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Biomedical subjects

W Hiddemann

Publications and source records attributed to W Hiddemann.

At least 253 records · Page 14Linked to original sources
PubMed1996

Response to cytarabine ocfosfate (YNK01) in a patient with chronic lymphocytic leukemia refractory to treatment with chlorambucil/prednisone, fludarabine, and prednimustine/mitoxantrone.

Cytarabine ocfosfate (YNK01) is a novel orally applicable prodrug of cytosine arabinoside. Recent pharmacokinetic studies have revealed a prolonged release of the cytotoxic agent cytosine arabinoside (araC) from hepatocytes into the systemic circulation, resulting in a half-life of approximately 24 h for araC. The specific pharmacokinetic characteristics of cytarabine ocfosfate lead to a prolonged exposure of leukemic cells to this antineoplasstic agent during the 14-day cycle. the oral applicability during outpatient treatment and the sustained antineoplastic activity of araC against slowly proliferating leukemic B-cells suggest that cytarabine ocfosfate might be a useful drug in the treatment of chronic lymphocytic leukemia. Four years after diagnosis of B-CLL, a 50-year-old patient was started on cytarabine ocfosfate. Sequentially, the patient's disease had proved refractory to treatment with chlorambucil/prednisone (31 months), fludarabine (5 months), and prednimustine/mitoxantrone (3 months). These established regimens were discontinued because of increasing lymphocytosis, significant thrombocytopenia, and progressive B-symptoms. Following three cycles of cytarabine ocfosfate B-symptoms resolved, lymphadenopathy disappeared, and thrombocytopenia was significantly reduced. The patient has been free of these symptoms on a dosage of 1500 mg cytarabine ocfosfate/day (cycle of 14 days with intervals of 14-21 days) for 24 months and remains in an ongoing partial remission.

Administration, Oral↗
PubMed1996

Induction of resistance to hexadecylphosphocholine in the highly sensitive human epidermoid tumour cell line KB.

Hexadecylphosphocholine (HePC, Miltefosine) is a representative of the group of alkyl-lysophosphocholines showing remarkable antitumoral activity in in vitro experiments and in experimental animal tumour models. The epidermoid tumour cell line KB, which is highly sensitive to HePC (half-maximal growth inhibiting concentration, IC50: 1.2 microM; half lethal concentration, LC50: 2.8 microM), was slowly adapted to increasing concentrations of HePC. After 14 months, the adaptation process was stopped at a concentration of 10 micrograms/ml (23.5 microM). At this point, the KB cells tolerated high doses of HePC (IC50: 41.2 microM; LC50: 87.1 microM). The resistant cells (KBr) also showed crossresistance to the other well studied ether-lysophospholipids, Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, OMG-3PC; ET18OCH3) and Ilmofosine (1 S-hexadecyl-2-methoxymethyl-rac-(1-thio-3-hydroxy)propyl-3-phosphocho lin e, BM 41.440). Comparison of the KB and KBr cells showed that total lipid phosphate, ether-lipid content, vinyl-ether-lipid content, protein content as well as cholesterol content were unchanged. Furthermore, no changes were observed in the lipid composition between KB and KBr cells. Uptake of choline was also unchanged in both cells, but the uptake of D-myo-inositol was lower by a factor of two in the KBr cells. However, in KB cells, the addition of HePC induced a 50% reduction of D-myo-inositol-uptake, whereas in KBr cells inositol uptake was unchanged. Differences in HePC uptake and HePC metabolism were apparent between the KB and KBr cell lines. KBr cells showed a 3-fold lower uptake for HePC and a 3- to 4-fold faster metabolism of HePC than KB cells. However, the amount of non-metabolised HePC after 2 days of incubation with 1 microgram/ml HePC (LC50: 1.2 microgram/ml) in KB cells was 3- to 4-fold lower than the amount of HePC in KBR cells at 10 micrograms/ml (LC50: 37 micrograms/ml), indicating that KBr cells can incorporate higher amounts of HePC than KB cells without adverse effects for cell growth and viability. This seems to indicate that mechanisms other than slower uptake and faster metabolism are involved in the induction of resistance to HePC in KBr cells.

