Epigenetic signature of ionising radiation in therapy-related AML patients
Therapy-related acute myeloid leukaemia (t-AML) is a late adverse effect of previous chemotherapy and/or radiotherapy (rt-AML) or immunosuppressive treatment. t-AMLs represent ~10-20% of all AML cases, are extremely aggressive and have a poor prognosis in comparison to de novo AML. We hypothesised that in rt-AML, exposure to radiation leads to genome-wide epigenetic modifications. An epigenome-wide association study was conducted, measuring over 850K methylation sites across the whole genome in 14 donors. We focused on 94K sites lying in CpG-rich gene promoter regions. Overall, we found genome-wide hypo-methylation in AML and identified specific genes with promoter hyper-methylation. Additionally, pyrosequencing was used to quantify the methylation in 24 samples. We confirmed that the promoters of the genes MEST and GATA5, both previously reported as tumour suppressors, were specifically hyper-methylated in rt-AML in comparison to control and other subtypes of t-AML. These may represent the epigenetic contribution to rt-AML development at the molecular level and be potential drug targets in rt-AML. The experiment was firstly done with the use of Illumina Infinium Human Methylation 450 K array for 12 patients: 5 de novo AMLs, 4 ct-AMLs and 3 rt-AMLs. Secondly, the experiment was repeated on Illumina Infinium Human Methylation EPIC Array with over 850K probes for the part of the abovementioned patients and healthy control. Data were collected in two batches: 3 de novo AML patients, 3 ct-AML patients and 2 rt-AML patients in the first batch, and 5 healthy donors, 2 de novo AML patients and 1 combined radio- and chemotherapy patient in the second batch.
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DNA quality was assessed by agarose gel electrophoresis and DNA quantity measured by NanoDrop 2000 (Thermo Fisher Scientific, Paisley, U.K.). The investigators were not blinded to allocation during experiments and outcome assessment. DNA was extracted from human bone marrow aspirate samples using the DNeasy® Blood & Tissue kit (Qiagen, Manchester, UK) according to the manufacturer's instructions. DNA quantity was measured by Quant-iT™ PicoGreen™ dsDNA Assay Kits (Thermo Fisher Scientific, Paisley, U.K.) and quality assessed by agarose gel electrophoresis using 1.3% agarose gel. 750ng DNA at a concentration of at least 20ng/ul was sent to NXT-DX (Ghent, Belgium) and run on the Human Methylation 450K BeadChip array for DNA methylation analysis. Samples were first sent for analysis using the 450K array, but due to discontinuation, some patient samples were repeated on the Infinium Human Methylation EPIC BeadChip array. 500ng DNA at a concentration of at least 20ng/ul was sent to NXT-DX and run on the EPIC array for DNA methylation analysis.