Management Issues in Paroxysmal Nocturnal Hemoglobinuria. Gabrielle Meyers, M. D. and Charles J. Parker, M. D.

Transcription

1 Management Issues in Paroxysmal Nocturnal Hemoglobinuria Gabrielle Meyers, M. D. and Charles J. Parker, M. D. Divisions of Hematology and Medical Oncology, University of Utah School of Medicine and VA Medical Center, Salt Lake City, Utah Running title: Treatment of PNH Correspondence Address: Charles J. Parker, M. D. Hematology/Oncology Section (111 H) VA Medical Center 500 Foothill Blvd Salt Lake City, Utah Tel: Fax: Page 1

2 Abstract Paroxysmal nocturnal hemoglobinuria (PNH) arises in the setting of bone marrow injury. Thus management decisions must take into account whether symptoms are a consequence of the underlying marrow failure or of the expansion of the clone of the PIG-A mutant, glycosyl phosphatidylinositol-anchored hematopoietic cells. The primary clinical manifestations of PNH are intravascular hemolysis and thrombophilia. Currently available options for treatment of the hemolysis of PNH are unsatisfactory, but the recent development of specific inhibitors of complement for use in human disease should make possible effective management of this pathology. The fundamental basis of the thrombophilia of PNH has not been elucidated. Currently, empiric anticoagulant therapy is the foundation for treatment of thromboembolic complications of PNH. The role of warfarin prophylaxis, however, remains an area of active debate. Pregnancy in a patient with PNH presents special concerns about fetal/maternal well being due to the high potential for thromboembolic complications. Bone marrow transplant can be considered curative, but the decision to recommend this treatment must take into account factors related both to PNH and to comorbid conditions. Refining the technology for both gene therapy by transducing stem cells with functional PIG-A and autotransplantation using stem cells selected for expression of glycosyl phosphatidylinositol anchored proteins remain challenges for the future. Page 2

3 Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired syndrome in which glycosyl phosphatidylinositol anchored proteins (GPI-AP) are deficient on the surface of blood cells. The GPI-AP deficient blood cells are the progeny of one or more mutant hematopoietic stem cells that have undergone clonal expansion. The gene that is mutant in PNH (PIG-A) is known (1). This knowledge explains the protein deficiencies of PNH (PIG-A is required for synthesis of the GPI anchor moiety) but does not provided an obvious explanation for the clonal expansion of the mutant stem cells. Arguably, PNH is not a primary disease but rather a secondary process that occurs because, under a specific (although as yet incompletely understood) set of circumstances, deficiency of one or more GPI-AP is advantageous. PNH is frequently associated with aplastic anemia and less frequently with myelodysplasia, (2-7) leading to the hypothesis of a conditional advantage for GPI-AP deficient hematopoietic stem cells in a specific set of bone marrow failure syndromes (6,8-10). Immune mechanisms that dominate (aplastic anemia) or contribute in some cases (myelodysplasia) to the pathophysiology may underlie the selection pressure (6,8-10). The concept of PNH as a secondary process is particularly relevant for this discussion because the coexistence of another pathological process must be taken into account when formulating a management plan. Hemolysis and Anemia The clinical hallmark of PNH is intravascular hemolysis resulting in hemoglobinuria. The hemolysis is a consequence of deficiency of two GPI-AP that normally protect erythrocytes Page 3

4 from complement-mediated lysis (Fig. 1) (11). Hemolysis is the only clinical manifestation of PNH that is unequivocally a consequence of deficiency of GPI-AP. The anemia of PNH is complex. Because PNH arises usually (if not invariably) in the setting of bone marrow injury, hemolysis may account for only part of a patient s anemia. The erythrocytes of PNH are a mosaic of normal and abnormal cells, and the portion of GPI-AP deficient RBC s varies greatly among patients (12,13). For example, in one patient, only 10% of the circulating RBC may be GPI-AP deficient while, in another case, 75% GPI-AP deficient erythrocytes may be observed. In the former, hemolysis would contribute only modestly to an observed anemia, while in the latter; a significant hemolytic component would be expected (12). Another complicating factor is that deficiency of GPI-AP may be partial rather than complete (Fig. 2). Erythrocytes that are completely deficient in GPI-AP are called PNH type III cells while those that are partially deficient (usually expressing ~10% of the normal amount of GPI- AP) are called PNH II cells (PNH I cells have normal expression of GPI-AP) (14) (Fig. 2). The distinction is clinically important because the partial expression of the GPI-anchored complement regulatory proteins is sufficient to protect PNH II cells from complement-mediated lysis in vivo (12). Thus even if a patient has a high proportion of PNH II cells, little intravascular hemolysis is observed. What is the contribution of hemolysis to the anemia? Prior to initiating therapy for anemia in a patient with PNH an effort should be made to determine how much of the anemia is a consequence of hemolysis and how much is due to impaired erythropoiesis (Table I). Review of the complete blood count (CBC) is informative because evidence of thrombocytopenia, leukopenia or both suggests significant impairment of hematopoiesis due to a stem cell abnormality. The capacity of the marrow to respond to the anemia can be inferred by the Page 4

