GBM Samples Research Articles

Human Mesenchymal glioblastomas are characterized by an increased immune cell presence compared to Proneural and Classical tumors

ABSTRACTGlioblastoma (GBM) is the most aggressive malignant primary brain tumor in adults, with a median survival of 14.6 months. Recent efforts have focused on identifying clinically relevant subgroups to improve our understanding of pathogenetic mechanisms and patient stratification. Concurrently, the role of immune cells in the tumor microenvironment has received increasing attention, especially T cells and tumor-associated macrophages (TAM). The latter are a mixed population of activated brain-resident microglia and infiltrating monocytes/monocyte-derived macrophages, both of which express ionized calcium-binding adapter molecule 1 (IBA1). This study investigated differences in immune cell subpopulations among distinct transcriptional subtypes of GBM. Human GBM samples were molecularly characterized and assigned to Proneural, Mesenchymal or Classical subtypes as defined by NanoString nCounter Technology. Subsequently, we performed and analyzed automated immunohistochemical stainings for TAM as well as specific T cell populations. The Mesenchymal subtype of GBM showed the highest presence of TAM, CD8+, CD3+ and FOXP3+ T cells, as compared to Proneural and Classical subtypes. High expression levels of the TAM-related gene AIF1, which encodes the TAM-specific protein IBA1, correlated with a worse prognosis in Proneural GBM, but conferred a survival benefit in Mesenchymal tumors. We used our data to construct a mathematical model that could reliably identify Mesenchymal GBM with high sensitivity using a combination of the aforementioned cell-specific IHC markers. In conclusion, we demonstrated that molecularly distinct GBM subtypes are characterized by profound differences in the composition of their immune microenvironment, which could potentially help to identify tumors amenable to immunotherapy.

Pro-tumoral Effects of Intra-tumoral Neutrophils in the Glioblastoma Microenvironment

Abstract INTRODUCTION The glioblastoma microenvironment contains immune cells, particularly well-defined macrophage populations; however, intratumoral neutrophils and their effects on GBM biology are under-characterized. While tumor-associated neutrophils (TANs) were initially thought as passive bystanders due to their short-lived nature, current investigation of TANs in other cancer types revealed distinct pro-tumoral roles. Therefore, we sought to characterize TANs in the glioblastoma microenvironment and define their oncologic effects. METHODS Following informed consent, patient-derived GBM samples were collected for flow cytometry of TANs, which were then used to produce conditioned media (CM) for in Vitro studies on tumor cell proliferation and ELISA quantification of TAN-secreted factors. Single-cell RNA sequencing was performed on TANs to identify pro-tumoral factors. RESULTS Flow cytometric analysis (CD11b+/CD15+ /CD66b+) indicated higher percentages of TAN-infiltration to glioblastoma compared to low-grade gliomas (1.76% [n = 13] vs 0.33% [n = 6], P = .03). Using the Transwell migration assay with glioblastoma tumor CM, we found recruitment of circulating neutrophils to tumor sites is mediated by leukotriene-B4 and that this chemoattraction can be blocked with an LtB4 receptor antagonist, LY293111. We then performed single-cell RNA sequencing of isolated TANs and identified upregulation of osteopontin. Osteopontin is linked to GBM stem cell-like phenotype maintenance, thus, we decided to investigate osteopontin as a possible driver of pro-tumoral signaling. Osteopontin concentration was significantly higher in TAN CM than in patient-matched peripheral blood neutrophil CM (3.2 ng/mL [n = 3] vs 0.02 ng/mL [n = 3], P < .05). In Vitro, TAN CM led to significantly increased GBM cell proliferation and increased stem marker expression (Nanog, Oct4, Sox2) when incubated with neurospheres from an established GBM line. Pro-tumoral effects were lost in presence of osteopontin-neutralizing antibodies. CONCLUSION This study elucidates a possible mechanism of neutrophil-mediated pro-tumoral signaling. We found that neutrophils are recruited to tumor sites and play a biologically relevant role in GBM cellular proliferation and maintenance of a stem cell phenotype via osteopontin secretion.

