Pediatric CNS Research Articles

IMG-05. USING CONVOLUTIONAL NEURAL NETWORKS TO IDENTIFY COMMON MOLECULAR ALTERATIONS IN PEDIATRIC BRAIN TUMORS

Abstract BACKGROUND Artificial intelligence (AI) and deep learning have had increased uses in healthcare. Initial studies have shown it is possible to predict multiple tumor types in adults and children using convolutional neural networks (CNN), a form of deep learning, using MRIs. In pediatric CNS tumors, molecular alterations are used in both diagnosis and treatment decisions. This project aims to verify that we can use deep learning to help diagnose and molecularly classify primary pediatric CNS tumors. Previously, we used Recurrent Health’s platform to predict high vs. low grade CNS lesions to >95% recall and 85% precision in pediatric patients. The objective of this study is to validate the AI’s ability to predict common genetic alterations with 90% accuracy. METHODS This retrospective study evaluated patients (ages 0-21) with histologically confirmed primary CNS tumors that underwent BRAF molecular analysis. We collected demographic data: age, tumor location, and presence of BRAF mutations/fusions. The data was divided into a training and validation set (80%/20% split). To determine the viability of models, we will measure specificity, sensitivity, and F1 Score (Sorenson-Dice coefficient) for predicting genetic alterations. Model score confidence was achieved through K-fold cross-validation and statistical power analysis with a finite population coefficient to determine the appropriate sample size. RESULTS Our retrospective test included 136 patients with multiple scans for training, model validation and testing. The CNN was able to predict presence of BRAF fusions with >95% recall/sensitivity, >76% precision/specificity and F1/accuracy >87% while predicting BRAFV600 mutations with >75% recall/sensitivity, >95% precision/specificity and F1/accuracy >85%. These metrics indicate strong discrimination in classification. CONCLUSIONS Preliminary data suggests the accuracy of the model in identifying the presence of BRAF mutations, providing a potential opportunity for a non-invasive tool to help aid in diagnosis and help guide initiation of targeted therapy.

MDB-102. CDK8 IS A DEPENDENCY IN GROUP 3 MEDULLOBLASTOMA AND COMBINES WITH BET BROMODOMAIN INHIBITION

Abstract Medulloblastoma (MB) is the most common pediatric CNS malignancy with varying prognoses depending on subgroup diagnosis. Group 3 MB displays the poorest overall prognosis, specifically in cases characterized by MYC hyperactivation. Current standard treatment can cause lifelong debilitating side effects, therefore the identification of targeted therapeutic agents will be critical in enhancing patient survival and bypassing therapy-induced aftereffects. We utilized the Pediatric Cancer Dependency Map to identify CDK8 as the most striking dependency in MB compared to all other pediatric malignancies. Further analysis revealed dependencies on its cognate cyclin, Cyclin C, and other members of the CDK8 kinase module (CKM). The CKM functions as a transcriptional regulator, phosphorylating RNAPol2 and other transcription factors. Interestingly, CDK8 dependency correlates with MYC amplification status in cell lines suggesting it to be a strong dependency in G3 MB. CDK8 expression, both RNA and protein, is highest in G3 tumors. We depleted CDK8 both genetically and pharmacologically and demonstrated downregulation of MYC expression in tumor cells and decreased cell survival. We next tested the efficacy of a CDK8 kinase inhibitor currently in clinical trials. Treatment with SEL-120 reduced cell proliferation and viability in vitro and inhibited tumor growth in vivo. This effect was limited to G3 models, as SEL120 showed little efficacy in SHH MB. Analysis of gene expression revealed that targeting CDK8 did not affect MYC-dependent gene expression but led to de-repression of differentiation genes regulated by PRC2. We further observed a global de-repression of H3K27me3 in cells treated with CDK8i. Finally, we tested the first ever blood-brain barrier penetrant CDK8i which mimics the activity of SEL-120 and potently synergizes with BETi, which antagonizes MYC signaling. In summary, we have identified and validated CDK8 as a novel target in G3 MB and identified a possible synergistic combination to move forward in clinical trials.

