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D Claxton

Publications and source records attributed to D Claxton.

At least 37 records · Page 2Linked to original sources
PubMed1996

Results of MDR-1 vector modification trial indicate that granulocyte/macrophage colony-forming unit cells do not contribute to posttransplant hematopoietic recovery following intensive systemic therapy.

To formally test the hypothesis that the granulocyte/macrophage colony-forming unit (GM-CFU) cells can contribute to early hematopoietic reconstitution immediately after transplant, the frequency of genetically modified GM-CFU after retroviral vector transduction was measured by a quantitative in situ polymerase chain reaction (PCR), which is specific for the multidrug resistance-1 (MDR-1) vector, and by a quantitative GM-CFU methylcellulose plating assay. The results of this analysis showed no difference between the transduction frequency in the products of two different transduction protocols: "suspension transduction" and "stromal growth factor transduction." However, when an analysis of the frequency of cells positive for the retroviral MDR-1 vector posttransplantation was carried out, 0 of 10 patients transplanted with cells transduced by the suspension method were positive for the vector MDR-1 posttransplant, whereas 5 of 8 patients transplanted with the cells transduced by the stromal growth factor method were positive for the MDR-1 vector transcription unit by in situ or in solution PCR assay (a difference that is significant at the P = 0.0065 level by the Fisher exact test). These data suggest that only very small subsets of the GM-CFU fraction of myeloid cells, if any, contribute to the repopulation of the hematopoietic tissues that occurs following intensive systemic therapy and transplantation of autologous hematopoietic cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Expression pattern of WT1 and GATA-1 in AML with chromosome 16q22 abnormalities.

WT1 is a tumor suppressor gene that can repress transcription of many growth-factor and growth-factor receptor genes. We quantitated WT1 expression levels in 62 acute myelogenous leukemia (AML) samples and found that 82% strongly expressed WT1. WT1 expression levels are highest in the undifferentiated and granulocytic French-American-British (FAB) subclasses and lower in the monocytic subclasses. WT1 was strongly expressed in normal CD34+ bone marrow (BM) stem cells but only weakly or not expressed in normal mature blood cells. This suggests that WT1 gene expression is associated with immature cells, which have high proliferative capacities. Previous studies of WT1 gene regulation showed that GATA-1 may regulate WT1 expression. To understand the relationship between WT1 and GATA-1 expression in leukemia, we examined the expression pattern of GATA-1 in the cells described above. Overall, AML samples expressed significant amounts of both WT1 and GATA-1. However, AML samples with 16q22 abnormalities, presumably interrupting the core binding factor (CBF) beta gene expressed lower than normal levels of GATA-1 but high levels of WT1. Our data suggest that the transcription factor CBF beta may be important for GATA-1 gene regulation. Thus, WT1 expression varied in different FAB subclasses, and GATA-1 expression was strongly affected by the presence of chromosome 16q22 abnormalities.

Chromosome Aberrations↗
PubMed1996

The gene product of CBFB-MYH11.

The inversion(16)(p13;q22) gives rise to chimeric transcripts CBFB-MYH11. To date however, no reports have described the full length coding sequence cloned from patient material or the protein product derived from transcripts. We describe here the cloning and sequencing of the coding region of the fusion gene (type A) from patient cells. The sequence is identical to the included portions of the normal constituent transcripts. We report the study of CBFB and CBFB-MYH11 protein using two anti-CBFB antisera. Twenty-two cases of inv(16) leukemia and a number of other cases of AML were examined. The predicted 70 kDa type A or 95 kDa type D CBFB-MYH11 peptide was detected in 20/22 cases of inv(16) AML. CBFB was expressed as a 21 kDa protein in all samples studied, including hematopoietic cell lines of all major lineages.

Amino Acid Sequence↗
PubMed1996

Allogeneic peripheral blood stem cell transplantation using normal patient-related pediatric donors.

