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Background: Risk assessment at diagnosis is crucial for neuroblastoma (NB) in order to address patients at high-risk to the most timely and appropriate treatments. 3-O-methyldopa (3-OMD), a direct metabolite of L-Dopa, is a promising biomarker of NB at diagnosis able to stratify high-risk patients., Methods: We show the development and validation of a method for measuring 3-OMD from dried plasma samples (DPS) and plasma using liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) on a Thermo Fisher Scientific Orbitrap Exploris 120., Results: The method was accurate and reproducible in the range 7.8-4000 ng/mL, from small amounts (50 mL) of plasma and DPS (obtained starting from 30 mL plasma). 3-OMD concentrations measured in plasma and DPS were highly correlated (R = 0.99 95 %CI 0.993-0.996). Differences of 3-OMD levels across stages L1 and M and L1 and L2 (p-value < 0.05) were statistically significant. Receiving Operator Curve (ROC) analysis showed that 3-OMD was able to discriminate patients at high-risk with high sensitivity and specificity both from plasma or DPS (AUC = 0.8295 %CI 0.71-0.94, P < 0.0001)., Conclusions: 3-OMD is confirmed as an interesting biomarker of high-risk NB. The described method is an added value for further prospective studies involving multiple sites. The stability of 3-OMD in DPS allows for easy shipment and storage at room temperature., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: 131 I-metaiodobenzylguanidine ( 131 I-mIBG) effectiveness in children with metastasised neuroblastoma (NB) is linked to the effective dose absorbed by the target; a target of 4 Gy whole-body dose threshold has been proposed. Achieving this dose often requires administering 131 I-mIBG twice back-to-back, which may cause haematological toxicity. In this study, we tried identifying the factors predicting the achievement of 4 Gy whole-body dose with a single radiopharmaceutical administration., Materials and Methods: Children affected by metastatic NB and treated with a high 131 I-mIBG activity (>450 MBq (megabecquerel)/kg) were evaluated retrospectively. Kinetics measurements were carried out at multiple time points to estimate the whole-body dose, which was compared with clinical and activity-related parameters., Results: Seventeen children (12 females, median age 3 years, age range: 1.5-6.9 years) were included. Eleven of them still bore the primary tumour. The median whole-body dose was 2.88 Gy (range: 1.63-4.22 Gy). Children with a 'bulky' primary (>30 mL) received a higher whole-body dose than those with smaller or surgically removed primaries (3.42 ± 0.74 vs. 2.48 ± 0.65 Gy, respectively, p = .016). Conversely, the correlation between activity/kg and the whole-body dose was moderate (R: 0.42, p = .093). In the multivariate analysis, the volume of the primary tumour was the most relevant predictor of the whole-body dose (p = .002)., Conclusions: These data suggest that the presence of a bulky primary tumour can significantly prolong the 131 I-mIBG biological half-life, effectively increasing the absorbed whole-body dose. This information could be used to model the administered activity, allowing to attain the target dose without needing a two-step radiopharmaceutical administration., (© 2023 Wiley Periodicals LLC.)

Background: 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., Methods: 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., Results: 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., Conclusions: 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., (© 2024. The Author(s).)

Background: Neuroblastoma is the most frequent extracranial solid tumour in children, accounting for ∼15% of deaths due to cancer in childhood. The most common clinical presentation are abdominal tumours. An altered gut microbiome composition has been linked to multiple cancer types, and reported in murine models of neuroblastoma. Whether children with neuroblastoma display alterations in gut microbiome composition remains unexplored., Methods: We assessed gut microbiome composition by shotgun metagenomic profiling in an observational cross-sectional study on 288 individuals, consisting of patients with a diagnosis of neuroblastoma at disease onset (N = 63), healthy controls matching the patients on the main covariates of microbiome composition (N = 94), healthy siblings of the patients (N = 13), mothers of patients (N = 59), and mothers of the controls (N = 59). We examined taxonomic and functional microbiome composition and mother-infant strain transmission patterns., Findings: Patients with neuroblastoma displayed alterations in gut microbiome composition characterised by reduced microbiome richness, decreased relative abundances of 18 species (including Phocaeicola dorei and Bifidobacterium bifidum), enriched protein fermentation and reduced carbohydrate fermentation potential. Using machine learning, we could successfully discriminate patients from controls (AUC = 82%). Healthy siblings did not display such alterations but resembled the healthy control group. No significant differences in maternal microbiome composition nor mother-to-offspring transmission were detected., Interpretation: Patients with neuroblastoma display alterations in taxonomic and functional gut microbiome composition, which cannot be traced to differential maternal seeding. Follow-up research should include investigating potential causal links., Funding: Italian Ministry of Health Ricerca Corrente and Ricerca Finalizzata 5 per mille (to MPonzoni); Fondazione Italiana Neuroblastoma (to MPonzoni); European Research Council (ERC-StG project MetaPG-716575 and ERC-CoG microTOUCH-101045015 to NS); the European H2020 program ONCOBIOME-825410 project (to NS); the National Cancer Institute of the National Institutes of Health 1U01CA230551 (to NS); the Premio Internazionale Lombardia e Ricerca 2019 (to NS); the MIUR Progetti di Ricerca di Rilevante Interesse Nazionale (PRIN) Bando 2017 Grant 2017J3E2W2 (to NS); EMBO ALTF 593-2020 and Knowledge Generation Project from the Spanish Ministry of Science and Innovation (PID2022-139328OA-I00) (to MV-C)., Competing Interests: Declaration of interests The authors report no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: Seek new candidate prognostic markers for neuroblastoma outcome, relapse or progression., Materials and Methods: In this multicentre and retrospective study, Random Forests coupled with recursive feature elimination techniques were applied to electronic records (55 clinical features) of 3034 neuroblastoma patients. To assess model performance and feature importance, dataset was split into a training set (80%) and a test set (20%)., Results: In the test set, the mean Matthews correlation coefficient for the Random Forests models was greater than 0.46. Feature importance analysis revealed that, together with maximum response to first-line treatment (D&#95;MAX&#95;RESP), time to maximum response to first-line treatment (TIME&#95;MAX&#95;RESP.days) is a relevant predictor of both patients' outcome and relapse\progression. We showed the prognostic value of the max response to first-line treatment in clinically relevant subsets of high-, intermediate-, and low-risk patients for both overall and relapse-free survival (Log-rank p-value<0.0001). In high-risk patients older than 18 months and stage 4 tumour achieving a complete response or very good partial response, patients who exhibited a D&#95;MAX&#95;RESP greater than 9 months showed a better prognosis with respect to patients achieving D&#95;MAX&#95;RESP earlier than 9 months (overall survival): hazard ratio 3.3 95% confidence interval 1.8-5.9, Log-rank p-value p < 0.0001; relapse-free survival: 3.2 95%CI 1.8-5.6, Log-rank p-value p < 0.0001)., Conclusion: Our findings evidence the emerging role of the TIME&#95;MAX&#95;RESP.days in addition to the D&#95;MAX&#95;RESP as relevant predictors of outcome and relapse\progression in neuroblastoma with potential clinical impact on the management and treatment of patients., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Neuroblastoma (NB) is the most common extracranial solid tumor during infancy, causing up to 10% of mortality in children; thus, identifying novel early and accurate diagnostic and prognostic biomarkers is mandatory. NB-derived exosomes carry proteins (Exo-prots) reflecting the status of the tumor cell of origin. The purpose of this study was to characterize, for the first time, the Exo-prots specifically expressed in NB patients associated with tumor phenotype and disease stage. We isolated exosomes from plasma specimens of 24 HR-NB patients and 24 low-risk (LR-NB) patients at diagnosis and of 24 age-matched healthy controls (CTRL). Exo-prot expression was measured by liquid chromatography-mass spectrometry. The data are available via ProteomeXchange (PXD042422). The NB patients had a different Exo-prot expression profile compared to the CTRL. The deregulated Exo-prots in the NB specimens acted mainly in the tumor-associated pathways. The HR-NB patients showed a different Exo-prot expression profile compared to the LR-NB patients, with the modulation of proteins involved in cell migration, proliferation and metastasis. NCAM, NCL, LUM and VASP demonstrated a diagnostic value in discriminating the NB patients from the CTRL; meanwhile, MYH9, FN1, CALR, AKAP12 and LTBP1 were able to differentiate between the HR-NB and LR-NB patients with high accuracy. Therefore, Exo-prots contribute to NB tumor development and to the aggressive metastatic NB phenotype.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Patients with relapsed/refractory disease have a poor prognosis, and additional therapeutic options are needed. Mutations and amplifications in the ALK (Anaplastic Lymphoma Kinase) gene constitute a key target for treatment. Our goal, within the Italian project of PeRsonalizEdMEdicine (PREME), was to evaluate the genomic status of patients with relapsed/refractory NB and to implement targeted therapies in those with targetable mutations. From November 2018 to November 2021, we performed Whole Exome Sequencing or Targeted Gene Panel Sequencing in relapsed/refractory NB patients in order to identify druggable variants. Activating mutations of ALK were identified in 8(28.57%) of 28 relapsed/refractory NB patients. The mutation p.F1174L was found in six patients, whereas p.R1275Q was found in one and the unknown mutation p.S104R in another. Three patients died before treatment could be started, while five patients received crizotinib: two in monotherapy (one with p.F1174L and the other with p.S104R) and three (with p.F1174L variant) in combination with chemotherapy. All treated patients showed a clinical improvement, and one had complete remission after two cycles of combined treatment. The most common treatment-related toxicities were hematological. ALK inhibitors may play an important role in the treatment of ALK-mutated NB patients.