Abstract 3954: Evaluating the compatibility of tumor treating electric fields with key anti-tumoral immune functions

Abstract Background: Combining Tumor Treating electrical Fields (TTFields) with immunotherapy is a rational approach due to their different mechanisms of action (MOA) and to TTFields’ ability to induce immunogenic cell death (ICD). Conversely, TTFields may interfere with immune functions critical for effective T cell responses. Methods: T cells from healthy donors’ peripheral blood or from viably dissociated glioblastoma samples were cultured under normal or TTFields conditions, with or without superantigen-stimulation. Eight-color flow cytometry was used to assess T cell responses by monitoring select pivotal antitumoral functions: proliferation (CFSE), IFNγ secretion, cytotoxic degranulation (CD107a), activation/exhaustion (PD1) and viability. Direct cytotoxicity was evaluated using chimeric antigen receptor (CAR) T cells. Results: The viability of stimulated T cells that attempted to proliferate decreased under TTFields, in line with TTFields’ MOA. Small or no reductions in viability were found in activated T cells that did not attempt to proliferate and in unstimulated T cells. The functionality of stimulated peripheral-blood T cells and tumor-infiltrating T cells (TILs) under TTFields was unhindered: T cells exhibited comparable PD1 upregulation, IFNγ secretion and CD107a expression as controls. T cell polyfunctionality, associated with effective antitumoral responses, was retained under TTFields conditions. PD1-expressing TILs, a subset containing most of the tumor antigen-specific TILs, exhibited unaltered viability and functionality under TTFields. CAR T-cells, which utilize the same killing machinery as unmodified T cells, exhibited unaltered cytotoxic capability under TTFields. Immunohistochemical evaluation of GBM samples before TTFields treatment and after recurrence showed that some patients had accommodated large increases in their CD8 and CD4 counts. RNA-Seq performed on GBM samples from 6 standardly-treated and 6 TTFields-treated patients before treatment and after recurrence. The data shows differential increases in TTFields-treated patients to controls, in the expression of immune genes associated with favorable prognosis (e.g. t-bet, NKG2D, ICOS-L, CD70) and concurrent decreases in genes associated with poor prognosis (e.g. IL4, TSLP, various complement genes). Conclusions: The preclinical data showed that all antitumoral T cell functions examined, but proliferation, were unhindered by TTFields. The clinical data showed that TTFields may shift treated tumors to a state more conducive of anti-tumoral immune responses. Our findings support the further preclinical and clinical investigation into combining TTFields with immunotherapy. Citation Format: Gil Diamant, Hadar Simchony, Tamar Shiloach, Anat Globerson-levin, Lital Gasri Plotnitsky, Zelig Eshhar, Niv Pencovich, Rachel Grossman, Zvi Ram, Ilan Volovitz. Evaluating the compatibility of tumor treating electric fields with key anti-tumoral immune functions [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 3954.

Genetic, Epigenetic, and Immunologic Profiling of MMR-Deficient Relapsed Glioblastoma.

In-depth characterization of recurrent glioblastoma (rGBM) might contribute to a better understanding of the mechanisms behind tumor progression and enable rGBM treatment with targeted drugs.Experimental Design: In this study, GBM samples were collected at diagnosis and recurrence from adult patients treated with Stupp protocol. Expression of mismatch repair (MMR) proteins was evaluated by IHC, followed by whole exome sequencing (WES) of tumor samples showing loss of MSH6 reactivity. Established genetic, epigenetic, and immunologic markers were assessed by standard methods and correlated with loss of MMR proteins and patient survival. Expression of MMR proteins was partially or completely lost in 25.9% rGBM samples. Specifically, 12 samples showed partial or total MSH6 expression reduction. Conversely, 96.4% of GBM samples at diagnosis expressed MMR markers. WES disclosed lack of variants in MMR genes in primary samples, whereas two MSH6-negative rGBM samples shared a c.3438+1G>A* splicing MSH6 variant with a potential loss of function effect. MSH6-negative rGBM specimens had high tumor mutational burden (TMB), but no microsatellite instability. In contrast, GBM samples with partial loss of MMR proteins disclosed low TMB. MMR-deficient rGBM showed significant telomere shortening and MGMT methylation and are characterized by highly heterogeneous MHC class I expression. Multilevel profiling of MMR-deficient rGBM uncovered hypermutated genotype uncoupled from enriched expression of immune-related markers. Assessment of MHC class I expression and TMB should be included in protocols aiming to identify rGBM patients potentially eligible for treatment with drugs targeting immune-checkpoint inhibitors.

