Neuroblastoma is the most common extracranial solid tumor in children. Amplification of the MYCN gene has been observed in approximately 20%-30% of tumors. It is strongly correlated with advanced-stage disease, rapid tumor progression, resistance to chemotherapy and poor outcomes independent of patient age and stage of advanced disease. MYCN amplification identifies high-risk patients. To assess neuroblastoma tumors with MYCN amplification we used paraffin-embedded tissue sections in 57 patients and intraoperative tumor imprints in 10 patients by fluorescence in situ hybridization (FISH). Positive results for MYCN amplification have been observed in twelve patients\' paraffin-embedded tissue sections and in three patients\' intraoperative tumor imprints, which represents 22.4% of all patients tested in the analysis. Fluorescence in situ hybridization is a highly sensitive and useful technique for detecting MYCN amplification on paraffin-embedded tissue sections of neuroblastoma tumors and intraoperative tumor imprints thus facilitating therapeutic decisions based on the presence or absence of this important biologic marker. The presence of structural changes, regardless of MYCN gene amplification status, influences the clinical behavior of neuroblastoma. High-Density SNP Arrays have emerged as the perfect tools for detecting these changes due to their exceptional accuracy, sensitivity and ability to analyze copy number and allele information. Consequently, they are proven to be highly valuable in the genomic diagnosis of immature neuroectodermal tumors.

BACKGROUND: Prognosis for patients with high-risk neuroblastoma (HR-NBL) is guarded despite aggressive therapy, and few studies have characterized outcomes after radiotherapy in relation to radiation treatment fields.
METHODS: Multi-institutional retrospective cohort of 293 patients with HR-NBL who received autologous stem cell transplant (ASCT) and EBRT between 1997-2021. LRR was defined as recurrence at the primary site or within one nodal echelon beyond disease present at diagnosis. Follow-up was defined from the end of EBRT. Event-free survival (EFS) and OS were analyzed by Kaplan-Meier method. Cumulative incidence of locoregional progression (CILP) was analyzed using competing risks of distant-only relapse and death with Gray\'s test.
RESULTS: Median follow-up was 7.0 years (range: 0.01-22.4). Five-year CILP, EFS, and OS were 11.9 %, 65.2 %, and 77.5 %, respectively. Of the 31 patients with LRR and imaging review, 15 (48.4 %) had in-field recurrences (>12 Gy), 6 (19.4 %) had marginal failures (≤12 Gy), and 10 (32.3 %) had both in-field and marginal recurrences. No patients receiving total body irradiation (12 Gy) experienced marginal-only failures (p = 0.069). On multivariable analyses, MYCN amplification had higher risk of LRR (HR: 2.42, 95 % CI: 1.06-5.50, p = 0.035) and post-consolidation isotretinoin and anti-GD2 antibody therapy (HR: 0.42, 95 % CI: 0.19-0.94, p = 0.035) had lower risk of LRR.
CONCLUSIONS: Despite EBRT, LRR remains a contributor to treatment failure in HR-NBL with approximately half of LRRs including a component of marginal failure. Future prospective studies are needed to explore whether radiation fields and doses should be defined based on molecular features such as MYCN amplification, and/or response to chemotherapy.

Tumor MYCN amplification is seen in high-risk neuroblastoma, yet direct targeting of this oncogenic transcription factor has been challenging. Here, we take advantage of the dependence of MYCN-amplified neuroblastoma cells on increased protein synthesis to inhibit the activity of eukaryotic translation initiation factor 4A1 (eIF4A1) using an amidino-rocaglate, CMLD012824. Consistent with the role of this RNA helicase in resolving structural barriers in 5\' untranslated regions (UTRs), CMLD012824 increased eIF4A1 affinity for polypurine-rich 5\' UTRs, including that of the MYCN and associated transcripts with critical roles in cell proliferation. CMLD012824-mediated clamping of eIF4A1 spanned the full lengths of mRNAs, while translational inhibition was mediated through 5\' UTR binding in a cap-dependent and -independent manner. Finally, CMLD012824 led to growth inhibition in MYCN-amplified neuroblastoma models without generalized toxicity. Our studies highlight the key role of eIF4A1 in MYCN-amplified neuroblastoma and demonstrate the therapeutic potential of disrupting its function.

