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Recent work has highlighted the tumor microenvironment as a central player in cancer. In particular, interactions between tumor and immune cells may help drive the development of brain tumors such as glioblastoma multiforme (GBM). Despite significant research into the molecular classification of glioblastoma, few studies have characterized in a comprehensive manner the immune infiltrate in situ and within different GBM subtypes. In this study, we use an unbiased, automated immunohistochemistry-based approach to determine the immune phenotype of the four GBM subtypes (classical, mesenchymal, neural and proneural) in a cohort of 98 patients. Tissue Micro Arrays (TMA) were stained for CD20 (B lymphocytes), CD5, CD3, CD4, CD8 (T lymphocytes), CD68 (microglia), and CD163 (bone marrow derived macrophages) antibodies. Using automated image analysis, the percentage of each immune population was calculated with respect to the total tumor cells. Mesenchymal GBMs displayed the highest percentage of microglia, macrophage, and lymphocyte infiltration. CD68 + and CD163 + cells were the most abundant cell populations in all four GBM subtypes, and a higher percentage of CD163 + cells was associated with a worse prognosis. We also compared our results to the relative composition of immune cell type infiltration (using RNA-seq data) across TCGA GBM tumors and validated our results obtained with immunohistochemistry with an external cohort and a different method. The results of this study offer a comprehensive analysis of the distribution and the infiltration of the immune components across the four commonly described GBM subgroups, setting the basis for a more detailed patient classification and new insights that may be used to better apply or design immunotherapies for GBM.

The Immunoglobulin superfamily (IgSF) is a heterogeneous and conserved family of adhesion proteins crucial during the development of the central nervous system including neuronal migration and synaptogenesis. The Immunoglobulin superfamily member 3 (IGSF3) is expressed in the developing brain and has been suggested to play a role during morphological development of the granule cells neurites in the cerebellum. In addition, a role for IGSF3 in supporting glioma progression has been recently demonstrated. Remaining unexplored is the physiological role of IGSF3 in regulating brain development, including neocortical development. We generated an Igsf3 knockout (KO) mouse using a CRISPR/Cas9-based approach and explored the function of Igsf3 in regulating cortical development. We found that Igsf3 largely co-localizes with other IgSF proteins during cortical development and despite its expression being developmentally regulated in neuronal progenitors and in postmitotic neurons, Igsf3 is not essential for brain development, neuronal migration, or neuronal maturation.

Structural variants (SVs) are increasingly recognized as important contributors to oncogenesis through their effects on 3D genome folding. Recent advances in whole-genome sequencing have enabled large-scale profiling of SVs across diverse tumors, yet experimental characterization of their individual impact on genome folding remains infeasible. Here, we leveraged a convolutional neural network, Akita, to predict disruptions in genome folding caused by somatic SVs identified in 61 tumor types from the Children’s Brain Tumor Network dataset. Our analysis reveals significant variability in SV-induced disruptions across tumor types, with the most disruptive SVs coming from lymphomas and sarcomas, metastatic tumors, and germline cell tumors. Dimensionality reduction of disruption scores identified five recurrently disrupted regions enriched for high-impact SVs across multiple tumors. Some of these regions are highly disrupted despite not being highly mutated, and harbor tumor-associated genes and transcriptional regulators. To further interpret the functional relevance of high-scoring SVs, we integrated epigenetic data and developed a modified Activity-by-Contact scoring approach to prioritize SVs with disrupted genome contacts at active enhancers. This method highlighted highly disruptive SVs near key oncogenes, as well as novel candidate loci potentially implicated in tumorigenesis. These findings highlight the utility of machine learning for identifying novel SVs, loci, and genetic mechanisms contributing to pediatric cancers. This framework provides a foundation for future studies linking SV-driven regulatory changes to cancer pathogenesis.

