Adult Glioblastoma Multiforme Research Articles

BIOS-02. RECAPITULATE TUMOR DIVERSION AND SPATIAL PATTERNING IN GLIOBLASTOMA BACKGROUND

Abstract BACKGROUND Intra-tumoral heterogeneity of glioblastoma multiforme (GBM) drives aggressive behavior and intrinsic therapeutic resistance. To investigate heterogeneity, high-resolution sequencing techniques provided us to feature tumor molecular subtyping and simulate differentiation. In our study, we proposed to dissect tumor heterogeneity in different anatomical regions by single cell transcriptome analysis. Material and Method We performed single-cell RNA sequencing on 10675 adult IDH-wild type GBM cells from six pairs of tumor core (central necrotic part of tumor) and tumor margin (junction between tumor and brain). Subtyping following RNA velocity was performed to investigate tumor diversity. Result Four subgroups of tumor cells (OPC-like (oligoprogenitor cell), NPC-like (neuroprogenitor cell), AC-like (astrocyte), and MES-like (mesenchymal) )were identified. The higher cell population of OPC/NPC at tumor margin and AC/MES at tumor core was found. The RNA velocity analysis showed the major diversity order was OPC at margin, OPC at core, NPC at margin, and AC with MES at core. CONCLUSION We explore the spatial pattern of tumorigenesis of GBM that OPC at margin part could be the tumor initiating cell. Further investigation of biological characteristics of OPC could be the strategies to block tumor differentiation and progression.

Identification of glioblastoma stem cell-associated lncRNAs using single-cell RNA sequencing datasets

Glioblastoma multiforme (GBM) is an aggressive, heterogeneous brain tumor in which glioblastoma stem cells (GSCs) are known culprits of therapy resistance. Long non-coding RNAs (lncRNAs) have been shown to play a critical role in both cancer and normal biology. A few studies have suggested that aberrant expression of lncRNAs is associated with GSCs. However, a comprehensive single-cell analysis of the GSC-associated lncRNA transcriptome has not been carried out. Here, we analyzed recently published single-cell RNA sequencing datasets of adult GBM tumors, GBM organoids, GSC-enriched GBM tumors, and developing human brain samples to identify lncRNAs highly expressed in GSCs. We further revealed that the GSC-specific lncRNAs GIHCG and LINC01563 promote proliferation, migration, and stemness in the GSC population. Together, this study identified a panel of uncharacterized GSC-enriched lncRNAs and set the stage for future in-depth studies to examine their role in GBM pathology and their potential as biomarkers and/or therapeutic targets in GBM.

51. Discerning cell types and states in spatial transcriptomics data using topic modelling

Glioblastoma multiforme (GBM) is the most common adult brain tumour. GBM cells include a diverse collection of normal and tumour cells with distinct molecular capabilities and with differential levels of sensitivity to treatment that led to treatment resistance. Spatial transcriptomics technologies (e.g. 10X Visium) have enabled analyzing tumour-normal cells interactions in a physical tissue context. Using Visium, a well-annotated cohort of adult GBM patient samples grown as patient-derived xenograft (PDX) models were profiled at different time points of the disease. However, the identification of cell types or states of Visium spots is a challenging problem as each spot contains a mixture of cells. Current tools for cell-type deconvolution are limited in discovering new novel cell types because they require a well-annotated reference dataset. Moreover, the PDX data has a mixture of human and mouse cell types at the same spot. In this work, we have applied topic modelling, a well-known probabilistic approach in natural language processing to discover the distribution of topics in language documents. Using the analogy between documents and spatial transcriptomic data, we were able to identify the main cell types in spatial PDX data without gene expression references dataset of tumour states, normal mouse, and immune cell types. Finally, we compared various probabilistic topic modelling algorithms to fit the PDX data, and to understand the cell-to-cell interactions between tumour and normal cells.

Progress and Viewpoints of Multifunctional Composite Nanomaterials for Glioblastoma Theranostics.

