Paroxysmal nocturnal hemoglobinuria: current issues in pathophysiology and treatment

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired genetic disorder of the bone marrow that produces intravascular hemolysis, proclivity to venous thrombosis, and hematopoietic failure. Mutation in the PIG-A gene in a stem cell aborts synthesis of glycosyl-phosphoinositol (GPI) anchors and therefore expression of all GPI-anchored proteins on the surface of progeny erythrocytes, leukocytes, and platelets. The hemolytic anemia of PNH is well understood, and erythrocyte susceptibility to complement may be treated with anti-C5a monoclonal antibody. The pathophysiology of PNH cell clonal expansion and its association with immune-mediated marrow failure are not understood, but PNH/aplasia responds to immunosuppressive regimens such as antithymocyte globulin and cyclosporine. The mechanism of thrombosis in PNH is also obscure, but frequently fatal clotting episodes may be prevented by Coumadin (Bristol-Myers Squibb Pharma Co., Wilmington, DE) prophylaxis.     

Acquired aplastic anemia

In aplastic anemia, hematopoiesis fails: Blood cell counts are extremely low, and the bone marrow appears empty. The pathophysiology of aplastic anemia is now believed to be immune-mediated, with active destruction of blood-forming cells by lymphocytes. The aberrant immune response may be triggered by environmental exposures, such as to chemicals and drugs or viral infections and, perhaps, endogenous antigens generated by genetically altered bone marrow cells. In patients with post-hepatitis aplastic anemia, antibodies to the known hepatitis viruses are absent; the unknown infectious agent may be more common in developing countries, where aplastic anemia occurs more frequently than it does in the West. The syndrome paroxysmal nocturnal hemoglobinuria (PNH) is intimately related to aplastic anemia because many patients with bone marrow failure have an increased population of abnormal cells. In PNH, an entire class of proteins is not displayed on the cell surface because of an acquired X-chromosome gene mutation. The PNH cells may have a selective advantage in resisting immune attack. In contrast, the disease myelodysplasia can be confused with aplasia and can also evolve from aplastic anemia. The occurrence of cytogenetic abnormalities in patients years after presentation implies that genomic instability is a feature of this immune-mediated disease. Aplastic anemia can be effectively treated by stem-cell transplantation or immunosuppressive therapy. Transplantation is curative but is best used for younger patients who have histocompatible sibling donors. Antithymocyte globulin and cyclosporine restore hematopoiesis in approximately two thirds of patients. However, recovery of blood cell count is often incomplete, recurrent pancytopenia requires retreatment, and some patients develop late complications (especially myelodysplasia).     

Paroxysmal nocturnal hemoglobinuria and myelodysplastic sydromes: clonal expansion of PIG-A-mutant hematopoietic cells in bone marrow failure

Paroxysmal hemoglobinuria clones can occur not only in bone marrow failure but also in myelodysplastic syndromes. In this perspective article, Dr. Young discusses the biologic and clinical significance of this association. See related article on page 29.Clones of paroxysmal nocturnal hemoglobinuria (PNH) cells – glycosylphosophoinositol (GPI)-anchored protein-deficient hematopoietic cells –can be detected by flow cytometry in patients with myelodysplastic sydromes (MDS). While the etiology of PNH is known to be a somatic mutation in the X-linked PIG-A gene, which abrogates GPI synthesis, the pathophysiology of PNH clonal expansion is not well understood. In frank PNH with clinical symptoms and signs of intravascular hemolysis and venous thrombosis, PNH cells dominate in the peripheral blood. Very small PNH clones can also be detected efficiently and routinely in patients with aplastic anemia and MDS. PNH clones emerge almost always in the setting of marrow failure, presumably immune-mediated hematopoietic destruction. Possible mechanisms for clonal expansion and clinical implications for the diagnosis, prognosis, and treatment of MDS are discussed.     