Background The prognosis for metastatic and recurrent tumors of the central nervous system (CNS) remains dismal, and the need for newer therapeutic targets and modalities is critical. The cell surface glycoprotein B7H3 is expressed on a range of solid tumors with a restricted expression on normal tissues. We hypothesized that compartmental radioimmunotherapy (cRIT) with the anti-B7H3 murine monoclonal antibody omburtamab injected intraventricularly could safely target CNS malignancies. Patients and methods We conducted a phase I trial of intraventricular 131 I-omburtamab using a standard 3 + 3 design. Eligibility criteria included adequate cerebrospinal fluid (CSF) flow, no major organ toxicity, and for patients > dose level 6, availability of autologous stem cells. Patients initially received 74 MBq radioiodinated omburtamab to evaluate dosimetry and biodistribution followed by therapeutic 131 I-omburtamab dose-escalated from 370 to 2960 MBq. Patients were monitored clinically and biochemically for toxicity graded using CTCAEv 3.0. Dosimetry was evaluated using serial CSF and blood sampling, and serial PET or gamma-camera scans. Patients could receive a second cycle in the absence of grade 3/4 non-hematologic toxicity or progressive disease. Results Thirty-eight patients received 100 radioiodinated omburtamab injections. Diagnoses included metastatic neuroblastoma ( n  = 16) and other B7H3-expressing solid tumors ( n  = 22). Thirty-five patients received at least 1 cycle of treatment with both dosimetry and therapy doses. Acute toxicities included < grade 4 self-limited headache, vomiting or fever, and biochemical abnormalities. Grade 3/4 thrombocytopenia was the most common hematologic toxicity. Recommended phase 2 dose was 1850 MBq/injection. The median radiation dose to the CSF and blood by sampling was 1.01 and 0.04 mGy/MBq, respectively, showing a consistently high therapeutic advantage for CSF. Major organ exposure was well below maximum tolerated levels. In patients developing antidrug antibodies, blood clearance, and therefore therapeutic index, was significantly increased. In patients receiving cRIT for neuroblastoma, survival was markedly increased (median PFS 7.5 years) compared to historical data. Conclusions cRIT with 131 I-omburtamab is safe, has favorable dosimetry and may have a therapeutic benefit as adjuvant therapy for B7-H3-expressing leptomeningeal metastases. Trial registration : clinicaltrials.gov NCT00089245, August 5, 2004.

Background: Excess flexion or extension during occipitocervical fusion (OCF) can lead to postoperative complications, such as dysphagia, respiratory problems, line of sight issues, and neck pain, but posterior fossa decompression (PFD) and OCF require different positions that require intraoperative manipulation. Objective: The objective of this study was to describe quantitative fluoroscopic morphometrics in Chiari malformation (CM) patients with symptoms of craniocervical instability (CCI) and demonstrate the intraoperative application of these measurements to achieve neutral craniocervical alignment while leveraging a single axis of motion with the Mayfield head clamp locking mechanism. Methods: A retrospective cohort study of patients with CM 1 and 1.5 and features of CCI who underwent PFD and OCF at a single-center institution from March 2015 to October 2020 was performed. Patient demographics, preoperative presentation, radiographic morphometrics, operative details, complications, and clinical outcomes were analyzed. Results: A total of 39 patients met the inclusion criteria, of which 37 patients (94.9%) did not require additional revision surgery after PFD and OCF. In this nonrevision cohort, preoperative to postoperative occipital to C2 angle (O-C2a) (13.5° ± 10.4° vs. 17.5° ± 10.1°, P = 0.047) and narrowest oropharyngeal airway space (nPAS) (10.9 ± 3.4 mm vs. 13.1 ± 4.8 mm, P = 0.007) increased significantly. These measurements were decreased in the two patients who required revision surgery due to postoperative dysphagia (mean difference - 16.6°° in O C2a and 12.8°° in occipital and external acoustic meatus to axis angle). Based on these results, these fluoroscopic morphometrics are intraoperatively assessed, utilizing a locking Mayfield head clamp repositioning maneuver to optimize craniocervical alignment prior to rod placement from the occipital plate to cervical screws. Conclusion: Establishing a preoperative baseline of reliable fluoroscopic morphometrics can guide surgeons intraoperatively in appropriate patient realignment during combined PFD and OCF, and may prevent postoperative complications.

