bluuebunny/crossref_metadata_2025 huggingface dataset:
BIOL-20. BIOPROCESSING OF SURGICAL PEDIATRIC BRAIN TUMOR SPECIMENS FOR GENOME-GUIDED PERSONALIZED DRUG TESTING
Authors: Nasajpour Emon , Tran Caitlynn , Garcia Cesar , Lyle Geoff , Guinle Maria I Barros , Bagley Bryce A , Lancero Hope , Gibson Eden E , Schouten Troy , Mahaney Kelly , Vogel Hannes , Learned Katrina , Vaske Olena , Grant Gerald A , Prolo Laura M , Petritsch Claudia K
Published: June 2023

Novel treatment approaches are urgently needed for pediatric central nervous system (CNS) tumors as they are the leading cause of cancer-related deaths in children. A lack of research materials impedes progress towards developing such therapies. Although various brain cancer biobanks exist, these rarely store viable patient-derived tissue and cells for live cell analyses, including cell fate assays and testing for drug sensitivities. We propose that a biorepository of viable cell dissociates and tissue broadly representing CNS tumor entities overcomes these limitations.

From a combination of molecular diagnoses and histopathologic assessment of over 90 samples collected, we generated a biorepository of roughly 35 distinct entities of pediatric CNS tumors in our Stanford neuro-bioprocessing cohort. Based on the Central Brain Tumor Registry of the United States, the proportion of subtypes represented in our cohort match that of the general population with exceptions. We established a standardized bioprocessing pipeline that can be used as a template for tissue collection and model development in the broader disease context and for multiple downstream applications. Our emphasis on cryostorage of viable cells and tissue facilitates ad hoc live cell analyses. In addition to gaining a better understanding of tumor pathophysiology, we attempted a rapid bed-to-bench-to-bedside approach using comparative RNA expression profile analyses in an individual patient’s pilocytic astrocytoma. Novel drug targets were identified by analyzing RNA transcripts that are highly expressed (outliers) compared with a compendium of 12,747 brain and non-brain tumor samples. We validated outliers as drug targets using acute cell isolates, and identified a novel target in recurrent low-grade glioma. These studies illustrate that biobanking of viable patient-derived material enables developing personalized therapies with the goal of improving patient outcomes. As demonstrated here, patient-derived acute cell isolates facilitate identifying drug vulnerability, creating an opportunity for more personalized treatment of patients with CNS tumors.
DIPG-01. LIN28B EXPRESSION IN DIFFUSE MIDLINE GLIOMA
Authors: Knowles Truman , An Shejuan , Huang Tina , Qi Jin , Saratsis Amanda
Published: June 2023

INTRODUCTION

Diffuse midline glioma (DMG) is the most lethal of all childhood cancers. LIN28B is an RNA binding protein expressed in a variety of cancers, which suppresses the let-7 family of microRNAs, which in turn suppresses a plethora of oncogenes. However, the role of LIN28B in DMG has not yet been explored. Therefore, we sought to determine the pattern of expression and potential function of LIN28B in DMG using rare DMG cell lines.

METHODS

All studies were performed on DMG cell lines (n=6) and controls (normal human astrocytes n=1, human neural stem cells n=1). RNA-Seq was performed using single-read sequencing at 50bp on the Illumina NextSeq 500 Sequencing System. Cells were then treated with DMSO or the LIN28B inhibitor, LI71, and submitted to functional in vitro studies, as well as RNA-Seq and Western Blot analysis. Functional pathways analysis was subsequently performed on resulting gene expression values with Ingenuity Pathways Analysis (Qiagen).

RESULTS

Differential LIN28B expression was demonstrated across cell lines, with DMG cells expressing greater LIN28B gene and protein levels compared to controls. LIN28B inhibition with small molecule inhibitor LI71 resulted in decreased cell proliferation and migration, in vitro. A total of 946 differentially expressed genes were identified between treatment groups (FC>2 or <-2, p<0.05). Functional pathways analysis of differentially expressed genes implicated Cancer and Neurological Disease as the top disease processes, with Cellular Movement as the top molecular process in cell lines expressing LIN28B. Further, inactivation of Myc signaling was implicated in cells treated with LI71.

CONCLUSIONS

Our data demonstrates increased LIN28B expression in DMG cell lines compared with controls, with oncogenic effects on cell proliferation and migration. Further investigation of the LIN28B-let7 signaling axis is warranted in order to further explore LIN28B as a novel therapeutic target in DMG.
DIPG-08. THE LANDSCAPE OF TUMOR CELL STATES AND SPATIAL ORGANIZATION IN H3-K27M MUTANT DIFFUSE MIDLINE GLIOMA ACROSS AGE AND LOCATION
Authors: Liu Ilon , Jiang Li , Samuelsson Erik , Salas Sergio Marco , Beck Alexander , Hack Olivia , Jeong Daeun , Shaw McKenzie , Englinger Bernhard , LaBelle Jenna , Mire Hafsa , Madlener Sibylle , Mayr Lisa , Quezada Michael , Trissal Maria , Panditharatna Eshini , Ernst Kati , Vogelzang Jayne , Gatesman Taylor , Halbert Matthew , Palova Hana , Vesela Petra , Sterba Jaroslav , Slaby Ondrej , Geyeregger Rene , Diaz Aaron , Findlay Izac , Dun Matt , Resnick Adam , Suva Mario , Jones David , Agnihotri Sameer , Ostlin Jessica , Koschmann Carl , Haberler Christine , Czech Thomas , Slavc Irene , Cotter Jennifer , Ligon Keith , Alexandrescu Sanda , Yung W K Alfred , Arrillaga-Romany Isabel , Gojo Johannes , Monje Michelle , Nilsson Mats , Filbin Mariella
Published: June 2023