Abstract A060: Targeting glioma-macrophage interplay via LOX in PTEN-deficient glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most lethal form of brain cancer in adults. The median survival of GBM patients is only about one year after initial diagnosis. Genomic profiling has stratified GBM into various subgroups, which are driven by specific genetic alternations of core signaling pathways, including RTK/RAS/PI3K/PTEN, P53/ARF/MDM2 and RB/CDKN2A pathways. However, targeted therapies, such as therapy against EGFR, have failed in the clinic, and no effective therapeutic drugs are available to target tumor suppressors. A key reason for therapeutic failure is inter- and intra-tumoral cancer cell genetic instability and heterogeneity, resulting in aberrant activation of multiple signaling pathways within and across tumors. Stromal/immune cells in the tumor microenvironment (TME) are genetically stable, which not only play a pivotal role in GBM progression by affecting multiple cancer hallmarks, but can also be educated by cancer cells. However, whether and how the behavior and function of specific stromal/immune cells in the TME are regulated by cancer cell with specific genetic alterations in GBM remain relatively undefined. Utilizing a large scale of bioinformatic analysis in TCGA GBM patients, we revealed that genetic alteration (deletion/mutation) of PTEN in GBM patients specifically triggers immune response by promoting macrophage recruitment, without affecting macroglia and other immune cells. Using unbiased transcriptome profiling following functional validation, we identified that lysyl oxidase (LOX) is preferentially secreted by PTEN-deficient cancer cells. In vitro transwell migration assay and in vivo Matrigel Plug assay demonstrated that LOX is a potent macrophage chemoattractant. Transcriptome profiling following Gene Set Enrichment Analysis (GSEA) and functional validation demonstrated that activation of SRC and AKT signaling pathways drives LOX upregulation in PTEN-deficient cancer cells. Genetic and pharmacologic inhibition of LOX in PTEN-deficient cancer cells does not affect tumor cell proliferation in vitro, but markedly inhibits macrophage density and tumor growth in vivo. Using the bioinformatics analysis in clinical GBM samples, we demonstrated that LOX is enriched in GBM patients with higher macrophage density, and that these patients show lower survival. Together, our findings highlight the significance of PTEN-LOX axis in macrophage infiltration in GBM, and demonstrate a possibility of improving GBM treatment by targeting this axis-mediated macrophage recruitment. Citation Format: Peiwen Chen, Alan Wang, Ronald DePinho. Targeting glioma-macrophage interplay via LOX in PTEN-deficient glioblastoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A060.

PO-105 CDH3/P-cadherin as a novel biomarker in glioblastoma: functional and prognostic insights

IntroductionGlioblastoma (GBM) is the most common and malignant primary brain tumour in adults. Patients diagnosed with GBM exhibit a poor prognosis with a median survival of approximately 15 months. P-cadherin (encoded by CDH3) is a classical cadherin that contributes for cell-cell adhesion by homophilic interactions, and is being studied and considered an attractive therapeutic target in several cancer types. Critically, its relevance in GBM is completely unknown. In this study, we investigate the functional roles and prognostic value of P-cadherin in this highly malignant form of gliomas.Material and methodsCDH3 mRNA expression was evaluated in glioma patients available in TCGA database, and at the protein level by immunohistochemistry in our dataset of glioma patients from Hospital Braga. CDH3/P-cadherin functional roles were assessed in vitro using silencing (in two primary GBM cell lines) and overexpression (a commercially available GBM cell line) models. In vitro functional assays included evaluation of cell viability (trypan blue and MTS), migration (wound healing), invasion (Matrigel chamber), and stemness features (neurosphere formation). In vivo, subcutaneous and orthotopic intracranial GBM xenografts were established in Nude and NOD-scid gamma (NSG) mice, respectively. CDH3 expression in GBM samples was evaluated by RT-qPCR and its relevance in patients’ prognosis was evaluated using the Log-rank test.Results and discussionsWe found CDH3 expression is higher in high-grade gliomas. CDH3-silencing and overexpression GBM models show CDH3/P-Cadherin regulates GBM cell viability, invasion, migration and stemness capacity. In vivo CDH3-overexpressing cells generate larger subcutaneous tumours, and when orthotopically injected cause shorter overall survival of mice. Concordantly, higher CDH3 expression is predictive of shorter overall and recurrence-free survivals in various cohorts of GBM patients.ConclusionTogether, our results show for the first time CDH3/P-cadherin is associated with GBM aggressiveness and patient prognosis, suggesting it may be an attractive therapeutic target in this deadly brain tumour.

