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Introduction: Scarcity of age appropriate formulations for orally administered medicines for paediatric malignancies is a critical and ongoing issue which necessitates urgent solutions. According to JARC Survey 27 % of oral medicines were never available in child-friendly formulations (Vassal et al., 2021 [2]). Pharmacists from the European Society of Oncology Pharmacy Global (ESOP) and the European Society for Pediatric Oncology (SIOPE) have collaborated to provide a document summarising literature data on this topic and a set of practical instructions on the preparation of extemporaneous oral liquid medicines. Material and methods: Literature review was conducted for the preparation of oral medicines for paediatric cancer, through Pubmed 2.0. A table was drawn up with necessary information for medicine preparation. We adapted the classification model of the molecule stability ranking of the International database of stability for injectable drugs, Stabilis® (Class A–C). Results: A total of 126 articles were selected and analysed. All commercially available marketed liquid oral medications used in paediatric cancer were overviewed using globally accessible drug databases and compiled in a table with appropriate indications. Based on the literature review, 28 formulations for 13 different active ingredients for chemotherapy and 35 formulations for 16 different active ingredients for supportive therapy were compiled. Conclusions: Development of child-appropriate formulations of anticancer medicines by the pharmaceutical industry should be incentivised towards marketing authorisation to enhance accessibility. The results of this study could help facilitate creation of European standards for extemporaneous preparation and persuade researchers in the field of paediatric oncology on the way forward.

Very rare cancers in children and adolescents pose a unique challenge for optimal care and research. Due to their rarity and diversity, there is a lack of standardized treatments and limited data to guide clinical decision-making. This paper aims to provide solutions for addressing these challenges through a multidisciplinary approach involving clinicians, researchers, and patient advocates. In recent years, members of the European Cooperative Study Group for Paediatric Rare Tumors (EXPeRT) and members of the European Society of Pediatric Oncology (SIOPE) clinical council worked within the EU Joint Action on Rare Cancers (JARC) and the Paed Can European Reference Network (ERN) on solutions to improve the care and research in the field. This includes a better classification, more financial resources, higher awareness and support by stakeholders, an international database, as well as harmonized diagnostic and therapeutic guidelines. Further research projects in the field of molecular biology, a better collaboration between medical oncologists and pediatricians, and a general strategy to facilitate access to modern therapies are urgently needed.

Abstract Background More effective incentives are needed to motivate paediatric oncology drug development, uncoupling it from dependency on adult drug development. Although the current European and North‐American legislations aim to promote drug development for paediatrics and rare diseases, children and adolescents with cancer have not benefited as expected from these initiatives and cancer remains the first cause of death by disease in children older than one. Drug development for childhood cancer remains dependent on adult cancer indications and their potential market. The balance between the investment needed to execute a Paediatric Investigation Plan (PIP) in Europe and an initial Paediatric Study Plan (iPSP) in the US, coupled with the potential financial reward has not been sufficiently attractive to incite the pharmaceutical industry to develop drugs for rare indications such as childhood cancer. Methods We propose changes in the timing and nature of the rewards within the European Paediatric Medicine Regulation (PMR) and Regulation on Orphan Medicinal Products (both currently under review), which would drive earlier initiation of paediatric oncology studies and provide incentives for drug development specifically for childhood indications. Results We suggest modifying the PMR to ensure mechanism‐of‐action driven mandatory PIP and reorganization of incentives to a stepwise and incremental approach. Interim and final deliverables should be defined within a PIP or iPSP, each attracting a reward on completion. A crucial change would be the introduction of the interim deliverable requiring production of paediatric data that inform the go/no‐go decisions on whether to take a drug forward to paediatric efficacy trials. Conclusion Additionally, to address the critical gap in the current framework where there is a complete lack of incentives to promote paediatric‐specific cancer drug development, we propose the introduction of early rewards in the Orphan Regulation, with a variant on the US‐Creating Hope Act and its priority review vouchers.

