Genome Wide Promoter Methylation Patterns Predict AML Subtype Outcomes and Identify Novel Pathways Characterizing Diagnostic and Relapsed Disease in Children

Abstract 1287

The NCI-directed TARGET AML initiative has provided an opportunity to evaluate the genomic, transcriptomic and epigenomic patterns of large numbers of pediatric patients with newly diagnosed and relapsed AML with the goal of identifying distinctive changes in pathways that are prognostic or can be therapeutically targeted. Genomic sequencing has revealed an important, but limited number of gene mutations, suggesting that epigenetic alterations may be required to explain the development and biological behavior of AML. We have analyzed genome-wide promoter methylation patterns using Illumina Infinium Methylation 27 arrays on 200 paired diagnostic (Dx) and remission (Rm) cases plus 47 case-matched relapsed (Rl) samples.

Marked differences in DNA methylation were observed in the AML compared to remission bone marrows. DNA methylation patterns could discriminate specific types of cytogenetically characterized AML, but no subset of specific methylation changes were identified that predicted outcome across the entire cohort irrespective of AML subtype. Due to the strong correlations of methylation pattern with cytogenetically defined subtypes, such a global signature for outcome would be expected to require either a very strong signal or very large sample size to detect a prognostic, methylation signature that was able to validated in heterogeneous cohorts. We therefore evaluated prognostic methylation features within each cytogenetic group. Using a linear model and empirical Bayes statistic, selective groups of high quality methylation probe signals revealed an association with survival that independently predicted outcome within each cytogenetic subtype. Using a top-scoring pair (TSP) algorithm for each karyotype subtype, five different TSPs each for Inv16, MLL, t(8;21) and normal karyotype subgroups were identified that predicted outcome with an accuracy of 100% except for the t(8;21) subtype in which accuracy of predicting outcome was 87%.

We used an alternative analytical approach to determine whether specific epigenetic promoter patterns were associated with diagnosis and/or relapse AML compared to remission bone marrows across all subtypes. These patterns could potentially be considered to be essential for AML development and/or progression. We divided patient data into balanced training and test sets. Using a novel pathway analysis based on outlier statistics, we identified methylation changes independent of cytogenetic subtype in gene promoters associated with specific cellular pathways. Cellular pathways of potential interest characterized by preferentially hypermethylated promoters of genes in the AML diagnostic samples compared to remission samples included Hedgehog, extracellular matrix receptor interactions, cell adhesion molecules, (NCAM/CAM) and phospholipase pathways. Those gene sets that showed hypomethylation in the AML diagnostic samples were selectively associated with cytochrome P450, tyrosine, and arachidonic acid metabolism. We generated a 10 gene signature from genes for two of the most significant pathways, i.e., the Hedgehog and cytochrome P450 metabolism pathways, using TSP and applied this TSP signature using a Fisher's exact test for both Dx vs Rm and Rl vs Rm samples (Table).

The confirmation of the signature in the independent test set validated the significance of the Hedgehog and P450 pathways in newly diagnosed and relapsed pediatric AML.

These data demonstrate the unique contribution of epigenetic changes as a prognostic indicator in subgroups of AML. Importantly, outlier analysis followed by pathway-based interrogation identified several key gene sets that are selectively epigenetically altered in an otherwise uncharacterized subset of pediatric AML. Of note, the hypermethylation and hypomethylation of gene promoters characterizing the Hedgehog and of promoters of cytochrome P450 pathways respectively was highly associated with AML diagnosis and relapse samples compared to remission marrows, suggesting a specific role for these pathways in the physiology and maintenance of pediatric AML. Work in progress is examining samples for mutations, gene copy number, expression and functional consequences of the pathways that are selectively altered by epigenetic mechanisms.