OTHR-29. A SINGLE INSTITUTION REVIEW REGARDING TRENDS IN THE MOLECULAR ANALYSIS OF PEDIATRIC CENTRAL NERVOUS SYSTEM (CNS) TUMORS AND GERMLINE ANALYSIS BETWEEN 2019 – 2023, EMPHASIZING THE IMPORTANCE OF EASY ACCESS TO COMPREHENSIVE TESTING

Abstract BACKGROUND Molecular characterization of pediatric CNS tumors improves diagnostic accuracy and guides treatment. Germline analysis is also important to uncover cancer predisposition syndromes, which influence the approach to treatment and tumor surveillance. The options for molecular assessment are influenced by availability, including access through a trial or insurance approval. METHODS A retrospective review of the molecular workup performed on all CNS tumors biopsied/resected at Children’s Minnesota from 2019 – 2023 was undertaken to look at trends in the specific tests performed over time and how the molecular results guided treatment. RESULTS Between 2019 – 2023, 220 patients at Children’s Minnesota were diagnosed with a CNS tumor. A total of 191 underwent surgery and 162 (85%) had sufficient tissue for molecular analysis. There was no apparent difference in the percentage of patients who underwent IHC/FISH/NGS between 2019 - 2023, but methylation profiling frequency increased over time (2019 2%, 2020 and 2021 10%, 2022 58% and 2023 71%). Frequency of germline analysis also increased (2019-2021 29%, 2022-52% and 2023-63%). Easy availability of comprehensive molecular and germline analysis for patients enrolled on COG-APEC14B1 is credited for these improving trends. Molecular analysis confirmed (59%) or supported (36%) the diagnosis, and guided treatment in 30% of cases (stratification of treatment in 89% of medulloblastoma and targeted treatment in 63% of high-grade glioma, 9% of medulloblastoma and 6% of low-grade glioma). Methylation profiling alone accurately diagnosed 79% of cases, including subtyping tumors. Germline analysis detected a pathogenic germline alteration in 14% of CNS tumor patients (NF1 5%; TP53 and BRIP1 1%; PALB1, NF2, TS2, SMARCB1, biallelic PMS2, CHEK2, FANCA, ORICH1 and SDHA 0.5%). CONCLUSION These findings demonstrate the importance of easy access to comprehensive molecular and germline analysis with quick turnaround time to provide the best diagnosis, prognosis and treatment options for pediatric CNS tumor patients.

DIPG-88. TARGETING THE ANTIOXIDANT RESPONSE ELEMENT AXIS SENSITIZES DIFFUSE MIDLINE GLIOMA CELLS TO ONC201

Abstract BACKGROUND Diffuse midline glioma (DMG) is a universally fatal CNS tumor, with a median overall survival of <12-months. Recent studies, including our own, have identified overexpression of the mitochondrial protease ClpP in DMG patient samples. ClpP plays a pivotal role in regulating energy production. Brain-penetrant ClpP agonists, including ONC201, ONC206, and TR107, induce non-specific degradation of the mitochondrial electron transport chain. However, there is considerable variability in the response of DMG cell lines to these therapies, with only about half reaching IC50. Notably, TP53-mutant cell lines exhibit reduced sensitivity. METHODS/RESULTS Further analysis using quantitative proteomics of TP53-mutant DMG cell lines exhibited hallmark activation of ClpP, increased mitochondrial stress and dysfunction. Importantly, ClpP agonism increased the activity of the transcription factor NRF2, driving expression of NQO1 (p=0.0017). NQO1 is a cytoprotective enzyme, promoting redox balance and cell survival. This highlights a potential adaptive response to ClpP agonist therapy in DMG cells. Analysis of DMG patient samples through publicly available RNA sequencing data identified ubiquitously high NRF2 expression compared to other pediatric CNS tumors. This led us to hypothesize that a clinically relevant, selective NQO1 inhibitor could elicit a strong response to ClpP agonism. Therefore, we examined ARQ761, a CNS-active therapy that disrupts NQO1 leading to the formation of an unstable hydroquinone, causing oxidative DNA damage and apoptosis. When ARQ761 was used concurrently with a ClpP agonist, the combination showed additive effects, average Bliss Score of 9.91 (n=4). Interestingly, administering ARQ761 24 h after ClpP agonist exposure combined synergistically, average Bliss Score of 15.87 (n=4). This synergy was further potentiated using ARQ761 48 h after ClpP agonism, when NQO1 expression peaked, resulting in an average Bliss Score of 20.30 (n=4). CONCLUSIONS This study has unveiled an intrinsic antioxidant defense mechanism in DMG, which facilitates evasion from treatments inducing oxidative stress. This defense mechanism appears particularly robust in cell lines harboring TP53-mutations, which are also known to exhibit resistance to radiotherapy.

