1. Paroxysmal Nocturnal Hemoglobinuria New ideas about an old disease Ahmad Shihada Silmi Msc, FIBMS Staff Specialist in Hematology Medical Technology Department Islamic University of Gaza

2. Objectives Try to answer some of the frequently asked questions about: Try to answer some of the frequently asked questions about: The cause of the PNH The cause of the PNH The clinical presentation of PNH The clinical presentation of PNH Diagnosing PNH Diagnosing PNH The complications of PNH The complications of PNH New treatments for PNH New treatments for PNH

3. What is PNH? A disorder of blood affecting all the cells which come the bone marrow. A disorder of blood affecting all the cells which come the bone marrow. Prevalence 1-4 per million The disease is quite rare, only 10, 000 patients in the US and Europe. The disease is quite rare, only 10, 000 patients in the US and Europe. There is no ethnic preference for the disorder. There is no ethnic preference for the disorder. It may present early or late in life. It may present early or late in life. The manifestations may be “classic” or obscure. The manifestations may be “classic” or obscure.

4. PNH Prognosis Significant morbidity and mortality Patients have lived for extended periods of time and have seen spontaneous recovery Report of 80 patients showed that 60% of deaths due to venous thrombosis or bleeding Retrospective study of 220 patients Kaplan-Meier survival 78, 65, and 48% at 5, 10, and 15 years 8 year rates of major complications of PNH (pancytopenia, thrombosis, MDS) 15, 28, and 5% Up to 5% of patients can develop acute leukemia; onset about 5 years after Dx of PNH Likely incident of leukemia lower than 5%

5. What is PNH Mutation? PNH is due to a mutation in a gene in a blood stem cell. PNH is due to a mutation in a gene in a blood stem cell. The gene is called the PIG-A gene and is located on the X chromosome. The gene is called the PIG-A gene (phosphatidylinositol glycan complementation group A) and is located on the X chromosome. >100 mutations in PIG - A gene known in PNH The mutations (mostly deletions or insertions) generally result in stop codons - yielding truncated proteins which may be non or partially functional - explains heterogeneity seen in PNH

6. What is PNH? In most cases of PNH, the change in the gene (mutation) is acquired, not something you are born with. When and why is unknown. In most cases of PNH, the change in the gene (mutation) is acquired, not something you are born with. When and why is unknown. The gene contains the genetic information for the GPI anchors which link proteins to the cell membrane The gene contains the genetic information for the GPI anchors which link proteins to the cell membrane

7. What is PNH? A mutation is a “mistake” or a “change” in the gene that arises during copying and is not corrected A mutation is a “mistake” or a “change” in the gene that arises during copying and is not corrected When the cell divides, the mutation is transmitted to daughter cells When the cell divides, the mutation is transmitted to daughter cells The effect of a mutation: The effect of a mutation: None None An altered protein (sickle hemoglobin) An altered protein (sickle hemoglobin) No protein is produced as in PNH, hemophilia etc No protein is produced as in PNH, hemophilia etc

8. Stem Cells Embryonic Stem Cell Somatic Stem Cells BloodMuscleNerveEtc. Egg or Sperm EggEgg Egg Sperm The Cells of Each Specific Organ

9. Stem Cells Embryonic Stem Cell Somatic Stem Cells BloodMuscleNerveEtc. Egg or Sperm EggEgg Egg Sperm The Cells of Each Specific Organ

10. Stem Cells Embryonic Stem Cell Somatic Stem Cells BloodMuscleNerveEtc. Egg or Sperm EggEgg Egg Sperm The Cells of Each Specific Organ

11. Stem Cells STEM CELL T LYMPHOCYTES B LYMPHOCYTES ERYTHROCYTES GRANULOCYTES MONOCYTES PLATELETS

12. Stem Cells STEM CELL T LYMPHOCYTES B LYMPHOCYTES ERYTHROCYTES GRANULOCYTES MONOCYTES PLATELETS ALTERED GENE

13. Stem Cells ABNORMAL CLONE NORMALCLONES

14. GPI-AP Biosynthesis: Involves 10 Steps and >20 Genes N N N PIG-A Endoplasmic Reticulum Plasma Membrane Raft GlcNAc-PI PI