The most common malignant tumor of the brain is glioblastoma multiforme (GBM) in adults. Many patients die shortly after diagnosis, and only 6% of patients survive more than 5 years. Moreover, the current average survival of malignant brain tumors is only about 15 months, and the recurrence rate within 2 years is almost 100%. Brain diseases are complicated to treat. The reason for this is that drugs are challenging to deliver to the brain because there is a blood–brain barrier (BBB) protection mechanism in the brain, which only allows water, oxygen, and blood sugar to enter the brain through blood vessels. Other chemicals cannot enter the brain due to their large size or are considered harmful substances. As a result, the efficacy of drugs for treating brain diseases is only about 30%, which cannot satisfy treatment expectations. Therefore, researchers have designed many types of nanoparticles and nanocomposites to fight against the most common malignant tumors in the brain, and they have been successful in animal experiments. This review will discuss the application of various nanocomposites in diagnosing and treating GBM. The topics include (1) the efficient and long-term tracking of brain images (magnetic resonance imaging, MRI, and near-infrared light (NIR)); (2) breaking through BBB for drug delivery; and (3) natural and chemical drugs equipped with nanomaterials. These multifunctional nanoparticles can overcome current difficulties and achieve progressive GBM treatment and diagnosis results.

An oncogenic splice variant of PDGFR\u03b1 in adult glioblastoma as a therapeutic target for selective CDK4/6 inhibitors

Understanding human genome alterations is necessary to optimize genome-based cancer therapeutics. However, some newly discovered mutations remain as variants of unknown significance (VUS). Here, the mutation c.1403A > G in exon 10 of the platelet-derived growth factor receptor-alpha (PDGFRA) gene, a VUS found in adult glioblastoma multiforme (GBM), was introduced in human embryonal kidney 293 T (HEK293T) cells using genome editing to investigate its potential oncogenic functions. Genome editing was performed using CRISPR/Cas9; the proliferation, drug sensitivity, and carcinogenic potential of genome-edited cells were investigated. We also investigated the mechanism underlying the observed phenotypes. Three GBM patients carrying the c.1403A > G mutation were studied to validate the in vitro results. The c.1403A > G mutation led to a splice variant (p.K455_N468delinsN) because of the generation of a 3’-acceptor splice site in exon 10. PDGFRA-mutated HEK293T cells exhibited a higher proliferative activity via PDGFRα and the cyclin-dependent kinase (CDK)4/CDK6-cyclin D1 signaling pathway in a ligand-independent manner. They showed higher sensitivity to multi-kinase, receptor tyrosine kinase, and CDK4/CDK6 inhibitors. Of the three GBM patients studied, two harbored the p.K455_N468delinsN splice variant. The splicing mutation c.1403A > G in PDGFRA is oncogenic in nature. Kinase inhibitors targeting PDGFRα and CDK4/CDK6 signaling should be evaluated for treating GBM patients harboring this mutation.

P15.02 Loss of Williams Syndrome Transcription Factor (WSTF) leads to improved temozolomide sensitivity in human glioblastoma cells irrespective of MGMT expression

