A low-cost, multiplexed digital high resolution melt platform for DNA methylation-based detection and identification of cancers in liquid biopsies

一种低成本、多重数字高分辨率熔解平台，用于液体活检中基于 DNA 甲基化的癌症检测和识别

• 批准号: 10697370
• 负责人: Thomas Russell Pisanic II
• 金额: $ 41.35万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-05 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10697370
• 关键词: Aberrant DNA Methylation; Algorithms; Alleles; Benchmarking; Biological Assay; Biological Markers; Blood Tests; Cancer Detection; Cancer Diagnostics; Cancer Patient; Cells; Cessation of life; Clinical; Code; Colon Carcinoma; Color; Complex; Control Locus; DNA; DNA Methylation; Detection; Development; Diagnostic; Diagnostic Procedure; Discrimination; Disease; Dyes; Early Diagnosis; Early identification; Endometrial Carcinoma; Fluorescence; Genetic Materials; Goals; Hematopoietic Neoplasms; Individual; Life; Logistic Regressions; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of ovary; Malignant neoplasm of pancreas; Methods; Methylation; Microfluidic Microchips; Microfluidics; Modeling; Mutation; Non-Invasive Detection; Patient-Focused Outcomes; Patients; Performance; Persons; Plasma; ROC Curve; Resolution; Sampling; Scheme; Sensitivity and Specificity; Serum; Site; Specificity; Technology; Time; Tissues; Tumor stage; Validation; Work; biomarker panel; bisulfite sequencing; cancer type; cell free DNA; clinical implementation; cohort; cost; cost effective; density; design; digital; digital assessment; early detection biomarkers; imager; improved; liquid biopsy; malignant breast neoplasm; melting; methylation biomarker; methylation pattern; neoplastic cell; next generation sequencing; novel; parallelization; rare cancer; ratiometric; single molecule; technology development; tool; tumor; tumor DNA

PROJECT SUMMARY This year alone, over 600,000 people in the U.S. will die from cancer, with each patient losing an average of 15.6 years of life. However, upwards of 25% of these deaths could likely be avoided if these cancers were detected at earlier stages. One particularly attractive approach for cancer diagnostics is the use of circulating cell-free DNA (cfDNA) from so-called “liquid-biopsies” of patient-derived serum/plasma as these samples are often enriched in genetic material from tissues, including tumors, located throughout the body. Nonetheless, tumor specific alterations, such as mutations and aberrant DNA methylation, are typically only present at extraordinarily low copy numbers (< 10 copies/ml) and fractional concentrations (< 0.1%) within a large background of healthy-tissue DNA. This issue in particular has proven problematic for current technologies and has thus far precluded development of a cfDNA diagnostic method that is simple, low-cost and, most importantly, able to detect cancer at stages sufficiently early to improve patient outcomes. In the present project, we aim to develop REM-DREAMing: a low-cost, highly-multiplexed digital methylation analysis platform that provides highly-sensitive and parallelized assessment of cfDNA methylation patterns to enable detection of rare tumor DNA, even from early-stage cancers. At the core of the REM-DREAMing platform is a unique, locus-specific DNA methylation assay, called DREAMing (Discrimination of Rare EpiAlleles by Melt), that has been successfully developed by our lab to provide detection and absolute quantification of cancer-specific DNA methylation even at extremely low fractions (<< 0.1%). Recently, we successfully incorporated the DREAMing assay into a massively-parallel digital microfluidic array to enable detection of a single copy of aberrantly-methylated DNA in a background of 2 million unmethylated alleles. Here, we propose to dramatically enhance the microfluidic DREAMing approach by significantly expanding its digitization power and incorporating novel, methylation-agnostic probes with a unique ratiometric fluorescence multiplexing scheme to achieve simultaneous digital assessment of a panel of 50 “cancer-detecting” and “cancer-identifying” methylation biomarkers, enabling liquid-biopsy-based detection and identification of early-stage cancers at a cost of only a few dollars per sample. To achieve this goal, we plan to accomplish the following aims: (1) Develop dual, 27-plex DREAMing assay panels targeting a panel of 50 pan-cancer-detecting and cancer-identifying methylation biomarkers. (2) Design, fabricate and validate a dual 400k-well, 4-color fluorescence-decoding dHRM platform to perform parallelized REM-DREAMing for simultaneous detection and identification of 50 methylation biomarkers. and (3) Assess and benchmark the ability of the REM-DREAMing platform to detect and identify six different cancer types from liquid biopsies.

项目摘要 仅今年，美国超过60万人就会死于癌症，每位患者平均失去 15。6年的生命。但是，如果这些死亡取消，可能会避免使用这些死亡的25％以上 我们在较早的阶段被发现。一种特别有吸引力的癌症诊断方法是使用 来自患者衍生的血清/血浆所谓的“液体 - 亲事件”的循环无细胞DNA（CFDNA） 这些样品通常富含来自组织的组织的遗传物质，包括肿瘤 身体。尽管如此，肿瘤特异性改变，例如突变和异常DNA甲基化，是 通常仅以极低的拷贝数（<10副本/毫升）和分数浓度存在 （<0.1％）在健康组织DNA的大背景下。特别是这个问题已证明有问题 对于当前的技术，到目前为止，已排除了CFDNA诊断方法的发展 简单，低成本，最重要的是，能够在阶段正确检测到癌症以改善患者 结果。 在本项目中，我们旨在开发重新启动：一个低成本，高度的数字数字 甲基化分析平台可提供高度敏感和平行的CFDNA评估 即使是从早期癌症中，甲基化模式也能够检测到罕见的肿瘤DNA。在核心 朗诵平台是一个独特的，特定于基因座的DNA甲基化测定法，称为梦想 （通过熔体歧视稀有的epiallles），这是我们实验室成功开发的 即使在极低的部分，癌症特异性DNA甲基化的检测和绝对定量 （<< 0.1％）。最近，我们成功地将梦境分析纳入了一个大规模的平行数字 微流体阵列可以在背景中检测单个异常甲基化DNA的副本 200万个未甲基等位基因。在这里，我们建议大大增强微流体梦想 通过显着扩展其数字化能力并结合新颖的甲基化不可能的方法来方法 独特的比率荧光多路复用方案的问题，以实现简单的数字 评估一个50个“癌症检测”和“癌症识别”甲基化生物标志物的评估，使得 基于液体生物的检测和对早期癌症的识别，只需几美元 样本。为了实现这一目标，我们计划实现以下目的：（1）开发双重27-plex 梦想测定面板针对50个Pan-Cantertecting和癌症识别的甲基化面板 生物标志物。 （2）设计，制造和验证双400k，4色荧光解码DHRM 进行平行化的rebreaming的平台，以简单检测和识别50 甲基化生物标志物。 （3）评估和基准重新启动平台的能力 从液体活检中检测并鉴定六种不同的癌症类型。