Cancer and Disease Epigenetics and Department of Paediatrics, University of Melbourne, Melbourne, Australia; current address: Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY;
National Measurement Institute, Sydney, Australia;
Clin Chem. 2014 Jul;60(7):995-1003. doi: 10.1373/clinchem.2013.219956. Epub 2014 May 14.
DNA methylation biomarkers capable of diagnosis and subtyping have been found for many cancers. Fifteen such markers have previously been identified for pediatric acute lymphoblastic leukemia (ALL). Validation of these markers is necessary to assess their clinical utility for molecular diagnostics. Substantial efficiencies could be achieved with these DNA methylation markers for disease tracking with potential to replace patient-specific genetic testing.
We evaluated DNA methylation of promoter regions of TLX3 (T-cell leukemia homeobox) and FOXE3 (forkhead box E3) in bone marrow biopsies from 197 patients classified as leukemic (n = 95) or clear of the disease (n = 102) by MALDI-TOF. Using a single nucleotide extension assay (methylSABER), we tested 10 bone marrow biopsies collected throughout the course of patient chemotherapy. Using reference materials, diagnostic thresholds and limits of detection were characterized for both methods.
Reliable detection of DNA methylation of TLX3 and FOXE3 segregated ALL from those clear of disease with minimal false-negative and false-positive results. The limit of detection with MALDI-TOF was 1000-5000 copies of methylated allele. For methylSABER, the limit of detection was 10 copies of methylated TLX3, which enabled monitoring of minimal residual disease in ALL patients.
Mass spectrometry procedures can be used to regionally multiplex and detect rare DNA methylation events, establish DNA methylation loci as clinically applicable biomarkers for disease diagnosis, and track pediatric ALL.
已经发现了许多癌症的能够用于诊断和亚型分类的 DNA 甲基化生物标志物。此前，已有 15 种此类生物标志物被鉴定用于小儿急性淋巴细胞白血病（ALL）。为了评估其用于分子诊断的临床实用性，需要对这些标志物进行验证。这些 DNA 甲基化标志物在疾病跟踪方面具有很高的效率，有可能替代针对患者个体的基因检测。
我们评估了通过 MALDI-TOF 对 197 名患者的骨髓活检样本中 TLX3（T 细胞白血病同源框）和 FOXE3（叉头框 E3）启动子区域的 DNA 甲基化。使用单核苷酸延伸检测（methylSABER），我们检测了 10 个在患者化疗过程中收集的骨髓活检样本。使用参考材料，对两种方法的诊断阈值和检测限进行了特征描述。
TLX3 和 FOXE3 的 DNA 甲基化的可靠检测将 ALL 与那些疾病清除的患者区分开来，假阴性和假阳性结果最小。MALDI-TOF 的检测限为 1000-5000 个甲基化等位基因拷贝。对于 methylSABER，检测限为 10 个甲基化 TLX3 拷贝，这使 ALL 患者的微小残留病监测成为可能。
质谱程序可用于区域性多重检测和检测罕见的 DNA 甲基化事件，将 DNA 甲基化位点确立为具有临床应用价值的疾病诊断生物标志物，并跟踪小儿 ALL。
