Jennifer A. Tooze

Multiparametric analysis of immature cell populations in umbilical cord blood and bone marrow.

Abstract: Adult stem cells are finding increased therapeutic potential not least in tissue regeneration protocols. The cell sources being proposed for such protocols include embryonic, umbilical cord blood (CB) and adult bone marrow (BM). Although embryonic sources are controversial, CB and marrow are available immediately. The appropriate cells of use in these sources are considered to be extremely rare and a characterisation of the starting cell source is important for the development of adult stem cell protocols and ex vivo expansion. Umbilical CB and BM mononuclear cells were labelled for the antigens CD34, CD133, CD117, CD164, Thy‐1 or CD38, and additional intracellular CD34 antigen. Three dimensional flow‐cytometric analyses were carried out together with dual laser confocal microscope analysis for antigen profile expression. Variable levels of immaturity were detected on CB and BM populations using internal and external CD34 antigen. For CB and BM cells, internal CD34 (intCD34+) could be detected on co‐expressing CD133+ cells before expression of external CD34 antigen (extCD34+). CD38 co‐expression analysis also showed that a small but distinct group of cells expressing low CD38 and no external CD34 antigen could be detected. Additional phenotyping of these cells using CD117, Thy‐1, CD164 and CD133 demonstrated variable primitive status detectable within the external CD34− population. Newly harvested primary CB and BM populations were shown to contain not only cellular populations of known standard sequential maturity but also populations of more extreme rarity. The presence of cells which lacked extracellular CD34 antigen, in both CB and BM, but which possessed CD133, has important implications for purification of human stem cells in clinical applications.

Clinical relevance of cytogenetic abnormalities at diagnosis of acquired aplastic anaemia in adults

The outcome of 81 adult aplastic anaemia patients who had successful cytogenetics at diagnosis and received immunosuppressive therapy was evaluated. Ten patients had an abnormal karyotype, six of which had a trisomy. Four of five evaluable patients with a trisomy responded. One patient with monosomy 7 achieved a complete response and later developed haemolytic paroxysmal nocturnal haemoglobinuria but no recurrence of monosomy 7. None of the patients with a non‐numerical karyotypic abnormality responded. No significant differences in survival or later clonal disorders were observed between patients with a normal karyotype and those with an abnormal karyotype.

Deficiency of glycosylphosphatidyl inositol‐anchored proteins in patients with aplastic anaemia does not affect response to immunosuppressive therapy

Deficient expression of glycosylphosphatidyl inositol (GPI)‐anchored proteins in aplastic anaemia (AA) patients has previously been reported to be associated with a poor response to immunosuppressive (IS) therapy. Here we report the response to IS therapy of 111 patients with AA and correlate this with GPI‐anchored protein expression on peripheral blood cells by flow cytometry. A GPI‐anchored protein deficient population was identified in 15% (17/111) of patients with AA who had a negative Hams test and no laboratory evidence of haemolysis. Patients were treated with antilymphocyte globulin and/or cyclosporin A, or oxymetholone. Bone marrow transplantation was performed in 12 patients, seven of whom had not responded to IS therapy. In patients tested for GPI‐anchored protein expression prior to IS therapy there was no difference in response rate to IS therapy between AA patients with a GPI‐anchored protein deficiency and those with normal GPI‐anchored protein expression (50% response rate versus 75%, respectively). Survival in these two groups was similar at 90% with follow‐up over 140 months from diagnosis. Eight of the 17 AA patients who developed a GPI‐anchored protein‐deficient population later went on to develop a positive Hams test. From this study we demonstrate a lower incidence of GPI‐anchored protein deficiency in AA patients compared with previous reports. In addition we have shown that the presence of a GPI‐anchored protein‐deficient cell population in patients with AA who have a negative Hams test is not a poor prognostic factor in terms of response and survival after IS therapy.

Myelodysplasia following aplastic anaemia–paroxysmal nocturnal haemoglobinuria syndrome after treatment with immunosuppression and G-CSF: evidence for the emergence of a separate clone

Myelodysplasia (MDS) and aplastic anaemia–paroxysmal nocturnal haemoglobinuria (AA/PNH) syndrome developed in a severe aplastic anaemia (AA) patient after treatment with immunosuppressive (IS) therapy. Glycosylphosphatidyl inositol (GPI)‐linked proteins were determined, and during the AA/PNH phase, a high proportion of neutrophils were found to be negative, without clinical evidence of haemolysis. However, MDS developed with cytogenetic abnormalities of monosomy 7, 9q− and a rearranged chromosome 6; the GPI‐linked protein negative cells were completely replaced by positively expressing cells. This represents the emergence of a GPI‐linked protein positive myelodysplasia clone arising separately from an AA/PNH clone.

Clonal Evolution Of Aplastic Anaemia To Myelodysplasia/Acute Myeloid Leukaemia and Paroxysmal Nocturnal Haemoglobinuria

Aplastic anaemia (AA) is a non-malignant haemopoietic disorder characterised by peripheral blood pancytopenia and a hypocellular bone marrow. Successful management of acquired AA including treatment with immunosuppressive agents, mainly antithymocyte globulin (ATG) and cyclosporin or allogeneic haemopoietic stem cell transplantation, has resulted in long-term survival of many patients. The later evolution of complicating clonal disorders such as paroxysmal nocturnal haemoglobinuria, myelodysplasia and acute myeloid leukaemia in patients treated with immunosuppressive therapy may be a manifestation of the natural history of the aplasia, the development of which may or may not be increased by immunosuppressive therapy. A persistent, profound deficiency and/or defect in the stem cell compartment, despite haematological recovery after immunosuppressive therapy, may create an unstable situation which predisposes to later clonal disorders. A review of the progression of AA to clonal disorders is now outlined.

