Neuroblastoma Patients Research Articles

Immune-oncology targets and therapeutic response of cell pyroptosis-related genes with prognostic implications in neuroblastoma.

Construction of a neuroblastoma (NB) prognostic predictive model based on pyroptosis-related genes (PRGs) to improve individualized management of NB patients. The NB cohort GSE49711 was obtained from the Gene Expression Omnibus (GEO) database, and a total of 498 patients were enrolled into the study, which were randomized into a training set and a test set at a ratio of 1:1, with 250 patients in the training set and 248 patients in the test set. A risk prediction model was constructed using the training set, and the GSE49711 cohort and test set were used as internal validation to verify the reliability of the model. Independent predictors associated with prognosis were screened using univariate and multivariate COX regression analyses, and risk score models were constructed. Single-cell gene set enrichment analysis (ssGSEA) was used to assess the relationship between PRGs and the tumor immune microenvironment. Nomograms were constructed to extend the clinical usability of the model and the reliability of the model was verified using ROC curves and calibration curves. Protein interaction networks of risk genes were mapped using the String database, and the expression of PRGs in NB cell lines was staged using the CCLE database. A prognostic model was first developed with the training set: the risk score formula was (-0.30 × GSDMB) + (-0.46 × IL-18) + (-0.21 × NLRP3) + (0.56 × AIM2). Patients were categorized into high- and low-risk groups based on the median risk score value. Survival analysis showed that NB patients in the high-risk group had a significantly lower survival rate than those in the low-risk group (P < 0.001). In both the GSE49711 overall cohort and the test cohort, survival analyses showed that patients in the high-risk group had significantly lower survival than those in the low-risk group (P < 0.001). Single-cell gene set enrichment analysis was used to assess the relationship between PRGs and the tumor immune microenvironment. Time-dependent ROC curves assessed the predictive performance of the nomogram in 5-, 7.5-, and 10-year survival with areas under the curve (AUC) of 0.843, 0.802 and 0.797, respectively. The calibration curves show good clinical predictive performance for nomograms. The results suggest that PRGs may serve as a novel prognostic marker for NB patients to provide new immunotherapeutic targets for the clinical treatment of NB patients.

Fine construction of gene coexpression network analysis using GTOM and RECODE detected a critical module of neuroblastoma stages 4 and 4S.

Stage 4 neuroblastoma (NBL), a solid tumor of childhood, has a poor prognosis. Despite intensive molecular genetic studies, no targetable gene abnormalities have been identified. Stage 4S NBL has a characteristic of spontaneous regression, and elucidation of the mechanistic differences between stages 4 and 4S may improve treatment. Conventional NBL studies have mainly focused on the detection of abnormalities in individual genes and have rarely examined abnormalities in gene networks. While the gene coexpression network is expected to contribute to the detection of network abnormalities, the fragility of the network due to data noise and the extraction of arbitrary topological structures for the high-dimensional network are issues. The present paper concerns the classification method of stages 4 and 4S NBL patients using highly accurate gene coexpression network analysis based on RNA-sequencing data of transcription factors (TFs). In particular, after applying a noise reduction method RECODE, generalized topological overlapping measure (GTOM), which weighs the connections of nodes in the network structure, succeeded in extracting a cluster of TFs that showed high classification performance for stages 4 and 4S. In addition, we investigated how these clusters correspond to clinical information and to TFs which control the normal adrenal tissue and NBL characters. A clustering method is presented for finding intermediate-scale clusters of TFs that give considerable separation performance for distinguishing between stages 4 and 4S. It is suggested that this method is useful as a way to extract factors that contribute to the separation of groups from multiple pieces of information such as gene expression levels.

