Screening DNA Methylation in Bodily Fluids for Early Cancer Diagnostics

癌症中研究最多的表观遗传学异常是与DNA高甲基化相关的基因沉默。 最近的研究表明，由启动子超甲基化导致的基因失活可能发生在癌症进展的早期阶段，甚至可能在检测到突变之前。使用DNA甲基化作为癌症生物标记物在早期诊断、高危个体评估和治疗后监测方面显示出巨大的希望。虽然利用体液进行非侵入性检测来检测癌症被临床医生视为圣杯，但检测包括血液、痰或粪便在内的体液中特定DNA甲基化变化的能力面临着更大的挑战，因为这些样本中可获得的DNA很少，而且这些样本中的肿瘤含量有限。因此，一项临床上有用的技术，允许检测体液中的DNA甲基化，将对癌症诊断和治疗产生重大影响。我们建议开发一个新的甲基化检测平台，该平台集成了涉及到的每个关键过程的改进方法：DNA提取、亚硫酸氢盐处理和甲基化检测。所有步骤最终将被集成并在一个微流体芯片上执行，利用超顺磁性粒子作为流体和分子操纵的公共载体，以及用于生物传感的量子点介导的荧光共振能量转移技术(QD-FRET)。这种方法将有助于高效的样品制备和对体液中DNA甲基化的灵敏检测。此外，所提出的技术可以进行集成和自动分析，最大限度地减少人工和时间，同时提供更具重复性的结果，适用于大规模筛查。将通过检测早期癌症患者和对照组的身体样本，包括血清/血浆、痰和粪便，来确定其在联合治疗前的潜在应用。 此外，在项目2、3和4中，将使用检测血液/血浆中癌症特异性甲基化来监测治疗反应，在检查癌症患者之前检查病情发展。

The most studied epigenetic abnormality in cancer is gene silencing associated with DNA hypermethylation. Recent studies demonstrate that gene inactivation by promoter hypermethylation can occur at early stages of cancer progression, perhaps even before mutations can be detected. Using DNA methylation as a cancer biomarker shows great promise for early diagnosis, assessments in high risk individuals, and post-therapy monitoring. While a noninvasive test with bodily fluids to detect cancer is seen as a holy grail by clinicians, the ability to detect specific DNA methylation changes in bodily fluids, including blood, sputum or stool represents a greater challenge, due to the small amounts of DNA available in these samples and the limited tumor content of such samples. Therefore, a clinically useful technology that allows for detection of DNA methylation in bodily fluids will have a substantial impact in both cancer diagnosis and management. We propose to develop a new methylation detection platform which integrates improved methods for each of the critical processes involved: DNA isolation, bisulfite treatment, and detection of methylation. All steps will ultimately be integrated and performed on a microfiuidic chip, utilizing superparamagnetic particles as a common carrier for fluidic and molecular manipulations and the quantum dots-mediated fluorescence resonance energy transfer technology (QD-FRET) for biosensing. This approach will facilitate highly efficient sample preparation and sensitive detection of DNA methylation in bodily fluids. In addition, the proposed technology can perform integrated and automatic analysis, minimizing manual labor and time while providing more reproducible results and being useful for a large scale screening. The potential use in pre-cUnical applications will be determined by testing bodily samples from patients with early stage cancers and controls, including serum/plasma, sputum and stool. In addition, the detection of cancer specific methylation in the blood/plasma will be used to monitor therapeutic response in Projects 2, 3 and 4, facihtating development prior to examination of cancer patients.