Antineoplastic Agents↗
PubMed1996

Expression of Bax in relation to Bcl-2 and other predictive parameters in breast cancer.

BACKGROUND: It has been suggested that modification of the physiological susceptibility to induction of programmed cell death in transformed cells contributes to the pathogenesis of cancer. One of the major regulators of cell death is the bcl-2 family. In breast cancer, altered expression of Bcl-2 has been described. Distribution of its counterpart Bax and the differential expression pattern have still to be evaluated. PATIENTS AND METHODS: Bax expression was investigated by immunohistochemistry in 122 primary breast cancers. Results were correlated with expression of Bcl-2 and other variables of predictive value. RESULTS: There was a positive association between Bax expression and histological grading, (over)expression of c-erbB-1 and -2 and proliferative activity. The correlation was most significant in cases where no concomitant Bcl-2 expression could be detected. In the same subgroup an inverse correlation with positivity for estrogen (and progesterone) receptors was observed. The presence of Bax was not significantly associated with either tumor type and size, nodal status or expression of c-erbB-3. CONCLUSION: Expression of Bax was coupled with negative histopathological features, especially when expression of Bcl-2 was downregulated concomitantly. It may appear that alterations of the differential Bax/Bcl-2 expression pattern take part in deregulation of proliferation and loss of differentiation, thus playing an important role in malignant progression.

Breast Neoplasms↗
PubMed1996

Increase of P-glycoprotein-mediated drug resistance by hsp 90 beta.

The expression of heat shock proteins hsp27, hsp60, hsp70, hsp90 alpha and hsp90 beta in extracts of three cell lines (LoVo DxR, KBChR8-5 and S180 DxR) expressing the MDR (multidrug resistance) positive phenotype as well as in the sensitive parental lines has been investigated. We present evidence that heat shock protein hsp90 beta is associated with the P-glycoprotein (Pgp or P170) one of the most prominent components of the drug resistance machinery. In the doxorubicin-resistant cell line LoVo DxR, but not in the sensitive parental line, hsp90 beta is expressed constitutively as shown by Northern blotting. The expression of hsp90 beta in the sensitive LoVo cell line, however, can be induced by exposure of the doxorubicin-sensitive parental cell line to different stress factors (dexamethasone, doxorubicin, heat treatment or cadmium chloride). We were able to demonstrate that hsp90 beta can be co-precipitated along with Pgp and vice versa. In native agarose gels both proteins migrated together as one single band as shown by Western blot analysis. This intracellular protein-protein interaction may present a mechanism for the modulation of Pgp function possibly by a stabilization of the protein which seems to be attributed to hsp90 beta (in the human colon carcinoma cell line and in the murine cell line S180). Antisense experiments with oligonucleotides directed against hsp90 beta and Pgp, respectively, showed a synergistic effect of the selected hsp90 beta antisense oligonucleotide in combination with the previously described Pgp antisense oligonucleotide in reducing the doxorubicin resistance. The hsp90 beta antisense oligonucleotide when applied in addition to the Pgp antisense oligonucleotide increased the doxorubicin sensitivity of the resistant human colon carcinoma cell line 2-fold. On the contrary, the hsp90 beta antisense oligonucleotide alone in contrast to the Pgp antisense oligonucleotide alone did not cause a reduction of the chemoresistance. Moreover, Pgp half-life was reduced in the presence of both antisense oligonucleotides as compared with an incubation with an anti-Pgp antisense oligonucleotide alone.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Optimizing therapy for acute myeloid leukemia based on differences in intracellular metabolism of cytosine arabinoside between leukemic blasts and normal mononuclear blood cells.