Automated and closed clinical-grade manufacturing protocol produces potent NK cells against neuroblastoma cells and AML blasts

Natural killer (NK) cells are a promising allogeneic immunotherapy option due to their natural ability to kill tumor cells, and due to their apparent safety. This study describes the development of a GMP-compliant manufacturing protocol for the local production of functionally potent NK cells tailored for high-risk acute myeloid leukemia (AML) and neuroblastoma (NBL) patients. Moreover, the quality control strategy and considerations for product batch specifications in early clinical development are described. The protocol is based on the CliniMACS Prodigy platform and Natural Killer Cell Transduction (NKCT) (Miltenyi Biotec). NK cells are isolated from leukapheresis through CD3 depletion and CD56 enrichment, followed by a 12-hour activation with IL-2 and IL-15 cytokines. Three CliniMACS Prodigy processes demonstrated the feasibility and consistency of the modified NKCT process. A three-step process without expansion, however, compromised the NK cell yield. T cells were depleted effectively, indicating excellent safety of the product. Characterization of the NK cells before and after cytokine activation revealed a notable increase in the expression of activation markers, particularly CD69, consistent with enhanced functionality. Intriguingly, the NK cells exhibited increased killing efficacy against patient-derived CD33 + AML blasts and NBL cells in vitro, suggesting a potential therapeutic benefit in AML and NBL.

Epac activation reduces trans-endothelial migration of undifferentiated neuroblastoma cells and cellular differentiation with a CDK inhibitor further enhances Epac effect.

Neuroblastoma (NB) is the most common solid extracranial neoplasm found in children and is derived from primitive sympathoadrenal neural precursor. The disease accounts for 15% of all cancer deaths in children. The mortality rate is high in patients presenting with a metastatic tumour even with extensive treatments. This signifies the need for further research towards the development of new additional therapies that can combat not only tumour growth but metastasis, especially amongst the high-risk groups. During metastasis, primary tumour cells become migratory and travel towards a capillary within the tumour. They then degrade the matrix surrounding the pericytes and endothelial cells traversing the endothelial barrier twice to establish a secondary. This led to the hypothesis that modulation of the endothelial cell junctional stability could have an influence on tumour metastasis. To test this hypothesis, agents that modulate endothelial permeability on NB cell line migration and invasion were assessed in vitro in a tissue culture model. The cAMP agonist and its antagonists were found to have no obvious effect on both SK-N-BE2C and SK-N-AS migration, invasion and proliferation. Next, NB cells were cocultured with HDMEC cells and live cell imaging was used to assess the effect of an Epac agonist on trans-endothelial cell migration of NB cells. Epac1 agonist remarkably reduced the trans-endothelial migration of both SK-N-BE2C and SK-N-AS cells. These results demonstrate that an Epac1 agonist may perhaps serve as an adjuvant to currently existing therapies for the high-risk NB patients.

STK33 as the functional substrate of miR-454-3p for suppression and apoptosis in neuroblastoma

miR-454-3p has been reported to be a tumor suppressive micro-RNA (miRNA) in multiple cancer types. We identified the kinase STK33 mRNA, which is a high-risk factor for survival in neuroblastoma (NB) patients, as being a substrate of miR-454-3p in NB. Even though STK33 is an attractive target for several cancers, development of inhibitors of STK33 has been challenging. For the various cell lines tested, we demonstrated reduced growth and viability with the miR-454-3p mimic. From among the candidate NB-associated miRNAs, miR-454-3p mimic and its antagonist had the most profound effect on STK33 mRNA and protein level changes. Under various conditions of growth and external stress for the cells, the RNA levels for miR-454-3p and STK33 also negatively correlated. Luciferase reporter assays demonstrated STK33 as a substrate for miR-454-3p, and recombinant versions of STK33 resistant to miR-454-3p significantly blunted the suppressive effect of the miR-454-3p and established STK33 as the major functional substrate of miR-454-3p. Overexpression of miR-454-3p or knockdown of STK33 mRNA promoted autophagy, and at the same time, increased the apoptotic markers in the tested NB cells, indicating a mechanism for the suppressive effect of the agents. Given the difficult-to-drug targets such as STK33 and the recent successes in RNA delivery methods for cancer treatment, it is thought that targeting cancer cells with a suppressive miRNA such as miR-454-3p for STK33-dependent cancer types may be an alternative means of NB therapy.

Zinc Finger Protein 536 is a Potential Prognostic Biomarker that Promotes Neuroblastoma Progression via VEGFR2-PI3K-AKT Pathway.