15. The Beginning of PNH The change that occurs in PNH stops the production of an anchor that ties protein molecules to the cell The change that occurs in PNH stops the production of an anchor that ties protein molecules to the cell Sometimes the stop is only partial and PNH II cells occur Sometimes the stop is only partial and PNH II cells occur

16. The GPI Anchor Defect in PNH 22 CH C=O O O O-P-O O O (18:0,1 ) (22:4,5) PNH

17. PIG - A gene codes for 60 kDa protein glycosyltransferase which effects the first step in the synthesis of the glycolipid GPI anchor (glycosylphosphatidylinositol). Results in clones lacking GPI anchor - in turn, attached proteins Pathogenesis - The Defect GPI Anchor PIG - A protein

18. What is PNH? As a result of the PIG-A mutation, there is little of no GPI anchor produced. As a result of the PIG-A mutation, there is little of no GPI anchor produced. PNH II cells- mild reduction PNH II cells- mild reduction PNH III cells- severely reduced. PNH III cells- severely reduced. When the anchor is reduced, certain proteins can’t attach to the cells. When the anchor is reduced, certain proteins can’t attach to the cells. The most important proteins for PNH are CD 59, CD55. The most important proteins for PNH are CD 59, CD55.

19. Proteins anchored by GPI Anchor and Surface Proteins Missing on PNH Blood Cells Antigen Expression Pattern Enzymes Acetylcholinesterase (AchE) Red blood cells Ecto-5'-nucleotidase (CD73) Some B- and T-lymphocytes Neutrophil alkaline phosphatase(NAP) Neutrophils ADP-rybosyl transferase Some T-lymphs, Neutrophils Adhesion molecules Blast-I/CD48 Lymphocytes Lymphocyte function- associated antigen-3(LFA-3 or CD58) All blood cells CD66bNeutrophils Complement regulating surface proteins Decay accelerating factor (DAF or CD55) All blood cells Homologous restriction factor, Membrance inhibitor of reactive lysis All blood cells (MIRL or CD59)

20. Surface Proteins Missing on PNH Blood Cells Antigen Expression Pattern Receptors Fc-  receptor III (Fc  Rlll or CD16) Neutrophils, NK-cells, macrophages, some T-lymphocytes Monocyte differentiation antigen Monocytes, macrophages (CD14) Urokinase-type Plasminogen Monocytes, granulocytes Activator Receptor (u-PAR, CD87) Blood group antigens Comer antigens (DAF) Red blood cells Yt antigens (AchE) Red blood cells Holley Gregory antigen Red blood cells John Milton Hagen antigen (JMH) Red blood cells, lymphocytes Dombrock reside Red blood cells Neutrophil antigens NB1/NB2 Neutrophils

21. Surface Proteins Missing on PNH Blood Cells Antigen Expression Pattern Other surface proteins of unknown functions CAMPATH-1 antigen (CDw52) Lymphocytes, monocytes CD24 B-lymphocytes, Neutrophils, eosinophils p5O-80Neutrophils GP500 Platelets GPI75 Platelets

22. Pathogenesis CD 55 Also known as decay accelerating factor Accelerates decay of enzyme that destroys red cell membranes CD55 inhibits the formation or destabilizes complement C3 convertase (C4bC2a) When GPI anchor absent, CD55 not available and RBC membranes hemolyze

23. Pathogenesis CD59 Also known as membrane inhibitor of reactive lysis, protectin, homologous restriction factor, and membrane attack complex inhibitory factor CD59 Protects the membrane from attack by the C5-C9 complex Absence or reduction of CD59 leads to increased hemolysis and perhaps thrombosis

24. Pathogenesis of CD55 & CD59 Inherited absences of both proteins in humans have been described Most inherited deficiencies of CD55 - no distinct clinical hemolytic syndrome Inherited absence of CD59 - produces a clinical disease similar to PNH with hemolysis and recurrent thrombotic events

25. Pathogenesis Many normal people have very small numbers (perhaps 6 per 1,000,000 bone marrow cells) Many normal people have very small numbers (perhaps 6 per 1,000,000 bone marrow cells) In PNH, the abnormal cells have an advantage and become a major population in the marrow and blood (anywhere from 1% to over 90%) In PNH, the abnormal cells have an advantage and become a major population in the marrow and blood (anywhere from 1% to over 90%) This may be a result of change in the immune system –inability to recognize something foreign. This may be a result of change in the immune system –inability to recognize something foreign. Or it may be related to aplastic anemia, a disease of poor production of blood from the marrow- WHY? Or it may be related to aplastic anemia, a disease of poor production of blood from the marrow- WHY?