Histone 3 lysine27-to-methionine mutations (H3-K27M) frequently occur in childhood diffuse midline gliomas (DMGs) of the pons, thalamus, and spinal cord, presumed to be driven by the specific spatiotemporal context of these midline locations during postnatal development. While most common in the pons and at mid-childhood ages, the same oncohistone mutation is recurrently detected in adult DMGs and throughout different midline regions. The potential heterogeneity of tumors at different ages and in different anatomical locations of the midline are vastly understudied. Through dissecting the transcriptomic, epigenomic and spatial architectures of a comprehensive cohort of patient H3-K27M DMGs - spanning the age range from 2-68 years and locations from spinal cord to thalamus - at single cell resolution, we delineate how age- and location-dependent contexts shape glioma cell-intrinsic and -extrinsic features in light of the shared driver mutation. We identify that oligodendrocyte precursor (OPC)-like cells constitute the stem-like compartment in H3-K27M DMGs across all clinico-anatomical groups, however, depending on location, display varying levels of maturity resembling less differentiated pre-OPCs or more mature OPCs further differentiated along the oligodendroglial lineage. We further demonstrate increased mesenchymal cell states in adult tumors, which we link to age-related differences in glioma-associated immune cell compartments, in particular an increase of macrophages in adult compared to pediatric tumors. Furthermore, we resolve the spatial organization of H3-K27M DMG cell types and states in intact patient tissues, identifying a local niche of the oligodendroglial lineage. Our study provides a powerful resource for rational modeling and therapeutic frameworks taking into account determinants of age and location in this lethal glioma group.
DIPG-24. BRD4 INHIBITION AS A RADIOSENSITIZER THROUGH BLOCKING DNA REPAIR FOR THE TREATMENT OF DIFFUSE MIDLINE GLIOMA
Authors: Watanabe Jun , Clutter Matt , Hoeman Christine , Amreena Sui , Abe Koki , Ishi Yukitomo , Uchida Eita , Becker Oren , Hashizume Rintaro
Published: June 2023

Diffuse intrinsic pontine glioma (DIPG) is one of the devastating childhood cancers. Radiation therapy (RT) remains the only effective treatment yet provides a 5-year survival rate of only 1%. Several clinical trials have attempted to enhance RT efficacy by combining it with radiosensitizing agents, though none have been successful in doing so. Given this, there is a critical need to identify effective therapeutics to enhance the RT anti-tumor activity in DIPG. Here, we identified BRD4 inhibitors as candidate radiosensitizers from a high throughput drug screening. DIPG cells show increased H3 K27 acetylation (H3K27ac) levels, which bind to BET bromodomain protein 4 (BRD4) and are strongly associated with active transcription. We tested two BRD4 inhibitors (BRD4i: AZD5153 and JQ-1) as well as genetic BRD4 depletion, and observed enhancement of radiation-induced DNA damage, confirming BRD4i as potential radiosensitizers in the treatment of DIPG. We evaluated the effects of BRD4i on gene expression using RNAseq, and observed significant inhibition of DNA-repair proteins such as BRCA1 and RAD51. CUT-RUN qPCR showed decreased H3K27ac at BRCA1 and RAD51 promoters. Combination of BRD4i and RT inhibited cell viability significantly using clonogenic survival assay, apoptosis assay, and also showed cell cycle arrest. Radiation-induced DNA double-strand break (DSB) repair was prolonged with BRD4i with high levels of gH2AX and 53BP1 likely due to inhibition of DNA-repair. In vivo studies revealed increased survival of animals treated with combination therapy of RT and BRD4i in comparison to either monotherapy. Together, these results highlight BRD4i as a potential radiosensitizer and provide a rationale for developing combination therapy with radiation in the treatment of DIPG.
DIPG-25. MAPPING TUMOR MICROENVIRONMENT USING EMERGING TECHNOLOGIES FOR PEDIATRIC BRAIN CANCERS
Authors: Laternser Sandra , Petrovic Antonela , Eze Augustine , Dettwiler Susanne , Prutek Fabiola , Contini Paola Agata , Morinigo Denise , Bornhorst Miriam , Rushing Elisabeth Jane , McDonough Elizabeth , Surrette Christine , Ginty Fiona , Minnis Hanna , Gartrell Robyn , Müller Sabine , Packer Roger , Nazarian Javad
Published: June 2023

Diffuse midline glioma (DMG) is a fatal childhood brain cancer with a survival rate of less than one year from diagnosis. Pharmacological approaches as well as immunotherapy have failed to make a clinical impact. Incomplete understanding of the tumor microenvironment (TME) and tumor associated antigens have contributed to the observed poor prognosis. We hypothesize that characterization of the DMG TME will identify biology-informed targeted treatments. We thus obtained postmortem specimens from 70 patients diagnosed with brain cancers, including 50 DMGs, 20 other types and 10 non-CNS-cancer patients. Up to four anatomical brain locations were selected including the primary tumor, metastatic and adjacent healthy sites. Formalin-fixed-paraffin-embedded (FFPE) specimens were processed for constructing a tissue microarray (TMA). The TMA was stained for a number of markers (H&E, H3K27M, H3K27me3, KI67) and was scored by a neuropathologist. We then used a multiplexed immunofluorescence (MxIF) technology, Cell DIVE™, to iteratively probe 33 biomarkers on a single tissue slide, focusing on immune cell type profiling and activation, and histone mutation status. Analysis of biomarker density and spatial relationships are underway. The main expressed immune markers across all patients were CD163, CD68, and CD8. CD8, a cytotoxic T-cell biomarker, was highly detected in pons, cerebellum, thalamus and the frontal lobe (tumor and healthy) of DMG patients and varied according to clinical intervention, including ONC201 treatment. Furthermore, in comparison to other tumors, DMGs exhibited a higher expression of CD3, T- cell marker, and CD4, a T-helper cell biomarker. Analysis of Iba1, a microglial marker, confirmed a higher difference in microglial activation in primary tumor compared to metastatic and adjacent healthy tissue. In contrast, CD68 was significantly increased in metastatic sites. Analysis is still ongoing. We report establishment of the most comprehensive TMA for pediatric brain tumors, which provides insights into TME comparing critically important clinical variables.
DIPG-52. EXTRAPONTINE PROGRESSION WITH STABLE PONTINE DISEASE POST-RADIOTHERAPY IN DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) ON A CONNECT TRIAL: IMPLICATIONS FOR RADIATION TREATMENT VOLUMES
Authors: Lazow Margot , Palmer Joshua , Stanek Joseph , Baxter Patricia , Thomas Diana , Salloum Ralph , de Blank Peter , Mikael Leonie , Kumar Shiva Senthil , Leonard Jeffrey , Mardis Elaine , Drissi Rachid , Fouladi Maryam , Rodriguez Diana
Published: June 2023