GCT-11. CENTRAL NERVOUS SYSTEM GERM TUMORS (CNS GCTS): RESULTS FROM A SINGLE INSTITUTION IN BRAZIL - SÃO PAULO UNIVERSITY

Abstract INTRODUCTION Central Nervous System Germ Tumors (CNS GCTs) are broadly classified as germinomatous (commonly called germinoma) and non-germinomatous germ cell tumors (NGGCTs) based on clinicopathological and laboratory characteristics, including tumor markers. OBJECTIVES Compare data from patients admitted from 2000 to 2023, with the usual presentation in the literature, identifying possible specific variables. METHODOLOGY Retrospective study involving 27 patients admitted to the institution without previous treatment. RESULTS In western countries, GCTs account for 3% to 4% of primary brain tumors in children, with a peak incidence between 10 and 19 years of age. In Japan and other Asian countries, one series reported that the incidence of GCTs in the CNS consists of approximately 15% of all pediatric CNS tumors. The genetic or environmental reasons for these differences remain unknown. In general, males have a higher incidence of GCT than females. Male patients have a preponderance of primary tumors of the pineal region. The data collected by our institution are compatible with those in the literature. CONCLUSION Our discussion and data analysis on GCTs are comparable to those presented in the literature, with the majority of cases treated being: adolescents between 10 and 15 years old, predominantly male and with a satisfactory response to therapy.

ETMR-07. OVEREXPRESSION OF THE PLAG-GENE FAMILY MEMBER PLAG1 DURING MURINE BRAIN DEVELOPMENT LEADS TO ABERRANT EXPRESSION OF IMPRINTED GENES AND THE FORMATION OF MIDLINE BRAIN TUMORS

Abstract BACKGROUND The transcription factor PLAG1, ordinarily active during development, has been identified as overexpressed in several pediatric brain tumor entities, including H3 K27-altered diffuse midline glioma. Additionally, our group recently identified a novel type of embryonic CNS tumors with amplification of the related genes PLAGL1 and PLAGL2. How this aberrant PLAG family gene activation drives tumor formation is unknown. We hypothesize that aberrant activity of these transcription factors impedes normal differentiation, thereby fostering neoplastic transformation. METHODS To investigate this, we utilize transgenic mouse models engineered to exhibit targeted PLAG1 overexpression and loss of Trp53 during brain development. Following, we use bulk and single-cell RNA-seq to examine underlying mechanisms and are working on whole genome CRISPR-Cas9 screens to detect genetic dependencies in derived tumorsphere cultures. RESULTS We found that transgenic mice develop brain tumors in the midline. RNA-seq of these tumors suggests the activation of developmental (homeobox transcription factors) and imprinted genes e.g. H19 and Dlk1, the latter being similarly activated in pediatric CNS tumors with PLAGL1/2 amplification. Using single-cell RNA-seq of the resultant tumors, we compared the gene expression profiles of the tumor cells to a developmental mouse brain atlas. Tumor cells best correlated to radial glia stem cells and glioblasts, supporting the hypothesis of a developmental block caused by PLAG1 overexpression. A subpopulation of the tumor cells seemed to escape the radial glia cell state but were then ultimately locked in the OPC state. A similar mechanism was observed in H3 K27M diffuse midline gliomas. CONCLUSION Collectively, we provide evidence that PLAG1 overexpression drives pediatric brain tumor formation by inhibiting normal cellular differentiation. The ongoing CRISPR screen in PLAG1 and PLAGL1 overexpressing murine cell lines, ChIP-seq analysis of the murine tumors, and comparisons with human data and hNSC models aims to unveil the specific molecular mechanisms underlying tumor progression.

EPID-09. CONGENITAL CENTRAL NERVOUS SYSTEM NEOPLASMS: A RETROSPECTIVE REVIEW FROM THEMARGARET HACKETT FAMILY PROGRAM CONSORTIUM