Abstract Many medicines are used “off‐label” in children outside the terms of the license. Feasible pediatric clinical trials are a challenge to design. Conect4children (c4c) is an Innovative Medicines Initiative project to set up a pan‐European pediatric clinical trial network aiming to facilitate the development of new medicines for children. To optimize pediatric trial development by promoting innovative trial design, c4c set up a European multidisciplinary advice service, including the voice of young patients and families, tailored to industry and academia. A network of experts was established to provide multidisciplinary advice to trial sponsors. Experts were selected to join clinical and innovative methodology expert groups. A patient and public involvement (PPI) database, to include the expert opinion of patients and parents/carers was formed. A stepwise process was developed: (1) sponsors contact c4c, (2) scoping interview takes place, (3) ad hoc advice group formed, (5) advice meeting held, and (6) advice report provided. Feedback on the process was collected. Twenty‐four clinical and innovative methodology expert groups (>400 experts) and a PPI database of 135 registrants were established. As of September 30, 2022, 36 advice requests were received, with 25 requests completed. Clinical and methodology experts and PPI representatives participated in several advice requests. Sponsors appreciated the advice quality and the multidisciplinary experts from different countries, including experts not known before. Experts and PPI participants were generally satisfied with the process. The c4c project has shown successful proof of concept for a service that presents a new framework to plan innovative and feasible pediatric trials.

Osteosarcoma is a rare bone cancer in adolescents and young adults with a dismal prognosis because of metastatic disease and chemoresistance. Despite multiple clinical trials, no improvement in outcome has occurred in decades. There is an urgent need to better understand resistant and metastatic disease and to generate in vivo models from relapsed tumors. We developed eight new patient-derived xenograft (PDX) subcutaneous and orthotopic/paratibial models derived from patients with recurrent osteosarcoma and compared the genetic and transcriptomic landscapes of the disease progression at diagnosis and relapse with the matching PDX. Whole exome sequencing showed that driver and copy-number alterations are conserved from diagnosis to relapse, with the emergence of somatic alterations of genes mostly involved in DNA repair, cell cycle checkpoints, and chromosome organization. All PDX patients conserve most of the genetic alterations identified at relapse. At the transcriptomic level, tumor cells maintain their ossification, chondrocytic, and trans-differentiation programs during progression and implantation in PDX models, as identified at the radiological and histological levels. A more complex phenotype, like the interaction with immune cells and osteoclasts or cancer testis antigen expression, seemed conserved and was hardly identifiable by histology. Despite NSG mouse immunodeficiency, four of the PDX models partially reconstructed the vascular and immune-microenvironment observed in patients, among which the macrophagic TREM2/TYROBP axis expression, recently linked to immunosuppression. Our multimodal analysis of osteosarcoma progression and PDX models is a valuable resource to understand resistance and metastatic spread mechanisms, as well as for the exploration of novel therapeutic strategies for advanced osteosarcoma.

Abstract Background Tropomyosin receptor kinase (TRK) fusion proteins resulting from neurotrophic tyrosine receptor kinase (NTRK) gene fusions are rare primary oncogenic drivers in a wide array of tumors. Larotrectinib is a first-in-class, highly selective, central nervous system-active TRK inhibitor approved by the US Food and Drug Administration (FDA), European Medicines Agency (EMA), and over 40 countries for the treatment of TRK fusion solid tumors in adult and pediatric patients. Due to the rarity of TRK fusion cancer, larotrectinib was granted accelerated approval based on a relatively small number of patients enrolled in three early phase trials. ON-TRK aims to evaluate the safety profile of larotrectinib in a broader population and over extended time periods. Methods ON-TRK is a prospective, non-interventional, open-label, multicenter, multi-cohort, post-approval study in adult and pediatric patients with locally advanced or metastatic TRK fusion cancer treated with larotrectinib that will describe the safety and effectiveness of larotrectinib in real-world practice conditions. Adult patients will be grouped by tumor type and followed for at least 2 years. Patients

