Solid Tumor In Children Research Articles

A Fast and Interactive Augmented Reality System for PET/CT-guided Intervention of Neuroblastoma.

Neuroblastoma is the most common type of extracranial solid tumor in children and can often result in death if not treated. High-intensity focused ultrasound (HIFU) is a non-invasive technique for treating tissue that is deep within the body. It avoids the use of ionizing radiation, avoiding long-term side-effects of these treatments. The goal of this project was to develop the rendering component of an augmented reality (AR) system with potential applications for image-guided HIFU treatment of neuroblastoma. Our project focuses on taking 3D models of neuroblastoma lesions obtained from PET/CT and displaying them in our AR system in near real-time for use by physicians. We used volume ray casting with raster graphics as our preferred rendering method, as it allows for the real-time editing of our 3D radiologic data. Some unique features of our AR system include intuitive hand gestures and virtual user interfaces that allow the user to interact with the rendered data and process PET/CT images for optimal visualization. We implemented the feature to set a custom transfer function, set custom intensity cutoff points, and region-of-interest extraction via cutting planes. In the future, we hope to incorporate this work as part of a complete system for focused ultrasound treatment by adding ultrasound simulation, visualization, and deformable registration.

Fertility preservation in pediatric solid tumors: A report from the Children's Oncology Group.

Treatment for childhood solid tumors may lead to an increased risk for gonadal dysfunction/infertility. Discussion of risk should occur at diagnosis, any changes in therapy, and during survivorship. Gonadotoxic therapies were abstracted from 32 Children's Oncology Group (COG) phase III, frontline solid tumor protocols, in use from 2000 to 2022. Risk for gonadal dysfunction/infertility was assessed based on gonadotoxic therapies, sex, and pubertal status and assigned as minimal, significant, and high following the Oncofertility Consortium Pediatric Initiative Network (PIN) risk stratification. Most protocols (65.6%, 21/32) contained at least one therapeutic arm with a high level of increased risk. Solid tumor therapies present challenges in risk stratification due to response-adjusted therapy and the need to account for radiation field in the risk assessment. This guide hopes to serve as a tool to assist in standardizing gonadotoxic risk assessments across disciplines and improve referral for fertility services and reproductive health counseling for patients receiving COG-based solid tumor therapy. Internationally, many solid tumor therapies follow similar paradigms to COG studies, and risk stratifications may be generalizable to similar styles of therapy. In addition, this model may be applied to other international groups with the goal of standardizing fertility assessments.

Abstract 132: DNA-PK inhibition to enhance etoposide therapy in neuroblastoma

Abstract Introduction. Neuroblastoma is the most common extracranial solid tumor in children and accounts for ~15% of all pediatric cancer deaths. DNA-PK, a key regulator of repair responses to double-strand DNA breakages induced by radiation or induction chemotherapy is used in the treatment of high-risk neuroblastomas. Here, we have demonstrated that high DNA-PK expression is associated with poor overall survival in a patient population, DNA-PK is highly activated in neuroblastoma cell lines, and inhibition of DNA-PK enhances therapeutic effects of DNA damaging therapies. Methods. DNA double-strand break damage was induced with etoposide, a topoisomerase II inhibitor. DNA-PK activity was inhibited with peposertib or DNA-PK siRNA. DNA-PK Ser2056 phosphorylation was analyzed in MYCN amplified (SK-NDZ, BE(2)-C, LAN-1) and MYCN non-amplified (SK-N-AS, SK-N-SH) cell lines. Clonogenic and proliferation assays were used to evaluate the efficacy of peposertib in combination with etoposide in BE-2-C and SK-N-DZ neuroblastoma cell lines. DSB-induced DNA repair was visualized with confocal laser scanning microscopy using γ-H2AX immunofluorescence. Western blot was used to confirm inhibition of DNA-PKcs (pDNA-PKcs S2056) and analyze cleaved PARP expression in combination groups. Results. DNA-PK inhibition resulted in enhancement of etoposide cytotoxic therapy (BE-2-C IC50 ET = 2.77 ± 0.2, ET+Pep 0.76 ± 0.04; SK-N-DZ IC50 ET = 0.55 ± 0.06, IC50 ET+Pep = 0.11 ± 0.02) in both BE-2-C and SK-N-DZ cell lines. Immunofluorescence analysis detected γH2AX foci increase in the etoposide and peposertib combination group. DNA-PK inhibition in combination with etoposide treatment significantly increased PARP cleavage in BE-2-C and SK-N-DZ cell lines. The effect of DNA-PK knockdown on neuroblastoma cell survival was even more pronounced compared to DNA-PK inhibition with peposertib. DNA-PK knockdown by siRNA alone increased PARP cleavage to the levels comparable to 2 µM etoposide treatment. The combination of etoposide and DNA-PK knockdown enhanced PARP cleavage even further in neuroblastoma cells. Conclusions. High DNA repair activity is commonly associated with cancer drug resistance. We have shown that DNA-PK activation is continuously supported in both MYCN amplified and MYCN non-amplified neuroblastoma cell lines. The importance of DNA-PK activity in neuroblastoma cell lines is confirmed with DNA-PK knockdown that leads to apoptosis in MYCN amplifiedneuroblastoma cell lines. In addition, the combination of DNA-PK inhibition or DNA-PK knockdown with DNA-damaging therapies stalled DNA damage response and enhanced neuroblastoma cell apoptosis. These findings demonstrate the significance of high DNA-PK activity in neuroblastoma cells and provide a potent therapeutic strategy to enhance neuroblastoma cell apoptosis with selective DNA-PK inhibition. Citation Format: Mahnaz Norouzi, Beibei Zhu, Tadahide Izumi, Eric J. Rellinger, B. Mark Evers, Piotr G. Rychahou. DNA-PK inhibition to enhance etoposide therapy in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 132.

