Paroxysmal Nocturnal Hemoglobinuria

2. Paroxysmal Nocturnal Hemoglobinuria BY Dr. HAYAM FATEHY ABDELHAY GHAZY Lecturer of medical oncology Mansoura University OCMU

3. Hemoglobinuria Alpha-Beta dimers MW 34,000 -- small enough to be filtered by glomerulus

4. Heamoglobinuria • Hemoglobinuria is a condition in which the oxygen transport protein hemoglobin is found in high concentrations in the urine. • The condition is often associated with hemolytic anemia, in which red blood cells are destroyed, thereby increasing levels of free plasma hemoglobin. • The excess hemoglobin is filtered by the kidneys, which release it into the urine, giving urine a red colour.

5. Classification of Hemolytic anemias I. Red cell abnormality (Intracorpuscular factors)A. Hereditary 1. Membrane defect (spherocytosis, elliptocytosis) 2. Metabolic defect (Glucoze-6-Phosphate-Dehydrogenaze (G6PD) deficiency, Pyruvate kinase (PK) deficiency) 3. Hemoglobinopathies (unstable hemoglobins, thalassemias, sickle cell anemia )B. Acquired1. Membrane abnormality-paroxysmal nocturnal hemoglobinuria (PNH)

6. II. Extracorpuscular factorsA. Immune hemolytic anemias 1. Autoimmune hemolytic anemia - caused by warm-reactive antibodies - caused by cold-reactive antibodies 2. Transfusion of incompatible blood B. Nonimmune hemolytic anemias 1. Chemicals 2. Bacterial infections, parasitic infections (malaria), venons 3. Hemolysis due to physical trauma - hemolytic - uremic syndrome (HUS) - thrombotic thrombocytopenic purpura (TTP) - prosthetic heart valves 4. Hypersplenism

7. G6PD deficiency Hexose monophosphate shunt Most common RBC enzyme defect, >50 variants X-linked Low glutathione due to low NADPH Oxidative lysis, Heinz bodies, spherocytic Primaquine, fava beans Pyruvate kinase deficiency Glycolysis Low RBC ATP level Non-spherocytic B12 and folate deficiency Macrocytic HJ bodies Hemoglobinopathies Poikilocytosis Abnormal Hb Metabolic Defect

8. Antibodies Autoimmune Isoimmune Drugs, antibiotics Fresh water Abnormal plasma lipids Acanthocytosis Venom Snake Spider Bee Extracorpuscular Factors

9. Trauma DIC Hemolytic uremic syndrome (HUS) TTP Angiopathy Heat Heart valves “March” hemoglobinuira Microorganisms Malaria Babesia Clostridium Gram negative endotoxin Extracorpuscular Factors

10. Mechanisms of hemolysis:- intravascular - extravascular

11. Intravascular hemolysis :- red cells destruction occurs in vascular space - clinical states associated with Intravascular hemolysis: acute hemolytic transfusion reactions severe and extensive burns paroxysmal nocturnal hemoglobinuria severe microangiopathic hemolysis physical trauma bacterial infections and parasitic infections (sepsis)

13. Intravascular hemolysis :- laboratory signs of intravascular hemolysis: indirect hyperbilirubinemia erythroid hyperplasia hemoglobinemia methemoalbuminemia hemoglobinuria absence or reduced of free serum haptoglobin hemosiderynuria

14. Extravascular hemolysis :- red cells destruction occurs in reticuloendothelial system - clinical states associated with extravascular hemolysis : autoimmune hemolysis delayed hemolytic transfusion reactions hemoglobinopathies hereditary spherocytosis hypersplenism hemolysis with liver disease- laboratory signs of extravascular hemolysis: indirect hyperbilirubinemia increased excretion of bilirubin by bile erythroid hyperplasia hemosiderosis

16. Paroxysmal Nocturnal Hemoglobinuria

17. Paroxysmal Nocturnal Hemoglobinuria (PNH) PNH is an acquired chronic hemolytic anemia which arises from a somatic mutation in a hematopoietic stem cell. Most hematopoitic cell lines may be affected by the intrinsic membrane defect. This defect renders the red cells highly susceptible to complement mediated lysis resulting in the characteristic hemolysis.

18. 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.