Successful allogeneic peripheral blood progenitor cell (PBPC) transplantation has recently been reported by several transplant centers. This is a first report describing allogeneic PBPC transplantation in five patients using related pediatric donors between the ages of 4 and 13 years. Donors underwent 3 or 4 days of rhG-CSF treatment (6 micrograms/kg q 12 h) for stem cell peripheralization prior to PBPC collection, which was performed by continuous-flow apheresis on day 4 or 5. Venous access was exclusively by ante-cubital veins. A median of 2.2 times (range 1.4-3.6) the donor's total blood volume (TBV) was processed per procedure. In cases where the donor's TBV was < 2 liters, the blood cell separator was primed with human serum albumin (HSA-5%), and anticoagulation was performed using a combination of heparin (pre-apheresis bolus + continuous infusion (CI)) and/or ACD-A (CI at a reduced rate). The median number of CD34+ cells collected per kg of donor body weight (b.w.) and per liter of donor blood processed during each procedure was 128 x 10(4) (range 58 x 10(4)-314 x 10(4)). Between one and two aphereses were sufficient to collect a safe CD34+ cell engraftment dose of 3 or 4 x 10(6)/kg of recipient b.w. Two PBPC recipients were parents, and three were siblings. After freezing and thawing, the median number of CD34+ cells per kg of recipient b.w. thawed and transfused was 8.5 x 10(6) (range 3.2 x 10(6)-9.7 x 10(6)). The time to PMN > 1000/microliters was between 10 and 16 days (four out of five evaluable patients), and platelets > 20000/microliters were reached between day 13 and 14 post-transplantation (three out of five evaluable patients). Two out of three evaluable patients developed grades one and three acute GVHD, and one out of three developed chronic GVHD. Two patients died of sepsis and VOD at day 10 and 19, respectively. Two adult patients are alive and in cytogenetic and molecular remission of CML at +339 and +227 days post-allotransplantation. One 3-year-old girl with hemophagocytic lymphohistiocytosis is in remission at +304 days post-transplantation. Using pediatric donors for allogeneic PBPC transplantation appears to be safe, yields a sufficient amount of progenitors for prompt engraftment, and results in clinical outcome similar to adult PBPC allotransplantation.

Adult↗
PubMed1995

Percentage of Philadelphia chromosome (Ph)-negative and Ph-positive cells found after autologous transplantation for chronic myelogenous leukemia depends on percentage of diploid cells induced by conventional-dose chemotherapy before collection of autologous cells.

We collected peripheral blood mononuclear cells and bone marrow cells soon after recovery from conventional-dose chemotherapy-induced myelosuppression and transplanted these cells into advanced chronic myelogenous leukemia (CML) patients after treating these patients with 120 mg/kg cyclophosphamide, 750 mg/m2 VP-16, and 1,020 cGy of total body irradiation (TBI). Of the 10 late chronic-phase patients and the eight accelerated-phase CML patients evaluable posttransplant, 90% and 87%, respectively, remain alive posttransplant, whereas none of the three blast crisis CML patients given this therapy remain alive posttransplant. We measured the percentage of Philadelphia chromosome (Ph)-negative cells in the autologous cells collected after conventional-dose chemotherapy-induced myelosuppression before autologous transplant and in the marrow of these same CML patients after autologous transplantation of these cells into recipients treated with the cyclophosphamide, VP-16, and TBI. A direct correlation (correlation coefficient = 0.91) was observed between the level of Ph+ cells in the transplanted cells and the percentage of Ph+ marrow cells after transplant in 21 patients so transplanted. The data show that the chance of generating cytogenetic remissions post-transplant depends on the percentage of diploid cells in the preparations of autologous cells used for transplant and the stage of disease of the patients at the time of collection of the autologous cells.

Adult↗
PubMed1995

Intravesicular carboprost for the treatment of hemorrhagic cystitis after marrow transplantation.

OBJECTIVES: To determine the minimal active dose and extent of activity of intravesicular carboprost for the treatment of hemorrhagic cystitis after marrow transplantation. METHODS: Twenty-four adults with grade 3 or 4 hemorrhagic cystitis were treated. All but 2 had failed other local therapy. Treatment was initiated at a median of 32 days post-transplant. Eleven patients received carboprost intravesicularly at 0.2 mg/dL for 60 minutes every 6 hours, and the dose was escalated every 24 hours until a dose of 1.0 mg/dL was reached unless a response was achieved. Thirteen additional patients were treated at an initial dose of 0.8 mg/dL, with escalation to 1.0 mg/dL after four doses in the absence of a response. RESULTS: Overall, 15 of the 24 patients responded. In the dose-escalation setting, 0.8 mg/dL was the minimal active dose. The total response rate was 62% with doses at or above 0.8 mg/dL and 18% at lower doses. All but one response occurred with 7 or fewer days of therapy, and 9 patients relapsed later. Four additional patients were salvaged following cystoscopy with clot evacuation with or without alum or formalin instillation. In all but 1 patient, bladder spasms developed during treatment with carboprost, but were not sufficiently severe to discontinue therapy. CONCLUSIONS: Intravesicular carboprost at 1.0 mg/dL every 6 hours for no more than 7 days should be considered for a randomized study for treatment of refractory hemorrhagic cystitis. Cystoscopic examination and evacuation of clots prior to therapy may be required to achieve the full benefit of this treatment.