Abstract 149: Modeling patient-specific CHEK2 genetic variants associated with high-risk neuroblastoma

Abstract Background: Neuroblastoma (NB) is the most common extra-cranial solid tumor in children and accounts for approximately 10% of childhood cancer deaths. Recently, next-generation sequencing of patients enrolled in the SickKids Cancer Sequencing (KiCS) program revealed somatic and germline alterations in genes involved in the DNA damage response (DDR). Multiple germline and somatic pathogenic variants in CHEK2 were detected in patients with NB. Aims and Methods: To better understand potential roles for CHEK2 in NB pathogenesis, we are modeling patient-derived CHEK2 variants in an established transgenic zebrafish model of MYCN-driven NB. Using a CRISPR/Cas9 “knock-in” approach, we have generated stable zebrafish lines that are homozygous and heterozygous for variants of interest with either null, heterozygous, or wild-type tp53 functional status. We aim to assess tumor incidence, growth, and metastasis using direct visualization of disseminated EGFP+ tumor cells, as compared to MYCN over-expression only control fish. Given potential drug sensitivities, we also plan to assess the efficacy of various DDR pathway inhibitors in combination with frontline chemotherapies, which are effective in the treatment of DDR-deficient adult cancers. Results: Targeting chek2 in a tp53 null background had no significant effect on tumor incidence and growth compared to tp53 mutant control animals (p=0.1570 log-rank test). This contrasts with other patient-specific DDR models including gene targeting brca2 (p=0.0235, log-rank test), atm (p<0.001, log-rank test), and bard1 (p<0.001, log-rank test), potentially highlighting Tp53-dependent roles for Chek2 in NB formation in vivo. Through functional characterization of patient-specific CHEK2 variants in NB progression, our work will provide important preclinical information that will inform future diagnostics and therapeutic strategies for patients with high-risk NB. Citation Format: Xueting Xiong, Sarah Cohen-Gogo, Anita Villani, Adam Shlien, Meredith S. Irwin, Madeline Hayes. Modeling patient-specific CHEK2 genetic variants associated with high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 149.

Serum cholesterol level as a predictive biomarker for prognosis of Neuroblastoma

BackgroundNeuroblastoma (NB), a type of solid tumor in children, has a poor prognosis. Few blood biomarkers can accurately predict the prognosis, including recurrence and survival, in children with NB. In this study, we found that the serum total cholesterol (Tchol) level was associated with the prognosis of patients through a retrospective study.MethodsMultivariate Cox regression model was used to identify the independent risk factors in the children with NB. Kaplan–Meier method was used to analyze the correlation between the common biomarkers, including the serum Tchol level, and the prognosis of the patients. ROC curves were used to predict the accuracy of the International Neuroblastoma Staging System (INSS) stage and Children’s Oncology Group (COG) risk stratification after adding the serum Tchol level.ResultsCompared with the other patients, serum Tchol level was significantly increased in the relapsed and died patients (P < 0.05). Subsequently, serum Tchol level was found as an independent risk factor to affect the outcome of patients (P < 0.05). Finally, we added serum Tchol level into traditional stage and risk classification system to form the new INSS stage and COG risk classification system. It was found that the areas under the ROC curve (AUC) of recurrence-free survival in the new INSS stage and COG risk classification system were increased to 0.691 (95%CI: 0.535–0.847) and 0.748 (95%CI: 0.622–0.874), respectively. Moreover, the AUC areas of overall survival in the new INSS stage and COG risk classification system were increased to 0.722 (95%CI: 0.561–0.883) and 0.668 (95%CI: 0.496–0.819), respectively.ConclusionWe found that serum Tchol level, a clinical biomarker, is a risk factor for recurrence and death among the children with NB. The serum Tchol level could significantly increase the accuracy of the prediction for NB prognosis.

Abstract 3615: Targeting high-risk neuroblastoma by TGFβ imprinted natural killer cells

