GBM Cases Research Articles

HGG-14. Molecular characterization of unique biological subgroups among H3 wild type high-grade gliomas

INTRODUCTION: Paediatric high-grade gliomas (HGG) are characterised by the aggressive biological behaviour with dismal prognosis of long-term survival 10-15%. Current molecular-biological diagnostic approaches allow for more precise characterization and determination of new unique subgroups of HGG. Our aim was to identify novel and rare HGG subgroups within our institution cohort. PATIENTS AND METHODS: Our reference centre patients′ cohort consisted of 97 clinically annotated patients with HGG diagnosed between 2000 and 2021. Sanger sequencing was used for screening of the most common HGG-related oncogenic drivers; furthermore we employed whole genome methylation array (Illumina Infinium MethylationEPIC BeadChip) and for selected samples RNA sequencing and expression profiling. RESULTS: Based on H3 status and previous radiotherapy we separated our HGG cases into the RIG, H3mut and H3wt groups. In contrast to H3mut(n=35) and RIG(n=11) that were uniformly fatal, H3wt group contained a proportion of long-term survivors. In the H3wt group we found patients carrying driver mutations in IDH1/2 (n=2) and BRAFV600E (7). Five young patients (under 3) consisted of 3 infant hemispheric gliomas (with NTRK and ROS1 fusions), one gliomatosis cerebri and one brainstem anaplastic astrocytoma with MYB/QKI fusion. We also identified a rare EWSR1-PATZ1 gene fusion in one patient. Importantly, long-term survivors recruited from these subgroups. On the contrary, four cases of MYCN GBM with poor prognosis presented in various locations: one disseminated, one gliomatosis cerebri and two with hemispheric tumour. We identified one patient with “hypermutated” glioblastoma and used targeted therapy with Nivolumab. In three samples of our patients with thalamic glioblastomas, we detected “loss of H3K27-trimethylation” caused by EZHIP overexpression. These tumours proved to be very aggressive with early metastatic recurrence and dismal prognosis. SUMMARY: Detailed characterization of H3 wild-type HGG is very important for further understanding of their biological behaviour, diagnostics, prognostication and identification of therapeutic targets.

Abstract IA013: Genetic heterogeneity and tumor microenvironment in glioblastoma

Abstract Glioblastoma (GBM), the most aggressive brain tumor, remains an unmet medical need. Despite aggressive chemotherapy, radiation and surgery, only 5.6% of patients survive beyond 5 years post-diagnosis. Targeted approaches have not been successful in this tumor type due to the extreme heterogeneity of GBM. Mosaic amplification of oncogenes suggests that multiple genetically distinct clones are present in each tumor. However, little is known about how different subpopulations of GBM cells interact with each other or with the surrounding tumor microenvironment (TME). To address this, we employed spatial protein profiling coupled with single-cell spatial maps of fluorescence in situ hybridization (FISH) for key oncogenes frequently amplified in GBM. Our analysis spanned 3-4 areas from 17 formalin-fixed paraffin-embedded (FFPE) GBM cases, 96 sub-regions of interest and total of 35,843 single nuclei. Digital spatial profiling for 79 protein markers associated with GBM biology, TME and neurobiology revealed high intra-tumor variability of vasculature, between distinct regions of the same tumor biopsy. The single-cell FISH signal quantification allowed us to classify tumors based on the relative frequency of cells harboring co-occurring of EGFR and CDK4 amplifications. Tumors with low odds ratio for EGFR and CDK4 co-amplification in a cell had a significantly higher EGFR copy number in cells harboring this amplification. Cell-to-cell variation in EGFR copy number was also much higher in these tumors, suggesting a possibility for extrachromosomal origin of this amplification. Interestingly, the tumors with high frequency of cells harboring co-amplification of EGFR and CDK4 exhibit higher infiltration by CD163+ immunosuppressive macrophages. Our results suggest that high throughput assessment of genomic alterations at the single cell level could provide a measure for predicting the immune state of GBM. Citation Format: Kacper A. Walentynowicz, Dalit Engelhardt, Shreya Yadav, Ugoma Onubogu, Roberto Salatino, Cristina Vincentelli, Thomas O. McDonald, Franziska Michor, Michalina Janiszewska. Genetic heterogeneity and tumor microenvironment in glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr IA013.