26. Pathogenesis - Clonal evolution and cellular selection Expansion of abnormal hematopoietic stem cell required for PNH disease expression Theories for expansion Blood cells lacking GPI-linked proteins have intrinsic ability to grow abnormally fast In vitro growth studies demonstrate that there are no differences in growth between normal progenitors and PNH phenotype progenitors In vivo - mice deficient for PIG -A gene also demonstrates no growth advantage to repopulation of BM. Additional environmental factors exert selective pressure in favor of expansion of GPI anchor deficient blood cells Close association with AA - PNH hematopoitic cells cells may be more resistant to the Immune System than normal hematopoitic cells. Evidence in AA is that the decrease in hematopoitic cells is due to increased apoptosis via cytotoxic T cells by direct cell contact or cytokines (escape via deficiency in GPI linked protein???)

27. PNH Clinical Features Aplastic Anemia Some PNH patients have aplastic anemia or a history of aplastic anemia Some PNH patients have aplastic anemia or a history of aplastic anemia Many PNH patients have evidence of a bone marrow that doesn’t work well or well enough to maintain normal blood counts Many PNH patients have evidence of a bone marrow that doesn’t work well or well enough to maintain normal blood counts Therefore, whatever causes aplastic anemia (immune suppression or dysregulation or damage to the stem cells) may allow PNH to develop Therefore, whatever causes aplastic anemia (immune suppression or dysregulation or damage to the stem cells) may allow PNH to develop

28. What is PNH? Complement Complement is a group of blood proteins that act together to help the body get rid of microbiological invaders Complement is a group of blood proteins that act together to help the body get rid of microbiological invaders One of the ways it does this is by penetrating the membrane (outside surface) of the invading bacteria or viruses. One of the ways it does this is by penetrating the membrane (outside surface) of the invading bacteria or viruses. When this happens to PNH blood cells, the cells are destroyed. When this happens to PNH blood cells, the cells are destroyed.

29. What is PNH? Complement circulates in an inactive form It is activated spontaneously and by a variety of events It is activated spontaneously and by a variety of events It is normally activated more at night It is normally activated more at night It is more active with infections, trauma, vaccinations, surgery, immune complexes, autoimmune diseases It is more active with infections, trauma, vaccinations, surgery, immune complexes, autoimmune diseases

30. What is PNH? Complement Complement activity is regulated by proteins in the blood and on the membranes of the cell. Complement activity is regulated by proteins in the blood and on the membranes of the cell. Proteins on the cell surface interfere with complement to prevent breakdown (lysis) of the cell membrane Proteins on the cell surface interfere with complement to prevent breakdown (lysis) of the cell membrane The most important of these is CD59, which is missing on the abnormal cells of PNH The most important of these is CD59, which is missing on the abnormal cells of PNH For this reason, PNH red cells are extremely sensitive to very small amounts of activated complement For this reason, PNH red cells are extremely sensitive to very small amounts of activated complement

31. C5b C5 C5a Adapted from Cellular and Molecular Immunology AK Abbas, AH Litchman and JS Pober, 3 rd Edition. 1991 WB Saunders; Philadelphia. C7 C8 C5b C7 C6 C7 C6 C5b,6,7 C8 C5b C6 C5b-8 C9 C7 C8 C5b C6 C9 C7 C8 C5b C6 C9 x 12 - 15 C5b-9 CD59 XX C5 convertase C5 convertase Absence of CD59 Allows Terminal Complement Complex Formation C3b Bb + C3

34. What is PNH? Complement successfully attacks the red cells and they break up (hemolysis) Complement successfully attacks the red cells and they break up (hemolysis) This releases hemoglobin (the red pigment in red cells) into the plasma This releases hemoglobin (the red pigment in red cells) into the plasma Causes anemia Causes anemia Pieces of the membrane come off. Pieces of the membrane come off. The white cells release granule contents and change to express other proteins. The white cells release granule contents and change to express other proteins. The platelets form vesicles (membrane blisters) and activate. The platelets form vesicles (membrane blisters) and activate.