MYCN (master regulator of cell cycle entry and proliferative metabolism) gene amplification defines a molecular subgroup of spinal cord ependymomas that show high-grade morphology and aggressive behavior. Demonstration of MYCN amplification by DNA methylation or fluorescence-in situ hybridization (FISH) is required for diagnosis. We aimed to (i) assess prevalence and clinicopathological features of MYCN-amplified spinal ependymomas and (ii) evaluate utility of immunohistochemistry (IHC) for MYCN protein as a surrogate for molecular testing. A combined retrospective-prospective study spanning 8 years was designed during which all spinal cord ependymomas with adequate tissue were subjected to MYCN FISH and MYCN IHC. Among 77 spinal cord ependymomas included, MYCN amplification was identified in 4 samples from 3 patients (3/74, 4%) including two (1st and 2nd recurrences) from the same patient. All patients were adults (median age at diagnosis of 32 years) including two females and one male. The index tumors were located in thoracic (n = 2) and lumbar (n = 1) spinal cord. One of the female patients had neurofibromatosis type 2 (NF2). All four tumors showed anaplastic histology. Diffuse expression of MYCN protein was seen in all four MYCN-amplified samples but in none of the non-amplified cases, thus showing 100% concordance with FISH results. On follow-up, the NF2 patient developed widespread spinal dissemination while another developed recurrence proximal to the site of previous excision. To conclude, MYCN-amplified spinal ependymomas are rare tumors, accounting for ~ 4% of spinal cord ependymomas. Within the limitation of small sample size, MYCN IHC showed excellent concordance with MYCN gene amplification.

OBJECTIVE: To investigate the predictive value of 2-deoxy-2-fluorine-18-fluoro-D-glucose positron emission tomography/computed tomography (18F-FDG PET/CT) parameters for MYCN amplification in high-risk neuroblastoma (HR-NB).
METHODS: A retrospective analysis was performed by reviewing 68 HR-NB patients who underwent MYCN testing and 18F-FDG PET/CT imaging at our hospital between January 2018 and December 2019. Based on the results of MYCN testing, patients were categorized into either the MYCN-amplified (MNA) or MYCN non-amplified (MYCN-NA) group. The 18F-FDG PET/CT parameters, including maximum standardized uptake value (SUVmax), mean standardized uptake value (SUVmean), peak standardized uptake value (SUVpeak), tumor metabolic volume (MTV), total lesion glycolysis (TLG), coefficient of variation (COV), and areas under the curve of cumulative SUV-volume histogram index (AUC-CSH index) were evaluated. Independent predictors were identified through univariate and multivariate logistic regression analyses, and their diagnostic performance was evaluated using the receiver-operating characteristic (ROC) curve.
RESULTS: Univariate logistic regression analysis revealed that SUVpeak was significantly associated with MYCN amplification. Multivariate logistic regression analysis showed that SUVpeak was an independent predictor of MYCN amplification in HR-NB [Odds ratio (OR) = 0.673, 95 % confidence interval (95 % CI): 0.494-0.917, P = 0.012]. ROC curve analysis demonstrated that the predictive model including SUVpeak had higher diagnostic performance [area under the curve (AUC): 0.790, 95 % CI: 0.677-0.881, sensitivity: 0.861, specificity: 0.591, positive predictive value (PPV): 0.820, negative predictive value (NPV): 0.722] compared to using SUVpeak alone (AUC: 0.640, 95 % CI: 0.514-0.752, sensitivity: 0.630, specificity: 0.682, PPV: 0.806, NPV: 0.469).
CONCLUSIONS: SUVpeak can predict the MYCN amplification in HR-NB patients. The predictive model constructed by combining SUVpeak and age can distinguish MYCN status in HR-NB non-invasively with superior efficacy compared to using SUVpeak alone.

