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Abstract The limited understanding of the molecular mechanism underlying MYCN-amplified (MNA) neuroblastoma (NB) has hindered the identification of effective therapeutic targets for MNA NB, contributing to its higher mortality rate compared to MYCN non-amplified (non-MNA) NB. Therefore, a comprehensive analysis integrating metabolomics and transcriptomics was conducted to systematically investigate the MNA NB. Metabolomics analysis utilized plasma samples from 28 MNA NB patients and 68 non-MNA NB patients, while transcriptomics analysis employed tissue samples from 15 MNA NB patients and 37 non-MNA NB patients. Notably, joint metabolomics and transcriptomics analysis was performed. A total of 46 metabolites exhibited alterations, with 21 displaying elevated levels and 25 demonstrating reduced levels in MNA NB. In addition, 884 mRNAs in MNA NB showed significant changes, among which 766 mRNAs were higher and 118 mRNAs were lower. Joint-pathway analysis revealed three aberrant pathways involving glycerolipid metabolism, purine metabolism, and lysine degradation. This study highlights the substantial differences in metabolomics and transcriptomics between MNA NB and non-MNA NB, identifying three abnormal metabolic pathways that may serve as potential targets for understanding the molecular mechanisms underlying MNA NB.

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.

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. [ABSTRACT FROM AUTHOR]

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: Neuroblastoma is the most common extracranial solid tumor in children. MYCN gene amplification (MNA) is an independent prognostic factor for rapid tumor progression and poor prognosis, regardless of age and clinical stage. Gain of the MYCN gene locus on the short arm of chromosome 2 can also be found in neuroblastoma.Method: In this retrospective descriptive analysis of genetic alterations in neuroblastoma tumor samples, both before and after standard chemotherapy, we examined the MYCN gene copy number status in 20 neuroblastic tumor samples using the fluorescence in situ hybridization (FISH) method. We also evaluated its relationship with clinical variables and tumor maturation after standard chemotherapy treatment. Additionally, we compared disease outcomes among different MYCN copy number categories.Results: Among the tumor samples, four (25%) exhibited increased MYCN copy numbers, 20% showed MYCN amplification, and 5% displayed MYCN gain. We observed decreased survival rates in advanced stages of neuroblastoma. Furthermore, in male patients, we noted an association between increased MYCN copy number and metastatic tumors.Conclusion: We found that increased MYCN copy number is moderately associated with an immature phenotype and correlated with lower event-free survival. However, we did not detect a statistically significant difference.

Background: Neuroblastoma is the most common extracranial solid tumor in children. MYCN gene amplification (MNA) is an independent prognostic factor for rapid tumor progression and poor prognosis, regardless of age and clinical stage. Gain of the MYCN gene locus on the short arm of chromosome 2 can also be found in neuroblastoma. Method: In this retrospective descriptive analysis of genetic alterations in neuroblastoma tumor samples, both before and after standard chemotherapy, we examined the MYCN gene copy number status in 20 neuroblastic tumor samples using the fluorescence in situ hybridization (FISH) method. We also evaluated its relationship with clinical variables and tumor maturation after standard chemotherapy treatment. Additionally, we compared disease outcomes among different MYCN copy number categories. Results: Among the tumor samples, four (25%) exhibited increased MYCN copy numbers, 20% showed MYCN amplification, and 5% displayed MYCN gain. We observed decreased survival rates in advanced stages of neuroblastoma. Furthermore, in male patients, we noted an association between increased MYCN copy number and metastatic tumors. Conclusion: We found that increased MYCN copy number is moderately associated with an immature phenotype and correlated with lower event-free survival. However, we did not detect a statistically significant difference. [ABSTRACT FROM AUTHOR]

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ABSTRACTDespite aggressive treatment, the 5-year event-free survival rate for children with high-risk neuroblastoma is

Background: 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. Methods: 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. Results: 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. Conclusions: 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. [ABSTRACT FROM AUTHOR]

Abstract Background MYCN amplification (MNA) has been proved to be related to poor prognosis in neuroblastoma (NBL), but the MYCN-related immune signatures and genes remain unclear. Methods Enrichment analysis was used to identify the significant enrichment pathways of differentially expressed immune-related genes (DEIRGs). Weight gene coexpression network analysis (WGCNA) was applied to reveal the correlation between these DEIRGs and MYCN status. Univariate and multivariate Cox analyses were used to construct risk model. The relevant fractions of immune cells were evaluated by CIBERSORT and single-sample gene set enrichment analysis (ssGSEA). Results Five genes, including CHGA, PTGER1, SHC3, PLXNC1, and TRIM55 were enrolled into the risk model. Kaplan–Meier survival analysis and receiver operating characteristic (ROC) curve showed that our model performed well in predicting the outcomes of NBL (3-years AUC = 0.720, 5-year AUC = 0.775, 10-years AUC = 0.782), which has been validated in the GSE49711 dataset and the E-MTAB-8248 dataset. By comparing with the tumor immune dysfunction and exclusion (TIDE) and tumor inflammation signature (TIS), we further proved that our model is reliable. Univariate and multivariate Cox regression analyses indicated that the risk score, age, and MYCN can serve as independent prognostic factors in the E-MATB-8248. Functional enrichment analysis showed the DEIRGs were enriched in leukocyte adhesion-related signaling pathways. Gene set enrichment analysis (GSEA) revealed the significantly enriched pathways of the five MYCN-related DEIRGs. The risk score was negatively correlated with the immune checkpoint CD274 (PD-L1) but no significant difference with the TMB. We also confirmed the prognostic value of our model in predicting immunotherapeutics. Conclusion We constructed and verified a signature based on DEIRG that related to MNA and predicted the survival of NBL based on relevant immune signatures. These findings could provide help for predicting prognosis and developing immunotherapy in NBL.