A functional role of S100A4/non-muscle myosin IIA axis for pro-tumorigenic vascular functions in glioblastoma

BackgroundGlioblastoma (GBM) is the most aggressive form of brain tumor and has vascular-rich features. The S100A4/non-muscle myosin IIA (NMIIA) axis contributes to aggressive phenotypes in a variety of human malignancies, but little is known about its involvement in GBM tumorigenesis. Herein, we examined the role of the S100A4/NMIIA axis during tumor progression and vasculogenesis in GBM.MethodsWe performed immunohistochemistry for S100A4, NMIIA, and two hypoxic markers, hypoxia-inducible factor-1α (HIF-1α) and carbonic anhydrase 9 (CA9), in samples from 94 GBM cases. The functional impact of S100A4 knockdown and hypoxia were also assessed using a GBM cell line.ResultsIn clinical GBM samples, overexpression of S100A4 and NMIIA was observed in both non-pseudopalisading (Ps) and Ps (-associated) perinecrotic lesions, consistent with stabilization of HIF-1α and CA9. CD34(+) microvascular densities (MVDs) and the interaction of S100A4 and NMIIA were significantly higher in non-Ps perinecrotic lesions compared to those in Ps perinecrotic areas. In non-Ps perinecrotic lesions, S100A4(+)/HIF-1α(−) GBM cells were recruited to the surface of preexisting host vessels in the vascular-rich areas. Elevated vascular endothelial growth factor A (VEGFA) mRNA expression was found in S100A4(+)/HIF-1α(+) GBM cells adjacent to the vascular-rich areas. In addition, GBM patients with high S100A4 protein expression had significantly worse OS and PFS than did patients with low S100A4 expression. Knockdown of S100A4 in the GBM cell line KS-1 decreased migration capability, concomitant with decreased Slug expression; the opposite effects were elicited by blebbistatin-dependent inhibition of NMIIA.ConclusionS100A4(+)/HIF-1α(−) GBM cells are recruited to (and migrate along) preexisting vessels through inhibition of NMIIA activity. This is likely stimulated by extracellular VEGF that is released by S100A4(+)/HIF-1α(+) tumor cells in non-Ps perinecrotic lesions. In turn, these events engender tumor progression via acceleration of pro-tumorigenic vascular functions.-mzznLaNnzTzVPc8r2eojtVideo abstract

OD170 - A novel deep learning model to differentiate IDH-mutant from IDH-wild type on cohort of GBM cases

OD170 - A novel deep learning model to differentiate IDH-mutant from IDH-wild type on cohort of GBM cases

CSIG-08. ABT-414 RESISTANT GBM TUMORS HARBOR THE TEK S466I MUTATION AND EXHIBIT INCREASED STEMNESS

Abstract Glioblastoma (GBM), the most common primary brain tumor in adults, remains uniformly fatal due to the lack of effective targeted therapies for this aggressive malignancy. Genomic amplification of epidermal growth factor receptor (EGFR) occurs in 40-60% of primary GBM. Half of all EGFR-amplified cases of GBM also harbor EGFRvIII, a constitutively active, truncated variant of EGFR. The incidence of EGFR alterations in GBM makes inhibition of EGFR/EGFRvIII an attractive therapeutic approach. Depatuxizumab mafodotin (ABT-414) is an antibody-drug conjugate comprised of ABT-806, a mAB against EGFR, and a cytotoxic payload (monomethyl auristatin F). ABT-414 therapy initially showed promising results as an EGFRvIII therapy in vivo and in vitro; however, ABT414 failed to provide a survival benefit in phase I/II clinical trials, potentially due to therapeutic resistance. In this study, we seek to investigate the mechanisms of resistance to ABT-414 by performing whole exome, transcriptome and single cell RNA seq on ABT-414 resistant GBM PDX tumors. Our data showed an enrichment of mutations unique to the ABT-414 resistant tumors, including a point mutation (S466I) in TEK/TIE2 transmembrane angiopoietin receptor. In vitro expression of TEK S466I in GBM cells showed an increase in activation of ERK and STAT3 and increased TEK/TIE2 immunoprecipitation with EGFR compared to the wild-type TEK receptor. Furthermore, gene ontology analysis reveals that ABT-414-treated flank tumors exhibit increased activation of extracellular matrix organization and CNS developmental processes compared to flank tumors in the control treatment groups. ABT-414-treated tumors also demonstrate increased expression of inhibitor of differentiation (ID)1 and ID3, associated with stem-like phenotype in glioblastoma. Taken together, our data indicate that resistance to ABT-414 is mediated by both de novo mutations not detected in the parent tumor and adaptive dysregulation of pathways which may lead to dedifferentiation and therapeutic resistance.