Abstract Background: Neuroblastoma (NB) is a common malignant extracranial solid tumor in children, responsible for 11% of cancer deaths in pediatric patients under 15 (Smith et al, Cancer, 2014). High-risk neuroblastoma is a metastatic disease in children aged 18 months or older with L2, M, or MS disease with amplification of the MYCN oncogene (Chuang Pediatr Blood Cancer 2021). Recent analyses by COG show that HR Neuroblastoma still has a 5-year event free survival of 51% despite an extensive treatment regimen (Steven et al, ASCO, 2022). To address improved tumor targeting and overcome antigen escape, chimeric antigen receptor (CAR) technology to Natural Killer (NK) cell therapy has been adapted (Chu/Cairo, et al, Frontiers in Oncology, 2019). TGFβ is a potent mechanism of the NK cell anti-tumor resistance and is present at elevated levels in the NB tumor microenvironment (TME) (Foltz, Cancers, 2018). Studies demonstrated that blockade of TGF-β signaling in NK cells caused the accumulation of NK cells that produce IFN-γ and neutralization of TGF-β prevented NKG2D downregulation and also restored NK cell anti-tumor reactivity (Nayyar/Chu/Cairo, et al Frontierls in Oncology, 2018). ROR1, a transmembrane protein is highly expressed on the majority of NB (Rebagay/Yan Front Oncol, 2012). Our group has successfully expanded functional and active peripheral blood NK cells (exPBNK) with irradiated feeder cells and electroporated CAR mRNA to exPBNK (Chu/Cairo, et al Cancer Immunol Res, 2015). Objective: To determine in vitro cytotoxicity against neuroblastoma cells by ex vivo expanded TGFβ imprinting (TGFβi) NK cells. Methods: TGFβi NK cells are generated by culturing expanded NK cells with irradiated K562-41BBL-mbIL21 in 50 IU/mL IL-2 plus 10 ng/mL TGFβ every 2-3 days for two weeks. NK and TGFβi NK cells specific in-vitro cytotoxicity were compared via luciferase reporter assay against ROR1+ NB cell lines as we previously described (Chu/Cairo, et al, JITC, 2022). Cytokine/chemokine levels secreted by NK and TGFβi NK cells were compared using ELISA assays (Chu/Cairo, et al, JITC, 2022). Results: We have successfully expanded TGFβi NK cells. These TGFβi NK cells showed significantly enhanced in vitro cytotoxicity against NB cells compared to NK cells under TGFβ environment (20+2.4% vs 0%; p&amp;lt;0.001) at E:T=3:1 and consistently, TGFβ-imprinting NK secreted significantly high level of TNF-alpha (393.7pg/ml+16.77pg/ml) against NB cells (p&amp;lt;0.001) compared to NK cells (79.7pg/ml+14.7pg/ml) under TGFβ environment. Conclusion: Our data indicate TGFβi NK cells may be a promising novel therapy to overcome the immunosuppression of TGFβ in the tumor microenvironment for pediatric NB tumors. We will further engineer these TGFβi NK cells to express anti-ROR1 CAR to enhance the targeting specificity against NB tumors and investigate the anti-tumor effects of anti-ROR1 CAR TGFβi NK cells against NB tumors in-vitro and in-vivo (Funded by U54 CA232561). Citation Format: Jessica A. Ayala-Cuesta, Yaya Chu, Keira Foley, Meijuan Tian, Kayleigh Klose, Alyssa S. Mendelowitz, Timothy P. Cripe, Dean A. Lee, Mitchell S. Cairo. Targeting high-risk neuroblastoma by TGFβ imprinted natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3615.

Abstract 2865: Efficacy of MDM2 and epigenetic inhibitors treatments for neuroblastoma

Abstract Pediatric Neuroblastoma (NB) is one of the most prevalent solid tumors in children that arise from embryonic neural crest cells and poses a significant challenge in the field of oncology. There are about 700 to 800 new cases of neuroblastoma diagnosed each year in the United States, and accounts for approximately 15% of all childhood cancer mortalities. Since this malignancy predominantly affects infants and young children, it is imperative to decipher the underlying molecular factors driving its pathogenesis. The novel molecular therapeutic approach includes the use of epigenetic modifiers as drugs. This approach aims to reverse significant epigenetic events underlying cancer pathogenesis, particularly abnormalities in histone modifications and DNA methylation. The reduction of both DNA and histone methylation levels leads to the reactivation of tumor suppressor genes and inhibits cancer cell proliferation and cell growth. In this study, we used selective and reversible small molecule agents such as MDM2 inhibitor (RG7388) and epigenetic modifiers (CM272 and SAHA) to block cell proliferation by inducing cell cycle arrest via enforcing Epigenetic alterations. Our initial experiments were designed to understand the effects of RG7388, CM272, and SAHA treatments on NB and their impact on cell cycle progression. It was hypothesized that the decrease in DNA methylation and increase in histone modification- augment p21 expression via the use of MDM2 inhibitors and epigenetic modifiers. To test this hypothesis, the NB cells (SK-N-SK and IMR-32) were treated with RG7388, CM272, and SAHA for 24 hours. The extent of Cell death was determined for NB cells by using the Trypan Blue Dye Exclusion (TBDE) method, and CellTiter-Glo® Luminescent Cell Viability Assay. Induction of apoptosis was determined by using the Immunofluorescence staining method with DEVD-amc substrate. The results of our study suggested that RG7388, CM272, and SAHA were able to induce cell death in the SK-N-SH cells following 24-hour treatment. Furthermore, we analyzed the levels of Tumor suppressor (p53), cell cycle arrest (p21, p27, AurkB, CDC25A, CyclinD) cell death (Bax, Bcl2, LC3, Beclin, PARP, Cleaved PARP, RIP1, RIP3, MLKL, p-AKT), and Epigenetic biomarker (DNMT1, DNMT3A, DNMT3B, acetyl histoneH3, H4, and H2b) protein expression levels using western blot analysis. The results showed significant elevation of p53, p21, p27, and acetylation of histone family biomarkers after treatment when compared with the control group. In summary, our results indicate that RG7388, CM272, and SAHA treatments can effectively induce cellular responses and prevent cell growth and progression by enforcing epigenetic changes in the NB cells. (This research was supported by the National Pediatric Cancer Foundation, Tampa, Florida, and the Royal Dames of Cancer Research Inc. of Ft. Lauderdale, Florida) Citation Format: Tahani Momenah, Umamaheswari Natarajan, Shyam Jaganathan, Appu Rathinavelu. Efficacy of MDM2 and epigenetic inhibitors treatments for neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2865.

