The Significance of Blast Multidrug Resistance Detection by Flow Cytometry in Diagnostics of Acute Leukemia
Miglė Janeliūnienė, Rėda Matuzevičienė, Zita Kučinskienė
Introduction. With a modern intensive chemotherapy most of the patients suffering from acute leukemia achieve a complete remission. However, some of them eventually relapse. One of the most significant reasons of relapse is a multidrug resistance of leukemic cells. Transmembrane pumps, such as p-gp and MRP1, ensuring the extrusion of cycto- toxic compounds from the cell play a very important role in the pathogenesis of the multidrug resistance. Increased expression of these proteins on the blast cells influences a remission rate, its length and survival of acute leukemia patients. The main goal of this study was to evaluate the impact of flow cytometric assessment of the multidrug resistance in the diagnostic algorithm of acute leukemia. We compared p-gp and MRP1 expression and functional activity in blast and other blood cell populations. We also determined the correlation between structural and functional parameters of multidrug resistance. We searched for links between parameters of multidrug resisiance and the origin and immuno - phenotype of acute leukemia as well, as for their relation to the level of minimal residual disease at different treatment timepoints.
Material and methods. 30 paiients with acute leukemia were enrolled: 19 adults (10 females, 9 males, mean 52.2 years old) and 11 children (10 boys, 1 girl, mean 54.8 months old). For each patient blast immunophenotype was established, multidrug resistance tests (p-gp expression and functional test) were carried out and minimal residual disease level was evaluated at 5 treatment time-points.
Results. We found statistically reliable differences of multidrug resistance parameters between blast, lymphocyte and monocyte populations (e.g., t-test result between blast and lymphocyte p-gp expression was 0.024). Although we did not find a correlation between p-gp and MRP1 expression and calcein assay results, correlation between individual elements of structural resistance was rather strong (Pearson correlation coefficient was 0.830). Patients with acute lymphoblastic leukemia (ALL) had a lower MRP1 expression than patients with acute myeloid leukemia (AML), e.g. in case of T-ALL its mean fluorescence intensity was 78.667 and in case of AML with myelodysplastic features it was even 167.250. Calcein index (relation between calcein value in blast and lymphocyte populations) was also lower in ALL group, e.g. in case of B-ALL it was 0.930, and in case of AML it was 1.600. We found a statistically significant correlation between HLA-DR expression and calcein index and between CD7 and p-gp expression in case of AML, also between CD38 expression and calcein value in case of B-ALL. Statistically reliable links between calcein value and minimal residual disease at any time-point of treatment were not proven. Corre i aiion was found beiween p-gp and MRP1 expression and minimal residual disease at days 29-35 and 79-90.
We suggest that the determination of the multidrug resistance protein expression could be introduced into routine diagnostics of acute leukemia. The value of functional tests should be investigated further and several detection methods should be compared. As inhibitors of multidrug resistance appear in the market, laboratory tests of multidrug resistance inhibition would be useful.
Keywords: multidrug resistance, acute leukemia, flow cytometry, calcein, p-gp, MRP1.