Pathophysiology,diagnosis, and treatment of paroxysmal nocturnal hemoglobinuria: a review

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder of the hematopoietic stem cell that makes blood cells more sensitive to the action of complement. Patients experience intravascular hemolysis, smooth muscle dystonia, renal failure, arterial and pulmonary hypertension, recurrent infectious diseases and an increased risk of notably dreadful thrombotic complications. The diagnosis is made by flow cytometry. Efforts have been recently performed to improve the sensitivity and the standardization of this technique. PNH is frequently associated with aplastic anemia or low‐risk myelodysplasia and may be asymptomatic. Management of the classical form of PNH has been dramatically revolutionized by the development of eculizumab, which brings benefits in terms of hemolysis, quality of life, renal function, thrombotic risk, and life expectancy. Prophylaxis and treatment of arterial and venous thrombosis currently remain a challenge in PNH.     

The molecular basis of paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disease characterized by chronic intravascular hemolysis, cytopenia due to bone marrow failure and increased tendency to thrombosis. All patients with PNH studied so far have a somatic mutation in an X-linked gene, called PIG-A (phosphatidyl inositol glycan complementation group A), which encodes for a protein involved in the biosynthesis of the glycosyl phosphatidylinositol (GPI) molecule, that serves as an anchor for many cell surface proteins. The mutation occurs in a hematopoietic stem cell and leads to a partial or total deficiency of the PIG-A protein with consequent impaired synthesis of the GPI anchor: as a result, a proportion of blood cells is deficient in all GPI-linked proteins. The mutations are spread all over the gene and in some patients more than one mutated clone have been identified. The absence of GPI-anchored proteins on PNH cells explains some of the clinical symptoms of the disease but not the mechanism that enables the PNH clone to expand in the bone marrow of patients. Both in vitro and in vivo experiments have shown that PIG-A inactivation per se does not confer a proliferative advantage to the mutated hematopoietic stem cell. Clinical observations have shown a close relationship between PNH and aplastic anemia. Taken together, these findings corroborate the hypothesis that one or more additional factors are needed for the expansion of the mutant clone. Selective damage to normal hematopoiesis could be the cause which enables the PNH clone(s) to proliferate.     

Late complications following treatment for severe aplastic anemia (SAA) with high-dose cyclophosphamide (Cy): follow-up of a randomized trial

High-dose cyclophosphamide (Cy) has been promoted as curative therapy for severe aplastic anemia (SAA). However, our randomized trial comparing antithymocyte globulin (ATG) and Cy was terminated early because of excess morbidity/early mortality in the Cy arm. We now report analysis of secondary endpoints at a median of 38 months. Relapse occurred in 6 (46%) of 13 responders in the ATG arm versus 2 (25%) of 8 in the Cy arm (P =.38). Five (31%) of 16 patients in the ATG arm and 4 (27%) of 15 patients in the Cy arm had evidence of paroxysmal nocturnal hemoglobinuria (PNH) at diagnosis, with no substantial change in the overall percentage of glycophosphatidyl inositol (GPI)-anchored protein-deficient neutrophils over extended follow-up in individual patients in either arm. Bone marrow cytogenetic abnormalities have been observed among surviving patients in both arms (2 of 14 ATG versus 1 of 12 Cy, P =.70). High-dose Cy does not prevent relapse or clonal evolution in SAA.     

Erythopoietin treatment during complement inhibition with eculizumab in a patient with paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by intravascular hemolysis leading to anemia and other clinical manifestations. Transfusions are often required to support hemoglobin at tolerable levels. A PNH patient with aplastic anemia was treated with the complement inhibitor eculizumab, followed by concurrent treatment with recombinant human erythropoietin (rHuEpo). Eculizumab alone reduced hemolysis, increased PNH red blood cell (RBC) mass, and decreased transfusions. Addition of rHuEpo during eculizumab therapy, enhanced erythropoiesis, further increased PNH RBC mass and hemoglobin levels, and rendered the patient transfusion independent for more than two years. These data show that driving erythropoiesis during eculizumab treatment provided further benefit to a patient with PNH and underlying bone marrow failure.     

