Junfeng Wang 12/10/2010

Transcription

1 Paroxysmal Nocturnal Hemoglobinuria. Junfeng Wang 12/10/2010

2 31 Year Old Female with 11-year History of PNH In 1989, she presented with bleeding & pancytopenia. BM x2 showed AA. Cytogenetic ti was normal. No responses to treatment with ATG, ATG/CSA and Deca-Durabolin. She then quickly became transfusion-dependent. WBC 2.5, HCT 22%, PLT 20K, Retic 1.8%. In 1991, PNH was diagnosed d based on (+) Ham s test. t In 1992, resolution of leukopenia and thrombocytopenia was recorded. In 1993, WBC 3.5, HCT24.9%, PLT 75K, Retic 13.7%, LDH Repeat BM showed erythroid hyperplasia. With time, she developed typical PNH crises, i.e. wine-colored urine, abdominal pain, and nausea/vomiting. She also had SVC syndrome attributed to line-associated thrombosis, and was started on anticoagulation. The worst crisis was in 1994, triggered by Pneumovax, complicated by hypotension, ARF, ARDS and seizure. Cr was 8.1. She was hospitalized for 5 months. Then, she had multiple attacks of ischemic duodenum & proximal jejunum confirmed by EGD with biopsy. Multiple hospitalizations and prolonged TPN required. The most severe occurred on 06/14/07 and complicated by ARF. 06/19/2007, she was started on eculizumab. Now self-limited hemolytic events requiring RBC transfusion about every 3-4 months. No recurrence of ischemic bowel since. On LMWH and ASA. 2010: WBC 2.53, HCT 31%, PLT 198K, Retic 9%

3 Notable Characteristics of the This Case Her classic clinical PNH symptoms are preceded by 2-3 years of history of AA which seems to have resolved spontaneously She has had recurrent GI symptoms, including abdominal pain with nausea and vomiting She has had multiple l thrombotic ti events including SVC syndrome She has had recurrent history of ischemic small intestine, mainly involving duodenum and proximal jejunum She has had good but not complete responses to eculizumab She still has residual hemolysis based on LDH level, requiring blood transfusion about once every 3-4 months

4 Structure and Biosynthetic Pathway of Mammalian GPI-APs Kinoshita et al. J. Biochem 2008,144:287

5 Proteins Involved in Biosynthesis of GPI-APs Kinoshita et al. J. Biochem 2008,144:287

6 Spectrum of Mutations in PIG-A Gene in PNH Luzzatto et al. Cell 1997,88:1

7 GPI-Anchored Proteins Missing on PNH Blood Cells 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 LFA 3 or CD58 All blood cells CD66b Neutrophils Complement regulating Decay accelerating factor (DAF or CD55) All blood cells surface proteins MIRL, or CD59 All blood cells Receptors Fc g receptor III (Fc g Rlll or CD16) Neutrophils, NK cells, macrophages, some T lymphocytes Monocyte differentiation antigen (CD14) Monocytes, macrophages u PAR, CD87 Monocytes, granulocytes Blood group antigens Comer antigens (DAF) Rdbl 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 Others NB1/NB2 Neutrophils CAMPATH 1 antigen (CDw52) Lymphocytes, monocytes CD24 B lymphocytes, Neutrophils, eosinophils p5o 80 Neutrophils GP500 Plateletsl GPI75 Platelets

8 Wagner et al. Nat Review Drug Disc 2010

9 Mechanism of Intravascular Hemolysis in PNH Patients (CD59) (CD59)

10 Clonal Mosaicism of PNH Reflects Genotypic Mosaicism Phenotypic designation Complement sensitivity GPI AP expression by flow cytometry PNH I 1 Normal None Type of PIG A mutation PNH II 2 4 x Dim Positive Partial inactivation of PIG A PNH III x Negative Complete inactivation of PIG A Borowitz et al. Cytometry Part B 78b:211 (2010)

11 Clinical Indications for PNH Testing (I-PIG) Intravascular hemolysis as evidenced d by hemoglobinuria i or elevated plasma hemoglobin Evidence of unexplained hemolysis with accompanying: 1. Iron-deficiency deficiency, OR 2. Abdominal pain or esophageal spasm, OR 3. Granulocytopenia and/or thrombocytopenia Other acquired Coomb s-negative, non-schistocytic, non-infectious hemolytic anemia Thrombosis with unusual features: 1. Unusual sites: Hepatic veins ( Budd-Chiari syndrome), Other intra-abdominal veins (portal, splenic, splachnic) Cerebral sinuses Dermal veins 2. With unexplained cytopenia Evidence of bone marrow failure: 1. Suspected or proven aplastic or hypoplastic anemia 2. Refractory cytopenia with unilineage dysplasia 3. Other cytopenias of unknown etiology after adequate workup Borowitz et al. Cytometry Part B 78b:211 (2010)

