Genomewide DNA Methylation Analysis Reveals Specific Signatures in Childhood B-Lymphoblastic Leukemias,

Abstract 3476

Childhood B-cell acute lymphoblastic leukemias (B-ALL) arise from diverse genetic events. Gene dysregulation through abnormal DNA methylation is also believed to play an important role in the tumorigenesis of B-ALL but has not been thoroughly investigated in a large set yet.

We performed a genome-wide methylation analysis for 238 childhood B-ALL patients, including 77 hyperdiploid and 64 t(12;21)(p13;q22) [ETV6/RUNX1–fusion] patients, using the Infinium HD450 Methylation array (Illumina). The methylation profile was compared with that of 24 normal precursor B-cell samples, including pro-B, pre-B-I, pre-B-II and immature B cells, which were obtained from 8 normal fetuses and separated by flow cytometry using a panel of antibodies targeting the CD antigens 3/13/19/33/34/56, and IgM antibodies. A total of 3,641 CpGs in 1,953 genes, including 1,007 polycomb target genes, were differently methylated in B-ALL compared to normal precursor B-cells (at a P value below 1.0×10⁻⁴; beta difference>0.5). The Ingenuity Pathway Analysis System demonstrated that genes involve in the G-protein coupled receptor signaling are significantly demethylated or hypermethylated (P=8.7×10⁻⁶), followed by genes in antigen presentation, B-cell development, cyclic adenosine monophosphate (cAMP)-mediated signaling, and calcium-induced T-lymphocyte apoptosis pathways (P=6.1×10⁻⁵, 6.1×10⁻⁵, 1.0×10⁻⁴, and 1.2×10⁻³, respectively). Gene Set Enrichment Analysis revealed that genes containing motifs for SP1, LEF1, MAZ, TCF3, MLLT7, NFAT, ETS2, MYOD1, FOXA1, and VSX1 molecules were significantly enriched (P<1.0×10⁻¹⁶).

Hyperdiploid patients had 37 significantly hypermethylated and 70 significantly demethylated genes compared to ETV6/RUNX1–fusion patients (at a P value below 1.0×10⁻⁴; beta difference>0.5). Many of those genes were involved in the molecular mechanism of cancer (P=6.9×10⁻⁵), with several genes showing large differences in methylation status between hyperdiploid and ETV6/RUNX1. Heatmap generation and clustering analysis identified a demethlyation cluster and a hypermethylation cluster specific for hyperdiploid, and a demethylation cluster for ETV6/RUNX1. In addition, we discovered evidence of a highly variable CpG cluster, by which each cytogenetics group could be divided into two separate subgroups. Some genes in the cluster are previously shown to have an important role in hematopoiesis. An integrative analysis using the preexisting expression database for B-ALL identified that differences in methylation beta values between hyperdiploid and ETV6/RUNX1 generally have an inverse correlation with differences in expression levels. By filtering both methylation and expression data, we could identify several genes that may potentially attribute to the biological difference between hyperdiploid and ETV6/RUNX1.

These data indicate that numerous genes are aberrantly hypermethylated or demethylated in B-ALL, with changes in some genes having a significant impact on the leukemogenesis as well as on the biologic differences in each B-ALL subgroup. Heterogeneity in methylation profile also appears to be present even within a well-established cytogenetics group.