Abstract Background Since pediatric cancer drug development is a global enterprise, we sought to provide an overview of the landscape of intercontinental clinical trials in pediatric oncology opened over the last decade. Methods ClinicalTrials.gov was systematically searched to identify all clinical therapeutic trials which opened between 2010 and 2020 and recruited pediatric patients (

Abstract Background Optimal Phase‐II design to evaluate new therapies in refractory/relapsed Ewing sarcomas (ES) remains imperfectly defined. Objectives Recurrent/refractory ES phase‐I/II trials analysis to improve trials design. Methods Comprehensive review of therapeutic trials registered on five databases (who.int/trialsearch, clinicaltrials.gov, clinicaltrialsregister.eu, e‐cancer.fr, and umin.ac.jp) and/or published in PubMed/ASCO/ESMO websites, between 2005 and 2018, using the criterion: (Ewing sarcoma OR bone sarcoma OR sarcoma) AND (Phase‐I or Phase‐II). Results The 146 trials identified (77 phase‐I/II, 67 phase‐II, and 2 phase‐II/III) tested targeted (34%), chemo‐ (23%), immune therapies (19%), or combined therapies (24%). Twenty‐three trials were ES specific and 48 had a specific ES stratum. Usually multicentric (88%), few trials were international (30%). Inclusion criteria cover the recurrent ES age range for only 12% of trials and allowed only accrual of measurable diseases (RECIST criteria). Single‐arm design was the most frequent (88%) testing mainly single drugs (61%), only 5% were randomized. Primary efficacy outcome was response rate (RR=CR+PR; Complete+Partial response) (n = 116/146; 79%), rarely progression‐free or overall survival (16% PFS and 3% OS). H0 and H1 hypotheses were variable (3%–25% and 20%–50%, respectively). The 62 published trials enrolled 827 ES patients. RR was poor (10%; 15 CR=1.7%, 68 PR=8.3%). Stable disease was the best response for 186 patients (25%). Median PFS/OS was of 1.9 (range 1.3–14.7) and 7.6 months (5–30), respectively. Eleven (18%) published trials were considered positive, with median RR/PFS/OS of 15% (7%–30%), 4.5 (1.3–10), and 16.6 months (6.9–30), respectively. Conclusion This review supports the need to develop the international randomized phase‐II trials across all age ranges with PFS as primary endpoint.

Abstract Background Neuroblastoma (NB) is a frequent pediatric tumor associated with poor prognosis. The disregulation of Bcl-2, an anti-apoptotic protein, is crucial for the tumoral development and chemoresistance. Autophagy is also implicated in tumor cell survival and chemoresistance. The aim of our study was to demonstrate therapeutic efficiency of GX 15–070, a pan-Bcl-2 family inhibitor, used alone and in combination with conventional drugs or with hydroxychloroquine (HCQ), an autophagy inhibitor. Methods Five neuroblastoma cell lines were tested for the cytotoxic activity of GX 15–070 alone or in combination with cisplatin, doxorubicin, HCQ or Z-VAD-FMK a broad-spectrum caspase inhibitor. Apoptosis and autophagy levels were studied by western-blot and FACS. Orthotopic injections were performed on NOD/LtSz-scid/IL-2Rgamma null mice that were treated with either GX 15–070 alone or in combination with HCQ. Results Synergistic cytotoxicity was observed for the drug combination in all of the 5 neuroblastoma cell lines tested, including MYCN amplified lines and in cancer stem cells. GX 15–070 significantly increased apoptosis and autophagy in neuroblastoma cells as evidenced by increased levels of the autophagy marker, LC3-II. Inhibition of autophagy by HCQ, further increased the cytotoxicity of this combinatorial treatment, suggesting that autophagy induced by these agent plays a cytoprotective role. In vivo, GX 15–070 combined with HCQ significantly decreased the growth of the tumor and the number of distant metastases. Conclusions Based on the synergistic effect of HCQ and GX 15–070 observed in this study, the combination of these two drugs may be utilized as a new therapeutic approach for neuroblastoma.