Abstract 6883: Investigation of tumor-immune interactions in pediatric sarcomas using single-cell multi-omics

Abstract Background: Pediatric sarcoma represents the second most common solid tumor in children. Despite advances in multimodal therapies, one-third of sarcoma patients do not survive the disease. Over the past few decades, the development of novel therapeutic strategies for sarcoma has been impeded by an insufficient understanding of the tumor microenvironment (TME). In particular, the potential of immunotherapy remains constrained by the gap of knowledge about the molecular and functional phenotypes of the immune cells in the sarcoma TME. Methods: To address this knowledge gap, we collected treatment-naïve tumor samples from 32 patients with five pediatric sarcoma subtypes. We employed a single-nuclei RNA+ATAC-Seq multi-omics assay to comprehensively characterize the transcriptomic and epigenomic heterogeneity of tumor and immune cells within the sarcoma TME. Results: Our analysis revealed that tumor-associated macrophages (TAMs) were the most abundant immune cell type in the sarcoma TME. We identified seven distinct TAM subpopulations, each exhibiting unique gene expression and chromatin accessibility patterns. Among these, TAM subsets with higher expression of SPP1 and C1QC genes displayed the strongest immunosuppressive potential. Transcriptional regulatory network (TRN) analysis identified transcription factors that were most likely to drive the phenotypes of SPP1+ and C1QC+ TAMs, including ELF2, JUND and RUNX1. Through cell-cell communication analysis, we discovered that SPP1+ TAMs interact with T cells via SPP1-CD44 signaling, while C1QC+ TAMs interact with NK cells through HLA-E-KLRC1 signaling, suggesting the potential involvement of these signaling pathways in TAM-mediated suppression of these cytotoxic lymphocytes. We prioritized ligand-receptor gene pairs, such as SEMA3D-NRP1 and LTBP3-ITGB5, which likely mediate the interaction between malignant sarcoma cells and SPP1+/C1QC+ TAMs based on their dominance across sarcoma subtypes and association with poorer clinical outcomes. On the other hand, our analysis revealed that sarcoma subtype-specific interactions between TAMs and malignant cells are common, highlighting the heterogeneity of signaling events across sarcoma subtypes. Conclusion and Significance: Our study presents a comprehensive single-cell multi-omics characterization of pediatric sarcomas and sheds light on its tumor microenvironment. By identifying unique markers for immunosuppressive TAM subpopulations and elucidating intercellular interactions promoting tumor development, this study proposes potential therapeutic targets for sarcoma treatment based on the role of TAMs in sarcoma TME. Citation Format: Zhan Zhang, Kyung Jin Ahn, Changya Chen, Rumeysa Biyik Sit, Hesham Mohei, Brian Lockhart, Malay Haldar, Kathrin Bernt, Vinodh Pillai, Kai Tan. Investigation of tumor-immune interactions in pediatric sarcomas using single-cell multi-omics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6883.

Abstract 153: Noncoding elements of MYCN mRNA are powerful drivers on oncogenicity in neuroblastoma

Abstract Precision in genetic modeling of oncogene-driven cancer is crucial for comprehending disease pathology. Traditionally, models focused on protein expression through open-reading-frame (ORF) transgenes. This approach overlooks post-transcriptional regulation in endogenous mRNAs, which include noncoding elements like 5’ and 3’ UTRs and introns. All patient oncogene mRNAs encompass these elements, and grasping the collective impact of noncoding and protein coding components may revolutionize our comprehension of cancer origins.Neuroblastoma (NB) is the most common extracranial solid tumor in children. Genetic amplification of MYCN (MA) in NB results in very high mRNA levels and drives poor prognosis. MYCN mRNA is targeted by multiple microRNAs, including let-7. We previously demonstrated that in MA disease the 3’ UTR of MYCN can sequester let-7, inhibiting its function. MYCN mRNA thus also influences genetic patterning in NB by relieving the selective pressure to lose chromosome arms harboring let-7 loci, an event frequent in MYCN non-amplified NB but exceedingly rare in MA disease. This work established for the first time that noncoding mRNA elements within an oncogenic mRNA can itself contribute to disease pathology. To further understand the roles of noncoding elements within MYCN mRNA, we generated BALB/c 3T3 fibroblasts and murine Neuro-2a cells that express the following transgenes: 1.MYCN-ORF: expresses only MYCN open reading frame sequence.2.MYCN-GL: intact genetic locus of MYCN, expresses full length MYCN mRNA.3.M/GFP-GL: variant of #2 where MYCN protein coding sequences are replaced by EGFP. All three constructs demonstrated significantly enhanced in vitro proliferation compared to GFP control cells. Notably, cells expressing MYCN-GL exhibited best growth in both cell types, despite possessing an intact 3’UTR. Unexpectedly, M/GFP-GL cells demonstrated comparable growth to MYCN-ORF in 3T3s and significantly outperformed MYCN-ORF in Neuro-2a cells. This observation not only validates our prior research on the oncogenic role of the MYCN 3’ UTR but also extends it by revealing direct contributions to growth advantages, occasionally surpassing even MYCN protein. Syngeneic animal studies mirrored these trends, with MYCN-GL and M/GFP-GL inducing nearly 100% tumor incidence, surpassing the 57% incidence associated with MYCN-ORF. Tumors induced by MYCN-GL and M/GFP-GL were more than twice the size of those induced by MYCN-ORF. These observations underscore that full-length MYCN mRNA, encompassing all noncoding elements, serves as a more potent driver of cellular growth and oncogenicity than only MYCN protein expression. These findings have exciting implications for understating neuroblastoma pathology and genetic patterns, with broader implications for other oncogene-driven cancers. We are currently generating transgenic mouse models based on the above three constructs. Citation Format: Vishwa Patel, Lorraine Rana Benhamou, Dr. John T. Powers. Noncoding elements of MYCN mRNA are powerful drivers on oncogenicity in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 153.

