FLT3-ITD and MLL-PTD Mutations Influence the Expression of MDR1, MRP1 and BCRP mRNA in Adult Acute Myeloid Leukemia

A number of molecular aberrations have been described in acute myeloid leukemia (AML). They disturb normal haematopoiesis and are response for leukaemogenic mechanism and drug resistance. Therefore, they seem to be of prime importance for treatment outcome. FMS-like tyrosine kinase 3- internal tandem duplication (FLT3-ITD) and mixed-lineage leukemia gene-partial tandem duplication (MLL-PTD) are leukemia associated aberrations which confer worse prognosis. Downstream regulatory targets of FLT3-ITD and MLL-PTD are not well defined.

The over-expression of ABC transport proteins that function as a membrane pump responsible for the efflux of a range of chemotherapeutic drugs are known mechanism behind multidrug drug resistance (MDR) in AML. The p-glycoprotein (Pgp)-product of MDR1 gene and multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP) encoded by the relevant genes negatively influence the results of AML therapy.

The aim of this study was to analyze the expression of the MDR-1, MRP-1, BCRP messenger RNA (mRNA) in relation to the response to induction chemotherapy and relapse and with pretreatment laboratory and clinical characteristics such as age (<60, ≥60 years), disease status (de novo/secondary), cytogenetics, FLT3-ITD and MLL-PTD mutational status, white blood cell count (WBC) and the type of AML according to the French-American-British (FAB) classification. Moreover, the influence of MDR genes on disease free survival (DFS) and overall survival (OS) were estimated.

A total of 185 adult consequent patients (88 females and 97 males) at a median age of 53,1 years (range 18,5–86,6 years) with newly diagnosed, previously untreated AML, as well as 40 healthy donors were included in this study. DNA and RNA were extracted from mononuclear cells of bone marrow (147 patients) or peripheral blood (38 patients) samples collected after approval from the ethics committee and informed consent from the patients. FLT3-ITD and MLL-PTD were detected with PCR method. The RQ-PCR method was performed for the assessment of expression of the MDR-1, MRP-1, BCRP and LRP mRNA and the results were presented as coefficients calculated with an intermediate method according to Pfaffl's rule. The relative expression ratio of a target gene was calculated based on efficiency and the Ct deviation of the healthy donor′s sample versus the patient′s sample, and expressed in comparison to a reference gene:

In univariate analysis, the high expression of MDR-1 mRNA (>0,13) was associated with outcome of induction therapy (p=0,06) and of BCRP mRNA (> 1,14) with high relapse rate (RR) (p=0,013). We found that high expression of MDR-1 (>0,13), MRP-1 mRNA (> 0,84) and BCRP mRNA (> 1,14) significantly influence DFS (p=0,059, 0,032 and 0,009, respectively) and OS (0,048, 0,014 and 0,059, respectively). Interestingly, high BCRP mRNA expression (>1,14) proved to be an independent prognostic factor for RR (p=0,01) and DFS (p=0,002) in the multivariate analysis. FLT3-ITD and MLL-PTD were detected in 53 (29%) and 44 (25%), respectively.

Interestingly, we found correlation between expression of MDR1, MRP1 and BCRP mRNA and FLT3-ITD and MLL-PTD mutational status in presented group of patients. The expression of MDR-1 mRNA was significantly higher in patients no harboring FLT3-ITD mutation (0,20 vs. 0,05, p= 0,0001) and ≥60 years old (0,30 vs. 0,10, p= 0,007). The higher expression of MRP-1 mRNA was found in patients with FLT3-ITD mutation (0,96 vs. 0,70, p= 0,002) and the expression of BCRP mRNA was significantly higher in patients with MLL-PTD (0,61 vs 0,38, p=0,03).

The combined analysis of MDR genes allowed different classes of AML to be identified, with distinct clinico-biological features. The high expression of MDR genes correlates with worse treatment outcome of patients with AML. It seems that especially BCRP is associated with clinical resistant disease in AML and influence the RR and DFS. Our study also shows that MDR gene expression in AML patients should be considered together with leukemia associated aberrations. The interesting correlation found between expression of MDR1, MRP1 and BCRP mRNA and FLT3-ITD and MLL-PTD in AML patients needs further investigation to determine its clinical value and made final conclusions.

No relevant conflicts of interest to declare.