Pediatric Glioblastoma Multiforme Research Articles

Deciphering the Role of microRNA Mediated Regulation of Coronin 1C in Glioblastoma Development and Metastasis.

Glioblastoma multiforme (GBM) is a highly heterogenic and malignant brain tumour with a median survival of 15 months. The initial identification of primary glioblastomas is often challenging. Coronin 1C (CORO1C) is a key player in actin rearrangement and cofilin dynamics, as well as enhancing the processes of neurite overgrowth and migration of brain tumour cells. Different bioinformatic databases were accessed to measure CORO1C expression at the mRNA and protein level in normal and malignant brains. CORO1C expression was observed in brain regions which have retained high synaptic plasticity and myelination properties. CORO1C was also expressed mainly within the hippocampus formation, including the Cornu Ammonis (CA) fields: CA1-CA4. Higher expression was also noticed in paediatric GBM in comparison to their adult counterparts. Pediatric cell populations were observed to have an increased log2 expression of CORO1C. Furthermore, 62 miRNAs were found to target the CORO1C gene. Of these, hsa-miR-34a-5p, hsa-miR-512-3p, hsa-miR-136-5p, hsa-miR-206, hsa-miR-128-3p, and hsa-miR-21-5p have shown to act as tumour suppressors or oncomiRs in different neoplasms, including GBM. The elevated expression of CORO1C in high grade metastatic brain malignancies, including GBM, suggests that this protein could have a clinical utility as a biomarker linked to an unfavorable outcome.

Abstract 3895: Moxidectin unravels the role of Hippo-YAP pathway in maintaining immunity of pediatric glioblastoma

Abstract Pediatric glioblastoma multiforme (GBM) is considered to be the second most lethal childhood cancer type after leukemia. Recent preclinical, clinical and genomic studies have highlighted upon the role of Hippo-Yap pathway in the progression of pediatric GBM. In addition, recent studies have established the role of YAP in creating immune suppressive tumor microenvironment (TME) facilitating drug resistance, recurrence and metastasis of GBM tumors. Herein, we report that ‘moxidectin’ an anti-helminthic drug inhibits the proliferation of SF268, SF295, SF188 and CT-2A Luc GBM cells by inducing apoptosis. Immunoblotting and immunofluorescence microscopy studies show that moxidectin mediates its effects by inhibiting MEK-ERK pathway, a regulator of Hippo-YAP signaling. As a result, moxidectin suppressed the nuclear translocation and transcriptional activity of YAP/TAZ-TEAD complex. Oral administration of 3.5mg/kg moxidectin suppressed the growth of GBM tumors by 90% in intracranial tumor model. Ex-vivo analysis of excised tumors confirmed the observations made in in vitro studies. We further conducted immunophenotyping on the excised tumors from both control and moxidectin treated mice to evaluate the effect of moxidectin on immune cell markers in TME and Tumor Draining Lymph Nodes (TDLNs). Our analysis indicated that treatment with moxidectin suppressed the immune suppressive TME created by GBM tumor. Interestingly, moxidectin enhanced antigen presentation in the TDLN. Indicating that it might play a role in activating the peripheral immune response. Moxidectin is an FDA approved drug and any findings from our research will promote its further investigation as a potential therapeutic agent for GBM patients. Citation Format: Itishree Kaushik, Shreyas Gaikwad, Sanjay K. Srivastava. Moxidectin unravels the role of Hippo-YAP pathway in maintaining immunity of pediatric glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3895.

