The Impact of FLT3-ITD Mutation on the Expression of Multidrug Resistance Genes MDR-1, MRP-1, BCRP and LRP In Adult Acute Myeloid Leukemia

Multidrug resistance (MDR) is a clinically relevant problem in the treatment of acute myeloid leukaemia (AML). From 20–50% of patients with AML are primarily resistant to induction chemotherapy and in the great majority of those who respond well to initial therapy the disease relapses. While some mechanisms of MDR have been well characterized, this phenomenon still remains incompletely understood. Three ATP-dependent drug transporting proteins: p-glycoprotein (Pgp; MDR-1), multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP) and one major vault protein-lung resistance-related protein (LRP) and the internal tandem duplication (ITD) in the receptor tyrosine kinase FLT3 have been found to have a strong correlation with MDR and poor outcome in AML. About 30% of AML cases harbor FLT3-ITD mutation. Mainly MDR-1 protein and FLT3-ITD mutation are targets for inhibition by numerous agents in clinical practice.

In this retrospective study we estimated expression of MDR genes encoding MDR-1, MRP-1, BCRP, LRP proteins and detected FLT3-ITD mutation in patients with AML. We divided all patients into two groups according to FLT3-ITD mutation and we compared expression of MDR genes and other prognostic factors in AML: age (<60 vs ≥60 years), cytogenetic/molecular aberrations (good, intermediate, poor), de novo/secondary AML, white blood cell count (WBC), extramedullary involvement, type of AML according to the FAB classification, CD34 expression on blast cells and myeloperoxidase activity (<50% of blast cells, ≥50%) between groups Moreover the rate of complete remissions (CR) and relapses and disease free survival (DFS) and overall survival (OS) in both groups were estimated.

A total of 126 adult patients at median age of 53 years (range 21–87) with newly diagnosed between June 2005 and March 2010, previously untreated AML and 40 healthy donors were included in this study. Diagnosis of AML was based on standard morphological and immunophenotypical criteria according to the WHO classification. Most of the patients were treated with anthracycline and cytarabine during the induction regime. There were 39 FLT3-ITD positive (30,4%) and 87 FLT3-ITD negative (69,6%) patients. DNA was extracted from bone marrow (90 patients) or peripheral blood (36 patients) samples and used to detect FLT3-ITD with PCR method. RNA was extracted from the same samples and following reverse transcription RQ-PCR method was performed for assessment of expression of MDR-1, MRP-1, BCRP and LRP genes. Parameters of RQ-PCR reaction were established based on the Europe Against Cancer protocol. Every tested sample was amplified simultaneously for the presence of control ABL gene for standardization of mRNA level for tested genes. Results of the expression of tested genes were presented as coefficients calculated with an intermediate method according to Pfaffl's rule.

To our surprise, significant higher expression of MDR-1 gene was found either in bone marrow or peripheral blood samples of the patients who do not harbor FLT3-ITD in contrast to the patients harboring this mutation (median 0,3025 vs 0,0756, p=0,003 total; median 0,2690 vs 0,0583, p= 0,002 bone marrow samples; median 0,5775 vs 0,0920, p=0,001 peripheral blood samples). For MRP-1, BCRP and LRP genes we did not find significant differences in their expression between two groups. When adjusted for other prognostic factors, only WBC was significantly higher in FLT3-ITD positive group of patients: 1–294×10⁹/l, median 74 vs 0–286×10⁹/l, median 6,5 in FLT3-ITD negative group of patients (p=0,0001). We did not observe significant differences in CR and relapses rates, DFS and OS between groups.

It seems that FLT3-ITD mutation may in some mechanisms influence MDR-1 gene expression We observed significant inhibition of MDR-1 expression in FLT3-ITD positive patients. Because risk factors for AML and outcome of AML therapy did not differ between groups it seems that FLT3-ITD mutation and high expression of MDR-1 gene are independent high risk factors which negatively influence results of AML therapy. These results might provide a powerful risk classification and useful information for treatment strategies including inhibitors of tyrosine kinase or MDR-1 protein.

This project was supported by grant of The Ministry of Science (N N402 208935).

No relevant conflicts of interest to declare.