Radiotherapy remains the only treatment modality proven to prolong survival and alleviate symptom burden for patients with DIPG. Upfront focal photon radiation to 54 Gy with 1-1.5cm margins is standard. Subventricular, leptomeningeal, and/or spinal dissemination is observed in up to 50% of cases at autopsy, with activation of metastasis-related pathways (eg, epithelial-mesenchymal transition) in a subset of tumors. Given potential for metastatic spread and critical implications for radiation field volumes, we analyzed the frequency of extrapontine disease progression with pontine tumor stability within a prospective cohort with standardized longitudinal imaging. In a CONNECT consortium-sponsored phase Ib trial with a radiosensitizing agent administered concurrently with focal radiation and adjuvantly, among 10 patients with DIPG who experienced progressive disease with available serial imaging, four (40%) had evidence of disease extension and/or non-contiguous dissemination with stable pontine tumor size at a median of 9 months (range: 5-15 months) from diagnosis. Specifically, extrapontine progression was observed in the spinal cord (n=3), cerebellum (n=2), basal ganglia (n=1), temporal lobe (n=1) and/or leptomeninges and ependymal surfaces (n=1), without radiographic pontine disease progression. Five of the remaining six patients (who came off treatment due to pontine progression) subsequently had radiographic evidence of extrapontine extension (cerebellar, spinal, thalamic, supratentorial). Comprehensive molecular profiling is underway on biopsy or autopsy specimens for three patients, focusing on metastasis pathways. In summary, within a small prospective cohort with longitudinal imaging, we observed extrapontine progression despite local pontine disease control following focal radiotherapy in 40% of patients with DIPG at a median of 9 months after diagnosis. Further research will be essential to identify biological predictors of dissemination and corroborate findings in a larger cohort, but results suggest at least a subset of patients with DIPG may benefit from larger radiation volume expansion into contiguous structures and white matter tracts.
EPEN-05. TRANSCRIPTIONAL FACTOR-DIRECTED EPIGENETIC REGULATION IN CANCER NEUROSCIENCE OF ZFTA-RELA EPENDYMOMA
Authors: Chen Hsiao-Chi , McDonald Malcolm , Varadharajan Srinidhi , Sardar Debosmita , Mack Stephen C , Deneen Benjamin
Published: June 2023

ZFTA-RELA (ZRFUS) ependymoma is a pediatric brain tumor with dismal 10-year progression free survival rates. Prior studies revealed low somatic mutation load in pediatric ependymoma compared to adult glioma, suggesting that developmental mechanisms play a key role during disease development. Using human tissues and mouse tumors generated from our autochthonous in utero electroporation (IUE) model, we identified a cohort of developmental transcription factors (TFs) linked to ZRFUS by investigating histone serotonylation changes with other epigenetic datasets. We further performed in vivo barcode-screening and identified the ETS variant transcription factor 5 (ETV5) is sufficient and required for disease progression. To further reveal the disease mechanism driven by ETV5, we applied transcriptomic, epigenetic, and proteomic analyses, and found ETV5 suppresses neuropeptide Y (NPY) expression in mouse ZRFUS tumors by binding with epigenetic modulator, chromobox 3 (CBX3). We further found NPY overexpression delays tumor progression and suppressed tumor-driven neuronal activity in mice. This study suggests developmental TF can shape tumor-promoting microenvironment during disease progression through epigenetic regulation.
EPEN-13. PATTERN OF RELAPSE IN POSTERIOR FOSSA EPENDYMOMA GROUP A
Authors: Bajin Inci Yaman , Zapotocky Michal , Bartels Ute , Tabori Uri , Huang Annie , Tsang Derek , Laperierre Normand , Dirks Peter , Laughlin Suzanne , Bouffet Eric , Ramaswamy Vijay
Published: June 2023

PURPOSE/OBJECTIVE

Posterior fossa ependymoma group A (PF-EPN-A) is a poor prognostic ependymoma subgroup, predominantly seen in infants and young children. Among all ependymomas, most of the recurrences are seen in patients with the PF-EPN-A subgroup. Despite advances in neurosurgery and postoperative adjuvant therapy, management of these tumors remains challenging, and recurrence occurs in over 50% of cases. In this study, we aimed to evaluate the pattern of relapse in PF-EPN-A.

MATERIALS AND METHODS

The clinical features, treatment strategies and relapse patterns of patients diagnosed and treated with PF-EPN-A at the Hospital for Sick Children, Toronto have been evaluated retrospectively.

RESULTS

Seventy-five patients were diagnosed with PF-EPN-A at the Hospital for Sick Children, Toronto from 01 June 1996 to 01 June 2022. All patients were treated with surgery +/- focal radiation +/- chemotherapy after diagnosis. Out of 75 patients, 38 (50.6%) had recurrent disease. Twenty-three patients (60.5%) had a local relapse while 15 patients (40.5) experienced a distant relapse. Out of 23 patients who had a local relapse, only 8 patients (34%) developed a local relapse after a gross total resection (GTR). Other 15 patients developed a local relapse after either subtotal (STR) or near total resection (nTR). Out of 15 patients who experienced a distant relapse, 10 patients (66%) developed a distant relapse after GTR. Time to relapse was mean 32 months (11-132 months) after GTR and 24 months (1-98 months) after STR /nTR.