Amplification of the MYCN oncogene is found in ~20% of neuroblastoma (NB) cases and correlates with high-risk disease and poor prognosis. Despite the plethora of studies describing the role of MYCN in NB, the exact molecular mechanisms underlying MYCN's contribution to high-risk disease are not completely understood. Herein, we implemented an integrative approach combining publicly available RNA-Seq and MYCN ChIP-Seq datasets derived from human NB cell lines to define biological processes directly regulated by MYCN in NB. Our approach revealed that MYCN-amplified NB cell lines, when compared to non-MYCN-amplified cell lines, are characterized by reduced expression of genes involved in NOTCH receptor processing, axoneme assembly, and membrane protein proteolysis. More specifically, we found genes encoding members of the γ-secretase complex, which is known for its ability to liberate several intracellular signaling molecules from membrane-bound proteins such as NOTCH receptors, to be down-regulated in MYCN-amplified NB cell lines. Analysis of MYCN ChIP-Seq data revealed an enrichment of MYCN binding at the transcription start sites of genes encoding γ-secretase complex subunits. Notably, using publicly available gene expression data from NB primary tumors, we revealed that the expression of γ-secretase subunits encoding genes and other components of the NOTCH signaling pathway was also reduced in MYCN-amplified tumors and correlated with worse overall survival in NB patients. Genetic or pharmacological depletion of MYCN in NB cell lines induced the expression of γ-secretase genes and NOTCH-target genes. Chemical inhibition of γ-secretase activity dampened the expression of NOTCH-target genes upon MYCN depletion in NB cells. In conclusion, this study defines a set of MYCN-regulated pathways that are specific to MYCN-amplified NB tumors, and it suggests a novel role for MYCN in the suppression of genes of the γ-secretase complex, with an impact on the NOTCH-target gene expression in MYCN-amplified NB. [ABSTRACT FROM AUTHOR]

Objectives The protein ALK is targeted for therapy in neuroblastoma, and ALK mutation confers a poor prognosis. We evaluated ALK in a cohort of patients with advanced neuroblastoma diagnosed by fine-needle aspiration biopsy (FNAB). Methods Fifty-four cases of neuroblastoma were evaluated for ALK protein expression by immunocytochemistry and ALK gene mutation by next-generation sequencing. MYCN amplification by fluorescence in situ hybridization, International Neuroblastoma Risk Group (INRG) staging, and risk assignment was performed, and patients were managed accordingly. All parameters were correlated with overall survival (OS). Results ALK protein showed cytoplasmic expression in 65% cases and did not correlate with MYCN amplification (P =.35), INRG groups (P =.52), and OS (P =.2); however, ALK-positive, poorly differentiated neuroblastoma showed better prognosis (P =.02). ALK negativity was associated with poor outcome by Cox proportional hazard model (hazard ratio, 2.36). Two patients showed ALK gene F1174L mutation with 8% and 54% allele frequency and high ALK protein expression; they died of disease 1 and 17 months following diagnosis, respectively. A novel IDH1 exon 4 mutation was also detected. Conclusions ALK expression is a promising prognostic and predictive marker in advanced neuroblastoma that can be evaluated in cell blocks from FNAB samples along with traditional prognostic parameters. ALK gene mutation confers a poor prognosis for patients with this disease. [ABSTRACT FROM AUTHOR]

Abstract Background 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. Methods 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. Results 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. Conclusions 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.

Despite aggressive treatment, the 5-year event-free survival rate for children with high-risk neuroblastoma is <50%. While most high-risk neuroblastoma patients initially respond to treatment, often with complete clinical remission, many eventually relapse with therapy-resistant tumors. Novel therapeutic alternatives that prevent the recurrence of therapy-resistant tumors are urgently needed. To understand the adaptation of neuroblastoma under therapy, we analyzed the transcriptomic landscape in 46 clinical tumor samples collected before (PRE) or after (POST) treatment from 22 neuroblastoma patients. RNA sequencing revealed that many of the top-upregulated biological processes in POST MYCN amplified (MNA+) tumors compared to PRE MNA+ tumors were immune-related, and there was a significant increase in numerous genes associated with macrophages. The infiltration of macrophages was corroborated by immunohistochemistry and spatial digital protein profiling. Moreover, POST MNA+ tumor cells were more immunogenic compared to PRE MNA+ tumor cells. To find support for the macrophage-induced outgrowth of certain subpopulations of immunogenic tumor cells following treatment, we examined the genetic landscape in multiple clinical PRE and POST tumor samples from nine neuroblastoma patients revealing a significant correlation between an increased amount of copy number aberrations (CNA) and macrophage infiltration in POST MNA+ tumor samples. Using an in vivo neuroblastoma patient-derived xenograft (PDX) chemotherapy model, we further show that inhibition of macrophage recruitment with anti-CSF1R treatment prevents the regrowth of MNA+ tumors following chemotherapy. Taken together, our work supports a therapeutic strategy for fighting the relapse of MNA+ neuroblastoma by targeting the immune microenvironment. [ABSTRACT FROM AUTHOR]

Abstract Background Previously, we had analyzed the prognosis of E2F transcription factors across adult tumor types. However, the expressions and prognosis of E2F transcription factors in pediatric neuroblastoma have not yet been fully studied. Methods The prognosis of E2F transcription factors was determined in four independent pediatric neuroblastoma cohorts from Therapeutically Applicable Research to Generate Effective Treatments (TARGET), Gene Expression Omnibus (GEO) and European ArrayExpres datasets using Kaplan–Meier and cox regression analysis. Results E2F regulated gene set was associated with the event free survival and the overall survival of neuroblastoma. E2F1 and E2F3 were prognostic factors in all four independent pediatric neuroblastoma cohorts. Over-expressions of E2F1 or E2F3 were correlated with the shorted event free survival and overall survival of neuroblastoma. Expression levels of E2F1 and E2F3 were higher in neuroblastoma patients with MYCN amplification or age at diagnosis ≥ 18 months. Moreover, the prognostic significance of E2F1 or E2F3 in neuroblastoma was independent of MYCN amplification and age of diagnosis. Combinations of E2F1, E2F3 with MYCN amplification or age of diagnosis achieved better prognosis of neuroblastoma. Identification of 234 genes were associated with E2F1 and E2F3 expressions in neuroblastoma and those genes were significantly enriched in cell cycle signaling pathway. Also, higher scores of cell cycle signaling pathway were correlated with the adverse prognosis of neuroblastoma. Conclusions E2F transcription factors E2F1 and E2F3 were prognostic makers of neuroblastoma.