Glioblastomas are among the deadliest human cancers and are highly vascularized. Angiogenesis is dynamic during brain development, almost quiescent in the adult brain but reactivated in vascular-dependent CNS pathologies, including brain tumors. The oncofetal axis describes the reactivation of fetal programs in tumors, but its relevance in endothelial and perivascular cells of the human brain vasculature in glial brain tumors is unexplored. Nucleolin is a regulator of cell proliferation and angiogenesis, but its roles in the brain vasculature remain unknown. Here, we studied the expression of Nucleolin in the neurovascular unit in human fetal brains, adult brains, and human gliomas in vivo as well as its effects on sprouting angiogenesis and endothelial metabolism in vitro. Nucleolin is highly expressed in endothelial and perivascular cells during brain development, downregulated in the adult brain, and upregulated in glioma. Moreover, Nucleolin expression correlated with glioma malignancy in vivo. In culture, siRNA-mediated Nucleolin knockdown reduced human brain endothelial cell (HCMEC) and HUVEC sprouting angiogenesis, proliferation, filopodia extension, and glucose metabolism. Furthermore, inhibition of Nucleolin with the aptamer AS1411 decreased brain endothelial cell proliferation in vitro. Mechanistically, Nucleolin knockdown in HCMECs and HUVECs uncovered regulation of angiogenesis involving VEGFR2 and of endothelial glycolysis. These findings identify Nucleolin as a neurodevelopmental factor reactivated in glioma that promotes sprouting angiogenesis and endothelial metabolism, characterizing Nucleolin as an oncofetal protein. Our findings have potential implications in the therapeutic targeting of glioma.

Alzheimer's disease (AD) is characterized by the age-related deposition of β -amyloid(Aβ) 40/42 peptide aggregates in vulnerable brain regions. Multiple levels of evidence implicate a central role for Aβ in the pathophysiology of AD. Aβ peptides are generated by the regulated cleavage of an ≈ 700-aa Aβ precursor protein (β APP). Full-length β APP can undergo proteolytic cleavage either within the Aβ domain to generate secreted sβ APPα or at the N- and C-terminal domain(s) of Aβ to generate amyloidogenic Aβ peptides. Several epidemiological studies have reported that estrogen replacement therapy protects against the development of AD in postmenopausal women. We previously reported that treating cultured neurons with 17β -estradiol reduced the secretion of Aβ 40/42 peptides, suggesting that estrogen replacement therapy may protect women against the development of AD by regulating β APP metabolism. Increasing evidence indicates that testosterone, especially bioavailable testosterone, decreases with age in older men and in postmenopausal women. We report here that treatment with testosterone increases the secretion of the nonamyloidogenic APP fragment, sβ APPα , and decreases the secretion of Aβ peptides from N2a cells and rat primary cerebrocortical neurons. These results raise the possibility that testosterone supplementation in elderly men may be protective in the treatment of AD.

Context: Multiple angles of the craniocervical junction (CCJ) are associated with pathological conditions and surgical outcomes, including the clivo-axial angle (CXA), clival slope (CS), and sagittal axis (XS). However, there are varying normative ranges reported and a paucity of data analyzing the effects of imaging modality, age, and gender on these angles. Setting and Design: A retrospective review of computed tomographic (CT) and magnetic resonance imaging (MRI) scans in fifty adults without CCJ pathology from 2014 to 2019. Methods: Age, gender, indication, and hours between scans were recorded. Two-blinded observers measured all angles. Analysis between angles from the same patient was performed using the Wilcoxon signed-rank test. Multivariable linear regression was used to test for associations between average angles and age or gender. Results: Average age and time between scans were 41.3 and 14.3 h, respectively, with 94% performed due to trauma. On CT, average CXA, CS, and XS were 162.1°, 118.4°, and 81.3°, respectively. On MRI, they were 159.8°, 117.2°, 85.3°, respectively. There were statistically significant differences between CXA and XS (P < 0.01) based on imaging modality. On CT, there was a significant increase in XS by 1.93°° and decrease in CS by 1.88°° and on MRI, there was a significant increase in CXA by 1.93°° and decrease in CS by 2.75°° corresponding with a 10-year advancement of age. Gender did not have an effect. Conclusion: There are significant differences in angular measurements of the CCJ between CT and MRI from the same patient, as well as changes in normative values based on age.

