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

> **NIH NIH R33** · JOHNS HOPKINS UNIVERSITY · 2022 · $433,765

## Abstract

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.

## Key facts

- **NIH application ID:** 10496926
- **Project number:** 1R33CA272321-01
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Thomas Russell Pisanic II
- **Activity code:** R33 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $433,765
- **Award type:** 1
- **Project period:** 2022-09-05 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10496926

## Citation

> US National Institutes of Health, RePORTER application 10496926, A low-cost, multiplexed digital high resolution melt platform for DNA methylation-based detection and identification of cancers in liquid biopsies (1R33CA272321-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10496926. Licensed CC0.

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