Peripheral neuroblastic tumors consist of a variety of neoplasms ranging from benign to highly malignant, with significant intertumoral and intratumoral heterogeneity. The current gold standard in classification, the International Neuroblastoma Pathology Committee classification, relies on morphologic features and patient age to assign favorable or unfavorable histology for each patient. The International Neuroblastoma Risk Group uses a subset of this pathologic data, along with patient age and several key molecular features, to assign one of several prognostic categories.

Inactivation of α-thalassemia/mental retardation X-linked (ATRX) represents a defining molecular feature in large subsets of malignant glioma. ATRX deficiency gives rise to abnormal G-quadruplex (G4) DNA secondary structures, enhancing replication stress and genomic instability. Building on earlier work, we evaluated the extent to which pharmacological G4 stabilization selectively enhances DNA damage and cell death in ATRX-deficient preclinical glioma models.

Using the G4 stabilizer CX-5461, we treated patient-derived glioma stem cells (GSCs) in vitro and GSC flank and intracranial murine xenografts in vivo to evaluate efficacy as both a single agent and in combination with ionizing radiation (IR), the latter a central element of current treatment standards.

CX-5461 promoted dose-sensitive lethality in ATRX-deficient GSCs relative to ATRX-intact controls. Mechanistic studies revealed that CX-5461 disrupted histone variant H3.3 deposition, enhanced replication stress and DNA damage, activated p53-independent apoptosis, and induced G2/M arrest to a greater extent in ATRX-deficient GSCs than in ATRX-intact counterparts. These data were corroborated in vivo, where CX-5461/IR treatment profoundly delayed tumor growth and prolonged survival in mice bearing ATRX-deficient flank xenografts. Histopathological analyses revealed decreased proliferation, increased apoptosis, and significant G4 induction, replication stress, and DNA damage in CX-5461-treated tumors, both alone and in combination with IR. Finally, despite suboptimal blood-brain-barrier penetration, systemic CX-5461 treatment induced tangible pharmacodynamic effects in ATRX-deficient intracranial GSC models.

In totality, our work substantively demonstrates efficacy and defines mechanisms of action for G4 stabilization as a novel therapeutic strategy targeting ATRX-deficient malignant glioma, laying the groundwork for clinical translation.

The clinical course of neuroblastoma is more heterogeneous than any other malignant disease. Many low-risk patients experience regression after limited or even no chemotherapy. However, more than half of high-risk patients die from disease despite intensive multimodal treatment. Precise disease characterization for each patient at diagnosis is key for risk-adapted treatment. The guidelines presented here incorporate results from national and international clinical trials to produce recommendations for diagnosing and treating neuroblastoma patients in German hospitals outside of clinical trials.

Neuroblastoma is one of the most common extracranial solid tumors in children and it frequently displays high heterogeneity throughout the course of the disease. It has previously been described those changes in the ATRX gene (Alpha Thalassemia/Mental Retardation, X-linked) are the most common recurring events in the indolent clinical subtype (∼30 %) of MYCN amplified neuroblastoma. There is no effective treatment for this type of neuroblastoma, which is associated with overall poor survival. On the other hand, few studies have detected an association between high-risk (stage IV) non-amplified MYCN neuroblastoma patients and mutant ATRX.

In this study, 37 tumor samples from Brazilian patients with stages I to IV MYCN non-amplified neuroblastoma, according to the International Neuroblastoma Staging System (INSS), were analyzed using the panel Oncomine™ Childhood Cancer Research Assay.

The authors found two older children (NB1 and NB2) with advanced MYCN non-amplified neuroblastoma carried each one of the two following novel nonsense ATRX variants (p.Gln1670* or p.Glu1984*). These variants created a stop codon in the helicase domain of the ATRX gene, leading to ATRX loss-of-function. These mutations were confirmed by Sanger sequencing and the protein loss-of-function was confirmed by immunohistochemistry. The finding of these heterozygous mutations in two patients with MYCN non-amplified neuroblastoma deserves further investigation. Thus, the authors analyzed each of these cases to better understand how these mutations may be related to disease severity and prognosis.

ATRX loss-of-function from p.Gln1670* or p.Glu1984* mutations turn MYCN non-amplified neuroblastoma more aggressive and similar to what is seen in MYCN amplified neuroblastoma. This information may help clinical decision-making and facilitate establishing an accurate prognosis for patients with MYCN non-amplified neuroblastoma.

Phaeochromocytoma, paraganglioma and neuroblastoma are catecholamine secreting neuroendocrine tumours. Biochemical screening for suspected cases of these tumours involves the measurement of catecholamines and their metabolites in either urine or plasma. The South African National Health Laboratory service (NHLS) measures urine fractionated metanephrines (UMF) and normetanephrines (UNF), urine vanillylmandelic acid (UVMA) and urine homovanillic acid (UHVA).

To analyse the demographic, biochemical and testing patterns of patients' UMF, UNF, UVMA and UHVA in the NHLS.

Data from January 2015 to December 2016 for all patients undergoing UMF, UNF, UVMA and UHVA testing was extracted from the NHLS central data warehouse. Neuroendocrine tumours were biochemically diagnosed when results were >2x multiples of the upper reference limits. Multiple testing was defined as ≥2 tests within a 14-day period. Ethnicity was determined through hot-deck imputation.

Biochemically abnormal test results were identified by UMF/UNF measurements in 98.2 % of cases. In 1.8 % of cases, the addition of UVMA resulted in a previously unidentified biochemical positive. Adult white and coloured populations have significantly less biochemically positive UMF results compared to the African population. Multiple testing resulted in discordant results for 12.8 % of UMF and 13.1 % of UNF testing.

UVMA testing for phaeochromocytoma and paraganglioma offers little benefit over testing with UMF alone. Requesting consecutive multiple samples is preferred, however, a single 24-h fractionated UMF/UNF is efficient and cost-effective for phaeochromocytoma and paraganglioma screening, with further testing recommended when clinically indicated. African individuals are more likely to have raised catecholamines and requires further investigation.