The increasing insights into the pharmacokinetics and the metabolism of arabinoside C (AraC) have improved the rationale for its application in leukemia therapy and have led to a pharmacologically directed design of antileukemic treatment. The current study aims at adding to this approach by detecting differences in the intracellular metabolism of AraC 5'-triphosphate (AraCTP) between leukemic and normal mononuclear blood cells. Measurements of intracellular AraCTP levels were complemented by determinations of plasma AraC and arabinoside uridine (AraU) concentrations and were performed in 26 patients with acute myeloid leukemia (AML) who were undergoing combination therapy, including high-dose (1.0 or 3.0 g/m2 x 2/day) AraC. Plasma AraC concentrations showed a linear relationship to the applied AraC dose but did not correlate with intracellular AraCTP levels. Substantial differences in AraCTP retention times were revealed, during 3-h infusions of either 1.0 or 3.0 g/m2 AraC in leukemic blasts from 10 patients with t1/2 values of 1.60-7.63 h (median, 2.42 h). In addition, AraCTP levels declined in only one patient by > 10% within the first hour after the end of therapy and remained constant or even increased up to 1.5-fold during a posttreatment period of 1-2.5 h in the other nine cases. In contrast, AraCTP retention times were relatively uniform in normal mononuclear blood cells from 11 patients, with t1/2 values of 3.34-5.29 h (median, 3.85 h). More importantly, AraCTP levels dropped by > 10% within the first hour after the end of the high-dose AraC infusion in eight of 11 cases. A posttherapeutic increase of > 10% was not observed in any patient. These differences in AraCTP pharmacokinetics between leukemic and normal blood cells provided the basis for a modified timing of AraC administration with the aim of selectively maintaining cytotoxic AraCTP levels in leukemic blasts while allowing an intermittent drop of AraCTP levels in normal cells. This modification may result in higher antileukemic activity without increasing the damaging effect on normal cells and may, thus, improve the therapeutic index for AraC.

Acute Disease↗
PubMed1996

Oral idarubicin pharmacokinetics--correlation of trough level with idarubicin area under curve.

Idarubicin is the first anthracycline that can be successfully administered via the oral route and thus may facilitate antineoplastic chemotherapy at an improved quality of life. These perspectives are somewhat hampered by the large variation in bioavailability between individual patients and the obvious requirement to monitor plasma concentration and area-under the curve values (AUC) for an appropriate adjustment of idarubicin dose. In this study we describe the pharmacokinetics of idarubicin and its main metabolite idarubicin in 12 patients after oral application of 20 mg/m2 idarubicin on 3 consecutive days and demonstrate that the 24-h trough levels shows high correlation with AUC and may thus allow a rapid and easy determination of individual drug concentrations and an appropriate dose adjustment. The average terminal half-life was 30.5h for idarubicin and 66.9 h for idarubicinol. The AUC for idarubicin and its main metabolite idarubicinol revealed a substantial interpatient variation with AUC values ranging from 25.7 to 114 ng x h/ml (average 58.1 ng x h/ml) for idarubicin and from 109.4 - 445.2 ng x h/ml (average 287.3 ng x h/ml) for idarubicinol. However, the ratio of idarubicin/idarubicinol differed only two-fold from 1:3.7 to 1:7.7 with an average of 1:5.1. Both idarubicin and idarubicinol concentrations were highly reproducible, however, upon measurements after repeated applications within individual patients. Moreover, idarubicinol and idarubicin AUCs showed a good correlation with r=0.78, indicating that the interindividual variations of idarubicin AUC reflects differences in absorptions rather than metabolism. In order to describe the interindividual bioavailability of idarubicin - represented by AUC - measurement of a single data point with a high correlation with the AUC would be ideal. Our study demonstrates that the 24-h trough level shows such an excellent correlation (r=0.96) with AUC, making it the perfect candidate for fast estimates of the individual bioavailability in a given patient. On this basis, the longitudinal measurement of the 24-h trough level may allow the assessment of the impact of interindividual variations in AUC of clinical outcome and toxicity.

Acute Disease↗
PubMed1996

[Low-grade malignant non-Hodgkin lymphomas--current status and trends in therapy].

