BACKGROUND: A major obstacle in translating the therapeutic potential of chimeric antigen receptor (CAR) T cells to children with central nervous system (CNS) tumors is the blood-brain barrier. To overcome this limitation, preclinical and clinical studies have supported the use of repeated, locoregional intracranial CAR T-cell delivery. However, there is limited literature available describing the process for the involvement of an investigational drug service (IDS) pharmacy, particularly in the setting of a children\'s hospital with outpatient dosing for CNS tumors.
OBJECTIVE: To describe Seattle Children\'s Hospital\'s experience in clinically producing CAR T cells and the implementation of IDS pharmacy practices used to deliver more than 300 intracranial CAR T-cell doses to children, as well as to share how we refined the processing techniques from CAR T-cell generation to the thawing of fractionated doses for intracranial delivery.
METHODS: Autologous CD4+ and CD8+ T cells were collected and transduced to express HER2, EGFR, or B7-H3-specific CAR T cells. Cryopreserved CAR T cells were thawed by the IDS pharmacy before intracranial delivery to patients with recurrent/refractory CNS tumors or with diffuse intrinsic pontine glioma/diffuse midline glioma.
RESULTS: The use of a thaw-and-dilute procedure for cryopreserved individual CAR T-cell doses provides reliable viability and is more efficient than typical thaw-and-wash protocols. Cell viability with the thaw-and-dilute protocol was approximately 75% and was always within 10% of the viability assessed at cryopreservation. Cell viability was preserved through 6 hours after thawing, which exceeded the 1-hour time frame from thawing to infusion.
CONCLUSIONS: As the field of adoptive immunotherapy grows and continues to bring hope to patients with fatal CNS malignancies, it is critical to focus on improving the preparatory steps for CAR T-cell delivery.

Medulloblastoma (MB) is a common malignant intracranial neoplasms in children. The treatment and prognosis of this tumor depends on histology and molecular subtypes. Survivin, implicated in various malignancies, may hold prognostic significance. We investigated survivin and p53 immunoreactivity in different histological subtypes in 20 MB cases from January 2018 to June 2021. Immunohistochemistry revealed survivin expression in 75% (15/20) of cases, with cytoplasmic (10 cases), nuclear (four cases), or combined expression (one case). p53 nuclear expression was present in 35% (7/20) of cases. Classical variant MB exhibited predominant p53 and cytoplasmic survivin expression. Given the association of survivin and p53 expression with poor prognosis, especially in the prevalent classical variant, targeted therapies may hold promise for MB treatment advancement.

Low-grade gliomas, especially glioneuronal tumors, are a common cause of epilepsy in children. Seizures associated with low-grade pediatric tumors are medically refractory and present a significant burden to patients. Often, morbidity and patients´ quality of life are determined rather by the control of seizures than the oncological process itself and the resolution of epilepsy represents an important part in the treatment of LGGs. The pathogenesis of tumor-related seizures in focal LGG tumors is multifactorial, and mechanisms differ probably among patients and tumor types. Pediatric low-grade tumors associated with epilepsy include a series of neoplasms that have a pure astrocytic or glioneuronal lineage. They are usually benign tumors with a neocortical localization typically in the temporal lobes, but also in other supratentorial locations. Gangliogliomas and dysembryoplastic neuroepithelial tumors (DNET) are the most common entities together with astrocytic gliomas (pilocytic astrocytomas and pleomorphic xanthoastrocytoma) and angiocentric gliomas, and dual pathology is found in up to 40% of glioneuronal tumors. The treatment of low-grade gliomas and associated epilepsy is based mainly on resection and the extent of surgery is the main predictor of postoperative seizure control in patients with a LGG. Long-term epilepsy-associated tumors (LEATs) tend to be well-circumscribed, and therefore, the chances for a complete resection and epilepsy control with a safe approach are very high. New treatments have emerged as alternatives to open microsurgical approaches, including laser thermal ablation or the use of BRAF inhibitors. Future advances in identifying seizure-related biomarkers and molecular tumor pathways will facilitate targeted treatment strategies that will have a deep impact both in oncologic and epilepsy outcomes.

