Prognosis of Pediatric Acute Myeloid Leukemia with KMT2A-MLLT3 According to DNA Methylation Patterns: Jccg JPLSG AML-05 Study

Introduction Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by cytogenetic abnormalities, including fusion genes, mutations, epigenetic modifications, and dysregulated gene expression. DNA methylation patterns have recently been appeared to be associated with molecular subtypes, chromosomal abnormalities, gene fusion, and AML prognosis. DNA methylation pattern of adult AML has also been proposed as a novel biomarker for AML prognosis. However, few studies have discussed the influence of DNA methylation status on the clinical features of pediatric AML. We analyzed genome-wide DNA methylation in 127 pediatric patients with AML to determine its association with clinical features, genetic alterations, and prognostic impact.

Methods Between 2006 and 2010, 443 pediatric patients with de novo AML (0-17 years) participated in the Japanese AML-05 trial of the Japanese Pediatric Leukemia/Lymphoma Study Group. Of these patients, 127 patients were enrolled in this study. Their cytogenetic features were as follows: normal karyotype, 26 patients; RUNX1-RUNX1T1, 11; CBFB-MYH11, 5; KMT2A rearrangement, 47 (22 of 47 had KMT2A-MLLT3); NUP98-NSD1, 8; NUP98-KDM5A, 5; CBFA2T3-GLIS2, 4; FUS-ERG, 4; and other cytogenetics, 18. This cohort included 35 patients with FLT3-internal tandem duplication (ITD), 9 with CEBPA biallelic mutations, 15 with high MECOM (also known as EVI1) expression, and 29 with high PRDM16 (also known as MEL1) expression. In all patients, genome-wide DNA methylation analysis using Infinium MethylationEPIC BeadChip (Illumina) was performed.

Results and Discussion Based on unsupervised hierarchical clustering, patients were divided into six clusters (cluster 1-6) associated with genetic alterations. Cluster 1 (n = 28) was characterized by the presence of KMT2A rearrangement with low MECOM expression. CBFA2T3-GLIS2 and NUP98-KDM5A considered as adverse prognostic factors were included in cluster 2 (n = 15). Cluster 3 (n = 16) comprised 11 RUNX1-RUNX1T1 and 5 CBFB-MYH11. No CBF-AML was included in other clusters. All nine patients with biallelic CEBPA mutations, which indicated a favorable outcome, were classified under cluster 4 (n = 18). Interestingly, all four patients with FUS-ERG were also classified under cluster 4. Clusters 5 (n = 15) and 6 (n = 35) comprised patients with molecular features related to adverse outcomes, such as FLT3-ITD, KMT2A-MLLT3 with high MECOM expression, and high PRDM16 expression. All seven patients with NUP98-NSD1 were classified under cluster 6. Patients with AML with KMT2A rearrangement showed a significant difference in DNA methylation levels at 500 CpG sites compared with those without. Patients were categorized into four clusters (clusters A-D) based on the methylation values of the 500 CpG sites that were significantly different between patients with AML with or without KMT2A rearrangement, and 96% (45 of 47) of patients with AML with KMT2A rearrangement were classified under clusters A (n = 32) or B (n = 13). The remaining two patients were classified under clusters C (n = 1) and D (n = 1). Interestingly, all 32 patients with AML with KMT2A rearrangement classified under cluster A showed low MECOM expression. Moreover, 22 patients with AML with KMT2A-MLLT3 were classified under clusters A (n = 14) and B (n = 8). Patients with KMT2A-MLLT3 in cluster B had a significantly worse prognosis than those in cluster A (5-year overall survival, 30% vs. 100%; P = 0.001, and 5-year event-free survival, 0% vs. 100%; P < 0.001). All eight patients with KMT2A-MLLT3 in cluster B had high MECOM expression, whereas all 14 patients in cluster A had low MECOM expression. These results indicate that the methylation pattern corresponded to MECOM expression. Our findings suggest that DNA methylation levels at specific CpG sites are useful biomarkers for the prognosis of pediatric patients with AML.

Tomizawa:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Horibe:Amgen Inc: Speakers Bureau; Chugai Pharmaceutical Co., Ltd.: Speakers Bureau; Novartis Japan: Speakers Bureau; Pfizer Japan Inc.: Consultancy; Kyowa Kirin Co.,Ltd.: Consultancy. Ogawa:2014-191287: Patents & Royalties; 15/353395 (US03): Patents & Royalties; 2015-239547: Patents & Royalties; Clinical Research Support Center Kyushu: Research Funding; Otsuka Pharmatheutical: Research Funding; MSD: Speakers Bureau; Kirin/Chugai: Speakers Bureau; The Chemo-Sero-Therapeutic Research Institute: Speakers Bureau; Astrazeneca: Speakers Bureau; DaiichiSankyo: Speakers Bureau; Nanpu Hospital: Research Funding; Sysmex: Honoraria; Novartis: Honoraria, Speakers Bureau; Chordia Threapeutics: Consultancy, Current equity holder in publicly-traded company, Research Funding; The Mitsubishi foundation: Honoraria; Astellas: Speakers Bureau; 62/187386 (US01): Patents & Royalties; ASAHI Genomics: Current equity holder in publicly-traded company; Esai Pharmatheutical: Consultancy; Pfaizer: Speakers Bureau; 2013-526957 (JP02): Patents & Royalties; 2013-096582 (JP01): Patents & Royalties; PCT/JP2014/062112 (WO01): Patents & Royalties.