Low-grade Non-Hodgkin Lymphomas [NHL] comprise a heterogenous group of disorders both in terms of their cellular and histological composition as in terms of their clinical course. The currently mostly applied classification systems, the Working Formulation and the Kiel Classification as well as the recently proposed Revised European American Lymphoma Classification, discriminate between low-, intermediate and high-grade subtypes. Low-grade NHL are characterized by a low to moderate proliferative activity and a long clinical course with median survival times ranging for approximately three years for centrocytic [CC] or mantle cell lymphomas [MCL] to five to eight years for centroblastic centrocytic [CB-CC] or follicular lymphomas [FL]. Recent cytogenetic and molecular biologic analyses indicate that these differences may result from distinct genetic abnormalities such as the translocation t [14; 18] which is frequently observed in FL-NHL and is associated with a bcl 2 overexpression, or the deregulation of the PRAD 1 in MCL-NHL induced by the translocation t [11; 14]. Therapy of low-grade lymphomas depends mainly on the extend of the disease. In the early stages I and II, at which approximately 15-20% of low-grade NHL are diagnosed, an extended field or a total nodal radiotherapy may be applied with curative intention. The treatment of patients with more advanced stages III and IV is controversial. The currently available information justifies a conservative approach by observing the natural course of the disease until therapeutic intervention is required due to the occurrence of B-symptoms, hematopoietic insufficient or lymphoma progression. Intensive chemotherapy seems not to translate into an improved disease-free or overall survival and can, therefore, not be recommended for first-line treatment. The most recent data of the German Low-Grade Lymphoma Study Group indicate, however, that after successful initial cytoreductive therapy maintenance treatment with interferon-alpha prolongs the disease-free interval and possibly also overall survival. New perspectives have arisen from the introduction of novel cytostatic agents such as purine analogues and the development of immunotoxins and antibody conjugated radioisotopes. Most promising at the present time appears the application of myelo-ablative radiochemotherapy followed by autologous peripheral blood stem cell transplantation which may provide a curative approach even for advanced stages of the disease.

Antineoplastic Agents↗
PubMed1996

Prednimustine, mitoxantrone (PmM) vs cyclophosphamide, vincristine, prednisone (COP) for the treatment of advanced low-grade non-Hodgkin's lymphoma. German Low-Grade Lymphoma Study Group.

The current study was initiated to compare the anti-lymphoma activity and side-effects of prednimustine/mitoxantrone (PmM) vs cyclophosphamide, vincristine, prednisone (COP) in patients with advanced low-grade non-Hodgkin's lymphomas in way of a prospective randomized multicenter trial. Two hundred and forty-six patients with stage III or IV centroblastic-centrocytic (CB-CC (Kiel-classification)) or follicle center lymphoma (FCL (REAL classification)) and centrocytic (CC) or mantle-cell-lymphoma (MCL) were randomized for therapy with either PmM or COP and are fully evaluable for response and toxicity. PmM consisted of prednimustine 100 mg/m2/day on days 1-5 and mitoxantrone 8 mg/m2 /day days 1 and 2, while COP comprised cyclophosphamide 400 mg/m2/day on days 1-5, vincristine 1.4 mg/m2/day on day 1 and prednisone 100 mg/m2/day on days 1-5. Both regimens were repeated for a total of six cycles followed by an additional two courses for consolidation in responding cases and a subsequent second randomization for interferon alpha maintenance vs observation only. Overall response rates were comparable with 83% complete and partial remissions after COP and 84% remissions after PmM. PmM revealed a significantly higher rate of complete remissions (36 vs 18%, P < 0.006), the majority being achieved after four courses. The more rapid and possibly also more effective reduction of the lymphoma cell mass by PmM resulted in a tendency to a longer event-free interval for patients achieving remissions after PmM as compared to COP with estimated median event-free intervals of 31 vs 14 months, respectively (P=0.04). Separate analysis of lymphoma subtypes showed a tendency to a lower rate of complete remission in CC or MCL as compared to CB-CC or FCL (16 vs 30%, P=0.12, NS) while overall response rates were in a similar range (81 vs 85%). In both subtypes, PmM induced a higher rate of complete remission while overall response rates were comparable after PmM or COP. Treatment associated side-effects comprised predominantly myelosuppression and granulocytopenia in particular which was more frequently observed after PmM than COP (43 vs 31 %, P < 0.0001). This difference was clinically irrelevant, however, since serious infectious complications were encountered in less than 3% of cycles after both regimens. COP therapy was associated with a significantly higher incidence and degree of hair loss and complete alopecia (31 vs 2%) as well as of peripheral neurotoxicity (23 vs 2%). These data show that both PmM and COP reveal a high anti-lymphoma activity in patients with advanced stage non-Hodgkin's lymphoma. PmM appears advantageous with a higher rate of complete remissions and a better tolerability with regard to secondary side-effects. A longer follow-up is needed to assess the long-term effects of initial treatment on disease-free and overall survival and the impact on additional maintenance therapy with interferon alpha.