MYCN proto-oncogene, bHLH transcription factor (MYCN) amplification is associated with aggressive retinoblastoma (RB) and neuroblastoma (NB) cancer recurrence that is resistant to chemotherapies. Therefore, there is an urgent need to identify new therapeutic tools. This study aimed to evaluate the potential repurposing of ceftriaxone for the treatment of MYCN-amplified RB and NB, based on the clinical observations that the drug was serendipitously found to decrease the volume of the MYCN-driven RB subtype. Using patient-derived tumor organoids and tumor cell lines, we demonstrated that ceftriaxone is a potent and selective growth inhibitor targeting MYCN-driven RB and NB cells. Profiling of drug-induced transcriptomic changes, cell-cycle progression, and apoptotic death indicated cell-cycle arrest and death of drug-treated MYCN-amplified tumor cells. Drug target identification, using an affinity-based proteomic and molecular docking approach, and functional studies of the target proteins revealed that ceftriaxone targeted DEAD-box helicase 3 X-linked (DDX3X), thereby inhibiting translation in MYCN-amplified tumors but not in MYCN-nonamplified cells. The data suggest the feasibility of repurposing ceftriaxone as an anticancer drug and provide insights into the mechanism of drug action, highlighting DDX3X as a potential target for treating MYCN-driven tumors.

BACKGROUND: Genetic 1p deletion is reported in 30% of all neuroblastoma and is associated with the unfavorable prognosis of neuroblastoma. The expressions and prognosis of 1p candidate genes in neuroblastoma are unclear.
METHODS: Public neuroblastoma cohorts were obtained for secondary analysis. The prognosis of 1p candidate genes in neuroblastoma was determined using Kaplan-Meier and cox regression analysis. The prediction of the nomogram model was determined using timeROC.
RESULTS: First, we confirmed the bad prognosis of 1p deletion in neuroblastoma. Moreover, zinc finger protein 436 (ZNF436) located at 1p36 region was down-regulated in 1p deleted neuroblastoma and higher ZNF436 expression was associated with the longer event free survival and overall survival of neuroblastoma. The expression levels of ZNF436 were lower in neuroblastoma patients with MYCN amplification or age at diagnosis ≥ 18months, or with stage 4 neuroblastoma. ZNF436 had robust predictive values of MYCN amplification and overall survival of neuroblastoma. Furthermore, the prognostic significance of ZNF436 in neuroblastoma was independent of MYCN amplification and age of diagnosis. Combinations of ZNF436 with MYCN amplification or age of diagnosis achieved better prognosis. At last, we constructed a nomogram risk model based on age, MYCN amplification and ZNF436. The nomogram model could predict the overall survival of neuroblastoma with high specificity and sensitivity.
CONCLUSIONS: Chromosome 1p36 candidate gene ZNF436 was a prognostic maker of neuroblastoma.

MYCN amplification as a common genetic alteration that correlates with a poor prognosis for neuroblastoma (NB) patients. However, given the challenge of directly targeting MYCN, indirect strategies to modulate MYCN by interfering with its cofactors are attractive in NB treatment. Although cyclin B1 interacting protein 1 (CCNB1IP1) has been found to be upregulated in MYCN-driven mouse NB tissues, its regulation with MYCN and collaboration in driving the biological behaviour of NB remains unknown.
To evaluate the expression and clinical significance of CCNB1IP1 in NB patients, public datasets, clinical NB samples and cell lines were explored. MTT, EdU incorporation, colony and tumour sphere formation assays, and a mouse xenograft tumour model were utilized to examine the biological function of CCNB1IP1. The reciprocal manipulation of CCNB1IP1 and MYCN and the underlying mechanisms involved were investigated by gain- and loss-of-function approaches, dual-luciferase assay, chromatin immunoprecipitation (CHIP) and co-immunoprecipitation (Co-IP) experiments.
CCNB1IP1 was upregulated in MYCN-amplified (MYCN-AM) NB cell lines and patients-derived tumour tissues, which was associated with poor prognosis. Phenotypic studies revealed that CCNB1IP1 facilitated the proliferation and tumourigenicity of NB cells in cooperation with MYCN in vitro and in vivo. Mechanistically, MYCN directly mediates the transcription of CCNB1IP1, which in turn attenuated the ubiquitination and degradation of MYCN protein, thus enhancing CCNB1IP1-MYCN cooperativity. Moreover, CCNB1IP1 competed with F box/WD-40 domain protein 7 (FBXW7) for MYCN binding and enabled MYCN-mediated tumourigenesis in a C-terminal domain-dependent manner.
Our study revealed a previously uncharacterized mechanism of CCNB1IP1-mediated MYCN protein stability and will provide new prospects for precise treatment of MYCN-AM NB based on MYCN-CCNB1IP1 interaction.