New Insights into Molecular Pathogenesis of Bone Marrow Failure in Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is caused by the clonal expansion of hematopoietic stem cells with mutations of the phosphatidylinositol glycan-class A gene (PIGA). PNH clones then fail to generate glycosylphosphatidylinositol (GPI) or to express a series of GPI-linked membrane proteins including complement-regulatory proteins, resulting in complement-mediated intravascular hemolysis and thrombosis. Bone marrow failure is another characteristic feature of PNH. It is currently considered that immune-mediated injury of hematopoietic cells is implicated in PNH marrow failure as well as in aplastic anemia, a well-known PNH-related disorder. There is increasing evidence that the autoimmune attack allows PNH clones to selectively survive in the injured marrow, leading to clinical manifestations characteristic of PNH. As candidate molecules that trigger the immune attack on marrow cells, stress-inducible membrane proteins and Wilms’ tumor protein WT1 have been proposed. Among the stress-inducible proteins, GPI-linked proteins, such as cytomegalovirus glycoprotein UL16-binding protein, are distinct candidates that not only induce immune attack, but also allow PNH clones to survive the attack. Here, we overview the current understanding of the molecular pathogenesis of bone marrow failure in PNH.     

Inefficient response of T lymphocytes to glycosylphosphatidylinositol anchor-negative cells: implications for paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder in which clonal cells defective in glycosylphosphatidylinositol (GPI) biosynthesis are expanded, leading to complement-mediated hemolysis. PNH is often associated with bone marrow suppressive conditions, such as aplastic anemia. One hypothetical mechanism for the clonal expansion of GPI(-) cells in PNH is that the mutant cells escape attack by autoreactive cytotoxic cells that are thought to be responsible for aplastic anemia. Here we studied 2 model systems. First, we made pairs of GPI(+) and GPI(-) EL4 cells that expressed major histocompatibility complex (MHC) class II molecules and various types of ovalbumin. When the GPI-anchored form of ovalbumin was expressed on GPI(+) and GPI(-) cells, only the GPI(+) cells presented ovalbumin to ovalbumin-specific CD4(+) T cells, indicating that if a putative autoantigen recognized by cytotoxic cells is a GPI-anchored protein, GPI(-) cells are less sensitive to cytotoxic cells. Second, antigen-specific as well as alloreactive CD4(+) T cells responded less efficiently to GPI(-) than GPI(+) cells in proliferation assays. In vivo, when GPI(-) and GPI(+) fetal liver cells, and CD4(+) T cells alloreactive to them, were cotransplanted into irradiated hosts, the contribution of GPI(-) cells in peripheral blood cells was significantly higher than that of GPI(+) cells. The results obtained with the second model suggest that certain GPI-anchored protein on target cells is important for recognition by T cells. These results provide the first experimental evidence for the hypothesis that GPI(-) cells escape from immunologic attack.     

Immune Pathogenesis of Paroxysmal Nocturnal Hemoglobinuria

Somatic mutation in the PIG-A gene is the initial event in the pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH), but the pathophysiologic mechanisms leading to clonal expansion remain unclear. The intricate association of PNH with immune-mediated bone marrow failure syndromes, including aplastic anemia (AA), suggests an immunologic selection process for the glycosylphosphatidyl-inositol (GPI)-deficient hematopoietic clone. The mechanism for the growth advantage of PNH cells may be related to the nature of the antigens targeted by the immune response or to the function of immunomodulatory GPI-anchored proteins on the surface of the hematopoietic target cells. Alternative theories of PNH evolution may include intrinsic properties of the mutated cells, but the experimental evidence is largely lacking. Elucidation of the pathogenesis of PNH may provide key information about the causes of idiopathic AA and help understand the regulation of the hematopoietic stem cell compartment.     