OBJECTIVES/GOALS: It has been previously shown that pediatric high-grade glioma (pHGG) survival is different between sexes. We set out to find out whether there are sex-specific differences in the genomic landscapes of pHGG that may underlie this sex disparity. METHODS/STUDY POPULATION: We downloaded Illumina 450k DNAm data from ArrayExpress and GeneExpressionOmnibus. The minfi package was used to process raw DNAm data. Sex chromosomes and CpGs that are common SNPs were removed. Surrogate variables (SVs) were estimated via the sva Bioconductor package. Differentially methylated CpGs were identified by fitting a multiple linear regression model for the DNAm level at each CpG, with independent variables being sex (a binary variable) and the estimated SVs. RNAseq data was downloaded from Cavatica, and differential gene expression analysis was carried out via the DESeq2 package. RESULTS/ANTICIPATED RESULTS: In the pediatric glioblastoma (GBM) DNAm data [58 female & 91 male IDH wt samples; ages 0.1–21 yrs;], we found 7,371 differentially methylated cytosines (DMCs) at FDR≤0.05. Of the DMCs, 289 had DNAm differences between male and female samples ≥10%. The majority of probes (68%) were in CpG islands, shelves, or shores. We also found 4 differentially methylated regions (DMRs) between sexes (FWER≤0.1). In the adult GBM DNAm samples [32 F & 32 M IDH wt samples; ages 22–75 yrs], we found only 117 DMCs at FDR≤0.05, and no DMRs. In the RNAseq dataset [68 F & 54 M pHGG samples, ages 0.08–30.6 yrs], we found 383 differentially expressed genes (at FDR≤0.05), and 16 of them (4%) overlapped a DMC. DISCUSSION/SIGNIFICANCE OF IMPACT: Our findings demonstrate that pHGG exhibits sex-specific methylome differences. Interestingly, this difference is greater in the pediatric population as compared to adults. The pHGG transcriptome also differs by sex, which may be related to differential DNAm in a minority of cases.

The excessive generation and accumulation of 40- and 42-aa β -amyloid peptides (Aβ40/Aβ42) in selectively vulnerable brain regions is a major neuropathological feature of Alzheimer's disease. Aβ , derived by proteolytic cleavage from the β -amyloid precursor protein (β APP) is normally secreted. However, recent evidence suggests that significant levels of Aβ also may remain inside cells. Here, we have investigated the subcellular compartments within which distinct amyloid species are generated and the compartments from which they are secreted. Three experimental approaches were used: (i) immunofluorescence performed in intact cortical neurons; (ii) sucrose gradient fractionation performed with mouse neuroblastoma cells stably expressing wild-type β APP695(N2a695); and (iii) cell-free reconstitution of Aβ generation and trafficking from N2a695cells. These studies demonstrate that: (i) Aβ40(Aβ1-40plus Aβx-40, where x is an NH2-terminal truncation) is generated exclusively within the trans-Golgi Network (TGN) and packaged into post-TGN secretory vesicles; (ii) Aβx-42is made and retained within the endoplasmic reticulum in an insoluble state; (iii) Aβ42(Aβ1-42plus Aβx-42) is made in the TGN and packaged into secretory vesicles; and (iv) the amyloid peptides formed in the TGN consist of two pools (a soluble population extractable with detergents and a detergent-insoluble form). The identification of the organelles in which distinct forms of Aβ are generated and from which they are secreted should facilitate the identification of the proteolytic enzymes responsible for their formation.