CONCLUSIONS

The modern management approach (more aggressive resection and early conformal radiotherapy) changed the relapse pattern and the classical dogma that “nearly all relapses in ependymoma are local’’ in modern series. This study supported the importance of GTR in the management of PF-EPN-A and also demonstrated that PF-EPN-A tends to recur more on distant sites rather than locally when the initial surgical intervention is a GTR.
METB-03. THE DISTINCT GENOMICS OF HIGH-GRADE INFANT GLIOMAS AMONG PEDIATRIC GLIOMAS HARBORING RECEPTOR TYROSINE KINASE FUSIONS
Authors: de Biagi-Junior Carlos A O , Eder Sebastian K , LaBelle Jenna , Filbin Mariella G
Published: June 2023

Gliomas account for about 60% of pediatric brain tumors, about half of which are classified as high-grade. Recent studies have shown that infants with high-grade gliomas (HGG) harboring receptor tyrosine kinase (RTK) fusions have a more favorable prognosis than pediatric HGG patients. The molecular mechanisms for this favorable, but from the histology divergent clinical outcome, are yet unknown. We analyzed n=18 brain tumor samples (n=7 infant, n=11 pediatric) of patients with low-grade glioma (LGGs) or HGG presenting with RTK fusions using single-cell RNA sequencing. Considering the different fusion genes (ALK n=6, NTRK n=8, and MET n=4), age groups, and glioma grades, we evaluated the metaprograms of tumor cells and their proportions using non-negative matrix factorization. Preliminary results show similar metaprograms comparing infant and pediatric LGG. Cells detected as MES-like did not occur in LGG but were found in several HGG samples. However, in HGG, the MES-like cells show different expression patterns for infant and pediatric samples. In addition, compared with pediatric HGG, infant HGG has a higher proportion of programs that are predominantly present in LGG, such as AC-like and NPC-like. These initial findings serve as a starting point for cell type-specific transcription factor regulatory networks and are an important step toward understanding the underlying biology of this distinct pediatric brain tumor entity.
RARE-04. DEVELOPMENT OF A PLATFORM TO PROFILE MICRORNA IN CEREBROSPINAL FLUID FROM PATIENTS WITH INTRACRANIAL GERM CELL TUMORS
Authors: Gell Joanna , Therieault Jody , Ng Patrick , Lau Ching
Published: June 2023

BACKGROUND

Current diagnosis of intracranial germ cell tumors (iGCTs) utilizes alpha fetoprotein (AFP) and chorionic gonadotropin (β-HCG) as biomarkers in the blood or cerebrospinal fluid (CSF). Two thirds of iGCTs are marker negative and AFP/HCG are not specific to iGCTs. Thus, there is a need to develop other biomarkers to improve the diagnosis of iGCT. Two microRNA (miRNA) clusters, miR-371-373 and miR-302/367 have previously been identified as biomarkers for extracranial GCTs and preliminary data from our lab and others confirmed their potential utility in iGCTs. However, the optimal way to profile miRNAs in CSF is not fully established. Two commonly used methods are real-time polymerase chain reaction (qPCR) or droplet digital polymerase chain reaction (ddPCR).

METHODS

CSF samples were obtained from biorepositories at Connecticut Children’s and through the Children’s Brain Tumor Network. Cell culture media from an embryonal carcinoma cell line was used as positive control. MicroRNA was extracted from 50uL of CSF, reverse transcribed into cDNA and pre-amplified. Samples were then diluted 1:5 for qPCR and 1:10 or 1:50 for ddPCR.

RESULTS

We profiled the CSF from 6 germinomas and 4 non-germinomatous, including two mature teratomas. Additionally, we profiled CSF from two non-iGCT brain tumors and two leukemia patients, as non-iGCT controls. For qPCR, raw CT values were evaluated with CT <35 considered detectable. Both platforms, qPCR and ddPCR were able to detect miR-371-373 and miR-302/367 in malignant iGCTs, including marker negative germinomas. miR-302/367 cluster showed varying expression in non-iGCT samples.

CONCLUSIONS

We were able to use low input (50uL) of CSF to identify markers of malignant iGCT, the microRNA clusters miR-371-373 and miR-302/367. Both platforms were able to identify the microRNAs. More samples will need to be profiled to evaluate which platform ddPCR or qPCR is best to move forward towards a clinically validated platform.
OUTC-08. IMPLEMENTING SOCIAL DETERMINANT OF HEALTH DATA COLLECTION FOR PEDIATRIC, ADOLESCENT AND YOUNG ADULT NEURO-ONCOLOGY PATIENTS IN THE CLINICAL TRIAL CONSORTIA SETTING: PROGRESS AND CHALLENGES
Authors: Puthenpura Vidya , Bona Kira , Cooney Tab , Coven Scott , Davidson Tom , Fouladi Maryam , Kline Cassie , Leary Sarah , Mueller Sabine , Green Adam , Marks Asher
Published: June 2023

Adverse social determinants of health (SDoH) are associated with decreased survival and increased long-term complications amongst pediatric and adolescent and young adult (AYA) neuro-oncology patients. These outcome disparities are in part due to inferior access to clinical trials and comprehensive, multidisciplinary care. SDoH including food insecurity, housing instability, inability to pay for utilities, and personal safety issues have been shown to negatively impact therapy adherence and exacerbate survival disparities. As such, efforts to improve health equity must include evaluating and addressing SDoH at diagnosis. Existing literature has been limited by a reliance on proxy measures of adverse SDOH such as neighborhood or census area-level socioeconomic status data, which poorly reflect individual circumstances. There is a complex interplay between neighborhood and personal socioeconomic status that has not been extensively studied and is poorly understood. In addition, socioeconomic status is only one component of SDoH. No validated tool currently exists to assess SDoH amongst pediatric and AYA cancer patients. It is imperative that multicenter clinical trials collect SDoH data to inform both trial design and interpretation of results. These data will be important to understanding and identifying patterns in tumor and toxicity incidence, as well as understanding the burden on families in adhering to trial therapies. Based on our previous work, we will present the importance of incorporating SDoH data collection within the clinical trial consortia setting. In addition, we will review the development of a comprehensive SDoH questionnaire and discuss facilitators and barriers to the incorporation of SDoH questionnaires into multicenter clinical trials and offer solutions based on our experience.
HGG-01. INFANT-TYPE HEMISPHERIC GLIOMA: NEW MOLECULAR ALTERATIONS AND PRECISION-MEDICINE TREATMENT
Authors: Hu Wanming , Yuan Li , Zeng Jing
Published: June 2023