Administration, Intravesical↗
PubMed1995

High levels of constitutive WAF1/Cip1 protein are associated with chemoresistance in acute myelogenous leukemia.

The WAF1/Cip1 gene product is an important regulator at the G1 checkpoint in the cell cycle. WAF1/Cip1 expression can be activated through p53-dependent and p53-independent pathways. The WAF1/Cip1 protein binds to cyclin-dependent kinase complexes and inhibits the kinase activity that is required for cell cycle progression. In this preliminary study, we analyzed with Western blot assays the steady-state levels of the WAF1/Cip1 protein in the leukemia cells of 100 untreated acute myelogenous leukemia (AML) patients. Normal bone marrow cells from six donors were used as a control. The results of these analyses showed that the levels of the WAF1/Cip1 protein were very low in normal marrow cells and in the leukemia cells of 83 AML patients. High levels of WAF1/Cip1 were detected in 17 patients; these patients with high WAF1/Cip1 levels were significantly less likely to achieve complete remission (41% versus 69%, P = 0.03) and were four times as likely to be resistant to therapy (47% versus 12%, P = 0.003) as patients with very low levels of WAF1/Cip1. Median survival was 38 weeks for patients having very low expression levels versus 11 weeks for patients having high expression levels (P = 0.04). The WAF1/Cip1 level was an independent predictor for response but not survival in a stepwise multivariate regression analysis. Southern blotting analyses did not detect deletion of the WAF1/Cip1 gene in the 12 negative WAF1/Cip1 AML samples tested. Also, the level of WAF1/Cip1 protein expression was not correlated with overexpression of cyclin D1, cyclin E, proliferating cell nuclear antigen, cyclin-dependent kinase 4, or p53 in the leukemia cells. However, the levels of cyclin D1, cyclin E, and cyclin-dependent kinase 4 were elevated in most of the AML samples compared with that in normal marrow. We hypothesize that high-level constitutively expressed WAF1/Cip1 in tumor cells may result in an indolent state that is refractory to chemotherapy drugs. We conclude that the WAF1/Cip1 expression level may be an important prognostic factor for response to therapy and survival in AML patients.

Adult↗
PubMed1994

Genetic marking shows that Ph+ cells present in autologous transplants of chronic myelogenous leukemia (CML) contribute to relapse after autologous bone marrow in CML.

Relapse after autologous bone marrow transplantation for chronic myelogenous leukemia (CML) can be due either to the persistence of leukemia cells in systemic tissues following preparative therapy, or due to the persistence of leukemia cells in the autologous marrow used to restore marrow function after intensive therapy. To help distinguish between these two possible causes of relapse, we used safety-modified retroviruses, which contain the bacterial resistance gene NEO, to mark autologous marrow cells that had been collected from patients early in the phase of hematopoietic recovery after in vivo chemotherapy. The cells were then subjected to ex vivo CD34 selection following collection and 30% of the bone marrow were exposed to a safety-modified virus. This marrow was infused after delivery of systemic therapy, which consisted of total body irradiation (1,020 cGy), cyclophosphamide (120 mg/kg), and VP-16 (750 mg/m2). RT PCR assays specific for the bacterial NEO mRNA, which was coded for by the virus, and the bcr-abl mRNA showed that in two evaluable CML patients transplanted with marked cells, sufficient numbers of leukemia cells remained in the infused marrow to contribute to systemic relapse. In addition, both normal and leukemic cells positive for the retroviral transgenome persisted in the systemic circulation of the patients for at least 280 days posttransplant showing that the infused marrow was responsible for the return of hematopoiesis following the preparative therapy. This observation shows that it is possible to use a replication-incompetent safety-modified retrovirus in order to introduce DNA sequences into the hematopoietic cells of patients undergoing autologous bone marrow transplantation. Moreover, this data suggested that additional fractionation procedures will be necessary to reduce the probability of relapse after bone marrow transplantation in at least the advanced stages of the disease in CML patients undergoing autologous bone marrow transplantation procedures.

Antigens, CD↗
PubMed1994

Consistent intergenic splicing and production of multiple transcripts between AML1 at 21q22 and unrelated genes at 3q26 in (3;21)(q26;q22) translocations.