The development of oral pediatric forms by pharmaceutical companies is still insufficient. In fact, many drugs used in paediatric oncology, such as temozolomide, are not labeled and adapted for paediatric use. Temozolomide (TMZ) is an alkylating agent used as the standard of care for many adult and pediatric brain tumours, such as neuroblastoma, glioblastoma and medulloblastoma. The present study was carried out to propose a suitable and palatable formulation of the oral liquid preparation of TMZ. The suspension is composed of TMZ suspended in SyrSpend SF pH 4, as well as TMZ crystallization stabilizing agents and sweetening agents. To reach this formulation, several taste-masking agents were evaluated. Here, we describe the method of preparation of the formation as well as the monocentric population treated with the formulation over a 5–year period. A 20 mg/mL TMZ suspension was developed. TMZ suspension is stable for 6 weeks, stored between 2 and 8 degrees, protected from light, and compatible with nasogastric tubes. Thirty-eight patients participated in the palatability study and choose cola flavour, and 104 patients were treated in Gustave Roussy with the developed suspension; no unexpected event was reported. To conclude, we propose here a new TMZ liquid formulation which is stable for at least 6 weeks and well-tolerated with extensive feedback.

Introduction: Despite initial benefit, virtually all patients suffering from EGFR-mutant NSCLC experience acquired resistance to tyrosine kinase inhibitors (TKIs), driven by multiple mechanisms. Recent reports have identified oncogenic kinase fusions as off-target resistance mechanisms; however, these alterations have been rarely investigated at EGFR TKIs progression. Methods: Patients with EGFR-mutated metastatic NSCLC (N = 62) with tissue and plasma biopsies at EGFR TKI progression between January 2015 and June 2019, at a French hospital and optionally before progression, were identified from the prospective MATCH-R study (NCT02517892). Postprogression biopsy samples were analyzed for gene fusions using targeted gene panel sequencing, whole-exome sequencing, RNA sequencing, and comparative genomic hybridization array. Results: Six gene fusions were detected in tumor progression biopsies under an EGFR TKI from 62 consecutive patients (9.7%) with EGFR-mutated advanced NSCLC. Among 31 patients progressing to first- or second-generation EGFR TKIs, one (3%) had an Eukaryotic translation initiation factor 4 gamma 2–GRB2 associated binding protein 1 (EIF4G2-GAB1) fusion. Among 31 patients progressing to the third-generation osimertinib, five (16%) presented oncogene fusions of fibroblast growth factor receptor 3–transforming acidic coiled-coil containing protein 3 (FGFR3-TACC3) (n = 2), kinesin family member 5B–Ret proto-oncogene (KIF5B-RET) (n = 1), striatin–anaplastic lymphoma kinase (STRN-ALK) (n = 1), and zinc finger DHHC-Type palmitoyltransferase 20–Thr790Met (ZDHHC20-BRAF) (n = 1) transcripts. Out of two patients that received osimertinib at first-line, one acquired an FGFR3-TACC3 fusion at progression. In all patients, fusions co-occurred with the original activating EGFR mutation; however, among four patients with an acquired T790M mutation, three (75%) lost the T790M mutation. Conclusions: Oncogenic fusions at the time of EGFR TKI resistance were identified at a relatively high frequency, mainly after the third-generation TKI osimertinib. Patients progressing to EGFR TKIs may have a new opportunity for targeted therapy when oncogenic fusions are identified.

