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.

项目总结