UNASSIGNED: Despite surgical resection, chemoradiation, and targeted therapy, brain tumors remain a leading cause of cancer-related death in children. Immunotherapy has shown some promise and is actively being investigated for treating childhood brain tumors. However, a critical step in advancing immunotherapy for these patients is to uncover targets that can be effectively translated into therapeutic interventions.
UNASSIGNED: In this study, our team performed a transcriptomic analysis across pediatric brain tumor types to identify potential targets for immunotherapy. Additionally, we assessed components that may impact patient response to immunotherapy, including the expression of genes essential for antigen processing and presentation, inhibitory ligands and receptors, interferon signature, and overall predicted T cell infiltration.
UNASSIGNED: We observed distinct expression patterns across tumor types. These included elevated expression of antigen genes and antigen processing machinery in some tumor types while other tumors had elevated inhibitory checkpoint receptors, known to be associated with response to checkpoint inhibitor immunotherapy.
UNASSIGNED: These findings suggest that pediatric brain tumors exhibit distinct potential for specific immunotherapies. We believe our findings can guide investigators in their assessment of appropriate immunotherapy classes and targets in pediatric brain tumors.

UNASSIGNED: Pleomorphic xanthoastrocytoma (PXA) is a rare brain tumor that accounts for <1% of all gliomas. An in-depth understanding of PXA\'s molecular makeup remains a work in progress due to its limited numbers globally. Separately, spontaneous intracranial hemorrhage (pICH) is an uncommon but potentially devastating emergency in young children, often caused by vascular malformations or underlying hematological conditions. We describe an interesting case of a toddler who presented with pICH, later found to have a PXA as the underlying cause of hemorrhage. Further molecular interrogation of the tumor revealed a neurotrophic tyrosine receptor kinase (NTRK) gene fusion and CDKN2A deletion more commonly seen in infantile high-grade gliomas. The unusual clinicopathological features of this case are discussed in corroboration with published literature.
UNASSIGNED: A previously well 2-year-old male presented with acute drowsiness and symptoms of increased intracranial pressure secondary to a large right frontoparietal intracerebral hematoma. He underwent an emergency craniotomy and partial evacuation of the hematoma for lifesaving measures. Follow-up neuroimaging reported a likely right intra-axial tumor with hemorrhagic components. Histology confirmed the tumor to be a PXA (WHO 2). Additional molecular investigations showed it was negative for BRAFV600E mutation but was positive for CDKN2A homozygous deletion and a unique neurotrophic tyrosine receptor kinase (NTRK) gene fusion. The patient subsequently underwent second-stage surgery to proceed with maximal safe resection of the remnant tumor, followed by the commencement of adjuvant chemotherapy.
UNASSIGNED: To date, there are very few pediatric cases of PXA that present with spontaneous pICH and whose tumors have undergone thorough molecular testing. Our patient\'s journey highlights the role of a dedicated multidisciplinary neuro-oncology team to guide optimal treatment.

BACKGROUND: Pediatric low-grade glioma incidence has been rising in the U.S., mirroring the rising rates of pediatric and maternal obesity. Recently, children of obese mothers were demonstrated to develop brain tumors at higher rates. Importantly, obesity in the U.S. is largely driven by diet, given the prevalence of high fat and high sugar (HFHS) food choices. Since high-fat diet exposure can increase embryonic neuroglial progenitor cell (NPC) proliferation, the potential cells of origin for low-grade glioma, we hypothesized that in utero exposure to an obesogenic diet would modify pediatric brain penetrance and latency by affecting the tumor cell of origin.
METHODS: We employed several murine models of the Neurofibromatosis type 1 (NF1) pediatric brain tumor predisposition syndrome, in which optic pathway gliomas (Nf1-OPGs) arise from NPCs in the embryonic third ventricular zone (TVZ). We exposed dams and offspring to an obesogenic HFHS diet or control chow and analysed fetal neurodevelopment at E19.5 and tumor formation at 6w-3mo.
RESULTS: Progeny from HFHS diet-exposed dams demonstrated increased TVZ NPC proliferation and glial differentiation. Dietary switch cohorts confirmed that these effects were dependent upon maternal diet, rather than maternal weight. Obesogenic diet (Ob) similarly accelerated glioma formation in a high-penetrance Nf1-OPG strain and increased glioma penetrance in two low-penetrance Nf1-OPG strains. In contrast, Ob exposure in the postnatal period alone did not recapitulate these effects.
CONCLUSIONS: These findings establish maternal obesogenic diet as a risk factor for murine Nf1-OPG formation, acting in part through in utero effects on the tumor cell of origin.

