Neuroblastoma Patients Research Articles

Joint metabolomics and transcriptomics analysis systematically reveal the impact of MYCN in neuroblastoma

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.

Current Knowledge and Perspectives of Immunotherapies for Neuroblastoma.

Neuroblastoma (NBL) cells highly express disialoganglioside GD2, which is restricted and weakly expressed in selected healthy cells, making it a desirable target of immunotherapy. Over the past two decades, application of dinutuximab, an anti-GD2 monoclonal antibody (mAb), has been one of the few new therapies to substantially improve outcomes to current levels. Given the persistent challenge of relapse and therapeutic resistance, there is an urgent need for new effective and tolerable treatment options for high-risk NBL. Recent breakthroughs in immune checkpoint inhibitor (ICI) therapeutics have not translated into high-risk NBL, like many other major pediatric solid tumors. Given the suppressed tumor microenvironment (TME), single ICIs like anti-CTLA4 and anti-PD1 have not demonstrated significant antitumor response rates. Meanwhile, emerging studies are reporting novel advancements in GD2-based therapies, targeted therapies, nanomedicines, and other immunotherapies such as adoptive transfer of natural killer (NK) cells and chimeric antigen receptors (CARs), and these hold interesting promise for the future of high-risk NBL patient care. Herein, we summarize the current state of the art in NBL therapeutic options and highlight the unique challenges posed by NBL that have limited the successful adoption of immune-modifying therapies. Through this review, we aim to direct the field's attention to opportunities that may benefit from a combination immunotherapy strategy.

Vitronectin Levels in the Plasma of Neuroblastoma Patients and Culture Media of 3D Models: A Prognostic Circulating Biomarker?

Vitronectin is a glycoprotein present in plasma and the extracellular matrix that is implicated in cell migration. The high amount of vitronectin found in neuroblastoma biopsies has been associated with poor prognosis. Moreover, increased vitronectin levels have been described in the plasma of patients with different cancers. Our aim was to assess vitronectin as a potential circulating biomarker of neuroblastoma prognosis. Vitronectin concentration was quantified using ELISA in culture media of four neuroblastoma cell lines grown in a monolayer and in 3D models, and in the plasma of 114 neuroblastoma patients. Three of the neuroblastoma cell lines secreted vitronectin to culture media when cultured in a monolayer and 3D models. Vitronectin release was higher by neuroblastoma cells cultured in 3D models than in the monolayer and was still elevated when cells were grown in 3D scaffolds with cross-linked vitronectin. Vitronectin secretion occurred independently of cell numbers in cultures. Its concentration in the plasma of neuroblastoma patients ranged between 52.4 and 870 µg/mL (median, 218 µg/mL). A ROC curve was used to establish a cutoff of 361 µg/mL, above which patients over 18 months old had worse prognosis (p = 0.0018). Vitronectin could be considered a new plasma prognostic biomarker in neuroblastoma and warrants confirmation in collaborative studies. Drugs inhibiting vitronectin interactions with cells and/or the extracellular matrix could represent a significant improvement in survival for neuroblastoma patients.

Validating process mining in pediatric oncology: Demonstrating protocol adherence and variability in neuroblastoma treatment.

166 Background: Neuroblastoma is the most common extracranial tumor in children. The mortality rate for patients with metastatic disease is over 50%. In the Netherlands, 30 new patients are diagnosed annually. Current clinical treatment guidelines are complex and include multiple treatment arms consisting of chemotherapy, surgery, radiotherapy, and immunotherapy (IT). Although poor patient health and complications are known to cause adaptations to the otherwise standardized treatment regimen, the extent to which these variations occur is unknown. This study aimed to validate the use of process mining, a data-driven technique, to retrospectively examine protocol adherence and variability within current clinical guidelines based upon the GPOH-DCOG NBL2009 neuroblastoma treatment protocol. Methods: Data was extracted from electronic health records, as well as laboratory, pathology, and pharmaceutical databases, to create individual event logs for each patient. Process mining was performed using a fuzzy mining algorithm. The treatment courses for all neuroblastoma patients were mapped. Commonalities and variations were highlighted, and the throughput time for key treatment phases was calculated. Subsequently, conformance with protocol was determined. Results: Analysis of 70 patients treated between 2018-2022 showed 62 distinct treatment processes, revealing significant variations in patient care. Patients with high-risk disease showed the least variation (n=47, 42 variants). Notably, 17 variants were identified among 18 patients who died, indicating a highly personalized approach in the advanced stages of the disease. Subgroup analysis of 17 high-risk patients who received IT consisting of alternating cycles of anti-GD2 antibody and retinoic acid showed 6 variants. 12/17 patients (70.6%) completed the full IT regimen. Other treatment adjustments included shortened anti-GD2 infusions for two patients (5 and 8 days versus the prescribed 10 days, respectively), potentially indicating treatment toxicity. One patient received a 15-day infusion, exceeding the prescribed limit of 11 days. The recorded interval between the administration of anti-GD2 and retinoic acid was a median 5.5 hours (range: 60 seconds – 6.4 days), deviating from the prescribed interval of 24 hours and warranting further investigation. Conclusions: Process mining is a valuable tool for analyzing protocol performance and adherence in neuroblastoma treatment. Despite standardized treatment protocols, significant heterogeneity was observed in the treatment of neuroblastoma patients, especially those with poor prognosis. Throughput time analysis revealed variabilities in the IT regimen that warrant further investigation. This study underscores the potential of data-driven approaches to optimize treatment protocols and support evidence-based practices in clinical oncology.

