5hmC as a novel liquid biopsy biomarker for colorectal cancer to enable detection and unveil tumor biology.

228 Background: 5-hydroxymethylcytosine (5hmC) is a DNA epigenetic modification which regulates gene expression and is associated with active transcription. 5hmC sequencing in cell-free DNA (cfDNA) enables assessment of gene activity through a liquid biopsy. We profiled 5hmC in plasma cfDNA to assess its potential as a liquid biopsy biomarker for colorectal cancer (CRC). Methods: Genome-wide 5hmC modifications were analyzed with a low-input whole-genome 5hmC sequencing method based on selective chemical labeling using cfDNA from: (i) a discovery cohort of 75 CRC patients and 150 non-cancer blood samples and (ii) a validation cohort of 72 CRC patients and 70 non-cancer blood samples. Differential 5hmC analysis between CRC and non-cancer samples was conducted using DESeq2. Low-pass whole genome sequencing (LP-WGS) was performed on all cfDNA samples. Tumor fraction was estimated using ichorCNA to classify samples into high ct-fraction CRC (ct-fraction ≥0.1) and low ct-fraction CRC (ct-fraction