5 reticulocyte count. An element of marrow failure is likely a contributing factor in a patient with PNH who has an anemia with a low reticulocyte count. Biochemical parameters of hemolysis should be assessed (Table I). The complementmediated hemolysis of PNH is an intravascular process. Consequently, serum lactate dehydrogenase (LDH) concentration is invariably abnormally high. Minimal elevation of serum LDH argues against hemolysis as a major contributing factor to anemia in a patient with PNH. The presence of urine hemosiderin suggests chronic intravascular hemolysis but provides no quantitative information. Determination by flow cytometry of the percentage of PNH I (RBC with normal expression of CD55 and CD59), PNH II (low expression of CD55 and CD59), and PNH III (absent expression of CD55 and CD59) erythrocytes is essential (Fig. 2). In patients with a small PNH III clone (< 20% of the total RBC population with the PNH III phenotype) or with mainly PNH II RBC, hemolysis is unlikely to contribute significantly to an observed anemia (12). The concentration of serum erythropoietin should be determined as chronic intravascular hemolysis may injure the kidneys (15) thereby negatively affecting erythropoietin production. In some instances, endogenous erythropoietin levels may be supernormal, but a response to pharmacological doses of the recombinant protein may still be observed (16-18). Chronic intravascular hemolysis can also lead to iron deficiency, making determination of iron stores necessary (19). Is treatment of hemolysis necessary? If the above assessment suggests that hemolysis contributes significantly to an observed anemia, the clinician must next decide if treatment is appropriate. Should a patient with active hemolysis, but with mild, asymptomatic anemia be treated? To answer this question, one must first answer another question: Is hemolysis per se Page 5

6 harmful? The answer to this question is an equivocal yes for the following reasons: (1) Patients with chronic hemolysis frequently complain of lethargy, malaise, and loss of sense of well being that diminishes significantly their quality of life. (2) There is evidence that chronic hemolysis has an untoward effect on renal function (15), although the clinical impact of the injury appears modest. (3) The dysphagia of PNH, a particularly distressing symptom, appears related to hemolysis (particularly during a hemolytic exacerbation) (20). (4) A correlation between thrombosis and hemolysis may exist. Treatment of the hemolysis of PNH. If a modality with a favorable therapeutic index were available, there would be little debate about whether treatment of hemolysis is appropriate. Although such an agent may be forthcoming (see below), current options are limited and the outcome of treatment is usually unsatisfactory. The use of corticosteroids to treat chronic hemolysis and acute hemolytic exacerbations is debated (19,21-23). Part of the debate is fueled by the empiric nature of the therapy, as there is no experimental data that provides a plausible explanation for why steroids should ameliorated complement-mediated hemolysis. Corticosteroids may have value in attenuating a hemolytic exacerbation associated with an inflammatory process, but their value in treating chronic hemolysis appears limited, and the harm that can accrue from the long-term use of even low doses cannot be overemphasized. Transfused RBC s have normal expression of CD55 and CD59 and therefore should survive normally in patients with PNH. Transfusion may further ameliorate hemolysis by suppressing erythropoiesis thereby reducing generation of GPI-AP deficient RBC s. The complications of chronic transfusion are well known. Concerns about inducing a hemolytic exacerbation as a consequence of infusion of small amounts of donor plasma that contaminates red cell preparations appear unwarranted (24). Iatrogenic hemochromatsis may be delayed in Page 6