Abstract Background This study aimed to better understand the prognostic effect of multiple genetic markers and identify more subpopulations at ultra high risk of poor outcome in bone marrow (BM) metastatic neuroblastoma (NB). Methods We screened the MYCN, 1p36 and 11q23 loss of heterozygosity (LOH) statuses of 154 patients by interphase fluorescence in situ hybridization of BM cells. The clinical characteristics of patients with the three markers and their associations with prognosis were analysed. Results MYCN amplification and LOH at 1p36 and 11q23 were identified in 16.2%, 33.1% and 30.5% of patients, respectively. There were strong associations between MYCN amplification and 1p36 LOH as well as 11q23 LOH. Both MYCN amplification and 1p36 LOH were strongly associated with high levels of lactate dehydrogenase (LDH) and neuron‐specific enolase, more than 3 metastatic organs, and more events. 11q23 LOH occurred mainly in patients older than 18 months, and those who had high LDH levels. In univariate analysis, patients with MYCN amplification had poorer prognosis than those without. Patients with 1p36 LOH had a 3‐year event‐free survival (EFS) and overall survival lower than those without. 11q23 LOH was associated with poorer EFS only for patients without MYCN amplification. In a multivariate model, MYCN amplification was independently associated with decreased EFS in all cohorts. 11q23 LOH was an independent prognostic factor for patients without MYCN amplification, whereas 1p36 LOH was not an independent marker regardless of MYCN amplification. Compared with all cohorts, patients with both MYCN amplification and 1p36 LOH had the worst outcome and clinical features. Conclusions Patients with both MYCN amplification and 1p36LOH had the worst survival rate, indicating an ultra high‐risk group. Our results may be applied in clinical practice for accurate risk stratification in future studies.

MYCN amplification (MYCN -amp) is a significant prognostic factor and early genetic marker of high-risk neuroblastoma (NB). MYCN induces the DNA damage response (DDR) and modulates the insensitivity of NB cells to Poly (ADP-ribose) polymerase (PARP) inhibitors. We previously reported that CCC-002, a DNA-alkylating agent conjugated with pyrrole-imidazole polyamide targeting MYCN , inhibits NB cell proliferation and induces DNA damage signaling. In this study, we investigated the synergistic effects of CCC-002 and PARP inhibitors on MYCN -amp NB cells. Combination treatment with PARP inhibitors significantly enhanced the sensitivity of MYCN -amp NB cells to CCC-002. DNA damage signals, such as phosphorylation of H2AX and RPA32 elicited after CCC-002 treatment, were further enhanced by PARP inhibitors, as detected through western blotting and immunofluorescence analyses. The potent cytotoxicity of this combination treatment was confirmed by the significant increase in the subG0-G1 phase. Although MYCN knockdown showed no synergistic effect with PARP inhibitors, fluorescence in situ hybridization and quantitative PCR analyses indicated that PARP inhibitors enhanced the effect of CCC-002 to reduce MYCN copy number and suppress its expression. Overall, our study provides novel insights into a therapeutic approach that combines CCC-002 and PARP inhibition to effectively induce DNA damage and apoptosis in MYCN -amp NB cells. [Display omitted] • PARP inhibitors enhance CCC-002 inhibitory effects on MYCN -amplified NB cell growth. • Copy number and expression of MYCN are reduced by the combined treatment of CCC-002 and PARP inhibitors. • CCC-002-induced DNA damage is key for PARP inhibitors' synergistic effects. • CCC-002 and PARP inhibitors cotreatment induces DNA damage and apoptosis. [ABSTRACT FROM AUTHOR]

Fan Li, Weiling Zhang, Huimin Hu, Yi Zhang, Jing Li, Dongsheng Huang Department of Pediatrics, Beijing Tongren Hospital, Capital Medical University, Beijing, 100730, People’s Republic of ChinaCorrespondence: Dongsheng HuangDepartment of Pediatrics, Beijing Tongren Hospital, Capital Medical University, Beijing, 100730, People’s Republic of ChinaTel +86 10-58266032Email hdsmed@126.comBackground: It is not clear which known adverse prognostic factors of neuroblastoma are closely associated with tumor recurrence after complete response. We analyzed the factors for post-remission recurrence in children with neuroblastoma through a retrospective study.Methods: A total of 179 children with neuroblastoma who achieved initial complete response were included in this study. Kaplan–Meier method and multivariate Cox regression model were used to assess the factors that may have impact on tumor recurrence after complete response.Results: The 5-year overall survival rates of the entire cohort (n = 179), recurrence group (n = 86) and non-recurrence group (n = 93) were 81.9%, 66.2%, and 98.7%, respectively. The 5-year recurrence-free survival (RFS) rates of the entire cohort and the high-risk cohort were 47.3% and 31.2%, respectively. RFSs were significantly reduced in children with age ≥ 18 months, INSS stage 4, unfavorable histology, bone marrow metastasis, osseous metastasis, serum NSE level ≥ 100 ng/mL, and serum LDH level ≥ 1400 U/L (P < 0.05). The independent risk factors for post-remission recurrence in the entire cohort were age ≥ 18 months, unfavorable histology, and serum LDH level ≥ 1400 U/L (P < 0.05). In the high-risk cohort, the independent risk factor for recurrence was serum LDH ≥ 1400 U/L (P < 0.05). Based on a new recurrence risk stratification, the 5-year RFSs of the children were 93.5%, 66.4%, and 22.5% in the low-risk, intermediate-risk, and high-risk groups, respectively. The area under the ROC curve of the new stratification was 0.773 (95% CI: 0.704− 0.842).Conclusion: Age ≥ 18 months, unfavorable histology, and serum LDH level ≥ 1400 U/L are independent risk factors for post-remission recurrence in children with neuroblastoma. A newly established recurrence risk stratification has diagnostic advantages in predicting risk of recurrence, which is especially suitable for low- and middle-income countries or regions.Keywords: age, MYCN amplification, histology, recurrence-free survival, serum lactate dehydrogenase, serum neuron-specific enolase

Background: MYCN amplification (MNA) has been proved to be related to poor prognosis in neuroblastoma (NBL), but the MYCN-related immune signatures and genes remain unclear. Methods: Enrichment analysis was used to identify the significant enrichment pathways of differentially expressed immune-related genes (DEIRGs). Weight gene coexpression network analysis (WGCNA) was applied to reveal the correlation between these DEIRGs and MYCN status. Univariate and multivariate Cox analyses were used to construct risk model. The relevant fractions of immune cells were evaluated by CIBERSORT and single-sample gene set enrichment analysis (ssGSEA). Results: Five genes, including CHGA, PTGER1, SHC3, PLXNC1, and TRIM55 were enrolled into the risk model. Kaplan–Meier survival analysis and receiver operating characteristic (ROC) curve showed that our model performed well in predicting the outcomes of NBL (3-years AUC = 0.720, 5-year AUC = 0.775, 10-years AUC = 0.782), which has been validated in the GSE49711 dataset and the E-MTAB-8248 dataset. By comparing with the tumor immune dysfunction and exclusion (TIDE) and tumor inflammation signature (TIS), we further proved that our model is reliable. Univariate and multivariate Cox regression analyses indicated that the risk score, age, and MYCN can serve as independent prognostic factors in the E-MATB-8248. Functional enrichment analysis showed the DEIRGs were enriched in leukocyte adhesion-related signaling pathways. Gene set enrichment analysis (GSEA) revealed the significantly enriched pathways of the five MYCN-related DEIRGs. The risk score was negatively correlated with the immune checkpoint CD274 (PD-L1) but no significant difference with the TMB. We also confirmed the prognostic value of our model in predicting immunotherapeutics. Conclusion: We constructed and verified a signature based on DEIRG that related to MNA and predicted the survival of NBL based on relevant immune signatures. These findings could provide help for predicting prognosis and developing immunotherapy in NBL. [ABSTRACT FROM AUTHOR]

