Search PubMed⌕ Search

Biomedical subjects

D C Linch

Publications and source records attributed to D C Linch.

At least 73 records · Page 4Linked to original sources
PubMed1998

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. Progenitor cell yields were determined by quantification of CD34+ cells and GM-CFC. Twelve patients (21%) failed to achieve the minimum progenitor cell requirements of 1 x 10(6)/kg CD34+ cells or 1 x 10(5)/kg GM-CFC in their pooled harvests and 40 patients (70%) failed to achieve the optimal harvest thresholds of 3.5 x 10(6)/kg CD34+ cells or 3.5 x 10(5)/kg GM-CFC. This high failure rate is significantly higher than that in patients with histologically aggressive NHL or Hodgkin's disease. A multivariate analysis was performed to identify factors contributing to the low stem cell yields in this group. This identified the time interval from the last chemotherapy to the priming chemotherapy as the most important predictive factor. With respect to CD34 and GM-CFC numbers, on the single harvest on the day the white cell count first exceeded 5 x 10(9)/l the P values were 0.0078 and 0.0065, respectively, and for the progenitor cell values on the pooled harvests the P values were 0.004 for CD34+ cells and 0.015 for GM-CFC. Progenitor cell yields may therefore be improved in patients with low grade lymphoma by harvesting at diagnosis if no marrow disease is present, or by delaying mobilization for 6 months post-chemotherapy in patients in first or subsequent remission.

Adult↗
PubMed1998

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.

Antigens, CD34↗
PubMed1998

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. In three consenting patients, purified CD34 cells were exposed to A12M1 MDR-1 retroviral supernatant for 6 h, cryopreserved then thawed and readministered following ablative chemotherapy. No delay in engraftment was observed, although one patient received additional back-up cells. Gene transfer was demonstrated by polymerase chain reaction (PCR) for vector-derived MDR-1 cDNA sequence in all cases. Analysis of peripheral blood and bone marrow cells after transplant has, however, shown no evidence of in vivo gene transfer with a follow-up of 12, 15 and 18 months. The effect of MDR-1 substrate drugs has not yet been tested as all patients remain in clinical and radiological remission of their lymphoma. These results confirm the difficulty of achieving in vivo gene transfer in human haemopoietic cells and indicate major logistical constraints in PBSC mobilisation in patients with relapsed and resistant disease in whom initial studies are appropriate.

Adolescent↗
PubMed1998

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. Interpretation of clonality requires knowledge of an individual's constitutive XCIP as skewing (>75% expression of one allele) occurs in a significant proportion of hematologically normal females, probably due to the stem cell pool size at the time of Lyonization. Furthermore, acquired skewing occurs with increasing age. For myeloid disorders in the young, T lymphocytes serve as a suitable control XCIP, but interpretation of imbalanced XCIPs in the elderly can be difficult. In AML, XCIPs at presentation are consistent with a clonal disorder whereas in remission comparison with normal controls suggests that true clonal remission is infrequent. Sequential analysis of samples may be helpful in some patients in order to determine evolving clonal populations.

Acute Disease↗
PubMed1998

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. DNA samples from 150 females were screened and 61 (41%) were found to be informative for the polymorphism. Reproducible results were obtained from clonal analysis of 56 RNA samples covering the complete spectrum of XCIP ratios. The values correlated closely with those obtained from the corresponding DNA samples analyzed using either PGK or HUMARA (r=0.98) provided that the number of amplification cycles was limited to 25 to reduce heteroduplex formation. However, sample purity is of greater importance than in the DNA-based assays as contaminating red cells and platelets will still contain the relevant transcripts and could influence the result.

Base Sequence↗
PubMed1998

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. Truncation mutations were detected in a minor percentage of transcripts from two other patients. In one patient the mutation has been constant at a low level (5-10% of total mRNA and 2/40 myeloid colonies) for 2 years. In the other patient the mutation was acquired, remained present at low levels for nearly 3 years and then spontaneously disappeared. Both patients had polyclonal haemopoiesis. We hypothesize that these mutations do not cause SCN, are randomly acquired with the mutant clone being expanded to detectable levels by high levels of exogenous or endogenous G-CSF, and may disappear by clonal succession. In a pre-leukaemic marrow the mutated subclone could achieve high levels, but this does not necessarily indicate a primary role of the mutant receptor in the leukaemogenic process.