Two genes have been implicated in leukemias of patients with abnormalities of chromosome 3, band q26: EVI1, which can be activated over long distances by chromosomal rearrangements involving 3q26, and EAP, a ribosomal gene that fuses with AML1 in a therapy-related myelodysplasia patient with a t(3;21)(q26.2;q22). AML1 was identified by its involvement in the t(8;21)(q22;q22) of acute myeloid leukemia. Here we report the consistent identification of fusion transcripts between AML1 and EAP or between AML1 and previously unidentified sequences that we named MDS1 (MDS-associated sequences) in the leukemic cells of four patients with therapy-related myelodysplasia/acute myeloid leukemia and in one patient with chronic myelogenous leukemia in blast crisis, all of whom had a t(3;21). In addition, we have identified a third chimeric transcript, AML1/EVI1, in one of the therapy-related acute myeloid leukemia patients. Pulsed-field gel electrophoresis established the order of the genes as EAP, the most telomeric, and EVI1, the most centromeric, gene. The results indicate that translocations could involve multiple genes and affect gene expression over long distances.

Amino Acid Sequence↗
PubMed1994

Consistent loss of the D5S89 locus mapping telomeric to the interleukin gene cluster and centromeric to EGR-1 in patients with 5q- chromosome.

Interstitial deletions of the long arm of chromosome 5 are common in a number of disorders of leukemic and preleukemic myeloid disorders. Although the limits of these deletions vary among patients, a region of cytogenetic overlap that includes band 5q31 is deleted consistently, suggesting loss of 5q31 loci critical for normal myeloid differentiation and leukemogenesis. An anonymous genomic DNA segment D5S89, previously mapped to 5q21-31, detects consistent loss of alleles in cases showing the 5q- chromosome at presentation or relapse. Analysis of a panel of natural-deletion somatic-cell hybrids in conjunction with irradiation hybrids containing fragments of human chromosome 5q shows that the D5S89 locus is telomeric to the interleukin (IL) genes (IL-3, IL-4, IL-5, IL-9, and granulocyte-macrophage colony-stimulating factor [GM-CSF]) and interferon response factor-1 (IRF-1) gene and centromeric to the early response transcription factor (early growth response gene-1 [EGR-1]) on 5q31. To further define the principal region of loss, we have isolated and characterized yeast artificial chromosomes (YACs) spanning D5S89. The presence of several CpG islands within the 300-kb YAC is suggestive of multiple transcription units. However, IL-4, IL-5, IRF-1, IL-3, GM-CSF, and EGR-1 genes were not detected in the YAC clone spanning D5S89, implying that none of these genes are in the vicinity of the D5S89 marker. Further characterization of these YACs should facilitate the isolation of novel candidate genes that may play a role in the evolution of the abnormal phenotype associated with 5q- chromosome.

Adult↗
PubMed1994

Development of a sensitive PCR to detect allele loss in a model hematopoietic neoplasm.

Loss or gain of an entire chromosome and interstitial deletions or amplifications are hallmarks of several hematopoietic neoplasms. These chromosomal anomalies can be identified by conventional cytogenetic analysis of bone marrow aspirates. We have developed a PCR-based assay to detect loss of chromosome 5q31 loci, in the model system of myeloid disorders with the 5q- chromosome (interstitial deletion of 5q), by taking advantage of a highly polymorphic dinucleotide repeat within the interleukin-9 (IL9) gene on 5q31. In a given sample, quantitation of amplification of individual alleles in a Phosphorimager allowed the representation of alleles to be expressed as a ratio of the larger to the smaller allele. Comparison of these ratios in paired DNA samples from Ficoll buoyant and pelletted fractions provides evidence for allele loss. Results presented here demonstrate that this technique of comparison of ratios of isotope incorporation could be expanded to Investigate any deletion or numerical abnormality in hematopoietic tumors.

Alleles↗
PubMed1994

Acanthamoeba meningoencephalitis after bone marrow transplantation.

Two patients presented with fever and nodular pulmonary infiltrates 9 and 6 months after marrow transplantation for leukemia. The second patient also had painful subcutaneous nodules that subsequently ulcerated. Both had a history of sinusitis and both had recently been treated with corticosteroids. During treatment with antibacterial and antifungal antibiotics, they developed rapid mental deterioration, coma and/or seizures. CT findings included hydrocephalus with extensive cortical and periventricular hypodensities in the first patient, and hydrocephalus with a cerebellar hemorrhage and edema in the second patient. Cerebrospinal fluid had a low glucose and elevated protein levels with few erythrocytes and little or no pleocytosis. Despite therapy with broad-spectrum antibiotics, including coverage for opportunistic infections, both patients died. Autopsy revealed Acanthamoeba species causing necrotizing meningoencephalitis, pneumonitis and adrenalitis in the first patient and causing necrotizing meningoencephalitis and dermatitis in the second patient. While these are the only reported cases of disseminated Acanthamoeba infection in marrow transplant recipients, a review of the literature suggests that this organism may be a new cause of opportunistic infections.

