Paroxysmal Nocturnal Hemoglobinuria

1. Paroxysmal Nocturnal Hemoglobinuria About the disease and the approaches to living with it. Gabrielle Meyers, MD

2. Outline • What is PNH? • How do you get it? • How do you diagnose it? • What are the things to worry about? • What are the treatments? Can this be cured?

3. Paroxysmal Nocturnal Hemoglobinuria • Now you know why we call it PNH for short. • Rare disease. • Caused from a defect in the production of GPI protein anchors on the surface of all blood cells produced by the PNH bone marrow stem cells • This is an acquired mutation of PIG-A, a gene on the X chromosome important in making GPI protein anchors. • Only blood cells have the defect. Defect makes the red cells in particular susceptible to destruction by the complement system.

4. PNH • There are 2 main ways to attach proteins to the surface of cells-either through transmembrane attachments or GPI-anchors. • Many proteins are attached to the surface by GPI anchors. • PIG-A gene is vital to the production of GPI anchors. • In PNH, you have a mutation in PIG-A so that it has reduced activity or no activity at all.

5. CD55 CD59 Normal Hematopoietic Cells GPI anchors Transmembrane protein Membrane lipid bilayer

6. PNH • The PIG-A mutation occurs in a bone marrow stem cell. All the blood cells made by this defective stem cell are deficient in GPI-anchored proteins. • There are key proteins on the surface of red blood cells that protect red cells from the activity of the complement system. • Complement system is a primitive immune system designed to attack foreign invaders • Kills them by poking holes in the membrane-what is called the Membrane Attack Complex (MAC)

7. MAC

8. PNH • PNH red cells are deficient in all GPI anchored protein, but 2 are important in protecting red cells from destruction: CD55 (DAF) and CD59 (MIRL). • Without these proteins, red cells don’t have their normal protection against the complement system. • In PNH, you have uncontrolled, complement mediated hemolysis (destruction of red cells). This happens all the time, and is accelerated when you have an event that activates the complement system (infection).

9. How do you get PNH? • This is an acquired disease. • We think PIG-A mutations can happen spontaneously, but unless the environment is supportive of those mutations they never develop into PNH. • In a bone marrow under attack or failing, PIG-A mutations have an advantage-they survive the attack better (for some reason). Therefore, the PNH cells have an advantage and can expand to become a significant portion of the bone marrow cells.

10. Two-Step Model of Developing PNH Normal Bone Marrow Normal bone marrow with normal HSC’s and rare PNH mutant HSC’s MARROW INJURY Step I. Clonal Selection Bone marrow damage (likely immune mediated) selects for clones. After selection, expansion of PIG-A mutant HSC’s varies depending on other characteristics of the affected cells. These other characteristics may be determined by genetic (mutational), epigenetic (nonmutational), or environmental factors. Step II. Clonal Dominance

11. How do we know PNH cells have an advantage? • PNH is found in diseases where the bone marrow is under attack or damaged: • Aplastic anemia (up to 60% of patients with aplastic anemia have a detectable PNH clone). • Myelodysplastic syndrome (MDS)-up to 20% of patients with MDS have an identifiable PNH clone • Other immune-mediated diseases: ITP • Blood cancers: leukemias-both chronic and acute Why the PNH cells have an advantage is unknown. Why the PNH cells expand to produce more blood cells is unknown.

12. How do you get PNH? • Patients with PNH often have a history of aplastic anemia or other bone marrow injury • PNH can come on later, after they have recovered from the initial bone marrow insult. • You can have a little or a lot of PNH cells, and that can effect how the disease impacts the health of the patient.

13. How do you diagnose PNH? • This is the easy part. It’s thinking about sending off the test that is the hold up in most cases. • You diagnose PNH by looking for the cells deficient in the GPI anchored proteins • Do flow cytometry, which uses a tag for certain proteins, and in PNH the cells will be missing those proteins (usually check for CD55, CD59 and others). • You do the test on peripheral blood.

14. Normal Peripheral Blood Sample PNH Peripheral Blood Sample Anti-DAF DAF

15. Red Cell Staining with anti-CD59 From Hall SE and Rosse WF, Blood 1996 ;87:5332

16. White Cell Staining with anti-CD59 From Hall SE and Rosse WF, Blood 1996; 87: 5332

17. What information from the flow test is important? • As mentioned before, you can have a little or a lot of PNH cells, and this impacts how you feel and the potential for complications. • The flow test tells you the size of the PNH clone, and whether you have more than 1 clone. • The flow test also tells you the extent of the deficiency in the PNH cells (type II or type III cells). • Flow tests should be followed over time to see what the PNH cells are doing (getting more, less, staying the same, etc).

