Neuroblastoma (NB) is the most common extracranial solid tumor during infancy, causing up to 10% of mortality in children; thus, identifying novel early and accurate diagnostic and prognostic biomarkers is mandatory. NB-derived exosomes carry proteins (Exo-prots) reflecting the status of the tumor cell of origin. The purpose of this study was to characterize, for the first time, the Exo-prots specifically expressed in NB patients associated with tumor phenotype and disease stage. We isolated exosomes from plasma specimens of 24 HR-NB patients and 24 low-risk (LR-NB) patients at diagnosis and of 24 age-matched healthy controls (CTRL). Exo-prot expression was measured by liquid chromatography–mass spectrometry. The data are available via ProteomeXchange (PXD042422). The NB patients had a different Exo-prot expression profile compared to the CTRL. The deregulated Exo-prots in the NB specimens acted mainly in the tumor-associated pathways. The HR-NB patients showed a different Exo-prot expression profile compared to the LR-NB patients, with the modulation of proteins involved in cell migration, proliferation and metastasis. NCAM, NCL, LUM and VASP demonstrated a diagnostic value in discriminating the NB patients from the CTRL; meanwhile, MYH9, FN1, CALR, AKAP12 and LTBP1 were able to differentiate between the HR-NB and LR-NB patients with high accuracy. Therefore, Exo-prots contribute to NB tumor development and to the aggressive metastatic NB phenotype.

Neuroblastoma (NB) is the most common solid extracranial paediatric tumour. Genome-wide association studies have driven the discovery of common risk variants, but no large study has investigated the contribution of rare variants to NB susceptibility. Here, we conducted a whole-exome sequencing (WES) of 664 NB cases and 822 controls and used independent validation datasets to identify genes with rare risk variants and involved pathways. WES was performed at 50× depth and variants were jointly called in cases and controls. We developed two models to identify mutations with high clinical impact (P/LP model) and to discover less penetrant risk mutations affecting non-canonical cancer pathways (RPV model). We performed a gene-level collapsing test using Firth's logistic regression in 242 selected cancer predisposition genes (CPGs) and a gene-sets burden analysis of biologically-informed pathways. Twelve percent of patients carried P/LP variants in CPGs and showed a significant enrichment (P = 2.3 × 10⁻⁴) compared to controls (6%). We identified P/LP variants in 45 CPGs enriched in homologous recombination (HR) pathway. The most P/LP enriched genes in NB were BRCA1, ALK and RAD51C. Additionally, we found higher RPV burden in gene-sets of neuron differentiation, neural tube development and synapse assembly, and in gene-sets associated with neurodevelopmental disorders (NDD). The high fraction of NB patients with P/LP variants indicates the need of genetic counselling. Furthermore, inherited rare variants predispose to NB development by affecting mechanisms related to HR and neurodevelopmental processes, and demonstrate that NDD genes are altered in NB at the germline level. Associazione Italiana per la Ricerca sul Cancro, Fondazione Italiana per la Lotta al Neuroblastoma, Associazione Oncologia Pediatrica e Neuroblastoma, Regione Campania, Associazione Giulio Adelfio onlus, and Italian Health Ministry.

Background: Neuroblastoma (NB) causes about 15% of cancer deaths in childhood. Recently, we suggested cell-surface nucleolin (NCL) as a novel target for preclinical therapy against NB. Methods: Here, a broad range of human NB cell lines were evaluated for NCL expression. PEGylated liposomal nanoparticles, co-encapsulating C6- or C18-ceramides and doxorubicin (DXR) and functionalized with the F3 peptide (F3-lipo[C6-DXR] or F3-lipo[C18-DXR]), were tested against NCL-expressing NB cell lines, grown in monolayers (2D) and as multicellular tumor spheroids (3D). Untargeted liposomes were used as the control. Cytotoxicity and apoptotic/necrotic deaths were evaluated. Results: All NB cell lines expressed cell-surface NCL. Compared to untargeted formulations, F3-lipo[C6-DXR] and F3-lipo[C18-DXR] showed enhanced cellular association and antitumor effects against NB cells. Compared to F3-lipo[C18-DXR], F3-lipo[C6-DXR] was significantly more effective in reducing 2D and 3D NB cell lines’ viability (2D: IC50 range 313–995 nM and 239–629 nM, respectively; 3D: IC50 range 202–416.2 nM and 62.61–398.6 nM, respectively) and in inducing apoptotic cell death. F3-lipo[C6-DXR] also led to a greater cytotoxicity compared to liposomal DXR alone, highlighting the benefit of co-encapsulation. Conclusions: NCL is a promising target in NB, and F3-targeted liposomes enable the selective delivery of their cargo. F3-lipo[C6-DXR] showed superior antitumor activity, supporting ceramide–DXR co-encapsulation as a potential treatment strategy, which needs to be further validated.

PURPOSE: Intermediate-risk neuroblastoma patients older than 18 months, with non– MYCN amplified, International Neuroblastoma Risk Group Staging System localized, unresectable or International Neuroblastoma Staging System stage 3 tumors, and unfavorable histology have inferior outcomes compared with other intermediate-risk patients. This study aimed to identify genetic prognostic biomarkers within this rare subgroup. METHODS: We conducted a large, international study including chromosomal copy number in all cases, next-generation DNA sequencing in most, and telomere maintenance mechanisms and gene expression in a subset, and correlated results with patient survival. RESULTS: Among 98 tumors, 9/98 (9.2%) had oncogene amplifications ( CDK4/MDM2/TERT coamplification (n = 1), CDK4/MDM2 coamplification (n = 4), CDK4 (n = 2), TERT (n = 1), and MYC (n = 1)), while 63/98 (64.3%) had typical segmental chromosomal aberrations (tSCAs). Patients with tumors with oncogene amplification had the worst 5-year event-free survival (EFS; 0%; P < .0001 log-rank test) and 5-year overall survival (OS; 44.4% [95% CI, 21.4 to 92.3]; P < .01 log-rank test). Patients with tumors harboring tSCAs had inferior EFS compared with those with numerical chromosomal aberrations only (51.7% [95% CI, 40.6 to 65.8] v 93.3% [95% CI, 81.5 to 100]; P < .01). Patients with p53 pathway tumor alterations (n = 10) had worse EFS than those without (0% v 61.1% [95% CI, 50.3 to 74.3]; P < .0001, log-rank test) and worse OS (26.7% [95% CI, 8.9 to 80.3] v 80.9% [95% CI, 71.8 to 91.3]; P < .001 log-rank test). Multivariable analysis identified tSCAs as an independent prognostic variable for EFS and oncogene amplification or p53 pathway abnormalities as independent prognostic variables for EFS and OS. CONCLUSION: Oncogene amplification and/or p53 pathway abnormalities and/or typical SCAs identify patients with intermediate-risk neuroblastoma with inferior outcome for whom intensified or alternative treatments should be considered.