Development, characterization, and inter-laboratory validation of methylated human cell free DNA candidate reference materials
- bgtaylor1
- Aug 11
- 2 min read
Date: | May 18, 2026 |
PMID: | |
Category: | N/A |
Authors: | Zhiyong He, Yves Konigshofer, Russell Garlick, Jayanthi Ramprakash, Madhumita Ramesh, Eric Hall, Adam Corner, Michelle Clarissa, Jocelyn H Wright, Victoria K Cannon, Ming Yu, William M Grady, Cecilia C S Yeung, Zachary Heimer, Zhili Wang, ShiPing Zou, Shidong Jia, Fang Liu, Giancarlo Bonora, Karol Bomsztyk, Daniel Mar, Kenneth D Cole, Hua-Jun He |
Abstract: |
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Background: Aberrant DNA methylation biomarkers have demonstrated potential for early cancer detection, multicancer detection, and determining the tissue of origin. Due to their stability, frequency, and accessibility in bodily fluids, circulating cell-free DNA (cfDNA) methylation is a promising biomarker in liquid biopsy. A reliable and quantifiable analysis of cfDNA methylation status is critical to its application. However, there are current challenges and a lack of consensus on measurement methods. To address this, we developed two candidate methylated cfDNA reference materials (RMs).
Methods: The National Institute of Standards and Technology (NIST) RM consists of five components, formulated by mixing in vitro methylated cfDNA simulant at fractions of 0%, 5%, 25%, 50%, and 100% with native-state cfDNA simulant derived from the GM24385 cell line. The LGC Clinical Diagnostics (LGC) RM consists of two components: non-methylated cfDNA simulant derived from GM24385 genomic DNA and whole genome amplification and methylated cfDNA produced by in vitro methylation of amplified material. The candidate RMs were characterized, and the methylation status of three targets was confirmed by droplet digital PCR (ddPCR) assays. To test the utility of these RMs, six laboratories participated in an interlaboratory study, each using their own lab-developed assays and methods, which included methylation-specific qPCR, nanoplate digital PCR (dPCR), ddPCR, matrix methylated DNA immunoprecipitation-based assays, and whole-genome bisulfite sequencing.
Results: The interlaboratory study results showed that the designed percentage of methylation was well correlated with the observed values across all participating labs, and good reproducibility was found for each individual method. However, slightly different methylation proportions associated with assay-specific biases were observed.
Conclusions: This study clearly demonstrates the value of candidate RMs as standards for evaluating assay performance, as well as for increasing confidence in reporting cfDNA methylation status for clinical applications.
Acknowledgements:
The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute, or the National Institute of Health.
The Translational and Basic Science Research in Early Lesions (TBEL) Research Consortia is supported and funded by grants from the National Cancer Institute and the National Institutes of Health under the following award numbers:
Project Number: | Awardee Organization |
U54CA274374 | Fred Hutchinson Cancer Center |
U54CA274375 | Houston Methodist Research Institute |
U54CA274370 | Johns Hopkins University |
U54CA274371 | UT MD Anderson Cancer Center |
U54CA274367 | Vanderbilt University Medical Center |
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