Integrated epigenetic and genetic analysis identifies markers of prognostic significance in pediatric acute myeloid leukemia

Abstract

Acute myeloid leukemia (AML) may be an epigenetically-driven malignancy because it harbors fewer genomic mutations than other cancers. In recent studies of AML in adults, DNA methylation patterns associate with clinical risk groups and prognosis. However, thorough evaluations of methylation in pediatric AML have not been done. Therefore, we performed an integrated analysis (IA) of the methylome and transcriptome with clinical outcome in 151 pediatric patients from the multi-center AML02 clinical trial discovery cohort. Intriguingly, reduced methylation and increased expression of DNMT3B was associated with worse clinical outcomes (IA p ≤ 10^-5; q ≤ 0.002). In particular, greater DNMT3B expression associated with worse minimal residual disease (MRD; p < 10^-5; q = 0.01), a greater rate of relapse or resistant disease (RR) (p = 0.00006; q = 0.06), and event-free survival (EFS; p = 0.00003; q = 0.04). Also, greater DNMT3B expression associated with greater genome-wide methylation burden (GWMB; R = 0.39; p = 10^-6) and greater GWMB associated with worse clinical outcomes (IA p < 10^-5). In an independent validation cohort of 132 similarly treated AAML0531 clinical trial patients, greater DNMT3B expression associated with greater GWMB, worse MRD, worse RR, and worse EFS (all p < 0.03); also, greater GWMB associated with worse MRD (p = 0.004) and EFS (p = 0.037). These results indicate that DNMT3B and GWMB may have a central role in the development and prognosis of pediatric AML.

Keywords: AML; leukemia; methylation; pediatrics.

Figure 1. Therapeutically beneficial and detrimental patterns of association detected by the CC-PROMISE procedure

Association Heatmap for top 50 genes by CC-PROMISE analysis. Each block of the heatmap illustrates the results of a statistical test of association. CC-PROMISE identified 11 genes for which increased expression associated with poorer prognosis (detrimental) and 39 for which increased expression associated with better prognosis (beneficial). The color indicates the direction of association (red = positive; blue = negative) and the intensity represents statistical significance on the log₁₀ p-value scale. Each row provides association analysis results for one gene. The columns provide results for the associations of methylation with expression (ME), expression and MRD (EMRD), expression and RR (ERR), methylation and MRD (MMRD), methylation and MRD (MMRD) and the integrated CC-PROMISE result (CCPR). MRD: Minimal Residual Disease; RR: Risk of relapse or resistant disease.

Figure 2. Association of DNMT3B methylation with MRD, risk of relapse and EFS in multicenter AML02 cohort

(A) Boxplot of DNMT3B methylation score by day 22 MRD status. (B) The cumulative incidence of risk of relapse and resistance disease (RR) in pediatric AML according to DNMT3B methylation score. (C) The cumulative incidence of event free survival (EFS) in pediatric AML according to DNMT3B methylation score.

Figure 3. Greater DNMT3B expression associates with worse clinical outcomes in the multicenter AML02 and COG-AAML0531 clinical trials

(A) Stacked bar plot showing proportion of AML02 patients who were MRD negative (blue), had MRD < 1% (yellow), or MRD > 1% (purple) with less than median DNMT3B expression (left column) or greater than median DNMT3B expression (right column). (B) Cumulative incidence of resistant disease or relapse among AML02 patients with less than median DNMT3B expression (black line) or greater than median DNMT3B expression (dashed red line). (C) Kaplan-Meier estimates of EFS for AML02 patients with low DNMT3B expression (black line) and high DNMT3B expression (dashed red line). Panels (D), (E), and (F) provide analogous results for AAML0531 (control arm) patients.

Figure 4. DNMT3B expression impacts genome wide methylation burden (GWMB) and GWMB is associated with outcome: AML02 cohort

(A) Scatterplot of DNMT3B expression with overall methylation burden (circle: low risk; triangle: standard risk; cross: high risk). (B) Bar plot of MRD according to GWMB. (C) The cumulative incidence of risk of relapse and resistance disease in pediatric AML according to GWMB. (D) The cumulative incidence of EFS in pediatric AML according to GWMB.

Figure 5. Overview of the current study

Integrated analysis of genome-wide methylation and gene- expression of leukemia cells obtained at diagnosis from patients enrolled in multicenter AML02 study identified 50 genes of significant prognostic value. DNMT3B is one of the top genes identified. Methylation of DNMT3B is potential regulator of own expression, and DNMT3B expression regulated genome-wide methylation burden, both of which are predictors of clinical outcome (AML02 study and validation in AAML0531). DNMT3B is correlated with expression and methylation levels of key candidate genes involved in various cellular pathways, which in turn are potential contributors to AML outcome. Also are shown genes of significance in AML as well as HOX family of genes with significant expression correlation with expression levels of DNMT3B.