Abstract BACKGROUND The prognosis for newly diagnosed adult glioblastoma multiforme (GBM) patients is poor even after standard therapy with a median survival of approximately 14–15 months. The DNA repair protein O 6 -methylguanine-DNA methyltransferase (MGMT) efficiently counteracts formation of the most lethal DNA adducts by temozolomide (TMZ) chemotherapy, and is thus associated with poor outcome in GBM patients. Williams Syndrome Transcription Factor (WSTF) has previously been suggested to regulate the DNA damage response pathway (DDR) in both an indirect (through chromatin remodeling together with SMARCA5 in the WICH complex) and direct manner (by phosphorylating H2AX at Tyr142). However, whether WSTF has any role in the development of resistance against chemotherapy through its ability to regulate the DDR in GBMs, is so far not known. In this study, we investigated whether loss of WSTF sensitizes different MGMT-proficient and -deficient GBM cell lines to TMZ treatment. MATERIAL AND METHODS We generated WSTF knockout clones from both MGMT-proficient (LN18, T98G) and -deficient GBM cell lines (U-251) using CRISPR/Cas9 gene-editing technology with lentiviral vectors. The PCR-based screening results combined with the T7 endonuclease mismatch assay for bi-allelic monoclonal knockouts were verified via sequencing and immunoblotting to identify candidate knockout clones. For each cell line, three knockout clones were chosen for further investigation. Colony formation assays were performed to determine the survival ability in response to TMZ treatment. Statistical analysis was performed using two-way ANOVA. RESULTS WSTF knockout clones showed a significant decrease in colony formation after TMZ-treatment compared to the corresponding WSTF-expressing control groups (non-target single guide RNA) (LN18: Clone 59 vs control: p= 0.0456, T98G: All three studied clones vs control: p <0.0001, U-251: Clone 7/35.1/70.2 vs control: p <0.0001/p= 0.0107/p= 0.0119). Furthermore, two out of three clones of T98G and U-251 (T98G Clone 13 and 128 vs control, p <0.0001, U-251 Clone 7 vs control, p= 0.0062; clone 70.2, p= 0.0052) showed significantly reduced plating efficiency compared to control cells. CONCLUSION WSTF is an important factor in both MGMT de- and proficient GBM cell lines for response against TMZ chemotherapy. The loss of WSTF leads to a significantly increased TMZ sensitivity in clinically relevant concentrations for all the studied cell lines. Ongoing studies are investigating the underlying mechanisms and potential alterations in the DDR pathway caused by WSTF loss.

Four specific biomarkers associated with the progression of glioblastoma multiforme in older adults identified using weighted gene co-expression network analysis

ABSTRACT Glioblastoma multiforme (GBM) is the most common primary intracranial malignancy in adults. Owing to individual tolerance and tumor heterogeneity, the therapy methods for young adults do not apply to older adults. The present study aimed to identify specific biomarkers for GBM in older adults using weighted gene co-expression network analysis (WGCNA). Gene expression profiles of older adults with GBM were downloaded from The Cancer Genome Atlas (TCGA) and set as a discovery cohort to construct WGCNA. Core genes of clinically significant modules were used to perform functional enrichment, protein-protein interaction, and Pearson correlation analyses. Gene expression profiles of young in TCGA and older GBM patients from our research group were set as verification cohorts for hub gene expression and diagnostic value. Four significant gene modules associated clinically with older adults with GBM were identified, whereas 251 genes were core genes with module membership>0.8 and gene significance>0.2. Ermin (ERMN), myelin-associated oligodendrocyte basic protein (MOBP), proteolipid protein 1 (PLP1), and oligodendrocytic myelin paranodal and inner loop protein (OPALIN) genes had significant relationships with the Karnofsky score (KPS) in older GBM patients. ERMN, MOBP, PLP1, and OPALIN had no relationship with KPS in young GBM patients. These genes were upregulated in GBM tissues from older patients with low but not high KPS and had high diagnostic value. In conclusion, ERMN, MOBP, PLP1, and OPALIN may serve as specific biomarkers for the progression of GBM in older adults.

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.