Purpose: To evaluate the prognostic value of texture analysis of the primary tumour with 18 fluorine-dihydroxyphenylalanine positron emission tomography/X-ray computed tomography ( 18 F-DOPA PET/CT) in patients affected by high-risk neuroblastoma (HR-NBL)., Methods: We retrospectively analysed 18 patients with HR-NBL, which had been prospectively enrolled in the course of a previous trial investigating the diagnostic role of 18 F-DOPA PET/CT at the time of the first onset. Texture analysis of the primary tumour was carried out on the PET images using LifeX. Conventional indices, histogram parameters, grey level co-occurrence (GLCM), run-length (GLRLM), neighbouring difference (NGLDM) and zone-length (GLZLM) matrices parameter were extracted; their values were compared with the overall metastatic load, expressed by means of whole-body metabolic burden (WBMB) score and the progression-free/overall survival (PFS and OS)., Results: There was a direct correlation between WBMB and radiomics parameter describing uptake intensity (SUV mean : p = .004) and voxel heterogeneity (entropy: p = .026; GLCM&#95;Contrast: p = .001). Conversely, texture indices of homogeneity showed an inverse correlation with WBMB (energy: p = .026; GLCM&#95;Homogeneity: p = .006). On the multivariate model, WBMB (p < .01) and the first standardised uptake value (SUV) quartile (p < .001) predicted PFS; OS was predicted by WBMB and the N-myc proto-oncogene protein (MYCN) amplification (p < .05) for both., Conclusions: Textural parameters describing heterogeneity and metabolic intensity of the primary HR-NBL are closely associated with its overall metastatic burden. In turn, the whole-body tumour load appears to be one of the most relevant predictors of progression-free and overall survival., (© 2022 Wiley Periodicals LLC.)

Background: Neuroblastoma (NB) represents the most frequent form of extra-cranial solid tumour of infants, responsible for 15% of childhood cancer deaths. Nucleolin (NCL) prognostic value in NB was investigated., Methods: NCL protein expression was retrospectively evaluated in tumour samples of NB patients at diagnosis and after chemotherapy. NCL prognostic value at mRNA level was assessed in a cohort of 20 patients with stage 4 NB (qPCR20, n=20, discovery dataset) and in the MultiPlatform786 including 786 patients of all stages (validation dataset). Overall and event-free survival curves were plotted by Kaplan-Meier method and compared by log-rank test., Findings: NCL protein, down-modulated after chemotherapy in association with features of neuroblastic differentiation,resulted statistically significantly overexpressed in NB tumours and higher in stage 4 compared to stage 1,2,3 patients. In the stage 4 patients cohort qPCR20, patients with high NCLmRNA expression revealed a statisticallysignificant lower survival probability than those with low NCL expression (OS: HR 4.1 95%CI 1.2-13.8;p=0.0215[Log-rank test], EFS: HR 4.1 95%CI 1.2-14.0, p=0.0197[Log-rank test]). In the MultiPlatform786 (n=786), multivariate analysis suggested thatNCL expression has a statistically significant prognostic value even in the model adjusted for established prognostic markers. NCL expression significantly stratified also patients with >18 months and stage 4 tumour (OS: HR 1.8 95%CI 1.2-2.7, p=0.0009[Log-rank test]; EFS: HR 1.7 95%CI 1.1-2.5, p=0.002[Log-rank test]), patients with>18 months stage 4 with MYCN non amplified tumour[EFS: HR 2.3 95%CI 1.2-4.7, p=0.01[Log-rank test]), and patients with MYCN non amplified and MYC high [OS: HR 11.9 95%CI 2.3-62.4, p=0.003[Log-rank test]; EFS: HR 7.2 95%CI 1.6-33.4, p=0.01[Log-rank test]). A statistically significant correlation between NCL and MYCN, MYC, and TERT was found in independent datasets (MultiPlatform786 (n=786) and Agilent394 (n=394). Gene set enrichment analysis revealed a statisticallysignificant positive enrichment of MYC target genes and genes involved in telomerase maintenance., Interpretation: NCL is a novel and independent (adjusting for age, INSS stage, and MYCN status) prognostic marker for NB., Funding: IMH-EuroNanoMed II-2015 and AIRC-IG., Competing Interests: Declaration of interests JNM is share-holder of TREAT U, SA. MC is Advisory Board Eusapharma member. The remaining authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Rationale: Therapy response evaluation by 18F-fluorodeoxyglucose PET/CT (FDG PET) has become a powerful tool for the discrimination of responders from non-responders in pediatric Hodgkin lymphoma (HL). Recently, volumetric analyses have been regarded as a valuable tool for disease prognostication and biological characterization in cancer. Given the multitude of methods available for volumetric analysis in HL, the AIEOP Hodgkin Lymphoma Study Group has designed a prospective analysis of the Italian cohort enrolled in the EuroNet-PHL-C2 trial. Methods: Primarily, the study aimed to compare the different segmentation techniques used for volumetric assessment in HL patients at baseline (PET1) and during therapy: early (PET2) and late assessment (PET3). Overall, 50 patients and 150 scans were investigated for the current analysis. A dedicated software was used to semi-automatically delineate contours of the lesions by using different threshold methods. More specifically, four methods were applied: (1) fixed 41% threshold of the maximum standardized uptake value (SUVmax) within the respective lymphoma site (V41%), (2) fixed absolute SUV threshold of 2.5 (V2.5); (3) SUVmax(lesion)/SUVmean liver >1.5 (Vliver); (4) adaptive method (AM). All parameters obtained from the different methods were analyzed with respect to response. Results: Among the different methods investigated, the strongest correlation was observed between AM and Vliver (rho > 0.9; p < 0.001 for SUVmean, MTV and TLG at all scan timing), along with V2.5 and AM or Vliver (rho 0.98, p < 0.001 for TLG at baseline; rho > 0.9; p < 0.001 for SUVmean, MTV and TLG at PET2 and PET3, respectively). To determine the best segmentation method, we applied logistic regression and correlated different results with Deauville scores at late evaluation. Logistic regression demonstrated that MTV (metabolic tumor volume) and TLG (total lesion glycolysis) computation according to V2.5 and Vliver significantly correlated to response to treatment (p = 0.01 and 0.04 for MTV and 0.03 and 0.04 for TLG, respectively). SUVmean also resulted in significant correlation as absolute value or variation. Conclusions: The best correlation for volumetric analysis was documented for AM and Vliver, followed by V2.5. The volumetric analyses obtained from V2.5 and Vliver significantly correlated to response to therapy, proving to be preferred thresholds in our pediatric HL cohort.

Background: Brentuximab vedotin (BV) is an antibody drug-conjugated anti-CD30 approved for the treatment of adult classical Hodgkin's lymphoma (HL), whereas it is considered as off-label indication in paediatrics. The aim of the study was to evaluate the safety and efficacy of BV to treat patients aged less than 18 years with refractory/relapsed HL., Materials and Methods: In this multicentre, retrospective study, 68 paediatric patients who received at least one dose of BV between November 2011 and August 2020 were enrolled. A median of nine doses of BV were administered as monotherapy (n = 31) or combined with other therapies (n = 37). BV was administrated alone as consolidation therapy after stem cell transplantation (SCT) in 12 patients, before SCT in 18 patients, whereas in 15 patients it was used before and after SCT as consolidation therapy. Median follow-up was 2.8 years (range: 0.6-8.9 years)., Results: The best response was observed in the 86% of patients; the overall response rate was 66%. The 3-year progression-free survival was 58%, whereas the overall survival was 75%. No statistically significant differences between patients treated with BV monotherapy or combination were highlighted. In multivariate analysis, patients with non-nodular sclerosis HL and not transplanted had an increased risk of failure. Overall, 46% of patients had grade 3-4 adverse events that led to BV discontinuation in five of them., Conclusion: In conclusion, our study confirms that BV was a safe and effective drug, able to induce complete remission, either as monotherapy or in association with standard therapy., (© 2022 Wiley Periodicals LLC.)

