5-hydroxymethylcytosine profiles in circulating cell-free DNA associate with disease status in patients with...

5-hydroxymethylcytosine (5-hmC) is a marker of open chromatin and active gene expression. We profiled 5-hmC using plasma-derived cell-free DNA (cfDNA) from patients with osteosarcoma to assess its utility as a biomarker of disease status. Genes with differential 5-hmC levels were identified from a Discovery cohort consisting of patients with osteosarcoma and healthy children. An independent Validation cohort was evaluated using these signature genes. Hierarchical clustering using 262 osteosarcoma signature genes identified in the Discovery cohort identified two clusters of samples in the Validation cohort. Cluster 1 contained 10 of 12 samples from patients with primary disease or osseous metastases, whereas Cluster 2 contained 26 of 33 samples from patients without active disease. Using a semi-quantitative osteosarcoma signature scoring system, the sensitivity and specificity to classify patients with active disease were 65% and 64%, respectively. This technique is feasible in this population, and further investigation with larger patient cohorts is warranted.