EXTH-12. INHIBITION OF EPIDERMAL GROWTH FACTOR RECEPTOR AND PLATELET-DERIVED GROWTH FACTOR RECEPTOR-ALPHA EXERTS SYNERGISTIC EFFICACY IN GLIOBLASTOMA

Abstract Epidermal growth factor receptor (EGFR) alterations, including amplification and activating mutations, occur in more than half of GBM cases. EGFR is located on Chr. 7, and Chr. 7 gain is one of the earliest events precipitating gliomagenesis. EGFR inhibitors, monoclonal antibodies, vaccines, and CAR-T cells have failed in GBM due to intrinsic heterogeneity and receptor tyrosine kinase (RTK) bypass pathways that mediate therapeutic resistance. New targeted therapeutic approaches to leverage synergistic combinations are desperately needed to improve GBM prognosis. Using the TCGA and other GBM databases, we previously demonstrated that PDGFRA amplification in patients with EGFR-amplified GBM carries significantly worse survival. EGFR and PDGFRA co-expression occur in more than one-third of GBM patients. The PDGFRA ligand PDGFA is also located on Chr. 7, and its expression is significantly increased with Chr. 7 gain and EGFR copy number increase. Therefore, Chr. 7 gain inherently leads to co-activation of both EGFR and PDGFRA signaling. We used patient-derived glioblastoma cells with Chr. 7 gain to test combined inhibition of EGFR and PDGFRA in vitro. We found that combined inhibition of both EGFR and PDGFRA using FDA-approved EGFR-targeted agents (Erlotinib, Gefitinib, Dacomitinib, Neratinib, and Osimertinib) and Crenolanib, respectively, leads to synergistic cytotoxicity in vitro. Inhibition of either EGFR or PDGFRA led to receptor cross-activation, and EGF and PDGF-AA-induced RTK activation was blocked by Neratinib and Crenolanib. Immunoprecipitation experiments and proximity ligation assays demonstrated that combined inhibition prevents EGFR and PDGFRA heterodimerization and pathways of therapeutic resistance. This combined inhibition led to decreased activation of downstream signaling pathways, including PI3K and MAPK. We show that combined inhibition of EGFR and PDGFRA exerts synergistic cytotoxicity in GBM and prevents resistance pathways that emerge during single-agent targeted therapy. These pathways are targetable with FDA-approved agents that could be used in patients with GBM with Chr. 7 gain.

A Rare Manifestation of a Presumed Non-Osteophilic Brain Neoplasm: Extensive Axial Skeletal Metastases From Glioblastoma With Primitive Neuronal Components.

BackgroundGlioblastoma multiforme (GBM) is the most common malignant tumor of the central nervous system. GBM with primitive neuronal component (GBM-PNC) is an aggressive variant identified in 0.5% of GBMs. Extracranial metastasis from GBM-PNC is a rare and challenging situation.MethodsA special case of early-onset GBM with systemic bone metastasis was enrolled. Clinical data, including patient characteristics, disease course, and serial radiological images were retrieved and analyzed. Tumor tissues were obtained by surgical resections and were made into formalin-fixed paraffin-embedded sections. Histopathological examinations and genetic testing were performed for both the primary and metastatic tumor specimens.ResultsA 20-year-old man suffered from GBM with acute intratumoral hemorrhage of the left temporal lobe. He was treated by gross total resection and chemoradiotherapy following the Stupp protocol. Seven months later, he returned with a five-week history of progressive neck pain and unsteady gait. The radiographic examinations identified vertebral collapse at C4 and C6. Similar osteolytic lesions were also observed at the thoracolumbar spine, pelvic, and left femur. Anterior spondylectomy of C4 and C6 was performed. The resected vertebral bodies were infiltrated with greyish, soft, and ill-defined tumor tissue. One month later, he developed mechanical low-back pain and paraplegia caused by thoracolumbar metastases. Another spine surgery was performed, including T10 total en-bloc spondylectomy, T7-9, L2-3, and L5-S1 laminectomy. After the operation, the patient’s neurological function and spinal stability remained stable. However, he finally succumbed to the rapidly increased tumor burden and died 15 months from onset because of cachexia and multiple organ failure. In addition to typical GBM morphology, the histological examinations identified monomorphic small-round cells with positive immunohistochemical staining of synaptophysin and CD99, indicating the coexistence of PNC. The next-generation sequencing detected pathogenic mutations in TP53 and DNMT3A. Based on above findings, a confirmed diagnosis of systemic metastases from GBM-PNC (IDH-wild type, WHO grade IV) was made.ConclusionsThe present case highlights the occurrence and severity of extensive axial skeletal metastases from GBM-PNC. This rare variant of GBM requires aggressive multimodal treatment including surgery and chemoradiotherapy targeting PNC. The pathological screening of PNC is recommended in patients with early-onset GBM and intratumoral hemorrhage. Surgery for spinal metastasis is appropriate in patients with chemoradioresistance and relatively good general status, with the objectives of restoring spinal stability and relieving spinal cord compression.