High-risk neuroblastomas (HR-NB) still have an unacceptable 5-year overall survival despite the aggressive therapy. This includes standardized immunotherapy combining autologous hemopoietic stem cell transplantation (HSCT) and the anti-GD2 mAb. The treatment did not significantly change for more than one decade, apart from the abandonment of IL-2, which demonstrated unacceptable toxicity. Of note, immunotherapy is a promising therapeutic option in cancer and could be optimized by several strategies. These include the HLA-haploidentical αβT/B-depleted HSCT, and the antibody targeting of novel NB-associated antigens such as B7-H3, and PD1. Other approaches could limit the immunoregulatory role of tumor-derived exosomes and potentiate the low antibody-dependent cell cytotoxicity of CD16 dim/neg NK cells, abundant in the early phase post-transplant. The latter effect could be obtained using multi-specific tools engaging activating NK receptors and tumor antigens, and possibly holding immunostimulatory cytokines in their construct. Finally, treatments also consider the infusion of novel engineered cytokines with scarce side effects, and cell effectors engineered with chimeric antigen receptors (CARs). Our review aims to discuss several promising strategies that could be successfully exploited to potentiate the NK-mediated surveillance of neuroblastoma, particularly in the HSCT setting. Many of these approaches are safe, feasible, and effective at pre-clinical and clinical levels.

Neuroblastoma (NB) is the most common extracranial malignant tumor in children. Although the survival rate of NB has improved over the years, the outcome of NB still remains poor for over 30% of cases. A more accurate risk stratification remains a key point in the study of NB and the availability of novel prognostic biomarkers of "high-risk" at diagnosis could help improving patient stratification and predicting outcome. In this paper we show a biomarker discovery approach applied to the plasma of 172 NB patients. Plasma samples from a first cohort of NB patients and age-matched healthy controls were used for untargeted metabolomics analysis based on high-resolution mass spectrometry (HRMS). Differential expression analysis highlighted a number of metabolites annotated with a high degree of identification. Among them, 3-O-methyldopa (3-O-MD) was validated in a second cohort of NB patients using a targeted metabolite profiling approach and its prognostic potential was also analyzed by survival analysis on patients with 3 years follow-up. High expression of 3-O-MD was associated with worse prognosis in the subset of patients with stage M tumor (log-rank p < 0.05) and, among them, it was confirmed as a prognostic factor able to stratify high-risk patients older than 18 months. 3-O-MD might be thus considered as a novel prognostic biomarker of NB eligible to be included at diagnosis among catecholamine metabolite panels in prospective clinical studies. Further studies are warranted to exploit other potential biomarkers highlighted using our approach., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Barco, Lavarello, Cangelosi, Morini, Eva, Oneto, Uva, Tripodi, Garaventa, Conte, Petretto and Cangemi.)

Neurotoxicity is an off-tumour, on-target side effect of GD2-directed immunotherapy with monoclonal antibodies. Here, we report the frequency, management and outcome of patients enrolled in two prospective clinical trials who experienced severe neurotoxicity during immunotherapy with the anti-GD2 antibody dinutuximab beta (DB) administered as short-term infusion (HR-NBL1/SIOPEN study, randomisation R2, EudraCT 2006-001489-17) or as long-term infusion (HR-NBL1/SIOPEN study, randomisation R4, EudraCT 2006-001489-17 and LTI/SIOPEN study, EudraCT 2009-018077-31), either alone or with subcutaneous interleukin-2 (scIL-2). The total number of patients included in this analysis was 1102. Overall, 44/1102 patients (4.0%) experienced Grade 3/4 neurotoxicities (HR-NBL1 R2, 21/406; HR-NBL1 R4, 8/408; LTI study, 15/288), including 27 patients with severe neurotoxicities (2.5%). Events occurred predominantly in patients receiving combined treatment with DB and scIL-2. Neurotoxicity was treated using dexamethasone, prednisolone, intravenous immunoglobulins and, in two patients, plasmapheresis, which was highly effective. While neurological recovery was observed in 16 of 21 patients with severe neurotoxicities, 5/1102 (0.45%) patients experienced persistent and severe neurological deficits. In conclusion, severe neurotoxicity is most commonly observed in patients receiving DB with scIL-2. Considering the lack of clinical benefit for IL-2 in clinical trials so far, the administration of IL-2 alongside DB is not recommended.

The objective of this study was to identify prognostic factors for children and adolescents with relapsed or progressive classical Hodgkin’s lymphoma (cHL) to design salvage therapy tailored to them. We analyzed a homogeneous pediatric population, diagnosed with progressive/relapsed cHL previously enrolled in two subsequent protocols of the Italian Association of Pediatric Hematology and Oncology in the period 1996−2016. There were 272 eligible patients, 17.5% of treated patients with cHL. Overall survival (OS) and event-free survival (EFS) after a 10-year follow-up were 65.3% and 53.3%, respectively. Patients with progressive disease (PD), advanced stage at recurrence, and ≥5 involved sites showed a significantly worse OS. PD, advanced stage, and extra-nodal involvement at recurrence were significantly associated with a poorer EFS. Multivariable analysis identified three categories for OS based on the type of recurrence and number of localizations: PD and ≥5 sites: OS 34%; PD and <5 sites: OS 56.5%; relapses: OS 73.6%. Four categories were obtained for EFS based on the type of recurrence and stage: PD and stage 3−4: EFS 25.5%; PD and stage 1−2: EFS 43%; relapse and stage 3−4: EFS 55.4%; relapse and stage 1−2: EFS 72.1%. Patients with PD, in advanced stage, or with ≥5 involved sites had a very poor survival and they should be considered refractory to first- and second-line standard chemotherapy. Probably, they should be considered for more innovative approaches since the first progression. Conversely, patients who relapsed later with localized disease had a better prognosis, and they could be considered for a conservative approach.

Thyroid nodule ultrasound-based risk stratification systems (US-RSSs) have been successfully used in adults to predict the likelihood of malignancies. However, their applicability to the paediatric population is unclear, especially in children with a history of radiation exposure, who are at a higher cancer risk. We tested the efficacy of three US-RSSs in this setting by retrospectively applying three classification systems (ACR-TIRADS, ATA and EU-TIRADS) to all paediatric patients referred for thyroid nodules and with a radiation exposure history. We compared the results with a reference standard (pathology or 36-month follow-up); sensitivity, specificity, positive and negative predictive values (PPV and NPV) and accuracy were calculated. A total of 52 patients were included; fourteen of them (27%) had papillary thyroid cancer (PTC) at the final histology. No significant differences across the US-RSSs were detected; specificity (range 95-97%) and NPV (range 88-93%) were particularly elevated. However, ACR-TIRADS, ATA and EU-TIRADS did not indicate the need for a biopsy in six (42.8%), seven (50%) and eight (57%) cases of PTC; in five cases, this lack of indication was due to a small (<1 cm) nodule size. In conclusion, US-RSSs show a high NPV and specificity in paediatric patients, whereas the cytology indication could be improved by reconsidering the dimensional criterion.

Purpose: Induction therapy is a critical component of the therapy of high-risk neuroblastoma. We aimed to assess if the Memorial Sloan Kettering Cancer Center (MSKCC) N5 induction regimen (MSKCC-N5) would improve metastatic complete response (mCR) rate and 3-year event-free survival (EFS) compared with rapid COJEC (rCOJEC; cisplatin [C], vincristine [O], carboplatin [J], etoposide [E], and cyclophosphamide [C])., Patients and Methods: Patients (age 1-20 years) with stage 4 neuroblastoma or stage 4/4s aged < 1 year with MYCN amplification were eligible for random assignment to rCOJEC or MSKCC-N5. Random assignment was stratified according to national group and metastatic sites. Following induction, therapy comprised primary tumor resection, high-dose busulfan and melphalan, radiotherapy to the primary tumor site, and isotretinoin with ch14.18/CHO (dinutuximab beta) antibody with or without interleukin-2 immunotherapy. The primary end points were mCR rate and 3-year EFS., Results: A total of six hundred thirty patients were randomly assigned to receive rCOJEC (n = 313) or MSKCC-N5 (n = 317). Median age at diagnosis was 3.2 years (range, 1 month to 20 years), and 16 were younger than 1 year of age with MYCN amplification. mCR rate following rCOJEC induction (32%, 86/272 evaluable patients) was not significantly different from 35% (99/281) with MSKCC-N5 ( P = .368), and 3-year EFS was 44% ± 3% for rCOJEC compared with 47% ± 3% for MSKCC-N5 ( P = .527). Three-year overall survival was 60% ± 3% for rCOJEC compared with 65% ± 3% for MSKCC-N5 ( P = .379). Toxic death rates with both regimens were 1%. However, nonhematologic CTC grade 3 and 4 toxicities were higher with MSKCC-N5: 68% (193/283) versus 48% (129/268) ( P < .001); infection 35% versus 25% ( P = .011); stomatitis 25% versus 3% ( P < .001); nausea and vomiting 17% versus 7% ( P < .001); and diarrhea 7% versus 3% ( P = .011)., Conclusion: No difference in outcome was observed between rCOJEC and MSKCC-N5; however, acute toxicity was less with rCOJEC, and therefore rCOJEC is the preferred induction regimen for International Society of Pediatric Oncology European Neuroblastoma Group., Competing Interests: Alberto GaraventaTravel, Accommodations, Expenses: Takeda Dominique Valteau-CouanetConsulting or Advisory Role: EUSA PharmaResearch Funding: Orphelia PharmaPatents, Royalties, Other Intellectual Property: Royalties from Apeiron to SIOPENTravel, Accommodations, Expenses: EUSA Pharma, Jazz Pharmaceuticals Martin ElliottConsulting or Advisory Role: Bayer Godfrey C. F. ChanLeadership: Xelllera, PengeniaConsulting or Advisory Role: Xellera, PengeniaSpeakers' Bureau: Xellera, PengeniaPatents, Royalties, Other Intellectual Property: Xellera, Pengenia Walentyna BalwierzHonoraria: Shire, Gilead Sciences, Novartis, AmgenConsulting or Advisory Role: Amgen, Novartis, Roche, TakedaTravel, Accommodations, Expenses: Jazz Pharmaceuticals, Shire, Roche, Servier Cormac OwensEmployment: Ymabs Therapeutics Inc Vassilios PapadakisHonoraria: EUSA Pharma, NovartisConsulting or Advisory Role: SOBITravel, Accommodations, Expenses: Sanofi, Gilead Sciences, Novartis, Astellas Pharma Toby TrahairStock and Other Ownership Interests: CSL Limited, Osteopore Ltd, Medical Developments International, Cochlear Limited, Sonic Healthcare Limited, Medibank Private LimitedConsulting or Advisory Role: IPSOSExpert Testimony: EUSA Pharma, KMC HealthcareUncompensated Relationships: EUSA Pharma, KMC Healthcare Gudrun SchleiermacherHonoraria: BMSResearch Funding: Bristol Myers Squibb, Pfizer, MSDavenir, RocheTravel, Accommodations, Expenses: Roche Andrew D. J. PearsonConsulting or Advisory Role: Lilly, Takeda, Merck, CelgeneTravel, Accommodations, Expenses: Lilly, Takeda, Merck, Celgene Ruth Lydia LadensteinHonoraria: Apeiron Biologics, Boehringer Ingelheim, EUSA PharmaConsulting or Advisory Role: Apeiron Biologics, Boehringer Ingelheim, EUSA PharmaResearch Funding: Apeiron Biologics, EUSA PharmaPatents, Royalties, Other Intellectual Property: Apeiron Biologics, EUSA PharmaExpert Testimony: Apeiron Biologics, EUSA PharmaTravel, Accommodations, Expenses: Apeiron Biologics, EUSA PharmaNo other potential conflicts of interest were reported.

