Stem cell transplantation for paroxysmal nocturnal haemoglobinuria in childhood

Paroxysmal nocturnal haemoglobinuria (PNH) is a clonal haematopoietic disorder characterized by chronic or intermittent intravascular haemolysis, variable cytopenia and an increased risk of thrombosis. Stem cell transplantation (SCT) is a curative therapeutic option, but its risks must be carefully weighed against the natural course of PNH. World-wide experience with SCT for PNH in the paediatric age group is scarce. We report on two adolescents suffering from PNH with life-threatening complications who were successfully transplanted from unrelated donors. Indications and techniques of SCT in childhood PNH are discussed and an overview of the literature is given.     

Deregulated expression of HMGA2 is implicated in clonal expansion of PIGA deficient cells in paroxysmal nocturnal haemoglobinuria

Patients with paroxysmal nocturnal haemoglobinuria (PNH) have expanded clonal cells bearing a somatic mutation in the PIGA gene. Our previous study on two PNH patients with chromosome 12 rearrangements demonstrated the involvement of HMGA2 expression in clonal expansion. The present study investigated HMGA2 expression in PNH patients without chromosomal abnormalities. The expression of short HMGA2 with latent exon was significantly high in peripheral blood cells from 18 of 24 patients. Over-expression of truncated HMGA2 in mouse bone marrow cells caused expansion in recipient mice. These results support the idea that deregulated expression of HMGA2 causes expansion of PNH cells.     

Genotypic, immunophenotypic and clinical features of Thai patients with paroxysmal nocturnal haemoglobinuria

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haematological disorder characterized by complement-mediated haemolytic anaemia caused by deficiency of glycosylphosphatidylinositol (GPI) anchored proteins. Somatic mutation of an X-linked gene, PIG-A, is responsible for the defect in biosynthesis of GPI-anchor. It appears that frequency of PNH differs geographically, and seems to be more frequent in some Asian countries, such as Thailand and China. We studied a group of 34 Thai patients with PNH to see whether the somatic mutations in PIG-A, extent of deficiency of GPI-anchored proteins (complete or partial) and complication with aplastic anaemia among Thai patients are different from those in other regions. We determined 37 PIG-A mutations in 33 patients (10 base substitutions, 14 single-base deletions, five multiple-base deletions, three single-base insertions, two multiple base insertions and three others) which were found to be similar to those found in European, American and Japanese patients. Most patients had cells with a complete deficiency of CD59 (type III cells), whereas 19% and 33% of the patients with reliable data for CD59 expression had partially deficient granulocytes and erythrocytes (type II cells), respectively. Most mutations resulted in a complete loss of function of PIG-A in accordance with the prevalent PNH III phenotype. 19 patients (51%) had aplastic anaemia; their PIG-A mutations were not different from those without pre-existing aplastic anaemia. These characteristics of Thai patients are similar to patients from other regions. There was some negative correlation between mean basal Hb concentration and percentage of CD59-negative granulocytes (r = -0. 374; P = 0.0476). In addition, patients with severe anaemia (basal Hb <7 g/dl) had a significantly higher percentage of affected granulocytes than those with mild anaemia (88.5 +/- 9.4 v 64.9 +/- 25.9; P = 0.01). The data suggest that the severity of anaemia in PNH depends partly on the size of the PNH clone.     

Eculizumab, a terminal complement inhibitor, improves anaemia in patients with paroxysmal nocturnal haemoglobinuria

In paroxysmal nocturnal haemoglobinuria (PNH), chronic destruction of PNH red blood cells (RBCs) by complement leads to anaemia and other serious morbidities. Eculizumab inhibits terminal complement-mediated PNH RBC destruction by targeting C5. In the phase III, double-blind, placebo-controlled, TRIUMPH study, eculizumab reduced haemolysis, stabilized haemoglobin levels, reduced transfusion requirements and improved fatigue in patients with PNH. Herein, we explored the effects of eculizumab on measures of anaemia in patients from the TRIUMPH study and the open-label SHEPHERD study, a more heterogeneous population. Eculizumab reduced haemolysis regardless of pretreatment transfusion requirements and regardless of whether or not patients became transfusion-dependent during treatment ( P  <   0·001). Reduction in haemolysis was associated with increased PNH RBC counts ( P  <   0·001) while reticulocyte counts remained elevated. Eculizumab-treated patients demonstrated significantly higher levels of haemoglobin as compared with placebo in TRIUMPH and relative to baseline levels in SHEPHERD ( P  <   0·001 for each study). Eculizumab lowered transfusion requirement across multiple pretreatment transfusion strata and eliminated transfusion support in a majority of both TRIUMPH and SHEPHERD patients ( P  <   0·001). Patients who required some transfusion support during treatment with eculizumab showed a reduction in haemolysis and transfusion requirements and an improvement in fatigue. Eculizumab reduces haemolysis and improves anaemia and fatigue, regardless of transfusion requirements.     

