Use of the haemopoietic progenitor cell count of the Sysmex SE-9500 to refine apheresis timing of peripheral blood stem cells.

The Sysmex SE-9500 automated cell counter provides an estimate of immature cells referred to as 'haemopoietic progenitor cells' (HPC). The aim of this study was to relate the HPC count to CD34+ cell levels in mobilized peripheral blood and to determine whether the HPC count was valuable in predicting apheresis yields of CD34+ cells. Studies were performed on 114 samples from 67 patients undergoing progenitor cell mobilization.

Primitive myeloid cells express high levels of phospholipase A(2) activity in the absence of leukotriene release: selective regulation by stem cell factor involving the MAP kinase pathway.

The activation of phospholipase A(2) (PLA(2)) with release of eicosanoids and prostanoids in mature myeloid cells and the augmentation (priming) of this activity by cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) are central to the inflammatory process. Yet, there are few data concerning PLA(2) activity and its regulation by growth factors in primary hematopoietic cells. We therefore analyzed the PLA(2) activity of mobilized human CD34 antigen-positive (CD34(+)) stem cells by quantitation of the extracellular release of (3)H-arachidonate.

Changes in signal transduction downstream from the granulocyte-macrophage colony-stimulating factor receptor during differentiation of primary hemopoietic cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifunctional cytokine, having different effects on primitive hemopoietic cells and terminally differentiated end-cells of the myeloid lineage. Human primitive hemopoietic cells (CD34+) were obtained from the peripheral blood after mobilization and induced to proliferate and then differentiate with a combination of cytokines in vitro. Cells at different time points were then used to analyze the expression of the GM-CSF receptor and GM-CSF mediated activation of the JAK 2-STAT 5 and MAP kinase pathways.

Peripheral blood stem cell versus autologous bone marrow transplantation for Hodgkin's disease: equivalent survival outcome in a single-centre matched-pair analysis.

A matched-pair retrospective analysis was used to compare 70 patients who had undergone peripheral blood stem cell transplantation (PBSCT) with 70 who had undergone autologous bone marrow transplantation (ABMT) for Hodgkin's disease. All transplants took place at a single centre using the same conditioning regimen (BEAM). Patients were matched for sex, previous chemotherapy and relapse status: factors which have previously been shown to have prognostic significance for transplant outcome in Hodgkin's disease at this centre.

Identification of the t(15;17) in AML FAB types other than M3: evaluation of the role of molecular screening for the PML/RARalpha rearrangement in newly diagnosed AML. The Medical Research Council (MRC) Adult Leukaemia Working Party.

Acute promyelocytic leukaemia (APL) is characterized by the t(15;17) leading to the formation of PML-RARalpha and RARalpha-PML fusion genes; this rearrangement has been considered both diagnostic for, and restricted to, this subtype of acute myeloid leukaemia (AML FAB M3). We describe two cases of AML with the t(15;17) associated with a PML/RARalpha rearrangement which lacked typical APL morphology, classified as FAB M1 and M2 respectively. In both cases morphological review revealed small populations of cells which exhibited some features associated with APL.

High dose therapy for Hodgkin's disease.

High dose therapy with haemopoietic stem cell support is now the standard approach to patients failing conventional dose chemotherapy for Hodgkin's disease (HD) although there is a lack of data from randomized trials substantiating this practise. There is even less data to justify transplanting patients in first complete remission although this might be appropriate for a small minority of patients. Approximately 40% of patients receiving high dose salvage therapy have achieved prolonged progression free survival with slightly better results in some centres.

A large proportion of patients with a diagnosis of essential thrombocythemia do not have a clonal disorder and may be at lower risk of thrombotic complications.

Essential thrombocythemia (ET) is traditionally considered to be a clonal disorder. No specific karyotypic abnormalities have been described, but the demonstration of clonality using X-chromosome inactivation patterns (XCIPs) has been used to differentiate ET from a non-clonal reactive thrombocytosis. However, these assays may be difficult to interpret, and contradictory results have been reported.

Quantification of X-chromosome inactivation patterns using RT-PCR of the polymorphic iduronate-2-sulphatase gene and correlation of the results obtained with DNA-based techniques.

Most studies using X-chromosome inactivation patterns (XCIPs) to determine clonality in females have used polymorphic DNA loci, but interpretation may be complicated by the complexity of the differential methylation patterns necessary to distinguish active and inactive X-chromosomes. Recent description of polymorphisms within the transcribed region of three X-chromosome genes has enabled XCIP analysis of allele expression at the RNA level, which should circumvent this problem. We report here a quantitative RT-PCR assay for one of these loci, the iduronate-2-sulphatase (IDS) gene, using a mismatch primer to introduce a Bc/l cutting site in one of the alleles.

