort of French PNH patients (n=415) de Latour RP et al.Blood 2008;112:3099–3106.

35. Thrombosis HillmenP et al.Blood 2007;110:4123–4128. Thrombosis Occurs in Both Typical and Atypical Sites* Myocardial Infarction/ unstable angina ARTERIAL Superficial vein thrombosis Cerebrovascular accident/ transient ischemic attack Cerebral/internal jugular thrombosis Pulmonary embolus Deep vein thrombosis* Hepatic/portal vein thrombosis VENOUS Mesenteric/splenic vein thrombosis *124 events, 63 patients *Includes 18.5% lower extremity and 14.5% other (inferior vena cava, bilateral lower extremity, pelvic, ureter, axillary, subclavian, and brachiocephalic veins).

36. Thrombosis PNH Patients are at Risk of Thrombosis Despite Anticoagulation or Minimal Transfusion Requirements (n=91) (n=22) HillmenP et al. Blood 2007;110:4123–4128.

37. Thrombosis Thrombosis Can Occur Regardless of Clone Size (n=43) Data from South Korean National Registry. Lee JW et al.Hematologica 2010;95(s2): Abstract 505.

38. Thrombosis Thrombosis in PNH Conclusions • 40–67% mortality in PNH results from thrombosis1 • Thrombosis is the leading cause of death in PNH2 • First TE increases risk for death 5- to 10-fold1 • LDH 1.5× ULN at diagnosis is associated with TE and mortality3 • DVT or PE most common clinical presentation1 • Arterial thromboses are also common1 • Anticoagulant therapy may not be adequate to control thrombosis in PNH1 • Clinical thrombosis evident in PNH patients: • No transfusion history1 • Smaller clone size4 1. Hillmen P et al.Blood 2007;110:12:4123–4128; 2. International PNH Group et al.Blood 2005;106:3699–3709; 3. Lee JW et al. Blood (ASH Annual Meeting Abstracts) Nov 2011;118:3166; 4. Lee JW et al.Hematologica2010;95(s2): Abstract 506.

39. Chronic Kidney Disease 1. Brodsky R. Hematology: Paroxysmal nocturnal hemoglobinuria. In: R Hoffman et al, eds. Hematology - Basic Principles and Practices. 4th ed. Philadelphia, PA: Elsevier Churchill Livingstone 2005;419–427; 2. Rother R et al.JAMA 2005;293:1653–1662; 3. Clark DA et al.Blood 1981;57:83–89; 4. Hillmen P et al.Am J Hematol 2010;85:553–559; 5. McKeage K. Drugs 2011;71:2327–2345. Kidney Pathology in PNH • Complement-mediated hemolysis and cell-free plasma hemoglobin lead to chronic kidney disease in PNH1–5 • Repetitive exposure of tissue to cell-free hemoglobin may lead to renal damage in PNH3,4 Micrograph of a Renal Biopsy from a PNH Patient, Indicative of Vascular Damage Normal tissue on the right Interstitial scarring on the left Clark DA, et al. Blood. 1981;57:83-89. Reprinted with Permission.

40. Chronic Kidney Disease 1.Hillmen P, et al. Am J Hematol 2010;85:553–559; 2. Brodsky R. Hematology: Paroxysmal nocturnal hemoglobinuria. In: R Hoffman et al, eds. Hematology - Basic Principles and Practices. 4th ed. Philadelphia, PA: Elsevier Churchill Livingstone 2005;419–427. 3. Hill A. et al. Blood 2006;108: Abstract 979. Chronic Kidney Disease: Morbidity and Mortality in PNH • Kidney failure is the cause of 8–18% of PNH-related deaths1 • 80% of PNH patients (median age of 31.5 years) had MRI evidence of significant renal hemosiderosis2,3 • 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

41. Chronic Kidney Disease 64% of PNH Patients Exhibit Clinical Chronic Kidney Disease (CKD) 1. HillmenP et al. AmJ Hematol 2010;85:553–559.

42. Chronic Kidney Disease Kidney Disease in PNH: Conclusions • Kidney failure is the cause of 8–18% of PNH-related deaths1 • Kidney disease in PNH is caused by complement-mediated hemolysis2,3 • 64% of patients with PNH exhibit chronic kidney disease at any one time4 • Kidney disease is underappreciated in PNH4 1. Nishimura JI et al. Medicine 2004;83:193–207; 2. Clark DA et al.Blood 1981;57:83–89; 3. McKeage K. Drugs 2011;71:2327–2345; 4. Hillmen P et al.Am J Hematol 2010;85:553–559; 5. Kim JSet al.Hematologica 2011;96(s2): Abstract 271.

43. Common Symptoms of PNH Clone Size Does Not Correlate to Symptom Severity 1. Urbano-Ispizua A et al. Hematologica 2010;95(s2): Abstract 1022.

44. Common Symptoms of PNH Common PNH Symptoms are Associated With TE P=0.0004 P=0.024 P=0.015 Elevated LDH (≥1.5× ULN) in combination with abdominal pain, chest pain, and dyspnea are associated with a higher risk of TE Study description: a retrospective analysis of the medical charts of 286 PNH patients in a South Korean National Registry. Lee JW et al. Hematologica2010;95(s2): Abstracts 505 and 506.

45. Common Symptoms of 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, 2012; Atlanta, GA. Abstract 1273. Chronic Complement-Mediated Hemolysis in Combination With Clinical Symptoms Increase the Risk of Thrombosis Odds Ratio for TE (Multivariate Analysis) The risk of TE in patients with LDH ≥1.5× ULN was 7.01 times greater than in patients with LDH <1.5× ULN (P=0.013).

46. 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 thrombosis2 • 66% of patients report shortness of breath3 • 59% of patients report abdominal pain4 • 97% of patients report fatigue1 • 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 activities1 • Clone size does not correlate to symptom severity5 1. Weitz I et al. Intern Med J. 2012; 2. Lee JW et al. Hematologica 2010;95(s2): Abstract 506. 3. Meyers G et al. Blood 2007;110: Abstract 3683; 4. Lee JW et al. Hematologica 2010;95(s2): Abstract 505; 5. Urbano-Ispizua A et al. Hematologica 2010;95(s2): Abstract 1022.

47. Early Identification of Patients at High Risk for PNH

48. Advancements in Treatment Options Warrant Early Diagnosis and Intervention • Early diagnosis is essential for improved patient prognosis: • PNH • Bone marrow failure1 • International PNH Registry • Provides evidence to inform clinical decision making • Over 2100 patients from 26 countries are currently enrolled in the PNH registry 1. Sugimori C et al. Blood 2006;107:1308–1314.

49. Two Independent International Groups Recommend Testing High-Risk Patients for PNH 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 Borowitz MJ et al. International Clinical Cytometry Society. Part B Clin Cytometry 2010;78B:211–230; International PNH Interest Group. Blood 2005;106:3699–3709.

50. Suggestions for PNH Testing by ICCS PNH Guidelines Borowitz MJ et al; Clinical Cytometry Society. Cytom B Clin Cytom 2010;78B:211–230