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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要今年美国将有超过 60 万人死于癌症，预计其中 25% 可能死于癌症。已通过早期检测来预防，例如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。然而，一些挑战阻碍了对大多数癌症，尤其是癌症的筛查的成功实施成像或组织活检不易获得的一种有前途的癌症诊断途径是。通过使用来自所谓“液体活检”或其他可获取的循环样本介质的循环DNA 在整个身体中从组织中收集遗传物质，包括肿瘤特异性分子。生物标志物，例如 DNA 突变和甲基化，可以在微创样本介质中找到，例如如血液、粪便和尿液，但通常仅以非常低的拷贝数（<10 拷贝/mL）和低拷贝数存在健康 DNA 的高背景中的分数（<0.1%）技术限制以及实际情况。目前可用的工具无法获得，阻碍了发现早期癌症的研究工作特定的 DNA 生物标记物组合和随后的临床实施可以改善患者的治疗结果。为了解决这个问题，我们之前开发了一个原型数字微流控平台，以促进高度敏感、通过高度并行的单分子低成本检测癌症特异性 DNA 甲基化模式在第一阶段 SBIR 提案中，我们将极大地扩展其功能。提高早期疾病检测的可及性并提高分析性能的平台通过显着提高其数字化能力，我们将开发一种高度复用范例。然后我们将使用多色条形码技术对生物标志物组进行检测和甲基化分析。这将检测方法整合到了微流体平台中，可以询问数百到数千个单一 DNA 通过将模板分子数字化成液滴来进行高通量单分子分析。平台将通过将成本降低至每次 25 美元以下，提高液体活检生物标志物研究的可及性样品和周转时间缩短至 4 小时，即使在高浓度环境下，该平台也能实现单拷贝检测。背景人群（<0.001% 敏感性），这对于早期疾病可能是必要的。第一阶段项目的工作将开发多重、多维分析的检测基础临床相关的生物标志物组（目标 1），该测定纳入高度并行化的液滴微流体平台（目标 2），并利用一组肺癌患者的血浆样本评估其临床可行性和控制（目标 3），这将为后续设计墨盒的第二阶段项目奠定基础。和仪器仪表，以实现可扩展的制造和用户友好性，并实现自动化、机器化学习图像和数据分析管道。