EXTH-34. ION CHANNELS AS A THERAPEUTIC TARGET IN PAEDIATRIC HIGH-GRADE GLIOMAS

Abstract Pediatric Glioblastoma Multiforme (GBM) is a leading cause of CNS cancer-related death in children. The scarcity of treatments for GBM has opened the field to new pathways in adults, such as tumour treating fields(TTF). Studies have identified the clinical benefit of electrotherapy in combination with chemotherapeutics, however the mechanistic action is unclear. Increasing evidence suggests that ion channels not only regulate electrical signalling of excitable cells, but are also crucial in the development/progression of brain tumours. Ion channels are essential in cell-cycle control, invasion and migration of cancer cells, therefore presenting as valuable therapeutic targets. Candidate ion channel genes(ICG) associated with high-grade glioma (HGG) were identified via analysis of inhouse and publicly available data sets. Expression patterns of selected ICGs were assessed along with clinician correlations.. Protein and RNA expression of target ICGs was assessed in pGBM cell lines/ TMAs. Finally, the Human ClariomTMS array was used for whole transcriptome gene expression analysis of paediatric GBM cell lines treated with tumour treating fields or low frequency electrical fields via deep brain stimulating electrodes. Paediatric brain tumour cells were exposed to genetic and pharmacological manipulation of CLIC1 and CLIC4 ion channels individually or in combination. We have shown that HGG exhibit increased expression of CLIC4 and CLIC1 at protein and RNA levels in both publically available data sets and inhouse cell lines / TMAs. Clinical correlation determined that high CLIC4 and CLIC1 expression was associated with poor overall survival. Further to this, DNA array analysis revealed a downregulation of CLIC1 and CLIC4 ion channels genes in KNS42 cells when treated with electrotherapy compared to untreated cells. Inhibition of CLIC1 and CLIC4 reduces Cl- flux across cell membranes and reduces cell proliferation. These data provide rationale that manipulation of ICGs will reduce the capacity of childhood brain tumours to proliferate and invade.

Primary and metastatic glioblastoma of the spine in the pediatric population: a systematic review.

Pediatric glioblastoma multiforme (GBM) involving the spine is an aggressive tumor with a poor quality of life for patients. Despite this, there is only a limited number of reports describing the outcomes of pediatric spinal GBMs, both as primary spinal GBMs and metastases from an intracranial tumor. Here, we performed an individual patient meta-analysis to characterize factors affecting prognosis of pediatric spinal GBM. MEDLINE, Embase, and the Cochrane databases were searched for published studies on GBMs involving the spine in pediatric patients (age ≤ 21 years old). Factors associated with the survival were assessed with multi-factor ANOVAs, Cox hazard regression, and Kaplan-Meier analyses. We extracted data on 61 patients with spinal GBM from 40 studies that met inclusion criteria. Median survival was significantly longer in the primary spinal GBM compared that those with metastatic GBM (11 vs 3 months, p < 0.001). However, median survival of metastatic GBM patients was 10 months following diagnosis of their primary brain tumor, which was not different from that of primary spinal GBM patients (p = 0.457). Among primary spinal GBM patients, chemotherapy (hazard ratio (HR) = 0.255 [0.106-0.615], p = 0.013) and extent of resection (HR = 0.582 [0.374-0.905], p = 0.016) conferred a significant survival benefit. Younger age (less than 14 years) was associated with longer survival in patients treated with chemotherapy than those who did not undergo chemotherapy (β = - 1.12, 95% CI [- 2.20, - 0.03], p < 0.05). In conclusion, survival after presentation of metastases from intracranial GBM is poor in the pediatric population. In patients with metastatic GBM, chemotherapy may have provided the most benefit in young patients, and its efficacy might have an association with extent of surgical resection.

PD-L1 Expression Correlated with p53 Expression in Pediatric Glioblastoma Multiforme.

High-grade gliomas are infrequent in the pediatric population compared to adults, nevertheless, mortality and morbidity caused by malignant gliomas in this group of patients remain significant. PD-L1 and PD-1 Immune checkpoints (IC) molecules maintain immunological balance between activation and suppression. Eighteen patients with a histopathological diagnosis of pediatric glioblastoma multiforme (GBM, WHO IV) were studied. In total, PD-L1 expression was detected in 8 patients (44%). The molecular aspect of IC and immunotherapy targeted on PD-1/PD-L1 axis in pediatric population may be a promising adjuvant therapy in pediatric glioblastoma multiform treatment, however, this subject requires further investigation.