Abstract 50: ALK inhibitors increase ALK expression and sensitize neuroblastoma cells to ALK.CAR-T cells

Abstract Neuroblastoma is the most common extracranial solid tumor of childhood, with limited success in treating refractory or relapsed cases using current therapies. Chimeric antigen receptor (CAR) T cell therapy targeting GD2 has shown promise in neuroblastoma treatment, but relapses are associated with loss of antigen expression. The selection of the best tumor antigen is critical for the therapeutic success of CAR-T cells in hematologic malignancies and solid tumors. The Anaplastic Lymphoma Kinase (ALK) receptor is expressed by most neuroblastomas while virtually absent in most normal tissues. ALK is an oncogenic driver in neuroblastoma and ALK inhibitors show promising clinical activity. All these features make ALK an attractive target for CAR-T cell therapy. We developed ALK.CAR-T cells that show potent efficacy in monotherapy against neuroblastoma with high ALK expression without toxicity. For neuroblastoma with low ALK expression, combination with ALK inhibitors specifically potentiates ALK.CAR-T cells but not GD2.CAR-T cells. In neuroblastoma cell lines and in a patient-derived xenograft (PDX), the combination of ALK inhibitors and ALK.CAR-T cells significantly reduced tumor growth and extended mice survival. Mechanistically, ALK inhibitors impair tumor growth and upregulate the expression of ALK, thereby facilitating the activity of ALK.CAR-T cells against neuroblastoma. Thus, while neither ALK inhibitors nor ALK.CAR-T cells will likely be sufficient as monotherapy in neuroblastoma with low ALK density, their combination specifically enhances therapeutic efficacy. These findings provide insights into the potential of ALK.CAR-T cells as a novel therapeutic strategy for neuroblastoma. A Phase I clinical trial to test ALK.CAR-T cells in combination with ALK TKIs in children with refractory/relapsed neuroblastoma is being implemented based on these results. Citation Format: Elisa Bergaggio, Wei-Tien Tai, Andrea Aroldi, Carmen Mecca, Elisa Landoni, Manuel Nüesch, Ines Mota, Jasna Metovic, Luca Molinaro, Leyuan Ma, Diego Alvarado, Chiara Ambrogio, Claudia Voena, Rafael B. Blasco, Tongqing Li, Daryl Klein, Darrell J. Irvine, Mauro Papotti, Barbara Savolodo, Gianpietro Dotti, Roberto Chiarle. ALK inhibitors increase ALK expression and sensitize neuroblastoma cells to ALK.CAR-T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 50.

Abstract 5122: Modulating anti-GD2 immunotherapy via dual DFMO and TGF-β inhibition in neuroblastoma

Abstract Neuroblastoma (NB) is an often-lethal childhood solid tumor. Anti-GD2 immunotherapy improves survival but not all responses are durable. Transforming growth factor-beta (TGFβ) has been implicated in solid tumor immunoresistance. High-risk NBs harbor activated MYC or MYCN to coordinately expand intratumoral polyamines (PA), oncometabolites with immunomodulatory roles, to support tumor progression. Difluoromethylornithine (DFMO) is an inhibitor of ornithine decarboxylase, the rate-limiting enzyme in PA synthesis, and has shown activity in preclinical NB models, including TH-MYCN+/+ transgenic mice, and is in clinical trials in children. We sought to augment anti-GD2 therapy using DFMO and the small molecule TGFβ-receptor inhibitor, galunisertib. We randomized TH-MYCN+/+ mice in two experiments (10 therapy arms; n=6-10 mice/arm) to receive combinations of DFMO, galunisertib, and anti-GD2 antibody m14G2a and to assess impact on survival. We measured 770 genes using nCounter Immuno-Oncology 360 (Nanostring), 36 cytokines using xMap bead-arrays (Luminex), and tumor-infiltrating leukocytes using flow cytometry in harvested tumors. Cell line TGFβ signaling was evaluated in tissue culture. In vitro, galunisertib consistently suppressed the upregulated TGFβ-Smad2/3 signaling cascade in MYCN-amplified neuroblastoma cell lines. In vivo, DFMO alone extends mouse survival and increases intra-tumoral neutrophils and natural killer (NK) cells. These NK cells increased surface expression of activation receptors like NKG2D (p&amp;lt;0.05), while tumor cells increased surface expression of NK-ligands. Tumor cells also expressed increased latency-associated peptide (LAP; p&amp;lt;0.05), a TGFβ precursor, suggesting NK cells were poised to kill but restrained by TGFβ. Cytotoxicity assays showed reversal of this inhibition with TGFβR1 inhibitor galunisertib in vitro. In a proof-of-concept trial, DFMO and galunisertib (DFMO/gal) demonstrated a clinical anti-tumor synergistic effect. DFMO/gal treated tumors had upregulated NK cell activity genes and downregulated TGFβ signaling genes. Cytokine analysis confirmed DFMO induced upregulation of TGFβ1 and TGFβ2 (p&amp;lt;0.01) that was modulated by galunisertib. IL6, GCSF, and GMCSF were markedly downregulated in tumors treated with both DFMO and galunisertib (p&amp;lt;0.05). An expanded trial showed DFMO/gal added to m14G2a showed no significant survival benefit over the DFMO and m14G2a combination. Ultimately, DFMO creates a pro-inflammatory cellular and cytokine milieu in TH-MYCN+/+ NBs. DFMO and TGFβR1 inhibition via galunisertib activate NK cells further and decrease the anti-inflammatory cytokine IL6. Ongoing studies are further characterizing the gene expression and cytokine levels of the NB TME under these treatment conditions. Citation Format: Om H. Gandhi, Christina S. Turn, Kangning Liu, Annette T. Vu, Hamid Bassiri, Michael D. Hogarty. Modulating anti-GD2 immunotherapy via dual DFMO and TGF-β inhibition in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5122.