Proton Radiation Therapy for Pediatric Skull Base Chondrosarcomas

<h3>Purpose/Objective(s)</h3> Skull base chondrosarcomas are extremely rare tumors, accounting for 6% of skull base (BOS) neoplasms and 0.15% of all intracranial tumors. There are very few studies discussing chondrosarcomas in pediatric population. We report outcomes for children and young adults with BOS chondrosarcomas treated with proton radiotherapy (PRT). <h3>Materials/Methods</h3> Between 1984 and 2018, pediatric patients were treated with PRT or combined proton/photon RT (comboRT) at a single institution. The primary endpoints were overall survival (OS) and progression free survival (PFS). Univariable analysis was used to identify prognostic factors for PFS. Cox proportional hazards regression and Kaplan Meier estimates were used for survival analyses. <h3>Results</h3> 65 patients were identified [37 females (57%) and 28 males (43%)]. Median age at diagnosis was 18 years (range, 6 -21). 3 patients presented with Ollier's disease. 63 patients (97%) were diagnosed with low-grade (grade 1-2/3) chondrosarcoma and 2 patients (3%) with a grade 3 chondrosarcoma. The majority of the patients (n = 42, 65%) had a mixed hyaline and myxoid chondrosarcoma, 10 (15%) were diagnosed with a myxoid subtype, 4 (6%) with a hyaline one, and 4 (6%) patients had a mesenchymal chondrosarcoma. All patients underwent surgical resection (range, 1-6). 7 patients (6%) underwent gross total resection, 51 patients (78%) underwent STR while 7 patients (6%) had biopsy only. 3 patients (4%) received pre-RT chemotherapy. All patients received post-operative radiation therapy. 22 (34%) patients developed progressive disease prior to RT. The median time from the time of diagnosis to RT was 8.6m (range, 1.2 - 113.7). 22 patients (34%) were treated with PRT and 43 (66%) patients received comboRT. Median prescription dose was 70 Gy (RBE) (range, 55.8 – 76.3) delivered in 1.8 - 2 Gy (RBE) fractions. The median maximum dose to the brainstem was 66.3 Gy (RBE) (range, 26 – 74.6). The median maximum dose to the optic chiasm was 62.5 Gy (RBE) (range, 3.8 – 66.1). At a median follow-up of 10 years (range, 0.6 –33.7) from the date of diagnosis, 8 patients recurred (4 local, 2 distant, 2 both local and distant). 4 patients died of disease. 4 patients died of other causes The 10-year OS and PFS rates were 91.9% (95% CI: 79.4 – 97.0) and 84.9% (95% CI: 70.2-92.7), respectively. On univariate analysis, high grade, mesenchymal chondrosarcoma, pre-RT chemo and larger tumor size were worse prognostic factors for PFS. RT was well tolerated, with most acute side effects limited to moderate erythema and epilation, otitis media, and nausea. Late toxicities include 1 case of RT-induced GBM of the brainstem, 1 case of radiation necrosis, 2 cases of RT-related vascular malformations, 4 cases of temporal lobe injury, and 1 case of RT ocular neuromyotonia. <h3>Conclusion</h3> This is the largest cohort of pediatric BOS chondrosarcomas in the literature to date. High dose PRT following surgical resection achieves excellent disease control with minimal toxicity.