7 patients with PNH due to iron loss as a consequence of hemoglobinuria (if the rate of intravascular hemolysis is sufficiently high) (19). Nonetheless iron overload remains an important concern in patients with PNH who require chronic transfusion. Complement inhibitors. Ideally, the hemolysis of PNH would be treated with a specific inhibitor of complement, and this topic has been the subject of a recent review (25). A number of complement inhibitors have been developed and some of these reagents are in phase II trails in humans. Recently, Hillmen and colleagues reported (in abstract form) (26), the preliminary results of a study using a humanized monoclonal antibody against complement C5 (Fig. 1). As anticipated, this reagent was highly effective in controlling the symptoms and signs of the hemolysis of PNH, and significant adverse events associated with the treatment were not observed. The major disadvantages to this treatment are that the reagent must be infused (in the study it was given weekly initially for four doses and then every other week for maintenance) and cost of chronic therapy. It remains uncertain whether concerns about potential adverse effects of chronic suppression of the complement system are justified (25). Even if untoward consequences of long-term use are identified, this reagent (and other complement inhibitors) (25) will certainly be of value in treating acute hemolytic exacerbations. Management of non-hemolytic anemia. Treatment of anemia that is primarily due to bone marrow failure should be aimed at the underlying disease (e. g. aplastic anemia or MDS). If absolute or relative erythropoietin deficiency is felt to contribute to the anemia, replacement with the recombinant protein is indicated. Hypothetically, erythropoietin supplementation could exacerbate hemolysis by stimulating erythropoiesis in the PIG-A mutant clone. Thus, patients should be monitored for evidence of worsening hemolysis when therapy with erythropoietin is initiated. Iron stores should be assessed as the efficacy of erythropoietin appears to be attenuated Page 7

8 if iron stores are inadequate (27). Iron repletion may initiate a hemolytic exacerbation (especially if iron stores are repleted acutely using infusional therapy) (19). The clinical significance of this potential problem, however, appears insufficient to deter replacement. Folate supplementation is also indicated in patients with significant hemolysis. Thrombosis In contrast to our thorough understanding of the basis of the hemolysis (Fig. 1), much less is known about the mechanisms underlying the thrombophilia of PNH (28). The PIG-A mutation arises in a hematopoietic stem cell. Thus platelets (and leukocytes) as well as erythrocytes are deficient in GPI-AP, and normal platelets express the complement regulatory proteins CD55 and CD59. Although platelet survival is normal (29), in vitro data suggests that deficiency of complement regulator proteins on platelets may contribute to the thrombophilia of PNH (30). In this case, complement activation induces release of microparticles that act as the nidus for formation of the prothrombinase complex. Microvesicles are also generated from erythrocytes as a consequence of complement-mediated lysis (31), and abnormal microvesiculation has been observed in vivo (32). Epidemiological data suggest that thromboembolic complications of PNH are more common in patients from Europe (33,34) and the United States (28,35) than in patients from Asia (36,37) and Mexico (38). This difference has been attributed by some to the proportion of GPI- AP deficient cells (39,40). PNH in patients from Asia and Mexico is dominated by sequelae of bone marrow failure (28), and these patients usually have a relatively low proportion of GPI-AP deficient peripheral blood cells. In contrast, hemolysis is often the dominant feature of PNH Page 8

9 patients from the United States and Europe due to a relatively large portion of GPI-AP deficient RBC s. The correlation between thrombophilia and the proportion of GPI-AP deficient blood cells is far from perfect, however. Further, a connection between thrombosis and hemolytic exacerbations has never been demonstrated. Even the role of platelet microparticles in thrombosis is debated (28). Other mechanisms have also been proposed as potential etiological factors for the thrombophilia of PNH including deficiency of the receptor for urokinase plasminogen activator, a GPI-AP expressed on platelets (28). Differences in genetic susceptibility could explain the variability in the incidence of thromboembolic events among patient with PNH, but to date, investigations aimed at addressing this hypothesis have shown no significant differences (41). One interpretation of a direct correlation between the proportion of GPI-AP deficient blood cells and thrombophilia is that thrombosis is causally related to lack of complement regulatory proteins on blood cells (particularly RBC s and platelets). Finding that the thrombophilia of PNH is a consequence of failure to regulate complement on hematopoietic cells would have significant therapeutic implications. In this case, treatment with complement inhibitory proteins would be expected to ameliorate the thromboembolic complications of PNH as well as the hemolysis. However, such treatment would likely have its greatest value in reducing the incidence of thromboembolic complications (i. e., prophylaxis) rather than in the management of an acute thromboembolic event. Prophylaxis. The issue of prophylaxis against thromboembolic disease in patients with PNH is an area of active debate. Current estimates of the risk of a thromboembolic event are suspect as randomized studies designed to address this critical issue are lacking (28). Further, Page 9

