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Biomedical subjects

D Crowther

Publications and source records attributed to D Crowther.

At least 55 records · Page 3Linked to original sources
PubMed1995

Molecular genetic analysis of a family with a history of Hodgkin's disease and dyschondrosteosis.

We describe a family in which two sisters with the autosomal dominant skeletal dysplasia, Leri-Weill dyschondrosteosis (LWD), developed Hodgkin's disease (HD) in late adolescence. In a preliminary attempt to identify HD susceptibility gene(s), HLA-typing and linkage analysis were carried out in the family. Using HLA molecular typing, both sisters were found to have inherited a variant of the HD-susceptibility allele, DPB1*0301, known as DPB1*2001. Following a previous report of a constitutional chromosome translocation (t(2q;8p)) in a family with LWD, preliminary linkage studies were carried out using chromosome 2q and 8p molecular markers. Regions covered by 7/10 chromosome 2 markers and 4/8 chromosome 8 markers were excluded as the location of a candidate LWD gene. Given the rarity of LWD and HD, their simultaneous occurrence is unlikely to have been due to chance. We suggest that a mutation in the LWD gene itself, or a gene closely linked to it, perhaps acting with increased susceptibility to infection conferred by DPB1*2001, resulted in HD in the two sisters.

Adolescent↗
PubMed1994

Relationships between the formation of O6-methyldeoxyguanosine by 1-p-carboxyl-3,3-dimethylphenyltriazene in DNA and O6-alkylguanine-DNA alkyltransferase in human peripheral leukocytes.

There is increasing experimental evidence to indicate that O6-methyldeoxyguanosine (O6-MedG) formation in DNA is a critical cytotoxic event following exposure to certain antitumor alkylating agents and that the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (ATase) can confer resistance to these agents. We recently demonstrated a wide interindividual variation in the depletion and subsequent regeneration of ATase in peripheral blood lymphocytes of patients treated with 24-h continuous infusion of 1-p-carboxyl-3,3-dimethylphenyltriazene (CB10-277) for metastatic melanoma. We have now measured the formation of O6-MedG in the DNA of peripheral leukocytes of nine patients receiving this treatment regimen. This lesion could be detected in DNA within 1 h and a progressive increase in adduct levels occurred during the CB10-277 infusion and for 24 h after completion. Considerable interindividual variation was observed in the peak O6-MedG levels, with values ranging from 3.0 to 23.8 mumol O6-MedG/mol deoxyguanosine (mean, 12.3 +/- 6.4 mumol O6-MedG/mol deoxyguanosine) following the first treatment cycle, possibly as a consequence of differences in the capacity of patients to metabolize CB10-277 to a methylating agent. There was, nevertheless, a clear temporal relationship between the progressive formation of leukocyte O6-MedG and lymphocyte ATase depletion. Repeated-measures regression showed that this was statistically significant (P < 0.001) during the CB10-277 infusion. A significant inverse correlation was also seen between pretreatment lymphocyte ATase activity and peak O6-MedG levels in leukocyte DNA (r = -0.73) and the area under the leukocyte O6-MedG concentration-time curve (r = -0.76). Metabolism of CB10-277 to a methylating agent could be one factor that combines with DNA repair capacity to determine clinical response, because the two responses observed in this series occurred in the two patients with the highest leukocyte O6-MedG levels and also the lowest pretreatment ATase activity. Hematological toxicity developed in the same two patients.

Adult↗
PubMed1994

Prevalence and diversity of constitutional mutations in the p53 gene among 21 Li-Fraumeni families.