The Pattern of Anemia in Pediatric Solid Tumors Prior to and after Chemotherapy- A Retrospective Cohort Study.

Solid pediatric tumors refer to cancers that affect children and adoles-cents, and they present unique challenges due to their distinct biological characteristics and their vulnerability to young patients. This study aims to shed light on addressing anemia and the causes of anemia in patients with solid pediatric tumors. This retrospective cohort comprised 200 healthy children as controls and 235 patients with solid tumors. The study was conducted at first Affiliated Hospital of Zhengzhou University between January 2020 and June 2023. We evaluated different parameters of blood components in controls and patients with solid tumors such as medulloblastoma, neuroblastoma, rhabdomyosarcoma, germ cell tumors, hepatoblastoma and nephroblastoma before and patients with only these tumors 3 weeks after the first cycle of chemotherapy. Further, we evaluated the relationship between serum ferritin and the weight of patients and assessed the relationship be-tween anemia and metastasis to the bone marrow in patients with neuroblastoma and hepatoblas-toma. We observed various combinations of derangements in blood parameters such as hemo-globin, red blood cells, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscu-lar hemoglobin concentration, hematocrit, red cell distribution width, white blood cells, and plate-let in medulloblastoma, neuroblastoma, rhabdomyosarcoma, germ cell tumors, hepatoblastoma and nephroblastoma before and 3 weeks after first cycle of chemotherapy. We found a significant correlation between serum ferritin levels and weight in neuroblastoma patients who are ≤ 2 years (p = 0.022). Involvement of tumor cells in bone marrow correlates with decreased Hb levels in both neuroblastoma (CI = 93.21-106.68, p = 0.001) and hepatoblastoma (CI = 113.36-121.00, p = 0.001). Anemia may manifest as an early symptom in neuroblastoma, hepatoblastoma, and nephroblastoma. Also, anemia may be worse in patients with neuroblastoma and hepatoblastoma after chemotherapy and might warrant anemia therapy.

Response-adapted consolidation therapy strategy for patients with metastatic high-risk neuroblastoma: Results of the SMC NB-2014 study.

Tandem high-dose chemotherapy and autologous stem cell transplantation (HDCT/auto-SCT) and incorporation of 131I-metaiodobenzylguanidine (131I-MIBG) treatment have shown positive outcomes in high-risk neuroblastoma. However, more optimized treatment strategies are still needed. The NB-2014 study was a nonrandomized, prospective trial that examined survival outcomes in metastatic high-risk neuroblastoma patients using response-adapted consolidation therapy. We used post-induction residual 123I-MIBG status at metastatic sites as a treatment response marker. Patients achieving complete resolution of MIBG uptake at metastatic sites underwent a reduced first HDCT/auto-SCT with a 20% dose reduction in HDCT. After the first HDCT/auto-SCT, patients with remaining MIBG uptake received dose-escalated (18mCi/kg) 131I-MIBG treatment. In contrast, those with complete resolution of MIBG at metastatic sites received a standard dose (12mCi/kg) of 131I-MIBG. We compared survival and toxicity outcomes with a historical control group from the NB-2009. Of 65 patients treated, 63% achieved complete resolution of MIBG uptake at metastatic sites following induction chemotherapy, while 29% of patients still had MIBG uptake at metastatic sites after the first HDCT/auto-SCT. The 3-year event-free survival (EFS) and overall survival (OS) rates were 68.2%±6.0% and 86.5%±4.5%, respectively. Compared to NB-2009, EFS was similar (p=.855); however, NB-2014 had a higher OS (p=.031), a lower cumulative incidence of treatment-related mortality (p=.036), and fewer acute and late toxicities. Our results suggest that response-adaptive consolidation therapy based on chemotherapy response at metastatic sites facilitates better treatment tailoring, and appears promising for patients with metastatic high-risk neuroblastoma.