Activating point mutations in the betaC chain of the GM-CSF, IL-3 and IL-5 receptors are not a major contributory factor in the pathogenesis of acute myeloid leukaemia.

A number of mutant growth factor receptors have been described which are constitutively activated and confer factor independence on growth factor dependent cells, possibly through constitutive dimerization in the absence of ligand or induction of a conformational change. Mutations in receptor chains may therefore contribute to the pathogenesis of haemopoietic malignancies, for example by causing constitutive receptor activation or uncontrolled downstream signalling. Since most of the activated mutants reported for the betaC chain of the GM-CSF/IL-3/IL-5 receptor involve point mutations or truncations of the extracellular domain, we have analysed the coding sequence of this region using RT-PCR-SSCP of RNA from blast cells of 31 patients with acute myeloid leukaemia (AML).

The activating splice mutation in intron 3 of the thrombopoietin gene is not found in patients with non-familial essential thrombocythaemia.

Essential thrombocythaemia (ET) is a condition of unknown aetiology characterized by sustained thrombocytosis in the absence of a detectable systemic cause. Although usually considered a clonal disease affecting myeloid cells, recent data indicate that a significant proportion of patients have polyclonal haemopoiesis. In some patients the thrombopoietin (TPO) levels are normal or raised.

A case of EBV-associated lymphoproliferative disease following high-dose therapy and CD34-purified autologous peripheral blood progenitor cell transplantation.

A fatal case of EBV-associated lymphoproliferative disorder arising after a CD34-selected autologous peripheral blood stem cell transplant is reported in a patient with multiple myeloma in first plateau phase. It is suggested that this is likely to be a consequence of the accessory cell depletion associated with the CD34+ cell purification and it is recommended that a source of autologous T cells is stored before transplantation to be used if a severe opportunistic infection or EBV lymphoma arises post-transplantation.

Outcome of secondary myeloid malignancy in Hodgkin's disease: the BNLI experience.

In Hodgkin's disease where the majority of patients are long-term survivors secondary myeloid malignancies are a well-documented complication. The survival of those who develop secondary myelodysplasia/acute myeloid leukaemia (MDS/AML) is historically said to be extremely poor. This study from the BNLI database of over 4900 patients with Hodgkin's disease reports long-term follow-up of 30 patients with secondary MDS/AML.

Progenitor cell yields are frequently poor in patients with histologically indolent lymphomas especially when mobilized within 6 months of previous chemotherapy.

High-dose therapy with peripheral blood stem cell (PBSC) support is a frequently used treatment option in younger patients with poor prognosis histologically indolent (low-grade) non-Hodgkin's lymphoma (NHL), usually at the time of second or subsequent response to conventional-dose therapy. We have undertaken PBSC collection in 57 patients with histologically indolent NHL mobilized with either cyclophosphamide 1.5 g/m2 or the ESHAP regimen, followed by daily G-CSF.

Feasibility of multidrug resistance (MDR-1) gene transfer in patients undergoing high-dose therapy and peripheral blood stem cell transplantation for lymphoma.

We have performed a pilot study of MDR-1 gene transfer in patients receiving CD34-selected peripheral blood stem cell (PBSC) transplant for lymphoma. To ensure minimum engraftment thresholds and facilitate CD34 purification, mobilisation of > 2 x 10(6) CD34 cells/kg was a condition for recruitment. Of 11 patients counselled for study entry, only five achieved this target in a single apheresis.

A study to determine whether trisomy 8, deleted 9q and trisomy 22 are markers of cryptic rearrangements of PML/RARalpha, AML1/ETO and CBFB/MYH11 respectively in acute myeloid leukaemia. MRC Adult Leukaemia Working Party. Medical Research Council.

Acute myeloid leukaemia (AML) patients with either a t(15;17), t(8;21) or inv(16) at diagnosis have 'good-risk' disease with a favourable response to therapy and improved survival. Detection of cryptic fusion genes created by these translocations has been reported where there is no cytogenetic evidence of the corresponding abnormality. It is likely that these cases share the same favourable prognosis.

Raised neutrophil phospholipase A2 activity and defective priming of NADPH oxidase and phospholipase A2 in sickle cell disease.