The entire coding sequence of the p53 gene was analysed for the presence of mutations in 12 families conforming to a restricted definition of Li-Fraumeni syndrome (classic LFS) and nine families with features of LFS conforming to a broader definition. Mutations were detected in seven families. Six were point mutations with one each affecting codons 175, 180, and 220 and three affecting codon 248. The seventh was a deletion/insertion mutation in exon 4. Germline mutations in p53 were a feature of families which included children with rhabdomyosarcoma and/or adrenal cortical carcinoma. Germline p53 mutations were detected in six of the nine families with such tumors. An analysis of these 7 mutations, together with 34 published examples, showed that more than one-half were transitions at CpG dinucleotides, suggesting that the majority of germline p53 mutations may arise as a result of spontaneous events. The most common cancers occurring in the 41 families with germline p53 mutations, in common with classic LFS, were bone and soft tissue sarcoma, breast cancer, brain tumors, leukemia, and adrenocortical carcinoma, although less than one-half of the probands with germline p53 mutations came from classic LFS families. More than one-half of the cancers overall and nearly one-third of the breast cancers were diagnosed before 30 years of age. These observations have important implications for asymptomatic carriers of germline p53 mutations, and there is a need for international collaboration in the development of protocols for the management of such families.

Base Sequence↗
PubMed1994

Immunomodulation in patients receiving intravenous Bryostatin 1 in a phase I clinical study: comparison with effects of Bryostatin 1 on lymphocyte function in vitro.

Bryostatin 1 is a protein kinase C activator that inhibits growth of tumour cells and activates lymphocytes in vitro, properties that have encouraged its use in phase 1 clinical studies as an anticancer agent. We investigated interleukin-2(IL-2)-induced proliferation and lymphokine-activated killer (LAK) cell activity in peripheral blood mononuclear cells (PBMC) from cancer patients receiving Bryostatin intravenously. After Bryostatin administration both LAK generation and proliferation were enhanced when patients' PBMC were stimulated with IL-2 in vitro. However, when normal donors' PBMC were cultured in vitro in the presence Bryostatin and IL-2, LAK induction was inhibited while IL-2-driven proliferation was increased. These effects were also seen following only 2 h exposure to Bryostatin and could be elicited by conditioned medium from Bryostatin-pretreated cells. Neither IL-4 nor interferon gamma was detected in the conditioned medium. Bryostatin in vitro was found to increase expression of IL-2 receptors on CD4+, CD8+ and CD56+ cells and augment the proportion of CD8+ cells in conjunction with IL-2. We conclude that Bryostatin in combination with IL-2 in vitro enhances proliferation and IL-2 receptor expression on lymphocytes, favouring CD8+ cells while suppressing the generation of LAK activity. Intravenous administration of Bryostatin increases the potential of IL-2 to induce proliferation and LAK activity in lymphocytes which, taken together with its putative direct antitumour effect, makes Bryostatin an interesting candidate for clinical trials in combination with IL-2.

Adjuvants, Immunologic↗
PubMed1994

Immunohistochemical detection of mutant p53 protein in epithelial ovarian cancer using polyclonal antibody CMI: correlation with histopathology and clinical features.

Approximately 30-50% of cases of ovarian adenocarcinoma harbour mutations in the p53 tumour-suppressor gene associated with elevated levels of the protein detected by immunohistochemical staining. To investigate any relation between the presence of mutant p53 and clinicopathological features of disease, we examined a series of 50 cases of epithelial ovarian adenocarcinoma for expression of p53 by immunohistological staining on fixed, paraffin-embedded tissue sections using the polyclonal antibody CM1, and by direct nucleotide sequencing of polymerase chain reaction-amplified DNA from selected cases. Of the 50 cases examined, 28 (56%) were p53 positive and there was no significant correlation between p53 status and differentiation stage, clinical (FIGO) stage, multidrug resistance (mdr-1 P-glycoprotein) expression or response to treatment. However, we observed a statistically significant difference between the high prevalence of p53-positive serous tumours (18 out of 23) and the lower prevalence of p53-positive cases in mucinous tumours (3 of 12) suggesting that factors related to disease aetiology, associated with these histological subtypes, may determine the prevalence of functional inactivation of the p53 tumour-suppressor gene in ovarian adenocarcinoma.

Base Sequence↗
PubMed1994

Establishment of a murine leukaemia cell line resistant to the growth-inhibitory effect of bryostatin 1.