ACTR-18. A PHASE IB/II CLINICAL TRIAL OF DODECAFLUOROPENTANE EMULSION (DDFPE) AS A RADIOSENSITIZER IN GLIOBLASTOMA

Abstract BACKGROUND Tumor hypoxia decreases the response of glioblastoma multiforme (GBM) to radiotherapy (RT) and chemotherapy (temozolomide[TMZ]). The purpose of this study was to evaluate the safety and efficacy of a novel oxygen therapeutic, dodecafluoropentane emulsion (DDFPe), in chemoradiation treatment of GBM and stratify the results based on predicted TMZ response. METHODS With ethics approval and informed consent, 11 adult GBM patients were enrolled. Patients were administered DDFPe via IV infusion (2% w/vol at doses of 0.05, 0.1 or 0.17 mL/kg) within 5–30 minutes prior to each 2 Gy fraction of RT (30 fractions over 6-weeks) while breathing supplemental oxygen. Patients also received standard concurrent and adjuvant TMZ. To evaluate the reversal of tumor hypoxia, patients underwent oxygen-sensitive (TOLD) MRI before and after DDFPe administration. Patients were also studied with serial MRI scans per standard of care and followed for survival. RESULTS The non-serious adverse events considered by the investigator to be possibly, probably, or definitely related to DDFPe administration included fatigue (n=4), headache (n=2) and decrease in platelet count (n=2). Serious adverse events included two patients with symptomatic radiation necrosis: one patient at each of dose levels 0.1 and 0.17 mL/kg. Enrollment continued at the 0.1 mL/kg dose without additional significant DDFPe-related toxicity. Historically, the average overall survival for GBM patients is about 14.6 months. The median overall survival for the study was 591 days, or 19.4 months. According to independent review of the serial MR images, the median time to progression was 555 days, or 18 months, compared to a historical control of 6.9 months. TOLD MRI showed a trend in improved tumor oxygenation. CONCLUSION Although small, this trial shows that DDFPe used as a radiosensitizer appears to be safe and may provide some survival benefit. The FDA has allowed a Phase II clinical trial to assess its effectiveness.

Abstract 4705: Single cell derived 3D organoids recapitulate the tumorigenic features of glioblastoma

Abstract In recent years, three-dimensional (3D) models of human brain derived from pluripotent embryonic and iPS cells have emerged as brain organoids, providing established models for brain development and genetic engineering. However, tumor organoids derived from adult glioblastoma multiforme (GBM) patients were seldomly formed. WHO grade IV GBM is a devastating locally invasive brain cancer with a median survival of 14.6 months. Standard of care for GBM includes surgical resection followed by radiotherapy plus concomitant and maintenance temozolomide (TMZ). Resistance to TMZ develops rapidly and neither dose-intensified TMZ nor anti-angiogenic approaches could improve survival. Genomic, transcriptomic and epigenetic profiling allowed classifying adult GBM into three groups: isocitrate dehydrogenase (IDH)-mutant (mut), 1p/19q co-deleted oligodendroglial GBM with best prognosis; IDH-mut, 1p/19q non-co-deleted astrocytic GBM with intermediate outcome; and IDH wild-type (WT) GBM with poor prognosis. Preclinical models that reflect these GBM profiles and heterogeneity are urgently needed to examine new therapies. Patient derived orthotopic xenografts (PDOXs) are thought to better mimic the GBM environment. Genetically engineered mouse models (GEMMs) make gliomas in mice with competent immune systems, but are laborious and expensive requiring compound genetics to mimic human GBM. Here, we describe a 3D serum-free organoid system in low-adherence plates derived from primary and sphere cultures that supports the long-term growth and expansion of GBM organoids for several months. We generated organoids from GBM with IDH-WT or IDH-Mut, EGFR amplification, +7q/-10q genotype, PTEN mutation, and EGFRvIII expression. GBM organoids are comprised of a heterogenous cell populations, thus mimicking the original tumor. GBM organoids could be generated from a single cell, therefore allowing to track intratumor heterogeneity. GBM organoids could be utilized for investigating aspects of GBM biology such as 3D cellular self-renewal showing expression of stem cell and differentiation targets, 3D cell cycle regulation including synchronized growth and phase cycling, 3D cell metabolism including intracellular adenosine triphosphate (ATP) level and protein synthesis rate, 3D cell invasiveness validated with IHC assays, and for evaluation of drug effects in the context of IDH, PI3K and EGFR aberrations. We have demonstrated that GBM sphere cells exhibit a solid growth pattern when implanted orthotopically into the mouse PDOXs. Since GBM organoids form tighter cell-cell contact, oxygen and nutrient gradients, we implanted GBM organoids in mouse orthotopic xenografts to demonstrate their tumor growth and invasive phenotypes. This human GBM organoid platform allows for novel preclinical therapeutic approaches to be assessed and provides personalized therapeutic options for individual GBM patients. Citation Format: Michelle Chadwick, Rachael Mfon, Kelly Jara, Shabbar Danish, Robert Aiken, Hatem E. Sabaawy. Single cell derived 3D organoids recapitulate the tumorigenic features of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4705.

Tumour treating fields in a combinational therapeutic approach.