BACKGROUND: Neuroblastoma (NB) is the most common extracranial solid tumour in childhood with a diverse clinical presentation and course. The early age of onset, high frequency of metastatic disease at diagnosis and tendency for spontaneous regression in infancy sets it apart from other childhood tumors. This heterogeneity is largely attributed to underlying genetic aberrations which are distinct in low-risk and high-risk NB. To this end, we sought to analyse our NB cases for the molecular alterations and find its correlation with clinical behaviour.
METHODS: NB cases (n = 50) diagnosed over last 7 years were retrospectively analysed for MYCN amplification (fluorescent-in-situ hybridization), TERT rearrangements (qRT-PCR), ATRX mutations (immunohistochemistry). These findings were correlated with demographic profiles, histologic features and clinical outcome.
RESULTS: Age ranged from 1 month to 30 years (mean 2.8 years) with male preponderance. Poorly differentiated subtype constituted the majority (64%), followed by differentiating (28%) and undifferentiated subtype (8%) which were equally distributed across all age groups. MYCN amplification, TERT-mRNA upregulation and ATRX mutations was observed in 30%, 42% and 24%, respectively. Cases with TERT-mRNA upregulation were distributed equally across all histological subtypes while those with ATRX mutations and MYCN amplification were frequent in poorly differentiated NB. ATRX mutation was mutually exclusive of TERT-mRNA upregulation and MYCN amplification. Kaplan-Meier analysis revealed significantly shorter overall and progression-free survival for tumors harboring MYCN amplification and TERT-mRNA upregulation, while that for ATRX mutant tumors was not significant.
CONCLUSIONS: Our results provide data indicating poor clinical outcome in NB carrying MYCN amplification and TERT-mRNA upregulation.

BACKGROUND: Ganglioneuroblastoma intermixed (GNBI) is classified as \"favorable\" histology by International Neuroblastoma Pathology Classification system. However, the International Neuroblastoma Risk Group (INRG) stratifies patients using wider clinicopathological and cytogenetic/molecular parameters. While the diagnosis of GNBI is typically made on resected tumor, it may sometimes be rendered on initial biopsy. We studied GNBI noted at diagnosis to evaluate its correlation with INRG staging and other clinicopathological and molecular features.
METHODS: In this retrospective study, clinical, radiological, pathological, cytogenetic, and molecular information from patients with GNBI at diagnosis seen between 1995 and 2021 was analyzed. INRG staging was performed.
RESULTS: Of the 15,827 neuroblastoma specimens, GNBI was noted in 237 patients. Of these, 53 had the initial pathological diagnosis of GNBI; median follow-up 3.5 (range: 0.2-14) years. Disease was locoregional in 41 (77%, 16 stage L1 and 25 L2); none relapsed. Twelve (23%) had metastatic disease at presentation; six (50%) relapsed, and two died of disease. MYCN was amplified in two metastatic tumors. Six of 31 (19%) tumors tested had recurrent cytogenetic abnormalities and nonrecurrent somatic gene mutations in 10/23 (43%). The presence of any adverse molecular/cytogenetic findings was associated with metastatic disease (p < .05). For patients with localized GNBI undergoing both biopsy and resection, GNBI was diagnosed in both in 17/19 (90%).
CONCLUSIONS: Localized GNBI at diagnosis has excellent long-term clinical outcome even without cytotoxic therapy. For localized GNBI, a biopsy sample is adequate to make the diagnosis. When associated with metastasis at diagnosis, prognosis is poorer, possibly due to associated adverse biological features.