Abstract Background Papillary renal cell carcinoma (pRCC) is the 2nd most frequent histological type of kidney cancer and accounts for approximately 15% of all renal cell carcinoma. It has a poorer prognosis than clear cell RCC (ccRCC) with a lack of standard treatments. Case presentation We report the case of a 51 year old man with a metastatic pRCC (hepatic dome and left colonic peritoneal carcinomatosis) progressive after sunitinib, with a MET amplification. The patient was enrolled in the UNICANCER-sponsored AcSé crizotinib trial (NCT02034981), designed to give an access to crizotinib for patients with tumors harboring a genomic alteration on one of the biological targets of the drug. With 2nd line crizotinib (250 mg twice/day), the patient had a very good tolerance, a partial response in the target lesions using RECIST 1.1, and a 19 months’ clinical efficacy. Conclusions In metastatic pRCC with a MET amplification, crizotinib maybe a potential met-inhibitory therapeutic option.

The multikinase inhibitor regorafenib (BAY 73-4506) exerts both anti-angiogenic and anti-tumorigenic activity in adult solid malignancies mainly advanced colorectal cancer and gastrointestinal stromal tumors. We intended to explore preclinically the potential of regorafenib against solid pediatric malignancies alone and in combination with anticancer agents to guide the pediatric development plan. In vitro effects on cell proliferation were screened against 33 solid tumor cell lines of the Innovative Therapies for Children with Cancer (ITCC) panel covering five pediatric solid malignancies. Regorafenib inhibited cell proliferation with a mean half maximal growth inhibition of 12.5 μmol/L (range 0.7 μmol/L to 28 μmol/L). In vivo, regorafenib was evaluated alone at 10 or 30 mg/kg/d or in combination with radiation, irinotecan or the mitogen-activated protein kinase kinase (MEK) inhibitor refametinib against various tumor types, including patient-derived brain tumor models with an amplified platelet-derived growth factor receptor A (PDGFRA) gene. Regorafenib alone significantly inhibited tumor growth in all xenografts derived from nervous system and connective tissue tumors. Enhanced effects were observed when regorafenib was combined with irradiation and irinotecan against PDGFRA amplified IGRG93 glioma and IGRM57 medulloblastoma respectively, resulting in 100% tumor regressions. Antitumor activity was associated with decreased tumor vascularization, inhibition of PDGFR signaling, and induction of apoptotic cell death. Our work demonstrates that regorafenib exhibits significant antitumor activity in a wide spectrum of preclinical pediatric models through inhibition of angiogenesis and induction of apoptosis. Furthermore, radio- and chemosensitizing effects were observed with DNA damaging agents in PDGFR amplified tumors.

Neuroblastoma is a pediatric cancer of the peripheral nervous system in which structural chromosome aberrations are emblematic of aggressive tumors. In this study, we performed an in-depth analysis of somatic rearrangements in two neuroblastoma cell lines and two primary tumors using paired-end sequencing of mate-pair libraries and RNA-seq. The cell lines presented with typical genetic alterations of neuroblastoma and the two tumors belong to the group of neuroblastoma exhibiting a profile of chromothripsis. Inter and intra-chromosomal rearrangements were identified in the four samples, allowing in particular characterization of unbalanced translocations at high resolution. Using complementary experiments, we further characterized 51 rearrangements at the base pair resolution that revealed 59 DNA junctions. In a subset of cases, complex rearrangements were observed with templated insertion of fragments of nearby sequences. Although we did not identify known particular motifs in the local environment of the breakpoints, we documented frequent microhomologies at the junctions in both chromothripsis and non-chromothripsis associated breakpoints. RNA-seq experiments confirmed expression of several predicted chimeric genes and genes with disrupted exon structure including ALK, NBAS, FHIT, PTPRD and ODZ4. Our study therefore indicates that both non-homologous end joining-mediated repair and replicative processes may account for genomic rearrangements in neuroblastoma. RNA-seq analysis allows the identification of the subset of abnormal transcripts expressed from genomic rearrangements that may be involved in neuroblastoma oncogenesis.