This case report details a rare presentation of neuroblastoma in an adult patient. The patient, a 22-year-old female, presented with a right-sided abdominal mass and discomfort for the last 12 months. Imaging studies revealed a huge adrenal mass occupying the right quadrant of the abdomen with displacement of the surrounding structures. Intraoperatively, the mass was adherent to the liver and very close to the inferior vena cava. The mass was resected completely without any intraoperative complications. The histopathological examination of the mass revealed a poorly differentiated neuroblastoma. This report highlights a rare presentation of neuroblastoma in the adult population., Competing Interests: Human subjects: Consent for treatment and open access publication was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Al Hashimi et al.)

Background: MYC/MYCN are the most frequent oncogene amplifications in medulloblastoma (MB) and its primary biomarkers of high-risk (HR) disease. However, while many patients' MYC(N)-amplified tumors are treatment-refractory, some achieve long-term survival. We therefore investigated clinicobiological heterogeneity within MYC(N)-amplified MB and determined its relevance for improved disease management., Methods: We characterized the clinical and molecular correlates of MYC- (MYC-MB; n = 64) and MYCN-amplified MBs (MYCN-MB; n = 95), drawn from >1600 diagnostic cases., Results: Most MYC-MBs were molecular group 3 (46/58; 79% assessable) and aged ≥3 years at diagnosis (44/64 [69%]). We identified a "canonical" very high-risk (VHR) MYC-amplified group (n = 51/62; 82%) with dismal survival irrespective of treatment (11% 5-year progression-free survival [PFS]), defined by co-occurrence with ≥1 additional established risk factor(s) (subtotal surgical-resection [STR], metastatic disease, LCA pathology), and commonly group 3/4 subgroup 2 with a high proportion of amplified cells. The majority of remaining noncanonical MYC-MBs survived (i.e. non-group 3/group 3 without other risk features; 11/62 (18%); 61% 5-year PFS). MYCN survival was primarily related to molecular group; MYCN-amplified SHH MB, and group 3/4 MB with additional risk factors, respectively defined VHR and HR groups (VHR, 39% [35/89]; 20% 5-year PFS/HR, 33% [29/89]; 46% 5-year PFS). Twenty-two out of 35 assessable MYCN-amplified SHH tumors harbored TP53 mutations; 9/12 (75%) with data were germline. MYCN-amplified group 3/4 MB with no other risk factors (28%; 25/89) had 70% 5-year PFS., Conclusions: MYC(N)-amplified MB displays significant clinicobiological heterogeneity. Diagnostics incorporating molecular groups, subgroups, and clinical factors enable their risk assessment. VHR "canonical" MYC tumors are essentially incurable and SHH-MYCN-amplified MBs fare extremely poorly (20% survival at 5 years); both require urgent development of alternative treatment strategies. Conventional risk-adapted therapies are appropriate for more responsive groups, such as noncanonical MYC and non-SHH-MYCN MB., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Neuroblastoma is the most common extracranial solid tumor found in children and despite intense multi-modal therapeutic approaches, low overall survival rates of high-risk patients persist. Tumors with heterozygous loss of chromosome 11q and MYCN amplification are two genetically distinct subsets of neuroblastoma that are associated with poor patient outcome. Using an isogenic 11q deleted model system and high-throughput drug screening, we identify checkpoint kinase 1 (CHK1) as a potential therapeutic target for 11q deleted neuroblastoma. Further investigation reveals MYCN amplification as a possible additional biomarker for CHK1 inhibition, independent of 11q loss. Overall, our study highlights the potential power of studying chromosomal aberrations to guide preclinical development of novel drug targets and combinations. Additionally, our study builds on the growing evidence that DNA damage repair and replication stress response pathways offer therapeutic vulnerabilities for the treatment of neuroblastoma. [ABSTRACT FROM AUTHOR]

Neuroblastoma is the most common extracranial solid tumor found in children and despite intense multi-modal therapeutic approaches, low overall survival rates of high-risk patients persist. Tumors with heterozygous loss of chromosome 11q and MYCN amplification are two genetically distinct subsets of neuroblastoma that are associated with poor patient outcome. Using an isogenic 11q deleted model system and high-throughput drug screening, we identify checkpoint kinase 1 (CHK1) as a potential therapeutic target for 11q deleted neuroblastoma. Further investigation reveals MYCN amplification as a possible additional biomarker for CHK1 inhibition, independent of 11q loss. Overall, our study highlights the potential power of studying chromosomal aberrations to guide preclinical development of novel drug targets and combinations. Additionally, our study builds on the growing evidence that DNA damage repair and replication stress response pathways offer therapeutic vulnerabilities for the treatment of neuroblastoma.