HGG-04. ZINC ENHANCES TEMOZOLOMIDE CYTOTOXICITY IN PEDIATRIC GLIOBLASTOMA MULTIFORME MODEL SYSTEM

BACKGROUNDTemozolomide (TMZ) is an alkylating agent that has become the mainstay treatment of the most malignant brain cancer, glioblastoma multiforme (GBM). Unfortunately only a limited number of patients respond to it positively. We have shown that zinc metal reestablishes chemosensitivity in adult GBM in vitro and also in vivo but this effect has not been tested with pediatric GBM.METHODSUsing Human pediatric glioblastoma cell lines- KNS42 (mutant p53/ MGMT [+]) and SF188 (mutant p53/ MGMT [-]), we investigated whether addition of zinc to TMZ enhances its cytotoxicity against GBM.RESULTS In vitro cell viability analysis showed that the cytotoxic activity of TMZ was substantially increased with addition of zinc and this response was accompanied by an elevation of p21, PUMA, BAX and a decrease in growth fraction as manifested by low ki67. Beta gal analysis showed that most of the remaining cells after the combination therapy are in senescence state. In order to eliminate the senescent population created as a result of the combined treatment of TMZ and Zinc, we decided to use a senolytic agent Navitoclax (ABT-263) that was demonstrated to be effective in reducing senescent cells by specific inhibition of Bcl-2, Bcl-XL and Bcl-w. Following the addition of Navitoclax to the combined treatment, SF188 cells, but not KNS42, show a significance reduction in viability compare to the combination treatment.CONCLUSIONSOur results suggest that zinc may serve as a potentiator of TMZ therapy in pediatric GBM patients and using a second hit with senolytic drug in some cases may be even more beneficial.

HGG-43. CONGENITAL GLIOBLASTOMA MULTIFORME: A CASE REPORT OF A RARE PEDIATRIC BRAIN TUMOR, MOLECULAR ANALYSIS, AND REVIEW OF THE LITERATURE

Congenital brain tumors are rare, accounting for less than 4% of all pediatric brain tumors. Congenital glioblastoma multiforme (GBM) is rarer still, accounting for 3–15% of congenital brain tumors. There is literature to suggest that these tumors differ from pediatric and adult GBM clinically and molecularly, and as such should be treated as their own distinct entity. Our case is a 4 week old male who initially presented to his pediatrician for enlarging head circumference and upward gaze palsy. An MRI was obtained revealing a right parietal mass. He underwent gross total resection the following day with pathology revealing glioblastoma, WHO grade IV. Further analysis revealed ATRX retained, p53 immunoreactivity in 15–20% of nuclei, IDH1 and IDH2 wildtype, MGMT promoter not methylated, H3K27M wildtype, no 1p and/or 19q deletion/codeletion. Interestingly, RNA analysis of his tumor detected the PPP1CB-ALK fusion transcript as well as amplification of the ALK gene. Co-occurrence of these mutations has been reported in a small number of pediatric glioblastoma patients and PPP1CB-ALK fusions are one of the most common receptor tyrosine kinase fusions in infantile gliomas. ALK rearrangements and amplifications suggest a potential therapeutic target with tyrosine kinase inhibitors in glioblastoma. This patient serves as an example of a rare congenital glioblastoma with unique molecular features that may suggest novel treatment opportunities. We present his clinical course along with a pertinent review of the literature.

DGCR5 suppresses the EMT of pediatric primary glioblastoma multiforme cell and serves as a prognostic biomarker.

Pediatric primary glioblastoma multiforme (GBM) is a common brain tumor among childhood. Nevertheless, the underlying mechanism of glioblastoma progresses remains to be illuminated. The current study explores the potential functions of long noncoding RNA (lncRNA) DGCR5 in the aggressiveness of GBM. The expression of DGCR5 was evaluated by quantitative real-time PCR (qRT-PCR). Immunoblotting was carried out to assess the protein expressions of N-cadherin and E-cadherin. Cell Counting Kit-8 (CCK-8), wound healing, transwell assay, and flow cytometry were applied to explore the roles of DGCR5 in glioblastoma cell malignant biological behaviors. LncRNA DGCR5 was down expressed in glioblastoma. Transfection of DGCR5 markedly repressed cell viability and colony formation ability of U87 and U251 cells. Additional, DGCR5 overexpression caused cell cycle arrest and increased cell apoptosis. Moreover, upregulation of DGCR5 suppressed the growth and promoted cell apoptosis of U87 cell in vivo. Finally, overexpression of DGCR5 impaired the migration, invasiveness, and reversed epithelial-to-mesenchymal transition (EMT) process of glioblastoma cell. LncRNA DGCR5 inhibited the proliferation, aggressiveness phenotypes, and EMT of glioblastoma cell.