Neuroblastoma (NB) is a well-known pediatric malignancy intertwined with neurodevelopment. Previously implicated in neuronal differentiation, Zinc Finger Protein 536 (ZNF536) has emerged as a promising prognostic and immune-related biomarker in our pan-cancer analysis. Single-cell RNA transcriptome sequencing, bulk transcriptome analysis, and immuno-histochemistry were used to assess ZNF536 expression and its association with prognosis. Cell proliferation, migration, invasion, and differentiation in ZNF536-knockdown NB cell lines were detected to evaluate the effect of ZNF536 on tumor cells. Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), a potential target of ZNF536, and its downstream PI3K/AKT signaling cascade were investigated using transcriptome sequencing, CUT&Tag, quantitative real-time PCR (qRT-PCR), and Western blotting. The role of ZNF536 in tumorigenesis and the potential regulation axis was evaluated in vivo using a BALB/c nude mouse xenograft tumor model. ZNF536 mRNA and protein expression were significantly higher in NB patients with poor prognosis. In vitro, ZNF536 knockdown curtailed proliferation, migration, and invasion of NB cells while fostering differentiation. ZNF536 regulated VEGFR2 expression, thus activating the PI3K-AKT pathway. In vivo, ZNF536 knockdown reduced tumor growth and proliferation via the VEGFR2-PI3K-AKT pathway. ZNF536 resulted as a novel prognostic biomarker in NB, promoting oncogenesis through VEGFR2-PI3K-AKT signaling axis modulation, suggesting its therapeutic potential in managing NB progression.

Rationale for irradiation of persisting oligo-skeletal metastases to improve survival of metastatic neuroblastoma patients with a poor response to chemotherapy: A retrospective study.

Persistent metaiodobenzylguanidine (mIBG)-positive skeletal metastases post induction in high-risk neuroblastoma correlate with a poor outcome. The aim of this study was to investigate the potential rationale for a prospective randomized study evaluating the impact on event-free survival of the irradiation of residual oligo-skeletal metastases. Patients over 1year with a stage M neuroblastoma treated between 2000 and 2020 at Gustave Roussy were identified. Patients with a positive mIBG scan at diagnosis and persistent skeletal metastases after high-dose chemotherapy (HDC) were included. Data were retrospectively collected and mIBG scans reviewed by two nuclear medicine physicians. Persistent skeletal uptake after HDC was observed in 30/201 patients (15%). Four patients reached a complete response at the end of maintenance treatment and did not relapse (median follow-up [FU] 8years [1.8-11.8]), while two patients had progressive disease during maintenance. Among the 24 patients with persistent skeletal uptakes at the end of treatment, seven had a persistent response (median FU 8.2years [4-15.6]). Median SIOPEN (International Society of Paediatric Oncology European Neuroblastoma) scores post consolidation and at the end of treatment were, respectively, 2 [1-6] and 2 [0-4] for patients with persistent responses compared to 4 [1-28] and 2 [1-17] for patients with progressive diseases. Median SIOPEN score at progression was 34 [2-56]. Our study underlines that only a minority of patients had persistent skeletal mIBG-positive scans after HDC. Recurrence mainly occurred in disease sites present at diagnosis that cleared with chemotherapy. On-therapy control of the disease is the main challenge. These results highlight the complexity of conducting a randomized study exploring this strategy.

Early results of treatment in accordance with the NB-HR-2018 protocol in patients with high-risk neuroblastoma in the Republic of Belarus

The development of new criteria for high-risk neuroblastoma treatment optimization and the introduction of new approaches to its management are a pressing problem in modern pediatric oncology. In this study, we aimed to develop and implement new high-risk and ultra-high-risk criteria, introduce tandem autologous hematopoietic stem cell transplantation (HSCT) as consolidation therapy for high-risk neuroblastoma patients as well as to assess patient tolerability of this treatment. The study was approved by the Independent Ethics Committee and the Scientific Council of the Belarusian Research Center for Pediatric Oncology, Hematology and Immunology. In 2018, a new protocol called NB-HR-2018 was developed and implemented at the Center for Pediatric Oncology, Hematology, and Immunology (Belarus) that included new criteria defining high-risk groups. Twenty-three patients were treated according to the new protocol, with 20 of them receiving autologous HSCT. The comparison group included 56 high-risk patients who had undergone treatment in accordance with the NB 2004 protocol. Tandem autoHSCT significantly reduces the rates of underlying disease relapse/progression (p = 0.047) and demonstrates better event-free survival rates (56 ± 12% vs 36 ± 6%; р = 0.445). The use of the new high-risk criteria and the new treatment method (tandem autologous HSCT) is concluded to be a reasonable approach since it significantly reduces disease relapse rates and is well tolerated by the patients.