Primary Extra-axial Glioblastoma: Case Report and Literature Review

AbstractGlioblastoma multiforme (GBM) is the most frequent and most aggressive primary brain tumor in adults, mainly located in the cerebral hemispheres. In the literature, few cases of primary GBM have been reported to have radiographic and intraoperative features of extra-axial lesions, leading to a diagnostic dilemma. Despite the advances in imaging modalities, the diagnosis of GBM can be challenging, and it is mainly based on the histopathologic confirmation of the excised tumor. We describe the case of a 76-year-old previously healthy female patient who presented to our hospital due to speech disturbances and cognitive impairment. The diagnosis of the tumor type on magnetic resonance imaging (MRI) was difficult, as the findings were suggestive of a malignant meningioma due to the heterogeneous enhancement of a dural-based mass with a dural tail sign. Moreover, the intraoperative findings revealed an extra-axial mass attached to the dura. A histological examination confirmed the diagnosis of glioblastoma with arachnoid infiltration. The patient underwent adjuvant radiotherapy and concomitant temozolomide treatment, she had clinical improvement postoperatively, and was stable during the six months of follow-up. Glioblastoma should be considered in the differential diagnosis of primary extra-axial mass with atypical and malignant features, especially in elderly patients.

Predictive Value of Blood Markers in Differentiating Healthy and Gbm Cases Using Artificial Neural Network

Predictive Value of Blood Markers in Differentiating Healthy and Gbm Cases Using Artificial Neural Network

Uncovering a Distinct Gene Signature in Endothelial Cells Associated With Contrast Enhancement in Glioblastoma.

PurposeGlioblastoma (GBM) is the most aggressive and lethal type of brain tumors. Magnetic resonance imaging (MRI) has been commonly used for GBM diagnosis. Contrast enhancement (CE) on T1-weighted sequences are presented in nearly all GBM as a result of high vascular permeability in glioblastomas. Although several radiomics studies indicated that CE is associated with distinct molecular signatures in tumors, the effects of vascular endothelial cells, the key component of blood brain barrier (BBB) controlling vascular permeability, on CE have not been thoroughly analyzed.MethodsEndothelial cell enriched genes have been identified using transcriptome data from 128 patients by a systematic method based on correlation analysis. Distinct endothelial cell enriched genes associated with CE were identified by analyzing difference of correlation score between CE-high and CE–low GBM cases. Immunohistochemical staining was performed on in-house patient cohort to validate the selected genes associated with CE. Moreover, a survival analysis was conducted to uncover the relation between CE and patient survival.ResultsWe illustrated that CE is associated with distinct vascular molecular imprints characterized by up-regulation of pro-inflammatory genes and deregulation of BBB related genes. Among them, PLVAP is up-regulated, whereas TJP1 and ABCG2 are down-regulated in the vasculature of GBM with high CE. In addition, we found that the high CE is associated with poor prognosis and GBM mesenchymal subtype.ConclusionWe provide an additional insight to reveal the molecular trait for CE in MRI images with special focus on vascular endothelial cells, linking CE with BBB disruption in the molecular level. This study provides a potential new direction that may be applied for the treatment optimization based on MRI features.

Glioblastoma in adolescents and young adults: An age-based comparative study from Jordan over a 17-year period

BackgroundGlioblastoma (GBM) is the most common primary brain tumor. Epidemiologic studies on GBM are rarely reported in the special age group of adolescents and young adults (AYA) in comparison to other age groups. We aim to present data on GBM in Jordan, with a focus on the AYA age, including the incidence, gender, location and outcome, as well as long term survival (LTS). MethodsData on GBM was requested from the Jordan Cancer Registry (JCR), and statistical analysis was performed. All data were retrospective and anonymized. ResultsEight hundred GBM cases were analyzed from 2000 to 2016 including 505 males (63.1 %). Males outnumbered females across are studied years (p-value <0.001). There were 49 pediatrics (0−14 years, 6.1 %), 125 AYA (15−39 years, 15.6 %), 358 adults (40−59 years, 44.8 %) and 268 elderly (60+ years, 33.5 %) cases. Supratentorial location predominated across all age groups (p-value <0.001). The preponderance of males and supratentorial tumors remained across the AYA age group in comparison to others. The median overall survival (OS) was 23.61 months. AYA age group had a better outcome in comparison to the adults/elderly age group (p-value< 0.001). LTS appear to be more common in the AYA age group (p-value 0.021). ConclusionsThis is the first comparative epidemiologic study of GBM in Jordan, focusing on the AYA age group. The AYA age group appears to be associated with a better outcome compared to older age groups, with more LTS compared to others.