Natural history of paroxysmal nocturnal haemoglobinuria using modern diagnostic assays

Paroxysmal nocturnal haemoglobinuria (PNH) is an uncommon, acquired disorder of blood cells caused by mutation of the phosphatidylinositol glycan class A (PIG-A) gene. The disease often manifests with haemoglobinuria, peripheral blood cytopenias, and venous thrombosis. The natural history of PNH has been documented in retrospective series; but there has only been one study that correlated the more sensitive and specific flow cytometric assays that have become available in the last decade with severe symptoms associated with PNH. In a retrospective analysis of 49 consecutive patients with PNH evaluated at Johns Hopkins, large PNH clones were associated with an increased risk for thrombosis as well as haemoglobinuria, abdominal pain, oesophageal spasm, and impotence. Of the 14 (29%) patients that developed thrombosis, nine died; six of these from complications related to thromboses. According to logistic regression modelling, for a 10% change in PNH clone size, the odds ratio for risk of thrombosis was estimated to be 1.64. No patient with <61% PNH granulocytes developed a thrombosis, whereas 12 of 22 patients (54.5%) with > or =61% PNH granulocytes manifested with thrombosis. These data not only confirm that the size of the PNH clone correlates with the risk for thrombosis, but they also suggest a correlation of PNH clone size to more symptomatic PNH.     

Tissue plasminogen activator for hepatic vein thrombosis in paroxysmal nocturnal haemoglobinuria

Abstract. Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder thought to arise in a multipotent haemopoietic stem cell. A distinct clinical feature is a tendency to thrombosis, with a particular predilection for the hepatic veins (Budd-Chiari syndrome). We report here on two patients with PNH who developed hepatic vein thrombosis (HVT) and who were treated with tissue plasminogen activator (t-PA). Both patients had a marked clinical and radiological improvement following the t-PA treatment and remain well over 2 years and 6 years after the treatment. This method of thrombolysis for HVT occurring in PNH has only been reported in two previous patients with limited follow-up. We suggest that this therapy is a useful first-line treatment for PNH patients who develop HVT.     

Myelodysplasia in a patient with pre-existing paroxysmal nocturnal haemoglobinuria: a clonal disease originating from within a clonal disease

SUMMARY. Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemolytic anaemia, clonal in nature, due to somatic mutation. PNH may evolve to aplastic anaemia; more rarely to a myelodysplastic syndrome (MDS) or to acute myeloid leukaemia (AML). We have studied a patient who suffered from PNH and later developed refractory anaemia with ringed sideroblasts (RARS) associated with trisomy 8. By testing peripheral blood cells with appropriate antibodies we have shown that all of the red cells, neutrophils and monocytes, as well as 20% of the lymphocytes, belonged to the PNH clone; in contrast, only 43% of neutrophils and 22% of monocytes belonged to the MDS clone. We infer that the MDS must have arisen from within the PNS clone.     

High-dose cyclophosphamide does not eradicate paroxysmal nocturnal haemoglobinuria haematopoiesis in mice carrying a Piga gene mutation

Recently, high-dose cyclophosphamide (HD CY) has been used in the treatment of aplastic anaemia. Several reports have suggested that the treatment may either eradicate or suppress mutant clonal haematopoiesis such as paroxysmal nocturnal haemoglobinuria (PNH). We therefore treated mice that have a proportion of blood cells deficient in GPI-anchor molecules (PIGA-) with HD CY, and monitored their peripheral blood counts during and after treatment. HD CY produced a transient myelosuppression; however, the contribution of PIGA- haematopoiesis to the peripheral blood remained unchanged, suggesting that HD CY is unlikely to eliminate an existing PNH clone in patients treated for aplastic anaemia.     

Clinical characteristics predict response to antithymocyte globulin in paroxysmal nocturnal haemoglobinuria

Seven patients with paroxysmal nocturnal haemoglobinuria (PNH) were treated with antithymocyte globulin (ATG). Each patient received ATG (20 mg/kg/d) for 8 d and prednisone to prevent or control serum sickness. Three patients experienced a sustained improvement in at least one peripheral blood cytopenia, including one patient who had a complete trilineage response. Several pre-treatment clinical features appeared to be associated with response to ATG. All responding patients had hypoproliferative features including depressed platelet counts (<30×10⁹/l), and a minor degree of chronic haemolysis as indicated by relatively low reticulocyte counts (<100×10⁹/l), lactate dehydrogenase (<1000 U/l) and total bilirubin (<17μmol/l) levels. Responding patients continued to have chronic low-grade haemolysis after their response to immunosuppression that was similar to that observed prior to treatment. The non-responding patients had a classic haemolytic form of PNH characterized by elevated reticulocyte counts (>100×10⁹/l), lactate dehydrogenase (>2000 U/l) and total bilirubin (>17μmol/l) levels.
The impaired haemopoiesis that occurs in hypoproliferative PNH may respond to ATG treatment, but the haemolytic component of the disease, and hence the PNH clone, is not altered by immunosuppressive therapy.     