Adult↗
PubMed1996

De novo AML with dysplastic hematopoiesis: cytogenetic and prognostic significance.

Dysplastic hematopoiesis is the morphological hallmark of myelodysplastic syndromes. Dysplastic features in one or more lineages are also found frequently in bone marrow aspirates from patients with de novo AML and have been associated with an unfavorable prognosis. We asked whether dyshematopoiesis is an independent prognostic factor or an indicator of unrecognized secondary leukemia, identified by characteristic chromosomal abnormalities. Bone marrow aspirates from 102 patients with newly diagnosed AML were analyzed. Morphological analysis was obtained in all patients, flow cytometric analysis in 96 and successful cytogenetic analysis in 65 bone marrow aspirates. Dysgranulopoiesis (DysG) was found in 55, dysmegakaryopoiesis (DysM) in 32 and dyserythropoiesis (DysE) in 23 patients. Decreased side scatter signals of neutrophils in the flow cytometric analysis (DysS) were detected in 32 patients. DysG and DysS showed a highly significant correlation (P = 0.0005). DysG was an adverse negative prognostic factor for remission rate and event-free survival (P = 0.04, P = 0.02). An unfavorable karyotype was associated with a significantly lower chance for event-free survival (P = 0.002). The incidence of an unfavorable karyotype was significantly higher in patients with DysG (P = 0.01), DysM (P = 0.02) and DysS (P = 0.01). In patients with an unfavorable karyotype, dysplasia had no additional prognostic influence, however, in patients with a normal, favorable or prognostically uncertain karyotype DysG remained a predictor of lower remission rate (P = 0.03). We conclude that dysgranulopoiesis, dysmegakaryopoiesis and decreased side scatter signals of neutrophils are indicators of secondary leukemias in bone marrow aspirates from patients with de novo AML.

Bone Marrow↗
PubMed1996

[Classification of malignant lymphomas--current status and clinical implications].

New techniques such as cytogenetics, molecular biology and immunology have provided new insights into the pathogenesis and development of malignant lymphomas. By this way they provided a new basis for a classification of lymphoid malignancies which is increasingly oriented on biology and pathogenesis. This process resulted in the 'Revised European-American Lymphoma' (R.E.A.L.) classification, which was recently proposed by an international group of pathologists. This classification comprises a preliminary end point of a continuing process which will require a reevaluation and adaptation to new knowledge in the future again. In order to facilitate the clinical use of this histopathologically and biologically oriented classification, a proposal for a clinical grouping was developed, which divides the R.E.A.L. lymphoma subtypes into the following categories: I. Indolent lymphomas (low risk group). II. Aggressive lymphomas (intermediate risk group). III. Very aggressive lymphomas (high risk group). IV. Morbus Hodgkin. This internationally developed consensus of a histopathologic classification and a clinical grouping is a substantial step forward towards an international cooperation in the management of malignant lymphomas. It is background for cooperative studies and justifies the hope that further advances will be achieved in the treatment of malignant lymphomas and that more effective strategies can be developed in the near future.

Cytogenetics↗
PubMed1996

Hexadecylphosphocholine inhibits phosphatidylinositol and phosphatidylcholine phospholipase C in human leukemia cells.