Patterns of glioblastoma treatment and survival over a 16-years period: pooled data from the German Cancer Registries

IntroductionGlioblastoma multiforme (GBM) is a primary malignant brain tumour characterized by a very low long-term survival. The aim of this study was to analyse the distribution of treatment modalities and their effect on survival for GBM cases diagnosed in Germany between 1999 and 2014.MethodsCases were pooled from the German Cancer Registries with International Classification of Diseases for Oncology, third edition (ICD-O-3) codes for GBM or giant-cell GBM. Three periods, first (January 1999–December 2005), second (January 2006–December 2010) and a third period (January 2011–December 2014) were defined. Kaplan–Meier plots with long-rank test compared median overall survival (OS) between groups. Survival differences were assessed with Cox proportional-hazards models adjusted for available confounders.ResultsIn total, 40,138 adult GBM cases were analysed, with a mean age at diagnosis 64.0 ± 12.4 years. GBM was more common in men (57.3%). The median OS was 10.0 (95% CI 9.0–10.0) months. There was an increase in 2-year survival, from 16.6% in the first to 19.3% in the third period. When stratified by age group, period and treatment modalities, there was an improved median OS after 2005 due to treatment advancements. Younger age, female sex, surgical resection, use of radiotherapy and chemotherapy, were independent factors associated with better survival.ConclusionThe inclusion of temozolomide chemotherapy has considerably improved median OS in the older age groups but had a lesser effect in the younger age group of cases. The analysis showed survival improvements for each treatment option over time.

Spinal cord glioblastoma during pregnancy: Case report

Spinal Cord Glioblastoma Multiforme (SCGBM) is a very rare, debilitating and often fatal tumor. Cases of intracranial GBM during pregnancy have been reported, and as other tumor occurring in this setting, it harbors a great dilemma to attending physicians and families. We report the first case of a SCGBM diagnosed during pregnancy and discuss its management and treatment.

PATH-08. EVALUATION OF MISMATCH REPAIR GENE EXPRESSION BY IMMUNOHISTOCHEMISTRY MAY DETECT EARLY PHASE OF MMR DEFICIENCY IN RECURRENT GLIOMAS

Abstract BACKGROUND Mismatch Repair (MMR) Deficiency is common in recurrent gliomas from IDH-mutant and IDH-wild-type tumors. Emergence of MMR deficit is strongly associated with the chemotherapy using TMZ, and it is considered to be a key mechanism of acquiring resistance to TMZ. Several studies showed MMR protein loss detected by immunohistochemistry was well concordant with molecular genetics sequencing data, and sometimes even more reliable in detecting MMR functional deficit. Here we evaluated Mismatch Repair protein expression by immunohistrochemistry for the pairs of primary and recurrent gliomas. METHODS We investigated the expression of 4 MMR proteins (MLH1/MSH2/MSH6/PMS2) in 37 cases of paired primary-recurrent gliomas (7 grade II &amp; III astrocytomas, 16 grade II &amp; III oligodendrogliomas, and 14 glioblastomas). Clinical information including history of chemo-radiotherapy after primary surgery were collected retrospectively. RESULTS Except for one case of GBM, all primary tumors retained 4 MMR proteins. 10 of 37 recurrent tumors lost one or more MMR protein expression (4 GBM 3 astrocytoma, 3 oligodendroglioma), including one case in which primary tumor already had MSH2/MSH6 loss. 2 recurrent GBM lost MMR protein expression homogenously, while in other 8 tumors, the patterns of MMR protein loss were heterogenous. 8 of 10 tumors that lost MMR protein expression were recurrence after TMZ treatment. CONCLUSION Although the interpretation of the heterogenous loss of MMR protein expression is somewhat difficult, the immunohistochemical evaluation of MMR proteins appears to be feasible, and may be able to detect early phase of MMR deficiency and the rise of hypermutator phenotype.

TAMI-40. TUMOR MICROBIOME AND GLIOBLASTOMA (GBM)

