Ghislaine Simonin

Adult biphenotypic acute leukaemia: an entity with poor prognosis which is related to unfavourable cytogenetics and P-glycoprotein over-expression.

Biphenotypic acute leukaemia (BAL) patients represented 8% of the 287 de novo consecutive adult acute leukaemias (23 BAL, 230 acute myeloid leukaemia (AML) and 34 acute lymphoblastic leukaemia (ALL)) referred to our department during the last 4‐year period. Of these 23 BAL patients, 14 patients showed myeloid morphology and nine cases lymphoid morphology according to FAB criteria. There were no differences between lymphoid and myeloid BAL according to clinical and biological presentation and treatment outcome. We confirm the poor prognosis of BAL when compared to AML or ALL seen during the same period of time, in terms of complete remission (47%, 62% and 82% respectively, BAL v AML, NS and BAL v ALL, P = 0.006) and 4‐year overall survival (8.1%, 25.8% and 23.8% respectively, BAL v AML, P = 0.05 and BAL v ALL, P = 0.003). Comparing adult BAL patients with AML patients, we found an increase in poor prognostic factors: CD34+ phenotype (82% v 60% respectively, P = 0.03), unfavourable karyotype (60% v 20%, P < 0.0001) and Pgp over‐expression by RT‐PCR (0.705 v 0.107, P < 0.0001) and flow cytometry (0.824 v 0.391, P = 0.0001). MRP and LRP were not found to be poor prognostic factors. Comparing BAL patients with ALL patients, we found also an increase in poor prognostic factors: age (51 v 39, P = 0.003) and CD34+ phenotype (82% v 50%, P = 0.02). We conclude that BAL patients need a more aggressive treatment procedure, including high‐dose AraC or the use of Pgp modulators for first‐line therapy.

Expression of the multidrug resistance-associated protein (MRP) mRNA and protein in normal peripheral blood and bone marrow haemopoietic cells

We studied the expression of multidrug resistance‐associated protein (MRP) in normal haemopoietic cells from peripheral blood and bone marrow. The MRP mRNA levels were estimated by RT/PCR and in situ hybridization (ISH) assay, and the protein levels by flow cytometry. 21 samples of peripheral blood and 21 samples of bone marrow (11 normal bone marrow donors, 10 patients in complete remission after chemotherapy for large cell lymphoma or acute myeloid leukaemia) were analysed. In peripheral blood the mean MRP mRNA level in CD3+ cells was statistically higher than in the other cells (3‐fold by the methods used). The levels of MRP in CD3+ varied from one individual to another (4.5–34.8 units by RT/PCR and 5–23 grains/cell by ISH); however, this was proportional to the variation in all the cell lineages of same individual (r = 0.84). In bone marrow the mean MRP levels of the various cell lineages (including CD34+) were similar to the basal level in HL60 cells. Individual expression levels were again variable; however, there was no difference between untreated normal bone marrow and post chemotherapy normal bone marrow. MRP protein expression was determined by flow cytometry with the monoclonal antibody MRPm6. The CD4+ lymphocytes exhibited a higher MRP protein expression than the other cell lineages, including CD8+ cells. There was a good correlation between the three methods used (RT/PCR and ISH, P = 0.0001, r = 0.87; RT/PCR and flow cytometry, P = 0.0001, r = 0.85; ISH and flow cytometry, P = 0.002, r = 0.67).

Lung resistance protein (LRP) gene expression in adult acute myeloid leukemia: a critical evaluation by three techniques

The role of LRP in clinical drug resistance in acute myeloid leukemia (AML) is controversial. We therefore compared multiple assays, including RT-PCR, immunocytochemistry (ICC) and flow cytometry (FC), in 10 cell lines and in 47 fresh and thawed AML cells in order to validate and to quantitate measures for LRP phenotype detection. We also compared different ways of expressing the results. Lastly, in cell lines, we analyzed the 50% lethal concentration (LC50), by MTT assay, of cisplatin which could estimate the functionality of LRP. The reproducibility of LRP detection measured by RT-PCR, ICC and FC was good. In the same way, within the same technique, there was good correlation between the different methods of expressing the results of LRP level. Therefore, the discrepancies noted with the three techniques used were neither a problem of reproducibility nor a problem of results expression. On the other hand, there was only a correlation between ICC and FC, and no correlation between RT-PCR and LRP protein detection techniques. Therefore, RT-PCR is probably not the optimal technique for LRP detection. We have shown in 10 cell lines a higher correlation between FC and LC50 of cisplatin than between ICC and LC50 of cisplatin and no correlation between RT-PCR and LC50 of cisplatin. For five patients, there was a dissociation between ICC and FC. Four patients were positive by FC and negative by ICC and only one patient was negative by FC and positive by ICC. Therefore, if in vitro resistance to cisplatin represents the functionality of LRP, we recommend the use of FC rather than ICC to detect LRP expression. Besides the measurement of LRP as a diagnostic tool in the evaluation of resistance to chemotherapy in patients with AML, we urgently need to establish a functional test in order to assess LRP activity.

