Pisanic Thomas R, Athamanolap Pornpat, Poh Weijie, Chen Chen, Hulbert Alicia, Brock Malcolm V, Herman James G, Wang Tza-Huei
Johns Hopkins Institute for NanoBioTechnology, Baltimore, MD 21218, USA.
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
Nucleic Acids Res. 2015 Dec 15;43(22):e154. doi: 10.1093/nar/gkv795. Epub 2015 Aug 24.
Many cancers comprise heterogeneous populations of cells at primary and metastatic sites throughout the body. The presence or emergence of distinct subclones with drug-resistant genetic and epigenetic phenotypes within these populations can greatly complicate therapeutic intervention. Liquid biopsies of peripheral blood from cancer patients have been suggested as an ideal means of sampling intratumor genetic and epigenetic heterogeneity for diagnostics, monitoring and therapeutic guidance. However, current molecular diagnostic and sequencing methods are not well suited to the routine assessment of epigenetic heterogeneity in difficult samples such as liquid biopsies that contain intrinsically low fractional concentrations of circulating tumor DNA (ctDNA) and rare epigenetic subclonal populations. Here we report an alternative approach, deemed DREAMing (Discrimination of Rare EpiAlleles by Melt), which uses semi-limiting dilution and precise melt curve analysis to distinguish and enumerate individual copies of epiallelic species at single-CpG-site resolution in fractions as low as 0.005%, providing facile and inexpensive ultrasensitive assessment of locus-specific epigenetic heterogeneity directly from liquid biopsies. The technique is demonstrated here for the evaluation of epigenetic heterogeneity at p14(ARF) and BRCA1 gene-promoter loci in liquid biopsies obtained from patients in association with non-small cell lung cancer (NSCLC) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), respectively.
许多癌症在全身的原发部位和转移部位都由异质性细胞群体组成。在这些群体中，具有耐药性遗传和表观遗传表型的不同亚克隆的存在或出现会使治疗干预变得极为复杂。癌症患者外周血的液体活检被认为是一种理想的方法，可用于采集肿瘤内的遗传和表观遗传异质性，以进行诊断、监测和治疗指导。然而，目前的分子诊断和测序方法并不适合对困难样本（如液体活检）中的表观遗传异质性进行常规评估，因为这些样本中循环肿瘤DNA（ctDNA）的固有浓度较低，且存在罕见的表观遗传亚克隆群体。在此，我们报告了一种名为DREAMing（通过熔解区分罕见表观等位基因）的替代方法，该方法使用半定量稀释和精确熔解曲线分析，以单CpG位点分辨率区分和计数低至0.005%的组分中表观等位基因物种的单个拷贝，可直接从液体活检中轻松、廉价地对位点特异性表观遗传异质性进行超灵敏评估。本文展示了该技术分别用于评估从非小细胞肺癌（NSCLC）患者和骨髓增生异常/骨髓增殖性肿瘤（MDS/MPN）患者获得的液体活检中p14（ARF）和BRCA1基因启动子位点的表观遗传异质性。