Abstract 4637: Tumor treating fields (TTFields) have antiproliferative effects on high-grade pediatric brain tumor cell lines

Abstract Introduction Approximately 400 children in the UK are diagnosed with a CNS tumor each year, which represents a quarter of childhood cancer cases in the UK. The 5-year survival rate of paediatric brain tumor patients is approximately 75%, but survival remains much worse for some brain tumor patients, particularly high-grade gliomas. Pre-clinical and clinical studies have demonstrated efficacy of TTFields as a treatment for high-grade glioma leading to the approval of TTFields for adult Glioblastoma multiforme (GBM) patients. The lack of overlapping toxicities associated with TTFields has made electric field therapy an attractive treatment strategy for poor prognosis paediatric brain tumors.Methods The inovitro is the laboratory based testing system used to deliver TTFields to cell cultures. inovitro was used to deliver TTFields over 72 hours at a range of clinically relevant frequencies (100-400kHz) to our panel of paediatric GBM, Medulloblastoma and Ependymoma cell lines. The effects of TTFields on cell viability and cell cycle was assessed using Resazurin based metabolic viability tests and flow cytometry. The determined optimal frequency for each cell line was also combined with a mitotic inhibitor to explore synergistic effects. Gene expression changes of TTFields treated cells was investigated. Results TTFields treatment reduces cell viability of all cell lines tested within the frequency panel. The optimum frequencies were between 100 and 400kHz for all cell lines tested. TTFields treatment had effects on cell cycling with increased G2 accumulation of treated cells being observed. Combining TTFields with mitotic inhibitors increased efficacy in an additive fashion. Conclusions This preliminary study has demonstrated efficacy of TTFields on paediatric brain tumor cell lines. The optimal frequencies for all the paediatric GBM cell lines used did not match the 200kHz as previously reported for U-87 MG cells. This report warrants further study into the potential for TTFields therapy for paediatric brain tumor patients. Citation Format: Joshua Branter, Maria Estevez-Cebrero, Richard Grundy, Surajit Basu, Stuart Smith. Tumor treating fields (TTFields) have antiproliferative effects on high-grade pediatric brain tumor cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4637.

Cell line-based xenograft mouse model of paediatric glioma stem cells mirrors the clinical course of the patient.

The leading cause of cancer-related mortality among children is brain tumour, and glioblastoma multiforme (GBM) has the worst prognosis. New treatments are urgently needed, but with few cases and clinical trials in children, pre-clinical models such as patient-derived tumour xenografts (PDTX) are important. To generate these, tumour tissue is transplanted into mice, but this yields highly variable results and requires serial passaging in mice, which is time-consuming and expensive. We therefore aimed to establish a cell line-based orthotopic mouse model representative of the patient tumour. Glioma stem cell (GSC) lines derived from paediatric GBM were orthotopically transplanted into immunodeficient mice. Overall survival data were collected and histological analysis of the resulting neoplasias was performed. Genome-wide DNA methylation arrays were used for methylation and copy-number alterations (CNA) profiling. All GSC lines initiated tumours on transplantation and the survival of the mice correlated well with the survival of the patients. Xenograft tumours presented histological hallmarks of GBM, and were also classified as GBM by methylation profiling. Each xenograft tumour clustered together with its respective injected GSC line and patient tumour based on the methylation data. We have established a robust and reproducible cell line-based xenograft paediatric GBM model. The xenograft tumours accurately reflected the patient tumours and mirrored the clinical course of the patient. This model can therefore be used to assess patient response in pre-clinical studies.

Concurrent Inhibition of Neurosphere and Monolayer Cells of Pediatric Glioblastoma by Aurora A Inhibitor MLN8237 Predicted Survival Extension in PDOX Models