Abstract INTRODUCTION Congenital intracranial neoplasms are a heterogeneous group of lesions that account for <2% of pediatric CNS tumors. Current understanding of histopathologic and survival outcomes in this population is limited. METHODS Pediatric patients (<3-years) with congenital neoplasms were retrospectively evaluated across five institutions. Demographic, prenatal care, clinical presentation, histopathologic characteristics, operative and adjuvant therapies, and survival outcomes were collected. RESULTS 323 patients met inclusion. The mean age at diagnosis was 1.5-years. Six patients were conceived through IVF. Eleven families reported abnormal prenatal ultrasounds, although only six were diagnosed prenatally. The most common histopathologies included low-grade astrocytoma (30.6%), ependymoma (15.8%), ATRT (11.5%), medulloblastoma (8.0%), and high-grade astrocytoma (2.8%). 48.9% of patients had low-grade neoplasms, and a third of patients (33.1%) had high-grade tumors. Patients commonly presented with hydrocephalus (59.1%), emesis (34.7%), ocular abnormalities (20.4%), macrocrania (18.0%), lethargy (14.6%), developmental delays (12.7%), and irritability (11.8%). All patients received surgical resection: 55.4% received gross-total (GTR) and 20.4% received subtotal resection. Concurrent treatment with adjuvant therapies occurred in a subset of patients, with 52.9% of patients receiving chemotherapy, 27.2% undergoing radiation, and 6.2% participating in a clinical trial. Patients with low-grade tumors had a rate of 88% progression-free (PFS) at 1-year, 74% PFS at 3-years, and 64% PFS at 5-years post-operative. Patients with high-grade neoplasms had worse PFS, with estimates at 59%, 40%, and 37% at 1-, 3-, and 5-years, respectively. There was statistically significant differences in PFS between patients with high- and low-grade neoplasms (p<0.0001). CONCLUSIONS This series consisted of very young pediatric patients across five participating institutions with diverse histopathologies that vary in frequency relative to the entire pediatric population. Lesions were rarely detected prenatally, despite their young age at diagnosis. GTR occurred in half of patients and subsequent management with adjuvant therapies was common in both sub-totally resected low-grade lesions and high-grade tumors.

Rapid DNA methylation-based classification of pediatric brain tumors from ultrasonic aspirate specimens

BackgroundAlthough cavitating ultrasonic aspirators are commonly used in neurosurgical procedures, the suitability of ultrasonic aspirator-derived tumor material for diagnostic procedures is still controversial. Here, we explore the feasibility of using ultrasonic aspirator-resected tumor tissue to classify otherwise discarded sample material by fast DNA methylation-based analysis using low pass nanopore whole genome sequencing.MethodsUltrasonic aspirator-derived specimens from pediatric patients undergoing brain tumor resection were subjected to low-pass nanopore whole genome sequencing. DNA methylation-based classification using a neural network classifier and copy number variation analysis were performed. Tumor purity was estimated from copy number profiles. Results were compared to microarray (EPIC)-based routine neuropathological histomorphological and molecular evaluation.Results19 samples with confirmed neuropathological diagnosis were evaluated. All samples were successfully sequenced and passed quality control for further analysis. DNA and sequencing characteristics from ultrasonic aspirator-derived specimens were comparable to routinely processed tumor tissue. Classification of both methods was concordant regarding methylation class in 17/19 (89%) cases. Application of a platform-specific threshold for nanopore-based classification ensured a specificity of 100%, whereas sensitivity was 79%. Copy number variation profiles were generated for all cases and matched EPIC results in 18/19 (95%) samples, even allowing the identification of diagnostically or therapeutically relevant genomic alterations.ConclusionMethylation-based classification of pediatric CNS tumors based on ultrasonic aspirator-reduced and otherwise discarded tissue is feasible using time- and cost-efficient nanopore sequencing.

Phase I Trial of GD2.CART Cells Augmented With Constitutive Interleukin-7 Receptor for Treatment of High-Grade Pediatric CNS Tumors.

