1. PNH A Review and Update October 2010

2. PNH What is PNH? What is PNH? What causes PNH? What causes PNH? What are the clinical symptoms of PNH? What are the clinical symptoms of PNH? What are the long-term risks and complications of PNH? What are the long-term risks and complications of PNH? How is PNH treated? How is PNH treated? What does the future hold? What does the future hold?

3. What is PNH? Paroxysmal – sudden onset Paroxysmal – sudden onset Nocturnal – occuring at night (or early in morning upon awakeneing) Nocturnal – occuring at night (or early in morning upon awakeneing) Hemoglobinuria Hemoglobinuria Despite the name, most patients do not present this way.

4. What is PNH? A rare and unusual acquired hematologic disorder characterized by A rare and unusual acquired hematologic disorder characterized by Intravascular hemolysisIntravascular hemolysis Bone marrow failure (cytopenias)Bone marrow failure (cytopenias) ThrombosisThrombosis There is great clinical heterogeneity amongst patients with PNH. There is great clinical heterogeneity amongst patients with PNH. A disease that has fascinated hematologists for a long time… A disease that has fascinated hematologists for a long time…

5. 1 st published case report of PNH - 1866 Gull WW. Guys Hospital Reports 12:381-392, 1866.

6. PNH- Historical Perspective

7. What is PNH? A rare and unusual acquired hematologic disorder characterized by A rare and unusual acquired hematologic disorder characterized by Intravascular hemolysisIntravascular hemolysis Bone marrow failure (cytopenias)Bone marrow failure (cytopenias) ThrombosisThrombosis

8. What causes PNH? PNH is due to a change (mutation) in a single gene in a bone marrow “stem” cell. PNH is due to a change (mutation) in a single gene in a bone marrow “stem” cell. What is a mutation?What is a mutation? What is a stem cell?What is a stem cell? PNH is due to a condition that allows this mutated cell to become the dominant cell in the bone marrow. PNH is due to a condition that allows this mutated cell to become the dominant cell in the bone marrow.

9. What is a mutation? A mutation is a “mistake” in a gene that arise when a cell divides and has to copy the DNA. This mistake is not corrected and is passed on to “daughter cells” and all subsequent cells. A mutation is a “mistake” in a gene that arise when a cell divides and has to copy the DNA. This mistake is not corrected and is passed on to “daughter cells” and all subsequent cells. Mutations can cause: Mutations can cause: No effectNo effect Complete absence of the protein produced by that geneComplete absence of the protein produced by that gene An altered protein with decreased or different functionAn altered protein with decreased or different function

10. What is a stem cell? A stem cell has two properties A stem cell has two properties Can divide to produce daughter cells and more stem cells (self renewal) forever.Can divide to produce daughter cells and more stem cells (self renewal) forever. Can “differentiate” (mature) into many different types of cells.Can “differentiate” (mature) into many different types of cells. Many different types of stem cells Many different types of stem cells EmbryonicEmbryonic Induced pluripotent stem cells (IPS)Induced pluripotent stem cells (IPS) Tissue specificTissue specific HematopoieticHematopoietic Many different sources of hematopoietic stem cells Many different sources of hematopoietic stem cells Bone marrowBone marrow Peripheral bloodPeripheral blood Umbilical cordUmbilical cord AutologousAutologous

11. Hematopoietic Stem Cells In PNH, a mutation occurs in a single gene (PIG-A) in a single hematopoitic stem cell X X X X X X X X X X

12. What causes PNH? The mutation in the PIG-A gene in a hematopoietic stem cell leads to a defect in the production of an anchor protein that ties other proteins to the cell surface. The mutation in the PIG-A gene in a hematopoietic stem cell leads to a defect in the production of an anchor protein that ties other proteins to the cell surface. Sometimes the mutation leads to a partial decrease in the amount of anchor protein that is made and the cells have a partial deficiency (Type II cells)Sometimes the mutation leads to a partial decrease in the amount of anchor protein that is made and the cells have a partial deficiency (Type II cells) Some patients have several stem cells with different mutations in PIG-A geneSome patients have several stem cells with different mutations in PIG-A gene C= O As p 2 CHCH CHCH C= O O CHCH 2 NHNH O PHOSPHATIDYL - INOSITOL ETHANOLAMINE GLYCAN CORE PROTEIN MEMBRANE C= O 2 O O=P= O O O CHCH CHCH 2 CHCH 2 NHNH CHCH 2 O 2 O O- P=O O-P- O O N ( 1- 2) ( 1- 6) ( 1- 4)

