5-Hydroxymethylcytosine Profiles in Circulating Cell-Free DNA Associate with Disease Burden in Children with Neuroblastoma

Clin Cancer Res. 2020 Mar 15;26(6):1309-1317. doi: 10.1158/1078-0432.CCR-19-2829. Epub 2019 Dec 18.


Purpose: 5-Hydroxymethylcytosine (5-hmC) is an epigenetic marker of open chromatin and active gene expression. We profiled 5-hmC with Nano-hmC-Seal technology using 10 ng of plasma-derived cell-free DNA (cfDNA) in blood samples from patients with neuroblastoma to determine its utility as a biomarker.

Experimental design: For the Discovery cohort, 100 5-hmC profiles were generated from 34 well children and 32 patients (27 high-risk, 2 intermediate-risk, and 3 low-risk) at various time points during the course of their disease. An independent Validation cohort encompassed 5-hmC cfDNA profiles (n = 29) generated from 21 patients (20 high-risk and 1 intermediate-risk). Metastatic burden was classified as high, moderate, low, or none per Curie metaiodobenzylguanidine scores and percentage of tumor cells in bone marrow. Genes with differential 5-hmC levels between samples according to metastatic burden were identified using DESeq2.

Results: Hierarchical clustering using 5-hmC levels of 347 genes identified from the Discovery cohort defined four clusters of samples that were confirmed in the Validation cohort and corresponded to high, high-moderate, moderate, and low/no metastatic burden. Samples from patients with increased metastatic burden had increased 5-hmC deposition on genes in neuronal stem cell maintenance and epigenetic regulatory pathways. Further, 5-hmC cfDNA profiles generated with 1,242 neuronal pathway genes were associated with subsequent relapse in the cluster of patients with predominantly low or no metastatic burden (sensitivity 65%, specificity 75.6%).

Conclusions: cfDNA 5-hmC profiles in children with neuroblastoma correlate with metastatic burden and warrants development as a biomarker of treatment response and outcome.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5-Methylcytosine / analogs & derivatives*
  • 5-Methylcytosine / metabolism
  • Adolescent
  • Adult
  • Biomarkers, Tumor / analysis*
  • Cell-Free Nucleic Acids / blood*
  • Child
  • Child, Preschool
  • DNA Methylation*
  • Epigenomics*
  • Female
  • Humans
  • Infant
  • Infant, Newborn
  • Male
  • Neoplasm Metastasis
  • Neuroblastoma / blood
  • Neuroblastoma / genetics
  • Neuroblastoma / pathology*
  • Prognosis
  • Young Adult


  • Biomarkers, Tumor
  • Cell-Free Nucleic Acids
  • 5-hydroxymethylcytosine
  • 5-Methylcytosine