T cells modified with chimeric antigen receptors (CARTs) have demonstrated efficacy for hematologic malignancies; however, benefit for patients with CNS tumors has been limited. To enhance T cell activity against GD2+ CNS malignancies, we modified GD2-directed CART cells (GD2.CARTs) with a constitutively active interleukin (IL)-7 receptor (C7R-GD2.CARTs). Patients age 1-21 years with H3K27-altered diffuse midline glioma (DMG) or other recurrent GD2-expressing CNS tumors were eligible for this phase I trial (ClinicalTrials.gov identifier: NCT04099797). All subjects received standard-of-care adjuvant radiation therapy or chemotherapy before study enrollment. The first treatment cohort received GD2.CARTs alone (1 × 107 cells/m2), and subsequent cohorts received C7R-GD2.CARTs at two dose levels (1 × 107 cells/m2; 3 × 107 cells/m2). Standard lymphodepletion with cyclophosphamide and fludarabine was included at all dose levels. Eleven patients (age 4-18 years) received therapy without dose-limiting toxicity. The GD2.CART cohort did not experience toxicity, but had disease progression after brief improvement of residual neurologic deficits (≤3 weeks). The C7R-GD2.CART cohort developed grade 1 tumor inflammation-associated neurotoxicity in seven of eight (88%) cases, controllable with anakinra. Cytokine release syndrome was observed in six of eight (75%, grade 1 in all but one patient) and associated with increased circulating IL-6 and IP-10 (P < .05). Patients receiving C7R-GD2.CARTs experienced temporary improvement from baseline neurologic deficits (range, 2 to >12 months), and seven of eight (88%) remained eligible for additional treatment cycles (range 2-4 cycles). Partial responses by iRANO criteria were observed in two of seven (29%) patients with DMG treated by C7R-GD2.CARTs. Intravenous GD2.CARTs with and without C7R were well tolerated. Patients treated with C7R-GD2.CARTs exhibited transient improvement of neurologic deficits and increased circulating cytokines/chemokines. Treatment with C7R-GD2.CARTs represents a novel approach warranting further investigation for children with these incurable CNS cancers.

Imaging Review of Pediatric Monogenic CNS Vasculopathy with Genetic Correlation.

Monogenic cerebral vasculopathy is a rare but progressively recognizable cause of pediatric cerebral vasculopathy manifesting as early as fetal life. These monogenic cerebral vasculopathies can be silent or manifest variably as fetal or neonatal distress, neurologic deficit, developmental delay, cerebral palsy, seizures, or stroke. The radiologic findings can be nonspecific, but the presence of disease-specific cerebral and extracerebral imaging features can point to a diagnosis and guide genetic testing, allowing targeted treatment. The authors review the existing literature describing the frequently encountered and rare monogenic cerebral vascular disorders affecting young patients and describe the relevant pathogenesis, with an attempt to categorize them based on the defective step in vascular homeostasis and/or signaling pathways and characteristic cerebrovascular imaging findings. The authors also highlight the role of imaging and a dedicated imaging protocol in identification of distinct cerebral and extracerebral findings crucial in the diagnostic algorithm and selection of genetic testing. Early and precise recognition of these entities allows timely intervention, preventing or delaying complications and thereby improving quality of life. It is also imperative to identify the specific pathogenic variant and pattern of inheritance for satisfactory genetic counseling and care of at-risk family members. Last, the authors present an image-based approach to these young-onset monogenic cerebral vasculopathies that is guided by the size and predominant radiologic characteristics of the affected vessel with reasonable overlap. ©RSNA, 2024 Test Your Knowledge questions for this article are available in the supplemental material.

Rapid identification of pediatric brain tumors with differential mobility spectrometry.

Brain tumors are a major source of disease burden in pediatric population, with the most common tumor types being pilocytic astrocytoma, ependymoma and medulloblastoma. In every tumor entity, surgery is the cornerstone of treatment, but the importance of gross-total resection and the corresponding patient prognosis is highly variant. However, real-time identification of pediatric CNS malignancies based on the histology of the frozen sections alone is especially troublesome. We propose a novel method based on differential mobility spectrometry (DMS) analysis for rapid identification of pediatric brain tumors. We prospectively obtained tumor samples from 15 pediatric patients (5 pilocytic astrocytomas, 5 ependymomas and 5 medulloblastomas). The samples were cut into 36 smaller specimens that were analyzed with the DMS. With linear discriminant analysis algorithm, a classification accuracy (CA) of 70% was reached. Additionally, a 75% CA was achieved in a pooled analysis of medulloblastoma vs. gliomas. Our results show that the DMS is able to differentiate most common pediatric brain tumor samples, thus making it a promising additional instrument for real-time brain tumor diagnostics.