Identification of a ferroptosis-related gene signature for the prognosis of pediatric neuroblastoma.

Ferroptosis-related genes are correlated with the prognosis of patients with neuroblastoma (NB) remains unknown. This study aims to establish a prognostic ferroptosis-related gene model for predicting prognostic value in pediatric NB patients. The gene expression array and clinical characteristics of NB were downloaded from a public database. Correlations between ferroptosis-related genes and drug responses were analyzed by Childhood Cancer Therapeutics. The prognostic model was constructed by least absolute shrinkage and selection operator (LASSO) Cox regression and was validated in NB patients from the ICGC cohort. The survival analysis was performed by Cox regression analysis. single-sample gene set enrichment analysis (ssGSEA) was used to quantify the immune cell infiltration correlation. Overall, 70 genes were identified as ferroptosis-related differentially expressed genes (DEGs) from 247 samples. Then, 13 ferroptosis-related genes were correlated with OS in the univariate Cox regression analysis. Five prognostic ferroptosis-related DEGs (pFR-DEGs) (STEAP3, MAP1LC3A, ULK2, MTOR and TUBE1), which were defined as the intersection of DEGs and prognostic ferroptosis-related genes, were identified and utilized to construct the prognostic signature. The correlation between five pFR-DEGs and drug responses was analyzed, and the box plots indicated that MTOR gene expression was highest, suggesting that MTOR expression is related to progressive NB disease. The receiver operating characteristic (ROC) curve showed that the model had moderate predictive power. The survival analysis indicated that the high-risk group had poor overall survival (OS) (P=2.087×10-06). Univariate and multivariate analyses identified the risk score as a significant prognostic risk factor [P=0.003, hazard ratio (HR) =1.933]. Immune cell infiltration correlation analysis showed that the high-risk group was related to more immune cells. The present study indicated a difference in ferroptosis-related gene expression between low- and high-risk NB patients. The ferroptosis-related signature could serve as a prognostic prediction tool. Additionally, immune infiltration might play an important role in different risk groups for NB patients.

A mitochondria-related genes associated neuroblastoma signature - based on bulk and single-cell transcriptome sequencing data analysis, and experimental validation.

Neuroblastoma (NB), characterized by its marked heterogeneity, is the most common extracranial solid tumor in children. The status and functionality of mitochondria are crucial in regulating NB cell behavior. While the significance of mitochondria-related genes (MRGs) in NB is still missing in key knowledge. This study leverages consensus clustering and machine learning algorithms to construct and validate an MRGs-related signature in NB. Single-cell data analysis and experimental validation were employed to characterize the pivotal role of FEN1 within NB cells. MRGs facilitated the classification of NB patients into 2 distinct clusters with considerable differences. The constructed MRGs-related signature and its quantitative indicators, mtScore and mtRisk, effectively characterize the MRGs-related patient clusters. Notably, the MRGs-related signature outperformed MYCN in predicting NB patient prognosis and was adept at representing the tumor microenvironment (TME), tumor cell stemness, and sensitivity to the chemotherapeutic agents Cisplatin, Topotecan, and Irinotecan. FEN1, identified as the most contributory gene within the MRGs-related signature, was found to play a crucial role in the communication between NB cells and the TME, and in the developmental trajectory of NB cells. Experimental validations confirmed FEN1's significant influence on NB cell proliferation, apoptosis, cell cycle, and invasiveness. The MRGs-related signature developed in this study offers a novel predictive tool for assessing NB patient prognosis, immune infiltration, stemness, and chemotherapeutic sensitivity. Our findings unveil the critical function of FEN1 in NB, suggesting its potential as a therapeutic target.