10 available data suggest that the incidence may vary significantly based on differences in disease presentation that appear to segregate geographically (discussed above). In patients from the United States and Europe, the lifetime risk of a thromboembolic event may be in the range of 20%-30% (28,35). This relatively high risk has lead some to advocate prophylaxis with warfarin (34). In contrast, the incidence of thromboembolic events among patients from Japan, China, and Mexico (36-38) may be significantly lower (in the range of 2%). In this setting, prophylaxis is difficult to justify. Due to the paucity of randomized studies, evidenced-based guidelines for antithrombotic prophylaxis cannot be formulated satisfactorily. Available data (and its limitations) should be discussed with each patient, and factors such as age, activity level, compliance, and comorbid conditions should enter into decision making about prophylaxis with warfarin. If effective, antiplatelet agents would offer a more favorable therapeutic index than warfarin, but in the absence of controlled trials, the role of platelet inhibitors in prophylaxis against the thrombophilia of PNH is undefined. Management of thromboembolic disease. Although arterial thrombosis may be observed, thromboembolic events in patients with PNH usually involve the venous system. Unusual sites of thrombosis are well described and include abdominal involvement, particularly the hepatic vein (Budd-Chiari syndrome), portal vein, and mesenteric veins (34). Cerebral vein thrombosis is a potentially devastating complication, and dermal vein occlusions are noteworthy as they are also observed in other thrombophilic conditions (20). Acute thrombotic events require anticoagulation with heparin. The effects of therapeutic doses of heparin on the complement system are incompletely understood (42) but probably have little clinical relevance. Thrombolytic therapy should be considered in patients with acute onset Page 10

11 of Budd-Chiari syndrome (43). Thrombocytopenia often complicates PNH, and this issue must be addressed when formulating a management plan. Thrombocytopenia is a relative but not an absolute contraindication to anticoagulation, and transfusions should be given to maintain the platelet count in a safe range rather than withholding therapy because of concerns about excess morbidity due to thrombocytopenia (35). Unless there are significant contraindications, we recommend that patients with PNH who experience a thromboembolic event be anticoagulated indefinitely. A recurrent event in a patient who is therapeutically anticoagulated warrants consideration of high-intensity warfarin therapy. Chronic anticoagulation with subcutaneous low molecular weight heparin should also be considered in this setting. For patients who are warfarin failures, the addition of anti-platelet agents to therapeutic anticoagulation may also be indicated depending on the presence and degree of thrombocytopenia. Recurrent, life-threatening thrombosis merits consideration for bone marrow transplantation in eligible patients (discussed below). Pregnancy. Clearly there are fetal and maternal risks when pregnancy occurs in a patient with PNH (35). Counseling patients with PNH about whether to become pregnant is a delicate matter that should incorporate factors such as the nature of the underlying bone marrow failure, comorbid conditions, history of thromboembolic complications, degree of thrombocytopenia, and extent of hemolysis. Despite the many concerns, successful outcomes appear to be the rule rather than the exception (35). Nonetheless, management is complicated and should involve the combined efforts of an experienced hematologist and an obstetrician specializing in high-risk pregnancy. Anticoagulation with heparin should begin in the first trimester. Coumadin is contraindicated because of potential teratogenic effects. Low molecular weight heparin has a Page 11