Paroxysmal nocturnal hemoglobinuria: An acquired genetic disease.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder characterized by an intravascular hemolytic anemia. Abnormal blood cells lack a series of glycosylphosphatidylinositol (GPI)-anchored proteins. The lack of GPI-anchored complement regulatory proteins, such as decay-accelerating factor (DAF) and CD59, results in complement-mediated hemolysis and hemoglobinuria. In the affected hematopoietic cells from patients with PNH, the first step in biosynthesis of the GPI anchor is defective. At least four genes are involved in this reaction step, and one of them, an X-linked gene termed PIG-A, is mutated in affected cells. The PIG-A gene is mutated in all patients with PNH reported to date. Here, we review recent advances in the understanding of the molecular pathogenesis of PNH.     

Paroxysmal nocturnal hemoglobinuria clones in severe aplastic anemia patients treated with horse anti-thymocyte globulin plus cyclosporine

Background

Clones of glycosylphosphatidylinositol-anchor protein-deficient cells are characteristic in paroxysmal nocturnal hemoglobinuria and are present in about 40–50% of patients with severe aplastic anemia. Flow cytometry has allowed for sensitive and precise measurement of glycosylphosphatidylinositol-anchor protein-deficient red blood cells and neutrophils in severe aplastic anemia.

Design and Methods

We conducted a retrospective analysis of paroxysmal nocturnal hemoglobinuria clones measured by flow cytometry in 207 consecutive severe aplastic anemia patients who received immunosuppressive therapy with a horse anti-thymocyte globulin plus cyclosporine regimen from 2000 to 2008.

Results

The presence of a glycosylphosphatidylinositol-anchor protein-deficient clone was detected in 83 (40%) patients pre-treatment, and the median clone size was 9.7% (interquartile range 3.5–29). In patients without a detectable clone pre-treatment, the appearance of a clone after immunosuppressive therapy was infrequent, and in most with a clone pre-treatment, clone size often decreased after immunosuppressive therapy. However, in 30 patients, an increase in clone size was observed after immunosuppressive therapy. The majority of patients with a paroxysmal nocturnal hemoglobinuria clone detected after immunosuppressive therapy did not have an elevated lactate dehydrogenase, nor did they experience hemolysis or thrombosis, and they did not require specific interventions with anticoagulation and/or eculizumab. Of the 7 patients who did require therapy for clinical paroxysmal nocturnal hemoglobinuria symptoms and signs, all had an elevated lactate dehydrogenase and a clone size greater than 50%. In all, 18 (8.6%) patients had a clone greater than 50% at any given time of sampling.

Conclusions

The presence of a paroxysmal nocturnal hemoglobinuria clone in severe aplastic anemia is associated with low morbidity and mortality, and specific measures to address clinical paroxysmal nocturnal hemoglobinuria are seldom required.     

Circulating PIG-A mutant T lymphocytes in healthy adults and patients with bone marrow failure syndromes

OBJECTIVE: Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematological disorder with acquired PIG-A gene mutations and absent surface expression of proteins utilizing glycosylphosphatidylinositol (GPI) anchors. PNH often follows aplastic anemia, suggesting PIG-A mutant cells have relative dominance over normal hematopoietic cells. Somatic PIG-A mutations could arise after aplasia, or healthy persons could have rare PIG-A mutant cells that expand under selection pressure. METHODS: We developed an in vitro negative selection method to isolate GPI-deficient T lymphocytes using aerolysin, an Aeromonas toxin that binds GPI anchors and induces cell lysis. Peripheral blood mononuclear cells (PBMC) from normal adults and patients with PNH or other bone marrow failure syndromes were analyzed. RESULTS: From healthy adults, 166 T lymphocyte clones with deficient GPI-linked surface protein expression (CD55, CD59) were isolated. The mean mutant frequency (M(f)) of aerolysin-resistant clones was 17.8 +/- 13.8 per 10(6) PBMC, range 5.0-59.6 per 10(6) cells. Clones had a Class A complementation defect and distinct PIG-A mutations. Patients with PNH had elevated aerolysin-resistant M(f) values averaging 19 x 10(-2), a 10,000-fold difference. Two patients with Fanconi anemia and two others with mild aplastic anemia had M(f) values less than 15 x 10(-6), but two with recovering aplastic anemia had M(f) values of 20 x 10(-4), representing an intermediate value between normal persons and PNH patients. CONCLUSION: Identification of PIG-A mutant T lymphocytes in healthy adults suggests PNH could develop following intense negative selection of hematopoiesis, with clonal outgrowth of naturally occurring PIG-A mutant stem cells.     

