Paroxysmal Nocturnal Hemoglobinuria PNH

1. Paroxysmal Nocturnal Hemoglobinuria (PNH)

2. Paroxysmal Nocturnal Hemoglobinuria (PNH) History Epidemiology Clinical Features Relationship to Aplastic Anemia other diseases Pathogenesis Laboratory Diagnosis Therapy

3. History

4. 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

5. Clinical Features Major symptoms (Hemolysis, Cytopenia, and tendency to thrombosis) chronic hemolysis with acute exacerbations (hallmark) most patient at some stage only 1/3 exhibit hemolysis at diagnosis Recurrent attacks of intravascular hemolysis are usually associated with; hemoglobinuria abdominal pain dysphagia

6. cytopenia (varying severity) isolated subclinical thrombocytopenia classical severe aplastic anemia tendency to thrombosis venous thrombosis (40%) of patients, main cause of morbidity Variable expression of above often causes considerable delay in the diagnosis Major cause of death venous thrombosis complications from progressive pancytopenia Clinical Features

7. 25% of PNH patients survive >25 years - one half of these go on to spontaneous remission Remission patients hematological values revert to normal no PHN rbcs or granulocytes detected PNH lymphocytes - still detected but no clinical consequence Higher incidence of acute leukemia (6%) �preleukemic condition� most likely bone marrow failure not PNH Clinical Features - Long term

8. AA described as pancytopenia with nonfunctioning bone marrow. Cytopenia in one or all cell lineages also common to PNH High percentage of patients with AA develop clinical PNH or have lab evidence of PNH abnormality at some point (52%) Supports the theory that bone marrow failure supports the abnormal PNH cells - more later Clinical Features - Relationship to aplastic anemia (AA)

9. 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

10. 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

11. PNH blood cells deficient in GPI anchor lack membrane proteins linked via the anchor Membrane proteins w/o anchor degraded in ER 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 - Schistosoma mansoni In Humans signal transduction, co-receptors advantage to this type of anchor? Pathogenesis - The DefectGPI Anchor deficiency

12. Proteins anchored by GPI Anchorand

15. 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

17. 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 PNH hematopoitic cells perferentially engraft SCID mice compared to phenotypically hematopoitic cells Close association with AA - PNH hematopoitic cells 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???)

18. 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

19. 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

20. PNH Diagnosis by Flow Cytometry

21. 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

22. PNH Diagnosis by Flow Cytometry

23. PNH Diagnosis by Flow Cytometry

24. 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 thecirculating clone can provide additional prognosticinformation. Theoretically - should be very valuable PNH Diagnosis by Flow Cytometry

25. Therapy Bone Marrow Transplantation Only curative treatment chronic condition (possiblity of spontaneous remission) - BMT should be avoided Immunosuppressive therapy Antilymphocyte globulin &/or cyclosporine A Does not alter proportion of PNH hemopoiesis Steroids - experimental - controlled studies ?? Growth Factors Some improvement no evidence that normal clones respond better than PNH clones