5-Hydroxymethylcytosine profiles in circulating cell-free DNA associate with disease burden in children with neuroblastoma

Mark A. Applebaum*, Erin K. Barr, Jason Karpus, Diana C. West-Szymanski, Meritxell Oliva, Elizabeth A. Sokol, Sheng Zhang, Zhou Zhang, Wei Zhang, Alexandre Chlenski, Helen R. Salwen, Emma Wilkinson, Marija Dobratic, Robert L. Grossman, Lucy A. Godley, Barbara E. Stranger, Chuan He, Susan L. Cohn

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

20 Scopus citations


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 ¼ 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.

Original languageEnglish (US)
Pages (from-to)1309-1317
Number of pages9
JournalClinical Cancer Research
Issue number6
StatePublished - Mar 15 2020

ASJC Scopus subject areas

  • General Medicine


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