Acute Disease↗
PubMed1998

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. Activation of MAP kinase (expressed as a proportion of total cellular MAP kinase) was maximal at 5 min and of similar magnitude in both cell types. There was, however, a marked difference in the later kinetics of activation, with the response being transient in the undifferentiated cells and disappearing within 15 min, whereas it was prolonged and persisted for at least 60 min in the differentiated cells. GM-CSF mediated activation of STAT 5 was markedly increased (15-20-fold) after differentiation of HL-60 cells but the kinetics of activation did not change. The increase in STAT 5 activation was not due to a change in total cellular STAT 5 expression but correlated with increased JAK-2 protein levels. These data show that in the HL-60 cell model, differentiation modulates the activation of signalling molecules downstream of the GM-CSF receptor.

Blotting, Western↗
PubMed1998

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. Secondary cytogenetic changes commonly associated with these rearrangements are +8 with t(15;17), del(9q) with t(8;21) and +22 with inv(16). These secondary abnormalities are also observed alone, raising the possibility that they may be markers of underlying cryptic rearrangements. In order to determine the frequency of these rearrangements in AML cases with +8, del(9q) or +22 we have performed an analysis of 63 such patients in whom there was no evidence of a t(15;17), t(8;21) or inv(16) by cytogenetics. No disease-related fusion transcripts were identified, indicating that the secondary changes are rarely markers for cryptic rearrangements.

Acute Disease↗
PubMed1998

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). Two point mutations detected were silent, C301-->T (Cys91) and C1306-->T (Ser426). The latter had previously been identified with an allele frequency of 0.13 in the general population. Two further point mutations detected led to amino acid substitutions, G773-->C (Glu249Gln), which is equivalent to the mouse sequences, and G962-->A (Asp312Asn), both of which were found at similar frequencies in normal controls. Activating mutations of the betaC chain which might contribute to the pathogenesis of the disease are therefore rare in AML.

Acute Disease↗
PubMed1998

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. Five patients have survived at least 5 yr (1>12 yr) from the time of diagnosis of AML. These patients were significantly younger (p=0.03) than those who succumbed to this complication and each also had standard or favourable risk cytogenetics. The actuarial 5- and 10-yr survival rates are 17.4% (7.7-34.9%, 95% CI) and 8.7% (1.9-31.7%, 95% CI), respectively. There is therefore a subgroup of patients who will achieve long-term survival despite the development of secondary myeloid malignancy.

Adolescent↗
PubMed1998

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). Between one and four aphereses were performed. RESULTS: In 51 of 324 patients, there was failure to obtain 1 x 10(6)/kg CD34+ cells. Twenty-three patients had greater than 1 x 10(5)/kg GM-CFC; 22 patients proceeded to high-dose therapy. Neutrophil recovery occurred within 21 days, but platelet independence was delayed (> 28 days) in eight patients. Of 28 patients with less than 1 x 10(5)/kg GM-CFC, six received high-dose therapy with PBSC alone and five had delayed engraftment. Twelve patients with less than 1 x 10(5)/kg GM-CFC received high-dose therapy supported by bone marrow collected after PBSC collection failure. Eleven patients were assessable for engraftment; four patients had slow (> 21 days) or delayed (> 28 days) neutrophil recovery and eight patients had delayed platelet recovery. In the group of patients who received less than 1 x 10(5)/kg GM-CFC, there were five procedure-related deaths. CONCLUSION: This study shows that delayed hematologic recovery is frequent if less than 1 x 10(6)/kg CD34+ cells are infused after high-dose therapy, particularly with GM-CFC less than 1 x 10(5)/kg. The procedure-related mortality in this latter group is high. In most patients whose PBSC collection contains less than 1 x 10(5)/kg GM-CFC, the use of bone marrow cells does not improve engraftment, which suggests that poor PBSC mobilization usually indicates poor marrow function.

Adolescent↗
PubMed1998

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. It has recently been reported that a variable number tandem repeat (VNTR) polymorphism in the IL-1 receptor antagonist (IL-1ra) gene is closely associated with the severity of many inflammatory and autoimmune diseases, and may also play a role in the pathogenesis of sAML. We sought to confirm this finding in a large group of patients classified as having sAML. We found no differences in either the genotypic or allele frequencies of the polymorphism studied when compared with those of normal controls or other haematological disorders. No differences were observed in allele frequencies between younger and older patients, or between those patients who had an antecedent myelodysplasia and those who had received prior chemotherapy or radiotherapy. We conclude that the described polymorphism in the IL-1ra gene is not associated with the development of sAML.