Intermittent painful crises due to vasoocclusion are the major clinical manifestation of sickle cell disease (SCD), but subclinical episodes may also occur. There is sparse evidence for the involvement of neutrophils in the pathophysiology of SCD, but production of cytokines by the damaged endothelium might influence neutrophil function and modulate responses to subsequent cytokine exposure. In addition, the activation of neutrophils in the microcirculation could itself exacerbate vasoocclusion.

Mutations of the granulocyte-colony stimulating factor receptor in patients with severe congenital neutropenia are not required for transformation to acute myeloid leukaemia and may be a bystander phenomenon.

Point mutations of the granulocyte-colony stimulating factor receptor (G-CSFR) resulting in an abnormally truncated receptor have been implicated in the pathogenesis of some cases of severe congenital neutropenia (SCN) and in the transformation of SCN to acute myeloid leukaemia (AML). We report here studies in 11 patients with SCN. No mutations were detected in the one patient who developed AML indicating that development of such mutations is not a prerequisite for transformation.

Differentiation-linked changes in granulocyte-macrophage colony-stimulating factor receptor mediated signalling in the HL-60 promyelocytic cell line.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the proliferation and maturation of immature myeloid progenitor cells and primes mature cell function in phagocytes. To investigate whether the biochemical events following the binding of GM-CSF to its receptor are differentiation dependent we analysed GM-CSF mediated activation of the JAK 2-STAT 5 and MAP kinase pathways in undifferentiated HL-60 cells and HL-60 cells induced to differentiate with dimethyl sulphoxide (DMSO) or retinoic acid (RA). GM-CSF stimulated MAP kinase activation in both the undifferentiated and differentiated HL-60 cells.

IL-1 receptor antagonist gene polymorphism in patients with secondary acute myeloid leukaemia.

Acute myeloid leukaemia (AML) may not only occur as a de novo disease but may evolve from a preceding myelodysplastic syndrome (MDS) or may result from therapy for a previous malignancy. These secondary acute myeloid leukaemias (sAML) possess some common biological and clinical features of the corresponding de novo disorders. The cytokine interleukin-1 (IL-1) is known to have a role in haematopoiesis, and modulation of its action might contribute to the deregulation of proliferation seen in leukaemia.

Back-up bone marrow is frequently ineffective in patients with poor peripheral-blood stem-cell mobilization.

Purpose: To assess hematologic recovery and procedure-related mortality in patients who received high-dose therapy with stem-cell support, in whom the peripheral-blood stem-cell (PBSC) collection fails (CD34+ cells < 1 x 10(6)/kg). The predictive value of granulocyte-monocyte colony-forming cell (GM-CFC) measurements and the value of bone marrow obtained after PBSC collection failure was assessed.

Patients And Methods: The study group comprised 324 consecutive patients mobilized with granulocyte colony-stimulating factor (G-CSF) and cyclophosphamide (273 patients), G-CSF with other chemotherapy (37 patients), and G-CSF alone (14 patients).

Clonality studies in acute myeloid leukemia.

The study of X-chromosome inactivation patterns (XCIPs) to determine tumor clonality was established by Fialkow using G6PD protein isoenzymes but was limited by the low frequency of heterozygotes. Analysis was extended to most females with the demonstration of differential DNA methylation patterns on active and inactive X-chromosome alleles and uses Southern blotting or PCR of either restriction enzyme polymorphisms, eg PGK, HPRT, or variable number tandem repeat sequences, eg M27beta, HUMARA. More recently RNA polymorphisms have been reported enabling direct analysis of expressed transcripts from the two alleles.

Optimal timing for collection of PBPC after glycosylated G-CSF administration.

Daily administration of granulocyte colony-stimulating Factor (G-CSF) results in progenitor cell mobilization with maximum blood levels achieved after 4-7 days. In this study the short-term effects of glycosylated G-CSF at a dose of 5 microg/kg s.c.

Transmigration of CD34+ cells across specialized and nonspecialized endothelium requires prior activation by growth factors and is mediated by PECAM-1 (CD31).

The transmigration of hematopoietic progenitor cells (HPCs) across vascular endothelium is a critical step in the homing of transplanted stem cells, but the molecular basis for this is unknown. We used mobilized peripheral blood CD34(+) selected cells and cultured bone marrow microvascular (BMECs) and human umbilical vein endothelial cells (HUVECs) to investigate the adhesion and transendothelial migration of HPCs. Colony-forming cells (CFCs) in freshly isolated CD34(+) cells showed high levels of adhesion to both forms of endothelium (28% +/- 4% and 38% +/- 6% of granulocyte-macrophage colony-forming cells [GM-CFCs] adhering to HUVECs and BMECs, respectively), but were unable to migrate to any significant extent across either (1.