DNA Methylation Clusters and Their Relation to Cytogenetic Features in Pediatric AML

Abstract

Acute Myeloid Leukemia (AML) is characterized by recurrent genetic and cytogenetic lesions that are utilized for risk stratification and for making treatment decisions. In recent years, methylation dysregulation has been extensively studied and associated with risk groups and prognosis in adult AML, however, such studies in pediatric AML are limited. Moreover, the mutations in epigenetic genes such as DNMT3A, IDH1 or IDH2 are almost absent or rare in pediatric patients as compared to their abundance in adult AML. In the current study, we evaluated methylation patterns that occur with or independent of the well-defined cytogenetic features in pediatric AML patients enrolled on multi-site AML02 clinical trial (NCT00136084). Our results demonstrate that unlike adult AML, cytosine DNA methylation does not result in significant unique clusters in pediatric AML, however, DNA methylation signatures correlated significantly with the most common and recurrent cytogenetic features. Paired evaluation of DNA methylation and expression identified genes and pathways of biological relevance that hold promise for novel therapeutic strategies. Our results further demonstrate that epigenetic signatures occur complimentary to the well-established chromosomal/mutational landscape, implying that dysregulation of oncogenes or tumor suppressors might be leveraging both genetic and epigenetic mechanisms to impact biological pathways critical for leukemogenesis.

Keywords: DNA methylation; acute myeloid leukemia; cytogenetics; pediatrics.

Figure 1

Heatmap representing top results for the unsupervised hierarchical clustering of methylation (500 probes) and expression (700 probes) probes representing different clusters across AML patients from AML02 clinical trial. For methylation, red depicts hypermethylation and blue corresponds to hypomethylation; for expression heat map at the bottom of the figure, red represents high expression and blue represents low expression. Cytogenetic features (as per color code on right), FLT3 status (FLT3-WT = blue; FLT3-ITD = yellow, FLT3-Mutation = red, FLT3-status not available = grey), FAB subtype (as per code on right); age group (<10 years = blue, >10 years = red), sex (male = blue, female = red); Day 22 MRD after induction 1 (negative = blue, positive = red, not available = grey), 3 year event free and 3 year overall survival are shown corresponding to the key on the right. For all the features, grey = data not available or censored <3 year for survival endpoints.

Figure 2

Association of the methylation clusters with (A) minimal residual disease after induction 1 (MRD1) and (B) Event free survival (EFS) and expression clusters with (C) MRD1 and (D) EFS in AML patients treated on AML02 clinical trial. The methylation and expression colors are as defined in Figure 1.

Figure 3

(A) Paired methylation and expression heatmap showing most significant features within each of the cytogenetic groups. Selected genes unique to different cytogenetic subgroups are shown in panels on right: (B) Inv(16); (C) t(8:21); (D) t(9:11); (E) other 11q23 MLL rearrangements; (F) normal cytogenetics and (G) Miscellaneous karyotype. Color for each cytogenetic group corresponds to the bar on top of the heatmaps.

Figure 4

Chromosome map depicting chromosomal location of the differential methylated genes unique to the cytogenetic features. Key to the cytogenetics—blue: inv(16); red: t(8:21); pink: miscellaneous; dark green: normal cytogenetics; green: t(9:11); and black: other MLL (11q23) rearranged AML.

Figure 5

Representation of selected pathways (A) Wnt/b-catenin, (B) mTOR, and (C) PIK3 signaling pathways representing genes identified within different cytogenetic groups. Arrow color corresponds to the respective cytogenetic feature.