13. What causes PNH? The lack of the GPI anchor protein leads to a lack of many proteins on the surface of affected blood cells. The lack of the GPI anchor protein leads to a lack of many proteins on the surface of affected blood cells. In PNH, the major two proteins lacking on the surface of the red cells are CD59 and CD55. In PNH, the major two proteins lacking on the surface of the red cells are CD59 and CD55. These proteins are important in protecting the red cells from complement. These proteins are important in protecting the red cells from complement. What is complement? (three slides later)What is complement? (three slides later)

14. What causes PNH? How do cells with a mutation take over the bone marrow? Normal people may carry cells in their bone marrow with a PIG-A mutation, usually at a very low level and probably not in a true stem cell. Normal people may carry cells in their bone marrow with a PIG-A mutation, usually at a very low level and probably not in a true stem cell. In PNH, something allows the abnormal cells to become the dominant cells and become the major population in the marrow (anywhere from 1 to over 90% - referred to as the clone size). In PNH, something allows the abnormal cells to become the dominant cells and become the major population in the marrow (anywhere from 1 to over 90% - referred to as the clone size). This “something” may be related to aplastic anemia, a disease of poor production of the bone marrowThis “something” may be related to aplastic anemia, a disease of poor production of the bone marrow

15. How is PNH related to aplastic anemia? Many patients with PNH have or will develop aplastic anemia, or have a history of having had aplastic anemia. Many patients with PNH have or will develop aplastic anemia, or have a history of having had aplastic anemia. Many PNH patients have evidence of poor production of cells by their bone marrow (Bone marrow failure) leading to low white cell counts or low platelet counts. Many PNH patients have evidence of poor production of cells by their bone marrow (Bone marrow failure) leading to low white cell counts or low platelet counts. The cause of aplastic anemia (immune assault?) may also play a role in the development of PNH. The cause of aplastic anemia (immune assault?) may also play a role in the development of PNH.

16. Models of pathogenesis Models of pathogenesis NormalAplasticPNHMDS MarrowAnemia_________________________ Immune Assault Immune Assault PNH (hemolysis) PNH (hypoplasia) Stromal cell Dysregulation, Immune Assault

17. What is Complement? Complement is a group of proteins that are part of our immune system. Complement is a group of proteins that are part of our immune system. Complement circulates in an inactive form. Complement circulates in an inactive form. A little bit of complement is always being activated spontaneously, especially at night. A little bit of complement is always being activated spontaneously, especially at night. Many different events can activate complement including trauma, infection, stress, etc. Many different events can activate complement including trauma, infection, stress, etc. Complement will attack certain bacteria by making pores in the surface of the bacteria. Complement will attack certain bacteria by making pores in the surface of the bacteria. In PNH, activated complement will attack red cells causing them to “lyse” (burst) In PNH, activated complement will attack red cells causing them to “lyse” (burst)

18. Terminal Complement Activation Renders RBCs Susceptible to Lysis Normal RBCs PNH RBC Intact RBC Complement Activation Lysed PNH RBCs and free hemoglobin in the plasma Lysed PNH RBCs and free hemoglobin in the plasma Chronic Hemolysis CD59

19. What happens when red cells lyse? The red cells are destroyed - anemia The red cells are destroyed - anemia Hemoglobin is released into the plasma (the fluid part of blood) Hemoglobin is released into the plasma (the fluid part of blood) Some of the hemoglobin passes through the kidneys and into the urine leading to the dark color of the urine Some of the hemoglobin passes through the kidneys and into the urine leading to the dark color of the urine Loss of ironLoss of iron May lead to kidney damage in the long runMay lead to kidney damage in the long run Free hemoglobin binds nitric oxide Free hemoglobin binds nitric oxide What is nitric oxide?What is nitric oxide?

