Epigenetic priming improves survival for pediatric Acute Myeloid Leukemia: Results from the multicenter randomized AML16 trial Free

Genome-wide loss of DNA methylation accompanied by hypermethylation of promoter-associated CpG-islands is a hallmark of cancer. DNA methylation signatures have been used to identify biologically distinct subtypes of acute myeloid leukemia (AML), including several genomically defined subsets, suggesting that aberrant DNA methylation patterns may partially reflect underlying pathogenic mechanisms. The dependency of AML cells on methylation signatures for viability and/or differentiation status, however, remains largely unknown. Our prior retrospective analysis of diagnostic pediatric AML specimens identified an association between lower total genome-wide methylation (TGWM) burdens and improved outcomes. Further, increased levels of 5-methylcytosine may contribute to leukemic resistance to the cytosine-analog cytarabine through epigenetic repression of nucleoside transporters. Thus, we hypothesized that five days of epigenetic priming with azacitidine (AZA) or decitabine (DAC) before each course of chemotherapy would improve outcomes by lowering TGWM. The AML16 clinical trial tested this hypothesis by evaluating the tolerability and efficacy of randomly assigned AZA or DAC priming in 200 pediatric AML patients enrolled at 10 U.S. centers (NCT03164057).

To establish tolerability, priming was limited to induction I and induction II during the initial phase of the study. Induction I included 5 days of priming followed by cytarabine, daunorubicin, and etoposide, and induction II included 5 days of priming before idarubicin, cytarabine, and fludarabine. Risk assignment and subsequent therapy were adapted based on molecular features and minimal residual disease (MRD) levels. Patients with high-risk (HR) disease and suitable donors underwent hematopoietic stem cell transplantation (HSCT) in first remission. Once priming prior to induction I and II were deemed tolerable, priming was expanded to all chemotherapy courses for patients not undergoing HSCT. A course was deemed tolerable if 15 or more of the first 25 evaluable patients completed it without experiencing an unacceptable adverse event (UAE), based on a multi-stage statistical design. The trial was approved by each participating IRB.

Patients were assigned to AZA priming for two courses (AZA₂, n=34), AZA priming for all courses (AZA_4/5, n=68), DAC priming for two courses (DAC₂, n=33), or DAC priming for all courses (DAC_4/5, n=65). Eleven unique chemotherapy courses with priming were deemed tolerable; no course was deemed intolerable. Across all patients, epigenetic priming improved outcomes compared to historical controls, with 3-year event free survival (EFS) of 75.8%, 57.9%, and 61.2% for patients enrolled on AML16, AML08, and AML02, respectively (p=0.0002). Low-risk (LR) and intermediate-risk (IR) patients had excellent outcomes with 3-year EFS of 95.5% and 86.4%, and overall survival (OS) of 100% and 94.6%, respectively. HR patients had a 3-year EFS of 58.0% and OS of 63.3%. Among molecular subsets, patients with LR mutations, including RUNX1::RUNX1T1, CBFB::MYH11, CEBPA and NPM1c had 3-year EFS and OS uniformly ≥90%. KMT2A rearranged (KMT2Ar) patients had a 3-year EFS and OS of 64.1% and 66.7%, respectively; among them, those with KMT2A-MLLT3 had the best outcomes, with 3-year EFS and OS of 85.7%.

The 3-year EFS was 69.6% and 82.1% (p=0.058) and 3-year OS was 74.4% and 88.5% (p=0.014) for AZA and DAC, respectively. LR and IR patients had excellent outcomes irrespective of the epigenetic priming agent, with 3-year EFS of 96.1%, 85.7%, 95%, 87.5% (p=0.6) and 3-year OS of 100%, 90.5%, 100%, and 100% (p=0.43) for LR AZA, IR AZA, LR DAC, and IR DAC, respectively. Among HR patients, DAC resulted in better outcomes, with a 3-year EFS of 67.3% vs. 50.5% with AZA (p=0.20) and OS of 70% vs. 53% (p=0.15). DAC was significantly better than AZA in a win-ratio (WR) analysis encompassing death, relapse, and response to induction I (WR = 1.77; 95% CI: 1.32, 2.39; p=0.0002). Five days of AZA and DAC priming reduced median TGWM by 0.39 and 0.50 M-value units, respectively (p=0.06). We conclude that epigenetic priming significantly improved outcomes in pediatric AML, with DAC demonstrating superiority over AZA. These results support the adoption of epigenetic priming as the standard of care for pediatric AML.