An integrated single-cell RNA-seq map of human neuroblastoma tumors and preclinical models uncovers divergent mesenchymal-like gene expression programs

BackgroundNeuroblastoma is a common pediatric cancer, where preclinical studies suggest that a mesenchymal-like gene expression program contributes to chemotherapy resistance. However, clinical outcomes remain poor, implying we need a better understanding of the relationship between patient tumor heterogeneity and preclinical models.ResultsHere, we generate single-cell RNA-seq maps of neuroblastoma cell lines, patient-derived xenograft models (PDX), and a genetically engineered mouse model (GEMM). We develop an unsupervised machine learning approach (“automatic consensus nonnegative matrix factorization” (acNMF)) to compare the gene expression programs found in preclinical models to a large cohort of patient tumors. We confirm a weakly expressed, mesenchymal-like program in otherwise adrenergic cancer cells in some pre-treated high-risk patient tumors, but this appears distinct from the presumptive drug-resistance mesenchymal programs evident in cell lines. Surprisingly, however, this weak-mesenchymal-like program is maintained in PDX and could be chemotherapy-induced in our GEMM after only 24 h, suggesting an uncharacterized therapy-escape mechanism.ConclusionsCollectively, our findings improve the understanding of how neuroblastoma patient tumor heterogeneity is reflected in preclinical models, provides a comprehensive integrated resource, and a generalizable set of computational methodologies for the joint analysis of clinical and pre-clinical single-cell RNA-seq datasets.

Neuroblastoma susceptibility and association of N7-methylguanosine modification gene polymorphisms: multi-center case-control study.

Neuroblastoma (NB) is a common extracranial solid malignancy in children. The N7-methylguanosine (m7G) modification gene METTL1/WDR4 polymorphisms may serve as promising molecular markers for identifying populations susceptible to NB. TaqMan probes was usded to genotype METTL1/WDR4 single nucleotide polymorphisms (SNPs) in 898 NB patients and 1734 healthy controls. A logistic regression model was utilized to calculate the odds ratio (OR) and 95% confidence interval (CI), evaluating the association between genotype polymorphisms and NB susceptibility. The analysis was also stratified by age, sex, tumor origin site, and clinical stage. Individual polymorphism of the METTL1/WDR4 gene investigated in this study did not show significant associations with NB susceptibility. However, combined genotype analysis revealed that carrying all 5 WDR4 protective genotypes was associated with a significantly lower NB risk compared to having 0-4 protective genotypes (AOR = 0.82, 95% CI = 0.69-0.96, P = 0.014). Further stratified analyses revealed that carrying 1-3 METTL1 risk genotypes, the WDR4 rs2156316 CG/GG genotype, the WDR4 rs2248490 CG/GG genotype, and having all five WDR4 protective genotypes were all significantly correlated with NB susceptibility in distinct subpopulations. In conclusion, our findings suggest significant associations between m7G modification gene METTL1/WDR4 SNPs and NB susceptibility in specific populations. Genetic variation in m7G modification gene is associated with susceptibility to NB. Single nucleotide polymorphisms in METTL1/WDR4 are associated with susceptibility to NB. Single nucleotide polymorphisms of METTL1/WDR4 can be used as a biomarker for screening NB susceptible populations.

An Enzymatically Activated and Catalytic Hairpin Assembly-Driven Intelligent AND-Gated DNA Network for Tumor Molecular Imaging.

Due to the potential off-tumor signal leakage and limited biomarker content, there is an urgent need for stimulus-responsive and amplification-based tumor molecular imaging strategies. Therefore, two tetrahedral framework DNA (tFNA-Hs), tFNA-H1AP, and tFNA-H2, were rationally engineered to form a polymeric tFNA network, termed an intelligent DNA network, in an AND-gated manner. The intelligent DNA network was designed for tumor-specific molecular imaging by leveraging the elevated expression of apurinic/apyrimidinic endonuclease 1 (APE1) in tumor cytoplasm instead of normal cells and the high expression of miRNA-21 in tumor cytoplasm. The activation of tFNA-H1AP can be achieved through specific recognition and cleavage by APE1, targeting the apurinic/apyrimidinic site (AP site) modified within the stem region of hairpin 1 (H1AP). Subsequently, miRNA-21 facilitates the hybridization of activated H1AP on tFNA-H1AP with hairpin 2 (H2) on tFNA-H2, triggering a catalytic hairpin assembly (CHA) reaction that opens the H1AP at the vertices of tFNA-H1AP to bind with H2 at the vertices of tFNA-H2 and generate fluorescence signals. Upon completion of hybridization, miRNA-21 is released, initiating the subsequent cycle of the CHA reaction. The AND-gated intelligent DNA network can achieve specific tumor molecular imaging in vivo and also enables risk stratification of neuroblastoma patients.

Clinical application of individualized tumor-informed circulating tumor DNA for therapeutic response and relapse prediction in patients with neuroblastoma.