Acanthamoeba↗
PubMed1993

Genetic therapy of human neoplastic disease.

Molecular biology has provided clinical investigators and basic scientists with the tools to identify those changes present within neoplastic hematopoietic and epithelial cells that lead to the evolution of unregulated patterns of cell growth. This information has made possible the development of therapy that involves genetic modification of either the normal hematopoietic cells (for chemoprotection), or the tumor cells themselves to suppress the growth of these cells. This article will summarize the clinical and laboratory data that is evolving in this area.

Animals↗
PubMed1992

Polyclonal hematopoiesis in interferon-induced cytogenetic remissions of chronic myelogenous leukemia.

Interferon (IFN) therapy of early chronic myelogenous leukemia (CML) frequently produces partial or complete cytogenetic remission of the disease. Patients with complete cytogenetic remission often continue on therapy for several years with bone marrow showing only diploid (normal) metaphases. We studied hematopoiesis in five female patients with major cytogenetic remissions from CML during IFN therapy. Clonality analysis using the BstXI PGK gene polymorphism showed that granulocytes were nonclonal in all patients during cytogenetic remission. BCR region studies showed rearrangement only in the one patient whose remission was incomplete at the time of sampling. Granulopoiesis is nonclonal in IFN-induced remissions of CML and may be derived from normal hematopoietic stem cells.

Clone Cells↗
PubMed1992

Use of cell-free retroviral vector preparations for transduction of cells from the marrow of chronic phase and blast crisis chronic myelogenous leukemia patients and from normal individuals.

Marrow cells were exposed to the LNL6 or G1N safety-modified variants of the N2 retrovirus, which contain the G418 bacterial resistance gene neo. The frequency of acquisition of the G418 resistance phenotype following exposure to LNL6 or G1N was compared among hematopoietic progenitor cells from the marrow of patients with chronic phase chronic myelogenous leukemia (CML), blast crisis CML, or from nonleukemic individuals. Under the conditions of our experiments, the myeloid committed progenitor cells from 3 of 6 nonleukemic individuals, 9 of 18 chronic-phase CML patients, and 2 of 4 blast crisis CML patients acquired resistance to at least 1 mg/ml G418 following incubation with cell-free supernatants from the PA317 LNL6 or PA317 G1N producer cell lines. Ten of the 32 colonies growing up in 0.8 mg/ml G418 from chronic-phase marrow exposed to LNL6 were shown to contain the neo gene by polymerase chain reaction (PCR) assay of DNA. These results were consistent with estimates of the transduction frequency based on acquisition of resistance to G418 as the number of colonies growing under G418 selection was always greater at 0.8 mg/ml G418 than at higher concentrations of G418 (1.0-1.4 mg/ml). The average transduction frequency at each G418 concentration (1.0, 1.2, and 1.4 mg/ml) in cells from blast crisis CML cells ranged from 2 to 14%, as measured by acquisition of G418 resistance. Chronic-phase CML showed slightly lower average frequencies of transduction (0.6-2.8% of the colonies are G418 resistant). The average transduction frequency of cells from nonleukemic marrow was as high as that seen from the marrow of chronic-phase CML individuals.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

Molecular analysis of retroviral transduction in chronic myelogenous leukemia.

We have developed a polymerase chain reaction (PCR) assay for detection of integrated retroviral transgenomes containing the neo G418 resistance gene in colonies (40 cells or more) grown in G418 selection after exposure to the neo-positive retrovirus LNL6. This assay also provides for simultaneous characterization of these colonies as belonging to a chronic myelogenous leukemic (bcr-abl positive) or nonleukemic population (bcr-abl negative). Using these techniques, we assessed transduction of the LNL6 retrovirus into the normal and leukemic cells of a blast-crisis chronic myelogenous leukemia (CML) patient. This work was designed to support the use of the LNL6 retroviral marker to help identify the origin of relapse after autologous marrow infusion. The data from these experiments show that the majority of CML blast crisis cells that, following exposure to the LNL6 virus, produce colonies under rigorous G418 selection are indeed transduced by the virus, as shown by the presence of the neo retroviral gene. Most of these colonies are also shown to be leukemic by PCR detection of the bcr-abl RNA. This demonstrates the feasibility of the study of CML marrow for retroviral marking. These procedures will be of use in establishing if relapse arises from leukemic blasts which contaminate purged autologous bone marrow infused following intensive therapy for leukemia.

Base Sequence↗