Simple and Accessible Microfluidic Platform for Single Molecule Sequence Profiling of Tumor-derived DNA within Liquid Biopsies

简单易用的微流体平台，用于液体活检中肿瘤来源 DNA 的单分子序列分析

• 批准号: 10699214
• 负责人: Christine O'Keefe
• 金额: $ 27.57万
• 依托单位: HYPERMELT, LLC
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10699214
• 关键词: Address; Adoption; Algorithms; Bar Codes; Binding; Biological Assay; Biological Markers; Biopsy; Blood; Buffers; Cancer Control; Cancer Detection; Cancer Diagnostics; Cancer Patient; Cause of Death; Cessation of life; Clinical; Clinical Sensitivity; Colonoscopy; Color; Colorectal Cancer; Complex; Cost Analysis; DNA; DNA Methylation; DNA Sequence Alteration; Data Analytics; Detection; Development; Diagnostic; Dimensions; Disease; Disease Progression; Early Diagnosis; Expenditure; Feces; Fluorescence; Formulation; Frequencies; Future; Genes; Genetic Materials; Genome; Goals; Hour; Image; Image Analysis; Individual; Laboratories; Laboratory Research; Machine Learning; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Malignant neoplasm of lung; Methods; Methylation; Microfluidics; Modification; Optics; Pap smear; Patient Care; Patient-Focused Outcomes; Patients; Performance; Persons; Phase; Plasma; Population; Procedures; Reaction; Research; Resolution; Resources; Sampling; Sensitivity and Specificity; Site; Small Business Innovation Research Grant; Specificity; System; Techniques; Technology; Thermodynamics; Time; Tissues; Tumor stage; Tumor-Derived; Uninsured; Urine; Work; biomarker panel; cancer biomarkers; cancer type; cell free DNA; circulating DNA; clinical implementation; clinically relevant; cohort; cost; cost effective; data analysis pipeline; design; detection platform; digital; digital platform; dimensional analysis; imager; improved; instrumentation; liquid biopsy; malignant breast neoplasm; manufacture; melting; methylation biomarker; methylation pattern; minimally invasive; molecular marker; mortality; multiplex assay; next generation sequencing; novel marker; parallelization; prevent; prototype; routine screening; screening; single molecule; standard of care; targeted biomarker; tool; tumor

Project Summary/ Abstract Over 600,000 people in the US will die from cancer this year. It is estimated that 25% of these deaths could have been prevented by detection in earlier stages. Implementation of minimally-invasive routine screening, such as pap smears for cervical cancer, has proven to be an effective approach for reducing cancer mortality. However, several challenges prevent successful implementation of screening in most cancers, especially ones which are not readily accessible for imaging or tissue biopsy. One promising avenue for cancer diagnostics is through use of circulating DNA from so-called “liquid biopsies” or other accessible sample media circulating throughout the body collecting genetic material from tissues, including tumors. Tumor-specific molecular biomarkers, such as DNA mutations and methylation, can be found in minimally-invasive sample media, such as blood, stool, and urine, but are typically only present in very low copy numbers (<10 copies/mL) and low fractions (<0.1%) among a high background of healthy DNA. Technical limitations as well as practical inaccessibility of currently available tools have precluded research efforts to discover early-stage cancer- specific DNA biomarker panels and subsequent clinical implementation that could improve patient outcomes. To address this, we previously developed a prototype digital microfluidic platform to facilitate highly sensitive, low-cost detection of cancer-specific DNA methylation patterns by highly parallelized single-molecule thermodynamic sequencing. In this Phase 1 SBIR proposal, we will greatly expand upon the capabilities of this platform to increase accessibility and improve analytical performance towards detection of early-stage disease by significantly increasing its digitization power. We will develop a high-degree multiplexing paradigm for detection and methylation profiling of biomarker panels using a multicolor barcoding technique. We will then incorporate this assay into a microfluidic platform that can interrogate hundreds to thousands of single DNA copies by digitizing template molecules into droplets for high-throughput single molecules analysis. The platform will increase accessibility for biomarker research from liquid biopsies by reducing costs to <$25 per sample and turnaround time to 4 hours. The platform will enable single-copy detection even among high background populations (<0.001% sensitivity), which may be necessary for early-stage disease. The proposed work in this Phase 1 project will develop the assay fundamentals for a multiplex, multidimensional analysis of a clinically relevant biomarker panel (Aim 1), incorporate this assay into a highly-parallelized droplet microfluidic platform (Aim 2), and assess its clinical feasibility with plasma samples from a cohort of lung cancer patients and controls (Aim 3). This will lay the groundwork for a subsequent Phase 2 project to design the cartridge and instrumentation for scalable manufacturing and user-friendliness and implement automated, machine- learning image and data analysis pipelines.

项目摘要/摘要 今年，美国有超过60万人死于癌症。据估计，这些死亡中有25％可以 在早期阶段被检测阻止了。实施最低侵入的常规筛选， 例如，用于宫颈癌的子宫颈抹片检查已被证明是降低癌症死亡率的有效方法。 但是，一些挑战阻止了大多数癌症的成功实施筛查 对于成像或组织活检而言，不容易获得。癌症诊断的一种承诺大道是 通过使用所谓的“液体活检”或其他可访问的样品介质循环的循环DNA 在整个身体中，从组织（包括肿瘤）收集遗传物质。肿瘤特异性分子 生物标志物，例如DNA突变和甲基化，可以在微创样品介质中找到，例如 作为血液，凳子和尿液 在健康DNA的高背景中的馏分（<0.1％）。技术限制和实用 当前可用工具的无法访问性排除了研究早期癌症的研究工作 - 特定的DNA生物标志物面板以及随后的临床实施，可以改善患者的预后。 为了解决这个问题，我们以前开发了一个原型数字微流体平台，以促进高度敏感的， 高度平行的单分子对癌症特异性DNA甲基化模式的低成本检测 热力学测序。在此阶段1 SBIR提案中，我们将大大扩展此功能 提高可访问性并提高分析性能以发现早期疾病的平台 通过显着增加其数字化功率。我们将开发一个高度的多路复用范式 使用多色条形码技术对生物标志物面板的检测和甲基化分析。然后我们会 将此测定纳入一个微流体平台，该平台可以询问数百至数千个单个DNA 通过将模板分子数字化成液滴进行副本，以进行高通量单分子分析。这 平台将通过将成本降低到每 / 25美元，从而使生物标志物研究的可访问性增加 样品和周转时间至4小时。该平台即使在高中也可以实现单拷贝检测 背景人群（<0.001％灵敏度），这对于早期疾病可能是必需的。提议 该阶段1项目的工作将开发用于多重，多维分析的测定基础。 临床上相关的生物标志物面板（AIM 1）将此测定纳入高度比较的液滴微流体中 平台（AIM 2），并评估其与来自肺癌患者队列的血浆样品的临床可行性 和控件（AIM 3）。这将为随后的2阶段项目设计墨盒的基础 以及用于可扩展制造和用户友好性的仪器，并实施自动化的机器 - 学习图像和数据分析管道。