Identification of Gene Expression Profiles in Acute Lymphoblastic Leukemia Cells That Discriminate Intracellular Thioguanine Nucleotide Accumulation in ALL Cells after In Vivo Treatment with Mercaptopurine.

Thioguanine nucleotides (TGN) are considered the principal active metabolites exerting the antileukemic effects of mercaptopurine (MP). Numerous clinical studies have reported substantial inter-patient variability in intracellular TGN concentrations during continuation therapy of acute lymphoblastic leukemia (ALL). To identify genes whose expression is related to the intracellular accumulation of TGN in leukemia cells after in vivo treatment with MP alone (MP) or in combination with MTX (MP+MTX), we used oligonucleotide microarrays (Affymetrixâ HG-U95Av2) to analyze the expression of approximately 9,670 genes in bone marrow leukemic blasts obtained at diagnosis from 82 children with ALL. TGN levels were determined in bone marrow aspirates of these patients 20 hours after mercaptopurine infusion (1 g/m2 I.V). Because, as previously reported, patients treated with MP alone achieved higher levels of intracellular TGN compared to those treated with the combination, we used Spearman’s rank correlation to identify genes associated with TGN levels separately for the 33 patients treated with MP alone and the 49 with the combination (MP: median TGN: 2.46 pmol/5x106 cells, range: 0.01–19.98; and MTX+MP: median TGN: 0.55 pmol/5x106 cells, range: 0.005–3.31). Hierarchical clustering using these selected probe sets clearly separated the 33 patients treated with MP alone into two major groups according to TGN concentration (< 2.46 and > 2.46 pmol/5x106 cells; n=60 genes) and two major branches were also found for patients treated with the combination (< 0.55 and > 0.55 pmol/5x106 cells; n=75 genes). Interestingly, there was no overlap between the two sets of genes, indicating that different genes influence the accumulation of TGN when this drug is given alone or in combination with MTX. The association between gene expression profiles and TGN levels determined by leave-one-out cross-validation using support vector machine (SVM) based on Spearman correlation, was rho=0.60 (p<0.001) for MP alone and rho=0.65 (p<0.001) for MTX+MP, with false discovery rate (FDR) computed using Storey’s q-value (MP: 50% true positive, MTX+MP: 70% true positive respectively). Genes highly associated with the post-treatment TGN level in ALL patients treated with MP alone encode transporters, enzymes involved in the MP metabolic pathway and cell proliferation. Genes associated with post-treatment levels of TGN after combined therapy have been implicated in protein and ATP biosynthesis. Together, these in vivo data provide new insights into the basis of inter-patient differences in TGN accumulation in ALL cells, revealing significant differences between treatment with MP alone or in combination with MTX.