Introduction

Infant-type hemispheric glioma, harboring alterations in the receptor tyrosine kinases ALK, ROS1, NTRK and MET, is a new subtype of Pediatric-type diffuse high-grade gliomas in the 2021 WHO classification of CNS tumors. It has important clinical therapeutic value with specialized therapeutic drugs. Here, we presented 3 cases of infant-type hemispheric glioma. Patient1 with EML4-ALK fusion which often appeared in lung cancer, the other 2 patients have new molecular alterations which has not been reported before (Patient2 has both NTRK1-TP53/TP53-NTRK1 fusions and p53 protein showed characteristic cytoplasm positive; Patient3 presented a brand-new ALK-QKI fusion combined with ALK mutation and focal SMARCB1 deletion. All these 3 cases received corresponding targeted therapy and have a good recovery and normal neurologic function till now.

Methods

Immunohistochemistry. Fluorescent in situ hybridization. Whole-transcriptome sequencing.

Results

Case 1: 8 months, male, right semiovale center occupation.

HISTOPATHOLOGY

High-grade neuroepithelial neoplasm. IHC: GFAP (only few cells+), Olig2(-), S100(+), CD56(+), Syn(-), NSE(focal+), NeuN(-), CD34(-), INI-1(+), Ki67(10%+). Characteristic Molecular Information: EML4-ALK fusion. Follow-up: 15 months, alive. Case 2: 3 years, female, insular lobe occupation.

HISTOPATHOLOGY

Gliosarcoma. IHC: GFAP(partly+), Olig2(partly+), Vimentin(+), P53(cytoplasm+), pan-TRK(+), Ki67(25%+). Reticular fiber staining showed biphasic tissue pattern with reticulin-rich sarcomatous and reticulin-free gliomatous elements. Characteristic Molecular Information: NTRK1-TP53 and TP53-NRTK1 fusion. Follow-up: 27 months, alive. Case 3: 3 years, male, left parietal occipital lobe occupation.

HISTOPATHOLOGY

GBM and AT/RT. IHC: GFAP(partly+), Olig2(partly+), INI-1(partly-), BRG1(+), SYN(-), CD34(-), BRAF(-), S100(-), CK(-), H3K27M(-), IDH1(-), P53(40%+), ATRX(+), pan-TRK(-), ALK(+), Ki67(30%+). Characteristic Molecular Information: ALK mutation, ALK-QKI fusion, RAD51C mutation and focal SMARCB1(INI-1) deletion. Follow-up: 14 months, alive. Conclusion: Infant-type hemispheric glioma is a special kind of glioma, which is particularly suitable for precision-medicine treatment approaches. Their overall survival is good compared with other three pHGG subtype.
HGG-08. COMBINATORIAL ACTIVITY OF CDK4/6-INHIBITORS AND TRAMETINIB IN PEDIATRIC HIGH-GRADE GLIOMA WITH BRAFV600E MUTATION AND HOMOZYGOUS CDKN2A DELETION
Authors: Mayr Lisa , Mager Leah , Gabler Lisa , Madlener Sibylle , Berger Julia , Kirchhofer Dominik , Lämmerer Anna , Bruckner Katharina , Lötsch Daniela , Berger Walter , Gojo Johannes
Published: June 2023

BRAFV600E mutations occur in 10-15% and homozygous CDKN2A deletions in approximately 40% of adolescent and pediatric high-grad glioma (HGG) and is associated with significantly decreased overall survival. Moreover, a subset of pHGG is defined by combined BRAFV600E mutation and concomitant CDKN2A homozygous deletion. Standard therapy has remained unchanged and consists of maximal safe resection and focal radiotherapy still resulting in poor overall survival. Therefore, new therapeutic approaches to increase patient survival are of utmost importance. Targeted therapy of BRAFV600E mutant HGG has emerged as effective treatment but remains challenging due to therapy resistance. CDK4/6 inhibitors represent a promising target in CDKN2A altered HGG. However, palbociclib demonstrated only low efficacy in HGG patients. Therefore, combination approaches may be more effective, and we investigated CDK4/6-inhibitors alone and in combination with trametinib in HGG cell models with BRAFV600E mutation and homozygous CDKN2A deletion.

Efficacy of CDK4/6-inhibitor monotherapy and combinatorial approach with trametinib was assessed by short- and long-term viability assays in four patient-derived HGG cell models with homozygous CDKN2A deletion and BRAFV600E mutation. Furthermore, effects of CDK4/6 inhibitor therapy alone or in combination with trametinib on downstream signaling pathways (MAPK, PI3K) were analyzed with Western blots.

Abemaciclib showed the highest activity in all cell models with IC50-values ranging from 0,5 – 2µM. Combined treatment approaches with trametinib showed synergistic effects across all cell models. Long-term viability assays revealed distinct sensitivity in the nanomolar range for CDK4/6-inhibitors and combined treatment with trametinib in all cell models. Exposure to abemaciclib and trametinib significantly decreased pRB, pERK, pS6, and pAKT protein expression levels when compared to monotherapy alone.