ATRX is one of the most frequently mutated genes in high-risk neuroblastoma. ATRX mutations are mutually exclusive with MYCN amplification and mark a recognizable patient subgroup, presenting in older children with chemotherapy-resistant, slowly progressive disease. The mechanisms underlying how ATRX mutations drive high-risk and difficult-to-treat neuroblastoma are still largely elusive. To unravel the role of ATRX in neuroblastoma, we generated isogenic neuroblastoma cell line models with ATRX loss-of-function and ATRX in-frame multi-exon deletions, representing different types of alterations found in patients. RNA-sequencing analysis consistently showed significant upregulation of inflammatory response pathways in the ATRX-altered cell lines. In vivo, ATRX alterations are consistently associated with macrophage infiltration across multiple xenograft models. Furthermore, ATRX alterations also result in upregulation of epithelial-to-mesenchymal transition pathways and a reduction in expression of adrenergic core-regulatory circuit genes. Consistent with this, bioinformatic analysis of previously published neuroblastoma patient data sets revealed that ATRX-altered neuroblastomas display an immunogenic phenotype and higher score of macrophages (with no distinction between M1 and M2 macrophage populations) and dendritic cells, but not lymphocytes. Histopathological assessment of diagnostic samples from patients with ATRX mutant disease confirmed these findings with significantly more macrophage infiltration compared to MYCN-amplified tumors. In conclusion, we show that gene expression and cell-state changes as a result of ATRX alterations associate with a characteristic immune cell infiltration in both in vivo models and patient samples. Together, this provides novel insight into mechanisms underlying the distinct clinical phenotype seen in this group of patients.

Neuroblastoma (NB) is the most common extracranial malignant solid tumor in children, accounting for >15 % of cancer-related deaths in children. We analyzed the epidemiological statistical indicators of neuroblastoma and other peripheral nervous system tumors patients from 1990 to 2021 in Global Burden of Disease (GBD) 2021 database, aiming to provide valuable insights for public health interventions and clinical practices.

Based on the GBD 2021 database, this study analyzed the incidence, mortality, prevalence, and Disability-Adjusted Life-Years (DALYs) of neuroblastoma and other peripheral nervous system tumors from 1990 to 2021, stratified by sociodemographic development index (SDI) and geographic regions. Cross-country inequalities analysis was conducted to quantify the SDI-related inequality of disease burden across countries. In addition, the average annual percentage change (AAPC) and Age-Period-Cohort (APC) model were used to evaluate the trend of disease burden, while the global burden of disease to 2035 was predicted by Bayesian Age-Period-Cohort (BAPC) model.

This study reported the disease burden of neuroblastoma and other peripheral nervous system tumors in GBD 2021 database for the first time. Globally, the incidence and mortality of neuroblastoma have increased year by year from 1990 to 2021, especially in regions with low SDI, such as South Asia and sub-Saharan Africa, where the burden of disease has increased significantly. Regions with high SDI, such as North America and Western Europe, have seen a reduction in disease burden due to higher levels of medical care and earlier diagnosis. The age distribution shows that children under 5 years of age are mainly affected, especially in low- and middle-income areas. In addition, the incidence is slightly higher in men than in women. The BAPC model predicts that the global incidence, mortality, and DALYs of neuroblastoma will continue to increase until 2035.

Significant regional and population variation in neuroblastoma and other peripheral nervous system tumors worldwide, with a particularly high disease burden in low SDI areas with limited medical resources. This trend highlights the urgent need for global public health interventions and resource allocation, particularly in low-income countries. Future research should focus on improving early diagnosis, risk stratification and target therapy in order to reduce the global burden of disease and improve patients' prognosis.

This study was supported by National Natural Science Foundation of China (No. 82293662, No 82172357 and No 81930066), Key project of Shanghai "Science and Technology Innovation Action Plan (22JC1402304) and Research fund of Shanghai Municipal Health Bureau (No. 2019cxjq03).

The heterogeneous prognosis in neuroblastoma, shaped by telomere maintenance mechanisms (TMMs), notably the alternative lengthening of telomeres (ALT) pathway, necessitates a refined risk classification for high-risk patients. Current systems often lack precision, hindering tailored treatment approaches. This individual participant data (IPD) meta-analysis of survival among ALT-positive patients aims to improve risk classification systems, enhancing therapeutic strategies and patient outcomes.

Following PRISMA-IPD guidelines, we conducted a comprehensive review of neuroblastoma patients retrieved from PubMed, Scopus, and Embase databases until March-2024. Patients were stratified into ALT-positive and TMM-negative subgroups. Overall and event-free survival probabilities were evaluated.

In our cohort of 293 patients (156 ALT-positive, 137 TMM-negative) obtained from eight different studies, ALT-positive individuals displayed lower survival rates than TMM-negative patients. Non-stage 4 ALT-positive patients had reduced overall and event-free survival probabilities compared to their TMM-negative counterparts, indicating potential misclassification. Stage 4 ALT-positive patients similarly showed poorer survival outcomes than non-stage 4 TMM-negative patients, underscoring the significance of ALT in patient prognosis.