Bryostatin 1 is a novel macrocyclic lactone activator of protein kinase C (PKC) which has clinical potential as an anti-cancer agent. The mechanism of action of this agent is unknown, but protein kinase C has been implicated. In order to investigate this possibility, we have developed P388 sublines resistant to bryostatin 1, by continuous challenge of the parent cell line with increasing incremental concentrations of the drug over 4 months. Cell lines were established at monthly intervals yielding four sublines: P388/BR/A, which were removed at 1 month; P388/BR/B, obtained after 2 months; P388/BR/C, obtained after 3 months; and P388/BR/D, which were established after 4 months. All four P388/BR sublines show an equal degree of resistance to the growth inhibitory effects of bryostatin 1, with a relative resistance ratio (RR) IC50 of approximately 4,000. The ability of the cytosol of cells to phosphorylate PKC-specific substrate is decreased by 41% for BR/A, 57% for BR/B 80% for BR/C and 94% for BR/D compared with the parental cell line, even when grown in the absence of bryostatin 1 for up to 4 weeks. Similar decreases are seen for cytosolic phorbol ester binding and whole-cell PKC isoenzyme expression. All four P388/BR sublines show high and equal levels of cross-resistance to the PKC activatory phorbol ester, phorbol 12-myristate 13-acetate (PMA). There is no loss of resistance to either bryostatin 1 or PMA up to 3 months after termination of exposure of the sublines to bryostatin 1. There was no significant degree of cross-resistance to daunorubicin in the bryosatin 1-resistant cell lines, P388/BR/A, B, C or D, when compared with the parent cell line, P388.

Animals↗
PubMed1994

Linkage studies in a Li-Fraumeni family with increased expression of p53 protein but no germline mutation in p53.

We report a family with the Li-Fraumeni syndrome (LFS) in whom we have been unable to detect a mutation in the coding sequence of the p53 gene. Analysis of linkage to three polymorphic markers within p53 enabled direct involvement of p53 to be excluded. This is the first example of a LFS family in whom exclusion of p53 has been possible. Four affected members of the family with sarcoma or premenopausal breast cancer showed increased expression of p53 protein in their normal tissues as detected by immunohistochemistry. It therefore appears that the LFS phenotype has been conferred by an aberrant gene, showing a dominant pattern of inheritance, which may be acting to compromise normal p53 function rather than by a mutation in p53 itself. In order to try to determine the chromosomal location of this putative gene, we have carried out studies of linkage to candidate loci. By these means we have excluded involvement of Rb1 and BRCA1 on chromosomes 13q and 17q respectively. The MDM2 oncogene on chromosome 12q was considered to be the prime candidate as MDM2 is amplified in sarcomas and the MDM2 product binds to p53. Furthermore, p53 mutation and amplification of MDM2 have been shown to be mutually exclusive events in tumour development. Linkage analysis to two polymorphic markers within MDM2 yielded a three-point LOD score of -5.4 at a recombination fraction theta equal to zero. Therefore MDM2 could be excluded. It is possible that the gene which is responsible for cancer susceptibility in this family, possibly via interaction with p53, will be important in the histogenesis of breast cancer in general. We are now carrying out further studies to locate and identify this gene.

Base Sequence↗
PubMed1994

Male fertility after VAPEC-B chemotherapy for Hodgkin's disease and non-Hodgkin's lymphoma.

Semen analysis was performed in 14 men a median of 13.5 months after completion of VAPEC-B chemotherapy for Hodgkin's disease or non-Hodgkin's lymphoma. Semen from 12 patients contained motile spermatozoa, and in nine cases the count was > 20 million ml-1. One patient was azoospermic (VAPEC-B followed by pelvic radiotherapy) and another had a count of 21 million ml-1 but sperm were non-motile. These findings suggest that, in the majority of cases, VAPEC-B chemotherapy does not cause permanent damage to the male germinal epithelium. A more detailed study of gonadal function in males and females before and after treatment with VAPEC-B for Hodgkin's disease is currently in progress.