Hexadecylphosphocholine (HePC) is the main representative of a new group of antineoplastic agents, the alkylphosphocholines. Besides remarkable antiproliferative properties on tumor cells in vitro and in vivo, HePC also induces differentiation and inhibits invasive growth of neoplastic cells. Knowledge of the molecular mechanisms by which HePC mediates its biological effects is poor. The observation that analogous substances, the alkyllysophospholipids, may interfere with lipid dependent intracellular signaling suggested similar mechanisms for HePC. We therefore investigated the effects of HePC on phospholipase C (PLC) activation in intact human leukemia cell lines. HePC inhibited fMLP induced phosphatidylinositol-specific PLC activation in HL60 cells and TNF-alpha induced activation of phosphatidylcholine-specific PLC in U937 cells. HePC reduced the number of TNF-alpha receptors on the surface of U937 cells by about 60%. Receptors for fMLP were not affected. Inhibition of TNF-alpha induced PC-PLC activation, however, seemed to be regulated at a post-receptor level as PLC inhibition and receptor occupancy did not correlate.

Antineoplastic Agents↗
PubMed1996

Induction of resistance in the human leukemia cell line HL60 towards hexadecylphosphocholine and other ether phospholipid analogues.

Hexadecylphosphocholine (HePC) is a new ether lipid analogue with remarkable antineoplastic activity in vitro and in vivo. As the precise molecular mechanism by which this substance and probably other ether lipids exert their biological effects is still not defined, we tried to approach this problem by generating a cell line resistant to the antiproliferative properties of HePC. This was successfully accomplished by slow adaptation over a period of 14 months, of the very sensitive human leukemia cell line HL60. HePC resistant HL60 cells (HL60R) tolerate 8- to 10-fold higher doses of HePC and are continuously cultured in medium containing 10 micrograms/ml of HePC. An immunophenotypic and karyotypic characterization of HL60 and HL60R cells showed only marginal differences between the two cell lines. Total phospholipids, total cholesterol, protein and vinyl ether lipid content were equal in both cells. A down-regulation of the ether lipid mass in HL60R of about 40% could reflect one mechanism of tolerance induction. Though HePC uptake in HL60R cells was significantly lower than in the parental line, steady state measurements of cellular HePC content revealed similar HePC content in the membranes at HePC concentrations that were cytotoxic for HL60 but did not affect HL60R. This observation indicates that uptake and cellular accumulation of HePC do not determine HePC resistance. The resistant HL60R cells also showed a considerable degree of cross-resistance to ether phospholipids ET-18-OCH3 and BM 41.440, suggesting a common mode of action for HePC and other ether lipid analogues.

Antigens, Surface↗
PubMed1995

Analysis of rearranged T-cell receptor beta-chain genes by polymerase chain reaction (PCR) DNA sequencing and automated high resolution PCR fragment analysis.

Polymerase chain reaction (PCR)-directed amplification and sequencing of rearranged immune genes for identification of clone-specific markers are increasingly being used in acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL) patients instead of the time consuming and labor intensive Southern analysis. In previous reports, no single common V beta and J beta sequence had been identified that allowed reliable amplification of the majority of rearranged T-cell antigen receptor (TCR)-beta V-D-J junctions at the DNA level because of the relatively large number of possible TCR-beta variable (V beta) and joining (J beta) gene segments involved in the rearrangement processes. In the present study we designed highly degenerate PCR primers directed against conserved sequences of the J beta genes. IN combination with a previously published consensus V beta primer, these J beta primers specifically amplify TCR- beta V-N(D)N-J junctions from genomic DNA. Using this approach we studied DNA extracted from biopsy material of nine patients with T-cell lymphoproliferative disorders, one c-ALL patient, and five patients with nonmalignant diseases. T-cell lines Molt 3, Jurkat, and HM 2 served as monoclonal controls. Individual PCR products were sequenced after cloning. The nucleotide sequences of 96 randomly chosen recombinant vectors were determined. In the polyclonal controls all analyzed clones differed in their TCR-beta V-N(D)N-J junctions. In the T-cell lines, in all of the T-cell malignancies, and in the c-ALL, monoclonal PCR products could be identified by demonstration of clonally restricted V-N(D)N-J junctions. The PCR results were confirmed by automated fluorescence quantification and size determination of PCR products after separation in a high-resolution polyacrylamide gel. The procedure allows rapid and specific characterization of clonal TCR-beta rearrangements from genomic DNA and will significantly simplify current experimental approaches to identify and to quantitate malignant T cells during initial staging and follow-up of T-lineage NHL and ALL patients.