20. What is nitric oxide? A gas produced by the body to regulate smooth muscle cells. A gas produced by the body to regulate smooth muscle cells. An increase in free nitric oxide causes smooth muscle cells to relax. A decrease causes smooth cells to contract. An increase in free nitric oxide causes smooth muscle cells to relax. A decrease causes smooth cells to contract. Smooth muscle cells are in many tissue Smooth muscle cells are in many tissue Blood vessel walls: ischemia, impotenceBlood vessel walls: ischemia, impotence Esophagus and GI tract: esophageal spasm, reflux, abdominal painEsophagus and GI tract: esophageal spasm, reflux, abdominal pain

21. What about thrombosis (blood clots) in PNH? Blood clots are a presenting sign in 10-20% of patients with PNH. Blood clots are a presenting sign in 10-20% of patients with PNH. Can occur in up to 40% of patients with PNH. Can occur in up to 40% of patients with PNH. Occur in unusual locations – veins of the liver (Budd-Chiari syndrome), spleen, brain, and skin. Occur in unusual locations – veins of the liver (Budd-Chiari syndrome), spleen, brain, and skin. Associated with a very bad prognosis Associated with a very bad prognosis Cause of these blood clots is unknown – possibly related to complement activation. Cause of these blood clots is unknown – possibly related to complement activation. Mesenteric / splenic 18% Hepatic / portal 16% PE 7% Cerebral 6% Superficial 4% Arterial / CVA 14% Arterial / MI 2% DVT: leg 18% DVT: other 15%

22. The clinical picture of PNH Hemolysis due to complement activation Hemolysis due to complement activation Anemia and fatigueAnemia and fatigue Hemoglobinuria, kidney damageHemoglobinuria, kidney damage Nitric oxide trapping >> Esophageal spasm, abdominal pain, pulmonary hypertension, impotence, fatigue?Nitric oxide trapping >> Esophageal spasm, abdominal pain, pulmonary hypertension, impotence, fatigue? Thrombosis – Cause of blood clots is still unknown Thrombosis – Cause of blood clots is still unknown Unusual sites of blood clotsUnusual sites of blood clots Bone marrow failure Bone marrow failure Decreased blood counts (cytopenias)Decreased blood counts (cytopenias)

23. Treatment of PNH Who needs to be treated? Who needs to be treated? With what? With what? Does everybody respond? Does everybody respond? What is the long term outlook? What is the long term outlook?

24. PNH – Who needs to be treated? Patients with blood clots Patients with blood clots Patients with symptomatic anemia from hemolysis Patients with symptomatic anemia from hemolysis Patients with severe bone marrow failure Patients with severe bone marrow failure Patients with hemolysis who are not symptomatic Patients with hemolysis who are not symptomatic Can we prevent long term complications such as blood clots, renal failure, or pulmonary hypertension?Can we prevent long term complications such as blood clots, renal failure, or pulmonary hypertension? The Treatment “Grey zone”

25. How do we treat PNH - hemolysis Transfusion Iron, folic acid Steroids Eculizumab (Soliris)

26. What does Eculizumab do? Quickly and effectively blocks complement activation at C5. Quickly and effectively blocks complement activation at C5. Blocks hemolysis and related effects Blocks hemolysis and related effects Stops hemoglobinuria Stops hemoglobinuria Markedly reduces transfusion requirements Markedly reduces transfusion requirements Hemoglobin / hematocrit may not return to “normal” Hemoglobin / hematocrit may not return to “normal”

27. Reduction in LDH During Eculizumab Treatment in TRIUMPH and SHEPHERD TRIUMPH – Placebo/extension TRIUMPH – SOLIRIS/extension SHEPHERD - SOLIRIS Time, Weeks Lactate Dehydrogenase (U/L) Data on file. Alexion Pharmaceuticals; 2007. TRIUMPH placebo patients switched to SOLIRIS after week 26. All TRIUMPH patients entered the long-term extension study.