Functional expression of the proton sensors ASIC1a, TMEM206, and OGR1 together with BKCa channels is associated with cell volume changes and cell death under strongly acidic conditions in DAOY medulloblastoma cells

Fast growing solid tumors are frequently surrounded by an acidic microenvironment. Tumor cells employ a variety of mechanisms to survive and proliferate under these harsh conditions. In that regard, acid-sensitive membrane receptors constitute a particularly interesting target, since they can affect cellular functions through ion flow and second messenger cascades. Our knowledge of these processes remains sparse, however, especially regarding medulloblastoma, the most common pediatric CNS malignancy. In this study, using RT-qPCR, whole-cell patch clamp, and Ca2+-imaging, we uncovered several ion channels and a G protein-coupled receptor, which were regulated directly or indirectly by low extracellular pH in DAOY and UW228 medulloblastoma cells. Acidification directly activated acid-sensing ion channel 1a (ASIC1a), the proton-activated Cl− channel (PAC, ASOR, or TMEM206), and the proton-activated G protein-coupled receptor OGR1. The resulting Ca2+ signal secondarily activated the large conductance calcium-activated potassium channel (BKCa). Our analyses uncover a complex relationship of these transmembrane proteins in DAOY cells that resulted in cell volume changes and induced cell death under strongly acidic conditions. Collectively, our results suggest that these ion channels in concert with OGR1 may shape the growth and evolution of medulloblastoma cells in their acidic microenvironment.

Abstract 1770: Right oncogene, wrong tumor - CBL mutations in pediatric CNS and solid tumors

Abstract The molecular analysis of individual patient tumors by personalized medicine programs like the Zero Childhood Cancer Program (ZERO) enables the detection of potentially targetable lesions, unlocking new therapeutic opportunities for patients. Somatic mutations in the E3-ubiquitin ligase, CBL, are known to activate receptor tyrosine kinases (RTKs) in cancer and have been exclusively characterized in hematological malignancies. This includes acute myeloid leukemia, where CBL mutations lead to FLT3 activation. Using molecular data from ZERO we have identified known and novel CBL variants in novel tumor contexts. Our findings raise the possibility that CBL mutation may be a marker of RTK activation in a range of paediatric cancer types and may represent a group of patients who could benefit from tyrosine kinase inhibitor (TKI) therapy. We analyzed whole genome and RNA sequencing data from the ZERO cohort and identified 26 somatic CBL variants in 22 individual patients, the majority of which were in CNS tumors (14 patients). In 8 patients with CNS tumors, we identified missense and splicing variants, which were either novel or not previously seen in this tumor type, that are predicted to have a functional impact on CBL E3 ligase activity due to their location in the linker region or RING finger domain of CBL. Interestingly, most of these patients did not fall into a pre-defined DNA methylation-based subtype classification of CNS tumors and had no significant upregulation of RTK genes. In addition to the novel variants, we identified an established oncogenic CBL deletion variant, CBL exon 8/9 deletion (CBL ex8/9Δ), in novel tumor types - neuroblastoma and a germ cell tumor. Functionally, we show that overexpression of CBL ex8/9Δ enhances cell proliferation and maintains EGFR signaling by blocking CBL mediated degradation of phosphorylated EGFR in neuroblastoma cells. In this study, we demonstrate that CBL is mutated in a range of pediatric cancer types beyond hematological malignancies and may represent a marker of RTK activation and a new target for high-risk patients with limited therapeutic options. Citation Format: Lauren M. Brown, Chelsea Mayoh, Pablo Acera Mateos, Antoine De Weck, Robert Salomon, Teresa Sadras, Neevika Manoharan, Marie Wong, Mark J. Cowley, Paul G. Ekert. Right oncogene, wrong tumor - CBL mutations in pediatric CNS and solid tumors [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 1770.

Abstract 2421: Targeted and low-pass whole genome sequencing of cerebrospinal fluid circulating tumor DNA for the diagnosis and monitoring of pediatric, adolescent and young adult CNS tumors

