A CpG island methylator phenotype in acute myeloid leukemia independent of IDH mutations and associated with a favorable outcome

Acute myeloid leukemia (AML) causes the most leukemia-related deaths in the United States. Although genetic changes are assessed in the clinic for risk stratification, current classifications are imperfect and epigenetic determinants of AML curability remain poorly understood. To address this gap in knowledge we performed genome-wide DNA methylation analysis using the next-generation sequencing-based Digital Restriction Enzyme Analysis of Methylation (DREAM) assay on 96 clinical AML samples, and 35 normal peripheral blood controls. We identified patterns of aberrant DNA hypermethylation in distinct subsets of cases, and these patterns were associated with unique clinical, and genetic features. Validation an extension of these findings was performed using 194 samples from The Cancer Genome Atlas (TCGA). Overall design: Digital restriction enzyme analysis of methylation (DREAM) was performed to determine the methylation profile of human genomic DNA from the bone marrow of 96 patients with acute myeloid leukemia (AML) and peripheral blood from 35 healthy controls. Briefly, genomic DNA was sequentially digested by SmaI and XmaI, which both recognize the sequence CCCGGG. SmaI is methylation sensitive, where XmaI is methylation insensitive. Distinct signatures, 5'-GGG at unmethylated sites or 5'-CCGGG at methylated sites were created by enzyme digestion and ultimately high througput sequencing was used to map these sites to the genome. Methylation ratios for each individual CCCGGG site were calculated as a proportion of methylated counts to the sum of unmethylated and methylated counts, and subsequently adjusted for differences in restriction enzyme efficiency using values obtained from spiked in standards.

急性髓系白血病（Acute myeloid leukemia, AML）是美国范围内白血病相关死亡的首要病因。尽管临床中会通过评估遗传变异对患者进行风险分层，但当前的分类方案仍存在缺陷，且AML治愈性的表观遗传决定因素仍未得到充分阐释。为填补这一认知空白，研究团队采用基于下一代测序的甲基化数字限制性酶分析（Digital Restriction Enzyme Analysis of Methylation, DREAM）技术，对96例临床AML样本及35例正常外周血对照开展全基因组DNA甲基化分析。研究人员在不同亚型的病例中鉴定出异常DNA高甲基化模式，且此类模式与独特的临床及遗传特征存在关联。随后研究团队利用癌症基因组图谱（The Cancer Genome Atlas, TCGA）的194例样本，对上述发现进行了验证与拓展。

本研究的整体实验设计如下：采用甲基化数字限制性酶分析（DREAM）技术，对96例急性髓系白血病患者的骨髓基因组DNA，以及35例健康对照的外周血基因组DNA进行甲基化谱分析。简言之，基因组DNA先后经均识别序列CCCGGG的SmaI与XmaI酶切：其中SmaI对甲基化敏感，而XmaI对甲基化不敏感。通过酶切可得到两种特征序列——未甲基化位点处的5'-GGG，以及甲基化位点处的5'-CCCGGG，最终通过高通量测序将这些位点定位至人类基因组。以甲基化读段数占总读段数（甲基化与未甲基化读段数之和）的比例，计算单个CCCGGG位点的甲基化比率，并通过加入的内参标准物得到的数值，校正限制性酶切效率的差异。