Assessment of 5-methylcytosine and 5-hydroxymethylcytosine technologies in contrived ctDNA-like samples.

Abstract

e15585 Background: Epigenomic changes to DNA are early driving events in tumorigenesis and are specific markers of the tumour and its microenvironment. Cell-free DNA that is released from the tumour microenvironment can potentially be used for the detection of tumour-specific epigenomic changes for cancer liquid biopsy testing. In this study, we assessed the robustness of 5-methylcytosine and 5-hydroxymethylcytosine technologies in contrived ctDNA-like samples from NSCLC. Additionally, we assessed the limit of detection of the tumour fraction within the tested dilution range looking at a subset of differential epigenomic marks. Methods: 4 NSCLC tumour and buffy coat DNA samples were commercially obtained and processed to generate contrived tumour DNA dilution mimicking circulating tumour DNA at varying tumour fractions. Samples were sent in triplicate to multiple epigenomic assay providers for analysis. The robustness i.e. reproducibility and limit-of-detection within the tested range was determined. Results: Technical replicates all passed method-specific quality controls and were highly reproducible. Cancer specific differentially-methylated or hydroxymethylated regions were determined for each individual tumour sample as a “sample-specific cancer signature”; when applied to the tumour dilution samples, these signatures were capable of distinguishing all tumour dilutions down to 0.05% from normal background DNA. Detection of differential methylation and hydroxymethylation regions was reproducible across technical replicates for all four NCSLC samples at > 0.05%. Generation of pooled methylation “signatures” derived from commercially available healthy plasma and cancer tissue datasets could still detect cancer DNA in these four NCSLC samples at > 0.1% tumour fraction. Conclusions: Our pilot study showed that all assessed technologies generated reproducible, robust data and could detect ctDNA at even clinically relevant levels for early stage cancer. One limitation of this study was the small number of “n” and the use of sonicated contrived sample that may induce artificial bias and reduce sensitivity highlighting a requirement for the development of reference material in the community. The ability to detect cancer-specific epigenomic changes from liquid biopsy with high sensitivity offers new promising avenues for early detection of cancer, which can increase the chances of successful treatment.