Adolescent↗
PubMed1994

Inactivation of O6-alkylguanine-DNA alkyltransferase in human peripheral blood mononuclear cells by temozolomide.

O6-alkylguanine-DNA-alkyltransferase (ATase) activity was measured in extracts of peripheral blood mononuclear cells (PMCs) taken from eight patients at various times during 5 days of oral treatment with temozolomide (150 mg m-2, days 1-5). Pretreatment ATase levels ranged from approximately 70 to 600 fmol per mg of protein. Depletion of PMC ATase was seen within 4 h of the first dose of temozolomide and had a median nadir of 52.9% and values ranging from 44.4% to 71.0% of pretreatment levels. There was a correlation between the extent of ATase depletion (pretreatment minus nadir level) and the pretreatment ATase level (r = 0.97). A progressive depletion of ATase was observed during the 5 days of continuous temozolomide therapy with median ATase activities of 66.3%, 52.5%, 39.5%, 30.5% and 28.9% of the pretreatment values at days 2, 3, 4, 5 and 6 respectively. This suggests that the schedule-dependent anti-tumour activity of temozolomide seen in experimental models and clinics may be related to a cumulative depletion of ATase.

Administration, Oral↗
PubMed1994

A reliable approach for sequencing clone-specific CDRIII regions in B-cell lymphoma.

We report a reliable approach for sequencing lymphoma-specific CDRIII regions. CDRIII regions present in DNA prepared from routinely fixed and paraffin-embedded diagnostic lymph node material were amplified by the use of consensus VH and JH primers via PCR. PCR products were subcloned directly, without purification or modification of PCR fragments. Only small amounts of miniprep plasmid DNA of recombinant clones were required for cycle sequencing, resulting in autoradiograms of high quality. The easy and reproducible method which we describe has enabled us to determine the lymphoma-specific CDRIII region in 7/11 high-grade non-Hodgkin's lymphomas as well as in 3/3 cases of ALL and 1/1 case of a centroblastic/centrocytic lymphoma. The obtained sequence data can serve to generate lymphoma-specific oligonucleotides, which then can be used as PCR primers or hybridization probes for the detection of minimal residual disease in individual patients.

Base Sequence↗
PubMed1994

Haemopoietic growth factor and dose intensity in high-grade and intermediate-grade lymphoma.

Here we review the evidence that cytotoxic dose intensity is an important determinant of outcome in high- and intermediate-grade non-Hodgkin's lymphoma. The results of retrospective analyses and prospective studies support this proposition but confirmatory studies are required. We discuss the role of haemopoietic growth factors and mobilized peripheral blood progenitor cells to increase dose intensity. Studies using these modalities will enable the importance of dose intensity to be tested.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1994

Report on a workshop convened to discuss the pathological and staging classifications of gastrointestinal tract lymphoma.

It was considered timely to review the pathological and staging classifications of GI tract lymphoma. This meeting specifically did not address the question of treatment; the management of GI tract lymphoma could perhaps form the basis for a further workshop. The following recommendations were made: to adopt the Isaacson histological classification, that all patients with GI tract lymphoma be investigated uniformly, to record the prognostic factors described above, to use the staging classification shown above. It is hoped that these recommendations will be taken into account in the design of future clinical trials of therapy for GI tract lymphoma.

Gastrointestinal Neoplasms↗
PubMed1994

Effects of erythropoietin on mobilisation of haemopoietic progenitor cells.

