DNA Methylation in Resistant Paediatric Acute Lymphoblastic Leukaemia.

Abstract Aberrant DNA methylation is a frequent phenomenon in paediatric acute lymphoblastic leukaemia (ALL). Increased methylation has been identified as an independent factor of poor prognosis in paediatric ALL patients. This study investigates the evolution of epigenetic changes in resistant and relapsed paediatric ALL to increase our understanding of the mechanisms of treatment failure in these patients. The primary objective is to establish global DNA methylation profile in ALL and whether changes in this profile are associated with chemo-resistance and relapse. We performed a genome-wide analysis of CpG island methylation in leukaemic blasts on a cohort of 22 paediatric ALL patients; 11 at presentation and 11 at relapse. Global methylation profiles were investigated using the recently developed CpG island array technology, which includes 12,192 sequenced CpG-island clones obtained from the Wellcome Trust Sanger Institute (Cambridge, UK). These clones have undergone comprehensive sequence analysis which validated the array content and confirmed that 80% of the clones have usable sequences that can be aligned to genomic DNA. The majority of CpG-island loci are localised to known distal promoter regions or close to a transcriptional start site. We have validated the application of this technology to leukaemia cells using established ALL and AML cell lines. Pairwise Pearson correlation coefficient, used to compare log2 ratios between triplicate experiments, revealed high correlation values between replicates (median=0.85), demonstrating significant reproducibility. Bisulphite sequencing was performed to confirm that this technology could reliably depict the actual methylation status of the CpG islands. We have evaluated CpG island methylation profiling in childhood ALL both at presentation and relapse. We report that ALL specific CpG island methylation patterns are emerging. We have identified a significant number of genes, involved in cell growth and differentiation and cell cycle regulation that are consistently differentially methylated between paediatric ALL patients and healthy controls. Expressly, a number of Hox genes and Hox-genes regulators, involved in cell differentiation are differentially methylated in leukaemic blasts compared to controls. Comparisons between methylation profiles at presentation and relapse demonstrate that overall genes that are methylated at presentation remain methylated in relapse; however there is an important subset of genes that become differentially methylated. At relapse, there is a significant increase in methylation levels of a set of pro-apoptotic and tumour suppressor genes, as well as several genes involved in drug metabolism. Interestingly, a group of genes involved in ErbB signalling pathway are differentially methylated in relapse, suggesting that epigenetic regulation of this pathway might be implicated in resistant disease. Our results suggest altered methylation status within a defined subset of genes may be implicated in relapse of ALL.