35. What is PNH? Normal red blood cells are protected from complement attack by a shield of terminal complement inhibitors Complement activation P P Without this protective complement inhibitor shield, PNH red blood cells are destroyed

36. What is PNH? Clinical Features Some of the hemoglobin passes through the kidneys and into the urine, causing red to dark brown urine (hemoglobinuria) Some of the hemoglobin passes through the kidneys and into the urine, causing red to dark brown urine (hemoglobinuria) This causes a loss of iron from the body This causes a loss of iron from the body In the long run, this may damage the kidney In the long run, this may damage the kidney Free hemoglobin binds nitric oxide causing vascular and smooth muscle spasm Free hemoglobin binds nitric oxide causing vascular and smooth muscle spasm Causes inflammation Causes inflammation

37. Action of Nitric Oxide (NO) Smooth Muscle Contraction Smooth Muscle Relaxation NO

38. Free Hemoglobin Binds NO Smooth Muscle Contraction Smooth Muscle Relaxation NO Free Hemoglobin

39. What Are The Effects of Nitric Oxide Trapping by Hemoglobin Spasm of the esophagus Spasm of the esophagus Abdominal pain Abdominal pain Erectile dysfunction Erectile dysfunction Other symptoms such as “fatigue” Other symptoms such as “fatigue”

40. What is PNH Clinical aspects Vascular (arterial constriction, HBP) Vascular (arterial constriction, HBP) Pulmonary artery pressure increase (PHTN) Pulmonary artery pressure increase (PHTN) Spasm of the esophagus Spasm of the esophagus Abdominal pain Abdominal pain Erectile dysfunction Erectile dysfunction Other symptoms such as “fatigue” Other symptoms such as “fatigue” Platelets are more “reactive” Platelets are more “reactive”

41. What is PNH? Clinical Features WBC: WBC: Granulocytes - release content stimulating inflammation Granulocytes - release content stimulating inflammation Monocytes - activate expressing TF which leads to blood clots. TF-Microvesicles Monocytes - activate expressing TF which leads to blood clots. TF-Microvesicles Platelets become “activated” Platelets become “activated” They stick together and form clumps They stick together and form clumps The membrane changes, allowing them to bind to monocytes The membrane changes, allowing them to bind to monocytes Pieces of the membrane come off (microvesicles) Pieces of the membrane come off (microvesicles)

43. What is PNH? Clinical Features Hemolytic anemia due to complement activation Hemolytic anemia due to complement activation Hemoglobinuria and, eventually, kidney damage Hemoglobinuria and, eventually, kidney damage Anemia to a variable degree Anemia to a variable degree Effects of NO depletion- HBP, smooth muscle dystonia, reduced blood flow to the kidney and lungs Effects of NO depletion- HBP, smooth muscle dystonia, reduced blood flow to the kidney and lungs Impaired bone marrow function Impaired bone marrow function

44. What is PNH? Clinical Features  Thrombosis (Blood clots)  Often in unusual places (liver veins, abdominal veins, cerebral veins, dermal veins)  Fatigue – overwhelming, poor correlation to level of hemoglobin  inflammation  anemia  Portal hypertensionPHTN).  Portal hypertension ( PHTN).