Treatment of paroxysmal nocturnal hemoglobinuria (PNH)

Paroxysmal nocturnal hemoglobinuria is an acquired clonal disorder of the hematopoietic stem cell in which intravascular hemolysis is due to an intrinsic defect in the membrane of red cells that makes them increasingly susceptible to lysis by complement. The phenotypic hallmark of PNH cells is an absence or marked deficiency of GPI-anchored proteins such as CD 59+, CD 55+ and others which normally protect cells from the action of complement. PHN is closely associated with aplastic anemia. Some degree of bone marrow failure is always present. Management of PNH is complicated by a highly variable clinical picture and course. Some patients have severe anemia aggravated by hemolytic crises and associated thromboses. Bone marrow failure is accompanied with frequent infections and hemorrhagic manifestations due to thrombocytopenia. With the exception of marrow transplantation, no definite therapy is available. In the exceptional circumstance in which the patient has a syngeneic twin, bone marrow transplantation is the most appropriate therapy for severe PNH because of absence of graft-versus-host disease. In general syngeneic transplantation without preconditioning has been unsuccessful because abnormal hematopoiesis returns. Allogeneic bone marrow transplantation has been used, but the transplant-associated morbidity and mortality are high due mainly to the fatal graft-versus-host disease and severe posttransplant marrow failure. Use of an unrelated donor transplant has to be considered as contraindicated. PNH is associated with striking predisposition to intravascular thrombosis which often involves the portal system or the brain. Fatal thromboses account for about 40-50% of all deaths in patients with PNH. The etiology of the thrombophilia in PNH is not fully clarified. Anticoagulation or thrombolytic therapy is required for treatment of venous thrombosis, the latter vena cava. Prophylactic anticoagulation in patients without contraindications such as severe thrombocytopenia seems to be justified. However, whether such therapy may be efficacious in reducing the incidence of thromboses or affect survival is conjectural. PNH patients have varying degree of platelet activation and some authors suggest that antiplatelet therapy might be efficacious in reducing the incidence and severity of venous thrombosis in PNH. Pregnancy is hazardous. Female patients should avoid the use of oral contraceptives. Pregnant patients require combined care of an experienced hematologist and obstetrician specialized in the management of high-risk pregnancies.     

Bone marrow failure in Shwachman-Diamond syndrome does not select for clonal haematopoiesis of the paroxysmal nocturnal haemoglobinuria phenotype

Bone marrow failure is believed to be the underlying condition that drives the expansion of the paroxysmal nocturnal haemoglobinuria (PNH) clone. Indeed, circulating PNH blood cells have been identified in patients with acquired aplastic anaemia and with hypoplastic myelodysplasia. Whether PNH blood cells are also present in patients with inherited aplastic anaemia has not been reported. We screened a large group of patients diagnosed with Shwachman-Diamond Syndrome (SDS) for PNH blood cells. None of the patients analysed had detectable circulating PNH blood cells, indicating that bone marrow failure in SDS does not select for PNH progenitor cells.     