Abstract Background MYCN amplification and age are two critical prognostic factors of pediatric neuroblastoma. Previously, we had revealed the prognosis of MYCN target genes. However, the prognostic effects of age related genes in neuroblastoma are unclear. Methods The prognostic significance of age and MYCN amplification was determined through multivariate cox regression and Kaplan-Meier survival analysis. Genes differentially expressed in MYCN non-amplified younger neuroblastoma patients were identified using Therapeutically Applicable Research to Generate Effective Treatments (TARGET) and Gene Expression Omnibus (GEO) datasets. The prognostic effects of age related genes ALCAM, CACNA2D3, DST, EPB41L4A and KIF1B in pediatric neuroblastoma patients were determined by Kaplan-Meier survival. Results In a pediatric pan-cancer analysis, age was associated with the overall survival of pediatric B-lineage acute lymphoblastic leukemia, neuroblastoma and wilms tumor in TARGET dataset. Moreover, the prognostic effects of age in neuroblastoma were validated using two independent neuroblastoma cohorts. Furthermore, age and MYCN amplification were independent prognostic factors in pediatric neuroblastoma. Compared with MYCN non-amplified older neuroblastoma patients, MYCN non-amplified younger neuroblastoma patients had better clinical outcomes. ALCAM, CACNA2D3, DST, EPB41L4A and KIF1B were highly expressed in MYCN non-amplified younger neuroblastoma patients. And the higher expression levels of ALCAM, CACNA2D3, DST, EPB41L4A or KIF1B were associated with better prognosis of MYCN non-amplified neuroblastoma patients. DST was an independent prognostic factor in MYCN non-amplified neuroblastoma patients and MYCN non-amplified neuroblastoma younger patients with higher DST expression levels had the best clinical overall survival. Conclusions Age related gene DST was an independent prognostic factor in MYCN non-amplified neuroblastoma. MYCN non-amplified younger neuroblastoma patients with higher DST expression levels had the best clinical overall survival.

• Patterns of locoregional relapse (LRR) for high-risk neuroblastoma after radiotherapy are not well-described. • Approximately half of LRRs in high-risk neuroblastoma include a component of marginal failure. • No patients receiving total body irradiation had a marginal-only failure. • On multivariable analyses, MYCN amplification had higher risk of LRR and post-consolidation isotretinoin and anti-GD2 antibody therapy had lower risk of LRR. 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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

Background: Previously, we had analyzed the prognosis of E2F transcription factors across adult tumor types. However, the expressions and prognosis of E2F transcription factors in pediatric neuroblastoma have not yet been fully studied.Methods: The prognosis of E2F transcription factors was determined in four independent pediatric neuroblastoma cohorts from Therapeutically Applicable Research to Generate Effective Treatments (TARGET), Gene Expression Omnibus (GEO) and European ArrayExpres datasets using Kaplan-Meier and cox regression analysis.Results: E2F regulated gene set was associated with the event free survival and the overall survival of neuroblastoma. E2F1 and E2F3 were prognostic factors in all four independent pediatric neuroblastoma cohorts. Over-expressions of E2F1 or E2F3 were correlated with the shorted event free survival and overall survival of neuroblastoma. Expression levels of E2F1 and E2F3 were higher in neuroblastoma patients with MYCN amplification or age at diagnosis ≥ 18 months. Moreover, the prognostic significance of E2F1 or E2F3 in neuroblastoma was independent of MYCN amplification and age of diagnosis. Combinations of E2F1, E2F3 with MYCN amplification or age of diagnosis achieved better prognosis of neuroblastoma. Identification of 234 genes were associated with E2F1 and E2F3 expressions in neuroblastoma and those genes were significantly enriched in cell cycle signaling pathway. Also, higher scores of cell cycle signaling pathway were correlated with the adverse prognosis of neuroblastoma.Conclusions: E2F transcription factors E2F1 and E2F3 were prognostic makers of neuroblastoma. [ABSTRACT FROM AUTHOR]

Simple Summary: The invasive nature of surgical biopsies prevents their sequential application to monitor disease. Single biopsies fail to reflect cancer dynamics, intratumor heterogeneity, and drug sensitivities that change over time. Detection and characterization of cell-free circulating tumor DNA in biofluids from patients with solid tumors may better support disease monitoring and provide advanced molecular information for clinical decision-making toward personalized medicine. Here, we investigated the cell-free DNA characteristics in blood, bone marrow, cerebrospinal fluid, and urine provided from 84 infants and children with low-, intermediate-, or high-risk neuroblastoma. We report characteristic size distribution and concentration patterns for each biofluid to provide information to support the development of successful liquid biopsy biobanking strategies. We investigate potential correlations between disease activity and cfDNA concentration and provide strong evidence that markers specific for neuroblastoma can be detected in very small blood volumes from infants. 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. [ABSTRACT FROM AUTHOR]

Neuroblastoma represents the most common and lethal solid tumour of early childhood. In view of variations in genetic elements, MYCN amplification is certainly the most prominent genetic factor occurring in 1/4 or 1/5 of children with neuroblastoma; however, overall, the pathogenesis of neuroblastoma remains to be resolved. Rare, sporadic infections with Epstein‑Barr, hepatis C virus and varicella‑zoster virus have been detected in children with neuroblastoma, while the presence of BK virus was initially claimed, but later falsified as a triggering factor for the development of high‑risk neuroblastoma. The proposed model by Professor Ugo Rovigatti, Professor of Molecular Biology at the University of Florence in Italy, is based upon infection with micro‑foci inducing virus and its potential tumorigenic role as trigger of i) high and persistent inflammation; ii) chromothripsis and genetic instability; and iii) in vitro cell transformation and in vivo carcinogenesis. Modelling is still under‑utilized and under-developed in clinical virology and cancer research; however, it is expected to play a significant role in the future, aiming to elucidate the cancer enigma. This article is based on a webinar on neuroblastoma in children, which was organised virtually on December 12, 2020, by the Institute of Paediatric Virology. [ABSTRACT FROM AUTHOR]

Background: This study aimed to better understand the prognostic effect of multiple genetic markers and identify more subpopulations at ultra high risk of poor outcome in bone marrow (BM) metastatic neuroblastoma (NB). Methods: We screened the MYCN, 1p36 and 11q23 loss of heterozygosity (LOH) statuses of 154 patients by interphase fluorescence in situ hybridization of BM cells. The clinical characteristics of patients with the three markers and their associations with prognosis were analysed. Results: MYCN amplification and LOH at 1p36 and 11q23 were identified in 16.2%, 33.1% and 30.5% of patients, respectively. There were strong associations between MYCN amplification and 1p36 LOH as well as 11q23 LOH. Both MYCN amplification and 1p36 LOH were strongly associated with high levels of lactate dehydrogenase (LDH) and neuron‐specific enolase, more than 3 metastatic organs, and more events. 11q23 LOH occurred mainly in patients older than 18 months, and those who had high LDH levels. In univariate analysis, patients with MYCN amplification had poorer prognosis than those without. Patients with 1p36 LOH had a 3‐year event‐free survival (EFS) and overall survival lower than those without. 11q23 LOH was associated with poorer EFS only for patients without MYCN amplification. In a multivariate model, MYCN amplification was independently associated with decreased EFS in all cohorts. 11q23 LOH was an independent prognostic factor for patients without MYCN amplification, whereas 1p36 LOH was not an independent marker regardless of MYCN amplification. Compared with all cohorts, patients with both MYCN amplification and 1p36 LOH had the worst outcome and clinical features. Conclusions: Patients with both MYCN amplification and 1p36LOH had the worst survival rate, indicating an ultra high‐risk group. Our results may be applied in clinical practice for accurate risk stratification in future studies. [ABSTRACT FROM AUTHOR]