The standard of care for patients with newly diagnosed Glioblastoma multiforme (GBM) has remained unchanged since 2005, with patients undergoing maximal surgical resection, followed by radiotherapy plus concomitant and maintenance Temozolomide. More recently, Tumour treating fields (TTFields) therapy has become FDA approved for adult recurrent and adult newly-diagnosed GBM following the EF-11 and EF-14 trials, respectively. TTFields is a non-invasive anticancer treatment which utilizes medium frequency alternating electric fields to target actively dividing cancerous cells. TTFields selectively targets cells within mitosis through interacting with key mitotic proteins to cause mitotic arrest and cell death. TTFields therapy presents itself as a candidate for the combinational therapy route due to the lack of overlapping toxicities associated with electric fields. Here we review current literature pertaining to TTFields in combination with alkylating agents, radiation, anti-angiogenics, mitotic inhibitors, immunotherapies, and also with novel agents. This review highlights the observed synergistic and additive effects of combining TTFields with various other therapies, as well highlighting the strategies relating to combinations with electric fields.

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.

Prognostic stratification of adult primary glioblastoma multiforme patients based on their tumor gene amplification profiles.

Several classification systems have been proposed to address genomic heterogeneity of glioblastoma multiforme, but they either showed limited prognostic value and/or are difficult to implement in routine diagnostics. Here we propose a prognostic stratification model for these primary tumors based on tumor gene amplification profiles, that might be easily implemented in routine diagnostics, and potentially improve the patients management. Gene amplification profiles were prospectively evaluated in 80 primary glioblastoma multiforme tumors using single-nucleotide polymorphism arrays and the results obtained validated in publicly available data from 267/347 cases. Gene amplification was detected in 45% of patients, and chromosome 7p11.2 including the EGFR gene, was the most frequently amplified chromosomal region – either alone (18%) or in combination with amplification of DNA sequences in other chromosomal regions (10% of cases). Other frequently amplified DNA sequences included regions in chromosomes 12q(10%), 4q12(7%) and 1q32.1(4%). Based on their gene amplification profiles, glioblastomas were subdivided into: i) tumors with no gene amplification (55%); ii) tumors with chromosome 7p/EGFR gene amplification (with or without amplification of other chromosomal regions) (38%); and iii) glioblastoma multiforme with a single (11%) or multiple (6%) amplified DNA sequences in chromosomal regions other than chromosome 7p. From the prognostic point of view, these amplification profiles showed a significant impact on overall survival of glioblastoma multiforme patients (p>0.001). Based on these gene amplification profiles, a risk-stratification scoring system was built for prognostic stratification of glioblastoma which might be easily implemented in routine diagnostics, and potentially contribute to improved patient management.

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.

An overview of crucial genes involved in stemness of glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and first leading cause of death in primary human brain cancers. Following current interventions, despite continuous advancements in medicine, the median survival time of patients is unfavorable regardless of radiotherapy, chemotherapy, or surgery. This poor outcome is referred to “cancer stem cell” (CSC) concept. According to it a minority of cells within the GBM mass originates from neural stem cells (NSCs) or alternatively hijack similar checkpoints in them that evolution has selected in order to prevent inappropriate neurogenic-to-gliogenic switch which is responsible for the growth of tumor and its resistance to existing therapies. New developments in non-surgical treatment of GBM are based on this knowledge, however, similarities in molecular pathways between GBM and glioma stem cells (GSCs) motive the race to identify and target GSC regulators with less and more harmful effect in normal adult NSC and GBM respectively. In it the implications of downstream stemness genes with modulatory roles for stemness state pathways are assumed importance. This review, embodies NSC-intrinsic genes by recent elucidated roles of importance for GBM to affix to the stemness. Therefor it could facilitate a holistic approach of molecular targeted therapy to compromise the formidable resistant GBM to eradication.

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.