BACKGROUND: Atypical Teratoid Rhabdoid Tumor (ATRT) is a rare, devastating, and largely incurable pediatric brain tumor. Although recent studies have uncovered three molecular subgroups of ATRTs with distinct disease patterns, and signaling features, the therapeutic profiles of ATRT subgroups remain incompletely elucidated.
METHODS: We examined the effect of 465 kinase inhibitors on a panel of ATRT subgroup-specific cell lines. We then applied multi-omics analyses to investigate the underlying molecular mechanism of kinase inhibitor efficacy in ATRT subgroups.
RESULTS: We observed that ATRT cell lines are broadly sensitive to inhibitors of the PI3K and MAPK signaling pathways, as well as CDKs, AURKA/B kinases, and PLK1. We identified two classes of multi-kinase inhibitors (MKIs) predominantly targeting receptors tyrosine kinase (RTKs) including PDGFR and EGFR/ERBB2 in MYC/TYR ATRT cells. The PDGFRB inhibitor, Dasatinib, synergistically affected MYC/TYR ATRT cell growth when combined with broad-acting PI3K and MAPK pathway inhibitors, including Rapamycin and Trametinib. We observed that MYC/TYR ATRT cells were also distinctly sensitive to various inhibitors of ERBB2 signaling. Transcriptional, H3K27Ac ChIPSeq, ATACSeq, and HiChIP analyses of primary MYC/TYR ATRTs revealed ERBB2 expression which correlated with differential methylation and activation of a distinct enhancer element by DNA looping. Significantly, we show the brain penetrant EGFR/ERBB2 inhibitor, Afatinib, specifically inhibited in vitro and in vivo growth of MYC/TYR ATRT cells.
CONCLUSIONS: Taken together our studies suggest combined treatments with PDGFR and ERBB2-directed TKIs with inhibitors of the PI3K and MAPK pathways as an important new therapeutic strategy for the MYC/TYR subgroup of ATRTs.

OBJECTIVE: To evaluate the amide proton transfer (APT), tumor blood flow (TBF), and apparent diffusion coefficient (ADC) combined diagnostic value for differentiating intracranial malignant tumors (MTs) from benign tumors (BTs) in young patients, as defined by the 2021 World Health Organization classification of central nervous system tumors.
METHODS: Fifteen patients with intracranial MTs and 10 patients with BTs aged 0-30 years underwent MRI with APT, pseudocontinuous arterial spin labeling (pCASL), and diffusion-weighted imaging. All tumors were evaluated through the use of histogram analysis and the Mann-Whitney U test to compare 10 parameters for each sequence between the groups. The diagnostic performance was evaluated using receiver operating characteristic (ROC) curve analysis.
RESULTS: The APT maximum, mean, 10th, 25th, 50th, 75th, and 90th percentiles were significantly higher in MTs than in BTs; the TBF minimum (min) was significantly lower in MTs than in BTs; TBF kurtosis was significantly higher in MTs than in BTs; the ADC min, 10th, and 25th percentiles were significantly lower in MTs than in BTs (all p < 0.05). The APT 50th percentile (0.900), TBF min (0.813), and ADC min (0.900) had the highest area under the curve (AUC) values of the parameters in each sequence. The AUC for the combination of these three parameters was 0.933.
CONCLUSIONS: The combination of APT, TBF, and ADC evaluated through histogram analysis may be useful for differentiating intracranial MTs from BTs in young patients.

OBJECTIVE: Survivors of pediatric brain tumors (SPBT) are at risk for social deficits, fewer friendships, and poor peer relations. SPBT also experience reduced brain connectivity via microstructural disruptions to white matter from neurological insults. Research with other populations implicates white matter connectivity as a key contributor to poor social functioning. This case-controlled diffusion-weighted imaging study evaluated structural connectivity in SPBT and typically developing controls (TDC) and associations between metrics of connectivity and social functioning.
METHODS: Diffusion weighted-imaging results from 19 SPBT and 19 TDC were analyzed using probabilistic white matter tractography. Survivors were at least 5 years post-diagnosis and 2 years off treatment. Graph theory statistics measured group differences across several connectivity metrics, including average strength, global efficiency, assortativity, clustering coefficient, modularity, and betweenness centrality. Analyses also evaluated the effects of neurological risk on connectivity among SPBT. Correlational analyses evaluated associations between connectivity and indices of social behavior.
RESULTS: SPBT demonstrated reduced global connectivity compared to TDC. Several medical factors (e.g., chemotherapy, recurrence, multimodal therapy) were related to decreased connectivity across metrics of integration (e.g., average strength, global efficiency) in SPBT. Connectivity metrics were related to peer relationship quality and social challenges in the SPBT group and to social challenges in the total sample.
CONCLUSIONS: Microstructural white matter connectivity is diminished in SPBT and related to neurological risk and peer relationship quality. Additional neuroimaging research is needed to evaluate associations between brain connectivity metrics and social functioning in SPBT.

UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.