Our study highlights poorer outcomes in ALT-positive neuroblastoma patients, emphasizing the need to integrate TMM status into international risk classification guidelines. Standardizing TMM assessment is key for refining treatment strategies, considering the unique biology of ALT-positive patients.

c-MYC is an important driver of high-risk neuroblastoma. A lack of c-MYC-driven genetically engineered mouse models (GEMM) has hampered the ability to better understand mechanisms of neuroblastoma oncogenesis and to develop effective therapies. In this study, we showed that conditional c-MYC induction via Cre recombinase driven by a tyrosine hydroxylase promoter led to a preponderance of PDX1+ somatostatinoma, a type of pancreatic neuroendocrine tumor. However, c-MYC activation via an improved Cre recombinase driven by a dopamine β-hydroxylase promoter resulted in neuroblastoma development. The c-MYC murine neuroblastoma tumors recapitulated the pathologic and genetic features of human neuroblastoma and responded to anti-GD2 immunotherapy and difluoromethylornithine, an FDA-approved inhibitor targeting the MYC transcriptional target ODC1. Thus, c-MYC overexpression results in different but related tumor types depending on the targeted cell. The GEMMs represent valuable tools for testing immunotherapies and targeted therapies for these diseases. Significance: The development of c-MYC-driven genetically engineered neuroblastoma and somatostatinoma mouse models provides useful tools for understanding the tumor cell origin and investigating treatment strategies.

Neuroblastoma, the most common extracranial solid tumor in children, is rare in adults. This study compares patient characteristics, disease patterns, and treatments among adults, adolescents, and children with neuroblastoma.

We queried the National Cancer Database (2004-2019) for neuroblastoma cases. Patient and tumor characteristics, treatments, and 5-year overall survival (5-OS) were compared between adults (≥ 18 years), adolescents (10-17 years), and children (0-9 years). Kaplan-Meier curves and Cox regression assessed survival differences.

Among 6350 neuroblastoma patients, 256 (4.0%) were adults, 222 (3.5%) were adolescents, and 5872 (92.5%) were children. Tumors were largest in adolescents (9.7 cm), followed by adults (8.0 cm) and children (6.7 cm) (p < 0.001). Adults were less likely to have tumors in the adrenal glands (34.0% vs. children: 54.7%, adolescents: 43.2%, p < 0.001) and had lower rates of metastasis (10.9% vs. 19.3% and 19.4%, p < 0.001). Compared to children, adults received less chemotherapy, immunotherapy, and bone marrow transplants (p < 0.001). 5-OS was worse in adults (65.8%), followed by adolescents (70.4%) and children (78.2%) (p < 0.001). After adjustment, adults (aHR: 2.27; 95% CI, 1.71-3.01) and adolescents (aHR: 2.02; 95% CI, 1.54-2.64) had higher hazards of death compared to children.

Adults and adolescents with neuroblastoma have distinct clinical features and lower survival than children, underscoring the need for tailored treatment approaches for older patients.

III.

High-risk neuroblastoma continues to show a very high mortality, with a 5-year survival rate of 50%. While MYCN amplification is the main genetic alteration associated with high-risk tumours, other molecular mechanisms, such as alterations in ATRX and TERT, remain poorly understood. ATRX and TERT biomarkers, which are associated with a more aggressive neuroblastoma pattern, should be considered for accurate prognostic stratification. We highlight the promising results of the clinical trial involving the combination of adavosertib and irinotecan, which encourages further clinical trials with adavosertib targeting NB with ATRX mutations. Preclinical results with BET inhibitors (OTX015 and AZD5153) and with 6-thio-2'-deoxyguanosine, targeting NB with TERT mutations, are promising. Both represent future therapeutic targets, emphasizing the need to prioritize research using these models.

Neuroblastoma, " a malignancy originating from neural crest cells, is most commonly diagnosed in children and adolescents. Polymorphisms within the long noncoding RNA (lncRNA) HOXA distal transcript antisense RNA (HOTTIP) are believed to have the capacity to alter an individual's susceptibility to various cancers. This study aimed to investigate the link between HOTTIP gene polymorphisms and neuroblastoma susceptibility. We identified the genotypes of two prevalent polymorphisms (rs3807598 and rs1859168) within the HOTTIP via the TaqMan assay in a cohort comprising 402 individuals diagnosed with neuroblastoma and 473 healthy controls. Logistic regression was used to evaluate the associations between the HOTTIP polymorphisms and the likelihood of neuroblastoma susceptibility. Additionally, the genotype-tissue expression (GTEx) database was used to investigate how these HOTTIP gene variations influence gene expression across different tissues. Our findings demonstrated a significant association between the rs1859168 C > A polymorphism and reduced neuroblastoma susceptibility (CA vs. CC: adjusted odds ratio (OR) = 0.55, 95% confidence interval (CI) = 0.40-0.74, P = 0.0001; CA/AA vs. CC: adjusted OR = 0.69, 95% CI = 0.53-0.91, P = 0.010). The additional stratified analysis revealed that the presence of rs1859168 CA/AA or two protective genotypes was associated with a lower susceptibility in specific subgroups, such as older children and girls. Expression quantitative trait locus (eQTL) analysis revealed that the rs1859168 CC genotype was related to high expression of the HOTTIP gene.

We found that HOTTIP gene polymorphisms were associated with a reduced likelihood of neuroblastoma in Chinese children. Further studies with large cohorts and diverse ethnicities are warranted to verify our results.

• Genetic variations can influence neuroblastoma susceptibility. HOTTIP gene polymorphisms may alter an individual's susceptibility to various cancers.

• HOTTIP gene polymorphisms were associated with a reduced risk of neuroblastoma in Chinese children.

Neuroblastoma (NB), a heterogenous pediatric tumor of the sympathetic nervous system, is the most common and deadly extracranial solid malignancy diagnosed in infants. Numerous efforts have been invested in understanding its origin and in development of novel curative targeted therapies. Here, we summarize the recent advances in the identification of the cell of origin and the genetic alterations occurring during development that contribute to NB. We discuss current treatment regimens, present and future directions for the identification of novel therapeutic metabolic targets, differentiation agents, as well as personalized combinatory therapies as potential approaches for improving the survival and quality of life of children with NB.