Simple Summary: Photothermal therapy (PTT) is an effective thermal therapy for treating tumors. PTT has been combined with immunotherapy in various preclinical cancer models showing promising treatment outcomes. However, in these studies, PTT has primarily been utilized for maximizing tumor cell death. Previously, we observed that based on the "thermal dose" applied, PTT can generate dramatically different responses from the immune system when tested in cellular and animal models. Here, we sought to provide a framework to systematically assess the effect of PTT-based thermal doses on the immunogenic correlates of treated tumors as a measure of the effectiveness of PTT in eliciting an antitumor immune response. In human neuroblastoma tumor cells in vitro, we determined specific phenotypic markers, which demonstrated that SH-SY5Y cells were more responsive to PTT-based thermal dose compared with LAN-1 cells, which possess a high-risk phenotype. Our findings suggest the importance of conducting tumor thermal dose-responsiveness studies in vitro as an early measure of PTT effectiveness against a specific tumor. Photothermal therapy (PTT) is an effective method for tumor eradication and has been successfully combined with immunotherapy. However, besides its cytotoxic effects, little is known about the effect of the PTT thermal dose on the immunogenicity of treated tumor cells. Therefore, we administered a range of thermal doses using Prussian blue nanoparticle-based photothermal therapy (PBNP-PTT) and assessed their effects on tumor cell death and concomitant immunogenicity correlates in two human neuroblastoma cell lines: SH-SY5Y (MYCN-non-amplified) and LAN-1 (MYCN-amplified). PBNP-PTT generated thermal dose-dependent tumor cell killing and immunogenic cell death (ICD) in both tumor lines in vitro. However, the effect of the thermal dose on ICD and the expression of costimulatory molecules, immune checkpoint molecules, major histocompatibility complexes, an NK cell-activating ligand, and a neuroblastoma-associated antigen were significantly more pronounced in SH-SY5Y cells compared with LAN-1 cells, consistent with the high-risk phenotype of LAN-1 cells. In functional co-culture studies in vitro, T cells exhibited significantly higher cytotoxicity toward SH-SY5Y cells relative to LAN-1 cells at equivalent thermal doses. This preliminary report suggests the importance of moving past the traditional focus of using PTT solely for tumor eradication to one that considers the immunogenic effects of PTT thermal dose to facilitate its success in cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Purpose: The proto-oncogene MYCN is considered a transcription factor involved in the regulation of neuroblastoma (NB) cell biology. Since minimally invasive-surgery represents a debated treatment of NB, we investigated CO2 effects on proliferative activity and apoptotic pathway in two NB cell lines, SH-SY5Y (MYCN-non-amplified) and IMR-32 (MYCN-amplified). Methods: SH-SY5Y and IMR-32 were exposed to CO2 (100%) at a pressure of 15 mmHg for 4 h and then moved to normal condition for 24 h. Cell proliferation, caspase 3 activity and transcript levels of BAX, BCL-2, cyclin B, cyclin D and MMP-2 were evaluated. Results: CO2 exposure caused a decrease in cell proliferation associated to increases in BAX/BCL-2 ratio and caspase 3 activity in SH-SY5Y, while opposite effects have been found in IMR-32. CO2 exposure induced a decrease of cyclin B1 in SH-SY5Y, while an increase in cyclin B1 and D1 was observed in IMR-32. A slight up-regulation of MMP-2 expression in SH-SY5Y and a significant increase of 2.2 folds in IMR-32 was observed (p < 0.05). Conclusions: Our results suggest that CO2 exposure may cause different effects on various NB cell lines, likely due to MYCN amplification status. Further in vitro and in vivo studies are needed to highlight the role of laparoscopy on NB behaviour. [ABSTRACT FROM AUTHOR]

Purpose: Medulloblastoma is the most frequent pediatric malignant brain tumor, and is divided into four main subgroups: WNT, SHH, group 3, and group 4. MYCN amplification is an important medulloblastoma prognostic biomarker. We aimed to molecular classify and predict MYCN amplification in a single assay. Methods: It was included 209 medulloblastomas from 205 patients (Brazil, Argentina, and Portugal), divided into training (n = 50) and validation (n = 159) sets. A nCounter assay was carried out using a custom panel for molecular classification, with additional genes, including MYCN. nSolver 4.0 software and the R environment were used for profiling and MYCN mRNA analysis. MYCN amplification by FISH was performed in 64 cases. Results: The 205 medulloblastomas were classified in SHH (44.9%), WNT (15.6%), group 3 (18.1%) and group 4 (21.4%). In the training set, MYCN amplification was detected in three SHH medulloblastomas by FISH, which showed significantly higher MYCN mRNA counts than non-FISH amplified cases, and a cutoff for MYCN amplification was established ( X ¯ + 4σ = 11,124.3). Applying this threshold value in the validation set, we identified MYCN mRNA counts above the cutoff in three cases, which were FISH validated. Conclusion: We successfully stratified medulloblastoma molecular subgroups and predicted MYCN amplification using a single nCounter assay without the requirement of additional biological tissue, costs, or bench time. [ABSTRACT FROM AUTHOR]

Purpose: Cyclin-dependent kinases (CDKs) that have critical roles in RNA polymerase II (Pol II)-mediated gene transcription are emerging as therapeutic targets in cancer. We have previously shown that THZ1, a covalent inhibitor of CDKs 7/12/13, leads to cytotoxicity in MYCN -amplified neuroblastoma through the downregulation of super-enhancer-associated transcriptional upregulation. Here we determined the effects of YKL-5-124, a novel covalent inhibitor with greater selectivity for CDK7 in neuroblastoma cells. Experimental Design: We tested YKL-5-124 in MYCN -amplified and nonamplified neuroblastoma cells individually and in combination with other inhibitors in cell line and animal models. Cell viability, target validation, effects on cell cycle and transcription were analyzed. Results: CDK7 inhibition with YKL-5-124 did not lead to significant cell death, but resulted in aberrant cell cycle progression especially in MYCN -amplified cells. Unlike THZ1, YKL-5-124 had minimal effects on Pol II C-terminal domain phosphorylation, but significantly inhibited that of the CDK1 and CDK2 cell cycle kinases. Combining YKL-5-124 with the BRD4 inhibitor JQ1 resulted in synergistic cytotoxicity. A distinct MYCN -gene expression signature associated with resistance to BRD4 inhibition was suppressed with the combination. The synergy between YKL-5-124 and JQ1 translated into significant tumor regression in cell line and patient-derived xenograft mouse models of neuroblastoma. Conclusions: The combination of CDK7 and BRD4 inhibition provides a therapeutic option for neuroblastoma and suggests that the addition of YKL-5-124 could improve the therapeutic efficacy of JQ1 and delay resistance to BRD4 inhibition. [ABSTRACT FROM AUTHOR]