Summarizing, combined treatment with CDK4/6-inhibitors and trametinib showed promising therapeutic effects on HGG models with homozygous CDKN2A deletion and BRAFV600E mutation. Currently, effects on cell cycle distribution and downstream molecular mechanisms are investigated to identify potential predictive biomarkers.
HGG-17. SINGLE-CELL AND SPATIAL ANALYSES DECIPHER THE UNIQUE INVASIVE GROWTH PATTERN OF GLIOMATOSIS CEREBRI
Authors: Crampsie Shauna , Liu Ilon , Petrilli Lucia Lisa , Mackay Alan , Spinozzi Giulio , Grabovska Yura , Hack Olivia A , Kessler Ketty , Temelso Sara , Carvalho Diana Martins , Burford Anna , Sejdiu Drenusha , Molinari Valeria , Shaw McKenzie L , Mire Hafsa M , Pericoli Giulia , Cerimele Alfredo , Barry Clémentine , Cocito Carolina , Debily Marie-Anne , Kergrohen Thomas , Ajlil Yassine , Beccaria Kévin , Varlet Pascale , Tauziède-Espariat Arnault , Mastronuzzi Angela , Carai Andrea , Giovannoni Isabella , Rossi Sabrina , Miele Evelina , Grill Jacques , Jacques Thomas S , Greenfield Jeffery P , Castel David , Vinci Maria , Filbin Mariella G , Jones Chris
Published: June 2023

Gliomatosis cerebri (GC) is a rare, lethal glioma that is radiologically diagnosed and characterised by its diffuse infiltration throughout the cerebral lobes of the brain. It is no longer recognised as a separate entity by the WHO classification, but its growth pattern and invasive phenotype differ from other types of glioma. In order to understand what underlies this unique presentation, we sought to unravel these complex tumours in children through single cell and spatial approaches. 26 paediatric cases (1.3-19 years, median=11.3) were collected for DNA methylation, whole exome sequencing and single cell RNA-sequencing. Of these, 16 cases were used for Imaging Mass Cytometry (IMC). 5 patient-derived cell lines were used to model migration and invasion in vitro, whereby we have also been able to explore gene expression differences associated with the invading cells by RNAseq. We analyzed the transcriptional landscape of all 26 GC patients at single-cell resolution, and identified shared tumor cellular programs of glial and neuronal lineage. We observed notable heterogeneity of neuronal lineage tumor cells that were composed of different states, such as metabolic active and synaptogenic states. We described tumor cell states associated with higher invasivity, as corroborated from integrated analysis with our in vitro invasion-associated gene expression profiles. Next, we stratified all tumors by their DNA methylation profiles, and defined 16 archetypal GC cases, for which we show specific transcriptional features. Furthermore, we analyzed multiple regions of interest within FFPE patient tissue sections by IMC and demonstrate spatial patterns in the interaction between the tumour and normal brain tissue interface, and inter-regional heterogeneity for stem, invasive and proliferative markers. Together, we dissect the characteristic invasive phenotype of GC at multiple molecular, spatial and functional layers, thereby providing the basis for modelling and therapeutic avenues much needed for this lethal disease.
HGG-18. SINGLE CELL RNA-SEQ ANALYSIS OF PEDIATRIC HIGH-GRADE GLIOMA PATIENT SAMPLES IDENTIFIES COMMON GLIAL AND IMMUNE CELL TYPES THAT HELP TO EXPLAIN HETEROGENEITY AND TUMORIGENESIS
Authors: DeSisto John , Donson Andrew , Griesinger Andrea , Fu Rui , Levy Jean Mulcahy , Foreman Nicholas , Vibhakar Rajeev , Green Adam
Published: June 2023

BACKGROUND

Pediatric high-grade gliomas (PHGG) are aggressive, undifferentiated central nervous system (CNS) tumors. PHGG comprises subtypes that differ phenotypically, histologically and by cellular composition. Despite intensive research, overall survival rates have remained for decades around 2% for diffuse midline glioma (DMG) and 20% for non-DMG PHGG. We hypothesized that PHGG’s heterogeneity, a key difficulty in devising therapies, results from functional and lineage differences in tumor cell types.

METHODS

Using 19 PHGG samples from our institution’s pediatric brain tumor bank, we constructed a single-cell RNA-Seq (scRNA-Seq) dataset that included tumor and immune cells, generated predicted cell types by mapping the scRNA-Seq data onto known cell types, and performed differential gene expression and geneset enrichment analyses. We acquired bulk RNA-Seq and DNA methylation data to characterize overall tumor properties.

RESULTS

Samples consisted of several principal tumor cell types, including cells with astrocyte characteristics, oligodendrocyte progenitor cell (OPC) characteristics, and cells that expressed OPC markers but had slight enrichment in mesenchymal and inflammatory gene expression (OPC-like/Mes cells). We also found a potential stemlike population that expressed neural stem cell markers. The predicted astrocytes, OPCs and OPC-like/Mes cells differed in their gene expression profiles, pathway enrichment, and tendency to proliferate as assessed from expression of cell cycle genes. A combined microglia/macrophage population, strongly enriched in mesenchymal and inflammatory gene expression, may serve to induce inflammatory gene expression in tumor cells. Key conclusions from the scRNA-Seq analysis were validated using multi-channel immunofluorescence staining.

