Screening DNA Methylation in Bodily Fluids for Early Cancer Diagnostics

癌症中研究最多的表观遗传异常是与DNA超甲基化相关的基因沉默。 最近的研究表明，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志物显示出对早期诊断、高风险个体评估和治疗后监测的巨大希望。虽然使用体液检测癌症的非侵入性测试被临床医生视为圣杯，但检测体液（包括血液，痰液或粪便）中特定DNA甲基化变化的能力代表了更大的挑战，因为这些样本中可用的DNA量很小，并且这些样本的肿瘤含量有限。因此，允许检测体液中DNA甲基化的临床有用技术将对癌症诊断和管理产生重大影响。我们建议开发一种新的甲基化检测平台，该平台集成了针对所涉及的每个关键过程的改进方法：DNA分离，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微流控芯片上进行，利用超顺磁性颗粒作为流体和分子操纵的共同载体，以及量子点介导的荧光共振能量转移技术（QD-FRET）用于生物传感。这种方法将有助于高效的样品制备和体液中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劳动和时间，同时提供更具重现性的结果，并可用于大规模筛选。将通过检测早期癌症患者和对照患者的身体样本（包括血清/血浆、痰液和粪便）来确定在临床前应用中的潜在用途。 此外，血液/血浆中癌症特异性甲基化的检测将用于监测项目2、3和4中的治疗反应，在检查癌症患者之前促进发展。

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.