Neuroblastoma (NB) is one of the deadliest pediatric cancers, accounting for 15% of deaths in childhood. Hypoxia is a condition of low oxygen tension occurring in solid tumors and has an unfavorable prognostic factor for NB. In the present study, we aimed to identify novel promising drugs for NB treatment. Connectivity Map (CMap), an online resource for drug repurposing, was used to identify connections between hypoxia-modulated genes in NB tumors and compounds. Two sets of 34 and 21 genes up- and down-regulated between hypoxic and normoxic primary NB tumors, respectively, were analyzed with CMap. The analysis reported a significant negative connectivity score across nine cell lines for 19 compounds mainly belonging to the class of PI3K/Akt/mTOR inhibitors. The gene expression profiles of NB cells cultured under hypoxic conditions and treated with the mTORC complex inhibitor PP242, referred to as the Mohlin dataset, was used to validate the CMap findings. A heat map representation of hypoxia-modulated genes in the Mohlin dataset and the gene set enrichment analysis (GSEA) showed an opposite regulation of these genes in the set of NB cells treated with the mTORC inhibitor PP242. In conclusion, our analysis identified inhibitors of the PI3K/Akt/mTOR signaling pathway as novel candidate compounds to treat NB patients with hypoxic tumors and a poor prognosis.

Background: Neuroblastoma (NB) represents the most frequent and aggressive form of extracranial solid tumor of infants. Nucleolin (NCL) is a protein overexpressed and partially localized on the cell surface of tumor cells of adult cancers. Little is known about NCL and pediatric tumors and nothing is reported about cell surface NCL and NB., Methods: NB cell lines, Schwannian stroma-poor NB tumors and bone marrow (BM)-infiltrating NB cells were evaluated for the expression of cell surface NCL by Flow Cytometry, Imaging Flow Cytometry and Immunohistochemistry analyses. The cytotoxic activity of doxorubicin (DXR)-loaded nanocarriers decorated with the NCL-recognizing F3 peptide (T-DXR) was evaluated in terms of inhibition of NB cell proliferation and induction of cell death in vitro, whereas metastatic and orthotopic animal models of NB were used to examine their in vivo anti-tumor potential., Results: NB cell lines, NB tumor cells (including patient-derived and Patient-Derived Xenografts-PDX) and 70% of BM-infiltrating NB cells show cell surface NCL expression. NCL staining was evident on both tumor and endothelial tumor cells in NB xenografts. F3 peptide-targeted nanoparticles, co-localizing with cell surface NCL, strongly associates with NB cells showing selective tumor cell internalization. T-DXR result significantly more effective, in terms of inhibition of cell proliferation and reduction of cell viability in vitro, and in terms of delay of tumor growth in all NB animal model tested, when compared to both control mice and those treated with the untargeted formulation., Conclusions: Our findings demonstrate that NCL could represent an innovative therapeutic cellular target for NB.

Extraosseous Ewing sarcoma of primary cardiac origin is an extremely rare variety among pediatric cardiac neoplasms. We report a case of extraosseous Ewing sarcoma of primary cardiac origin in a 9-year-old girl, treated with debulking surgery, adjuvant chemotherapy, and radiotherapy., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.)

Background: High-risk neuroblastomas (HR-NBs) are rare, aggressive pediatric cancers characterized by resistance to therapy and relapse in more than 30% of cases, despite using an aggressive therapeutic protocol including targeting of GD2. The mechanisms responsible for therapy resistance are unclear and might include the presence of GD2neg/low NB variants and/or the expression of immune checkpoint ligands such as B7-H3., Method: Here, we describe a multiparametric flow cytometry (MFC) combining the acquisition of 10 6 nucleated singlets, Syto16pos CD45neg CD56pos cells, and the analysis of GD2 and B7-H3 surface expression. 41 bone marrow (BM) aspirates from 25 patients with NB, at the onset or relapse, are analyzed, comparing results with cytomorphological analysis (CA) and/or immunohistochemistry (IHC). Spike in experiments assesses the sensitivity of MFC. Kaplan-Meier analysis on 498 primary NBs selects novel prognostic markers possibly integrating the MFC panel., Results: No false positive are detected, and MFC shows high sensitivity (0.0005%). Optimized MFC identifies CD45negCD56pos NB cells in 11 out of 12 (91.6%) of BM indicated as infiltrated by CA, 7 of which coexpress high levels of GD2 and B7-H3. MFC detects CD45negCD56posGD2neg/low NB variants expressing high surface levels of B7-H3 in two patients with HR-NB (stage M) diagnosed at 53 and 139 months of age. One of them has a non-MYCN amplified tumor with unusual THpos PHOX2Bneg phenotype, which relapsed 141 months post-diagnosis with BM infiltration and a humerus lesion. All GD2neg/low NB variants are detected in patients at relapse. Kaplan-Meier analysis highlights an interesting dichotomous prognostic value of MML5, ULBPs, PVR, B7-H6, and CD47, ligands involved in NB recognition by the immune system., Conclusions: Our study validates a sensitive MFC analysis providing information on GD2 and B7-H3 surface expression and allowing fast, specific and sensitive evaluation of BM tumor burden. With other routinely used diagnostic and prognostic tools, MFC can improve diagnosis, prognosis, orienting novel personalized treatments in patients with GD2low/neg NB, who might benefit from innovative therapies combining B7-H3 targeting., Competing Interests: Competing interests: CB and RC are coinventors of the patent: 'Therapeutic and diagnostic methods and compositions targeting 4IgB7H3 and its counterpart NK cell receptor' (Application number 11186925.1 e 05775603.3)., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Neuroblastoma (NB) is an aggressive infancy tumor, leading cause of death among preschool age diseases. Here we focused on characterization of exosomal DNA (exo-DNA) isolated from plasma cell-derived exosomes of neuroblastoma patients, and its potential use for detection of somatic mutations present in the parental tumor cells. Exosomes are small extracellular membrane vesicles secreted by most cells, playing an important role in intercellular communications. Using an enzymatic method, we provided evidence for the presence of double-stranded DNA in the NB exosomes. Moreover, by whole exome sequencing, we demonstrated that NB exo-DNA represents the entire exome and that it carries tumor-specific genetic mutations, including those occurring on known oncogenes and tumor suppressor genes in neuroblastoma ( ALK , CHD5 , SHANK2 , PHOX2B , TERT , FGFR1 , and BRAF ). NB exo-DNA can be useful to identify variants responsible for acquired resistance, such as mutations of ALK , TP53 , and RAS / MAPK genes that appear in relapsed patients. The possibility to isolate and to enrich NB derived exosomes from plasma using surface markers, and the quick and easy extraction of exo-DNA, gives this methodology a translational potential in the clinic. Exo-DNA can be an attractive non-invasive biomarker for NB molecular diagnostic, especially when tissue biopsy cannot be easily available.