45. MONOCYTES PLATELET Endothelial cells C5b-9 HEMOLYSIS COMPLEMENT INJURY TF THROMBIN GENERATION COMPLEMENT Thrombosis in PNH Pathophysiology Thrombosis in PNH Pathophysiology TF = Tissue factor. NO PS CYTOKINES IL-6 INFLAMMATION C5a

46. Laboratory Evaluation of PNH Acidified Serum Test (Ham Test 1939) Acidified serum activates alternative complement pathway resulting in lysis of patient’s rbcs May be positive in congenitial dyserythropoietic anemia Still in use today Sucrose Hemolysis Test (1970) 10% sucrose provides low ionic strength which promotes complement binding resulting in lysis of patient’s rbcs May be positive in megaloblastic anemia, autoimmune hemolytic anemia, others Less specific than Ham test

47. PNH Diagnosis by Flow Cytometry (1986) Considered method of choice for diagnosis of PNH (1996) Detects actual PNH clones lacking GPI anchored proteins More sensitive and specific than Ham and sucrose hemolysis test Laboratory Evaluation of PNH

48. PNH Diagnosis by Flow Cytometry AntigenCell LineageFunction CD14monocytesLPS receptor, MDF CD16neutrophilsFc  III receptor CD24neutrophilsB-cell differentiation marker CD55all lineagesDAF CD58all lineagespossible adhesion CD59all lineagesMIRL, HRF, protectin CD66bneutrophilsCEA-related glycoprotein Of the long list of GPI anchored protein, monoclonal antibodies to the following antigens have been used in the diagnosis of PNH The most useful Abs are to CD14, 16, 55, 59, and 66. Are all required? Probably not - more studies needed

49. Antigen expression is generally categorized into three antigen density groups type I Normal Ag expression type II Intermediate Ag expression type III No Ag expression Patient samples that demonstrate cell populations with diminished or absent GPI-linked proteins (Type II or III cells) with multiple antibodies are considered to be consistent with PNH. Should examine multiple lineages (ie granulocytes & monocytes) PNH Diagnosis by Flow Cytometry

50. Examples of variable GPI linked CD59 expression on granulocytes on four PNH patients

51. PNH Diagnosis by Flow Cytometry Example of variable expression of several GPI linked Ags on several lineages From Purdue Cytometry CD-ROM vol3 97

52. Flow Cytometry is method of choice but only supportive for/against diagnosis More studies are needed to better define whether the type (I, II, or III), cell lineage, and size of the circulating clone can provide additional prognostic information. Theoretically - should be very valuable PNH Diagnosis by Flow Cytometry

53. Historical Management Options for PNH Transfusions Transfusions Anticoagulants Anticoagulants Supplements Supplements Folic acid Folic acid Iron Iron Erythropoiesis stimulating agents Erythropoiesis stimulating agents Steroids/androgen hormones Steroids/androgen hormones Allogeneic bone marrow transplant (limited eligibility ) Allogeneic bone marrow transplant (limited eligibility ) Generally conservative, supportive, and dependent on symptom severity 1,2

54. What is Soliris ® ? Monoclonal antibody (protein) that blocks complement at C5 preventing the formation of the terminal complement complex Monoclonal antibody (protein) that blocks complement at C5 preventing the formation of the terminal complement complex Quickly and markedly reduces hemolysis Quickly and markedly reduces hemolysis Stops hemoglobinuria Stops hemoglobinuria Increases hemoglobin level Increases hemoglobin level Reduces transfusions Reduces transfusions Hematocrit may not be quite normal Hematocrit may not be quite normal

55. SOLIRIS Blocks Terminal Complement LectinClassicalAlternative C3 C3a C3b C5 Proximal Terminal Microorganisms Antigen-Antibody Complexes Constitutive/ Microorganisms Figueroa, et al. Clin Microbiol Rev. 1991;4:359-395. Walport. N Engl J Med. 2001;344:1058. SOLIRIS™ (eculizumab) [package insert]. Alexion Pharmaceuticals; 2007. C5b-9 Cause of Hemolysis in PNH C5a C5b SOLIRIS Proximal functions of complement remain intact Weak anaphylatoxin Immune complex and apoptotic body clearance Microbial opsonization Terminal complement activity is blocked SOLIRIS binds with high affinity to C5

56. Reduction in LDH During Soliris ® Treatment in TRIUMPH and SHEPHERD Time, Weeks Lactate Dehydrogenase (U/L) TRIUMPH placebo patients switched to SOLIRIS after week 26. All TRIUMPH patients entered the long term extension study. 0 500 1000 1500 2000 2500 3000 01020304050 TRIUMPH – Placebo/extension TRIUMPH – SOLIRIS/extension SHEPHERD – SOLIRIS PI: All patients sustained a reduction in intravascular hemolysis over a total SOLIRIS exposure time ranging from 10 to 54 months.

