Liquid biopsy strategies in pediatric patients are challenging due to low body weight. This study investigated cfDNA size distribution and concentration in blood, bone marrow, cerebrospinal fluid, and urine from 84 patients with neuroblastoma classified as low (n = 28), intermediate (n = 6), or high risk (n = 50) to provide key data for liquid biopsy biobanking strategies. The average volume of blood and bone marrow plasma provided ranged between 1 and 2 mL. Analysis of 637 DNA electropherograms obtained by Agilent TapeStation measurement revealed five different major profiles and characteristic DNA size distribution patterns for each of the biofluids. The proportion of samples containing primarily cfDNA was, at 85.5%, the highest for blood plasma. The median cfDNA concentration amounted to 6.28 ng/mL (blood plasma), 58.2 ng/mL (bone marrow plasma), 0.08 ng/mL (cerebrospinal fluid), and 0.49 ng/mL (urine) in samples. Meta-analysis of the dataset demonstrated that multiple cfDNA-based assays employing the same biofluid sample optimally require sampling volumes of 1 mL for blood and bone marrow plasma, 2 mL for cerebrospinal fluid, and as large as possible for urine samples. A favorable response to treatment was associated with a rapid decrease in blood-based cfDNA concentration in patients with high-risk neuroblastoma. Blood-based cfDNA concentration was not sufficient as a single parameter to indicate high-risk disease recurrence. We provide proof of concept that monitoring neuroblastoma-specific markers in very small blood volumes from infants is feasible.

Telomerase is reactivated by genomic TERT rearrangements in ∼30% of diagnosed high-risk neuroblastomas. Dismal patient prognosis results if the RAS/MAPK/ALK signaling transduction network also harbors mutations. We present a liquid biopsy–based monitoring strategy for this particularly vulnerable pediatric patient subgroup for which real-time molecular diagnostic tools are limited to date. Droplet digital PCR assays quantifying patient-individualized TERT rearrangement breakpoint copies, ALK copy numbers, and allelic ALK p.R1275Q mutation frequencies were applied to longitudinally collected liquid biopsies (peripheral blood and bone marrow [BM] plasma, n = 44 biosamples), the mononuclear cell fraction from BM and matched tumor samples. Marker detection was compared with current gold-standard diagnostics. Reanalysis of whole-genome and targeted panel sequencing data from 169 patients identified 64 TERT-rearranged neuroblastoma samples collected at initial and/or relapse diagnosis from 55 patients (254 total TERT rearrangement events). Detection and quantification of unique TERT rearrangement breakpoints in as little as 1 ng of total cell-free DNA in peripheral blood plasma improved therapy response assessment and early relapse detection in individual patients. Proof-of-concept is provided for minimal residual disease detection in the BM niche, from which relapses frequently arise, by analyzing unique TERT rearrangement breakpoints in BM plasma–derived cell-free DNA. TERT rearrangement breakpoints, as a single marker or combined with mutations in the RAS/MAPK/ALK signaling transduction network, can serve as robust and highly sensitive biomarkers for disease activity and spatially and temporally resolve disease better than current gold-standard diagnostics in individual patients with TERT-driven neuroblastoma. Significance: Real-time molecular monitoring of TERT-rearranged high-risk neuroblastoma is an unmet clinical need. We tested liquid biopsy-based assays for patient-individualized TERT breakpoint sequences to monitor disease in pediatric patients. Our digital PCR approach provides high resolution of spatial and temporal disease quantification in individual patients and is applicable for clinical routine.

Background/Objectives: Neuroblastoma cells are phenotypically plastic, transitioning between mesenchymal and adrenergic states. Core functional genes (e.g., YAP1) mark the mesenchymal state, which is linked to unfavorable prognosis. We and others previously demonstrated relapse-specific Hippo-YAP pathway activation in matched primary/relapsed neuroblastomas. Here we explored the role of YAP1 in neuroblastoma aggressiveness and response to therapy. Methods: RT-qPCR and immunoblotting assessed YAP1 expression in neuroblastoma cell lines. RNA-sequencing detected YAP1-dependent gene signatures in Tet-ON SK-N-AS and SH-EP neuroblastoma cell models expressing wildtype YAP1 or constitutively activated YAP1^S127A. Data from cell models were compared with our published YAP1 expression data from neuroblastomas. Efficacy of commonly used chemotherapeutics was comparatively analyzed in the cell models. Results: YAP1 expression showed marked variability across a panel of neuroblastoma cell lines, assessed by mRNA analysis in 10 cell lines and protein analysis in a subset of 9 cell lines. RNA sequencing in constitutively activated YAP1^S127A mutant and wildtype YAP1 models detected 2162 and 1837 significantly differentially expressed genes in the SK-N-AS and SH-EP backgrounds, respectively. Continuously activating YAP1 in SK-N-AS cells upregulated mesenchymal signature genes and mesenchymal-associated transcription factors. Gene expression influenced by YAP1 activity in the cell models significantly overlapped with YAP1-associated genes (e.g., CYR61 and SPRY4) in published tumor data. Functionally, YAP1^S127A expression rendered neuroblastoma cells resistant to chemotherapy. Conclusions: Findings corroborate the idea of a mechanistic role for YAP1 in neuroblastoma adrenergic to mesenchymal reprogramming and therapy resistance. The YAP1-mediated plastic switch towards a mesenchymal expression state in neuroblastoma cells may provide the molecular basis for novel therapeutic avenues.