CONCLUSIONS

PHGG comprises a small stemlike population and varying proportions of proliferating glial lineage tumor cells. CNS-resident microglia/macrophages may trigger mesenchymal gene expression in PHGG. We will use our results to investigate the roles of PHGG’s diverse tumor cell populations in resistance to radiotherapy and to develop treatments to target individual cell types comprising PHGG.
HGG-28. INTERIM ANALYSIS OF THE GCC1949 PHASE 2 TRIAL USING INDOXIMOD-BASED CHEMO-IMMUNOTHERAPY FOR PATIENTS WITH PEDIATRIC BRAIN TUMORS
Authors: Johnson Theodore S , Pacholczyk Rafal , Aguilera Dolly , Berrong Zuzana , Castellino Robert C , Chi Susan , Creager Julianne , Eaton Bree R , Fangusaro Jason R , Huang Chenbin , Hummel Trent , Ingerski Lisa , Kennedy Eugene P , Ring Eric , Sadek Ramses F , Satpathy Sarthak , Schniederjan Matthew , Thomas Beena E , Yeo Kee Kiat , Bhasin Manoj , MacDonald Tobey J , Munn David H
Published: June 2023

Brain tumors are the leading cause of cancer-related death in children. We report interim results of an ongoing multi-institutional phase 2 trial (NCT04049669) of the IDO pathway-inhibitor indoximod utilized in a chemo-immunotherapy regimen for patients 3-21 years of age with either recurrent malignant brain cancer or newly-diagnosed diffuse intrinsic pontine glioma (DIPG). Patients are treated with oral indoximod (38.4 mg/kg/day, divided BID) plus oral temozolomide (200 mg/m2/day for 5 days) in 28-day cycles. Patients for whom radiation therapy is planned as standard-of-care receive indoximod during the up-front radiation regimen. During treatment, palliative radiation, surgery, or dexamethasone are allowed as needed for clinical management. To date, 53 patients have been treated. Estimated median follow-up time was 23 months (range 0.2-35.5 months), and indoximod-based therapy was well tolerated. Estimated median overall survival (OS) was 15 months for the entire mixed population (n= 53, 95%-C.I. 11.5-23.2 months); 23.8 months for recurrent ependymoma (n=26); 13.5 months for recurrent medulloblastoma (n=12); 5.8 months for recurrent glioblastoma/diffuse midline glioma (GBM/DMG, n=9, excludes DIPG); and 9.6 months for newly-diagnosed DIPG (n=6). For the 39 response-evaluable patients to date (those with measurable disease and at least one follow-up MRI on-therapy), objective response in any single lesion by pediatric RANO criteria occurred in 20/39 patients (9/17 ependymoma, 9/11 medulloblastoma, 0/6 GBM/DMG, 2/5 DIPG). Lesion response was associated with significantly better overall survival (estimated median OS 23.2 months, n=20) compared to patients without lesion response (median OS 6.6 months, n=19) (p=0.0005). Single-cell RNA sequencing of serial peripheral blood samples allowed identification of expanded T cell clones, which was correlated with survival. Updated results from the first 53 patients will be presented. These data are replicating results of the preceding phase 1 study (NCT02502708) and provide preliminary evidence of anti-tumor activity in pediatric brain tumors.
IMG-08. VOLUMETRIC MEASUREMENT OF TUMOR SIZE OUTPERFORMS STANDARD TWO-DIMENSIONAL METHOD IN EARLY PREDICTION OF TUMOR PROGRESSION IN PEDIATRIC GLIOMA
Authors: Khalili Nastaran , Kazerooni Anahita Fathi , Bagheri Sina , Familiar Ariana , Viswanathan Karthik , Anderson Hannah , Haldar Debanjan , Ware Jeffrey B , Vossough Arastoo , Nabavizadeh Ali
Published: June 2023

BACKGROUND

Pediatric brain tumors (PBTs) are the leading cause of cancer-related death in children. Currently, based on the RAPNO guidelines, two-dimensional (2D) measurements are used to detect progression. However, due to the complex radiographic appearance of many PBTs, 2D measurements may not accurately reflect tumor growth. Here, we compared 3D, 2D, and qualitative radiologists’ interpretations in a group of PBTs to determine tumor progression.

METHODS

Six PBT patients (5 low-grade glioma, 1 ependymoma, age range at the time of baseline scan: 23-191 months) with an average of 5 imaging time points who had at least one episode of progression with subsequent surgery were included. Segmentation was performed on either T1 post-contrast or FLAIR, whichever that best identified the tumor. For 2D measurements, the two largest perpendicular diameters in a section that included the largest tumor component were measured. For the volumetric assessment, the tumors were manually segmented, and the volume was computed using the ITK-SNAP software. In multifocal tumors, only the tumor component that showed progression and underwent resection was included. A 25% and 41% increase in tumor size was considered as progression on 2D and 3D measurements, respectively. The time to progression (TTP) based on 2D vs 3D assessment as well as the official pediatric neuroradiologist interpretation based on the electronic health record report was compared.

RESULTS

In three of six patients, volumetric segmentation detected tumor progression at an earlier time point compared to 2D measurements and radiologists’ interpretation (median TTP: 134 compared to 503 for 2D and radiologist interpretation). For these three patients, the TTP based on 3D vs 2D assessment was: 252 vs 1299 days, 134 vs 503 days, and 80 vs 255 days.

CONCLUSION

Volumetric measurements can determine tumor progression earlier than the current standard method of 2D measurements and qualitative interpretation of radiologists in PBTs.
IMG-10. MAGNETIC RESONANCE FUNCTIONAL IMAGING BIOMARKER FOR DISTINGUISHING HIGH GRADE FROM LOW GRADE PEDIATRIC BRAIN TUMORS
Authors: Kelly Teresa Gross , Schmainda Kathleen , Prah Melissa , Jeffrey Knipstein , Maheshwari Mohit , Lew Sean , Baker Marjorie Bessette
Published: June 2023