12 hypothetical advantage over unfractionated heparin because of the lower incidence of druginduced thrombocytopenia. Careful monitoring of the platelet count is warranted. Hemolysis may worsen during pregnancy. Corticosteroids are not recommended because of potential adverse effects, but judicious use of transfused blood products is an acceptable alternative. Anticoagulation should continue for at least 30 days post-partum, as thrombosis during the puerperium is a major concern. If found to be safe, complement inhibitors may prove to be the treatment of choice during pregnancy and the puerperium as they may ameliorate both hemolysis and thrombosis. Bone Marrow Failure Although the apparent severity is highly variable, all patients with PNH have evidence of bone marrow insufficiency during the course of their illness. Our view is that the outgrowth of PIG-A mutant, GPI-AP deficient stem cells is a response to a specific type of bone marrow injury. According to this perspective, the clinical manifestations of bone marrow failure (thrombocytopenia, leukopenia, and hypoproliferative anemia) are separate and distinct from the clinical manifestations of PNH (intravascular hemolysis, thrombophilia, and dysphagia) and should be managed separately. Thus, when immunosuppressive therapy is used in a patient with PNH, therapy is being aimed at treatment of bone marrow failure rather than at the consequences of GPI-AP deficiency affecting a proportion of the hematopoietic cells (i. e. PNH). Clinical and laboratory evidence of PNH may first appear during the recovery phase of aplastic anemia (2,44,45). It seems unlikely, however, that the treatment (usually immunosuppressive therapy using anti-thymocyte globulin and cyclosporin) per se causes expansion of the PIG-A mutant clone (7). Rather, clonal expansion may be a consequence of Page 12

13 either clonal evolution or a general depression of hematopoiesis. Other clonal myelopathies (e. g., acute leukemia, and myelodysplasia) are also seen in this setting (45), suggesting that genetic instability may be a component of the process underlying the bone marrow failure (46). High dose cyclophosphamide appears to be effective therapy for treatment of aplastic anemia (47), however, concerns about morbidity and mortality due to prolonged bone marrow suppression have tempered enthusiasm for this management approach (48). Also debated is whether relapses and secondary clonal myelopathies occur less frequently with high dose cyclophosphamide than with more standard immunosuppressive therapy (47,49). Withholding immunosuppressive therapy in patients with aplastic anemia because of concerns about inducing or exacerbating PNH, however, appears unjustified (7). This review does not include a discussion of the use of androgens in the treatment of PNH (20) because this class of agents is aimed at ameliorating symptoms of bone marrow failure (by stimulating hematopoiesis) rather than at affecting the pathology related to the PNH clone. Bone Marrow Transplantation. When considering bone marrow transplantation in a patient with PNH, it is again important to determine if the treatment is being aimed at symptoms of bone marrow failure (thrombocytopenia, leukopenia, and hypoproliferative anemia) or at symptoms of PNH (intravascular hemolysis or thrombophilia). The major distinction is that in bone marrow failure, the goal is restoration of hematopoiesis, whereas in PNH, there are two goals. The first goal is elimination of the PIG-A mutant clone or clones. The second goal is abrogation of the process that provides the basis for the conditional growth or survival advantage for the GPI-AP deficient Page 13

14 stem cells. The conditioning regiment used for myeloablative transplants appears sufficient to accomplish both goals. On the other hand, recurrent disease is observed in syngeneic transplants without preconditioning (50). Although there appears to be a hypothetical disadvantage to nonmyeloablative regimens for treatment of PNH, successful outcomes have been reported, and a recent preliminary study indicates that the GPI-AP deficient cells can be successfully eradicated using this approach (51). Interestingly, graft-versus-host disease appears to contribute significantly to the elimination of the GPI-AP deficient stem cell population (51). Evidence is insufficient to propose specific guidelines for stem cell or bone marrow transplant in patients with PNH (52). Candidates include patients with recurrent, life-threatening thromboembolic disease and patients with intractable, transfusion dependent hemolytic anemia. This latter group may eventually be eliminated if treatment with specific inhibitors of complement becomes practical. (This therapy may also eliminate the former group if the thrombophilia of PNH proves to be complement-mediated or complement-dependent). The final decision about transplant should be made on an individual basis and should take into consideration availability of a donor (sibling vs. matched unrelated), nature of the underlying bone marrow disease (aplastic anemia vs. myelodysplasia) age, severity of symptoms and comorbidity. There is no transplant related morbidity that is clearly particular to PNH. Can GPI-AP positive stem cells be separated from GPI-AP negative stem cells and used for autologous transplantation? Hematopoietic stem cells as defined by the phenotype (CD34 + CD38 - ) express GPI-AP including CD55 and CD59. Thus, it is conceivable that (using fluorescence activated cell sorting) the GPI-AP positive population could be separated from the GPI-AP negative population and used for marrow rescue following myeloablative therapy (53). There are two potential problems with this approach, however. First, it may be difficult to obtain Page 14