Diffuse intrinsic pontine glioma (DIPG) is one of the most frequent malignant pediatric brain tumor and its prognosis is universaly fatal. No significant improvement has been made in last thirty years over the standard treatment with radiotherapy. To address the paucity of understanding of DIPGs, we have carried out integrated molecular profiling of a large series of samples obtained with stereotactic biopsy at diagnosis. While chromosomal imbalances did not distinguish DIPG and supratentorial tumors on CGHarrays, gene expression profiling revealed clear differences between them, with brainstem gliomas resembling midline/thalamic tumours, indicating a closely-related origin. Two distinct subgroups of DIPG were identified. The first subgroup displayed mesenchymal and pro-angiogenic characteristics, with stem cell markers enrichment consistent with the possibility to grow tumor stem cells from these biopsies. The other subgroup displayed oligodendroglial features, and appeared largely driven by PDGFRA, in particular through amplification and/or novel missense mutations in the extracellular domain. Patients in this later group had a significantly worse outcome with an hazard ratio for early deaths, ie before 10 months, 8 fold greater that the ones in the other subgroup (p = 0.041, Cox regression model). The worse outcome of patients with the oligodendroglial type of tumors was confirmed on a series of 55 paraffin-embedded biopsy samples at diagnosis (median OS of 7.73 versus 12.37 months, p = 0.045, log-rank test). Two distinct transcriptional subclasses of DIPG with specific genomic alterations can be defined at diagnosis by oligodendroglial differentiation or mesenchymal transition, respectively. Classifying these tumors by signal transduction pathway activation and by mutation in pathway member genes may be particularily valuable for the development of targeted therapies.

Neuroblastoma (NB) is a typical childhood and heterogeneous neoplasm for which efficient targeted therapies for high-risk tumors are not yet identified. The chemokine CXCL12, and its receptors CXCR4 and CXCR7 have been involved in tumor progression and dissemination. While CXCR4 expression is associated to undifferentiated tumors and poor prognosis, the role of CXCR7, the recently identified second CXCL12 receptor, has not yet been elucidated in NB. In this report, CXCR7 and CXCL12 expressions were evaluated using a tissue micro-array including 156 primary and 56 metastatic NB tissues. CXCL12 was found to be highly associated to NB vascular and stromal structures. In contrast to CXCR4, CXCR7 expression was low in undifferentiated tumors, while its expression was stronger in matured tissues and specifically associated to differentiated neural tumor cells. As determined by RT-PCR, CXCR7 expression was mainly detected in N-and S-type NB cell lines, and was slightly induced upon NB cell differentiation in vitro. The relative roles of the two CXCL12 receptors were further assessed by overexpressing CXCR7 or CXCR4 receptor alone, or in combination, in the IGR-NB8 and the SH-SY5Y NB cell lines. In vitro functional analyses indicated that, in response to their common ligand, both receptors induced activation of ERK1/2 cascade, but not Akt pathway. CXCR7 strongly reduced in vitro growth, in contrast to CXCR4, and impaired CXCR4/CXCL12-mediated chemotaxis. Subcutaneous implantation of CXCR7-expressing NB cells showed that CXCR7 also significantly reduced in vivo growth. Moreover, CXCR7 affected CXCR4-mediated orthotopic growth in a CXCL12-producing environment. In such model, CXCR7, in association with CXCR4, did not induce NB cell metastatic dissemination. In conclusion, the CXCR7 and CXCR4 receptors revealed specific expression patterns and distinct functional roles in NB. Our data suggest that CXCR7 elicits anti-tumorigenic functions, and may act as a regulator of CXCR4/CXCL12-mediated signaling in NB.

