| 1. |
- Möllgård, Lars
(författare)
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Drug resistance in acute myeloid leukemia : pharmacokinetic and in vitro studies
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Chemotherapy has improved clinical outcome in adult acute myeloid leukemia (AML) but still the majority of the patients die of their disease. One important explanation is transport mediated drug resistance resulting in refractory or relapsing disease. P-glycoprotein (Pgp) is located in the membrane and can extrude cytostatic drugs. This will cause a reduction of intracellular concentrations of the drug and reduce the effect on the leukemic cells. Down regulation of the enzyme topoisomerase II alpha (topo II alpha) is another resistance mechanism. The aim of the first part of this thesis was to study effects of interaction with transport mediated resistance. In a phase I-II pharmacokinetic study 22 de novo AML patients were included and received a high single dose of mitoxantrone (30 or 40 mg/m2) in combination with ara-C. In the leukemic cells we found a high accumulation of mitoxantrone which, in contrast to plasma, remained stable during the 48 hours studied. Compared with previous results with mitoxantrone 12 mg /m2 the area under the curve (AUC) for intracellular concentrations was increased by 150% (30 mg/m2) and 260% (40 mg/m2) respectively. The toxicity was acceptable but a prolonged duration of neutropenia was noted. Several studies have shown that Pgp can be blocked and in the next study 10 AML patients were included and given a continuos infusion of daunorubicin in combination with the Pgp modulator PSC 833. The intracellular in vivo concentrations of daunorubicin in Pgp positive leukemic cells increased as well as the ratio of the AUC for daunorubicin in leukemic cells to the AUC in plasma suggesting that the mechanism was a specific inhibition of PgP by the modulator. Another aspect of drug resistance and cytostatic treatment is the potential interaction of Pgp, and drugs used in the supportive care of AML patients. 21 different drugs were tested in a Pgp expressing leukemia cell line and the change in intracellular rhodamine 123 was measured. However, in this model we found no significant interactions of the tested drugs on Pgp. In the second part of the thesis we studied two different chemosensitivity assays and markers for drug resistance. In the bioluminiscence ATP chemosensitivity assay cell death is measured as reduced ATP levels. 83 samples from 77 AML patients were included and the in vitro effect of six different cytostatic drugs were tested. We found that in vitro sensitivity to daunorubicin was associated with complete remission and prolonged disease free survival. One problem with in vitro assays is the possible contamination of non malignant cells which can interfere with the results. Therefore we established a new assay were selected myeloid cells were analysed using flow cytometry. 63 samples from 60 AML patients in different stages of their disease were tested. The method was feasible and we found correlations to clinical parameters and Pgp expression. Topoisomerase II alpha is a well known target enzyme for many cytostatic drugs. In this final investigation the expression of the enzyme was studied in different phases of the cell cycle in 25 acute leukemia patients. In contrast to normal cells we found that topo II alpha was expressed in GO/G1 phase. A low expression of topo II alpha in the GO/G1 phase seemed to be associated with resistant disease, which suggests that topo II alpha expression in the different cell cycle phases may have a predictive value. In conclusion a high single dose or the inhibition of Pgp increased the intracellular concentrations in vivo of mitoxantrone and daunorubicin respectively. Drugs used in the supportive care of AML patients did not interact with Pgp in vitro. In vitro sensitivity to daunorubicin was associated with both short and long term outcome using the bioluminiscence ATP assay. In vitro chemosensitivity testing of selected myeloid cells and low expression of topo II alpha in the GO/G1 phase, using flow cytometry techniques, correlated to clinical outcome but further studies are needed to evaluate the predictive value.
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| 2. |
- Möllgård, Lars, et al.
(författare)
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In vitro chemosensitivity testing of selected myeloid cells in acute myeloid leukemia
- 2003
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Ingår i: Leukemia and Lymphoma. - : Taylor & Frances healthsciences. - 1042-8194 .- 1029-2403. ; 44:5, s. 783-789
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Tidskriftsartikel (refereegranskat)abstract
- In several studies different chemosensitivity assays have been examined in acute myeloid leukemia (AML). Some have shown that in vitro chemosensitivity testing is an independent prognostic factor but so far no one has been able to show that the use of these methods can improve treatment outcome. In an attempt to improve in vitro chemosensitivity testing in AML we wanted to establish and evaluate a new flow cytometry chemosensitivity assay. After 4 days of incubation viable mononuclear myeloid cells were identified by the exclusion of propidium iodide in CD13 or CD33 positive cells. Sixty-eight samples from 64 AML patients were included. In this study, we showed that the flow cytometry method is feasible in AML and we also found some correlations to clinical data. The secondary AML at diagnosis showed an in vitro resistance to etoposide and amsacrine that was significantly higher compared to de novo AML at diagnosis (p = 0.04 and p = 0.02). When AML patients at diagnosis were compared to resistant disease/relapse patients there was a significantly higher effect of ara-C in the diagnosis group (p = 0.03). Responders and non-responders were compared in vitro but we found no significant differences. In vitro mitoxantrone was more effective in multidrug resistance (MDR) negative cells compared to MDR positive cells (p < 0.01). This new method is feasible and makes it possible to selectively evaluate the effect of cytotoxic drugs in myeloid cells. Further studies with a larger group of patients are needed to evaluate the predictive value of the assay.
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| 3. |
- Uggla, Bertil, 1962-, et al.
(författare)
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Expression of topoisomerase IIalpha in the G0/G1 cell cycle phase of fresh leukemic cells
- 2001
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Ingår i: Leukemia Research. - Oxford, United Kingdom : Elsevier. - 0145-2126 .- 1873-5835. ; 25:11, s. 961-966
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Tidskriftsartikel (refereegranskat)abstract
- Topoisomerase IIalpha (topoII alpha) is the target enzyme for several antineoplastic drugs. Correlation between low expression of topo IIalpha and drug resistance has been shown in vitro, but there is limited evidence of a correlation to initial response to treatment or to overall prognosis. Normal cells express topo IIalpha in S/G2/M phase of the cell cycle but not in G0/G1 phase. However, some data suggest that topo IIalpha could be expressed in G0/G1 phase in malignant cells. We have investigated the expression of topo IIalpha in leukemic cells from 25 patients with acute leukemia by flow cytometry, separating cells of different cell cycle phases. We demonstrated that 9/25 samples showed >50% positive cells in G0/G1, and another five samples showed >20%. This finding could possibly provide an explanation to previous difficulties in correlating topo IIalpha expression with clinical outcome. Six of eight patients, where >20% of the cells in G0/G1 were positive for topo IIalpha, entered CR, compared to one of five patients with <20% topo IIalpha positive cells in G0/G1. We suggest that topo IIalpha expression in G0/G1 in leukemic cells may be of predictive value for clinical response to cytostatic drugs.
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