Identification of Genetic Markers for the Outcome of Patients with Acute Myeloid Leukemia Treated with Cytarabine.

Cytotoxic nucleoside analogues are largely used in the treatment of hematological malignancies and solid tumors. Their mechanism of action, based on interference with the metabolism of physiological nucleosides, targets well known gene products involved in transmembrane transport and intracellular metabolism. Several studies have shown correlations between the expression level of some of these genes and the clinical response to nucleoside analogue-based treatments. We proposed to study the potential involvement of genomic sequence variation in inter-individual heterogeneity of the expression level of six genes playing a role in the metabolism and the mechanism of action of cytarabine, a nucleoside analogue used for the treatment of acute myeloid leukemia (AML). We also studied the role of selected SNPs in the clinical outcome of cytarabine-treated AML-patients. We genotyped exonic SNPs in the genes CDA, DCK, NT5C2, NT5C3, SLC29A1 and TP53 in leucoblasts from patients with AML at diagnosis (n=83), and used these as markers to study whether these genes are subject to differential allelic expression. Using only heterozygous samples and a method based on high resolution melting curve analysis, we observed differential allelic expression for CDA (17/26 assessed samples), DCK (3/8 assessed samples), NT5C2 (2/23 assessed samples), NT5C3 (11/27 assessed samples, 4 with monoallelic expression) and TP53 (3/19 assessed samples). In order to find candidate causal variants, we used bioinformatics tools to identify SNPs located in evolutionarily conserved transcription factor binding sites. Eleven SNPs in the 5′ region of CDA, NT5C2, NT5C3 and TP53 have been genotyped, but no variant has yet been confirmed as responsible for differential allelic expression in any gene. Preliminary statistical analysis of clinical data from 72 patients hinted at correlation between some of the genotyped SNPs and clinical outcome: i.e. increased time to relapse after complete response for CDA rs2072671 CC and AC carriers (n=28) as compared to AA homozygotes (n=11, p=0.048); shorter time to complete response for TT homozygotes (n=11) for NT5C3 rs3750117 than for CC and CT carriers (n=56, p=0.009); and shorter time to complete response for AA homozygotes (n=8) for NT5C3 rs7778958 than for GG and GA carriers (n=58, p=0.007). In addition, a correlation was found between rs10883841 in NT5C2 and the overall gene expression level determined by relative quantitative RT-PCR (CC: 50.5 ± 17.4, n=48 vs. CT: 17.3 ± 6.1, n=15; p=0.043). We have shown that several genes involved in the cellular response to the cytotoxic nucleoside analogue cytarabine present differential allelic expression in leucoblasts. This result indicates that genomic variants are at least partially responsible for the variation in gene expression levels observed between cytarabine-treated AML-patients. Further, we identified SNPs that can be used as markers for the clinical outcome of cytarabine treated AML patients. Ongoing research should identify the causal variants for the differential allelic expression and other SNPs that are possible predictive factors for the outcome of this treatment.