Abstract 2299: More information from limited DNA: simultaneous measurement of genetics, 5hmC and 5mC in cell-free DNA
Abstract Liquid biopsy for profiling of cell free DNA (cfDNA) in blood holds huge promise to transform how we experience and manage cancer by early detection and identification of residual disease and subtype. While early work in liquid biopsy focused on the identification of actionable somatic vari...
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| Published in: | Cancer research (Chicago, Ill.) Vol. 84; no. 6_Supplement; p. 2299 |
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| Main Authors: | , , , , , , , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
22-03-2024
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| Online Access: | Get full text |
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| Summary: | Abstract
Liquid biopsy for profiling of cell free DNA (cfDNA) in blood holds huge promise to transform how we experience and manage cancer by early detection and identification of residual disease and subtype. While early work in liquid biopsy focused on the identification of actionable somatic variations at specific loci, the past decade has seen an expansion into non-genetic features, notably methylation. 5-methylcytosine (5-mC) profiles of cancer are differential from non-cancer at many more loci and so provide a stronger signal. Moreover, recent research has suggested that 5-hydroxymethylcytosine (5-hmC) profiles in cfDNA can be a marker for early cancer. However, a standard blood draw yields an average of only 10ng of cfDNA, presenting the dilemma of how to use limited sample to obtain maximum information. Existing methods for measuring methylation using next-generation sequencing cannot distinguish between 5-mC and 5-hmC and are limited in their ability to detect mutations, with approaches that measure both 5-mC and 5-hmC requiring two separate workflows. Hence, to measure complete genetics, 5-mC and 5-hmC using existing technologies requires three separate workflows, each of which require separate DNA input. We will present a technology which sequences at single base resolution the complete genetic sequence of input DNA fragments integrated with the modification status (unmodified, 5-mC or 5-hmC) for each CpG from low nanogram input quantities of DNA. A hairpin construct is used to create a copy of the original DNA strand. Enzymatic conversion followed by next-generation sequencing enables coupled decoding of bases across the original and copy strand, uniquely reporting one of A, C, G, T, 5-mC, or 5-hmC for each position in the input DNA fragment. Using this technology, we generated whole genome 6-letter data (measuring A, C, G, T, 5-mC, and 5-hmC at single-base resolution across the genome) on cell-free DNA extracted from plasma of healthy volunteers and patients with colorectal cancer at various stages of progression. We demonstrate how the technology can be used to compare 5-mC and 5-hmC methylomic profiles, genomics, and fragmentomics, across different stages of colorectal cancer. In particular, we show how disease progression can be marked by changes in 5-mC and 5-hmC across several loci. We propose that the ability to measure 5-mC and 5-hmC at high accuracy and single base resolution, alongside genomic and fragmentomic profiles, from a limited quantity of DNA will enable greater insight into the ctDNA in plasma and help advance the field of liquid biopsy towards fulfilling its promise.
Citation Format: Tom J. Charlesworth, Fabio Puddu, Robert Crawford, Annelie Johansson, Ermira Lleshi, Aurelie Modat, Jamie Scotcher, Michael Wilson, Nicholas Harding, Jean Teyssandier, Jens Fullgrabe, Walraj Gosal, Paidi Creed. More information from limited DNA: simultaneous measurement of genetics, 5hmC and 5mC in cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2299. |
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| ISSN: | 1538-7445 1538-7445 |
| DOI: | 10.1158/1538-7445.AM2024-2299 |