18. Who Should be Tested for PNH? • Patients with unexplained hemolytic anemia • Patients with bone marrow failure, including aplastic anemia and MDS • Patients with hemoglobinuria • Patients with unusual/repetitive thrombosis, and arterial thrombosis otherwise unexplained. • Patients with episodic swallowing problems or abdominal pain of unclear etiology with associated hemolysis

19. What are the symptoms and complications of PNH? • Everything relates to either the hemolysis or the damaged bone marrow. • Most patients have a mixture of symptoms-daily symptoms and then worsening of symptoms during paroxysms (attacks of increased hemolysis due to complement activation). • Complications can be life-threatening

20. FATIGUE Anemia Decreased stamina Shortness of breath Abdominal pain Back pain Difficulty swallowing Chest pain Erectile dysfunction Decreased libido or interest in intimacy Headaches Swelling related to blood clots Increased risk for infections Increased risk for bleeding Depression, frustration, feeling lack of control over life Weight changes, body changes PNH symptoms

21. PNH Complications • Bone marrow failure-all the blood counts are low, bone marrow is not producing cells as it should be. • Clots! • Infections-either related to disease or complications of treatment (prednisone, eculizumab) • Bleeding-either from low blood counts or blood thinners for treatment/prevention of clots • Risk of blood transfusions-luckily the blood product pool is extremely safe at this time • Cardiopulmonary issues related to nitric oxide scavenging by free hemoglobin-can cause high pressure in the lung system (pulmonary hypertension) that can damage the heart. Some can be reversed by eculizumab. • Pregnancy

22. Implications of Living with PNH • Quality of life issues • Financial impacts • Ability to work • Costs of treatments/medical care • Unpredictability • Fear of complications • Am I going to clot (again)? • Burden of treatments • Blood transfusions • Eculizumab • Bone marrow transplantation

23. What are the treatments? Cures? • Treatment options depend on certain factors • What is the clone size? • How does the marrow function? What are all the blood counts? • Clot risk • Short-term vs. long-term treatment • Blood transfusions and pulse prednisone often used in short-term • Long-term-vitamin replacement, low-dose prednisone, eculizumab, bone marrow transplant • Cures? Yes, with bone marrow transplant • Control? Yes.

24. Eculizumab • First/only drug targeted to PNH • Monoclonal antibody against complement protein 5 (C5). Binds this protein and halts the rest of the complement cascade. • “Protects” PNH cells from destruction by halting the complement cascade. • Very effective at reducing hemolysis, reducing transfusion needs, improving QOL. Early evidence suggests it may reduce clots.

26. Pro’s Very effective at reducing hemolysis Well tolerated Improvements in QOL, reduction in transfusions proven Reduction in burden of disease Infusion weekly X5, then every 2 weeks Probable reduction in clots Con’s $$$$ Infusion weekly X5, then every 2 weeks Infection risk: meningococcal meningitis Burden of treatment Plan for lifetime therapy Does not improve other blood counts Eculizumab-Pro’s and Con’s

27. Eculizumab in Pregnancy • Pregnancy in PNH is very risky for both the mother and fetus, due to risk of clotting, infection, and fetal loss. • Is eculizumab safe in pregnancy? Could this help reduce the risks of pregnancy? • Report of 7 pregnancies in patients that received eculizumab at some point during pregnancy.

28. Outcomes in Pregnancy with Eculizumab

29. Bone Marrow Transplant for PNH • This is very effective at curing PNH • Risks include toxicity from the transplant and graft vs. host disease (GVHD) • In patients with aplastic anemia or MDS/leukemia and PNH transplant is driven by other disease • Transplantation continues to improve over time, in particular transplants from unrelated donors

30. From Italian group, 26 transplants for PNH 1988-2006 Matched sibling 10 year survival 65% All transplants 10 year survival 56%

31. Classical PNH High clone size (>50%) Risk of clot highest Bone marrow functions well and tries to keep up (high reticulocyte count) May or may not need red cell transfusions Treatments Folic acid 3-5 mg per day Iron supplements Prophylactic coumadin? Transfusions as needed Eculizumab Bone marrow transplant Prednisone Treatment Options

32. PNH/Aplastic Anemia In addition to anemia, have low platelet count and/or low white count Bone marrow production is deficient Clone size may be large or small Clotting risk may be less (still increased compared to baseline) Treatments Immunosuppression (cyclosporine, ATG) Danazol (marrow stimulator) Folic acid and iron less important, but need to make sure adequate Erythropoietin supplementation Bone marrow transplant Prednisone Eculizumab (?) Treatment Options

33. Where is the field moving? • Many research questions still to be answered: • Why do PNH cells survive immune mediated insults better? • Why clotting? • Why does the PNH clone expand? • Better treatments? • Improvement in supportive care and transplantation

34. Happy to answer questions.Thank you Gabrielle Meyers Email: meyersg@ohsu.edu