12 Proportion of PNH Patients Preceded by AA History AA No. of Series preceded PNH % References PNH patients British Hillman et al NEJM 1995 French Socie et al. Lancet 1996 French 2008 update de Latour et al. Blood 2008 Duke series Nishimura et al. Medicine 2004 Japanese Nishimura et al. Medicine 2004 JHH Moyo et al. BJH 2004 Total

13 Proportion of AA Patients Who Develop Classic PNH Series No. of AA Classica Patients l PNH % References NHLBI Scheinbert et al. Haematologica 2010 Japan Sugimori et al. BJH 2009 Germany Frickhofen et al. Blood 2003 Brazil Medeiros et al ASH 52 Abs# 2234 Total

14 Emergence and Fate of PNH Cells in Bone Marrow Failure Sugimori et al. BJH 2009;147:102

15 EXPLORE (EXamination of PNH, by Level Of CD59 on REd and white blood cells) The first large multicenter study of PNH cells in AA, MDS, or other BMF syndromes. High-sensitivity flow cytometry with antibodies (CD59, CD24, and CD14) and FLAER. The primary endpoint was percentage of patients who had a PNH WBC clone >=1%. Interium Analysis: Patients Total 5,212 AA 413 (7.9%) MDS 4,500 (86.3%) Other BMF 356 (6.8%) 80.0% 70.0% Other BMF 7% Median PNH clone size 60.0% AA 11.1% MDS 16.3% 50.0% MDS 85% Other BMF 32.6% % of Patients with PNH clone >=1% AA 24.5% MDS 1.2% Other BMF 4.6% % of Patients with PNH clone >=0.01% AA 70% MDS 55% Other BMF 55% 40.0% 30.0% 20.0% 10.0% 0.0% >=1% >=0.01% Median clone size AA 8% AA MDS Other BMF N. Galili et al. JCO 2009, 27: 7082

16 Thrombosis in PNH Patients Series Pts with No. thrombosis Patients % References JHH Moyo et al. BLH 2004 TRIUMPH Hillmen et al NEJM 2006 London Hillmen et al NEJM 1995 Hillman s Lab Hall et al Blood 2003 French Socie et al. Lancet 1996 French 2008 update de LaTour et al. Blood 2008 Combined

17 Cumulative Incidence of Thrombosis in PNH Patients Soce et al. Lancet 1996;348:573 delatour et al. Blood 2008; 112:3099 Moyo et al. BJH 2004;126:133 Hall et al. HTVB 2003;103:3587

18 Thrombosis in PNH: Sites, Risks, Outcome n (%) Deaths RR*(CI) P-value Year Reported Before (14.4) (30.2) ( ) ns (55.7) ( ) 0.01 Venous Events Hepatic veins 147(40.7) 47 1 Deep Vein 54(14.9) 1 CNS vein 51(14.0) 17 Portal vein 37(10.2) 7 Pulmonary embolism 26(7.2) 10 Inferior vena cava 26(7.2) 7 Mesenteric veins 26(7.2) 10 CNS sinuses 25(6.9) 9 Splenic vein 21(5.8) 3 Renal vein 12(3.3) 5 Skin 2(0.6) - Arterial Events CNS arteries 18(4.9) ( ) ns Coronary arteries 12(3.31) 31) 7 hepatic artery 5(1.4) 1 Aorta 1(0.3) 0 Mesenteric arteries 2(0.6) 0 MEDLINE Database reviewed; 339 cases of PNH with thrombosis identified. *RR: adjusted relative risk for death Ziakas et al. J Thromb Haemost 2007;5:642

19 Multivariate Analysis of the RR RR* 95% CI P-value Venous thrombosis Mesenteric veins Pulmonary embolism Hepatic veins <0.001 CNS veins CNS sinus ns Renal vein ns Portal vein ns Inferior vena cava ns Splenic vein ns Deep veins ns Arterial thrombosis Coronary arteries < CNS arteries Hepatic artery ns MEDLINE Database reviewed; 339 cases of PNH with thrombosis identified. *RR: adjusted relative risk for death Ziakas et al. J Thromb Haemost 2007;5:642