The impact of exercise on pediatric tumor biology is essentially unknown. We explored the effects of regular exercise on tumor proteome profile (as assessed with liquid chromatography with tandem mass spectrometry) in a mouse model of one of the most aggressive childhood malignancies, high-risk neuroblastoma (HR-NB). Tumor samples of 14 male mice (aged 6-8 wk) that were randomly allocated into an exercise (5-wk combined aerobic and resistance training) or nonexercise control group (6 and 8 mice/group, respectively) were analyzed. The Search Tool for the Retrieval of Interacting Genes/Proteins database was used to generate a protein-protein interaction (PPI) network and enrichment analyses. The Systems Biology Triangle (SBT) algorithm was applied for analyses at the functional category level. Tumors of exercised mice showed a higher and lower abundance of 101 and 150 proteins, respectively, than controls [false discovery rate (FDR) < 0.05]. These proteins were enriched in metabolic pathways, amino acid metabolism, regulation of hormone levels, and peroxisome proliferator-activated receptor signaling (FDR < 0.05). The SBT algorithm indicated that 184 and 126 categories showed a lower and higher abundance, respectively, in the tumors of exercised mice (FDR < 0.01). Categories with lower abundance were involved in energy production, whereas those with higher abundance were related to transcription/translation, apoptosis, and tumor suppression. Regular exercise altered the abundance of hundreds of intratumoral proteins and molecular pathways, particularly those involved in energy metabolism, apoptosis, and tumor suppression. These findings provide preliminary evidence of the molecular mechanisms underlying the potential effects of exercise in HR-NB.NEW & NOTEWORTHY We used liquid chromatography with tandem mass spectrometry to explore the impact of a 5-wk exercise intervention on the tumor proteome profile in a mouse model of one of the most aggressive childhood malignancies, high-risk neuroblastoma. Exercise altered the abundance of hundreds of proteins and pathways, particularly those involved in energy metabolism and tumor suppression. These molecular changes could mediate, at least partly, the potential antitumorigenic effects of exercise.

Cancer immunotherapies produce remarkable results in B cell malignancies; however, optimal cell surface targets for many solid cancers remain elusive. Here, we present an integrative proteomic, transcriptomic, and epigenomic analysis of tumor and normal tissues to identify biologically relevant cell surface immunotherapeutic targets for neuroblastoma, an often-fatal childhood cancer. Proteogenomic analyses reveal sixty high-confidence candidate immunotherapeutic targets, and we prioritize delta-like canonical notch ligand 1 (DLK1) for further study. High expression of DLK1 directly correlates with a super-enhancer. Immunofluorescence, flow cytometry, and immunohistochemistry show robust cell surface expression of DLK1. Short hairpin RNA mediated silencing of DLK1 in neuroblastoma cells results in increased cellular differentiation. ADCT-701, a DLK1-targeting antibody-drug conjugate (ADC), shows potent and specific cytotoxicity in DLK1-expressing neuroblastoma xenograft models. Since high DLK1 expression is found in several adult and pediatric cancers, our study demonstrates the utility of a proteogenomic approach and credentials DLK1 as an immunotherapeutic target.

Telomere maintenance in neuroblastoma is linked to poor outcome and caused by either telomerase reverse transcriptase (TERT) activation or through alternative lengthening of telomeres (ALT). In contrast to TERT activation, commonly caused by genomic rearrangements or MYCN amplification, ALT is less well understood. Alterations at the ATRX locus are key drivers of ALT but only present in ∼50% of ALT tumors. To identify potential new pathways to telomere maintenance, we investigate allele-specific gene dosage effects from whole genomes and transcriptomes in 115 primary neuroblastomas. We show that copy-number dosage deregulates telomere maintenance, genomic stability, and neuronal pathways and identify upregulation of variants of histone H3 and H2A as a potential alternative pathway to ALT. We investigate the interplay between TERT activation, overexpression and copy-number dosage and reveal loss of imprinting at the RTL1 gene associated with poor clinical outcome. These results highlight the importance of gene dosage in key oncogenic mechanisms in neuroblastoma.

The transcriptional regulator ATRX, a genetic factor, is associated with a range of disabilities, including intellectual, hematopoietic, skeletal, facial, and urogenital disabilities. ATRX mutations substantially contribute to the pathogenesis of ATRX syndrome and are frequently detected in gliomas and many other cancers. These mutations disrupt the organization, subcellular localization, and transcriptional activity of ATRX, leading to chromosomal instability and affecting interactions with key regulatory proteins such as DAXX, EZH2, and TERRA. ATRX also functions as a transcriptional regulator involved in the pathogenesis of neuronal disorders and various diseases. In conclusion, ATRX is a central protein whose abnormalities lead to multiple diseases.

Neuroblastoma (NB), a prevalent extracranial solid tumor in children, stems from the neural crest. Omics technologies are extensively employed in NB, and We analyzed published articles on NB omics to understand the research trends and hot topics in NB omics.

We collected all articles related to NB omics published from 2005 to 2023 from the Web of Science Core Collection database. Subsequently, we conducted analyses using VOSviewer, CiteSpace, Bibliometrix, and the Bibliometric online analysis platform (https://bibliometric.com/ ).

We included a total of 514 articles in our analysis. The increasing number of publications in this field since 2020 indicates growing attention to NB omics, gradually entering a mature development stage. These articles span 50 countries and 1,000 institutions, involving 3,669 authors and 292 journals. The United States has the highest publication output and collaboration with other countries, with Germany being the most frequent collaborator. Capital Medical University and the German Cancer Research Center are the institutions with the highest publication count. The Journal of Proteome Research and the Journal of Biological Chemistry are the most prolific journal and most co-cited journal, respectively. Wang, W, and Maris, JM are the scholars with the highest publication count and co-citations in this field. "Neuroblastoma" and "Expression" are the most frequent keywords, while "classification," "Metabolism," "Cancer," and "Diagnosis" are recent key terms. The article titled "Neuroblastoma" by John M. Maris is the most cited reference in this analysis.

The continuous growth in NB omics research underscores its increasing significance in the scientific community. Omics technologies have facilitated the identification of potential biomarkers, advancements in personalized medicine, and the development of novel therapeutic strategies. Despite these advancements, the field faces significant challenges, including tumor heterogeneity, data standardization issues, and the translation of research findings into clinical practice.

