Azacytidine as a Maintenance Therapy in Elderly AML Progressively Demethylates CpG Sites within the p16 Gene

Abstract

Acute myeloid leukemia (AML) accounts for ~30% of adult leukaemia cases and is expected to increase as the population ages, due to median age of onset at ~60 years old. Recent evidence suggests that DNA methylation is actively involved in AML and myelodysplastic syndrome (MDS). Tumor suppressor genes, such as p16, have been shown to be silenced by methylation in AML. However, epigenetic events such as DNA methylation are reversible and therefore targets for chemotherapeutic intervention. It has been reported that ~30% of MDS patients with an abnormal karyotype show normalization of their methylation status after receiving a demethylating drug during early stages of their therapy.The UK NCRI AML16 programme for elderly patients (>60 years old at diagnosis) with AML and high risk MDS has several therapeutic questions for patients considered fit for intensive treatment, one of which is to compare the use of azacytidine demethylation maintenance treatment with no maintenance therapy.Samples were obtained from patients entered into the AML16 trial, at diagnosis and from patients entered into the intensive arm of the trial who were randomized to receive azacytidine maintenance therapy were analyzed for the alterations for genomic methylation.Pyrosequencing was used to determine methylation within 17 CpG sites within p16, MLH1, and MGMT whilst LINE1 was used as a measure of global methylation. To date, approximately 714 patients have been entered into AML16. Of these 195 diagnostic samples have been analyzed, of which 103 were in the intensive arm of the trial. At the second randomization stage, 34 patient samples were analyzed and a further 26 samples were obtained following 3, 6 or 9 courses of azacytidine therapy.Statistical comparison of the methylation levels at each individual CpG or for the averaged CpG in each gene studies indicated that there was no difference whether the sample was derived from bone marrow or peripheral blood. This allowed the direct comparison of peripheral blood samples obtained at 2nd randomization and during azacytidine maintenance courses. Differential levels of methylation at individual CpG within the gene were seen at diagnosis. Higher levels of average p16 methylation were observed in the AML patients when compared to a small cohort of “well elderly” individuals.No difference was noted in the individual or averaged CpG methylation status for MGMT or LINE1 during the maintenance course of azacytidine. However, the methylation status of the CpG sites within the p16 and MLH1 genes reduced during maintenance by a median of 19% and 25% respectively. However, the number of patients completing three courses of azacytidine was only about 20% of those entering the intensive arm of AML16, however sequential samples from the same individual also showed demethylation of the CpG sites in p16 and MLH1. This study shows that azacytidine maintenance therapy in elderly AML patients does reduce the methylation status of some genes whilst others genes show no response. This is being investigated further using arrays containing 12,000 CpG sites which will be correlated with gene expression microarrays on the diagnostic samples from AML16.