Purpose: Pediatric glioblastoma multiforme (pGBM) is a highly aggressive tumor in need of novel therapies. Our objective was to demonstrate the therapeutic efficacy of MLN8237 (alisertib), an orally available selective inhibitor of Aurora A kinase (AURKA), and to evaluate which in vitro model system (monolayer or neurosphere) can predict therapeutic efficacy in vivoExperimental Design: AURKA mRNA expressions were screened with qRT-PCR. In vitro antitumor effects were examined in three matching pairs of monolayer and neurosphere lines established from patient-derived orthotopic xenograft (PDOX) models of the untreated (IC-4687GBM), recurrent (IC-3752GBM), and terminal (IC-R0315GBM) tumors, and in vivo therapeutic efficacy through log rank analysis of survival times in two models (IC-4687GBM and IC-R0315GBM) following MLN8237 treatment (30 mg/kg/day, orally, 12 days). Drug concentrations in vivo and mechanism of action and resistance were also investigated.Results: AURKA mRNA overexpression was detected in 14 pGBM tumors, 10 PDOX models, and 6 cultured pGBM lines as compared with 11 low-grade gliomas and normal brains. MLN8237 penetrated into pGBM xenografts in mouse brains. Significant extension of survival times were achieved in IC-4687GBM of which both neurosphere and monolayer were inhibited in vitro, but not in IC-R0315GBM of which only neurosphere cells responded (similar to IC-3752GBM). Apoptosis-mediated MLN8237 induced cell death, and the presence of AURKA-negative and CD133+ cells appears to have contributed to in vivo therapy resistance.Conclusions: MLN8237 successfully targeted AURKA in a subset of pGBMs. Our data suggest that combination therapy should aim at AURKA-negative and/or CD133+ pGBM cells to prevent tumor recurrence. Clin Cancer Res; 24(9); 2159-70. ©2018 AACR.

Tumour Treating Fields (TTFields) have anti-proliferative effects on paediatric brain tumour cell lines at clinically deliverable field settings

Approximately 400 children in the UK are diagnosed with a CNS tumour each, which represents a quarter of childhood cancer cases in the UK. The overall 5-year survival rate of peadiatric brain tumour patients is roughly 75%, however survival still remains poor for some brain tumours, particularly high grade gliomas. Pre-clinical studies have shown efficacy of TTFields on high grade glioma cell lines, while TTFields have only been approved for adult Glioblastoma multiforme (GBM) patients in combination with the Stupp treatment protocol. The lack of overlapping toxicities associated with OptuneTM has made TTFields an attractive treatment strategy for poor prognosis brain tumours. The InovitroTM system is the laboratory testing system used to develop the clinically approved Optune system. InovitroTM was used to deliver TTFields over 72 hours at a range of clinically relevant frequencies (100-400kHz) and intensities (1-3V/cm) to a panel of paediatric GBM, Medulloblastoma and Ependymoma cell lines. The effects of TTFields on cell viability and cell cycle was assessed using metabolic viability tests (Presto blue), cell proliferation assays and cell cycle analysis using flow cytometry. TTFields treatment reduces cell viability of all paediatric brain tumour cell lines tested within the frequency titration. The optimum frequencies were between 200 and 300kHz for all cell lines tested. TTFields treatment had significant effects on cell cycling relative to control cells. This preliminary study has confirmed the efficacy of electric fields on paediatric brain tumour cell lines. The optimum frequencies for the paediatric GBM cell lines did not uniformly fall into the 200kHz range as previously reported for glioma cell lines. This report warrants further study into the potential for TTFields therapy for paediatric patients.

Pediatric glioblastoma cells inhibit neurogenesis and promote astrogenesis, phenotypic transformation and migration of human neural progenitor cells within cocultures

Neural progenitor cell (NPC) fate is influenced by a variety of biological cues elicited from the surrounding microenvironment and recent studies suggest their possible role in pediatric glioblastoma multiforme (GBM) development. Since a few GBM cells also display NPC characteristics, it is not clear whether NPCs transform to tumor cell phenotype leading to the onset of GBM formation, or NPCs migrate to developing tumor sites in response to paracrine signaling from GBM cells. Elucidating the paracrine interactions between GBM cells and NPCs in vivo is challenging due to the inherent complexity of the CNS. Here, we investigated the interactions between human NPCs (ReNcell) and human pediatric GBM-derived cells (SJ-GBM2) using a Transwell® coculture setup to assess the effects of GBM cells on ReNcells (cytokine and chemokine release, viability, phenotype, differentiation, migration). Standalone ReNcell or GBM cultures served as controls. Qualitative and quantitative results from ELISA®, Live/Dead® and BrdU assays, immunofluorescence labeling, western blot analysis, and scratch test suggests that although ReNcell viability remained unaffected in the presence of pediatric GBM cells, their morphology, phenotype, differentiation patterns, neurite outgrowth, migration patterns (average speed, distance, number of cells) and GSK-3β expression were significantly influenced. The cumulative distance migrated by the cells in each condition was fit to Furth's formula, derived formally from Ornstein-Uhlenbeck process. ReNcell differentiation into neural lineage was compromised and astrogenesis promoted within cocultures. Such coculture platform could be extended to identify the specific molecules contributing to the observed phenomena, to investigate whether NPCs could be transplanted to replace lesions of excised tumor sites, and to elucidate the underlying molecular pathways involved in GBM-NPC interactions within the tumor microenvironment.

