5-Hydroxymethylcytosine Profiling of Cell-Free DNA Identifies Bivalent Genes That Are Prognostic of Survival in High-Risk Neuroblastoma.

PURPOSE: Neuroblastoma is the most common extracranial solid tumor in childhood. We previously showed that circulating cell-free DNA (cfDNA) and tumor biopsy derived 5-hydroxymethylcytosime (5-hmC) profiles identified patients with neuroblastoma who experienced subsequent relapse. Here, we hypothesized that 5-hmC modifications selectively enriched in cfDNA compared with tumor biopsy samples would identify epigenetic changes associated with aggressive tumor behavior and identify novel biomarkers of outcome in patients with high-risk neuroblastoma. METHODS: 5-hmC profiles from cfDNA (n = 64) and tumor biopsies (n = 48) were compared. Two neuroblastoma cell lines underwent chromatin immunoprecipitation followed by sequencing (ChIP-Seq) for H3K27me3, H3K4me3, and H3K27ac; kethoxal-associated single-stranded DNA sequencing; hmC-Seal for 5-hmC; and RNA-sequencing (RNA-Seq). Genes enriched for both H3K27me3 and H3K4me3 in the included cell lines were defined as bivalent. Using bivalent genes defined in vitro, a bivalent signature was established in three publicly available cohorts of patients with neuroblastoma through gene set variation analysis. Differences between tumors with high or low bivalent signatures were assessed by the Kaplan-Meier method and Cox proportional hazards models. RESULTS: In cfDNA compared with tumor biopsy derived 5-hmC profiles, we found increased 5-hmC deposition on Polycomb Repressive Complex 2 target genes, a finding previously described in the context of bivalent genes. We identified 313 genes that bore bivalent chromatin marks, were enriched for mediators of neuronal differentiation, and were transcriptionally repressed across a panel of heterogeneous neuroblastoma cell lines. In three distinct clinical cohorts, low bivalent signature was significantly and independently associated with worse clinical outcome in patients with high-risk neuroblastoma. CONCLUSION: Low expression of bivalent genes is a biomarker of worse outcome in patients with high-risk neuroblastoma.

5-hydroxymethylcytosine profiling of cell-free DNA identifies bivalent genes that are prognostic of survival in high-risk neuroblastoma.

Neuroblastoma is the most common extra-cranial solid tumor in childhood and epigenetic dysregulation is a key driver of this embryonal disease. In cell-free DNA from neuroblastoma patients with high-risk disease, we found increased 5-hydroxymethylcytosine (5-hmC) deposition on Polycomb Repressive Complex 2 (PRC2) target genes, a finding previously described in the context of bivalent genes. As bivalent genes, defined as genes bearing both activating (H3K4me3) and repressive (H3K27me3) chromatin modifications, have been shown to play an important role in development and cancer, we investigated the potential role of bivalent genes in maintaining a de-differentiated state in neuroblastoma and their potential use as a biomarker. We identified 313 genes that bore bivalent chromatin marks, were enriched for mediators of neuronal differentiation, and were transcriptionally repressed across a panel of heterogenous neuroblastoma cell lines. Through gene set variance analysis, we developed a clinically implementable bivalent signature. In three distinct clinical cohorts, low bivalent signature was significantly and independently associated with worse clinical outcome in high-risk neuroblastoma patients. Thus, low expression of bivalent genes is a biomarker of ultra-high-risk disease and may represent a therapeutic opportunity in neuroblastoma.