Neuroblastoma (NB) is an aggressive childhood cancer requiring intensive multimodal therapies in high-risk (HRNB) patients. Currently, invasive surgical biopsies are required to classify NB risk group and assign treatment based on the tumour genetic profile. Circulating tumour DNA (ctDNA) obtained from blood samples can be used to identify tumour biomarkers. Here we applied targeted next-generation sequencing (tNGS) using a panel of 42 genes to analyse 32 NB ctDNA samples for the presence of single-nucleotide variants and copy number changes from 28 patients in all NB risk groups. In two additional ctDNA samples, droplet digital PCR was used to detect hotspot ALK variants. Pathogenic mutations with a variant allele frequency (VAF) > 1% were identified in 13/32 (41%) ctDNA samples. ALK and PTPN11 were the most frequent, each being detected in 4/32 (13%) samples, together with oncogene amplifications. Targeted NGS of ctDNA detected actionable variants, including those absent in the diagnostic primary tumour due to spatial and temporal heterogeneity. Our findings confirm the usefulness of ctDNA in detecting genetic abnormalities in NB.

Background: Neuroblastoma (NB) is the most common extracranial paediatric solid cancer, with a 50% survival rate for high-risk patients. Circulating tumour cells (CTCs) are malignant cells shed by the primary tumour and metastatic sites that circulate in the bloodstream. CTCs form clusters with themselves (homotypic) or other cell types (heterotypic). Objectives: To use previously generated ImageStreamX Imaging Flow Cytometer data from blood samples from 24 patients across all NB risk groups, to examine for the presence of CTC clusters. Methods: Immunofluorescence and brightfield morphology were used to identify clusters followed by analysis using IDEAS image analysis software. Results: The mean number of clusters detected per sample was 87 (range, 0–725). Of the clusters detected, 1967/2094 (93.9%) were heterotypic and only 127/2094 (6.1%) were homotypic and found in 6/24 patients. Interestingly, in 3/24 patients, at least one cluster (median, 2 and range, 1–18) was found, but no single CTCs were detected. Clusters mostly comprised two cells (62.8%), with the maximum number of cells in homotypic and heterotypic clusters being four and eight, respectively. Conclusions: These results highlight that imaging flow cytometry can be used to detect and characterise CTC clusters in peripheral blood samples from NB patients, leading to further research exploring the composition and role of CTC clusters in NB metastasis.