137 Novel Genome-wide CRISPR Screen for Glioblastoma Invasion

Abstract INTRODUCTION Among high-grade pediatric brain tumors, glioblastoma multiforme (GBM) is particularly difficult to treat. Invasion of GBM cells into normal brain parenchyma renders malignant cells inaccessible to surgical intervention and poised to drive tumor recurrence. In order to better understand the molecular mechanisms and signaling pathways underlying pediatric GBM cell invasion we performed a genome-wide CRISPR/Cas9 loss of function screen for genes that inhibit invasion. METHODS CRISPR/Cas9 is an emerging technology for genome editing that can be used to knock out genes that are targeted by short RNA guide sequences (sgRNAs). First, we made an immortalized human glioma line that stably expresses the RNA-guided DNA endonuclease, Cas9, and showed that Cas9 is functional in these cells. We then used a pooled approach to knock-out individual genes on a genome-wide scale in the Cas9 GBM cell line. Pooled cells were subjected to a Boyden chamber invasion assay which separated the cell populations based on invasive phenotype. To determine which genes were enriched in the invasive cell fraction compared to the noninvasive cell population we used next generation sequencing of the sgRNAs isolated and amplified from each group. RESULTS &gt;Analysis of sgRNA enrichment in invasive compared to noninvasive cell populations identified specific genes expected to be altered in migration, such as those involved with actin based cell projections, as well as novel candidate genes. We are currently testing the necessity and sufficiency of these genes for glioma invasion in vitro and in vivo using a xenograft mouse model. CONCLUSION In this study, we identified critical genes necessary for GBM invasion using a novel genome-wide knock-out approach. Once validated, the genes of interest may serve as prognostic surveillance markers or targets for therapeutic intervention in pediatric glioblastoma.

Variegated Colors of Pediatric Glioblastoma Multiforme: What to Expect?

Malignant gliomas account for 35-45% of primary brain tumors; among these glioblastoma multiforme (GBM) is the most common adult brain tumor constituting approximately 85%. Its incidence is quite less in the pediatric population and treatment of these patients is particularly challenging. Exposure to ionizing radiation is the only environmental factor found to have any significant association with GBM. Several genetic alterations associated with GBM in adults have been well documented such as epidermal growth factor receptor amplification, overexpression of mouse double minute 2 homolog also known as E3 ubiquitin-protein ligase, Phosphatase and tensin homolog gene mutation, loss of heterozygosity of chromosome 10p and isocitrate dehydrogenase-1 mutation. However, data on genetic mutations in pediatric GBM is still lacking. Exophytic brain stem gliomas are rare tumors and are usually associated with a poor prognosis. The most effective treatment in achieving long-term survival in such patients, is surgical excision of the tumor and then chemoradiotherapy followed by adjuvant chemotherapy by temozolomide. This schedule is the standard treatment for GBM patients. In view of the rarity of pediatric GBM, we report here a case of pontine GBM in a 5-year-old girl.