Peripheral blood progenitor cell (PBPC) mobilising regimens that do not include cytotoxic drugs are needed if PBPC are to be used for allogeneic transplantation. We have studied the effects of erythropoietin on bone marrow and circulating haemopoietic progenitors. Eleven patients with untreated lymphoma received epoetin-alpha 300 or 450 iu/kg subcutaneously (sc) thrice weekly for 2 weeks. Their bone marrows and peripheral bloods were normal at entry. There were no differences between dose levels. Peripheral blood colony-forming cell (CFC) numbers increased fivefold over baseline (p = 0.003) including a sevenfold increase in GM-CFC (p = 0.003). CD34-positive cell numbers increased 4.6-fold (P < 0.01). Maximal CFC release was seen at days 5-8. No consistent change in megakaryocyte numbers was seen. The increase in numbers of cells capable of long-term haemopoiesis was not significant. The ratio of myeloid:erythroid cells in bone marrow reduced from 2.7 to 0.86 after erythropoietin treatment (p = 0.047). In bone marrow the only significant rise in CFCs was in erythroid progenitors. Erythropoietin alone resulted in a modest increase in PBPC that is unlikely to be useful for transplantation. The effects of erythropoietin in combination with other cytokines or with cytotoxic drugs remain to be explored.

Adult↗
PubMed1993

Peripheral blood progenitor cell transplantation in lymphoma and leukemia using a single apheresis.

Myeloablative treatment and peripheral blood progenitor cell (PBPC) transplantation are increasingly used for lymphomas and leukemias. We have sought to optimize conditions for priming, collection, and engraftment of the leukapheresis product. Fifty-four consecutive adult patients were eligible, 31 with high-grade non-Hodgkin's lymphoma of poor prognosis, 12 with Hodgkin's disease in chemosensitive relapse, and 11 with poor prognosis acute lymphoblastic leukemia. Filgrastim was administered after routine chemotherapy with VAPEC-B or HiCCOM to mobilize PBPC. A rapidly increasing white blood cell count was used to predict the time of peak PBPC release and plan leukapheresis. Forty-five patients underwent leukapheresis. A median of 14 L of blood was processed at a single apheresis. A median of 2.4 x 10(8)/kg mononuclear cells (MNCs), 1.04 x 10(6)/kg granulocyte-macrophage colony-forming cells (GM-CFCs), and 10.6 x 10(6)/kg CD34+ cells were obtained. Slightly fewer MNCs were obtained from the heavily pretreated Hodgkin's disease group. There were no other significant differences in the size or composition of the leukapheresis harvest in the three patient groups. Forty patients underwent high-dose therapy and PBPC transplantation. Filgrastim was administered by daily subcutaneous injection until the absolute neutrophil count was > or = 1 x 10(9)/L for 2 consecutive days. Rapid and sustained hematopoietic engraftment occurred in all patients. The median time to achieve a neutrophil count > or = 0.5 x 10(9)/L was 9 days (range, 8 to 16 days); to achieve a platelet count > or = 20 x 10(9)/L was 10 days (range, 6 to 88 days); and to achieve a platelet count > or = 50 x 10(9)/L was 15.5 days (range, 10 to 100 days). Neutrophil recovery was faster than that of a historical control group treated with autologous bone marrow transplantation and filgrastim, but platelet recovery times were halved in the PBPC group. There was no secondary engraftment failure. Requirements for blood and platelet transfusions, antibiotic use, and parenteral nutrition were similar in the three patient groups. The median number of days in the hospital was 13 (range, 10 to 55) in the PBPC patients, compared with 19 (range, 14 to 51) in the historical controls. Leukapheresis yields (MNC, GM-CFC, and CD34+ cell numbers) were not useful for predicting the times to engraftment. We have shown that sufficient PBPC for transplantation can be obtained at a single leukapheresis after mobilization with routine chemotherapy and filgrastim in patients with non-Hodgkin's lymphoma, Hodgkin's disease, and acute lymphoblastic leukemia, even those heavily pretreated.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMed1993

Transplantation potential of hematopoietic cells released into the circulation during routine chemotherapy for non-Hodgkin's lymphoma.