Introduction: Despite the progress in current treatments, the event-free survival of high-risk neuroblastoma (HR-NB) patients does not exceed 40%-50%, and the prognosis of refractory or relapsed patients is poor, still representing a challenge for pediatric oncologist. Therapeutic Iodine-131 meta-iodobenzylguanidine (Th- 131 I-MIBG) is a recognized safe and potentially effective treatment for NB., Materials: This retrospective study reports the outcomes of 28 MIBG-avid NB patients with advanced disease either refractory or relapsed, which was undertaken from 1996 to 2014. Th- 131 I-MIBG was administered shortly before (median: 17 days) high-dose chemotherapy with busulfan and melphalan (HD-BuMel) and autologous stem cell rescue (ASCR) at the Gaslini Institute in Genoa, with the aim of analyzing the feasibility, safety, and efficacy of this approach., Results: Engraftment occurred in all patients after a median of 14 (11-29) and 30 days (13-80) from ASCR for neutrophils and platelets, respectively. No treatment-related deaths were observed. The main high-grade (3-4) toxicity observed was oral and gastrointestinal mucositis in 78.6% and 7.1% of patients, respectively, whereas high-grade hepatic toxicity was observed in 10.7%. Two patients developed veno-occlusive-disease (7.1%), completely responsive to defibrotide. Hypothyroidism was the main late complication that occurred in nine patients (31.1%). After Th- 131 MIBG and HD-BuMel, 19 patients (67.8%) showed an improvement in disease status. Over a median follow-up of 15.9 years, the three-year and five-year overall survival (OS) probabilities were 53% (CI 0.33-0.69) and 41% (CI 0.22-0.59), and the three-year and five-year rates of cumulative risk of progression/relapse were 64% (CI 0.47-0.81) and 73% (CI 0.55-0.88), respectively. MYCN amplification emerged as the only risk factor significantly associated with OS (HR, 3.58;P = 0.041)., Conclusion: Th- 131 I-MIBG administered shortly before HD-BuMel is a safe and effective regimen for patients with advanced MIBG-avid NB. These patients should be managed in centers with proven expertise., (© 2020 Wiley Periodicals LLC.)

Purpose: To clarify the role of primary tumor resection in stage 4S neuroblastoma., Methods: We investigated a cohort of 172 infants diagnosed with stage 4S neuroblastoma between 1994 and 2013. Of 160 evaluable patients, 62 underwent upfront resection of the primary tumor and 98 did not., Results: Five-year progression-free and overall survival were significantly better in those who had undergone upfront surgery (83.6% vs 64.2% and 96.8% vs 85.7%, respectively). One post-operative death and four non-fatal complications occurred in the resection group. Three patients who had not undergone resection died of chemotherapy-related toxicity. Thirteen patients underwent late surgery to remove a residual tumor, without complications: all but one alive. Outcomes were better in patients diagnosed from 2000 onwards., Conclusion: Infants diagnosed with stage 4S neuroblastoma who underwent upfront tumor resection had a better outcome. However, this result cannot be definitely attributed to surgery, since these patients were selected on the basis of their favorable presenting features. Although the question of whether to operate or not at disease onset is still unsolved, this study confirms the importance of obtaining enough adequate tumor tissue to enable histological and biological studies to properly address treatment, to achieve the best possible outcome.

The biological and clinical heterogeneity of neuroblastoma (NB) demands novel biomarkers and therapeutic targets in order to drive the most appropriate treatment for each patient. Hypoxia is a condition of low-oxygen tension occurring in poorly vascularized tumor tissues. In this study, we aimed to assess the role of hypoxia in the pathogenesis of NB and at developing a new clinically relevant hypoxia-based predictor of outcome. We analyzed the gene expression profiles of 1882 untreated NB primary tumors collected at diagnosis and belonging to four existing data sets. Analyses took advantage of machine learning methods. We identified NB-hop, a seven-gene hypoxia biomarker, as a predictor of NB patient prognosis, which is able to discriminate between two populations of patients with unfavorable or favorable outcome on a molecular basis. NB-hop retained its prognostic value in a multivariate model adjusted for established risk factors and was able to additionally stratify clinically relevant groups of patients. Tumors with an unfavorable NB-hop expression showed a significant association with telomerase activation and a hypoxic, immunosuppressive, poorly differentiated, and apoptosis-resistant tumor microenvironment. NB-hop defines a new population of NB patients with hypoxic tumors and unfavorable prognosis and it represents a critical factor for the stratification and treatment of NB patients.

Adolescents and young adults (AYAs) represent a distinct group of patients. The objectives of this study were: To compare adolescent prognosis to that of younger children; to compare the results achieved with the two consecutive protocols in both age groups; to analyze clinical characteristics of children and adolescents. Between 1996 and 2017, 1759 patients aged <18 years were evaluable for the study. Five hundred and sixty patients were treated with the MH'96 protocol and 1199 with the LH2004 protocol. Four hundred and eighty-two were adolescents aged ≥15 years. Patients in both age groups showed very favorable prognoses. In particular, OS improved with the LH2004 protocol, especially in the adolescent group and in the low risk group, where radiation therapy was spared. Adolescent characteristics differed significantly from the children's according to sex, histology, and the presence of symptoms. Remarkable is the decrease both in mixed cellularity in the children and in low stages in both age groups in the LH2004 protocol with respect to MH'96 protocol. Based on our experience, adopting pediatric protocols for AYA does not compromise patient outcomes.

Patients with high-risk neuroblastoma (HR-NB) often initially respond to therapy, but afterward they become resistant and disease recurred. Unfortunately, it does not exist one or more specific chromosome defects associated with relapse or refractory NB. Recently, genomic evidence from primary tumors indicated that the distal region of chromosome 6q is loss in HR-NB patients with fatal outcome. We identified a minimal common region of loss of chromosome 6q27 spanning an area of 2.09 Mb by high-resolution DNA copy number data of a small cohort of HR-NB samples carrying 6q loss. This region of loss harbored five genes T, SFT2D1, RPS6KA2, FGFR1OP , and UNC93A . We found that low SFT2D1, RPS6KA2 , and FGFR1OP gene expression predicted poor outcome in HR-NB patients using public R2 Platform. Further functional studies will be essential to confirm the presumed tumor suppressor gene(s) located within 6q27 region. These results suggest that SFT2D1, RPS6KA2 , and FGFR1OP genes may be responsible for poor prognosis of HR-NB tumors with 6q27 loss, and their haploinsufficiency may be crucial in accelerating tumor progression.

Neuroblastoma (NB) is the most common extracranial malignant tumor of childhood and is characterized by a broad heterogeneity in clinical presentation and evolution. Recent advances in pangenomic analysis of NB have revealed different recurrent chromosomal aberrations. Indeed, it is now well established that the overall genomic profile is important for treatment stratification. In previous studies, 11 genes were shown to be recurrently amplified (ODC1, ALK, GREB1, NTSR2, LIN28B, MDM2, CDK4, MYEOV, CCND1, TERT, and MYC) besides MYCN, with poor survival of NB patients harboring these amplifications being suggested. Genomic profiles of 628 NB samples analyzed by array-comparative genome hybridization (a-CGH) were re-examined to identify gene amplifications other them MYCN amplification. Clinical data were retrospectively collected. We additionally evaluated the association of FRS2 gene expression with NB patient outcome using the public R2 Platform. We found eight NB samples with high grade amplification of one or two loci on chromosome arm 12q. The regional amplifications were located on bands 12q13.3-q14.1 and 12q15-q21.1 involving the genes CDK4, MDM2, and the potential oncogenic gene FRS2. The CDK4, MDM2, and FRS2 loci were coamplified in 8/8 samples. The 12q amplifications were associated with very poor prognosis and atypical clinical features of NB patients. Further functional and clinical investigations are needed to confirm or refute these associations., (© 2019 Wiley Periodicals, Inc.)