Aplastic anaemia and paroxysmal nocturnal haemoglobinuria: a study of the GPI-anchored proteins on human platelets

Twenty-six consecutive patients with acquired aplastic anaemia (AA) and nine patients with de novo paroxysmal nocturnal haemoglobinuria (PNH) were included in this study. In these 35 patients a GPI-anchored molecule defect at the platelet surface was investigated by flow-cytometry. Platelets from eight out of the nine patients with de novo PNH were found to be deficient for the GPI-anchored molecule CD55, CD58 and CD59. We also detected a GPI-anchored molecule defect on monocytes, granulocytes, and erythrocytes in all patients with de novo PNH. Among the 26 AA patients, a GPI defect was detected on platelets in five patients. Interestingly, these five patients were also found to have a GPI-anchored molecule defect on erythrocytes, whereas in 10 patients the GPI-anchored molecule defect was only detected on monocyte and polymorphonuclear (PMN) cells.     

Cytogenetic and morphological abnormalities in paroxysmal nocturnal haemoglobinuria

Paroxysmal nocturnal haemoglobinuria (PNH) is characterized by the expansion of a haematopoietic stem cell clone with a PIG-A mutation (the PNH clone) in an environment in which normal stem cells are lost or failing: it has been hypothesized that this abnormal marrow environment provides a relative advantage to the PNH clone. In patients with PNH, generally, the karyotype of bone marrow cells has been reported to be normal, unlike in myelodysplastic syndrome (MDS), another clonal condition in which cytogenetic abnormalities are regarded as diagnostic. In a retrospective review of 46 patients with a PNH clone, we found a karyotypic abnormality in 11 (24%). Upon follow-up, the proportion of cells with abnormal karyotype decreased significantly in seven of these 11 patients. Abnormal morphological bone marrow features reminiscent of MDS were common in PNH, regardless of the karyotype. However, none of our patients developed excess blasts or leukaemia. We conclude that in patients with PNH cytogenetically abnormal clones are not necessarily malignant and may not be predictive of evolution to leukaemia.     

Recent developments in the understanding and management of paroxysmal nocturnal haemoglobinuria

Paroxysmal nocturnal haemoglobinuria (PNH) has been recognised as a discrete disease entity since 1882. Approximately a half of patients will eventually die as a result of having PNH. Many of the symptoms of PNH, including recurrent abdominal pain, dysphagia, severe lethargy and erectile dysfunction, result from intravascular haemolysis with absorption of nitric oxide by free haemoglobin from the plasma. These symptoms, as well as the occurrence of thrombosis and aplasia, significantly affect patients' quality of life; thrombosis is the leading cause of premature mortality. The syndrome of haemolytic-anaemia-associated pulmonary hypertension has been further identified in PNH patients. There is currently an air of excitement surrounding therapies for PNH as recent therapeutic developments, particularly the use of the complement inhibitor eculizumab, promise to radically alter the symptomatology and natural history of haemolytic PNH.     

Elevated circulating endothelial membrane microparticles in paroxysmal nocturnal haemoglobinuria

We analysed endothelial cell membrane microparticles (ECMP) in the peripheral blood of patients with paroxysmal nocturnal haemoglobinuria (PNH) (n = 9), aplastic anaemia (AA) (n = 10), sickle cell disease (SCD) (n = 8), and healthy donors (HD) (n = 11). There was no clinically manifested thrombosis in the PNH or AA group, except one cured thrombophlebitis (PNH), while all SCD patients had a history of vaso-occlusive crises. We used three-colour flow cytometry with blood cell-specific antibodies and antibodies to endothelial antigens CD105 and CD144. Phosphatidylserine-positive microparticles were detected using the annexin V-binding (AVB) assay. The population of CD105+AVB+ ECMP was significantly (P < 0.05) higher in SCD (median: 0.568 x 10(9)/l; 25-75th percentile range: 0.351-0.976 x 10(9)/l) and PNH (0.401 x 10(9)/l; 0.19-0.441 x 10(9)/l) patients when compared with AA (0.122 x 10(9)/l; 0.061-0.172 x 10(9)/l) or HD (0.180 x 10(9)/l; 0.137-0.217 x 10(9)/l) group. Even more pronounced differences were observed in ECMP exhibiting a marker of inflammatory stimulation CD54 (CD105+CD54+). Similarly, ECMP that exhibited endothelial specific and proteolysis-sensitive antigen CD144 were increased in SCD and PNH, but not in AA. Elevated CD54+ ECMP may reflect the inflammatory status of endothelial cells in SCD and PNH, while CD144+ ECMP could indicate continuous endothelial stimulation and/or injury. Analysis of circulating ECMP appears promising to provide useful information on the status of the vascular endothelium in PNH and SCD.     