Detection of amplification of the MYCN gene is essential for determining optimal treatment and estimating prognosis of patients with neuroblastoma (NB). DNA FISH with neuroblastoma tissues or patient‐derived bone marrow cells is the standard clinical practice for the detection of MYCN amplification. As tumor cells may often be unavailable, we developed a method to detect MYCN amplification in the plasma of patients with neuroblastoma. Taking single‐copy NAGK DNA as reference, we used real‐time quantitative PCR (qPCR) to determine the MYCN/NAGK ratio in the plasma of 115 patients diagnosed with NB. An increased MYCN/NAGK ratio in the plasma was consistent with MYCN amplification as assessed by DNA FISH. The AUC for a MYCN/NAGK ratio equal to 6.965 was 0.943, with 86% sensitivity and 100% specificity. Beyond the threshold of 6.965, the MYCN/NAGK ratio correlated with a heavier tumor burden. Event‐free and overall survival of two years were significantly shortened in stage 4 patients with a MYCN/NAGK ratio higher than 6.965. Plasma MYCN/NAGK ratios increased in patients with progressive disease and relapse. Thus, we conclude that the determination of the plasma MYCN/NAGK ratio by qPCR is a noninvasive and reproducible method to measure MYCN amplification in patients with NB.

Abstract We report a novel group of clinically aggressive spinal cord ependymomas characterized by Grade III histology, MYCN amplification, an absence of NF2 alterations or other recurrent pathogenic mutations, and a unique methylation classifier profile. Seven cases were found to have MYCN amplification in the course of routine mutational profiling of 552 patients with central nervous system tumors between December 2016 and July of 2019 and an eighth patient was identified from an unrelated set of cases. Methylation array analysis revealed that none of the 8 cases clustered with any of the nine previously described ependymoma methylation subgroups, and 7 of 8 formed their own tight unique cluster. Histologically all cases showed grade III features, and all demonstrated aggressive clinical behavior. These findings are presented in the context of data from three other studies describing similar cases. Therefore, a combined total of 27 MYCN amplified spinal cord ependymoma cases have now been reported in the literature, warranting their consideration as a distinctive subtype of spinal cord ependymoma (SP-EPN-MYCN) with their unique molecular characteristics and aggressive clinical behavior.

Abstract Introduction Neuroblastoma (NB) is the most common extracranial solid tumor among children. The 5‐year event‐free survival rate for high‐risk (HR) NB is still poor, especially for patients with advanced NB with MYCN gene amplification. Chimeric antigen receptor T (CAR‐T) cell therapy is a new treatment for HR‐NB. Case presentation A 55‐month‐old boy with stage IV HR‐NB received 4th‐generation CAR‐T cells that target disialoganglioside GD2, as consolidation maintenance treatment after intensive chemotherapy, surgery, and autologous stem‐cell transplantation. As of February 2019, his CAR‐T follow‐up time was 37.5 months, indicating prolonged survival. Cranial MRI and ultrasound showed no mass; 123I‐metaiodobenzylguanidine (123I‐MIBG) scan was negative. Conclusion GD2‐CAR‐T cells may be an effective treatment option for NB patients with MYCN amplification.

PurposeThis study aimed to assess the predictive ability of 18F-FDG PET/CT radiomic features for MYCN, 1p and 11q abnormalities in NB.MethodOne hundred and twenty-two pediatric patients (median age 3. 2 years, range, 0.2–9.8 years) with NB were retrospectively enrolled. Significant features by multivariable logistic regression were retained to establish a clinical model (C_model), which included clinical characteristics. 18F-FDG PET/CT radiomic features were extracted by Computational Environment for Radiological Research. The least absolute shrinkage and selection operator (LASSO) regression was used to select radiomic features and build models (R-model). The predictive performance of models constructed by clinical characteristic (C_model), radiomic signature (R_model), and their combinations (CR_model) were compared using receiver operating curves (ROCs). Nomograms based on the radiomic score (rad-score) and clinical parameters were developed.ResultsThe patients were classified into a training set (n = 86) and a test set (n = 36). Accordingly, 6, 8, and 7 radiomic features were selected to establish R_models for predicting MYCN, 1p and 11q status. The R_models showed a strong power for identifying these aberrations, with area under ROC curves (AUCs) of 0.96, 0.89, and 0.89 in the training set and 0.92, 0.85, and 0.84 in the test set. When combining clinical characteristics and radiomic signature, the AUCs increased to 0.98, 0.91, and 0.93 in the training set and 0.96, 0.88, and 0.89 in the test set. The CR_models had the greatest performance for MYCN, 1p and 11q predictions (P < 0.05).ConclusionsThe pre-therapy 18F-FDG PET/CT radiomics is able to predict MYCN amplification and 1p and 11 aberrations in pediatric NB, thus aiding tumor stage, risk stratification and disease management in the clinical practice.

PurposeCyclin-dependent kinases (CDKs) that have critical roles in RNA polymerase II (Pol II)-mediated gene transcription are emerging as therapeutic targets in cancer. We have previously shown that THZ1, a covalent inhibitor of CDKs 7/12/13, leads to cytotoxicity in MYCN-amplified neuroblastoma through the downregulation of super-enhancer-associated transcriptional upregulation. Here we determined the effects of YKL-5-124, a novel covalent inhibitor with greater selectivity for CDK7 in neuroblastoma cells.Experimental DesignWe tested YKL-5-124 in MYCN-amplified and nonamplified neuroblastoma cells individually and in combination with other inhibitors in cell line and animal models. Cell viability, target validation, effects on cell cycle and transcription were analyzed.ResultsCDK7 inhibition with YKL-5-124 did not lead to significant cell death, but resulted in aberrant cell cycle progression especially in MYCN-amplified cells. Unlike THZ1, YKL-5-124 had minimal effects on Pol II C-terminal domain phosphorylation, but significantly inhibited that of the CDK1 and CDK2 cell cycle kinases. Combining YKL-5-124 with the BRD4 inhibitor JQ1 resulted in synergistic cytotoxicity. A distinct MYCN-gene expression signature associated with resistance to BRD4 inhibition was suppressed with the combination. The synergy between YKL-5-124 and JQ1 translated into significant tumor regression in cell line and patient-derived xenograft mouse models of neuroblastoma.ConclusionsThe combination of CDK7 and BRD4 inhibition provides a therapeutic option for neuroblastoma and suggests that the addition of YKL-5-124 could improve the therapeutic efficacy of JQ1 and delay resistance to BRD4 inhibition.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Winnicka, Skowera, Stelmach, Styka and Lejman.)