15 a sufficient quantity of GPI-AP positive stem cells to insure engraftment (particularly in patients whose underlying bone marrow abnormality is aplastic or hypoplastic anemia). Second, the GPI-AP positive stem cells may not be normal. For example, when PNH arises in the setting of myelodysplasia, the GPI-AP positive stem cells may have karyotypic abnormalities (54). Even cells with a normal karyotype might have clinically important genetic defects that are undetectable by available techniques. Given these limitations, autotransplant using selected stem cells is at present a hypothetical rather than a practical alternative to allotransplant. Preclinical work on this approach to management, however, should continue. Gene therapy. The deficient expression of GPI-AP can be corrected by transducing mutant cells with vector containing functional PIG-A (55). Thus, it is conceivable that gene therapy may eventually be an option for treatment of PNH. If so, it will be necessary to eliminate first both the residual PIG-A mutant stem cells and the underlying condition that provides the conditional growth or survival advantage to the GPI-AP deficient cells. Myeloablative conditioning regimens similar to those used in preparation for allotransplantation would likely accomplish these goals. Laboratory PNH. Available techniques for identifying GPI-AP deficient cells are extremely sensitive. Standard flow cytometry can reliably detect populations of 3% GPI-AP deficient cells (56) and more sensitive methods can detect populations of < 1% (6,57). Whether detection of such small populations has clinical or therapeutic implications is currently unclear. Conceivably, the presence of GPI-AP deficient cells may have prognostic significance. For example a patient with myelodysplasia and a population of GPI-AP deficient cells might be more likely to respond Page 15

16 to immunosuppressive therapy (6). In support of the concept the presence of GPI-AP deficient cells has prognostic significance, the risk of mortality appear lower in patients with PNH who have a history of aplastic anemia (33). Well-designed prospective clinical studies are needed to address the implications of laboratory PNH. Dysphagia Symptoms of esophageal spasm complicate hemolytic exacerbations in patients with PNH (20). The basis of the esophageal dysfunction is not clearly understood although sequestration of endothelial relaxing factor (nitric oxide) by free hemoglobin has been suggested (20). Whether enhancement of nitric oxide activity by using sildenafil citrate (Viagra) ameliorates the dysphagia (and male impotence) (20) of PNH has not been investigated formally. Ultimately, treatment of the hemolysis with specific complement inhibitors (26) may control the dysphagia of PNH. Page 16

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20 26. Hillmen P, Hall C, Marsh J, et al. Eculizumab, a C5 complement-blocking antibody, abolishes hemolysis and renders hemolytic patients with paroxysmal nocturnal hemoglobinuria (PNH) transfusion independent. Blood. 2002;100:44a 27. Goodnough LT, Skikne B, Brugnara C. Erythropoietin, iron, and erythropoiesis. Blood. 2000;96: Sloand EM, Young NS. Thrombotic Complications in PNH. In: Young NS, Moss, J., ed. Paroxysmal Nocturnal Hemoglobinuria and the Glycosylphosphatidylinositol-Linked Proteins. San Diego: Academic Press; 2000: Devine DV, Siegel RS, Rosse WF. Interactions of the platelets in paroxysmal nocturnal hemoglobinuria with complement. Relationship to defects in the regulation of complement and to platelet survival in vivo. J Clin Invest. 1987;79: Wiedmer T, Hall SE, Ortel TL, Kane WH, Rosse WF, Sims PJ. Complement-induced vesiculation and exposure of membrane prothrombinase sites in platelets of paroxysmal nocturnal hemoglobinuria. Blood. 1993;82: Ninomiya H, Kawashima Y, Hasegawa Y, Nagasawa T. Complement-induced procoagulant alteration of red blood cell membranes with microvesicle formation in paroxysmal nocturnal haemoglobinuria (PNH): implication for thrombogenesis in PNH. Br J Haematol. 1999;106: Hugel B, Socie G, Vu T, et al. Elevated levels of circulating procoagulant microparticles in patients with paroxysmal nocturnal hemoglobinuria and aplastic anemia. Blood. 1999;93: Socie G, Mary JY, de Gramont A, et al. Paroxysmal nocturnal haemoglobinuria: long-term follow-up and prognostic factors. French Society of Haematology. Lancet. 1996;348: Page 20