This study aims to show that the ratio between perfusion MRI (RCBV) and diffusion MRI (ADC - apparent diffusion coefficient) is more effective in distinguishing low-grade from high-grade pediatric brain tumors than either parameter (RCBV or ADC) alone. After IRB approval was obtained, an initial pilot prospective study was performed that included 38 patients (newborn - 18 years) (22 females and 16 males). All MRI exams were performed on a 1.5 T or 3.0 T scanner. Surgically resected tumors were classified as low grade (WHO I, II) or high grade (WHO III, IV). ROIs were selected using tumor boundaries delineated on the FLAIR sequence, excluding perilesional edema. Both standardized and normalized perfusion RCBV (sRCBV, nRCBV) as well as diffusion (ADC) mean values were acquired. Then sRCBV/ADC ratios were calculated. Statistical analysis included the Wilcoxon-Mann-Whitney t-test using an alpha=0.05 as the level of significance. The results of this preliminary study revealed that the sRCBV/ADC ratio calculations were more effective at separating high grade from low grade tumors than were RCBV or ADC values alone. As a result of this original pilot study, the calculated sRCBV/ADC ratio has occasionally been used as a clinical tool at our institution. In order to validate the results from this original pilot study, we have collected additional retrospective data to increase sample size. We plan to share the results of this additional data in order to validate that the sRCBV/ADC ratio is an easy-to-use imaging biomarker that can effectively separate high grade from low grade pediatric brain tumors. This information is useful for patient care in that it can potentially help predict prognosis which is essential for parental counseling, and potentially reduce the need for invasive tests and biopsies.
LGG-12. CLINICAL AND MOLECULAR FEATURES OF DISSEMINATED PEDIATRIC LOW-GRADE GLIOMA
Authors: Levine Adrian , Li Cyril , Haizel-Cobbina Joseline , Nobre Liana , Bennett Julie , Dewan Michael , Tabori Uri , Hawkins Cynthia
Published: June 2023

Although most pediatric LGG (PLGG) have excellent long-term survival, there is a subset of cases that disseminate throughout the neuraxis (DLGG) that have very poor outcomes. The reason for this aggressive behavior is unknown but we hypothesize that distinct and specific biological mechanisms underlie the metastatic ability. The methylation-based class of diffuse leptomeningeal glioneuronal tumor (DLGNT) is characterized by MAPK pathway activating fusions with chromosome 1p loss, 19q loss, and/or 1q gain. However, it is unknown what proportion of DLGG match the DLGNT group, and which other methylations classes are at risk of metastasis. To improve our understanding of this rare patient population, we created an international DLGG consortium. Data from the first 68 cases shows a broad age distribution and no sex predilection. As expected, DLGG has much worse prognosis than the overall PLGG population. Virtually all DLGG progress at 5 years, compared to a quarter of other PLGG, and DLGG are 5-times more likely to die at 10 years. We observe three patterns of dissemination – 35% present with a localized mass and have secondary dissemination, 50% with disseminated tumor and a clear dominant mass, and 15% with disseminated disease without a dominant mass. In 47 patients with molecular testing, BRAF fusions accounted for 64% of driver alterations. Additional alterations were identified less frequently, including BRAF V600E (9%), FGFR1 alterations (9%), and KRAS mutations (4%). In 25 patients with methylation profiling, 10 (40%) successfully classified with a calibrated score above 0.8, illustrating that many cases do not fit a defined methylation group. The most common methylation classification was pilocytic astrocytoma (5 patients), and surprisingly only two patients classified as DLGNT. In sum this study illustrates the variable clinical behaviour associated with metastasis in PLGG and expands the range of molecular driver alterations, including ones previously unreported in DLGNT.
LGG-17. EPIGENETICALLY DEFINED ANGIOCENTRIC GLIOMAS MAY LACK ANGIOCENTRIC GROWTH AND INSTEAD SHOW A VARIETY OF GROWTH PATTERNS
Authors: Stegat Lotte , Rohwer Jenny L , Stichel Damian , Schrimpf Daniel , Coras Roland , Pagès Mélanie , Tauziède-Espariat Arnault , Varlet Pascale , Hans Volkmar H , Meyer Jochen , Schittenhelm Jens , Staszewski Ori , Cheesman Edmund , Glatzel Markus , Schmid Simone , Wesseling Pieter , Korshunov Andrey , Sexton-Oates Alexandra , Schüller Ulrich , Kõrgvee Lenne-Triin , Mueller Sabine , Olar Adriana , Snuderl Matija , Schweizer Leonille , Aronica Eleonora , Sahm Felix , von Deimling Andreas , Blumcke Ingmar , Jones David T W , Capper David , Wefers Annika K
Published: June 2023

Angiocentric glioma mainly occurs in children and young adults. It is associated with a good prognosis (CNS WHO grade 1). Molecularly, most angiocentric gliomas have a MYB-QKI fusion. Histologically, angiocentric gliomas were initially defined by an angiocentric growth of the tumor cells. However, we noticed that epigenetically defined angiocentric gliomas often get different diagnoses based on their histology as they may lack this angiocentric growth pattern. We collected 48 epigenetically defined angiocentric gliomas from 46 patients with sufficient tissue for histological and molecular analyses (DNA methylation analyses; RNA sequencing of a subset; 37 supratentorial and 11 infratentorial cases). The classification was done using unsupervised hierarchical cluster analyses from DNA methylation data and the brain tumor classifiers v11b4 and v12.5 (www.molecularneuropathology.org). Angiocentric gliomas were epigenetically distinct from the diffuse astrocytomas, MYB- or MYBL1-altered. A MYB-QKI fusion was detected in 69% (n = 18/26) of angiocentric gliomas, confirming the diagnosis. Other fusions detected were fusions of MYB with intergenic sites, mainly close to QKI (19%; n = 5/26), and MYBL1-QKI (8%; n = 2/26). We then did a histological workup of these cases. Only 75% showed the typical angiocentric growth pattern that often was not very pronounced. Many angiocentric gliomas displayed an unspecific growth while others resembled pilocytic astrocytoma or ependymoma. Hence, about 59% of angiocentric gliomas were initially misdiagnosed without molecular analyses (supratentorial 50%, infratentorial 100%). We obtained similar results expanding the cohort with further epigenetically defined angiocentric gliomas for which no tissue for a histological re-evaluation was available (total 68 cases; 50 supratentorial, 12 infratentorial; 65 patients). In this expanded cohort, 54% of cases were initially misdiagnosed (45% of supratentorial and 100% of infratentorial cases). In summary, we show that angiocentric glioma often does not show the typical angiocentric growth pattern. Thus, molecular analyses are needed for a reliable diagnosis.