The genetic aetiology and the molecular mechanisms that characterize high-risk neuroblastoma are still little understood. The majority of high-risk neuroblastoma patients do not take advantage of current induction therapy. So far, one of the main reasons liable for cancer therapeutic failure is the acquisition of resistance to cytotoxic anticancer drugs, because of the DNA repair system of tumour cells. PARP1 is one of the main DNA damage sensors involved in the DNA repair system and genomic stability. We observed that high PARP1 mRNA level is associated with unfavourable prognosis in 3 public gene expression NB patients' datasets and in 20 neuroblastomas analysed by qRT-PCR. Among 4983 SNPs in PARP1, we selected two potential functional SNPs. We investigated the association of rs907187, in PARP1 promoter, and rs2048426 in non-coding region with response chemotherapy in 121 Italian patients with high-risk NB. Results showed that minor G allele of rs907187 associated with induction response of patients (P = .02) and with decrease PARP1 mRNA levels in NB cell line (P = .003). Furthermore, rs907187 was predicted to alter the binding site of E2F1 transcription factor. Specifically, allele G had low binding affinity with E2F1 whose expression positively correlates with PARP1 expression and associated with poor prognosis of patients with NB. By contrast, we did not find genetic association for the SNP rs2048426. These data reveal rs907187 as a novel potential risk variant associated with the failure of induction therapy for high-risk NB., (© 2020 Università degli Studi di Napoli Federico II. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Our purpose was to evaluate the diagnostic role of 18 F-3,4-dihydroxyphenylalanine (DOPA) PET/CT at the time of staging in children with neuroblastoma and to investigate its ability to assess treatment response. We also investigated the prognostic value of 18 F-DOPA PET/CT at the same time points. Methods: We enrolled children with neuroblastoma at onset. Before and after induction chemotherapy, all patients underwent 18 F-DOPA PET/CT and 123 I-metaiodobenzylguanidine (MIBG) scanning plus SPECT/CT. 18 F-DOPA PET/CT results were compared with those of 123 I-MIBG whole-body scanning (WBS). For each modality, patient-based analysis and lesion-based analysis were performed and sensitivity was calculated. We applied scoring systems to 123 I-MIBG scanning and 18 F-DOPA PET/CT (i.e., 123 I-MIBG WBS score and whole-body metabolic burden [WBMB], respectively) and evaluated the association between these parameters, the principal neuroblastoma risk factors, and outcome. Results: We enrolled 16 high-risk and 2 intermediate-risk neuroblastoma patients. On patient-based analysis, sensitivity in detecting primary tumors, soft-tissue metastases, and bone or bone-marrow metastases was 83%, 50%, and 92%, respectively, for 123 I-MIBG WBS versus 94%, 92%, and 100%, respectively, for 18 F-DOPA PET/CT. On lesion-based analysis, the sensitivity of 18 F-DOPA PET/CT in detecting soft-tissue and bone or bone-marrow metastases was 86% and 99%, respectively-significantly higher than that of 123 I-MIBG WBS, at 41% and 93%, respectively. After therapy, on patient-based analysis, the sensitivity in detecting primary tumors, soft-tissue metastases, and bone or bone-marrow metastases was 72%, 33%, and 38%, respectively, for 123 I-MIBG WBS versus 83%, 75% and 54%, respectively, for 18 F-DOPA PET/CT. On lesion-based analysis, the sensitivity of 18 F-DOPA PET/CT in detecting soft-tissue and bone or bone-marrow metastases was 77% and 86%, respectively-significantly higher than that of 123 I-MIBG WBS, at 28% and 69%, respectively. During follow-up, 8 cases of disease progression and 5 deaths occurred. On multivariate analysis, only posttherapeutic 18 F-DOPA WBMB (>7.5) was associated with progression-free survival. Conclusion: 18 F-DOPA PET/CT is more sensitive than 123 I-MIBG WBS in staging neuroblastoma patients and evaluating disease persistence after chemotherapy. In a time-to-event analysis, posttherapeutic 18 F-DOPA WBMB remained the only risk factor associated with disease progression., (© 2020 by the Society of Nuclear Medicine and Molecular Imaging.)

To explore the effects of immunotherapy in the International Society of Paediatric Oncology Europe Neuroblastoma Group SIOPEN high-risk neuroblastoma 1 trial (HR-NBL1 trial), two cohorts were studied: one prior to and one after the introduction of dinutuximab beta. All patients received standard induction and high-dose therapy (HDT) with autologous stem cell rescue (ASCR); the local control comprised surgery and radiotherapy to the primary tumour site, followed by isotretinoin. A landmark timepoint of 109 days, resulting from the median time between ASCR and initiation of immunotherapy, was used to define patients' eligibility in the pre-immunotherapy analysis cohort. Median follow-up was 5.8 years (inter-quartile range (IQR): 4.2-8.2 years) for 844 eligible patients balanced for risk factors, such as age, sex, stage 4, MYCN amplification and response prior to HDT. The five-year event-free and overall survival (95% confidence interval (CI) of 466 patients not receiving immunotherapy was 42% (38-47%) and 50% (46-55%) but was 57% (51-62%) and 64% (59-69%) for 378 patients receiving immunotherapy ( p < 0.001). A multivariate analysis identified absence of immunotherapy (p = 0.0002, hazard ratio (HR) 1.573); type of HDT ( p = 0.0029, HR 1.431); less than complete response prior to maintenance therapy ( p = 0.0043, HR 1.494) and >1 metastatic compartment at diagnosis ( p < 0.001, HR 2.665) as risk factors for relapse or progression. Results suggest an important role for dinutuximab beta-based immunotherapy within the treatment concepts applied in HR-NBL1/SIOPEN., Competing Interests: The academic data supported APEIRON to obtain the dinutuximab beta product licensure in May 2017 in the European Union (EMA). SIOPEN and CCRI established a contract with APEIRON regarding the provision of academic data. Ruth Ladenstein and Holger Lode acted as consultants for APEIRON on behalf of SIOPEN for the ch14.18/CHO development. The other authors declare no conflict of interest.

Despite intensive treatment, 50% of children with high-risk neuroblastoma (HR-NB) succumb to their disease. Progression through current trials evaluating the efficacy of new treatments for children with HR disease usually depends on an inadequate response to induction chemotherapy, assessed using imaging modalities. In this study, we sought to identify circulating biomarkers that might be detected in a simple blood sample to predict patient response to induction chemotherapy. Since exosomes released by tumor cells can drive tumor growth and chemoresistance, we tested the hypothesis that exosomal microRNA (exo-miRNAs) in blood might predict response to induction chemotherapy. The exo-miRNAs expression profile in plasma samples collected from children treated in HR-NBL-1/SIOPEN before and after induction chemotherapy was compared to identify a three exo-miRs signature that could discriminate between poor and good responders. Exo-miRNAs expression also provided a chemoresistance index predicting the good or poor prognosis of HR-NB patients., Competing Interests: The authors declare no conflict of interest.

Neuroblastoma (NB) is an aggressive, relapse-prone infancy tumor of the sympathetic nervous system and is the leading cause of death among preschool age diseases, so the search for novel therapeutic targets is crucial. Golgi phosphoprotein 3 (GOLPH3) has been reported to be involved in the development, and in the DNA damage response, of various human cancers. Golgi dispersal is a common feature of DNA damage response in mammalian cells. Understanding how cells react to DNA damage is essential in order to recognize the systems used to escape from elimination. We induced DNA damage in two human neuroblastoma cell lines by curcumin. The exposure of neuroblastoma cells to curcumin induced: (a) up-regulation of GOLPH3 + cells; (b) augmentation of double-strand breaks; (c) Golgi fragmentation and dispersal throughout the cytoplasm; (d) increase of apoptosis and autophagy; (e) increased expression of TPX2 oncoprotein, able to repair DNA damage. Primary neuroblastoma samples analysis confirmed these observations. Our findings suggest that GOLPH3 expression levels may represent a clinical marker of neuroblastoma patients' responsiveness to DNA damaging therapies-and of possible resistance to them. Novel molecules able to interfere with GOLPH3 and TPX2 pathways may have therapeutic benefits when used in combination with standard DNA damaging therapeutic agents in neuroblastoma.

Background: Neuroblastoma (NB), a pediatric tumor of the sympathetic nervous system, is characterized by very frequent chromosomal aberrations at the onset of the disease. Identification of further risk factors for relapse, which could lead to increased survival and potentially reduced late effects among survivors, is still urgently needed. Segmental chromosome aberrations (SCA) are associated with poor prognosis, whereas numerical whole-chromosome aberrations (NCA) are found in patients with a good prognosis; however, a small percentage of the latter patients (10%-15%) subsequently relapse and/or die of disease., Procedure: DNA copy-number data from 174 NB patients with an NCA genomic profile were analyzed. Association between NCA and event-free survival (EFS) was investigated by the Kaplan-Meier estimator and prognostic decision tree (DT)., Results: DT identified 65 patients with normal chromosome X and an excellent five-year EFS (100%) independently from the stage at diagnosis. The association between poor EFS and whole chromosome X alterations was confirmed after stratification into two groups of different expected prognosis and by internal validation via bootstrap analysis. Furthermore, the association was also observed in an independent cohort of NB patients extracted from the data set of the National Cancer Institute TARGET Project for Neuroblastoma, but sample size was small (n = 75) and statistical significance was not achieved., Conclusions: Loss of whole chromosome X may represent a new prognostic marker for NB patients with an NCA genomic profile. If confirmed by further studies, this finding could indicate that such patients should be reclassified as intermediate risk and treated accordingly., (© 2019 Wiley Periodicals, Inc.)