10053 Background: Patients with high-risk neuroblastoma (NB) generally present with widely metastatic disease and often relapse despite intensive therapy. At present, circulating tumor DNA (ctDNA)-based minimal residual disease (MRD) has been studied in various tumors and proven to be valuable in guiding treatment and predicting recurrence, but its application in NB has rarely been reported. This study aimed to assess the effectiveness of tumor-informed ctDNA monitoring for evaluating the response to induction therapy in NB patients, as well as its role in predicting the risk of relapse. Methods: We conducted a prospective cohort study of NB patients. Primary tumor samples were collected at baseline and blood samples were collected sequentially from baseline until disease progression or the last follow-up. Tumor tissue samples were subjected to whole exome sequencing (WES), and clonal mutations were selected for the individualized MRD panel. Sequencing was conducted at OrigiMed, a College of American Pathologists accredited and Clinical Laboratory Improvement Amendments certified laboratory. Results: Our cohort included 43 individuals diagnosed with NB, with a median age of 3.5 years (0.42-40 years). WES revealed that 75% (2054/2753) of the mutated genes were unique to each patient, and 98.9% (704/712) of the selected tumor-informed single nucleotide variants were variants of unknown significance, suggesting that individualized tumor-informed MRD (n = 23) is superior to fixed tumor-agnostic panel (n = 20) MRD in NB. A successful individualized MRD panel was constructed for 53.5% (23/43) of patients, with a remarkable 92.3% success rate for those older than 5 years (p < 0.01). The initial positivity rate for ctDNA detection was 68%, with the initial positivity rate being associated with the stage (p < 0.01). During treatment and follow-up, 0/6 of the patients whose ctDNA levels remained negative experienced a relapse, whereas 2/5 patients who had a negative conversion or an increase in positivity experienced a relapse. In addition, ctDNA changes matched radiological evaluations in 94% of patients. Notably, ctDNA was detected prior to radiological relapse, with a lead time of 6 months in one patient. Patients can be divided into four groups according to the mutation landscape: ALK mutation, MYCN amplification, chromosome amplification, and other groups. Among them, the rate of ctDNA clearance was the highest in the chromosome amplification group, indicating that patients in this group were the most sensitive to chemotherapy. Conclusions: Individualized tumor-informed ctDNA monitoring shows promise for evaluating therapeutic response and forecasting relapse in NB patients. This approach proves more effective than fixed tumor-agnostic panel-based MRD due to high genetic mutation uniqueness. Clinical trial information: NCT05076071 .

Efficacy and safety of the use of dinutuximab in newly diagnosed or relapse high risk neuroblastoma: Single arm systematic review and meta-analysis.

10044 Background: Patients with high-risk Neuroblastoma have a poor prognosis, with 43% experiencing a 3-year survival rate in newly diagnosed cases and a 21% 1-year event-free survival in relapsed patients. Dinutuximab, an immunotherapy targeting anti-disialoganglioside (GD2), emerges as a promising treatment for high-risk Neuroblastoma. This single arm meta-analysis aims to assess the efficacy and safety of dinutuximab, providing insights into its impact on survival rates among neuroblastoma patients. Methods: We systematically searched Pubmed, Cochrane and EMBASE for clinical trials between 1988 to 2023 utilizing Dinutuximab (ch14.18 or ch14.18/CHO) in patients with newly diagnosed or relapsed/refractory high-risk neuroblastoma. We pooled the prevalence and the 95% confidence intervals (CI) for the outcomes of interest: overall survival (OS), event-free survival (EFS) and progression-free survival (PFS), objective response rate (ORR), and treatment-emergent adverse events (TEAE). Heterogeneity was assessed using I² analysis and all statistical analyses were employed using R software version 4.3.2 and a random-effects model. Results: The analysis included 2214 patients from 13 clinical trials, comprising four phase III trials and six phase II. Six studies included Interleukin 2 (IL-2). Among the patients, 364 (16.5%) had relapsed or refractory disease. Dinutuximab was applied as post-consolidation treatment in 1809 patients (81.1%). The age of patients ranged from 1 to 21 years. In a pooled analysis, the 3-year OS rate was 69% (CI 0.62-0.76; I² = 85%), and the 3-year EFS rate was 61% (CI 0.52-0.69; I² = 77%). In the group of newly diagnosed patients, the 3-year EFS rate was 62% (CI 0.58-0.66; I² = 52%) and 3-year OS rate of 74% (CI 0.69-0.79; I² = 71%). Among patients with relapse, there was a 3-year OS rate of 57% (CI 0.50-0.64; I² = 0%). The overall ORR was 47% (CI 0.37-0.57; I² = 69%), of which 38 newly diagnosed patients using Dinutuximab during induction chemotherapy showed an ORR of 86%. TEAEs of grade ≥ 3 were 70% (CI 0.44-0.88; I² = 94%), and in the subgroup without IL-2 use, it was 43% (CI 0.29-0.57; I² = 85%). Conclusions: This single-arm meta-analysis suggests that both newly diagnosed or relapsed/refractory high-risk Neuroblastoma patients benefit from Dinutuximab, with a promising impact on 3-year OS and EFS. Dinutuximab appears to be a safer option; however, concomitant use of IL2 is associated with a higher rate of adverse effects.

