CD34-related coexpression of MDR1 and BCRP indicates a clinically resistant phenotype in patients with acute myeloid leukemia (AML) of older age

A group of 154 patients with AML aged 60 years or older were included in the present study. All patients were enrolled between May 1997 and February 1999 in an international, multigroup, randomized phase 3 trial performed under auspices of the Dutch–Belgian Hemato-Oncology Cooperative Group and the UK Medical Research Council [14]. In that trial, 419 eligible white patients ≥60 years with previously untreated de novo and secondary AML (M0–M2 and M4–M7 according to the French–American–British [FAB] classification [15]) were randomized to receive two cycles of induction chemotherapy consisting of daunorubicin (DNR) and cytarabine (ara-C) with or without the P-gp inhibitor PSC-833 (Valspodar, Amdray®; Novartis Pharma, Basle, Switzerland). Patients in both arms in complete remission after these two cycles were to receive one consolidation consisting of ara-C, MXT, and etoposide. Inclusion criteria, clinical characteristics, treatment, and outcome of the phase 3 trial have been previously reported [14].

Bone marrow (BM) aspirates had been collected at diagnosis for the analysis of P-gp function and expression, as described previously [14]. Selection of patients for our study was based on availability of sufficient purified AML blast samples in our tissue bank, which was the case for 154 patients.

This study was approved by the ethics committees of the participating institutions and was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients before randomization.

BM aspirates were obtained in heparinized tubes. Mononuclear BM cells were collected by Ficoll Hypaque density gradient centrifugation (density 1.077 g/m³; Pharmacia, Uppsala, Sweden). To obtain purified samples with more than 85% of blasts, T-cell depletion and adherence depletion was performed as previously described [16]. Cells were cryopreserved in Dulbecco modified Eagle medium (DMEM; Gibco, Paisley, UK) supplemented with 10% dimethyl sulfoxide (Merck, Darmstadt, Germany) and 20% fetal calf serum (FCS; Gibco) and stored in liquid nitrogen. On the day of the experiments, BM cells were thawed. Cells were washed and resuspended in DMEM supplemented with 10% FCS. Before RNA and DNA isolation, cells were washed with phosphate-buffered saline (Gibco).

The drug resistance proteins were analyzed using the methods that we previously reported [11]. In brief, total RNA was isolated using the TRISOLV™ extraction as described by the manufacturer (Biotecx, Houston, TX). RNA was aliquoted and stored at −80°C. RNA samples were analyzed for RNA integrity by gel electrophoresis. cDNA was synthesized by the use of the TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA), diluted, aliquoted, and stored at −80°C. Quantitative RT-PCR was used to measure the mRNA expression levels of MDR1, MRP1, LRP, and BCRP by Taqman-chemistry on an ABI PRISM 7700 sequence detector (Applied Biosystems) using two endogenous reference genes, i.e., glyceraldehyde-3-phosphate dehydrogenase and porphobilinogen deaminase.

The clinical endpoints have been defined previously [14]. In brief, complete response (CR) was defined as a normocellular BM with <5% blasts, no Auer rods, and no evidence of extramedullary involvement. Because data on peripheral blood recovery within 60 days were not always available, they were not considered as a criterium for CR. Patients who relapsed or died within 28 days after CR were considered as not having achieved a CR. Event-free survival (EFS) was calculated from the date of randomization until no CR on induction therapy, relapse after CR, or death in CR, whichever came first. Patients who did not reach CR were considered failure for EFS at 1 day after randomization. Disease-free survival (DFS) was determined for all patients who achieved CR on induction therapy and was calculated from the date of CR until relapse or death, whichever came first. Overall survival (OS) was measured from randomization until death from any cause. Patients who were still alive at the date of last contact were then censored.

mRNA data were obtained from a subset of 154 patients with sufficient BM samples in our tissue bank available for analysis. Baseline parameters of interest were MDR1, MRP1, LRP, and BCRP mRNA expression. Clinical endpoints were CR rate, EFS, DFS, and OS.

Baseline characteristics of patients with or without mRNA expression data available were compared using the Fisher exact test or the Pearson χ ² test in case of discrete variables, whichever appropriate, or the Wilcoxon rank-sum test in case of continuous variables. The association between patient baseline characteristics and mRNA expression levels was analyzed using the Pearson χ ² test of the Spearman rank correlation test, whichever was appropriate. The prognostic value of mRNA levels with respect to CR rate was determined using logistic regression [17] whereas the impact of MDR1, MRP1, LRP, and BCRP on EFS, DFS, and OS was analyzed with Cox regression analysis [18]. For this purpose, the natural logarithm of the mRNA expression levels of the four resistance genes were included in the analyses because of the very skewed distribution of the original mRNA levels. In addition, the outcome of patients with coexpression of MDR1 and BCRP was evaluated to confirm the poor prognosis of AML with MDR1/BCRP coexpression reported by Benderra et al. [19] in patients with a median age of 45 years or older. These patients were defined as having mRNA levels of these two drug resistance genes equal to or higher than the median. Their outcome was compared to the other patients with at least one of the MDR1 and BCRP mRNA expression levels below the median. Logistic regression and Cox regression analyses were performed unadjusted, as well as adjusted for other prognostic factors, i.e., secondary AML, natural logarithm of WBC count, square root of percentage CD34⁺ cells, and cytogenetic risk (favorable/intermediate versus unfavorable versus unknown), as well as for treatment arm in the phase 3 trial, as about half of the patients had been randomized to receive PSC-833 in addition to their chemotherapy. Kaplan–Meier curves [20] were generated to illustrate survival and were compared using the log-rank test [21]. All reported p values are two-sided and, in view of the exploratory nature of these analyses, were calculated without adjustment for multiple testing. p values ≤ 0.05 were considered statistically significant.