Abstract 5467: Identification of possible new treatment options for rare pediatric CNS tumors using a panel of validated and characterized PDX models

Abstract Central nervous system (CNS) malignancies are the most frequent group of childhood solid tumors. While pediatric mortality due to CNS tumors has decreased over the past 40 years, however, there has been no significant change in brain and other CNS tumor mortality in children and adolescents since 2007*. Advances in research and innovative therapies are crucial to improving outcomes and reducing mortality. Collaborative efforts between researchers, healthcare professionals, and the pharmaceutical industry play a pivotal role in developing more effective treatments for these indications. One important piece in this endeavor is the availability of well characterized preclinical models for rigorous preclinical testing. In the framework of the ITCCP4 consortium (www.ITCCP4.eu) our group determined the pharmacological profile of ten orthotopically implanted pediatric brain tumor PDX models. The panel comprises amongst others four CNS-BCOR models, one ZFTA::RELA ependymoma model and two models of PFA ependymoma. We tested the models in a single mouse trial format with 16 arms covering standard of care therapy including radiation as well as targeted agents. All tumors were intracranially implanted into immune-compromised NSG mice. Tumor load was determined using a fluorescence based in vivo imaging technology based on the lentiviral transient transduction of PDX cells with iRFP713 prior to implantation into the brain. In addition, body weight and neurological scoring were monitored to determine the overall condition of the animals. At the end of the study brain tissue was harvested and tumor load confirmed by immunohistochemistry. Vismodegib, a first in class inhibitor of the hedgehog signaling pathway, was the most efficacious treatment with a significantly extended overall survival (Log Rank, p&amp;lt; 0.001) and a reduced tumor load compared to untreated control arms. The effect was most prominent in the subclass of CNS-BCOR models leading to growth delay or stable disease. Several targeted agents such as Ipatasertib (AKT inhibitor), Idasanutlin (MDM2-antagonist) and standard of care such as Carboplatin and Radiotherapy induced stable disease as well but had no significant effect on overall survival. The positive results for Vismodegib, Ipatasertib as well as Idasanutlin will be validated in a smaller subset of models in a conventional set-up to confirm the current dataset. In summary, the single mouse trial format using intracranially implanted PDX models proved to be a feasible tool to identify most promising drug candidates for pediatric ependymoma. *Ostrom QT, Price M, Ryan K, Edelson J, Neff C, Cioffi G, et al.. CBTRUS statistical report: pediatric brain tumor foundation childhood and adolescent primary brain and other central nervous system tumors diagnosed in the United States in 2014-2018. Neuro-Oncology (2022) 24(Supplement_3):iii1-38. Citation Format: Eva Oswald, Dorothee Lenhard, Kanstantsin Lashuk, Olivier Delattre, Didier Surdez, Johannes Gojo, Daniela Lötsch-Gojo, Walter Berger, Stefan Pfister, Julia B. Schueler. Identification of possible new treatment options for rare pediatric CNS tumors using a panel of validated and characterized PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5467.

Prevention of cisplatin-induced hearing loss in children: achievements and challenges for evidence-based implementation of sodium Thiosulfate.

Ototoxicity is a devastating direct, irreversible side effect of platinum use in children with cancer, with its consequent effect on speech, language and social development, quality of life and adult productivity. Cisplatin, an essential chemotherapeutic agent for the treatment of solid tumors in children, is a DNA cross-linking agent. Which causes hearing loss in 50-70% of cisplatin treated children. Fortunately, to prevent hearing loss, sodium thiosulfate (STS), which binds to cisplatin, and reduces the superoxides in both tumor and outer hair cells of the cochlea has now been discovered to be an effective and safe otoprotectant if administered correctly. The aim of this perspective paper is to explore the key safety issues and challenges important for pediatric oncologists and pharmacists when considering the clinical use of STS as an otoprotectant for children and adolescents receiving cisplatin. These include: the choice of the formulation; the timing, both that of the STS in relation to cisplatin as well as the timing of the cisplatin infusion itself; the dosing; the challenge left by the definition of localized versus disseminated disease and the difference in indication for STS, between cisplatin treated patients and those receiving another platinum chemotherapeutic agent, carboplatin.