Enzymatically Cyclic Activated Biosensor Based on a Tetrahedral DNA Framework for Precise Tumor in Situ Molecular Imaging.

The development of stimulus-responsive and amplification-based strategies is crucial for achieving improved spatial specificity and enhanced sensitivity in tumor molecular imaging, addressing challenges such as off-tumor signal leakage and limited biomarker content. Therefore, a cyclically activated enzymatic biosensor based on the modification of an AP site within a tetrahedral framework DNA (AP-tFNA) was rationally developed for tumor cell-specific molecular imaging using the endogenous enzyme apurinic/apyrimidinic endonuclease 1 (APE1) as a target, exhibiting superior spatial specificity and high sensitivity. APE1, which predominantly localizes within the nucleus in normal cells but exhibits cytosolic and nucleus expression in cancer cells, can specifically recognize and cleave the AP site in AP-tFNA, resulting in the separation of the fluorophore and quenching group, thereby inducing a fluorescence signal. Additionally, upon completion of the excision of one AP site in AP-tFNA, APE1 is released, thereby initiating a subsequent cycle of hydrolytic cleavage reactions. The experimental results demonstrated that AP-tFNA enables precise differentiation of tumor cells both in vitro and in vivo. In particular, the AP-tFNA can monitor drug resistance in neuroblastoma cells and classify the risk for neuroblastoma patients at the clinical plasma level.

Repurposing of c-MET Inhibitor Tivantinib Inhibits Pediatric Neuroblastoma Cellular Growth

Background: Dysregulation of receptor tyrosine kinase c-MET is known to promote tumor development by stimulating oncogenic signaling pathways in different cancers, including pediatric neuroblastoma (NB). NB is an extracranial solid pediatric cancer that accounts for almost 15% of all pediatric cancer-related deaths, with less than a 50% long-term survival rate. Results: In this study, we analyzed a large cohort of primary NB patient data and revealed that high MET expression strongly correlates with poor overall survival, disease progression, relapse, and high MYCN levels in NB patients. To determine the effects of c-MET in NB, we repurposed a small molecule inhibitor, tivantinib, and found that c-MET inhibition significantly inhibits NB cellular growth. Tivantinib significantly blocks NB cell proliferation and 3D spheroid tumor formation and growth in different MYCN-amplified and MYCN-non-amplified NB cell lines. Furthermore, tivantinib blocks the cell cycle at the G2/M phase transition and induces apoptosis in different NB cell lines. As expected, c-MET inhibition by tivantinib inhibits the expression of multiple genes in PI3K, STAT, and Ras cell signaling pathways. Conclusions: Overall, our data indicate that c-MET directly regulates NB growth and 3D spheroid growth, and c-MET inhibition by tivantinib may be an effective therapeutic approach for high-risk NB. Further developing c-MET targeted therapeutic approaches and combining them with current therapies may pave the way for effectively translating novel therapies for NB and other c-MET-driven cancers.

Investigation of Cell Mechanics and Migration on DDR2-Expressing Neuroblastoma Cell Line.

Neuroblastoma is a devastating disease accounting for ~15% of all childhood cancer deaths. Collagen content and fiber association within the tumor stroma influence tumor progression and metastasis. High expression levels of collagen receptor kinase, Discoidin domain receptor II (DDR2), are associated with the poor survival of neuroblastoma patients. Additionally, cancer cells generate and sustain mechanical forces within their environment as a part of their normal physiology. Despite this, evidence regarding whether collagen-activated DDR2 signaling dysregulates these migration forces is still elusive. To address these questions, a novel shRNA DDR2 knockdown neuroblastoma cell line (SH-SY5Y) was engineered to evaluate the consequence of DDR2 on cellular mechanics. Atomic force microscopy (AFM) and traction force microscopy (TFM) were utilized to unveil the biophysical altercations. DDR2 downregulation was found to significantly reduce proliferation, cell stiffness, and cellular elongation. Additionally, DDR2-downregulated cells had decreased traction forces when plated on collagen-coated elastic substrates. Together, these results highlight the important role that DDR2 has in reducing migration mechanics in neuroblastoma and suggest DDR2 may be a promising novel target for future therapies.