Introduction: A substantial number of patients with neuroblastoma (NB) have increased excretion of catecholamines and metanephrines. Here, we have investigated the diagnostic role of plasma free metanephrines (PFM), metanephrine (MN), normetanephrine (NMN) and 3-methoxytyramine (3MT) for NB, the most common extra-cranial solid tumour in children., Methods: PFM were quantified by using a commercial IVD-CE LC-MS/MS method on a TSQ Quantiva coupled to an Ultimate 3000. The method was further validated on 103 samples from pediatric subjects (54 patients with histologically confirmed NB and 49 age and sex matched controls). Correlations between PFM concentrations with clinical factors were tested. We directly compared MN, NMN, and 3MT concentrations in matched plasma and urine samples of NB patients (n = 29)., Results: 3MT and NMN showed an excellent diagnostic performance with very high specificity (100% and 95.8%, respectively) and sensitivity (88.2% and 80.4%). ROC curves were obtained (AUC of 0.93 and 0.91 for 3MT and NMN, respectively) and optimal cut-offs that could discriminate between controls and NB patients were defined. A positive correlation between NMN levels in urine and plasma (p = .0017) was found., Discussion: The determination of plasma 3MT and NMN should be taken in consideration as a new diagnostic tool for NB. Validation in prospective clinical studies in comparison to urinary catecholamines and metanephrines is warranted., (Copyright © 2019 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Introduction: Advances in paediatric oncology led to the increase in long-term survival, revealing the burden of therapy-related long-term side effects. We evaluated overall and cause-specific mortality in a large cohort of Italian childhood cancer survivors (CCSs) and adolescent cancer survivors identified through the off-therapy registry., Materials and Methods: CCSs alive 5 years after cancer diagnosis occurring between 1960 and 1999 were eligible; the last follow-up was between 2011 and 2014. Outcomes were reported as standardised mortality ratios (SMRs) and absolute excess risks (AERs)., Results: Among 12,214 CCSs, 1113 (9.1%) deaths occurred. Survival at 35 years since diagnosis was 87% (95% confidence interval [CI]: 86-88) and at 45 years was 81% (95% CI: 77-84). CCSs had an 11-fold increased risk of death (SMR 95% CI: 10.7-12), corresponding to an AER of 48 (95% CI: 45-51). Mortality decreased by 60% for survivors treated most recently (1990-1999). The most frequent causes of death were recurrence of the original cancer (56%), a subsequent neoplasm (19%) and cardiovascular diseases (5.8%). Among those who survived at least 15 years after diagnosis, a secondary malignancy was the leading cause of death., Conclusions: This study confirms the impact of recent advances in anticancer therapy in reducing mortality, mainly attributable to recurrence but also to other causes. However, overall mortality continues to be higher than in the general population. A long-term follow-up is needed to prevent late mortality due to secondary neoplasms and non-neoplastic causes in CCSs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Many studies have reported a more favorable outcome in younger patients with Hodgkin lymphoma (HL). The aims of this study were to find an appropriate age cutoff able to identify low-risk children and to describe the natural history of 135 very young patients affected by classic HL (cHL). The best age cutoff was identified at 7 years of age. EFS (p = .0451) and PFS (p = .00921) were significantly better in the group of younger patients. The OS rate at 10 years was 97.0% in the younger group and 92.5% in the older one (p = .0448). However, age was not found to be an independent prognostic factor in multivariate analysis and the better prognosis in younger patients seems to be related to more favorable disease characteristics at presentation.

Background: Infants diagnosed with stage 4 s neuroblastoma commonly experience spontaneous disease regression, with few succumbing without response to therapy. We analyzed a large cohort of such infants enrolled in the Italian Neuroblastoma Registry to detect changes over time in presenting features, treatment and outcome., Methods: Of 3355 subjects aged 0-18 years with previously untreated neuroblastoma diagnosed between 1979 and 2013, a total of 280 infants (8.3%) had stage 4 s characteristics, 268 of whom were eligible for analyses. Three treatment eras were identified on the basis of based diagnostic and chemotherapy adopted. Group 1 patients received upfront chemotherapy; Group 2 and 3 patients underwent observation in the absence of life-threatening symptoms (LTS), except for Group 3 patients with amplified MYCN gene, who received more aggressive therapy., Results: The three groups were comparable, with few exceptions. Ten-year overall survival significantly increased from 76.9 to 89.7% and was worse for male gender, age 0-29 days and presence of selected LTS on diagnosis, elevated LDH, and abnormal biologic features. Infants who underwent primary resection ± chemotherapy did significantly better. On multivariate analysis, treatment eras and the association of hepatomegaly to dyspnea were independently associated with worse outcome., Conclusions: Our data confirm that stage 4 s neuroblastoma is curable in nearly 90% of cases. Hepatomegaly associated to dyspnea was the most important independent risk factor. The cure rate could be further increased through timely identification of patients at risk who might benefit from surgical techniques, such as intra-arterial chemoembolization and/or liver transplantation, which must be carried out in institutions with specific expertise.

Purpose: We present the results of an investigation of the role of FDG PET in response evaluation of bulky masses in paediatric patients with Hodgkin's lymphoma (HL) enrolled in the Italian AIEOP-LH2004 trial., Methods: We analysed data derived from 703 patients (388 male, 315 female; mean age 13 years) with HL and enrolled in 41 different Italian centres from March 2004 to September 2012, all treated with the AIEOP-LH2004 protocol. The cohort comprised 309 patients with a bulky mass, of whom 263 were evaluated with FDG PET at baseline and after four cycles of chemotherapy. Responses were determined according to combined functional and morphological criteria. Patients were followed up for a mean period of 43 months and for each child we calculated time-to-progression (TTP) and relapse rates considering clinical monitoring, and instrumental and histological data as the reference standard. Statistical analyses were performed for FDG PET and morphological responses with respect to TTP. Multivariate analysis was used to define independent predictive factors., Results: Overall, response evaluation revealed 238 PET-negative patients (90.5%) and 25 PET-positive patients (9.5%), with a significant difference in TTP between these groups (mean TTP: 32.67 months for negative scans, 23.8 months for positive scans; p < 0.0001, log-rank test). In the same cohort, computed tomography showed a complete response (CR) in 85 patients (32.3%), progressive disease (PD) in 6 patients (2.3%), and a partial response (PR) in 165 patients (62.7%), with a significant difference in TTP between patients with CR and patients with PD (31.1 months and 7.9 months, respectively; p < 0.001, log-rank test). Similarly, there was a significant difference in relapse rates between PET-positive and PET-negative patients (p = 0000). In patients with PR, there was also a significant difference in TTP between PET-positive and PET-negative patients (24.6 months and 34.9 months, respectively; p < 0.0001). In the multivariate analysis with correction for multiple testing, only the PET result was an independent predictive factor in both the entire cohort of patients and the subgroup showing PR on CT (p < 0.01)., Conclusion: After four cycles of chemotherapy, FDG PET response assessment in paediatric HL patients with a bulky mass is a good predictor of TTP and disease outcome. Moreover, in patients with a PR on CT, PET was able to differentiate those with a longer TTP. In paediatric HL patients with a bulky mass and in patients with a PR on CT, response on FDG PET was an independent predictive factor.