BackgroundChildren with ependymoma may experience a relapse in up to 50% of cases depending on the extent of resection. Key biological events associated with recurrence are unknown.Methodology/principal findingsTo discover the biology behind the recurrence of ependymomas, we performed CGHarray and a dual-color gene expression microarray analysis of 17 tumors at diagnosis co-hybridized with the corresponding 27 first or subsequent relapses from the same patient. As treatment and location had only limited influence on specific gene expression changes at relapse, we established a common signature for relapse. Eighty-seven genes showed an absolute fold change ≥2 in at least 50% of relapses and were defined as the gene expression signature of ependymoma recurrence. The most frequently upregulated genes are involved in the kinetochore (ASPM, KIF11) or in neural development (CD133, Wnt and Notch pathways). Metallothionein (MT) genes were downregulated in up to 80% of the recurrences. Quantitative PCR for ASPM, KIF11 and MT3 plus immunohistochemistry for ASPM and MT3 confirmed the microarray results. Immunohistochemistry on an independent series of 24 tumor pairs at diagnosis and at relapse confirmed the decrease of MT3 expression at recurrence in 17/24 tumor pairs (p = 0.002). Conversely, ASPM expression was more frequently positive at relapse (87.5% vs 37.5%, p = 0.03). Loss or deletion of the MT genes cluster was never observed at relapse. Promoter sequencing after bisulfite treatment of DNA from primary tumors and recurrences as well as treatment of short-term ependymoma cells cultures with a demethylating agent showed that methylation was not involved in MT3 downregulation. However, in vitro treatment with a histone deacetylase inhibitor or zinc restored MT3 expression.Conclusions/significanceThe most frequent molecular events associated with ependymoma recurrence were over-expression of kinetochore proteins and down-regulation of metallothioneins. Metallothionein-3 expression is epigenetically controlled and can be restored in vitro by histone deacetylase inhibitors.

Although paediatric high grade gliomas resemble their adult counterparts in many ways, there appear to be distinct clinical and biological differences. One important factor hampering the development of new targeted therapies is the relative lack of cell lines derived from childhood glioma patients, as it is unclear whether the well-established adult lines commonly used are representative of the underlying molecular genetics of childhood tumours. We have carried out a detailed molecular and phenotypic characterisation of a series of paediatric high grade glioma cell lines in comparison to routinely used adult lines.All lines proliferate as adherent monolayers and express glial markers. Copy number profiling revealed complex genomes including amplification and deletions of genes known to be pivotal in core glioblastoma signalling pathways. Expression profiling identified 93 differentially expressed genes which were able to distinguish between the adult and paediatric high grade cell lines, including a number of kinases and co-ordinated sets of genes associated with DNA integrity and the immune response.These data demonstrate that glioma cell lines derived from paediatric patients show key molecular differences to those from adults, some of which are well known, whilst others may provide novel targets for evaluation in primary tumours. We thus provide the rationale and demonstrate the practicability of using paediatric glioma cell lines for preclinical and mechanistic studies.

Neuroblastoma (NB) is a heterogeneous, and particularly malignant childhood neoplasm in its higher stages, with a propensity to form metastasis in selected organs, in particular liver and bone marrow, and for which there is still no efficient treatment available beyond surgery. Recent evidence indicates that the CXCR4/CXCL12 chemokine/receptor axis may be involved in promoting NB invasion and metastasis. In this study, we explored the potential role of CXCR4 in the malignant behaviour of NB, using a combination of in vitro functional analyses and in vivo growth and metastasis assessment in an orthotopic NB mouse model. We show here that CXCR4 overexpression in non-metastatic CXCR4-negative NB cells IGR-NB8 and in moderately metastatic, CXCR4 expressing NB cells IGR-N91, strongly increased tumour growth of primary tumours and liver metastases, without altering the frequency or the pattern of metastasis. Moreover shRNA-mediated knock-down experiments confirmed our observations by showing that silencing CXCR4 in NB cells impairs in vitro and almost abrogates in vivo growth. High levels of CXCL12 were detected in the mouse adrenal gland (the primary tumour site), and in the liver suggesting a paracrine effect of host-derived CXCL12 on NB growth. In conclusion, this study reveals a yet unreported NB-specific predominant growth and survival-promoting role of CXCR4, which warrants a critical reconsideration of the role of CXCR4 in the malignant behaviour of NB and other cancers.