Role of Genetic Polymorphisms -238 G>A and -308 G>A, and Serum TNF-α Levels in a Cohort of Mexican Pediatric Neuroblastoma Patients: Preliminary Study.

The results of in vitro and in vivo studies have shown the pro-tumor effects of TNF-α, and this cytokine's increased expression is associated with poor prognosis in patients with some types of cancer. Our study objective was to evaluate the possible association of TNF-α genetic polymorphisms and serum levels with susceptibility and prognosis in a cohort of Mexican patients with NB. We performed PCR-RFLP and ELISA methods to analyze the genetics of these SNPs and determine serum concentrations, respectively. The distribution of the -308 G>A and -238 G>A polymorphisms TNFα genotypes was considerably different between patients with NB and the control group. The SNP rs1800629 GG/GA genotypes were associated with a decreased risk of NB (OR = 0.1, 95% CI = 0.03-0.393, p = 0.001) compared with the AA genotype, which was associated with susceptibility to NB (OR = 2.89, 95% CI = 1.45-5.76, p = 0.003) and related to unfavorable histology and high-risk NB. The rs361525 polymorphism GG genotype was associated with a lower risk of developing NB compared with the GA and AA genotypes (OR = 0.2, 95% CI = 0.068-0.63, p = 0.006). Circulating TNF-α serum concentrations were significantly different (p < 0.001) between patients with NB and healthy controls; however, we found no relationship between the analyzed TNF-α serum levels and SNP genotypes. We found associations between the rs1800629AA genotype and lower event-free survival (p = 0.026); SNP rs361525 and TNF-α levels were not associated with survival in patients with NB. Our results suggest the TNF-α SNP rs1800629 as a probable factor of NB susceptibility. The -308 G/A polymorphism AA genotype has a probable role in promoting NB development and poor prognosis associated with unfavorable histology, high-risk tumors, and lower EFS in Mexican patients with NB. It should be noted that it is important to conduct research on a larger scale, through inter-institutional studies, to further evaluate the contribution of TNF-α genetic polymorphisms to the risk and prognosis of NB.

Systematic Analysis of miR-506-3p Target Genes Identified Key Mediators of Its Differentiation-Inducing Function.

Background/Objectives: miR-506-3p has been demonstrated to be a strong inducer of neuroblastoma cell differentiation, highlighting the potential of applying miR-506-3p mimics to neuroblastoma differentiation therapy. However, the target genes of miR-506-3p that mediate its differentiation-inducing function have not been fully defined. This study aims to comprehensively investigate the targetome of miR-506-3p regarding its role in regulating neuroblastoma cell differentiation. Methods: We combined gene expression profiling and functional high-content screening (HCS) to identify miR-506-3p target genes that have differentiation-modulating functions. For evaluating the potential clinical relevance of the identified genes, we analyzed the correlations of gene expressions with neuroblastoma patient survival. Results: We identified a group of 19 target genes with their knockdown significantly inducing cell differentiation, suggesting that these genes play a key role in mediating the differentiation-inducing activity of miR-506-3p. We observed significant correlations of higher mRNA levels with lower patient survival with 13 of the 19 genes, suggesting that overexpression of these 13 genes plays important roles in promoting neuroblastoma development by disrupting the cell differentiation pathways. Conclusions: Through this study, we identified novel target genes of miR-506-3p that function as strong modulators of neuroblastoma cell differentiation. Our findings represent a significant advancement in understanding the mechanisms by which miR-506-3p induces neuroblastoma cell differentiation. Future investigations of the identified 13 genes are needed to fully define their functions and mechanisms in controlling neuroblastoma cell differentiation, the understanding of which may reveal additional targets for developing novel differentiation therapeutic agents.