Automation↗
PubMed1995

Evidence for malignant transformation in acute myeloid leukemia at the level of early hematopoietic stem cells by cytogenetic analysis of CD34+ subpopulations.

Acute myeloid leukemia (AML) is a heterogenous disease according to morphology, immunophenotype, and genetics. The retained capacity of differentiation is the basis for the phenotypic classification of the bulk population of leukemic blasts and the identification of distinct subpopulations. Within the hierarchy of hematopoietic development and differentiation it is still unknown at which stage the malignant transformation occurs. It was our aim to analyze the potential involvement of cells with the immunophenotype of pluripotent stem cells in the leukemic process by the use of cytogenetic and cell sorting techniques. Cytogenetic analyses of bone marrow aspirates were performed in 13 patients with AML (11 de novo and 2 secondary) and showed karyotype abnormalities in 10 cases [2q+, +4, 6p, t(6:9), 7, +8 in 1 patient each and inv(16) in 4 patients each]. Aliquots of the samples were fractionated by fluorescence-activated cell sorting of CD34+ cells. Two subpopulations, CD34+/CD38- (early hematopoietic stem cells) and CD34+/CD38+ (more mature progenitor cells), were screened for karyotype aberations as a marker for leukemic cells. Clonal abnormalities and evaluable metaphases were found in 8 highly purified CD34+/CD38- populations and in 9 of the CD34+/CD38- specimens, respectively. In the majority of cases (CD34+/CD38-, 6 of 8 informative samples; CD34+/CD38+, 5 of 9 informative samples), the highly purified CD34+ specimens also contained cytogenetically normal cells. Secondary, progression-associated chromosomal changes (+8, 12) were identified in the CD34+/CD38- cells of 2 patients. We conclude that clonal karyotypic abnormalities are frequently found in the stem cell-like (CD34+/CD38-) and more mature (CD34+/CD38+) populations of patients with AML, irrespective of the phenotype of the bulk population of leukemic blasts and of the primary or secondary character of the leukemia. Our data suggest that, in AML, malignant transformation as well as disease progression may occur at the level of CD34+/CD38- cells with multilineage potential.

Acute Disease↗
PubMed1995

Highly sensitive coupled-column high-performance liquid chromatographic method for the separation and quantitation of the diastereomers of leucovorin and 5-methyltetrahydrofolate in serum and urine.

A column-switching chiral HPLC assay was developed that allows the separation and quantitation of the diastereomers of leucovorin (LV, 5-formyltetrahydrofolic acid) and its metabolite 5-methyltetrahydrofolate (METHF) in serum and urine by means of fluorescence detection. The analysis procedure consists of an on-line concentration of the folates in the HPLC system which is followed by the elution and separation of folates on an achiral 3-microns Microbore C18 column in (6R,S)-LV and (6R,S)-METHF. (6R,S)-LV and (6R,S)-METHF are subsequently transferred on-line onto a chiral 7-microns bovine serum albumin column through a Rheodyne valve system and are separated into their diastereometers. Time of analysis is 70 min. Detection limit is 5 ng/ml for each diastereometer. The within-day variation ranges between 3.2 and 15.8% in relation to the measured concentration. Between-day variation is 4.4-12.1% for a concentration of 100 ng/ml for each diastereometer. (6R,S)-LV and (6S)-LV pharmacokinetics were assessed by analyzing serum and urine samples of four-healthy volunteers.

Antineoplastic Agents↗