Acute Disease↗
PubMed1998

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. Recently a mutation has been described in the TPO gene in familial thrombocythaemia that results in elevated TPO levels. We have therefore screened 51 patients diagnosed with non-familial ET for the presence of this mutation, but it was not detected in any patient. The constitutional presence of this mutation is therefore unlikely to contribute to the pathogenesis of ET.

Adolescent↗
PubMed1997

Characterization of cell cycle status and E2F complexes in mobilized CD34+ cells before and after cytokine stimulation.

Mobilized peripheral blood progenitors (CD34+ cells) have been shown to be either in the G0 or G1 phase of the cell cycle. In this study, it is shown that they are small cells with low protein content suggestive of G0. Support for this is provided by showing that the principal E2F complex consists of hypophosphorylated p130, E2F-4, and DP-1. The E2F-4 is more highly phosphorylated than in quiescent T cells. In response to cytokines in vitro, the CD34+ cells start to enter G1 within 8 hours and enter S-phase at about 48 hours. As cells enter G1, E2F-4 is dephosphorylated to several hypophosphorylated forms and three new DNA-binding complexes appear, including one containing E2F-4, DP-1, and p107. We suggest that mobilized CD34+ cells may be maintained in G0 by p130, E2F-4, and DP-1 and the coordinate dephosphorylation of E2F-4 and hyperphosphorylation of p130 may be central to the initiation of proliferation.

Antigens, CD34↗
PubMed1997

Evaluation of clinical scale CD34+ cell purification: experience of 71 immunoaffinity column procedures.

Seventy-one mobilised PBSC collections were subject to CD34+ cell purification using the CEPRATE SC stem cell concentration system. The overall median purity of CD34+ cells was 69% (6-93%). CD34+ cell, and GM-CFC recoveries were 52% (8-107%) and 36% (3-118%). Purity was logarithmically related to the input percentage of CD34+ cells and starting requirements were established of 1% CD34 cell content for optimal purity and a minimum of 2 x 10(6)/kg CD34+ cells to ensure recovery of our minimum engraftment threshold of 1 x 10(6)/kg CD34+ cells. Reduction of the washing steps reduced non-specific cell losses and shortened the procedure but did not affect progenitor cell recovery. Purified CD34+ cells were reinfused following high-dose therapy in 35 patients. The median time to neutrophil recovery of 0.5 x 10(9)/l was 12 (10-23) days and to the attainment of platelet independence was 13 (7-100) days. The risks of delayed platelet recovery were related to the CD34+ cell dose infused and were identical to the risks when non-purified PBSC collections were used. In conclusion, purification of CD34+ cells using the CEPRATE device is reliable and the purified product results in prompt engraftment. The cell losses that occur do however restrict its use in many patients.

Adult↗
PubMed1997

Crossover study of the haematological effects and pharmacokinetics of glycosylated and non-glycosylated G-CSF in healthy volunteers.

A cross-over study of glycosylated and non-glycosylated G-CSF was performed in 20 healthy male volunteers to compare the effects of the different forms of G-CSF, the extent of inter-individual progenitor cell mobilization and to determine whether any differences observed were related to the serum concentrations of G-CSF attained. The peak WBC achieved during 6 d of G-CSF administration at a dose of 5 microg/kg/d was significantly higher with the glycosylated than the non-glycosylated product (P = 0.02) as was the peak level of granulocyte-monocyte colony forming cells (GM-CFC) (P=0.03). The average GM-CFC count on days 5, 6 and 7 was 28% higher with the glycosylated product (P=0.003). Serum concentrations of G-CSF achieved were significantly higher with the non-glycosylated G-CSF, however, suggesting that the difference in bio-efficacy was not due to a difference in G-CSF stability. Marked inter-individual variation in progenitor mobilization was observed, but this was not related to serum G-CSF levels. The G-CSF concentrations on day 6 were approximately one third of those on day 1, with both forms of G-CSF.

Adjuvants, Immunologic↗