Background: Immunotherapy with the chimeric anti-GD2 monoclonal antibody dinutuximab, combined with alternating granulocyte-macrophage colony-stimulating factor and intravenous interleukin-2 (IL-2), improves survival in patients with high-risk neuroblastoma. We aimed to assess event-free survival after treatment with ch14.18/CHO (dinutuximab beta) and subcutaneous IL-2, compared with dinutuximab beta alone in children and young people with high-risk neuroblastoma., Methods: We did an international, open-label, phase 3, randomised, controlled trial in patients with high-risk neuroblastoma at 104 institutions in 12 countries. Eligible patients were aged 1-20 years and had MYCN-amplified neuroblastoma with stages 2, 3, or 4S, or stage 4 neuroblastoma of any MYCN status, according to the International Neuroblastoma Staging System. Patients were eligible if they had been enrolled at diagnosis in the HR-NBL1/SIOPEN trial, had completed the multidrug induction regimen (cisplatin, carboplatin, cyclophosphamide, vincristine, and etoposide, with or without topotecan, vincristine, and doxorubicin), had achieved a disease response that fulfilled prespecified criteria, had received high-dose therapy (busulfan and melphalan or carboplatin, etoposide, and melphalan) and had received radiotherapy to the primary tumour site. In this component of the trial, patients were randomly assigned (1:1) to receive dinutuximab beta (20 mg/m 2 per day as an 8 h infusion for 5 consecutive days) or dinutuximab beta plus subcutaneous IL-2 (6 × 10 6 IU/m 2 per day on days 1-5 and days 8-12 of each cycle) with the minimisation method to balance randomisation for national groups and type of high-dose therapy. All participants received oral isotretinoin (160 mg/m 2 per day for 2 weeks) before the first immunotherapy cycle and after each immunotherapy cycle, for six cycles. The primary endpoint was 3-year event-free survival, analysed by intention to treat. This trial was registered with ClinicalTrials.gov, number NCT01704716, and EudraCT, number 2006-001489-17, and recruitment to this randomisation is closed., Findings: Between Oct 22, 2009, and Aug 12, 2013, 422 patients were eligible to participate in the immunotherapy randomisation, of whom 406 (96%) were randomly assigned to a treatment group (n=200 to dinutuximab beta and n=206 to dinutuximab beta with subcutaneous IL-2). Median follow-up was 4·7 years (IQR 3·9-5·3). Because of toxicity, 117 (62%) of 188 patients assigned to dinutuximab beta and subcutaneous IL-2 received their allocated treatment, by contrast with 160 (87%) of 183 patients who received dinutuximab beta alone (p<0·0001). 3-year event-free survival was 56% (95% CI 49-63) with dinutuximab beta (83 patients had an event) and 60% (53-66) with dinutuximab beta and subcutaneous IL-2 (80 patients had an event; p=0·76). Four patients died of toxicity (n=2 in each group); one patient in each group while receiving immunotherapy (n=1 congestive heart failure and pulmonary hypertension due to capillary leak syndrome; n=1 infection-related acute respiratory distress syndrome), and one patient in each group after five cycles of immunotherapy (n=1 fungal infection and multi-organ failure; n=1 pulmonary fibrosis). The most common grade 3-4 adverse events were hypersensitivity reactions (19 [10%] of 185 patients in the dinutuximab beta group vs 39 [20%] of 191 patients in the dinutuximab plus subcutaneous IL-2 group), capillary leak (five [4%] of 119 vs 19 [15%] of 125), fever (25 [14%] of 185 vs 76 [40%] of 190), infection (47 [25%] of 185 vs 64 [33%] of 191), immunotherapy-related pain (19 [16%] of 122 vs 32 [26%] of 124), and impaired general condition (30 [16%] of 185 vs 78 [41%] of 192)., Interpretation: There is no evidence that addition of subcutaneous IL-2 to immunotherapy with dinutuximab beta, given as an 8 h infusion, improved outcomes in patients with high-risk neuroblastoma who had responded to standard induction and consolidation treatment. Subcutaneous IL-2 with dinutuximab beta was associated with greater toxicity than dinutuximab beta alone. Dinutuximab beta and isotretinoin without subcutaneous IL-2 should thus be considered the standard of care until results of ongoing randomised trials using a modified schedule of dinutuximab beta and subcutaneous IL-2 are available., Funding: European Commission 5th Frame Work Grant, St. Anna Kinderkrebsforschung, Fondation ARC pour la recherche sur le Cancer., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Risk stratification is crucial to treatment decision-making in neuroblastoma. This study aimed to explore factors present at diagnosis affecting outcome in patients aged ≥18 months with metastatic neuroblastoma and to develop a simple risk score for prognostication., Procedure: Data were derived from the European high-risk neuroblastoma 1 (HR-NBL1)/International Society for Paediatric Oncology European Neuroblastoma (SIOPEN) trial with analysis restricted to patients aged ≥18 months with metastatic disease and treated prior to the introduction of immunotherapy. Primary endpoint was 5-year event-free survival (EFS). Prognostic factors assessed were sex, age, tumour MYCN amplification (MNA) status, serum lactate dehydrogenase (LDH)/ferritin, primary tumour and metastatic sites. Factors significant in univariate analysis were incorporated into a multi-variable model and an additive scoring system developed based on estimated log-cumulative hazard ratios., Results: The cohort included 1053 patients with median follow-up 5.5 years and EFS 27 ± 1%. In univariate analyses, age; serum LDH and ferritin; involvement of bone marrow, bone, liver or lung; and >1 metastatic system/compartment were associated with worse EFS. Tumour MNA was not associated with worse EFS. A multi-variable model and risk score incorporating age (>5 years, 2 points), serum LDH (>1250 U/L, 1 point) and number of metastatic systems (>1, 2 points) were developed. EFS was significantly correlated with risk score: EFS 52 ± 9% for score = 0 versus 6 ± 3% for score = 5 (P < 0.0001)., Conclusions: A simple score can identify an "ultra-high risk" (UHR) cohort (score = 5) comprising 8% of patients with 5-year EFS <10%. These patients appear not to benefit from induction therapy and could potentially be directed earlier to alternative experimental therapies in future trials., (© 2018 Wiley Periodicals, Inc.)

Identify a subset of early-stage HL children (GR1) curable with limited chemotherapy+/-radiotherapy; improve outcome of intermediate (GR2) and high-risk (GR3) patients; establish impact of response to chemotherapy evaluated with conventional imaging (CI). One hundred and sixty GR1-patients received 3ABVD + involved-field (IF) low-dose (LD) (20 Gy) irradiation if mediastinal mass or partial response (PR) after chemotherapy. Eighty-five GR2- and 315 GR3-patients received 4 and 6 COPP/ABV + IFRT, respectively. The 63 GR1 patients spared from radiotherapy had 15-year survival and EFS of 100 and 84.5%, respectively. The GR2 and GR3 15-year FFP were 84.7 and 78.6%, respectively. No different prognosis for patients in CR or PR evaluated during and after chemotherapy was observed. In conclusion, low-risk patients in CR may be successfully treated with radiation-free, low-intensity chemotherapy. Good, but less satisfactory, results were registered in GR2 and GR3. Response evaluated with CI is not a prognostic factor, but permits identification of low-risk patients who can avoid radiotherapy.

GD₂-directed immunotherapies improve survival of high-risk neuroblastoma (NB) patients (pts). Treatment with chimeric anti-GD₂ antibodies (Ab), such as ch14.18, can induce development of human anti-chimeric Ab (HACA). Here, we report HACA effects on ch14.18/CHO pharmacokinetics, pharmacodynamics and pain intensity in pts treated by long-term infusion (LTI) of ch14.18/CHO combined with IL-2. 124 pts received up to 5 cycles of ch14.18/CHO 10 days (d) infusion (10 mg/m²/d; d8⁻18) combined with s.c. IL-2 (6 × 10⁶ IU/m²/d; d1⁻5, d8⁻12). HACA, treatment toxicity, ch14.18/CHO levels, Ab-dependent cellular- (ADCC) and complement-dependent cytotoxicity (CDC) were assessed using respective validated assays. HACA-negative pts showed a steadily decreased pain in cycle 1 (74% pts without morphine by d5 of LTI) with further decrease in subsequent cycles. Ch14.18/CHO peak concentrations of 11.26 ± 0.50 µg/mL found in cycle 1 were further elevated in subsequent cycles and resulted in robust GD₂-specific CDC and ADCC. Development of HACA (21% of pts) resulted in strong reduction of ch14.18/CHO levels, abrogated CDC and ADCC. Surprisingly, no difference in pain toxicity between HACA-positive and -negative pts was found. In conclusion, ch14.18/CHO LTI combined with IL-2 results in strong activation of Ab effector functions. Importantly, HACA response abrogated CDC but did not affect pain intensity indicating CDC-independent pain induction.

Several studies support the notion that the kinase inhibitor Imatinib mesylate exerts off-target effects on cells of the immune system. After our first report of continuous daily oral administration in subjects with relapsed/refractory neuroblastoma (NB, EudraCT: 2005-005778-63), here we update the clinical information and report additional information on potential surrogate markers for prediction of efficacy. Peripheral blood (PB) samples collected at study entry and after the first and second cycle of Imatinib mesylate treatment were tested for IFN- γ , TNF-α, TGF-β, IL-10, CXCL12 and soluble (s) B7-H6 plasma levels. In addition, paired PB and bone marrow (BM) samples collected at study entry and after the second Imatinib cycle were evaluated for CXCL12, CXCR4 and NKp30 isoform mRNA levels. Correlation between each parameter level and response/outcome was then evaluated. Out of the six subjects still alive at the time of the first report, thee died after additional therapy, two for NB progression and one for a second malignancy. Three are presently alive and cured from NB at 10 years after the first Imatinib cycle. Of these, one achieved complete response (CR) during Imatinib treatment and never relapsed, one had a local relapse removed by surgery and the third received TVD as rescue therapy. Response and outcome were associated with low Imatinib exposure, whereas none of the tested immunological and molecular parameters was predictive of response/outcome. However, after Imatinib treatment NKp30 isoform mRNA levels significantly increase in BM samples, indicating that Imatinib mesylate exerted an off-target effect on NK cells in vivo . Imatinib mesylate efficacy in relapsed/refractory NB has been confirmed at a longer follow-up, supporting its inclusion in new Phase II trials for these subjects, that should envisage collection of samples to evaluate the predictive power of other potential surrogate markers of efficacy.

Introduction: Diffuse or massive tumors involving the perineal and pelvic compartments may require aggressive surgical treatment in children. The authors, propose and present their results using a Posterior Sagittal Anorectal Mobilization (PSAM) METHODS: The oncologic patient is placed in a prone position with the pelvis elevated, without a previous colostomy. A 10 Ch bladder catheter is positioned. A midline sagittal incision is performed from the coccyx to the posterior margin of the anus. Fine electrocautery is used to obtain an accurate dissection all around the anal margin itself. The incisional so involves the midline dissection into the perineal body. Reconstruction is achieved by the classical pelvic-perineal anatomical structure approximation., Results: The authors describe for the first time a novel posterior anorectal mobilization technique for four children with pelvic-perineal tumors. No intraoperative or post-operative complications occurred. The oldest boy presented at follow-up a complete normal faecal and urinary continence (Krickenbeck criteria)., Conclusion: The presented technical approach, defined as PSAM, is completely suitable for surgical resection of perineal-pelvic tumors, without the need of more invasive operations including transpubic dissection and/or anorectal split. Moreover, nervous and muscular sphincter structures are easily identified and preserved., (© 2018 Wiley Periodicals, Inc.)