Telomeres are a protective cap that prevents chromosome ends from being recognized as double-stranded breaks. In somatic cells, telomeres shorten with each cell division due to the end replication problem, which eventually leads to senescence, a checkpoint proposed to prevent uncontrolled cell growth. Tumor cells avoid telomere shortening by activating one of two telomere maintenance mechanisms (TMMs): telomerase reactivation or alternative lengthening of telomeres (ALT). TMMs are a viable target for cancer treatment as they are not active in normal, differentiated cells. Whereas there is a telomerase inhibitor currently undergoing clinical trials, there are no known ALT inhibitors in development, partially because the complex ALT pathway is still poorly understood. For cancers such as neuroblastoma and osteosarcoma, the ALT-positive status is associated with an aggressive phenotype and few therapeutic options. Thus, methods that characterize the key biological pathways driving ALT will provide important mechanistic insight. We have developed a first-in-class phenotypic high-throughput screen to identify small-molecule inhibitors of ALT. Our screen measures relative C-circle level, an ALT-specific biomarker, to detect changes in ALT activity induced by compound treatment. To investigate epigenetic mechanisms that contribute to ALT, we screened osteosarcoma and neuroblastoma cells against an epigenetic-targeted compound library. Hits included compounds that target chromatin-regulating proteins and DNA damage repair pathways. Overall, the high-throughput C-circle assay will help expand the repertoire of potential ALT-specific therapeutic targets and increase our understanding of ALT biology.

The purpose of the study was to design a pan-cancer gene panel for childhood malignancies and validate it using clinically characterized patient samples.

In addition to 5,275 coding exons, SJPedPanel also covers 297 introns for fusions/structural variations and 7,590 polymorphic sites for copy-number alterations. Capture uniformity and limit of detection are determined by targeted sequencing of cell lines using dilution experiment. We validate its coverage by in silico analysis of an established real-time clinical genomics (RTCG) cohort of 253 patients. We further validate its performance by targeted resequencing of 113 patient samples from the RTCG cohort. We demonstrate its power in analyzing low tumor burden specimens using morphologic remission and monitoring samples.

Among the 485 pathogenic variants reported in RTCG cohort, SJPedPanel covered 86% of variants, including 82% of 90 rearrangements responsible for fusion oncoproteins. In our targeted resequencing cohort, 91% of 389 pathogenic variants are detected. The gene panel enabled us to detect ∼95% of variants at allele fraction (AF) 0.5%, whereas the detection rate is ∼80% at AF 0.2%. The panel detected low-frequency driver alterations from morphologic leukemia remission samples and relapse-enriched alterations from monitoring samples, demonstrating its power for cancer monitoring and early detection.

SJPedPanel enables the cost-effective detection of clinically relevant genetic alterations including rearrangements responsible for subtype-defining fusions by targeted sequencing of ∼0.15% of human genome for childhood malignancies. It will enhance the analysis of specimens with low tumor burdens for cancer monitoring and early detection.

Neuroblastoma is the most prevalent solid tumor in early childhood, with a 5-year overall survival rate of 40-60% in high-risk cases. Therefore, the identification of novel biomarkers for the diagnosis, prognosis, and therapy of neuroblastoma is crucial for improving the clinical outcomes of these patients. In this study, we conducted the whole-exome sequencing of 48 freshly frozen tumor samples obtained from the Biobank. Somatic variants were identified and selected using a bioinformatics analysis pipeline. The mutational signatures were determined using the Mutalisk online tool. Cancer driver genes and druggable mutations were predicted using the Cancer Genome Interpreter. The most common mutational signature was single base substitution 5. MUC4, MUC16, and FLG were identified as the most frequently mutated genes. Using the Cancer Genome Interpreter, we identified five recurrent cancer driver mutations spanning MUC16, MUC4, ALK, and CTNND1, with the latter being novel and containing a missense mutation, R439C. We also identified 11 putative actionable mutations including NF1 Q1798*, Q2616*, and S636X, ALK F1174L and R1275Q, SETD2 P10L and Q1829E, BRCA1 R612S, NOTCH1 D1670V, ATR S1372L, and FGFR1 N577K. Our findings provide a comprehensive overview of the novel information relevant to the underlying molecular pathogenesis and therapeutic targets of neuroblastoma.

Neuroblastoma is a pediatric malignancy with heterogeneous clinical outcomes. Our aim was to identify prognostic genetic markers for patients with neuroblastoma, who were treated with the Taiwan Pediatric Oncology Group (TPOG) neuroblastoma N2002 protocol, to improve risk stratification and inform treatment.

Our analysis was based on 53 primary neuroblastoma specimens, diagnosed pre-chemotherapy, and 11 paired tumor relapse specimens. Deep sequencing of 113 target genes was performed using a custom panel. Multiplex ligation-dependent probe amplification was performed to identify clinical outcomes related to copy-number variations.

We identified 128 variations associated with survival, with the number of variations being higher in the relapse than that in the diagnostic specimen (p = .03). The risk of event and mortality was higher among patients with a tumor mutational burden ≥10 than that in patients with a lower burden (p < .0001). Multivariate analysis identified tumor mutational burden, MYCN amplification, and chromosome 3p deletion as significant prognostic factors, independent of age at diagnosis, sex, and tumor stage. The 5-year event-free survival and overall survival rate was lower among patients with high tumor burden than in patients with low tumor burden. Furthermore, there was no survival of patients with an ALK F1147L variation at 5 years after diagnosis.

Genome sequencing to determine the tumor mutational burden and ALK variations can improve the risk classification of neuroblastoma and inform treatment.

Neuroblastoma (NB) is the most common solid tumour in childhood, and rises in the sympathetic nervous system. Here, we addressed the in silico analysis of the association between the expression of H2AFX gene involved in DNA damage response, and the survival of a cohort of 786 NB patients.