Prognostic factors in high-grade pediatric osteosarcoma among children and young adults: Greek Nationwide Registry for Childhood Hematological Malignancies and Solid Tumors (NARECHEM-ST) data along with a systematic review and meta-analysis

The 5-year overall survival of children and adolescents with osteosarcoma has been in plateau during the last 30 years. The present systematic review (1976–2023) and meta-analysis aimed to explore factors implicated in the prognosis of children and young adults with high-grade osteosarcoma. Original studies including patients ≤30 years and the Nationwide Registry for Childhood Hematological Malignancies and Solid Tumors (NARECHEM-ST) data (2010–2021) referred to children ≤14 years were analysed. Individual participant data (IPD) and summary estimates were used to assess the n-year survival rates, as well as the association of risk factors with overall survival (OS) and event-free survival (EFS). IPD and the n-year survival rates were pooled using Kaplan-Meier and Cox regression models, and random effects models, respectively.Data from 8412 patients, including 46 publications, NARECHEM-ST data, and 277 IPD from 10 studies were analysed. The summary 5-year OS rate was 64% [95% confidence interval (95%CI): 62%-66%, 37 studies, 6661 patients] and the EFS was 52% (95%CI: 49%-56%, 30 studies, 5010 patients). The survival rates generally differed in the pre-specified subgroups. Limb-salvage surgery showed a higher 5-year OS rate (69%) versus amputation (47%). Good responders had higher OS rates at 3 years (94%) and 5 years (81%), compared to poor responders at 3 years (66%), and 5 years (56%). Patients with metastatic disease had a higher risk of death [Hazard Ratio (HR): 3.60, 95%CI: 2.52, 5.15, 11 studies]. Sex did not have an impact on EFS (HR females/males: 0.90, 95%CI: 0.54, 1.48, 3 studies), whereas age>18 years seems to adversely affect EFS (HR 18+/<10 years: 1.36, 95%CI: 1.09, 1.86, 3 studies).Our results summarize the collective experience on prognostic factors of high-grade osteosarcoma among children and young adults. Poor response to neoadjuvant chemotherapy and metastatic disease at diagnosis were confirmed as primary risk factors of poor outcome. International collaboration of osteosarcoma study groups is essential to improve survival.

BAP31 Promotes Angiogenesis via Galectin-3 Upregulation in Neuroblastoma.

Neuroblastoma (NB) is one of the highly vascularized childhood solid tumors, and understanding the molecular mechanisms underlying angiogenesis in NB is crucial for developing effective therapeutic strategies. B-cell receptor-associated protein 31 (BAP31) has been implicated in tumor progression, but its role in angiogenesis remains unexplored. This study investigated BAP31 modulation of pro-angiogenic factors in SH-SY5Y NB cells. Through protein overexpression, knockdown, antibody blocking, and quantification experiments, we demonstrated that overexpression of BAP31 led to increased levels of vascular endothelial growth factor A (VEGFA) and Galectin-3 (GAL-3), which are known to promote angiogenesis. Conditioned medium derived from BAP31-overexpressing neuroblastoma cells stimulated migration and tube formation in endothelial cells, indicating its pro-angiogenic properties. Also, we demonstrated that BAP31 enhances capillary tube formation by regulating hypoxia-inducible factor 1 alpha (HIF-1α) and its downstream target, GAL-3. Furthermore, GAL-3 downstream proteins, Jagged 1 and VEGF receptor 2 (VEGFR2), were up-regulated, and blocking GAL-3 partially inhibited the BAP31-induced tube formation. These findings suggest that BAP31 promotes angiogenesis in NB by modulating GAL-3 and VEGF signaling, thereby shaping the tumor microenvironment. This study provides novel insights into the pro-angiogenic role of BAP31 in NB.

Use of modern three-dimensional imaging models to guide surgical planning for local control of pediatric extracranial solid tumors.

In complex pediatric surgical oncology, surgical planning is contingent upon data gathered from preoperative imaging. Three-dimensional (3D) modeling and printing has been shown to be beneficial for adult presurgical planning, though pediatric literature is less robust. The study reviews our institutional experience with the use of 3D image segmentation and printed models in approaching resection of extracranial solid tumors in children. This is a single institutional series from 2021 to 2023. Models were based on computed tomography and magnetic resonance imaging studies, optimized for 3D imaging. The feasibility and creation of the models is reviewed, including specific techniques, software, and printing materials from our institution. Clinical implications for surgical planning are also described, along with detailed preoperative and intraoperative images. 3D modeling and printing was performed for four pediatric patients diagnosed with extracranial solid tumors. Diagnoses included Ewing sarcoma, hepatoblastoma, synovial sarcoma, and osteosarcoma. No intraoperative complications or discrepancies with the preoperative 3D-printed model were noted. No evidence of local recurrence was identified in any patient thus far. Our institutional series demonstrates a wide spectrum of clinical application for 3D modeling and printing technology within pediatric surgical oncology. This technology may aid in surgical planning for both resection and reconstruction, can be applied to a diverse breadth of diagnoses, and may potentially augment patient and/or family education about their condition.