20 The mechanism of thrombosis in PNH is unclear One explanation is complement-mediated attack on CD55 and CD59 deficient platelets which results in a phospholipid-rich milieu and the generation of factors Va, Xa and prothrombin complex (Hugel et al Blood 1999;93:3451) ) Complement-mediated damage may lead to increased leukocyte derived tissue factor levels in plasma, which activates the intrinsic coagulation pathway (Liebman et al. Thromb Res 2003;111:235) Venous endothelium in PNH may also be deficient in DAF and susceptible to complement-mediatedmediated damage [Asch et al. J Exp Med 1986;163:221]. Release of free hemoglobin by chronic hemolysis leading to NO depletion, and subsequently endothelial dysfunction and platelet activation (Schafer et al. Circulation 2004;109:1819; Reiter et al. Nat Med 2002;8:1383). Propensity towards thrombosis appears roughly proportional to the size of the PNH clone (Hall et al. Blood 2003;102;3587; Moyo et al. BJH 2004;126:133).

21 Membrane Particles are Increased in PNH Patients Hugel et al. Blood 1999;93:3451 Simak et al. et al. Exp Hematol 2008;36:1616

22 Plasma supar and Clot-free Survival in PNH Patients Sloand et al. Exp Hematol 2008;36:1616

23 PNH Platelets and Granulocytes: Sources of Plasma supar Platelets WBC Sloand et al. Exp Hematol 2008;36:1616

24 Hypothetical Mechanism for thrombosis in PNH Sloand et al. Exp Hematol 2008;36:1616

25 Survival Curve of PNH (Pre-Eculizumab) % Years of Follow-up Hillmen et al NEJM 1995;333:1253 Socie. et al. Lancet 1996;348:573 Nishimura et al. Medicine 2004,83:193 Moyo et al. BJH 2004;126:133 delatour et al. Blood 2008; 112:3099

26 Multivariate Analysis of Risk Factors Affecting Survival Socie. Lancet 1996;348:573

27 Eculizumab: Pilot Study in 11 PNH Patients Serum LDH Incidence of Paroxysms Hillmen et al NEJM 2004;350:552

28 Eculizumab: TRIUMPH Trial in 87 PNH Patients Eculizumab: iv 600 mg weekly x4 wks, followed 1 week later by a 900-mg, then 900 mg every other week through h week 26 Hillmen et al NEJM 2006;355:1233

29 Eculizumab: SHEPHARD Trial in 97 PNH Patients Brodsky et al. Blood 2008;111:1840

30 Long-Term Safety and Efficacy of Sustained Eculizumab Treatment in Patients with PNH: Survival Curve N=195 from Pilot,TRIUMPH, SHEPHERD and Extension studies Brodsky et al ASH 52th Annual Meeting, Poster Abs# 639

31 Long-Term Safety and Efficacy of Sustained Eculizumab Treatment in Patients with PNH: LDH Curve N=195 from Pilot,TRIUMPH, SHEPHERD and Extension studies Brodsky et al ASH 52th Annual Meeting, Poster Abs# 639

32 Long-Term Safety and Efficacy of Sustained Eculizumab Treatment in Patients with PNH: Thrombosis Events Pre- Treatment During Treatment Number of patients 25 7 No of Events Patients Years Rate/100 patient years Brodsky et al ASH 52 Meeting, Poster Abs# 639

33 Long-Term Safety and Efficacy of Sustained Eculizumab Treatment in Patients with PNH: Serious Infections Events N(%) Mean Onset (Days*) Meningococcal sepsis 2(1.0%) 385 Pyrexia 9(4.6%) 446 Cellulitis 2(1.0%) 235 Viral Gastroenteritis 2(1.0%) 419 Lower respiratory tract infection 3(1.5%) 833 Pneumonia 2(1.0%) 456 Sepsis 2(1.0%) 604 Septic shock 2(1.0%) 312 Urinary tract infection 3(1.5%) 878 Viral infection 6(3.1%) 477 * From 1st day of eculizumab treatment Brodsky et al ASH 52 Meeting, Poster Abs# 639

34 UK Leads PNH Center Experience with Eculizumab in PNH PNH Patients (40M/39F) May 2002-July Indications: thrombosis renal failure, transfusiondependent hemolysis 3. Mean treatment period; 39 months (1-98) Kelly et al ASH 52 Meeting, Poster Abs# 639

35 UK Leads PNH Center Experience with Eculizumab in PNH Improvement of Thrombosis and Blood Transfusion Requirement 34 thrombotic episodes in 21/79 patients prior to eculizumab. Only 2 thrombosis since treatment Primary prophylaxis with warfarin stopped in 21 patients with no thrombosis occurring since treatment 7 patients had thrombosis within 12 months prior and no further thrombosis after treatment. Mean of 19.9 units blood transfused (0-156) in the 12 months before, Of the 64 patients on eculizumab for at least a year, 43 (67%) have been transfusion-independent for >12 months. 21 patients still need transfusions, but the mean requirement has decreased from 24.6 (4-52) units in the 12 months before to 14.6 (2-50) units in the last 12 months. Kelly et al ASH 52 Meeting, Poster Abs# 639