Identification and validation of a novel five-gene signature in high-risk MYCN-not-amplified neuroblastoma

ObjectiveHigh-risk neuroblastoma patients often have poor outcomes despite multi-treatment options. The risk stratification of high-risk MYCN-not-amplified (HR-MYCN-NA) patients remains difficult. This study aims to identify a gene set signature that can help further stratify HR-MYCN-NA patients for a potential personalized therapeutic strategy.MethodsThree microarrays and one single-cell RNA sequence dataset were acquired and analyzed. Firstly, the prognostic-related genes (PRGs) in HR-MYCN-NA tumor cells were identified using TARGET-NB and GSE137804 datasets. Then, the prognostic model was established by LASSO-Cox regression, and verified in external cohort (GSE49710, GSE45547). Moreover, a time-dependent receiver operating characteristic curve (ROC) and area under the ROC (AUC) was used to assess survival prediction. A nomogram was established to predict the 1-, 3- and 5-year overall survival (OS) of HR-MYCN-NA patients.ResultsIn the training set, a five-PRGs signature, which include GAL, GFRA3, MARCKS, PSMD13, and ZNHIT3 genes, was identified and successfully stratified HR-MYCN-NA patients into ultra-high risk (UHR) and high-risk (HR) subtypes (HR = 4.29, P < 0.001). ROC curve analysis confirmed its predictive power (AUC = 0.74–0.82), suggesting a good predictive efficacy. Consistently, high-risk scores also predicted worse OS (HR = 2, P = 0.033) in the external validation dataset (AUC = 0.67–0.71). Moreover, the overall C-index of the nomogram was 0.75 (P < 0.001), which indicated good agreement between the observed and predicted survival rates. Further integrating the five PRGs signature with clinical factors, these 5 gene signature (HR = 4.45, P < 0.001) and tumor grade (HR = 4.15, P = 0.02) were found to be independent prognostic factors for HR-MYCN-NA patients.ConclusionThe novel five PRGs signature could well predict the survival of HR-MYCN-NA patients, which may provide constructive information for these subsets.

Neuroblastoma with high ASPM reveals pronounced heterogeneity and poor prognosis

ObjectiveWe explored the preliminary value of abnormal spindle-like microcephaly- associated (ASPM) protein in aiding precise risk sub-stratification, prediction of metabolic heterogeneity, and prognosis of neuroblastoma (NB).MethodsThis retrospective study enrolled newly diagnosed patients with NB who underwent positron emission tomography/computed tomography (PET/CT) before therapy, and tumor tissue was collected after surgery. Regression analysis was used to evaluate ASPM expression and risk stratification in patients with NB. The expression levels of ASPM, clinical information, and PET/CT text features were analyzed using univariate and multivariate survival analyses. Finally, a correlation analysis was used to explore the relationship between ASPM and tumor metabolic heterogeneity.ResultsThere were 48 patients with NB in this study (35 boys and 13 girls); 22 patients progressed and 16 died. We found that the level of ASPM was highly associated with risk stratification (OR = 5.295, 95%IC: 1.348–41.722, p = 0.021). Patients with NB and high-risk stratification with high ASPM level had a lower 3-year progression-free survival (PFS) rate (14.28%) and 1-year PFS rate (57.14%) than those with low ASPM level (57.14% and 93.75%, respectively). Using univariate and multivariate survival analyses, this study revealed that ASPM and LDH were independent risk factors for both PFS and overall survival (OS), whales GLZLM_ZLNU was only a risk factor for PFS.ConclusionASPM holds promise as a novel biomarker for refining current risk stratification and predicting prognosis in neuroblastoma. Elevated levels of ASPM, LDH, and GLZLM_ZLNU may be associated with poorer survival outcomes in neuroblastoma patients.

Epigenetic regulation of cell state by H2AFY governs immunogenicity in high-risk neuroblastoma.

Childhood neuroblastoma with MYCN-amplification is classified as high-risk and often relapses after intensive treatments. Immune checkpoint blockade therapy against the PD-1/L1 axis shows limited efficacy in neuroblastoma patients and the cancer intrinsic immune regulatory network is poorly understood. Here, we leverage genome-wide CRISPR/Cas9 screens and identify H2AFY as a resistance gene to the clinically approved PD-1 blocking antibody, nivolumab. Analysis of single-cell RNA sequencing datasets reveals that H2AFY mRNA is enriched in adrenergic cancer cells and is associated with worse patient survival. Genetic deletion of H2afy in MYCN-driven neuroblastoma cells reverts in vivo resistance to PD-1 blockade by eliciting activation of the adaptive and innate immunity. Mapping of the epigenetic and translational landscape demonstrates that H2afy deletion promotes cell transition to a mesenchymal-like state. With a multi-omics approach, we uncover H2AFY-associated genes that are functionally relevant and prognostic in patients. Altogether, our study elucidates the role of H2AFY as an epigenetic gatekeeper for cell states and immunogenicity in high-risk neuroblastoma.