Background: Neuroblastoma is the most common extracranial solid tumor in childhood. Amplification of MYCN in neuroblastoma is a predictor of poor prognosis. Materials and methods: DNA methylation data from the TARGET data matrix were stratified into MYCN amplified and non-amplified groups. Differential methylation analysis, clustering, recursive feature elimination (RFE), machine learning (ML), Cox regression analysis and Kaplan-Meier estimates were performed. Results and Conclusion: 663 CpGs were differentially methylated between the two groups. A total of 25 CpGs were selected by RFE for clustering and ML, and a 100% clustering accuracy was obtained. ML validation on three external datasets produced high accuracy scores of 100%, 97% and 93%. Eight survival-associated CpGs were also identified. Therapeutic interventions may need to be targeted to patient subgroups. [ABSTRACT FROM AUTHOR]

Background: Neuroblastoma (NB), the most common solid tumor in children, exhibits vastly different genomic abnormalities and clinical behaviors. While significant progress has been made on the research of relations between clinical manifestations and genetic abnormalities, it remains a major challenge to predict the prognosis of patients to facilitate personalized treatments.Materials and Methods: Six data sets of gene expression and related clinical data were downloaded from the Gene Expression Omnibus (GEO) database, ArrayExpress database, and Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database. According to the presence or absence of MYCN amplification, patients were divided into two groups. Differentially expressed genes (DEGs) were identified between the two groups. Enrichment analyses of these DEGs were performed to dig further into the molecular mechanism of NB. Stepwise Cox regression analyses were used to establish a five-gene prognostic signature whose predictive performance was further evaluated by external validation. Multivariate Cox regression analyses were used to explore independent prognostic factors for NB. The relevance of immunity was evaluated by using algorithms, and a nomogram was constructed.Results: A five-gene signature comprising CPLX3, GDPD5, SPAG6, NXPH1, and AHI1 was established. The five-gene signature had good performance in predicting survival and was demonstrated to be superior to International Neuroblastoma Staging System (INSS) staging and the MYCN amplification status. Finally, a nomogram based on the five-gene signature was established, and its clinical efficacy was demonstrated.Conclusion: Collectively, our study developed a novel five-gene signature and successfully built a prognostic nomogram that accurately predicted survival in NB. The findings presented here could help to stratify patients into subgroups and determine the optimal individualized therapy.

A combination therapy using Prussian blue nanoparticles (PBNP) as photothermal therapy (PTT) agents coated with CpG oligodeoxynucleotides, an immunologic adjuvant, as a nanoimmunotherapy (CpG‐PBNP‐PTT) for neuroblastoma (NB) is described. NB driven by MYCN amplification confers high risk and correlates with a dismal prognosis, accounting for the majority of NB‐related mortality. The efficacy of the CpG‐PBNP‐PTT nanoimmunotherapy in a clinically relevant, TH‐MYCN murine NB model (9464D) overexpressing MYCN is tested. When administered to 9464D NB cells in vitro, CpG‐PBNP‐PTT triggers thermal dose‐dependent immunogenic cell death and tumor cell priming for immune recognition in vitro, measured by the expression of specific costimulatory and antigen‐presenting molecules. In vivo, intratumorally administered CpG‐PBNP‐PTT generates complete tumor regression and significantly higher long‐term survival compared to controls. Furthermore, CpG‐PBNP‐PTT‐treated mice reject tumor rechallenge. Ex vivo studies confirm these therapeutic responses result from the generation of robust T cell‐mediated immunological memory. Consequently, in a synchronous 9464D tumor model, CpG‐PBNP‐PTT induces complete tumor regression on the treated flank and significantly slows tumor progression on the untreated flank, improving animal survival. These findings demonstrate that localized administration of the CpG‐PBNP‐PTT nanoimmunotherapy drives potent systemic T cell responses in solid tumors such as NB and therefore has therapeutic implications for NB.

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. [Display omitted] • MYCN-amplified neuroblastomas have preferentially increased expression of eIF4A1 • Amidino-rocaglates augment eIF4A1 binding across the full length of cellular mRNAs • MYCN mRNA is directly inhibited by amidino-rocaglates • Polypurine ranking of mRNA 5′ UTRs predicts amidino-rocaglate sensitivity in neuroblastoma MYCN amplification is a key driver of high-risk neuroblastoma. Volegova et al. capitalize on the dependence of MYCN-amplified neuroblastoma cells on increased protein synthesis by using amidino-rocaglates to inhibit translation initiation factor eIF4A1, resulting in direct targeting of the MYCN mRNA and selective cytotoxicity in animal models. [ABSTRACT FROM AUTHOR]

Abstract Background Interphase fluorescence in situ hybridization (FISH) of bone marrow cells has been confirmed to be a direct and valid method to assess the v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) amplification in patients with bone marrow metastatic neuroblastoma. MYCN amplification alone, however, is insufficient for pretreatment risk stratification. Chromosome band 11q23 deletion has recently been included in the risk stratification of neuroblastoma. In the present study, we aimed to evaluate the biological characteristics and prognostic impact of 11q23 deletion and MYCN amplification in patients with bone marrow metastatic neuroblastoma. Methods We analyzed the MYCN and 11q23 statuses of 101 patients with bone marrow metastatic neuroblastoma using interphase FISH of bone marrow cells. We specifically compared the biological characteristics and prognostic impact of both aberrations. Results MYCN amplification and 11q23 deletion were seen in 12 (11.9%) and 40 (39.6%) patients. The two markers were mutually exclusive. MYCN amplification occurred mainly in patients with high lactate dehydrogenase (LDH) and high neuron-specific enolase (NSE) levels (both P