Clinical and molecular aspects of 23 patients affected by paroxysmal nocturnal hemoglobinuria

We reviewed clinical and molecular data of 23 consecutive unrelated patients affected by paroxysmal nocturnal hemoglobinuria (PNH) (19 with hemolytic PNH, 3 with aplastic anemia/PNH, and 1 with myelodysplasia/PNH syndrome) with a mean follow-up of 11.8 years. Five patients had thrombotic episodes, and 10 needed regular blood transfusions; 2 died for cerebral hemorrhage and kidney failure, and 2 spontaneously recovered from PNH. Twenty different PIG-A gene mutations were detected in 21/23 patients: 15 frameshift, 1 splicing, 2 nonsense, and 2 missense mutations. Two mutations (DelG341 and IVS2 +1g-a) were detected twice. A PIG-A mutated clone was also revealed in the two patients in complete clinical remission. One patient with aplastic anemia/PNH syndrome was treated with two courses of antilymphocyte globulin and cyclosporin with partial sustained response. Six patients were given rHu-EPO 150 U/kg/day s.c. for at least 6 months: one became transfusion-independent for 8 months and then discontinued treatment for clinical complications; one displayed a mean rise of Hb of 1.5 g/dL and is currently maintaining Hb levels higher than 9 g/dL after 54 months of therapy. Mutation specific quantitative-competitive PCR showed that the rise of hemoglobin was related to an increase of PIG-A negative molecules, suggesting that the efficacy of rHu-EPO therapy may be due to the stimulation of the abnormal clone.     

The aplastic anemia-paroxysmal nocturnal hemoglobinuria syndrome

Occasionally it is difficult to differentiate paroxysmal nocturnal hemoglobinuria (PNH) from idiopathic aplastic anemia in patients who present with pancytopenia and an aplastic bone marrow. Patients with PNH may not have an abnormal acid hemolysis test, and patients with aplastic anemia may present with evidence of abnormal sucrose lysis, acid hemolysis, and antibody-mediated complement hemolysis. Demonstration of a population of red blood cells which are highly susceptible to antibody-mediated complement lysis makes a diagnosis of PNH probable. Donor red blood cell survival studies, which distinguish intracorpuscular from extracorpuscular hemolytic disorders, permit differentiation of the two disorders.     

Molecular Genetics of Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by the clonal expansion of glycosylphosphatidylinositol (GPI)-deficient cells that leads to complement-mediated hemolysis. A somatic mutation in the PIG-A gene involved in GPI biosynthesis causes a deficiency of GPI-anchored proteins. However, it is evident that the clonal expansion of GPI-deficient cells is not caused by only the PIG-A mutation and that other changes should be involved in the development of PNH. Some patients with aplastic anemia (AA) develop PNH. Furthermore, it has been reported that most patients with AA and refractory anemia (RA) who carry HLA-DRB1*15 and show a good response to immunosuppressive therapies have an expanded population of GPI-deficient clones. This finding, together with recent data showing resistance of GPI-deficient cells to cytotoxic cells, suggests that GPI-deficient cells escape immunologic attack and are positively selected in the autoimmune environment. However, GPI-deficient clones found in AA and RA are generally small and do not increase to near-complete dominance. Therefore, 1 or more additional genetic abnormalities that confer the growth phenotype on GPI-deficient cells are probably required for fully developed PNH or so-called florid PNH. The next 10 years should witness the discovery of the molecular mechanisms of immunologic selection and the identification of abnormalities involved in the further clonal expansion of PNH cells.     

Myeloblastic leukemoid reaction in paroxysmal nocturnal hemoglobinuria associated with myelodysplasia.

Paroxysmal nocturnal hemoglobinuria (PNH) has been observed to evolve into myelofibrosis and acute myeloid leukemia. Myeloblastic leukemoid reaction has not been described in PNH. We described a patient with PNH with myelodysplasia and septicemia. The marrow aspirates showed a picture of myeloblastosis which subsided when sepsis was controlled. The myeloblastic leukemoid reaction in our patient related to overwhelming sepsis, splenectomy and overt hemolysis.