Primitive hematopoietic cells released into the peripheral blood (PB) were studied in 50 patients with high-grade non-Hodgkin's lymphoma enrolled in a phase III trial of intensive weekly chemotherapy (VAPEC-B) alone or with granulocyte colony-stimulating factor (G-CSF). Mononuclear cells numbers were monitored and their in vitro growth potential assessed in clonogenic progenitor cell assays and in long-term culture. Total colony-forming cells (granulocyte-macrophage [GM], burst-forming unit, erythroid [BFU-E], Mix-CFC) were increased 40-fold (median) over baseline with chemotherapy alone and 106-fold with chemotherapy and G-CSF after the final dose. CD34+ cells were increased to a median of 4%, equivalent to that in normal bone marrow (BM) controls. Circulating colony-forming cell levels were maximal when the recovering total white blood cell (WBC) count reached 5 to 10 x 10(9)/L. The timing of the maximum was reproducible in individual patients. Therefore the WBC count can be used as a guide to the timing of leukapheresis. PB cells from normal controls' and patients' prechemotherapy were unable to sustain hemopoiesis in two-stage long-term cultures. In contrast, PB cells collected from patients primed with chemotherapy alone or chemotherapy with G-CSF at the time of predicted maximal colony-forming cell release were able to generate and sustain hematopoiesis in long-term cultures at a level comparable or superior to normal BM. These findings indicate that the use of G-CSF after routine outpatient chemotherapy stimulates maximal release of primitive hemopoietic cells into the circulation, including colony-forming cells and long-term culture-initiating cells. Their numbers are comparable with those in normal BM and are such that a single leukapheresis will usually yield enough cells for hemopoietic reconstitution after myeloablative chemotherapy.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1993

Expression of O6-alkylguanine-DNA-alkyltransferase in situ in ovarian and Hodgkin's tumours.

The cellular expression of O6-alkylguanine-DNA-alkyltransferase (ATase) may be an important factor in determining tumour sensitivity to certain alkylating agents. In a comparative study, we have examined the inter- and intracellular distribution of ATase in tumour biopsies of a series of patients with Hodgkin's disease and ovarian cancer using a rabbit antihuman ATase antiserum. The antibody recognises the ATase protein on western blots of cell-free extracts of a number of ovarian tumours with ATase activities varying from 20 to 420 fmol/mg protein as determined by in vitro assay and there was a linear correlation between ATase activity and the intensity of the band on western blots (r = 0.993). Immunohistochemical staining was seen in all of the ovarian tumours examined and was confined to the nucleus. This is in contrast to the Hodgkin's tissue, where staining was much reduced and present in both nuclei and cytoplasm. The results suggest that in ovarian tumours the general resistance to nitrosourea chemotherapy may be related to the high cellular expression of ATase protein: this is in contrast to the more chemosensitive Hodgkin's disease. This raises the possibility that it might be feasible to predict sensitivity or resistance to these alkylating agents by immunohistochemical staining of tumour or tissue specimens.

Adult↗
PubMed1993

Recombinant human interleukin-4 (rhu IL-4) administered by the intravenous and subcutaneous routes in patients with advanced cancer--a phase I toxicity study and pharmacokinetic analysis.

19 patients with advanced cancer were entered into a phase I study of recombinant human interleukin-4 (rhu IL-4). The predominant clinical side-effects included flu-like symptoms, gastrointestinal upset, lethargy and transient hypotension. In addition, there were several cases of capillary leak syndrome. 2 cases of gastrointestinal haemorrhage occurred; this was life threatening in 1 patient. The maximum tolerated dose (MTD) was 400 micrograms/m2/day. Biochemical toxicity was limited to asymptomatic elevation of liver enzymes suggesting IL-4 induced liver damage. Pharmacokinetic analysis following the intravenous bolus injection has shown that IL-4 is rapidly cleared (mean T1/2 = 19 +/- 8.7 min) from a small compartment (mean Vd = 4.9 +/- 3.68 l) probably indicating that IL-4 is retained in the systemic circulation or at most the extracellular fluid volume. 2 patients with non-Hodgkin lymphomas (NHL) showed a transient response to IL-4 whilst a third patient with NHL showed transient disease progression.

Adult↗