Evaluation of a novel antibody to define histone 3.3 G34R mutant brain tumours

Missense somatic mutations affecting histone H3.1 and H3.3 proteins are now accepted as the hallmark of paediatric diffuse intrinsic pontine gliomas (DIPG), non-brain stem paediatric high grade gliomas (pHGG) as well as a subset of adult glioblastoma multiforme (GBM). Different mutations give rise to one of three amino acid substitutions at two critical positions within the histone tails, K27M, G34R/V. Several studies have highlighted gene expression and epigenetic changes associated with histone H3 mutations; however their precise roles in tumourigenesis remain incompletely understood. Determining how such amino acid substitutions in a protein affect its properties can be challenging because of difficulties in detecting and tracking mutant proteins within cells and tissues. Here we describe a strategy for the generation of antibodies to discriminate G34R and G34V mutant histone H3 proteins from their wild-type counterparts. Antibodies were validated by western blotting and immunocytochemistry, using recombinant H3.3 proteins and paediatric GBM cell lines. The H3-G34R antibody demonstrated a high degree of selectivity towards its target sequence. Accordingly, immunostaining on a cohort of 22 formalin-fixed paraffin embedded tumours with a previously known H3.3 G34R mutation status, detected successfully the corresponding mutant protein in 11/11 G34R cases. Since there was a high concordance between genotype and immunohistochemical analysis of G34R mutant tumour samples, we analysed a series of tissue microarrays (TMAs) to assess the specificity of the antibody in a range of paediatric brain tumours, and noted immunoreactivity in 2/634 cases. Importantly, we describe the generation and validation of highly specific antibodies for G34 mutations. Overall our work adds to an extremely valuable portfolio of antibodies, not only for histopathologic detection of tumour-associated mutant histone sequences, but also facilitating the study of spatial/anatomical aspects of tumour formation and the identification of downstream targets and pathways in malignant glioma progression.

A systematic review of overall survival in pediatric primary glioblastoma multiforme of the spinal cord.

OBJECTIVE The incidence of primary spinal cord glioblastoma multiforme (GBM) in the pediatric age group is very rare. Only a few case series and case reports have been published in the literature; therefore, overall survival (OS) outcome and the as-yet poorly defined management options are not discussed in detail. The authors performed a cumulative survival analysis of all reported cases of pediatric spinal cord GBM to identify the predictive factors related to final survival outcome. METHODS A comprehensive search for relevant articles was performed on PubMed's electronic database MEDLINE for the period from 1950 to 2015 using the search words "malignant spinal cord tumor" and "spinal glioblastoma multiforme." This study was limited to patients younger than 18 years of age. Survival rates for children with various tumor locations and treatments were collected from the published articles and analyzed. RESULTS After an extensive literature search, 29 articles met the study inclusion criteria. From the detailed information in these articles, the authors found 53 children eligible for the survival analysis. The majority (45%) of the children were more than 12 years old. Thirty-four percent of the cases were between 7 and 12 years of age, and 21% were younger than 7 years. In the Kaplan-Meier survival analysis, children younger than 7 years of age had better survival (13 months) than the children older than 7 years (7-12 years: 10 months, > 12 years: 9 months; p = 0.01, log-rank test). Fifty-five percent of the children were female and 45% were male. A cervical tumor location (32%) was the most common, followed by thoracic (28.3%). Cervicothoracic (18.9%) and conus (18.8%) tumor locations shared the same percentage of cases. Cervical tumors had a worse outcome than tumors in other locations (p = 0.003, log-rank test). The most common presenting symptom was limb weakness (53%), followed by sensory disturbances (25%). Median OS was 10 months. The addition of adjuvant therapy (radiotherapy [RT] and/or chemotherapy [CT]) after surgery significantly improved OS (p = 0.01, log-rank test). Children who underwent gross-total resection and RT had better outcomes than those who underwent subtotal resection and RT (p = 0.04, log-rank test). Cerebrospinal fluid spread, hydrocephalus, brain metastasis, and spinal metastasis were not correlated with OS in primary spinal GBM. CONCLUSIONS Adjuvant therapy after surgery had a beneficial effect on overall outcome of spinal GBM in the pediatric age group. Gross-total resection followed by RT produced a better outcome than subtotal resection with RT. Further large-scale prospective study is required to establish the genetic and molecular factors related to OS in primary GBM of the spinal cord in pediatric patients.

Rapid Neurological Recovery Following Partial Surgical Resection of Spinal Glioblastoma Multiforme in a Pediatric Patient Presenting With Complete Paraplegia

Pediatric spinal cord glioblastoma multiforme is a rare entity with a poor prognosis often presenting with lower extremity weakness or paralysis. Previous literature suggests that aggressive surgical resection may provide overall survival benefit; however, there is limited concurrent analysis demonstrating neurological recovery following surgical resection. We report the case of a 9-year-old boy who presented with complete paraplegia and regained the ability to ambulate independently following subtotal surgical resection, radiation, and chemotherapy. The case demonstrates the balance between meaningful neurological recovery and overall survival when deciding on the extent of resection in cases of pediatric spinal glioblastoma multiforme.