Phase I study of safety and efficacy of allogeneic natural killer cell therapy in relapsed/refractory neuroblastomas post autologous hematopoietic stem cell transplantation

Despite low incidence, neuroblastoma, an immunologically cold tumor, is the most common extracranial solid neoplasm in pediatrics. In relapsed/refractory cases, the benefits of autologous hematopoietic stem cell transplantation (auto-HSCT) and other therapies are limited. Natural killer (NK) cells apply cytotoxicity against tumor cells independently of antigen-presenting cells and the adaptive immune system. The primary endpoint of this trial was to assess the safety of the injection of allogenic, ex vivo-expanded and primed NK cells in relapsed/refractory neuroblastoma patients after auto-HSCT. The secondary endpoint included the efficacy of this intervention in controlling tumors. NK cells were isolated and primed ex vivo (by adding interleukin [IL]-2, IL-15, and IL-21) in a GMP-compliant CliniMACS system and administered to four patients with relapsed/refractory MYCN-positive neuroblastoma. NK cell injections (1 and 5 × 107 cells/kg in the first and second injections, respectively) were safe, and no acute or sub-acute adverse events were observed. During the follow-up period, one complete response (CR) and one partial response (PR) were observed, while two cases exhibited progressive disease (PD). In follow-up evaluations, two died due to disease progression, including the case with a PR. The patient with CR had regular growth at the 31-month follow-up, and another patient with PD is still alive and receiving chemotherapies 20 months after therapy. This therapy is an appealing and feasible approach for managing refractory neuroblastomas post-HSCT. Further studies are needed to explore its efficacy with higher doses and more frequent administrations for high-risk neuroblastomas and other immunologically cold tumors.Trial registration number: irct.behdasht.gov.ir (Iranian Registry of Clinical Trials, No. IRCT20201202049568N1).

Overexpression of H2AFX gene in neuroblastoma is associated with worse prognosis.

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 etal., 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.

1030P Survival rates in high-risk neuroblastoma patients undergoing anti-GD2 immunotherapy: A single arm meta-analysis and systemic review

1030P Survival rates in high-risk neuroblastoma patients undergoing anti-GD2 immunotherapy: A single arm meta-analysis and systemic review

The deubiquitinase USP44 enhances cisplatin chemosensitivity through stabilizing STUB1 to promote LRPPRC degradation in neuroblastoma.

Dysregulated deubiquitinating enzymes (DUBs) execute as intrinsic oncogenes or tumor suppressors and are involved in chemoresistance in cancers. However, the functions and exact molecular mechanisms remain largely unclear in neuroblastoma. Here, a R2 screening strategy based on the standard deviation values was used to identify the most important DUB, USP44, in neuroblastoma with stage 4. We validated the role of USP44 regulation upon cisplatin treatment in vitro and in vivo experiments, revealing the molecular mechanisms associated with USP44 regulation and cisplatin sensitivity in neuroblastoma. We found that low USP44 expression was associated with an inferior prognosis in neuroblastoma patients. Overexpression of USP44 enhanced neuroblastoma cell sensitivity to cisplatin in vitro and in vivo. Mechanistically, USP44 recruited and stabilized the E3 ubiquitin ligase STUB1 by removing its K48-linked polyubiquitin chains at Lys30, and STUB1 further reinforced the K48-linked polyubiquitination of LRPPRC at Lys453 and promoted its protein degradation, thus enhancing the accumulation of mitochondrial reactive oxygen species (mROS), in turn facilitating neuroblastoma cell apoptosis and cisplatin sensitivity. Additionally, overexpression of LRPPRC reversed the promoting effect of USP44 on cell apoptosis in cisplatin-treated neuroblastoma cells. Our findings demonstrate that the USP44-STUB1-LRPPRC axis plays a pivotal role in neuroblastoma chemoresistance and provides potential targets for neuroblastoma therapy and prognostication.