Abstract Molecular analysis of CNS tumors is essential to their diagnosis and guides individualized treatment for patients, but tissue biopsy of primary and/or relapsed lesions to allow for this characterization often carries intolerable risk. The analysis of genetic material in cerebrospinal fluid (CSF), known as liquid biopsy, can allow for minimally invasive tumor identification and diagnosis. Furthermore, liquid biopsy can serve as a more sensitive test for residual disease than imaging or cytologic analyses. We aimed to implement CSF liquid biopsy at Canada’s largest pediatric hospital. &amp;gt;150 CSF samples were collected from CNS tumor patients with a median age of 9. The most common tumor types were low-grade gliomas (40%), high-grade gliomas (21%) and medulloblastomas (MB) (17%). The majority were collected via lumbar puncture (46%), external ventricular drain (24%) and intraoperatively (22%). The most frequent collection times were diagnosis (53%) and progression/relapse (28%). Cell-free DNA was extracted, and circulating tumor DNA was analyzed using low-pass whole genome sequencing and a targeted hybridization capture glioma gene panel for select cases. Pathogenic mutations and copy number alterations (CNA) were called using an in-house bioinformatics pipeline. Somatic alterations were detected in 57% of samples from patients with pathologically confirmed CNS tumors. CNAs were identified in 5/7 MB patients at the time of pre-treatment staging lumbar puncture. We also detected CNAs in several MB patients during treatment and at progression/relapse, including a patient with disseminated MB who was mid-induction chemotherapy with 1 suspicious cell in their CSF, highlighting our ability to detect minimal residual disease. Among samples with no previous biopsy, we identified tumor DNA in 29%, often providing diagnostic clarity and guiding management. For example, we identified a targetable FGFR1 N546D missense mutation in a patient with an unbiopsied suprasellar lesion. We also diagnosed recurrent MB in a patient 6 years off treatment with a new ambiguous nodule in their tumor bed by detecting an isochromosome 17q CNA. Overall, our liquid biopsy platform is feasible and can yield clinically impactful results. Next steps include refining our assays to increase their sensitivity and systematically evaluating our platform’s efficacy for monitoring response to treatment and tumor surveillance. Citation Format: Ian Burns, Liana Nobre, Yoshiko Nakano, Michelle Ku, Monique Johnson, Javal Sheth, Logine Negm, Chantel Cacciotti, Mansuba Rana, Richard Yuditskiy, Andrew Bondoc, Robert Siddaway, Uri Tabori, Cynthia Hawkins. Targeted and low-pass whole genome sequencing of cerebrospinal fluid circulating tumor DNA for the diagnosis and monitoring of pediatric, adolescent and young adult CNS tumors [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 2421.

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.

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.

Next-generation sequencing for pediatric CNS tumors: does it add value in a middle-income country setup?

Advances in molecular diagnostics led to improved targeted interventions in the treatment of pediatric CNS tumors. However, the capacity to test for these is limited in LMICs, and thus their value needs exploration. We reviewed our experience with NGS testing (TruSight RNA Pan-Cancer-seq panel) for pediatric CNS tumors at KHCC/Jordan (March/2022-April/2023). Paraffin blocks' scrolls were shipped to the SickKids laboratory based on the multidisciplinary clinic (MDC) recommendations. We reviewed the patients' characteristics, the tumor types, and the NGS results' impact on treatment. Of 237 patients discussed during the MDC meetings, 32 patients (14%) were included. They were 16 boys and 16 girls; the median age at time of testing was 9.5 years (range, 0.9-21.9 years). There were 21 samples sent at diagnosis and 11 upon tumor progression. The main diagnoses were low-grade-glioma (15), high-grade-glioma (10), and other histologies (7). Reasons to request NGS included searching for a targetable alteration (20) and to better characterize the tumor behavior (12). The median turnaround time from samples' shipment to receiving the results was 23.5 days (range, 15-49 days) with a median laboratory processing time of 16 days (range, 8-39 days) at a cost of US$1,000/sample. There were 19 (59%) tumors that had targetable alterations (FGFR/MAPK pathway inhibitors (14), checkpoint inhibitors (2), NTRK inhibitors (2), and one with PI3K inhibitor or IDH1 inhibitor). Two rare BRAF mutations were identified (BRAFp.G469A, BRAFp.K601E). One tumor diagnosed initially as undifferentiated round cell sarcoma harbored NAB2::STAT6 fusion and was reclassified as an aggressive metastatic solitary fibrous tumor. Another tumor initially diagnosed as grade 2 astroblastoma grade 2 was reclassified as low-grade-glioma in the absence of MN1 alteration. NGS failed to help characterize a tumor that was diagnosed histologically as small round blue cell tumor. Nine patients received targeted therapy; dabrafenib/trametinib (6), pembrolizumab (2), and entrectinib (1), mostly upon tumor progression (7). In this highly selective cohort, a high percentage of targetable mutations was identified facilitating targeted therapies. Outsourcing of NGS testing was feasible; however, criteria for case selection are needed. In addition, local capacity-building in conducting the test, interpretation of the results, and access to "new drugs" continue to be a challenge in LMICs.