Overall survival prediction and risk stratification in patients with neuroblastoma through machine learning in the large multi-institutional PRIMAGE cohort.

10054 Background: Neuroblastoma (NB) is the most prevalent solid cancer in childhood in which imaging plays a pivotal role at every step of the patient's journey. This investigation aimed to develop a machine learning model using clinical, molecular (MycN amplification), and magnetic resonance (MR) radiomics features at diagnosis to predict patient’s overall survival (OS) and improve their risk stratification. Methods: A database comprising clinical, molecular, and International Neuroblastoma Risk Group (INRG) staging on 513 patients with accessible MR imaging (discovery cohort) was employed for model training, validation, and testing. An additional 22 patients from non-discovery cohort hospitals served as an external validation group. Tumor segmentations of NB were manually and semi-automatically performed on corresponding T2-weighted MR images by an experienced radiologist. In total, 107 radiomics features were extracted and harmonized across MR scan manufacturers and magnetic field strengths using the nested ComBat methodology to correct both batch effects. These radiomics features, combined with clinical and molecular data, were utilized as input for the models. A nested cross-validation approach was employed for model development to determine the optimal preprocessing, machine learning algorithm, and model configuration. Results: The discovery cohort yielded a C-index of 0.788 ± 0.049 in the test partitions, with a random survival forest (RSF) exhibiting the best performance. In the validation cohort, a C-index of 0.934 was achieved. Interpretability analysis identified lesion heterogeneity, size, and molecular variables as crucial factors in OS prediction. The model demonstrated superior predictive performance and patient stratification compared to conventional staging systems in both cohorts. Conclusions: The RSF predictive model exhibited high performance, emphasizing the significant contribution of radiomics features and alignment with established clinical and molecular variables. The model stratified NB patients into low-, intermediate-, and high-risk categories and suggests that radiomics features potentially could improve current risk stratification systems. Additional external validation is warranted as this may present new evidence for enhancing patient care and clinical decision-making for NB patients.

Preliminary experience of ex-vivo expanded allogeneic universal donor TGFβi NK cell infusions in combination with irinotecan, temozolomide, and dinutuximab in patients with relapsed or refractory neuroblastoma: The Allo - STING trial.

10005 Background: Neuroblastoma (NBL) is the most common extra-cranial solid tumor in children and outcomes for children with relapsed/refractory (RR) NBL remain dismal. While the combination of chemoimmunotherapy (CIT) with the anti-GD2 antibody dinutuximab as per COG protocol ANBL1221 has improved responses, this regimen is still not curative. Dinutuximab can recruit NK cells for anticancer activity, however, NK cells are depleted in NBL patients undergoing chemotherapy. We devised a method to expand allogeneic NK cells ex vivo to improve effector function and render the NK cells resistant to TGFβ-induced suppression (TGFβi). These TGFβi NK cells are expanded from a universal donor (UD) pool, enabling multiple cycles of “off-the-shelf” high-dose therapy. We hypothesize that adoptive transfer of UD-TGFβi NK cells to patients with RR NBL sequentially following CIT is safe and improves outcomes compared to CIT alone. Methods: This Phase I/II study evaluates the safety and tolerability of UD-TGFβi NK cells in combination with CIT and compares anti-cancer efficacy to CIT alone. Eligibility includes age < 30 years, RR or progressive NBL, and prior treatment with at least 4 cycles of induction chemotherapy. The treatment protocol consists of 21-day cycles of CIT as per COG protocol ANBL1221 with UD-TGFβi NK cells (1x108 cells/kg) infused on day 8. Up to six cycles of treatment are permitted. Overall response rates are defined using the Revised International NBL Response Criteria, with 95% confidence intervals compared to the results from ANBL1221. Progression-free and overall survival will be estimated by the Kaplan Meier method. Results: Four subjects (7 – 11 years) have been enrolled. All patients presented with high risk NBL: three with relapsed disease and one with refractory disease. All received at least one prior salvage therapy. Three patients have completed all 6 cycles of protocol therapy, and one remains on treatment. 20 NK cell infusions have been administered to date. All NK cell infusions occurred within the protocol specified window. One subject experienced grade 1 fever with two NK cell infusions. There have been no other adverse events attributable to the NK cells. Adverse events attributable to CIT were similar to those described in COG ANBL 1221. Treatment delays for cytopenias due to CIT occurred in two patients. Three patients achieved a partial response. A fourth patient attained prolonged stable disease (9 months) but experienced a skeletal recurrence. Conclusions: UD allo-TGFβi NK cells can be safely and feasibly administered to children with RR NBL after treatment with CIT, with early objective responses observed in the preliminary cohort of patients. Ongoing studies will assess markers of response, NK cell persistence, pharmacokinetics and pharmacodynamics. Clinical trial information: NCT04211675 .

