Digital Detection of Tumor-Derived Circulating Methylated DNA

肿瘤来源的循环甲基化 DNA 的数字检测

基本信息

批准号：
9124834
负责人：
Tza-Huei Jeff Wang
金额：
$ 21.14万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9124834
关键词：
Address Alleles Azacitidine Base Sequence Biological Assay Biological Markers Blood Blood Circulation Blood Plasma Volume Cancer Detection Cancer Patient Cells Clinical Clinical Sensitivity Coupling CpG Islands CpG dinucleotide DNA DNA Methylation Detection Diagnostic Encapsulated Epigenetic Process Fluorescence Gene Mutation Genes Genetic Goals Health Heterogeneity Hypermethylation Malignant Neoplasms Malignant neoplasm of lung Methods Methylation Microfluidic Microchips Microfluidics Modeling Monitor Oncogenes Patient-Focused Outcomes Patients Performance Plasma Preparation Procedures Process Promoter Regions Reaction Reagent Reporting Resolution Sampling Screening for cancer Sensitivity and Specificity Silicon Dioxide Stratification Surface Tension Techniques Technology Testing Time Tube Tumor Suppressor Genes Tumor-Derived Variant Work base bisulfite bisulfite sequencing cancer biomarkers cancer type circulating DNA clinical application cohort companion diagnostics design digital evaporation gain of function instrument loss of function meetings melting methylation pattern prognostic programs promoter pyrosequencing screening single molecule success superparamagnetic beads synthetic construct theranostics tumor tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): Aberrantly methylated DNA is found abundantly in all forms of cancer and the tumors that produce them. In fact, it has been estimated that within the cells of every tumor are several hundreds of aberrantly methylated CpG islands, many of which are promoters of tumor suppressor genes. The ability to detect and quantify promoter methylation will allow much more refined diagnostic and prognostic stratification. Recently, several groups including ours have reported the detection of tumor-associated methylated DNA circulating in serum/plasma. The use of circulating methylated DNA is a particularly attractive for screening and companion diagnostics for cancer, as serum/plasma is obtained through a simple, relatively noninvasive procedure. Currently, detection of circulating methylated DNA is mainly performed using bisulfite-based methods such as methylation specific PCR (MSP) due to their high sensitivity and specificity. However, clinical applications of these tests have been encumbered by a number of hurdles, resulting in highly variable success. For any bisulfite based methods, the process of DNA extraction and bisulfite conversion involves several disconnected steps on different platforms, resulting in substantial sample loss. Furthermore, MSP is designed to detect a specific methylation pattern; however, the promoter methylation patterns may be highly variable in tumors, compromising its clinical sensitivity. While sequence-based methods including bisulfite sequencing and pyrosequencing can be used to analyze methylation heterogeneity, these methods do not have the requisite sensitivity to detect the extremely low ratios (<0.1%) of methylated epialleles present in the bloodstream. In order to address these issues, we propose a streamlined methylation detection platform combining a silica paramagnetic bead (SSB)-based method for processing circulating DNA from large volume plasma samples to maximize assay fidelity and a microfluidic digital high resolution melt (HRM) approach for detecting and quantifying heterogeneous promoter epialleles at the single molecule level. A microfluidic device will be developed to facilitate high fidelity digital PCR and HRM in 1.6x106 microfluidic reaction chambers. A melt curve analysis program will be developed for analyzing the specific methylation allele in each reaction chamber according to the respective melt profile. The platform will be validated using both synthetic control samples and clinical samples from lung cancer patients undergoing epigenetic therapy. The proposed technology will enable detection and quantification of heterogeneous methylation with a low LOD (<10 copies of methylated DNA in ≥ 2 ml plasma sample), high sensitivity (1/100,000; methylated/unmethylated alleles) and a wide dynamic range (7 orders of magnitude), a level of performance unattainable by any other existing technologies (MSP, real- time qMSP and sequencing).

描述（由申请人提供）：异常甲基化 DNA 在所有形式的癌症和产生它们的肿瘤中大量存在。事实上，据估计，每个肿瘤的细胞内都有数百个异常甲基化 CpG 岛，其中许多是异常甲基化的 CpG 岛。检测和量化启动子甲基化的能力将允许更精细的诊断和预后分层。最近，包括我们在内的几个小组已经报告了肿瘤相关的检测。血清/血浆中循环的甲基化 DNA 对于循环甲基化 DNA 的使用特别有吸引力。癌症的筛查和伴随诊断，因为血清/血浆是通过简单、相对无创的程序获得的，目前，循环甲基化 DNA 的检测主要使用基于亚硫酸氢盐的方法进行，例如甲基化特异性 PCR (MSP)，因为它们具有高灵敏度和稳定性。然而，这些测试的临床应用受到许多障碍的阻碍，导致任何基于亚硫酸氢盐的方法的成功率差异很大，DNA 提取和亚硫酸氢盐转化的过程涉及不同的几个互不相关的步骤。此外，MSP 旨在检测特定的甲基化模式；然而，肿瘤中的启动子甲基化模式可能高度可变，从而损害其临床敏感性，而基于序列的方法（包括亚硫酸氢盐测序和焦磷酸测序）可能会受到影响。用于分析甲基化异质性，这些方法不具备检测血液中存在的极低比例（<0.1%）甲基化表观等位基因所需的灵敏度。为了解决这些问题，我们提出了一种简化的甲基化检测平台，该平台结合了基于二氧化硅顺磁珠 (SSB) 的方法（用于处理来自大量血浆样本的循环 DNA，以最大限度地提高检测保真度）和微流体数字高熔体分辨率 (HRM) 方法（用于检测和定量异质启动子）将开发单分子水平的表观等位基因以促进高保真数字PCR和将开发 1.6x106 微流体反应室中的 HRM，用于根据各自的熔解曲线分析每个反应室中的特定甲基化等位基因。该平台将使用来自肺癌患者的合成对照样品和临床样品进行验证。所提出的技术将能够以低 LOD（≥ 2 ml 血浆样本中甲基化 DNA 的拷贝数少于 10 个）、高灵敏度检测和定量异质甲基化。（1/100,000；甲基化/非甲基化等位基因）和宽动态范围（7 个数量级），这是任何其他现有技术（MSP、实时 qMSP 和测序）无法达到的性能水平。