57. 4 47755885841181111111111111 Pre-eculizumab Post-eculizumab Urine score 2 weeks before & after Eculizumab 1

58. Effect of Soliris ® on Ability to Maintain a Good Hemoglobin P<0.000000001 SOLIRISPlacebo Hemoglobin Stabilization (%) Hemoglobin Stabilization (%) 0 10 20 30 40 50

59. Effect of Soliris® on Transfusion in PNH P<0.0000001 Transfused Units/Patient (median) Transfused Units/Patient (median) Soliris ® Placebo 0 2 4 6 8 10

60. What is the effect of Soliris ® in PNH Stops the symptoms associated with hemolysis Stops the symptoms associated with hemolysis “Fatigue” “Fatigue” Esophageal and abdominal spasm Esophageal and abdominal spasm Erectile dysfunction Erectile dysfunction Improves sense of well being Improves sense of well being Reduced the need for transfusion Reduced the need for transfusion Appears to reduce thrombosis (blood clots) Appears to reduce thrombosis (blood clots)

61. Effect of Eculizumab on the blood clots in PNH Equalized Patient Years (195) Patients on Antithrombotics n=103 (P < 0.000000001 91% reduction in TE event rate with Eculizumab 92% reduction in TE events with Eculizumab Pre-Eculizumab Post-Eculizumab Pre-EculizumabPost-Eculizumab

62. What is the Effect of Soliris ® ? Improves kidney function reduced hemoglobinuria and iron deposition reduced hemoglobinuria and iron deposition Reduced thrombosis Reduced thrombosis Improves hypertension May in part be due to availability of nitric oxide

63. Side Effects of Soliris ® Treatment Susceptibility to sepsis by meningococcal organism Susceptibility to sepsis by meningococcal organism All patients must be vaccinated at least 2 weeks before starting Soliris All patients must be vaccinated at least 2 weeks before starting Soliris All patients must know to seek medical help at once when fever happens All patients must know to seek medical help at once when fever happens All patients must carry cards describing this complication All patients must carry cards describing this complication Headache – first week or 2 Headache – first week or 2 Cost Cost Inconvenience Inconvenience Must be given every 12-14 days by vein Must be given every 12-14 days by vein

64. What Soliris ® Cannot Do Does not appear to improve impaired bone marrow function Does not appear to improve impaired bone marrow function Low white count or low platelet count may persist in some patients, especially if it is due to aplastic anemia Low white count or low platelet count may persist in some patients, especially if it is due to aplastic anemia Other treatments may be indicated Other treatments may be indicated Bone marrow transplantation Bone marrow transplantation immunosuppressives immunosuppressives

65. When is Soliris Ineffective or Less Effective  Patient has been incorrectly diagnosed with PNH.  Patient has a very small PNH clone (less than 10%) bone marrow failure- AA bone marrow failure- AA  Breakthrough – infections, increased clearance, delayed dosing  Extravascular hemolysis

66. Which PNH Patients are Candidates for Soliris? Patients with hemolysis (LDH ) Patients with hemolysis (LDH ) Patients with large CD 59 deficient clones- RBC, WBC Patients with large CD 59 deficient clones- RBC, WBC Patients with hemolysis with evidence of renal impairement GRF<90, proteinuria etc Patients with hemolysis with evidence of renal impairement GRF<90, proteinuria etc Patients with history of thrombosis Patients with history of thrombosis Patients with hemolysis and elevated Ddimers Patients with hemolysis and elevated Ddimers Patients with hemolysis with fatigue Patients with hemolysis with fatigue Patients with hemolysis and transfusion dependence Patients with hemolysis and transfusion dependence Patients prior to ?BMT, surgery, ???pregnancy Patients prior to ?BMT, surgery, ???pregnancy

67. Who Should Get Soliris ® ? In all cases, the decision should be made by a physician that understand PNH, its complications and its treatment in conjunction with the patient, whose life is affected by the disorder.