Feasibility of Circulating 5-Hydroxymethylcytosine Profiling in Osteosarcoma

Aims: To examine the utility and performance of 5-hydroxymethycytosine (5-hmC) profiling of cell-free DNA (cfDNA) as a biomarker of disease in patients with osteosarcoma. Population: A Discovery cohort consisting of 5 patients with primary, localized osteosarcoma and an independent Validation cohort consisting of 17 patients with osteosarcoma. Serial cfDNA samples were collected for the majority of patients.Molecular Technologies Employed: Nano-hmC-Seal libraries were constructed from 1-10 ng of cfDNA. Briefly, cfDNA 5-hmC marks were subjected to enzymatic modification with UDP-N3-glucose, followed by chemical modification with biotin. Biotin-labeled cfDNA fragments were then pulled down using streptavidin beads, and PCR amplified to construct sequencing libraries. Findings: Using genes with differential 5-hmC deposition between osteosarcoma patients at diagnosis in the Discovery cohort and well children, hierarchal clustering identified two clusters of samples in the Validation cohort that were associated with disease status. Using a semi-quantitative osteosarcoma signature scoring system, the sensitivity and specificity to identify patients with active disease were 65% and 64%, respectively. Lung/soft tissue metastases were not readily identified. Despite limitations, 5-hmC profiling of cfDNA is feasible as a technique and further investigation with larger patient cohorts is warranted.]]>
Patients with biopsy-proven osteosarcoma and a minimum of one peripheral blood sample obtained for cfDNA analysis were included in the present study. There were no specific exclusion criteria for the study. ]]>