Genome wide gene expression array analysis in T-ALL

In short: The objective with the gene expression array (Illumina HT-12 v.4) analysis of 17 T-ALL samples was to correlate gene expression levels with DNA promoter methylation status. Manuscript Abstract: Background: Treatment of pediatric T-cell acute lymphoblastic leukemia (T-ALL) has improved, but there is a considerable fraction of patients experiencing a poor outcome. There is a need for better prognostic markers and aberrant DNA methylation is a candidate in other malignancies, but its potential prognostic significance in T-ALL is hitherto undecided. Design and Methods: Genome wide promoter DNA methylation analysis was performed in pediatric T-ALL samples (n=43) using arrays covering >27000 CpG sites. Clinical outcome was evaluated in relation to methylation status and compared with a contemporary T-ALL group not tested for methylation (n= 32). Results: Based on CpG island methylator phenotype (CIMP), T-ALL samples were subgrouped as CIMP+ (high methylation) and CIMP- (low methylation). CIMP- T-ALL patients had significantly worse overall and event free survival (p=0.02 and p=0.001, respectively) compared to CIMP+ cases. CIMP status was an independent factor for survival in multivariate analysis including age, gender and white blood cell count. Analysis of differently methylated genes in the CIMP subgroups showed an overrepresentation of transcription factors, ligands and polycomb target genes. Conclusions: We identified global promoter methylation profiling as being of relevance for subgrouping and prognostication of pediatric T-ALL. RNA was extracted with TRIZOL according to manufacturer instructions. Total RNA was amplified bu the Illumina TotalPrep RNA Amplification kit. Gene expression array analysis was performed on the 17/43 pediatric T-ALL samples from which RNA was availible. 2 control samples of stimulted T-cells were included and one replicate.

简言之：针对17例T细胞急性淋巴细胞白血病（T-cell acute lymphoblastic leukemia, 以下简称T-ALL）样本开展的Illumina HT-12 v.4版基因表达微阵列（gene expression array）分析，核心目标为探究基因表达水平与DNA启动子甲基化状态之间的关联。

论文摘要：
背景：儿童T-ALL的治疗方案已取得显著进展，但仍有相当比例的患者预后不良。目前亟需更为精准的预后标志物，而异常DNA甲基化在其他恶性肿瘤中已被证实为潜在预后靶点，但该指标在T-ALL中的潜在预后价值迄今尚未明确。
研究设计与方法：对43例儿童T-ALL样本开展全基因组启动子DNA甲基化分析，所用芯片覆盖超过27000个CpG位点。研究人员评估了甲基化状态与临床预后的相关性，并将结果与同期未进行甲基化检测的32例T-ALL患者队列进行对比。
研究结果：基于CpG岛甲基化表型（CpG island methylator phenotype, 以下简称CIMP），T-ALL样本被划分为CIMP+（高甲基化）与CIMP-（低甲基化）两个亚组。与CIMP+患者相比，CIMP- T-ALL患者的总生存期与无事件生存期（event free survival）均显著更差（分别为p=0.02与p=0.001）。在纳入年龄、性别与白细胞计数的多因素分析（multivariate analysis）中，CIMP状态是影响患者生存的独立危险因素。对CIMP亚组间差异甲基化基因的富集分析显示，转录因子（transcription factors）、配体（ligands）及多梳蛋白靶基因（polycomb target genes）的占比显著偏高。
研究结论：本研究证实，全基因组启动子甲基化谱可用于儿童T-ALL的亚组分型与预后评估。按照试剂盒说明书，使用TRIZOL试剂提取总RNA，随后采用Illumina TotalPrep RNA扩增试剂盒完成总RNA扩增。本研究对43例T-ALL样本中可提取到RNA的17例样本开展基因表达微阵列分析，纳入了2例活化T细胞对照样本，并设置1份生物学重复。