Abstract 1351: Alternative splicing of neurofibromin 1 is associated with elevated MAPK activity and poor prognosis in glioma

Abstract High-grade gliomas (HGG) are invasive with poor prognosis regardless of age: diffuse intrinsic pontine gliomas (DIPG), arising in the brainstem, are almost universally fatal and the leading cause of brain-tumor death in children; while adult anaplastic astrocytoma and glioblastoma multiforme (GBM) have median survivals of 1-3 years. The mutational spectra of adult and pediatric HGG differ, with pediatric tumours containing recurrent mutations of H3F3A and HIST1H3B. However, alterations leading to RAS/MAPK/PI3K pathway activation, including PDGFRA amplification, EGFRvIII, BRAF-V600E, NF1 deletion, are frequently found, although not all tumours will have mutations in this pathway. The neurofibromin 1 (NF1) gene negatively regulates RAS signalling by stimulating RAS-GTP turnover, thereby leading to RAS-inactivation. The two major isoforms, NF1-I and NF1-II, differ only by inclusion of the 21 aa exon23a in the GAP-related domain of NF1-II. Exon23a-inclusion has been shown to render NF1 10 times less active towards RAS, leading to elevated MAPK signalling. The brain expresses predominantly NF1-I, while the major isoform elsewhere is NF1-II. Here we used RNA-Seq to identify genes alternatively spliced between DIPG and normal brain, identifying an isoform switch from NF1-I in normal brain to NF1-II; we additionally found the same isoform switch in the TCGA adult GBM and LGG cohorts. For both GBM and LGG, RAS/MAPK/PI3K wild-type tumors with elevated NF1-II conferred significantly reduced patient survival compared to RAS/MAPK/PI3K mutant tumors. NF1-exon23a inclusion is known to be repressed in cell model systems by the CELF and ELAV-like families of splice regulators. We further show that members of these gene families are downregulated in HGG. Together, our results indicate a novel mechanism by which gliomas can activate signaling downstream from RAS independent of mutations and tumor grade, which promotes tumorigenesis by regulating pathways such as proliferation and invasion. Citation Format: Robert Siddaway, Arun Ramani, Man Yu, Michael Brudno, Cynthia Hawkins. Alternative splicing of neurofibromin 1 is associated with elevated MAPK activity and poor prognosis in glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1351. doi:10.1158/1538-7445.AM2017-1351

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 phase Ib/II clinical trial of a novel oxygen therapeutic in chemoradiation of glioblastoma.

2561 Background: Tumor hypoxia limits the response of glioblastoma multiforme (GBM) to radiotherapy (RT) and chemotherapy (temozolomide[TMZ]). Additionally, patient biomarkers are strong predictors of responsiveness to TMZ. The purpose of this study is to evaluate the use of a novel oxygen therapeutic, dodecafluoropentane emulsion (DDFPe), in chemoradiation treatment of GBM and stratify the results based on predicted TMZ response. Methods: 11 adult GBM patients have been enrolled. Patients were administered DDFPe via IV infusion (2% w/vol at doses of 0.05, 0.1 or 0.17 mL/kg) an hour prior to each 2 Gy fraction of RT (30 fractions over 6-weeks) with supplemental oxygen. Patients also received standard concurrent and adjuvant TMZ. To confirm tumor re-oxygenation, patients underwent TOLD MRI before and after DDFPe administration. Archived tumor specimens were studied with GliomaSTRAT assay for methylated genes predictive of response. Patients were also studied with serial MRI scans per standard of care and followed for survival. Results: There were minimal acute adverse events related to DDFPe administration. One patient at each of dose levels 0.1 and 0.17 mL/kg developed symptomatic radiation necrosis, which was judged to be related to DDFPe. Enrollment has continued at the 0.1 mL/kg dose without additional significant DDFPe-related toxicity. TOLD MRI showed significant decrease in T1of tumor tissue consistent with tumor re-oxygenation (p=0.015) with no significant change in oxygenation of normal brain tissue. Historically, the average overall survival for GBM patients, for both TMZ responders and non-responders, is about 14.6 months. The progress chart for patient overall survival is listed below, 4 patients have died. All other patients are alive. Conclusions: DDFPe is a promising oxygen therapeutic for reversing tumor hypoxia. A Phase II study is being planned to assess its effectiveness. Clinical trial information: NCT02189109. [Table: see text]