Abstract Bacteria have been found to play major roles in many physiologic/disease processes including cancer. The presence of bacteria within brain tumors has never been explored. The aim of this study was to examine the microbiome of Glioblastoma. A cohort of 40 glioblastoma samples (FFPE), from two medical centers served for DNA extraction using a specialized extraction protocol that includes a bead beating step to ensure complete bacterial DNA recovery. A set of negative controls was introduced at different steps of the assay to identify and monitor contaminating bacterial DNA. We measured the levels of bacterial DNA in the samples using a RT-qPCR assay, amplifying the bacterial 16S rRNA gene and detected bacterial DNA in over 40% of the samples. To characterize the bacterial taxa that are present in GBM tumors, we applied 16S DNA sequencing on the samples. After implementing a stringent set of filters on the sequencing data, eliminating contaminating signal, we detected a total of 22 bacterial taxa in GBM tumors. To visualize bacteria in GBM tissues and learn about their localization within the tissue we used immunohistochemistry staining with anti-lipopolysaccharide (LPS) and anti-lipoteichoic acid (LTA) antibodies detecting gram negative and gram positive bacteria (correspondingly). Bacteria were also visualized by staining bacterial RNA using a 16S rRNA in situ hybridization assay. Staining of a human GBM tissue microarray (TMA) containing 32 cases of GBM showed that the majority of cases stained positive for LPS and ~40% were positive for 16S rRNA staining. Bacterial LPS and 16S rRNA were localized mainly inside the tumor cells. Our study demonstrates, for the first time, that bacteria or bacterial components are present in human Glioblastoma tumors. We are currently expanding our study cohort in order to better define the bacteria found within glioblastoma samples and assess their possible effects

TMOD-11. APPLICATION OF A NOVEL TRANSGENIC AND SYNGENEIC MOUSE MODELS OF HUMAN CLASSICAL GLIOBLASTOMA FOR DRUG SCREENING AND PRE-CLINICAL TRIALS

Abstract Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults with 1 year median survival. The classical subtype of GBM occurs in about 10% of GBM cases and has the worst clinical outcome. The most frequent genetic alteration associated with classical GBM is amplification in the epidermal growth factor receptor (EGFR) and homozygous deletion in CDKN2A (Cyclin Dependent Kinase Inhibitor 2A). We generated a transgenic mouse harboring EGFRvIII, a constitutively active mutant of EGFR, knock in and homozygous deletion in p19 ARF, an alternative spliced form of the CDKN2a gene, restricted to glial cells. Transgenic mice develop tumors as early as 1 month and 73% of mice die by 6 months of age. MRIs reveal that mice develop infiltrative tumor cells throughout the brain and craniospinal junction with some infiltration within the leptomeninges that can result in hydrocephalus. Histology shows that 72% of tumors are high grade gliomas and some tumors demonstrate classical histological features of human GBM such as pseudopallisading necrosis, hyper vascularity and cellular atypia. We generated primary cell lines from transgenic mice that retained EGFRVIII expression and P19 ARF deletion and formed allografts in syngeneic mouse brains. Epigenetic modifications are considered a key mechanism in GBM development. We have therefore evaluated the efficacy of a panel of 42 compounds that target various epigenetic proteins including the readers, writers, and editors of several histone marks available from the Structural Genomics Consortium in primary cell lines derived from our mouse model. The primary cell lines were exposed to epigenetic drugs showing different responses to the same epigenetic modifiers. Key candidate drugs will further be tested pre-clinically in our syngeneic mouse model. In summary, we developed a novel transgenic and syngeneic mouse model for human classical GBM that can be used for drug screening and pre-clinical trials.

Abstract IA10: Uncovering the ecology of glioblastoma with spatial digital profiling and single cell genotyping

Abstract Glioblastoma (GBM) remains one of the biggest challenges in oncology, with median survival of ~15 months. Extreme heterogeneity of the GBM cells within each tumor results in resistance to most of the available therapies. Understanding the relationships between the heterogenous populations of GBM cells and the diverse niches they rely on is key to developing more effective treatment for this aggressive brain tumor. Analysis of the spatial localization of cells with different properties can shed light on their microenvironmental preferences, interactions and potential vulnerabilities. Therefore, we set out to profile 20 GBM cases using methods allowing for single-cell assessment of select genomic features and protein marker expression in intact tissue samples from each tumor. We constructed a tissue microarray (TMA), containing four distant areas from each tumor block to represent the intratumor heterogeneity within each tumor fragment. Based on multicolor immunofluorescent staining with markers of glial cells, immune cells and vasculature, we selected 96 regions of interest, which were then subject to digital spatial profiling for 90 protein markers associated with glioblastoma biology, neurobiology and immunology. Pearson’s correlation and Shannon index calculation based on our profiling results showed that majority of the regions cluster by their tumor of origin, however several highly diverse tumors were also identified. To link the diversity of the protein expression found with the digital spatial profiling with underlying genomic alterations, using the same TMA slide, we performed multiplexed fluorescence in situ hybridization for genes most frequently amplified in GBM. Automated quantification allowed us to collect information from 38,000 individual cells. Based on the presence or absence of the amplifications each cell was assigned a genotype. Since mutation in hTERT promoter has been reported as one of the drivers of GBM, we used STAR-FISH, an in situ single-cell mutation detection assay, to record the frequency and spatial localization of cells harboring this mutation on our TMA. Our datasets provide a unique opportunity to link single-cell genetic diversity to the phenotypic heterogeneity of the tumor cells and their microenvironment. Spatial localization of cells with distinct genotypes allows us to further dissect the interactions between the distinct subclones of GBM cells and uncover their novel dependencies. Citation Format: Kacper A. Walentynowicz, Shreya Yadav, Cristina Vincentelli, Michalina Janiszewska. Uncovering the ecology of glioblastoma with spatial digital profiling and single cell genotyping [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA10.