21 34. Hillmen P, Lewis SM, Bessler M, Luzzatto L, Dacie JV. Natural history of paroxysmal nocturnal hemoglobinuria. N Engl J Med. 1995;333: Ray JG, Burows RF, Ginsberg JS, Burrows EA. Paroxysmal nocturnal hemoglobinuria and the risk of venous thrombosis: review and recommendations for management of the pregnant and nonpregnant patient. Haemostasis. 2000;30: Fujioka S, Takayoshi T. Prognostic features of paroxysmal nocturnal hemoglobinuria in Japan. Acta Haematol Japan. 1989;52: Le XF, Yang TY, Yang XY, Wang XM. Characteristics of paroxysmal nocturnal hemoglobinuria in China. Clinical analysis of 476 cases. Chin Med J (Engl). 1990;103: Gongora Bianchi RA. Paroxysmal nocturnal hemoglobinuria: the mexican experience. Rev Invest Clin. 1997;49:85S-88S 39. Moyo VM, Mukhina GL, Garrett ES, Brodsky RA. Correlation between thrombosis and the percentage of glycosylphosphatidylinositol (GPI) anchor deficient cells in PNH. Blood. 2002;100:230a 40. Hall C, Richards S, Hillmen P. Primary prohylaxis with warfarin prevents thrombosis in paroxysmal nocturnal haemoglobinuria (PNH). Blood. 2001;98:220a 41. Nafa K, Bessler M, Mason P, et al. Factor V Leiden mutation investigated by amplification created restriction enzyme site (ACRES) in PNH patients with and without thrombosis. Haematologica. 1996;81: Ninomiya H, Kawashima Y, Nagasawa T. Inhibition of complement-mediated haemolysis in paroxysmal nocturnal haemoglobinuria by heparin or low-molecular weight heparin. Br J Haematol. 2000;109: Page 21

22 43. McMullin MF, Hillmen P, Jackson J, Ganly P, Luzzatto L. Tissue plasminogen activator for hepatic vein thrombosis in paroxysmal nocturnal haemoglobinuria. J Intern Med. 1994;235: de Planque MM, Bacigalupo A, Wursch A, et al. Long-term follow-up of severe aplastic anaemia patients treated with antithymocyte globulin. Severe Aplastic Anaemia Working Party of the European Cooperative Group for Bone Marrow Transplantation (EBMT). Br J Haematol. 1989;73: Tichelli A, Gratwohl A, Nissen C, Speck B. Late clonal complications in severe aplastic anemia. Leuk Lymphoma. 1994;12: Horikawa K, Kawaguchi T, Ishihara S, et al. Frequent detection of T cells with mutations of the hypoxanthine-guanine phosphoribosyl transferase gene in patients with paroxysmal nocturnal hemoglobinuria. Blood. 2002;99: Brodsky RA, Sensenbrenner LL, Smith BD, et al. Durable treatment-free remission after high-dose cyclophosphamide therapy for previously untreated severe aplastic anemia. Ann Intern Med. 2001;135: Tisdale JF, Dunn DE, Geller N, et al. High-dose cyclophosphamide in severe aplastic anaemia: a randomised trial. Lancet. 2000;356: Tisdale JF, Maciejewski JP, Nunez O, Rosenfeld SJ, Young NS. Late complications following treatment for severe aplastic anemia (SAA) with high-dose cyclophosphamide (Cy): follow-up of a randomized trial. Blood. 2002;100: Endo M, Beatty PG, Vreeke TM, Wittwer CT, Singh SP, Parker CJ. Syngeneic bone marrow transplantation without conditioning in a patient with paroxysmal nocturnal hemoglobinuria: in vivo evidence that the mutant stem cells have a survival advantage. Blood. 1996;88: Page 22

23 51. Takahashi Y, McCoy JP, Carvallo C, Rivera C, Igarashi, T., Srinivasan, R., Young NS, Childs R. PNH cells are not resistant to allogeneic immune attack and can be eradicated following nonmyeloablative allogeneic stem cell transplantation (NST). Blood. 2002;100:9a 52. Araten DJ, Luzzatto L. Allogeneic bone marrow transplantation for paroxysmal nocturnal hemoglobinuria. Haematologica. 2000;85: Prince GM, Nguyen M, Lazarus HM, Brodsky RA, Terstappen LW, Medof ME. Peripheral blood harvest of unaffected CD34+ CD38- hematopoietic precursors in paroxysmal nocturnal hemoglobinuria. Blood. 1995;86: Sloand EM, Fuhrer M, Maciejewski JP, et al. When cytogenetic abnromalities occur in patients with paroxysmal nocturnal hemoglobinuira (PNH), they primarily affect the glycosylphosphatidylinositol (GPI)-positive cell clones. Blood. 2002;100:229a 55. Nishimura J, Phillips KL, Ware RE, et al. Efficient retrovirus-mediated PIG-A gene transfer and stable restoration of GPI-anchored protein expression in cells with the PNH phenotype. Blood. 2001;97: Hall SE, Rosse WF. The use of monoclonal antibodies and flow cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria. Blood. 1996;87: Brodsky RA, Mukhina GL, Li S, et al. Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin. Am J Clin Pathol. 2000;114: Page 23