SESN1 functions as a new tumor suppressor gene via Toll-like receptor signaling pathway in neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor in children, with a 5-year survival rate of <50% in high-risk patients. MYCN amplification is an important factor that influences the survival rate of high-risk patients. Our results indicated MYCN regulates the expression of SESN1. Therefore, this study aimed to investigate the role and mechanisms of SESN1 in NB. siRNAs or overexpression plasmids were used to change MYCN, SESN1, or MyD88's expression. The role of SESN1 in NB cell proliferation, migration, and invasion was elucidated. Xenograft mice models were built to evaluate SESN1's effect invivo. The correlation between SESN1 expression and clinicopathological data of patients with NB was analyzed. RNA-Seq was done to explore SESN1's downstream targets. SESN1 was regulated by MYCN in NB cells. Knockdown SESN1 promoted NB cell proliferation, cell migration, and cell invasion, and overexpressing SESN1 had opposite functions. Knockdown SESN1 promoted tumor growth and shortened tumor-bearing mice survival time. Low expression of SESN1 had a positive correlation with poor prognosis in patients with NB. RNA-Seq showed that Toll-like receptor (TLR) signaling pathway, and PD-L1 expression and PD-1 checkpoint pathway in cancer were potential downstream targets of SESN1. Knockdown MyD88 or TLRs inhibitor HCQ reversed the effect of knockdown SESN1 in NB cells. High expression of SESN1 was significantly associated with a higher immune score and indicated an active immune microenvironment for patients with NB. SESN1 functions as a new tumor suppressor gene via TLR signaling pathway in NB.

Twinning to reduce research and innovation inequalities in paediatric solid tumours across Europe

Inequalities in research and innovations affect childhood cancer survival across Europe. Vilnius University Hospital Santaros Klinikos (VULSK, the coordinator) and eight research-intensive institutions from seven European countries implemented the TREL project (Twinning in Research and Education to improve survival in childhood solid tumours in Lithuania) supported by the Horizon 2020 Widening programme. TREL aimed to enhance translational, clinical, and survivorship research in paediatric CNS, neuroblastoma, and renal tumours to improve future treatment outcomes in Lithuania. From January 2021 to December 2023, 49 VULSK professionals and 55 peers from partner institutions collaborated in this twinning program. Achievements after three years were: nine educational events, the initiation of basic and clinical research on fertility preservation, ten VULSK researchers joining international research groups, six signed agreements to participate in international academic clinical trials and the implementation of the European Survivorship Passport. Thirty patients received individual treatment recommendations following multidisciplinary discussions with experts from partner institutions. Twenty-five rare genetic variants were classified by the twinning bioinformatician teams with direct consequences on patient management. In conclusion, coordination of the Horizon 2020 project enhanced VULKS’s research capacities, networking channels and attractiveness for industry and academia-initiated innovative actions that will improve survival rates in the long run.

Development of a high-throughput screening platform to identify new therapeutic agents for Medulloblastoma Group 3

Pediatric brain tumors (PBTs) represent about 25 % of all pediatric cancers and are the most common solid tumors in children and adolescents. Medulloblastoma (MB) is the most frequently occurring malignant PBT, accounting for almost 10 % of all pediatric cancer deaths. MB Group 3 (MB G3) accounts for 25–30 % of all MB cases and has the worst outcome, particularly when associated with MYC amplification. However, no targeted treatments for this group have been developed so far.Here we describe a unique high throughput screening (HTS) platform specifically designed to identify new therapies for MB G3. The platform incorporates optimized and validated 2D and 3D efficacy and toxicity models, that account for tumor heterogenicity, limited efficacy and unacceptable toxicity from the very early stage of drug discovery. The platform has been validated by conducting a pilot HTS campaign with a 1280 lead-like compound library. Results showed 8 active compounds, targeting MB reported targets and several are currently approved or in clinical trials for pediatric patients with PBTs, including MB. Moreover, hits were combined to avoid tumor resistance, identifying 3 synergistic pairs, one of which is currently under clinical study for recurrent MB and other PBTs.

A Potential Prognostic Gene Signature Associated with p53-Dependent NTRK1 Activation and Increased Survival of Neuroblastoma Patients.

Neuroblastoma is the most common extracranial solid tumour in children, comprising close to 10% of childhood cancer-related deaths. We have demonstrated that activation of NTRK1 by TP53 repression of PTPN6 expression is significantly associated with favourable survival in neuroblastoma. The molecular mechanisms by which this activation elicits cell molecular changes need to be determined. This is critical to identify dependable biomarkers for the early detection and prognosis of tumours, and for the development of personalised treatment. In this investigation we have identified and validated a gene signature for the prognosis of neuroblastoma using genes differentially expressed upon activation of the NTRK1-PTPN6-TP53 module. A random survival forest model was used to construct a gene signature, which was then assessed across validation datasets using Kaplan-Meier analysis and ROC curves. The analysis demonstrated that high BASP1, CD9, DLG2, FNBP1, FRMD3, IL11RA, ISGF10, IQCE, KCNQ3, and TOX2, and low BSG/CD147, CCDC125, GABRB3, GNB2L1/RACK1 HAPLN4, HEBP2, and HSD17B12 expression was significantly associated with favourable patient event-free survival (EFS). The gene signature was associated with favourable tumour histology and NTRK1-PTPN6-TP53 module activation. Importantly, all genes were significantly associated with favourable EFS in an independent manner. Six of the signature genes, BSG/CD147, GNB2L1/RACK1, TXNDC5, FNPB1, B3GAT1, and IGSF10, play a role in cell differentiation. Our findings strongly suggest that the identified gene signature is a potential prognostic biomarker and therapeutic target for neuroblastoma patients and that it is associated with neuroblastoma cell differentiation through the activation of the NTRK1-PTPN6-TP53 module.