Sarcopenia with decreased total psoas muscle area in children with high-risk neuroblastoma

BackgroundWe calculated psoas muscle area (PMA) z-scores in high-risk neuroblastoma patients undergoing treatment to examine the clinical significance of sarcopenia in this cohort. MethodsWe analyzed retrospective data from patients aged 0–18 who were diagnosed with abdominal neuroblastoma between 2005 and 2019 at Samsung Medical Center. Patients categorized as high-risk undergone induction chemotherapy, neuroblastoma excision, and tandem high-dose chemotherapy with autologous stem cell transplantation (HDCT/auto-SCT) were selected. L3-4 lumbar levels on axial CT images were identified and we measured the areas of the left and right psoas muscles to determine tPMA. Total PMA z-scores were calculated using an open online tool. ResultsThere were 45 boys and 25 girls with a mean age of 3.86 years. CT images taken at initial diagnosis and after tandem HDCT/auto-SCT were selected to calculate tPMA z-scores. Mean elapsed time between the two measurements was 12.91 ± 1.73 months. Mean tPMA z-score significantly decreased from −0.21 ± 1.29 to −0.66 ± 0.97 (p = 0.022). Length of hospital stay was significantly longer in the group of patients whose tPMA z-scores decreased by more than .45 (177.62 ± 28.82 days vs. 165.75 ± 21.34 days, p = 0.049). Presence of sarcopenia at initial diagnosis was a significant risk factor for bacterial infection during neuroblastoma treatment.Conclusion: tPMA z-scores in high-risk neuroblastoma patients decreased significantly following a treatment regimen that included induction chemotherapy, tumor resection surgery, and HDCT/auto-SCT. A greater decrease in tPMA z-score was associated with longer hospital stay during treatment.

The relationship between contrast-enhanced computed tomography radiomics features and mitosis karyorrhexis index in neuroblastoma

ObjectiveMitosis karyorrhexis index (MKI) can reflect the proliferation status of neuroblastoma cells. This study aimed to investigate the contrast-enhanced computed tomography (CECT) radiomics features associated with the MKI status in neuroblastoma.Materials and methods246 neuroblastoma patients were retrospectively included and divided into three groups: low-MKI, intermediate-MKI, and high-MKI. They were randomly stratified into a training set and a testing set at a ratio of 8:2. Tumor regions of interest were delineated on arterial-phase CECT images, and radiomics features were extracted. After reducing the dimensionality of the radiomics features, a random forest algorithm was employed to establish a three-class classification model to predict MKI status.ResultsThe classification model consisted of 5 radiomics features. The mean area under the curve (AUC) of the classification model was 0.916 (95% confidence interval (CI) 0.913–0.921) in the training set and 0.858 (95% CI 0.841–0.864) in the testing set. Specifically, the classification model achieved AUCs of 0.928 (95% CI 0.927–0.934), 0.915 (95% CI 0.912–0.919), and 0.901 (95% CI 0.900–0.909) for predicting low-MKI, intermediate-MKI, and high-MKI, respectively, in the training set. In the testing set, the classification model achieved AUCs of 0.873 (95% CI 0.859–0.882), 0.860 (95% CI 0.852–0.872), and 0.820 (95% CI 0.813–0.839) for predicting low-MKI, intermediate-MKI, and high-MKI, respectively.ConclusionsCECT radiomics features were found to be correlated with MKI status and are helpful for reflecting the proliferation status of neuroblastoma cells.

Distinct Expression Profiles of Neuroblastoma-Associated mRNAs in Peripheral Blood and Bone Marrow of Non-High-Risk and High-Risk Neuroblastoma Patients.