Pediatric bacteremia and CNS infections associated with &lt;i&gt;klebsiella pneumoniae&lt;/i&gt;: molecular genetic characteristics and clinical features

Klebsiella pneumoniae is one of the most significant and life-threatening pathogen of nosocomial infections. This opportunistic microorganism can cause infections of the bloodstream, respiratory tract, urinary tract, skin and soft tissues, inflammation of meninges of the brain and spinal cord, leading to elevated hospital mortality. The purpose of our study was a retrospective analysis of molecular genetic characteristics of K. pneumoniae isolated from blood and liquor samples as well as to describe clinical features in bacteremia and CNS infections. According to the results of assessed clinical data, K. pneumoniae isolates were selected from 64 children suffered from surgical pathology (congenital heart defects — 30%, abdominal pathology — 39%, severe combined trauma — 12%) and somatic diseases accompanied by antibacterial and/or glucocorticosteroid therapy — 14%. The minimum suppressive concentrations of antibiotics were determined by the broth micro-dilution method. Carbapenemases were detected by real time polymerase chain reaction. Virulence genes and capsule serotypes K1/K2 were assessed by multiplex PCR. Biofilms were grown using flat-bottomed polystyrene plates, followed by coloring, fixation, elution and data detection. The population diversity was assessed by multilocus sequence typing. Bacteremia and CNS infections associated with K. pneumoniae were fatal in 25% of cases. A substantial portion of the isolates demonstrated the phenotype of extremely drug resistance (XDR) — 43%, the phenotype of multidrug resistance (MDR) was shown in 16% of the isolates. The blaCTX-M cephalosporinase gene was found in 85% of the strains. The main determinant of resistance to carbapenems was the blaOXA-48 gene (33%); the blaNDM gene was detected in 9% of strains. The combination of blaOXA-48 and blaNDM was found in 7% of isolates. The study of biofilm production showed that moderate ability to form biofilms was shown in 61%, strong — 21%, and weak — 15% isolates. Two isolates (3%) did not form biofilms. The virulence genes entB and mrkD were detected in 100% of isolates, ybtS — in 78%. The iutA gene was found in 18% of the strains. Two isolates showed the presence of the kfu gene. Seven isolates belonged to the K2 serotype. 27 different genotypes were found in K. pneumoniae isolates examined. The most common were: ST307 — 21%, ST395 — 12%, ST48 — 7%, ST39 — 6% and ST29 — 6%. Infections of the bloodstream and central nervous system associated with K. pneumoniae have great importance in clinical practice. This microorganism is able to long persist on biotic and abiotic surfaces, has a wide natural and acquired resistance to antibiotics.

Feasibility and antitumour activity of the FGFR inhibitor erdafitnib in three paediatric CNS tumour patients.

Alterations of the fibroblast growth factor (FGF) signalling pathway are increasingly recognized as frequent oncogenic drivers of paediatric brain tumours. We report on three patients treated with the selective FGFR1-4 inhibitor erdafitinib. Two patients were diagnosed with a posterior fossa ependymoma group A (PFA EPN) and one with a low-grade glioma (LGG), harbouring FGFR3/FGFR1 overexpression and an FGFR1 internal tandem duplication (ITD), respectively. While both EPN patients did not respond to erdafitinib treatment, the FGFR1-ITD-harbouring tumour showed a significant decrease in tumour volume and contrast enhancement throughout treatment. The tumour remained stable 6months after treatment discontinuation.

Germline susceptibility from broad genomic profiling of pediatric brain cancers.

Identifying germline predisposition in CNS malignancies is of increasing clinical importance, as it contributes to diagnosis and prognosis, and determines aspects of treatment. The inclusion of germline testing has historically been limited due to challenges surrounding access to genetic counseling, complexity in acquiring a germline comparator specimen, concerns about the impact of findings, or cost considerations. These limitations were further defined by the breadth and scope of clinical testing to precisely identify complex variants as well as concerns regarding the clinical interpretation of variants including those of uncertain significance. In the course of conducting an IRB-approved protocol that performed genomic, transcriptomic and methylation-based characterization of pediatric CNS malignancies, we cataloged germline predisposition to cancer based on paired exome capture sequencing, coupled with computational analyses to identify variants in known cancer predisposition genes and interpret them relative to established clinical guidelines. In certain cases, these findings refined diagnosis or prognosis or provided important information for treatment planning. We outline our aggregate findings on cancer predisposition within this cohort which identified 16% of individuals (27 of 168) harboring a variant predicting cancer susceptibility and contextualize the impact of these results in terms of treatment-related aspects of precision oncology.