Mark A. Applebaum

Background Cell‐free DNA (cfDNA) profiles of 5‐hydroxymethylcytosine (5‐hmC), an epigenetic marker of open chromatin and active gene expression, are correlated with metastatic disease burden in patients with neuroblastoma. Neuroblastoma tumors are comprised of adrenergic (ADRN) and mesenchymal (MES) cells, and the relative abundance of each in tumor biopsies has prognostic implications. We hypothesized that ADRN and MES‐specific signatures could be quantified in cfDNA 5‐hmC profiles and would augment the detection of metastatic burden in patients with neuroblastoma. Methods We previously performed an integrative analysis to identify ADRN and MES‐specific genes ( n = 373 and n = 159, respectively). Purified DNA from cell lines was serial diluted with healthy donor cfDNA. Using Gene Set Variation Analysis (GSVA), ADRN and MES signatures were optimized. We then quantified signature scores, and our prior neuroblastoma signature, in cfDNA from 84 samples from 46 high‐risk patients including 21 patients with serial samples. Results Samples from patients with higher metastatic burden had increased GSVA scores for both ADRN and MES gene signatures ( p < .001). While ADRN and MES signature scores tracked together in serially collected samples, we identified instances of patients with increases in either MES or ADRN score at relapse. Conclusions While it is feasible to identify ADRN and MES signatures using 5‐hmC profiles of cfDNA from neuroblastoma patients and correlate these signatures to metastatic burden, additional data are needed to determine the optimal strategies for clinical implementation. Prospective evaluation in larger cohorts is ongoing.