In silico gene expression was retrieved from the publicly available dataset summarised by Cangelosi et al., including 13,696 gene expression profiles of 786 NB tumours at onset of disease. The prognostic value of H2AFX (H2A histone family member X) gene expression for event-free survival (EFS) and overall survival (OS) was evaluated by Kaplan-Meier and Cox regression analysis. The main results were validated on another openly accessible in silico database (NRC-283) containing 13,489 gene expressions in 283 NB patients. The expression of H2AFX protein was then tested by immunofluorescence on 48 primary NB samples of different tumour stages. H2AFX activity as an oncogene has been further validated in vitro by silencing the molecule in two NB cell lines, characterised by MYCN amplified or not, and performing cell growth and migration assays.

A strong inverse association between H2AFX expression and patients' survival was observed and confirmed by immunofluorescence results on primary NB tissue sections. Cox regression analysis also disclosed H2AFX as an independent predictor of EFS and OS. The gene-silencing experiments strongly suggested an oncogenic role for H2AFX on NB cells, regardless of MYCN amplification.

H2AFX is a prognostic marker for unfavourable NB and could be considered a target for therapeutic interventions.

Adult- and adolescent-onset neuroblastomas are rare, with no established therapy. In addition, rare pheochromocytomas may harbor neuroblastic components. This study was designed to collect epidemiological, diagnostic and therapeutic data in order to better define the characteristics of malignant peripheral neuroblastic tumors (MPNT) and composite pheochromocytomas (CP) with MPNT.

Fifty-nine adults and adolescents (aged over 15 years) diagnosed with a peripheral or composite neuroblastic tumor, who were treated in one of 17 institutions between 2000 and 2020, were retrospectively studied.

Eighteen patients with neuroblastoma (NB) or ganglioneuroblastoma (GNB) had locoregional disease, and 28 patients had metastatic stage 4 NB. Among the 13 patients with CP, 12 had locoregional disease. Fifty-eight percent of the population were adolescents and young adults under 24 years of age. The probability of 5-year event-free survival (EFS) was 40% (confidence interval: 27%-53%).

Outcomes were better for patients with localized tumor than for patients with metastases. For patients with localized tumor, in terms of survival, surgical treatment was the best therapeutic option. Multimodal treatment with chemotherapy, surgery, radiotherapy, and immunotherapy-based maintenance allowed long-term survival for some patients. Adolescent- and adult-onset neuroblastoma appeared to have specific characteristics associated with poorer outcomes compared to pediatric neuroblastoma. Nevertheless, complete disease control improved survival. The presence of a neuroblastic component in pheochromocytoma should be considered when making therapeutic management decisions. The development of specific tools/resources (Tumor Referral Board, Registry, biology, and trials with new agents or strategies) may help to improve outcomes for patients.

Acquiring a telomere maintenance mechanism is a hallmark of high-risk neuroblastoma and commonly occurs by expressing telomerase (TERT). Telomerase-negative neuroblastoma has long telomeres and utilizes the telomerase-independent alternative lengthening of telomeres (ALT) mechanism. Conversely, no discernable telomere maintenance mechanism is detected in a fraction of neuroblastoma with long telomeres. Here, we show, unlike most cancers, DNA of the TERT promoter is broadly hypomethylated in neuroblastoma. In telomerase-positive neuroblastoma cells, the hypomethylated DNA promoter is approximately 1.5 kb. The TERT locus shows active chromatin marks with low enrichment for the repressive mark, H3K27me3. MYCN, a commonly amplified oncogene in neuroblstoma, binds to the promoter and induces TERT expression. Strikingly, in neuroblastoma with long telomeres, the hypomethylated region spans the entire TERT locus, including multiple nearby genes with enrichment for the repressive H3K27me3 chromatin mark. Furthermore, subtelomeric regions showed enrichment of repressive chromatin marks in neuroblastomas with long telomeres relative to those with short telomeres. These repressive marks were even more evident at the genic loci, suggesting a telomere position effect (TPE). Inhibiting H3K27 methylation by three different EZH2 inhibitors induced the expression of TERT in cell lines with long telomeres and H3K27me3 marks in the promoter region. EZH2 inhibition facilitated MYCN binding to the TERT promoter in neuroblastoma cells with long telomeres. Taken together, these data suggest that epigenetic regulation of TERT expression differs in neuroblastoma depending on the telomere maintenance status, and H3K27 methylation is important in repressing TERT expression in neuroblastoma with long telomeres.

The epigenetic landscape of the TERT locus is unique in neuroblastoma. The DNA at the TERT locus, unlike other cancer cells and similar to normal cells, are hypomethylated in telomerase-positive neuroblastoma cells. The TERT locus is repressed by polycomb repressive complex-2 complex in neuroblastoma cells that have long telomeres and do not express TERT. Long telomeres in neuroblastoma cells are also associated with repressive chromatin states at the chromosomal termini, suggesting TPE.

Neuroblastoma (NB) is one of the leading causes of cancer-associated death in children. MYCN amplification is a prominent genetic marker for NB, and its targeting to halt NB progression is difficult to achieve. Therefore, an in-depth understanding of the molecular interactome of NB is needed to improve treatment outcomes. Analysis of NB multi-omics unravels valuable insight into the interplay between MYCN transcriptional and miRNA post-transcriptional modulation. Moreover, it aids in the identification of various miRNAs that participate in NB development and progression. This study proposes an integrated computational framework with three levels of high-throughput NB data (mRNA-seq, miRNA-seq, and methylation array). Similarity Network Fusion (SNF) and ranked SNF methods were utilized to identify essential genes and miRNAs. The specified genes included both miRNA-target genes and transcription factors (TFs). The interactions between TFs and miRNAs and between miRNAs and their target genes were retrieved where a regulatory network was developed. Finally, an interaction network-based analysis was performed to identify candidate biomarkers. The candidate biomarkers were further analyzed for their potential use in prognosis and diagnosis. The candidate biomarkers included three TFs and seven miRNAs. Four biomarkers have been previously studied and tested in NB, while the remaining identified biomarkers have known roles in other types of cancer. Although the specific molecular role is yet to be addressed, most identified biomarkers possess evidence of involvement in NB tumorigenesis. Analyzing cellular interactome to identify potential biomarkers is a promising approach that can contribute to optimizing efficient therapeutic regimens to target NB vulnerabilities.