36 RBC PNH Clone before and after Eculizumab Therapy Before After Borowitz et al. Cytometry Part B 78b:211 (2010)

37 Eculizumab Unmasks CD55 Deficiency in PNH Patients Normal Volunteer Before Eculizumab During Eculizumab Before Eculizumab During Eculizumab During Eculizumab During Eculizumab Hill et al. Haematologica 2010; 95:567

38 Extravascular Hemolysis in Eculizumab treated PNH Patients King College Hospital, London UK 26 PNH patients treated with eculizumab as compared with 22 patients untreated non-transfusion dependent patients DAT was positive in 21/26 during eculizumab treatmetn, but was positive in only 2/24 untreated patients 20/21 DAT positive patients had varying degree C3d positivity There was a reduction in transfusion requirements during eculizumab treatment in both DAT positive and DAT negative group Eculizumab is effective in resolution of intravascular haemolysis, but unmaking of low level C3d mediated haemolysis. The significance is still not entirely clear Kulasekararaj et al ASH 52 Meeting, Poster Abs# 4240

39 PIG-A Mutation Occurs in Multiple Potential HSC in PNH G6PD studies in female patients with PNH (Oni et al. Blood 1970) Matching mutations have been found in multiple lineage blood cells, including granulocytes, monocytes and lymphocytes. This implies that PNH is a clonal HSC disease (Takeda et al Cell 1993, and etc). More than one clones have been reported in PNH, but usually one of the clones dominants, similarly dominant multiple clones also have been reported (Bessler et al. Lancet 1994, and Napa et al 1995) Four PIG-A mutant clones have been found the hematopoetic progenitor colonies/bursts, three of which were also found in B cell lines and/or granulocytes (Nishimura et al Blood 1997, 89:3470).

40 Mutation-Driven Growth Advantage in PNH Mutant Clone Rearrangement of Chr 12 reported in 2 PNH patients, resulting in dysregulation of HMGA2 expression Expression of HMGA2 in PB but not in BM from PNH patients was significantly higher than that from normal volunteers In mouse transplanted with BM cells transfected with retrovirus vectors, pmys-hmga2-ires-egfp or pmy-ires-egfp as a control, % HMGA2 expressing cells in PB higher than control However, can not account for vast majority of PNH patient, as no HMGA2 over-expression has been found in 42 PNH patients. Inoue et al Blood 2006;4232; Murakami et al ASH 52th Meeting, Poster Abs# 4242; Kelly et al. Blood 2007;110: Abs #3671

41 Conditional Growth Advantage (Luzzatto-Young Hypothesis) Autoreactive T cells against HSC attacks normal GPI + HSC cells while sparing GPI - HSC because lack of GPI-AP renders them invulnerable. Close relationship with AA, MDS PNH patients CD8+CD57+ T cells are oligoclonal T cells from PNH patients showed a set of highly homologous TCR molecules (Gargiulo et al. Blood; 109:5036). Increased frequency of T cells expressing activating NK cell receptors with a CD8 + effector-memory phenotype and with different cytotoxicity towards GPI + and GPI - hematopoietic cell lines(bijnen et al ASH 52 Abs# 2235) However few PNH patients responded to immunosuppressive therapy not However, few PNH patients responded to immunosuppressive therapy not c/w this hypothesis ( Paquette BJH 96:92, 1997)

42 Pure Neutral Stochastic Drift (Dingli-Luzzatto-Pacheco Hypothesis) PIG-A mutations in HSCs occur spontaneously, and given the stochastic nature of hematopoiesis, even if we assume neutral coevolution of PIG-A mutated HSCs and normal HSCs, PNH would develop whenever the clone of PIG-A mutated HSCs becomes sufficiently large Dingli et al. PNAS 2008;105:18496

43 Nature s Gene Therapy, a Blessing in Disguise, No matter what mechanism that lead to PNH clonal expansion, this timely event, despite the associated complications, may have in reality saved her life, unlike other AA patients. Lussatto et al. Cell 1997;88:1 Hillmen et al. J Clin Path Mol Path 2002:55:45

44 MCQ Nocturnal hemoglobinuria can be accounted for by: 1. Diurnal hormonal changes 2. Mild acidemia overnight 3. Overnight concentration of free hemoglobin 4. Morning postural changes 5. Unknown