Differentially Expressed Long Non-Coding RNAs Were Predicted to Be Involved in the Control of Signaling Pathways in Pediatric Astrocytoma.

Expression changes for long non-coding RNAs (lncRNAs) have been identified in adult glioblastoma multiforme (GBM) and in a mixture of adult and pediatric astrocytoma. Since adult and pediatric astrocytomas are molecularly different, the mixture of both could mask specific features in each. We determined the global expression patterns of lncRNAs and messenger RNA (mRNAs) in pediatric astrocytoma of different histological grades. Transcript expression changes were determined with an HTA 2.0 array. lncRNA interactions with microRNAs and mRNAs were predicted by using an algorithm and the LncTar tool, respectively. Interactomes were constructed with the HIPPIE database and visualized with the Cytoscape platform. The array showed expression changes in 156 and 207 lncRNAs in tumors (versus the control) and in pediatric GBM (versus low-grade astrocytoma), respectively. Predictions identified lncRNAs that have putative microRNA binding sites, which might suggest that they function as sponges in these tumors. Also, lncRNAs were shown to interact with many mRNAs, such as Pleckstrin homology-like domain, family A, member 1 (PHLDA1) and sulfatase 2 (SULF2). For example, qPCR found long intergenic non-coding RNA regulator of reprogramming (linc-RoR) expression levels upregulated in pediatric GBM when they were compared with control tissues or with low-grade tumors. Meanwhile, PHLDA1 and ELAV-like RNA binding protein 1 (ELAV1) showed expression changes in tumors relative to the control. Our data showed many lncRNAs with expression changes in pediatric astrocytoma, which might be involved in the regulation of different signaling pathways.

P01.04 Adult and paediatric GBM cells show differential phenotypic responses to external lipoprotein deprivation

AbstractIntroduction:Glioblastoma multiforme (GBM) is a highly aggressive CNS neoplasm histologically characterised by mitotic atypia and microvascular proliferation. Aberrant activation of angiogenesis during tumour growth results in an improperly formed tumour vasculature, leading to gradients of oxygen and nutrients within the tumour microenvironment. Transport of lipids in blood is mediated via lipoprotein complexes. Adult GBM cells demonstrate a preferential uptake of lipoproteins to meet metabolic demands over de novo lipid synthesis. We investigated the growth and morphological responses of paediatric GBM cells under lipoprotein-deplete conditions.Materials and Methods:Two paediatric GBM cell lines (KNS42 and SF188) and one paediatric grade I pilocytic astrocytoma (Res186) cell line were cultured under lipoprotein-replete and –deplete conditions over 7 days. Cell viability was quantitatively assessed using the resazurin-based PrestoBlue reagent and was complemented with qualitative cell density assessment through crystal violet staining. Experiments were repeated using the adult GBM U87 cell line and patient-derived tumour cells (GIN-8) isolated from the invasive margin of an adult GBM.Results:Cell viability and density assays revealed reduced exponential growth rates of KNS42 and SF188 cells grown in lipoprotein deficient media compared to cells cultured under lipoprotein-replete conditions. In contrast, Res186, U87 and GIN-8 cells demonstrated cytostatic growth patterns after 3 days culture under lipoprotein-deplete conditions. Accompanying the growth responses of Res186 and U87 cells were morphological changes reminiscent of differentiation (process elongation), a phenotype not observed for KNS42 and SF188 cells.Conclusions:Evaluation of growth under lipoprotein–deplete conditions revealed an enhanced capacity of paediatric GBM cells to proliferate under acute metabolic stress compared to adult GBM and grade I paediatric glioma cells. This is likely reflective of differences at the genetic level and/or respective cell-of-origin. We will discuss metabolomic and transcriptomic strategies identifying signalling pathways mediating adaptive responses to metabolic stress that will be assessed in 3D culture models. Growth of GBM cells under lipoprotein-deplete conditions likely recapitulates selective pressures within the tumour microenvironment in vivo, revealing essential metabolic pathways that will inform next-generation targeted treatment strategies.