Glyceraldehyde (GA) is a three-carbon monosaccharide that can be present in cells as a by-product of fructose metabolism. Bruno Mendel and Otto Warburg showed that the application of GA to cancer cells inhibits glycolysis and their growth. However, the molecular mechanism by which this occurred was not clarified. We describe a novel multi-modal mechanism by which the L-isomer of GA (L-GA) inhibits neuroblastoma cell growth. L-GA induces significant changes in the metabolic profile, promotes oxidative stress and hinders nucleotide biosynthesis. GC-MS and 13C-labeling was employed to measure the flow of carbon through glycolytic intermediates under L-GA treatment. It was found that L-GA is a potent inhibitor of glycolysis due to its proposed targeting of NAD(H)-dependent reactions. This results in growth inhibition, apoptosis and a redox crisis in neuroblastoma cells. It was confirmed that the redox mechanisms were modulated via L-GA by proteomic analysis. Analysis of nucleotide pools in L-GA-treated cells depicted a previously unreported observation, in which nucleotide biosynthesis is significantly inhibited. The inhibitory action of L-GA was partially relieved with the co-application of the antioxidant N-acetyl-cysteine. We present novel evidence for a simple sugar that inhibits cancer cell proliferation via dysregulating its fragile homeostatic environment.

The 5-methylcytosine (m5C) is the key chemical modification in RNAs. As one of the demethylases in m5C, TET2 has been shown as a tumor suppressor. However, the impact of TET2 gene polymorphisms on neuroblastoma has not been elucidated. 402 neuroblastoma patients and 473 controls were genotyped for TET2 gene polymorphisms using the TaqMan method. The impact of TET2 gene polymorphisms on neuroblastoma susceptibility was determined using multivariate logistic regression analysis. We also adopted genotype-tissue expression database to explore the impact of TET2 gene polymorphisms on the expression of host and nearby genes. We used the R2 platform and Sangerbox tool to analyze the relationship between gene expression and neuroblastoma risk and prognosis through non-parametric testing and Kaplan-Meier analysis, respectively. We found the TET2 gene polymorphisms (rs10007915 G > C and rs7670522 A > C) and the combined 2-5 risk genotypes can significantly increase neuroblastoma risk. Stratification analysis showed that these significant associations were more prominent in certain subgroups. TET2 rs10007915 G > C and rs7670522 A > C are significantly associated with reduced expression of TET2 mRNA. Moreover, lower expression of TET2 gene is associated with high risk, MYCN amplification, and poor prognosis of neuroblastoma. The rs10007915 G > C and rs7670522 A > C are significantly related to the increased expression of inorganic pyrophosphatase 2 mRNA, and higher expression of PPA2 gene is associated with high risk, MYCN amplification, and poor prognosis of neuroblastomas. In summary, TET2 rs10007915 G > C and rs7670522 A > C significantly confer neuroblastoma susceptibility, and further research is needed to investigate the underlying mechanisms.

The MYC proto-oncogenes (c-MYC, MYCN , MYCL ) are among the most deregulated oncogenic drivers in human malignancies including high-risk neuroblastoma, 50% of which are MYCN -amplified. Genetically engineered mouse models (GEMMs) based on the MYCN transgene have greatly expanded the understanding of neuroblastoma biology and are powerful tools for testing new therapies. However, a lack of c-MYC-driven GEMMs has hampered the ability to better understand mechanisms of neuroblastoma oncogenesis and therapy development given that c-MYC is also an important driver of many high-risk neuroblastomas. In this study, we report two transgenic murine neuroendocrine models driven by conditional c-MYC induction in tyrosine hydroxylase (Th) and dopamine β-hydroxylase (Dbh)-expressing cells. c-MYC induction in Th-expressing cells leads to a preponderance of Pdx1 + somatostatinomas, a type of pancreatic neuroendocrine tumor (PNET), resembling human somatostatinoma with highly expressed gene signatures of δ cells and potassium channels. In contrast, c-MYC induction in Dbh-expressing cells leads to onset of neuroblastomas, showing a better transforming capacity than MYCN in a comparable C57BL/6 genetic background. The c-MYC murine neuroblastoma tumors recapitulate the pathologic and genetic features of human neuroblastoma, express GD2, and respond to anti-GD2 immunotherapy. This model also responds to DFMO, an FDA-approved inhibitor targeting ODC1, which is a known MYC transcriptional target. Thus, establishing c-MYC-overexpressing GEMMs resulted in different but related tumor types depending on the targeted cell and provide useful tools for testing immunotherapies and targeted therapies for these diseases.