24 Figure Legends Figure 1. The hemolytic anemia of PNH is complement-mediated. There is continuous lowgrade activation of the alternative pathway of complement due to spontaneous formation of the C3 convertase (the tick-over phenomenon ). The C3 convertase is comprised of activated C3 (C3b), activated factor B (Bb), and properdin (P). Activated factor B is the enzymatic subunit of the complex and properdin stabilizes the complex. The C3b component of the complex binds native C3 and positions it for cleavage by Bb. Enzymatic activation of C3 results in generation of the anaphylatoxin C3a. The C5 convertase consists of two molecules of activated C3 (C3b) along with activated factor B (Bb) and properdin. The second C3b molecule is the binding site for C5, positioning it for cleavage by Bb. The C5a peptide that is generated as a consequence of enzymatic activation of C5 mediates a number of proinflammatory events including chemotaxis, smooth muscle contraction, vasodilitation, and histamine release. The membrane attack complex (MAC) consists of 1 molecule each of C5b, C6, C7, and C8 and multiple molecules of C9 (C5b- 9 n ). The MAC forms the donut lesion seen by electron microscopy on erythrocytes that have undergone complement-mediated lysis. The pore that is formed by the MAC disrupts the integrity of the red cell membrane resulting in osmotic lysis. DAF (CD55) is a GPI-AP that controls the formation and stability of both the C3 and the C5 convertase. CR1 (CD35) also controls the formation and stability of both convertases. CR1 is a transmembrane protein. Although CR1 is a potent inhibitor of complement, it is present in relatively low density on erythrocytes, making DAF the primary RBC regulator of C3/C5 convertase activity. MIRL (CD59) inhibits formation of the MAC by binding to C8 and preventing C9 multiplicity. MIRL (like DAF) is GPI-anchored and is consequently deficient on the erythrocytes of PNH. Page 24

25 The lightening bolt symbol indicates the site at which Eculizumab, a humanized anti-c5 monoclonal antibody, acts. By blocking C5, the antibody prevents formation of the MAC, thereby preventing complement-mediated lysis. Eculizumab is the first complement inhibitor used to treat the hemolysis of PNH in humans. Figure 2. Mosaicism in PNH. The histogram was generated by flow cytometric analysis of peripheral blood erythrocytes using monoclonal anti-cd59 as the primary antibody. Three distinct populations of cells (PNH III, PNH II, and PNH I) are apparent. Substituting isotypematched non-immune mouse IgG for anti-cd59 established the position of the dotted blue line. Thus, the PNH III population is defined by absence of expression of GPI-AP. The PNH II population expresses approximately 10% of the normal amount of GPI-AP as a result of a missense mutation affecting PIG-A (Endo et al.). The PNH I population has normal expression of GPI-AP. In this case 7% of the circulating RBC s were PNH III, 70% were PNH II, and 23% were PNH I. Because of the low percentage of PNH III cells and the relatively high percentage of PNH II cells, hemolysis in this patient is mild. Phenotypic mosaicism resulting from genotypic mosaicism is common in PNH and plays an important role in the clinical characteristics of the disease. Page 25

26 Table I. Information useful for managing anemia in patients with PNH Parameter Complete blood count Reticulocyte count Serum lactate dehydrogenase, bilirubin (total and direct fractions), haptoglobin Urine hemosiderin Flow cytometric analysis of erythrocytes for expression of GPI-AP * Serum erythropoietin concentration Serum blood urea nitrogen (BUN) and creatinine Serum iron studies (iron concentration, total iron binding capacity, ferritin concentration) * This analysis should be used to determine the percentage of PNH I, PNH II, and PNH III cells (see Fig. 2 for an example). Page 26