Association of clonal T-cell large granular lymphocyte disease and paroxysmal nocturnal haemoglobinuria (PNH): further evidence for a pathogenetic link between T cells, aplastic anaemia and PNH

There is mounting evidence to suggest that T-cell-mediated suppression of haemopoiesis is a pathogenetic mechanism in three bone marrow failure syndromes: aplastic anaemia (AA), paroxysmal nocturnal haemoglobinuria (PNH) and myelodysplasia (MDS). T-cell microclones can be detected by sensitive polymerase chain reaction (PCR)-based methods in all three disorders. Recently, larger clonal populations of T-cell large granular lymphocytes (T-LGLs) have been observed in some patients with AA and MDS. Here, we report the development of a large clonal T-LGL population in a patient with bona fide PNH. In this patient, we defined part of the sequence of the T-cell receptor (TCR) beta-chain gene, and we have shown that the large T-LGL population emerged from a background of multiple smaller T-cell clones. Thus, T-LGL clones in AA, MDS and PNH probably expand as a result of antigenic stimulation. It is postulated that the antigen driving clonal T-cell proliferations in these disorders exists on haemopoietic stem cells.     

Detection of GPI-anchored protein-deficient cells in patients with aplastic anaemia and evidence for clonal expansion during the clinical course

The incidence of patients with aplastic anaemia (AA) who show a deficiency of glycosylphosphatidylinositol (GPI)-anchored proteins on their peripheral blood (PB) or bone marrow (BM) cells is higher than that previously reported. We analysed the expression of CD55 or CD59 on PB and BM cells and those of colonies and bursts formed in cultures with marrow cells from AA patients. 4/21 (19%) AA cases later developed paroxysmal nocturnal haemoglobinuria (PNH). 7/17 (41. 2%) AA cases showed a subpopulation without GPI-anchored proteins. The defect characteristic for PNH was observed only on the colonies/bursts but not on the PB or BM cells in three cases. We found the affected colonies/bursts in cultures using thawed marrow cells which had been cryopreserved at the diagnosis of aplasia in one patient who developed PNH 3 years later. Two different mutations of the PIG-A gene were found in the colonies/bursts at the time of AA. The nucleotide sequences were identical between the colonies/bursts at the time of AA and those after the transition to PNH. However, one of the mutations was detected only at the haemopoietic progenitor level but not in PB granulocytes. There may be latent subclones with PNH abnormalities which could expand to the level of clinical detection, or which do not progress and remain indolent for a relatively long time, implying the different extents of clonal expansion among the affected progenitors.     

Increased frequency of somatic mutations at glycophorin A loci in patients with aplastic anaemia, myelodysplastic syndrome and paroxysmal nocturnal haemoglobinuria

Paroxysmal nocturnal haemoglobinuria (PNH), aplastic anaemia (AA) and myelodysplastic syndrome (MDS) are haemopoietic stem cell disorders. These disorders have some features in common, and a percentage of cases progress to acute leukaemia. We speculated that changes in gene stability are involved in the pathogenesis of these haemopoietic stem cell disorders. Therefore we investigated in vivo mutation frequencies in these disorders by erythrocyte glycophorin A (GPA) mutation assay. The assay enumerates NO or NN variant cells in 10⁶ erythrocytes of the MN type using a flowcytometric technique. Patients undergoing chemotherapy known to be at risk of hypermutageneity were also studied. Events exceeding the 95th percentile of healthy donors (≧ 32 and 34 events, respectively for NO and NN variants) were defined as abnormal. Abnormal events in the NO variants were found in three out of seven patients undergoing chemotherapy, two out of nine patients with AA, two out of seven patients with MDS, and four out of nine patients with PNH. Abnormal events in the NN variants were found in three out of seven patients undergoing chemotherapy, two out of nine patients with AA, one out of seven patients with MDS, and two out of nine patients with PNH. These results suggest that not only PIG-A, but also other genes including the GPA gene, are hypermutable in haemopoietic stem cell disorders, and that mutagenic pressure and/or gene instability can contribute to the pathogenesis of these disorders.     

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.