Characterisation of histological, immunohistochemical and molecular prognostic and predictive biomarkers has contributed significantly to precision medicine and better outcomes in the management of paediatric solid tumours. Prognostic biomarkers allow predictions to be made regarding a tumour's aggressiveness and clinical course, whereas predictive biomarkers help determine responses to a specific treatment. This review summarises prognostic biomarkers currently used in the more common paediatric solid tumours, with a brief commentary on the most relevant less common predictive biomarkers. MYCN amplification is the most important genetic alteration in neuroblastoma prognosis, and the histological classification devised by Shimada in 1999 is still used in routine diagnosis. Moreover, a new subgrouping of unfavourable histology neuroblastoma enables immunohistochemical characterisation of tumours with markedly different genetic features and prognosis. The predominant histology and commonly observed cytogenetic abnormalities are recognised outcome predictors in Wilms tumour. Evaluation for anaplasia, which is tightly associated with TP53 gene mutations and poor outcomes, is central in both the International Society of Paediatric Oncology and the Children's Oncology Group approaches to disease classification. Characterisation of distinct genotype-phenotype subclasses and critical mutations has expanded overall understanding of hepatoblastoma outcomes. The C1 subclass hepatoblastoma and CTNNB1 mutations are associated with good prognosis. In contrast, the C2 subclass, NFE2L2 mutations, TERT promoter mutations and high expression of oncofetal proteins and stem cell markers are associated with poor outcomes. Risk stratification in sarcomas is highly variable depending on the entity. The prognosis of rhabdomyosarcoma, for example, primarily depends on histological and molecular characteristics. Advances in our understanding of clinically significant biomarkers will translate into more precise diagnoses, improved risk stratification and more effective and less toxic treatment in this challenging group of patients.

To develop and validate an 18F-FDG PET/CT-based radiomics nomogram for differentiating early relapse and late relapse of intermediate- and high-risk neuroblastoma (NB).

A total of eighty-five patients with relapsed NB who underwent 18F-FDG PET/CT were retrospectively evaluated. All selected patients were randomly assigned to the training set and the validation set in a 7:3 ratio. Tumors were segmented using the 3D slicer, followed by radiomics features extraction. Features selection was performed using random forest, and the radiomics score was constructed by logistic regression analysis. Clinical risk factors were identified, and the clinical model was constructed using logistic regression analysis. A radiomics nomogram was constructed by combining the radiomics score and clinical risk factors, and its performance was evaluated by receiver operating characteristic (ROC) curves, calibration curves, and decision curve analysis (DCA).

Finally, the 12 most important radiomics features were used for modeling, with an area under the curve (AUC) of 0.835 and 0.824 in the training and validation sets, respectively. Age at diagnosis and International Neuroblastoma Pathology Classification were determined as clinical risk factors to construct the clinical model. In addition, the nomogram achieved an AUC of 0.902 and 0.889 for identifying early relapse in the training and validation sets, respectively, which is higher than the clinical model (AUC of 0.712 and 0.588, respectively). The predicted early relapse and actual early relapse in the calibration curves were in good agreement. The DCA showed that the radiomics nomogram was clinically useful.

Our 18F-FDG PET/CT-based radiomics nomogram can well predict early relapse and late relapse of intermediate- and high-risk NB, which contributes to follow-up and management in clinical practice.

MYC genes regulate ornithine decarboxylase (Odc) to increase intratumoral polyamines. We conducted a Phase I trial [NCT02030964] to determine the maximum tolerated dose (MTD) of DFMO, an Odc inhibitor, with celecoxib, cyclophosphamide and topotecan.

Patients 2-30 years of age with relapsed/refractory high-risk neuroblastoma received oral DFMO at doses up to 9000 mg/m2/day, with celecoxib (500 mg/m2 daily), cyclophosphamide (250 mg/m2/day) and topotecan (0.75 mg/m2/day) IV for 5 days, for up to one year with G-CSF support.

Twenty-four patients (median age, 6.8 years) received 136 courses. Slow platelet recovery with 21-day courses (dose-levels 1 and 2) led to subsequent dose-levels using 28-day courses (dose-levels 2a-4a). There were three course-1 dose-limiting toxicities (DLTs; hematologic; anorexia; transaminases), and 23 serious adverse events (78% fever-related). Five patients (21%) completed 1-year of therapy. Nine stopped for PD, 2 for DLT, 8 by choice. Best overall response included two PR and four MR. Median time-to-progression was 19.8 months, and 3 patients remained progression-free at >4 years without receiving additional therapy. The MTD of DFMO with this regimen was 6750 mg/m2/day.

High-dose DFMO is tolerable when added to chemotherapy in heavily pre-treated patients. A randomized Phase 2 trial of DFMO added to chemoimmunotherapy is ongoing [NCT03794349].

Neuroblastoma (NB) represents the most frequent and aggressive form of extracranial solid tumor of infants. Although the overall survival of patients with NB has improved in the last years, more than 50% of high-risk patients still undergo a relapse. Thus, in the era of precision/personalized medicine, the need for high-risk NB patient-specific therapies is urgent.

Within the PeRsonalizEd Medicine (PREME) program, patient-derived NB tumors and bone marrow (BM)-infiltrating NB cells, derived from either iliac crests or tumor bone lesions, underwent to histological and to flow cytometry immunophenotyping, respectively. BM samples containing a NB cells infiltration from 1 to 50 percent, underwent to a subsequent NB cells enrichment using immune-magnetic manipulation. Then, NB samples were used for the identification of actionable targets and for the generation of 3D/tumor-spheres and Patient-Derived Xenografts (PDX) and Cell PDX (CPDX) preclinical models.

Eighty-four percent of NB-patients showed potentially therapeutically targetable somatic alterations (including point mutations, copy number variations and mRNA over-expression). Sixty-six percent of samples showed alterations, graded as "very high priority", that are validated to be directly targetable by an approved drug or an investigational agent. A molecular targeted therapy was applied for four patients, while a genetic counseling was suggested to two patients having one pathogenic germline variant in known cancer predisposition genes. Out of eleven samples implanted in mice, five gave rise to (C)PDX, all preserved in a local PDX Bio-bank. Interestingly, comparing all molecular alterations and histological and immunophenotypic features among the original patient's tumors and PDX/CPDX up to second generation, a high grade of similarity was observed. Notably, also 3D models conserved immunophenotypic features and molecular alterations of the original tumors.

PREME confirms the possibility of identifying targetable genomic alterations in NB, indeed, a molecular targeted therapy was applied to four NB patients. PREME paves the way to the creation of clinically relevant repositories of faithful patient-derived (C)PDX and 3D models, on which testing precision, NB standard-of-care and experimental medicines.