28. SHEPHERD: Eculizumab Reduced Transfusions Transfusion Requirements 12 Months Prior to Treatment Young, et al. Blood. 2006;108:971. (n = 97)(n = 21)(n = 47)(n = 15)(n = 14) * † ‡ § *

29. SHEPHERD: Eculizumab Improved Fatigue FACIT-F = Functional Assessment of Chronic Illness Therapy ‑ Fatigue instrument. *P<0.001. P value compared with baseline based on signed rank test. * * * * * ** * * * Improvement Young, et al. Blood. 2006;108:971.

30. Renal Function with Eculizumab in Different Baseline Populations – 12 Months Hillmen et al. Blood. 2007 Abstract 3678; High Incidence of Progression to Chronic Renal Insufficiency in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH). 58.1 23.4 76.9 35.2 71.4 20.5 6.7 5.2 2.6 0 10 20 30 40 50 60 70 80 90 Segment of PNH Population Proportion of Patients (%) P<0.001P=0.02P<0.001 No ChangeImprovementWorsening Overall (n=179) Stage 1 – 2 (n=77) Stage 3 - 5 (n=39)

31. Change in BNP during eculizumab treatment PHT with NT-proBNP ≥160 pg/mL 1 Eculizumab vs placebo (P<0.001) 50% reduction 14% increase Baseline Week 26 52.5 39.4 26.3 43.8 0 10 20 30 40 50 60 Placebo Eculizumab Treatment group: TRIUMPH (n=73) Proportion of patients with evidence of PHT 1 Machado RF et al. JAMA 2006; 296: 310-318; Hill A et al. Blood (ASH Annual Meeting Abstracts) 2008; 112: abstract 486

32. What eculizumab does not do Probably does not help bone marrow failure (improve other low blood counts) Probably does not help bone marrow failure (improve other low blood counts) Completely correct anemia Completely correct anemia Teach you how to play the violin if you have never played before Teach you how to play the violin if you have never played before

33. Downside of Eculizumab treatment Increased risk of meningococcal infections Increased risk of meningococcal infections All patients must be vaccinatedAll patients must be vaccinated All patients educated on signs and symptoms of meningitis and what to doAll patients educated on signs and symptoms of meningitis and what to do All patients given cards describing thisAll patients given cards describing this Cost Cost Possible coating of red cells with C3 complement leading to their destruction (extravascular) and anemia Possible coating of red cells with C3 complement leading to their destruction (extravascular) and anemia Inconvenience Inconvenience Must be given intravenously every 12-14 daysMust be given intravenously every 12-14 days

34. How do we treat or prevent blood clots? Coumadin prophylaxis Acute treatment with lytic agents (clot busters) Anticoagulation therapy Bone marrow transplantation Eculizumab

35. Effect of Eculizumab on Thrombosis 92% Fewer thrombotic events with SOLIRIS treatment 92% Fewer thrombotic events with SOLIRIS treatment There were fewer thrombotic events with SOLIRIS treatment than during the same period of time prior to treatment. Hillmen P, et al. Blood. 2007;110: 4123-4128 39 3 0 5 10 15 20 25 30 35 40 45 Pre-SOLIRIS TreatmentSOLIRIS Treatment Thrombotic Events (#) P=0.0001

36. How do we treat bone marrow failure? Stimulating agents such as erythropoietin Immunosuppressive agents (ATG, cyclosporine A) Bone marrow transplantation

37. Where are we going? Improve current therapy Improve current therapy Oral eculizumabOral eculizumab Increase treatment intervalsIncrease treatment intervals Find other ways to inhibit complement Find other ways to inhibit complement Understand how PNH cells take over the bone marrow so we can reverse this process (Restore normal stem cells) Understand how PNH cells take over the bone marrow so we can reverse this process (Restore normal stem cells) Gene therapy Gene therapy Stem cell transplants Stem cell transplants