Effect of the multidrug inhibitor GG918 on drug sensitivity of human leukemic cells

The drug GG918 has been specifically developed for overcoming MDR phenotype and is now in use in clinical trials. In this study, the effects of GG918 on leukemic cell were investigated using a 3 day MTT assay. Results showed that, in a highly resistant P-gp(+) leukemic cell line, 0.1 μ M of GG918 gives rise to a 40-fold sensitization to daunorubicin (DNR) (residual resistance: 2.1), a 57-fold sensitization to mitoxantrone (residual resistance: 1.5), and a 3.3-fold sensitization to idarubicin (residual resistance: 2.9). When human AB serum was added to the incubation medium, 1 μ M GG918 was needed to observe the full P-gp modulation potency described above. The effect of 1 μ M of GG918 was tested on 27 samples of poor prognosis acute leukemia (25 AML, two ALL). DNR sensitization (using the MTT assay) and modulation of rhodamine 123 uptake were monitored and used as criteria for comparing the in vitro modulation potency of this new compound to the potency of 10 μ M of verapamil, which was used as reference. A good correlation (r = 0.8, P = 0.001) was observed between the results of the two tests. Eleven out of the 26 cases tested were MDR1 (+) (42%), and showed a higher IC50 for DNR than the negative cases (861 ± 1284 n Mvs 187 + 246 n M, P = 0.05). GG918 was able to modulate the in vitro resistance to DNR in eight cases (seven MDR1(+), no MDR1(−), one non-tested). Verapamil did not increase DNR toxicity in four of these eight cases, but was more efficient in one other MDR1(+) case. In conclusion, the DNR sensitivity of the majority of the fresh AML samples expressing P-gp could be modulated in vitro by 1 μ M of GG918.

Both P-gp and MRP contribute to drug resistance in AML

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Expression of genes implicated in multidrug resistance in acute lymphoblastic leukemia in India.

Abstract In order to investigate the phenomenon of multidrug resistance as a possible mechanism for poor response to treatment in patients with acute lymphoblastic leukemia (ALL) from India, a series of 32 cases of de novo untreated ALLs were analyzed by a cDNA-PCR approach to estimate the relative mRNA levels of the MDR-associated genes encoding MDR1, MRP, GSTπ, and GSTμ. The expression of β2 microglobulin served as an internal standard. Quantifiable transcripts were observed in 20 patients for MRP, in 5 for MDR1, in 24 for GSTπ, and in 19 for GSTμ. The values ranged from undetectable to 132% of the control A549 cell line for MRP, undetectable to 49% of the HL60/DNR control cell line for MDR1, undetectable to 268% of A549 control cell line for GSTπ, and undetectable to 247% of A549 control cell line for GSTμ mRNA. Increased MRP levels were associated with increased GSTπ and GSTμ levels (p<0.01 for both), and increased levels of MDR1 were associated with increased GSTπ levels (p<0.05). The present observations showed no correlation between the MDR1 and MRP values with treatment outcome, in terms of either achieving a complete remission or predilection to early relapse. In view of some recent studies that envisage MRP as an energy-dependent pump involved in the efflux of GSH conjugates, the simultaneous up-regulation of transcription of all these genes might well be part of an integrated detoxification response that has been switched on after exposure to an environmental stress.

Both Pgp and MRP1 Activities Using Calcein-AM Contribute to Drug Resistance in AML

Thirteen cell lines with different levels of Pgp and MRP1 expression were used to assess the ability of calcein-AM uptake and calcein efflux to measure Pgp and MRP1 functions, respectively. There was a good correlation between MRP1 expression and the modulatory effect of probenecid (a specific modulator of MRP1) on the calcein efflux (r = 0.91, p = 0.0003) and between Pgp expression and the modulatory effect of CsA on calcein-AM uptake (r = 0.96, p < 0.0001). On light of the high correlations for both proteins, we tested calcein-AM uptake and efflux in fresh myeloid leukemic cells. In 53 AML patients, there was also a good correlation between MRP1 expression (measured by RT/PCR and by MRPm6 expression by flow cytometry) and the modulatory effect of probenecid on the calcein fluorescence (r = 0.92, p < 0.0001) and between Pgp expression as measured by UIC2 antibody binding on flow cytometry and the modulatory effect of CsA on calcein-AM uptake (r = 0.83, p < 0.0001). Pgp activity was higher in CD34+ leukemia than in CD34- leukemia (2.26 +/- 1.50 vs 1.46 +/- 1.21 respectively, p = 0.003) and MRP1 activity was higher in CD34- leukemia than in CD34+ leukemia (1.77 +/- 0.40 vs 1.4 +/- 0.29 respectively, p = 0.004). Pgp expression and activity (p = 0.004 and p = 0.01, respectively), MRP1 activity (p = 0.03) but not MRP1 expression were prognostic factors for achievement of CR. The effect of probenecid and CsA together were higher than the effect of either probenecid or CsA alone on calcein-AM uptake. These results suggest that functional testing (with calcein-AM +/- modulators) for the presence of both MRP1 and Pgp activities is of prognostic value and that MRP1 contributes to drug resistance in AML.