Whole genome and transcriptome analysis of genetic determinants associated with cyclophosphamide in vitro in pediatric acute leukemias

Abstract

Introduction: The main objective was to implement the determinations of the ex vivo resistance to cyclophosphamide and to identify the genetic profile for pediatric patients with acute leukemias. Methods: In order to determine the ex vivo drug resistance profile, MTT cytotoxicity assay was performed on mononuclear cells. Gene expression profiles were prepared on the basis of cRNA hybridization to oligonucleotide arrays of the human genome (Affymetrix). We performed also array-based comparative genomic hybridization using a SurePrint G3 Human CGH Microarray. Data was analyzed by bioinformatics tools. Verification of the relative expression level of 20 genes was carried out by qRT- PCR. Results: We observed a multitude of differentially expressed genes, e.g. ANXA1 (FC=3,04), BCL2A1 (FC=2,69), SERPINA1 (FC=2,12), DHRS7 (FC=2,13), PCDH9 (FC=- 4,58), TTC28 (FC=-2,25) and DUSP1 (FC=-2,91). The expression of genes that code for inflammation mediated by chemokine and cytokine signaling, Wnt, angiogenesis and integrin signaling and T cell activation pathways genes affect the sensitivity of leukemic blasts to cyclophosphamide. Transcriptome level changes are associated with chromosomal aberrations, especially located on chromosomes 8, 10, 14, 15, 16 and 22. Conclusion: Our work delineated genes with differentiated expression and recurrent copy number changes, and revealed novel amplified loci and frequent deletions in resistant to cyclophosphamide cells, which may guide future work aimed at identifying the relevant target genes. In particular, deletion seems to be a frequent mechanism of IFIT3 gene inactivation. ANXA1, SERPINA1, TCF7 and BCL2A1 may also be included among the candidate genes of resistance (Ontological analysis).