19. History Investigator Year Contribution Gull 1866 Described nocturnal and paroxysmal nature of “intermittent haematinuria” in a young tanner. Strubing 1882 Distinguished PNH from paroxysmal cold haemoglobinuria and march haemoglobinuria. Attributed the problem to the red cells. van den Burgh 1911 Red cells lysed in acidified serum. Suggested a role for complement. Enneking 1928 Coined the name “paroxysmal nocturnal haemoglobinuria”. Marchiafava 1928- Described perpetual hemosiderinemia in absence of and Micheli 1931 hemolysis. Their names became eponymous for PNH in Europe. Ham 1937- Identified the role of complement in lysis of PNH red 1939 cells. Developed the acidified serum test, also called the Ham test, which is still used to diagnose PNH. Demonstrated that only a portion of PNH red cells are abnormally sensitive to complement. Davitz 1986 Suggests defect in membrane protein anchoring system responsible Hall & Rosse 1996 Flow cytometry for the diagnosis of PNH

20. Natural History of PNH 1. Hillmen P, Lewis SM, Bessler M et al. New England Journal of Medicine 1995;333:1253-8 2. Socie G, Mary JY, Gramont A et al. Lancet 1996;348:573-7 3. Peffault de Latour R, Mary JY, Salanoubat C et al. Blood 2008; Jun 5 4. Nishimura J, Kanakura Y, Ware RE et al. Medicine 2004;83:193-207

21. Epidemiology • Rare disease - • frequency unknown • thought to be on the same order as aplastic anemia (2-6 per million) • Median age at diagnosis • ~ 35 yrs • PNH reported at extremes of age • Female:Male ratio = 1.2:1.0 • No increased risk of PNH in patient relatives • Median Survival after diagnosis ~ 10-15 yrs

22. Paroxysmal Nocturnal Hemoglobinuria • 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.

23. 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

24. 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.

25. PNH is a disease of chronic hemolysis During chronic hemolysis, excess free hemoglobin depletes plasma Nitric Oxide (NO), which may play an important role in normal platelet function. It is believed that NO may down regulate platelet aggregation, adhesion, and regulating molecules in the coagulation cascade. Therefore, NO depletion may lead to platelet activation and aggregation.

26. Consequences of Hemolysis • Even in the absence of symptoms, hemolysis is ongoing and destructive7

27. Pathogenesis - The Defect • Defect - Somatic mutation of PIG-A gene (phosphatidylinositol glycan complementation group A) located on the X chromosome in a clone of a hematopoietic stem cell • >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

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

29. Pathogenesis - The Defect GPI Anchor • 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 PIG - A protein

30. Pathogenesis - The DefectGPI Anchor deficiency • PNH blood cells deficient in GPI anchor lack membrane proteins linked via the anchor • Severity & size of deficiency - variable - clinical/diagnostic implications • GPI anchor highly conserved in all eukaryotic cells • Variant surface proteins of Trypanosomes - GPI linked • Shed by cleavage of GPI anchor - immune system avoid • Swapping GPI linked proteins - CD55 complement resistance - Schistosomamansoni • In Humans • signal transduction, co-receptors • advantage to this type of anchor?

31. Proteins anchored by GPI Anchorand 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 CD66b Neutrophils 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)

32. 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

33. 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-80 Neutrophils GP500 Platelets GPI75 Platelets

34. Proteins Deficient from PNH Blood Cells CD55 CD58 CD59 PrPC AChE JMH Ag Dombroch HG Ag CD24CD55 CD58 CD59 CD48 PrPC CD73 CD108 B cells Haematopoietic Stem Cell T cells RBC CD55 CD58* CD59 CD48 CD52 CD87 CD108 PrPc ADP-RT CD73 CD90 CD109 CD16* CD55 CD58 CD59 CD109 PrPC GP500 Gova/b CD59, CD90, CD109 Platelets NK cells CD55 CD58* CD59 CD14 CD16 CD24 CD48 CD66b CD66c CD87 CD109 CD157 LAPNB1 PrPC p50-80 GPI-80 ADP-RT NA1/NA2 CD55 CD58 CD59 CD48 CD52 PrPc CD16 Monocytes PMN CD14 CD55 CD58* CD59 CD48 CD52 CD87 CD109 CD157 Group 8 PrPC GPI-80 CD16 (Courtesy of Lucio Luzzatto)

35. Pathogenesis - Functional consequences of lack of GPI linked proteins • In vivo function of many of these membrane proteins not fully understood • However, CD55 and CD59 functions are well known • CD55 (decay accelerating factor) inhibits the formation or destabilizes complement C3 convertase (C4bC2a) • CD59 (membrane inhibitor of reactive lysis, protectin, homologous restriction factor) Protects the membrane from attack by the C5-C9 complex • 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

36. Mechanism for hemolysis in PNH via lack of CD59 (CD59) (CD59)

37. 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.

38. Additional environmental factors exert selective pressure in favor of expansion of GPI anchor deficient blood cells • PNH hematopoitic cells perferentially engraft SCID mice • compared to phenotypically hematopoiticcells • Close association with AA - PNH hematopoitic cells may be more resistant to the IS 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???)

40. 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).

41. 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.

42. 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.

43. GPI-linked antigen Immune attack via GPI-linked antigen (aplastic anaemia) Relative Growth Advantage in PNH Normal stem cells GPI-deficient (PNH) stem cells

44. Intense growth factor driven expansion Relative Growth Advantage in PNH

45. Relative Growth Advantage in PNH

46. 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.

47. 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

49. 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

50. Chronic hemolysis is the underlying cause of progressive morbidities in PNH.9