Non-high-risk (non-HR) neuroblastoma (NB) patients have excellent outcomes, with more than a 90% survival rate, whereas HR NB patients expect less than a 50% survival rate. Metastatic disease is the principal cause of death among both non-HR and HR NB patients. Previous studies have reported the significant but limited prognostic value of quantitative PCR (qPCR)-based assays, measuring overlapping but different sets of neuroblastoma-associated mRNAs (NB-mRNAs), to detect metastatic disease in both non-HR and HR patient samples. A droplet digital PCR (ddPCR)-based assay measuring seven NB-mRNAs (CRMP1, DBH, DDC, GAP43, ISL1, PHOX2B, and TH mRNAs) was recently developed and exhibited a better prognostic value for HR patient samples than qPCR-based assays. However, it remained to be tested on non-HR patient samples. In the present study, we employed the ddPCR-based assay to study peripheral blood (PB) and bone marrow (BM) samples collected at diagnosis from eight non-HR and eleven HR cases and characterized the expression profiles of NB-mRNAs. The most highly expressed NB-mRNAs in PB and BM differed between non-HR and HR cases, with the CRMP1 mRNA being predominant in non-HR cases and the GAP43 mRNA in HR cases. The levels of NB-mRNAs in PB and BM were 5 to 1000 times lower in non-HR cases than in HR cases. The PB to BM ratio of NB-mRNAs was 10 to 100 times higher in non-HR cases compared to HR cases. The present case series suggests that non-HR and HR NB patients have the distinct expression profiles of NB-mRNAs in their PB and BM.

Dual roles of HK3 in regulating the network between tumor cells and tumor-associated macrophages in neuroblastoma

Neuroblastoma (NB) is the most common and deadliest extracranial solid tumor in children. Targeting tumor-associated macrophages (TAMs) is a strategy for attenuating tumor-promoting states. The crosstalk between cancer cells and TAMs plays a pivotal role in mediating tumor progression in NB. The overexpression of Hexokinase-3 (HK3), a pivotal enzyme in glucose metabolism, has been associated with poor prognosis in NB patients. Furthermore, it correlates with the infiltration of M2-like macrophages within NB tumors, indicating its significant involvement in tumor progression. Therefore, HK3 not only directly regulates the malignant biological behaviors of tumor cells, such as proliferation, migration, and invasion, but also recruits and polarizes M2-like macrophages through the PI3K/AKT-CXCL14 axis in neuroblastoma. The secretion of lactate and histone lactylation alterations within tumor cells accompanies this interaction. Additionally, elevated expression of HK3 in M2-TAMs was found at the same time. Modulating HK3 within M2-TAMs alters the biological behavior of tumor cells, as demonstrated by our in vitro studies. This study highlights the pivotal role of HK3 in the progression of NB malignancy and its intricate regulatory network with M2-TAMs. It establishes HK3 as a promising dual-functional biomarker and therapeutic target in combating neuroblastoma.

CD20highCD138low tumor-infiltrating lymphocytes predominantly related to cytokine‒cytokine receptor interactions are associated with favorable outcomes in neuroblastoma patients

Recent advances have revealed that the role of the immune system is prominent in the antitumor response. In the present study, it is aimed to provide an expression profile of tumor-infiltrating lymphocytes (TILs), including mature B cells, plasma cells, and their clinical relevance in neuroblastoma. The expression of CD20 and CD138 was analyzed in the Cangelosi786 dataset (n = 769) as a training dataset and in our cohort (n = 120) as a validation cohort. CD20 high expression was positively associated with favorable overall survival (OS) and event-free survival (EFS) (OS: P < 0.001; EFS: P < 0.001) in the training dataset, whereas CD138 high expression was associated with poor OS and EFS (OS: P < 0.001; EFS: P < 0.001) in both the training and validation datasets. Accordingly, a combined pattern of CD20 and CD138 expression was developed, whereby neuroblastoma patients with CD20highCD138low expression had a consistently favorable OS and EFS compared with those with CD20lowCD138high expression in both the training and validation cohorts (P < 0.0001 and P < 0.01, respectively). Examination of potential molecular functions revealed that signaling pathways, including cytokine‒cytokine receptor interactions, chemokine, and the NF-kappa B signaling pathways, were involved. Differentially expressed genes, such as BMP7, IL7R, BIRC3, CCR7, CXCR5, CCL21, and CCL19, predominantly play important roles in predicting the survival of neuroblastoma patients. Our study proposes that a new combination of CD20 and CD138 signatures is associated with neuroblastoma patient survival. The related signaling pathways reflect the close associations among the number of TILs, cytokine abundance and patient outcomes and provide therapeutic insights into neuroblastoma.