Glycosylphosphatidyl-inositol (GPI)-linked protein deficiency on the platelets of patients with aplastic anaemia and paroxysmal nocturnal haemoglobinuria: two distinct patterns correlating with expression on neutrophils.

Deficiencies in glycosylphosphatidyl‐inositol (GPI)‐linked proteins on erythrocytes and leucocytes in patients with paroxysmal nocturnal haemoglobinuria (PNH) are well known; however, expression on platelets in these patients is less well documented. We have studied CD55 and CD59 on the platelets of PNH and aplastic anaemia (AA) patients using flow cytometry. In all cases of PNH, CD55 and CD59 negative populations of platelets were detected with single or bimodal distribution and these results showed close correlation with the CD55 and CD59 patterns of neutrophils. Previous published studies have not demonstrated this distribution. We suggest that our findings may be due to the methodology used.

Evaluation of the haemopoietic reservoir in de novo haemolytic paroxysmal nocturnal haemoglobinuria

Summary. Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder of the haemopoietic stem cell (HSC). The pathogenetic link with bone marrow failure is well recognized; however, the process of clonal expansion of the glycosylphosphatidylinositol (GPI)‐deficient cells over normal haemopoiesis remains unclear. We have carried out detailed analysis of the stem cell population in 10 patients with de novo haemolytic PNH using the long‐term culture‐initiating cells (LTC‐IC) assay in parallel with measurements of CD34+ cells and mature haemopoietic progenitors, granulocyte–macrophage colony‐forming unit (CFU‐GM) and CFU‐erythroid [burst‐forming units erythroid (BFU‐E) + CFU granulocyte/erythroid/macrophage/megakaryocyte (GEMM)]. All patients had hypercellular bone marrows with erythroid hyperplasia, normal blood counts or mild peripheral blood cytopenias, increased reticulocyte counts and evidence of deficient GPI‐anchored proteins. We found a significant reduction in the LTC‐IC frequency in the CD34+ compartment of PNH patients (mean 2, range 1·3–3·0; n = 6) compared with normal donors (mean 13, range 5·2–45·5; n = 21) (P < 0·0001). Furthermore, there was a significant reduction in the erythroid compartment [CFU‐E/105 bone marrow mononuclear cells (BMMC) and CFU‐E/105 CD34+ cells] of PNH patients, but no significant difference in the granulocyte–monocyte precursors (CFU‐GM/105 BMMC or CFU‐GM/105 CD34+ cells) compared with normal donors, suggesting that there is a defect in the early stem cell pool in PNH patients without clinical or haematological evidence of bone marrow failure.

Absence of N-RAS point mutations in peripheral blood cells of patients with aplastic anaemia and paroxysmal nocturnal haemoglobinurea.

Summary. The myelodysplastic syndromes (MDS) have a significant frequency of evolution into acute myeloid leukaemia (AML). Approximately 30% of MDS patients show activating mutations of the N‐RAS proto‐oncogene, and these patients are at increased risk of leukaemic evolution. Long‐term survivors of aplastic anaemia (AA) and paroxysmal nocturnal haemoglobinurea (PNH) are also at significant risk of developing AML. We have screened peripheral blood DNA from 42 AA patients and 15 PNH patients for the presence of N‐RAS point mutations. No mutations were detected in these samples, indicating that the mechanisms of evolution into AML may be different from those in MDS.

Differential apoptosis and Fas expression on GPI-negative and GPI-positive stem cells: a mechanism for the evolution of paroxysmal nocturnal haemoglobinuria.

Summary. Paroxysmal nocturnal haemoglobinuria (PNH) has a dual pathogenesis. PIG‐A mutations generate clones of haemopoietic stem cells (HSC) lacking glycosylphosphatidylinositol (GPI)‐anchored proteins and, secondly, these clones expand because of a selective advantage related to bone marrow failure. The first aspect has been elucidated in detail, but the mechanisms leading to clonal expansion are not well understood. We have previously shown that apoptosis and Fas expression in HSC play a role in bone marrow failure during aplastic anaemia. We have now investigated apoptosis in PNH. Ten patients were studied. Apoptosis, measured by flow cytometry, was significantly higher among CD34+ cells from patients compared with healthy controls. Fas expression was also increased. Cells that were stained for CD34, CD59 and apoptosis showed a significantly lower apoptosis in CD34+/CD59− compared with CD34+/CD59+ cells from the same patient. In three patients, staining for CD34, CD59 and Fas revealed lower Fas expression on CD34+/CD59− cells. Differential apoptosis of CD34+/CD59− HSC may be sufficient in itself to explain the expansion of PNH clones in the context of aplastic anaemia. In addition to demonstrating a basic mechanism underlying PNH clonal expansion, these results suggest further hypotheses for the evolution of PNH, based on the direct or indirect resistance of GPI‐negative HSC to pro‐inflammatory cytokines.

Paroxysmal nocturnal haemoglobinuria due to an 88 bp direct tandem repeat insertion in the PIG‐A gene

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired stem cell abnormality which frequently develops in patients with aplastic anaemia. The disease is due to somatic mutations in the PIG‐A gene, and a variety of mutations have been reported. The majority are point mutations, or small insertions and deletions resulting in a frameshift. Previous insertions reported have all been within the range of 1–10 bp. We describe here a patient with PNH due to a large insertion of 88 bp; DNA sequencing showed this to be a tandem repeat of PIG‐A sequences. The same mutation could be found in granulocytes and lymphocytes, indicating a pluripotent stem cell origin.