Effect of surgery and chemotherapy on long-term survival in infants with congenital glioblastoma: an integrated survival analysis.

Glioblastoma (GBM) during infancy is rare, and the clinical outcomes of congenital GBM are not well understood. Correspondingly, the aim of this study was to present a long-term survivor case from the authors' institution, and establish an integrated cohort of cases across the published literature to better understand the clinical course of this disease in this setting. The authors report the outcomes of an institutional case of congenital GBM diagnosed within the first 3 months of life, and performed a comprehensive literature search for published cases from 2000 onward for an integrated survival analysis. All cases were integrated into 1 cohort, and Kaplan-Meier estimations, Fisher's exact test, and logistic regression were used to interrogate the data. The integrated cohort of 40 congenital GBM cases consisted of 23 (58%) females and 17 (42%) males born at a median gestational age of 38 weeks (range 22-40 weeks). Estimates of overall survival (OS) at 1 month was 67%, at 1 year it was 59%, and at 10 years it was 45%, with statistically superior outcomes for subgroups in which patients survived to be treated by resection and chemotherapy. In the overall cohort, multivariable analysis confirmed resection (p < 0.01) and chemotherapy (p < 0.01) as independent predictors of superior OS. Gestational age > 38 weeks (p < 0.01), Apgar scores ≥ 7 at 5 minutes (p < 0.01), absence of prenatal hydrocephalus (p < 0.01), and vaginal delivery (p < 0.01) were associated with greater odds of surgical diagnosis versus autopsy diagnosis. Congenital GBM can deviate from the expected poor prognosis of adult GBM in terms of OS. Both resection and chemotherapy confer statistically superior prognostic advantages in those patients who survive within the immediate postnatal period, and should be first-line considerations in the initial management of this rare disease.

Abstract 807: Predictive biomarkers for Bevacizumab activity in recurrent glioblastoma: A single institution study

Abstract Glioblastoma Multiforme (GBM) is an aggressive type of astrocytoma and the most common malignant brain tumor in adults. Bevacizumab is a recombinant monoclonal antibody that targets VEGF-A. VEGF is regulated by the hypoxia-inducible-factor (HIF-1α) pathway, with the tumor suppressor gene TP53 as one of the key regulating factors. Our study examines the relevance of various genetic mutations of the HIF-1α pathway and their predictive significance in patients diagnosed with recurrent GBM and treated with bevacizumab. A total of 41 patients diagnosed with recurrent GBM and treated with Bevacizumab at Virginia Commonwealth University from January 1990 through November 2018 were included in our study. Our study cohort consists of 56.1% males and 43.9% females, with an average age of 59.9 (SD 12.1), and 68.3% were Caucasian. The majority (90%) of the cohort were classified as de novo cases of GBM. The overall survival (OS) was 172.5 days (95% CI: 122.0, 218.5), with a minimum of 11 days and maximum of 904 days measured from the date of the first bevacizumab treatment to the date of death. The majority of patients (70.7%) responded to treatment, with 61% exhibiting a partial response and 9.8% exhibiting a complete response. The median first response to treatment (FRT) was 34 days (95% CI: 21, 40), with a minimum of 2 days and maximum of 76. All patients died during the follow-up period. Survival analyses for OS, along with FRT, were both performed with Kaplan-Meier curves generated individually. We have examined the clinical characteristics of our study cohort and are currently examining the genetic characteristics of the tumors in our cohort. At the time of the conference we will have updated genomic and radiographic data in addition to the current detailed clinical data. Citation Format: Marcin Dragan, Matthew Carli, Mark Malkin, Victor Yazbeck. Predictive biomarkers for Bevacizumab activity in recurrent glioblastoma: A single institution study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 807.