PO-338 Recurrent glioblastoma: a complex scenario dominated by loss of MMR proteins

IntroductionGlioblastoma (GBM) is the most common primary brain tumour in adults and the Stupp protocol represents the standard of care. However, the tumour invariably relapses suggesting marked intra-tumour genetic heterogeneity enabling rapid adaptation to therapy. In-depth characterisation of recurrent GBM (rGBM) might contribute to better understand mechanisms behind tumour progression and enable rGBM treatment with targeted drugs.Material and methodsMatched GBM samples have been collected at diagnosis and recurrence from adult patients (n=57) treated with the Stupp protocol. Expression of mismatch repair (MMR) proteins (MLH1, PMS2, MSH2, MSH6) was evaluated by IHC, followed by exome sequencing of 3 pairs showing loss of MSH6 reactivity as well as of 3 MSH6 positive pairs. In addition, established genetic and epigenetic markers of GBM were investigated along with their correlation with loss of MMR proteins and patients’ survival.Results and discussionsAccording to IHC results, 13 out of 52 rGBM samples (25%) lacked expression of MMR proteins. In particular, 11 among the 13 samples (85%) showed partial or total reduction of MSH6 expression. Conversely, almost all GBM samples at diagnosis (96.4%) stained positive for the 4 MMR markers. Consistent with IHC data, exome sequencing disclosed lack of variants in MMR genes in primary samples whereas rGBM samples lacking MSH6 expression were mutated in the abovementioned genes and shared a c.3438+1G>A* splicing variant in MSH6 with a potential loss of function effect. Moreover, MSH6 negative relapsed specimens were characterised by 30 to 100-fold more variants compared to the matched primary ones and lacked microsatellite instability. Notably, MMR deficiency was associated with significant telomere shortening. Conversely, the tumour pairs expressing MMR proteins showed an almost comparable number of mutations in primary versus relapsed samples and absence of variants in MMR genes both in the initial tumours and in their recurrent counterpart.ConclusionOur study shows that IHC staining is a valuable tool to identify a subset of rGBM patients with alterations in MMR genes linked to high mutational burden and, hence, potentially eligible for drugs targeting immune checkpoint inhibitors.

Abstract 1075: CD99 expression in astrocytomas and functional analysis in glioblastoma cell line

Abstract Glioblastoma (GBM) is the most frequent high grade brain tumor in adults. Treatment consists of a combination of maximal surgical resection, radiotherapy and chemotherapy, but prognosis is still poor. Therefore, new therapeutic strategies with other biologically-based therapies is essential to improve the survival of patients with GBM, currently restricted to 15-17 months. This urgent need for better therapies has motivated many studies to pursuit new targets in GBM. Our laboratory demonstrated higher expression of CD99 mRNA in GBM samples when compared to lower grade astrocytomas and non-neoplastic brain tissue. CD99 is a membrane molecule and acts as a key regulator in several biological processes, including cell adhesion, migration, apoptosis, differentiation of T cells and thymocytes, diapedesis of lymphocytes to inflamed vascular endothelium, maintenance of cellular morphology and regulation of intracellular membrane protein trafficking. CD99 expression has been described in many types of cancers, with distinct function and signaling pathways, and its role is not completely elucidated yet, including in GBM.We confirmed the higher CD99 expression in TCGA dataset of GBM cases when compared to grade II and III astrocytomas. Furthermore, amongst GBM cases, Proneural molecular subtype presented lower CD99 expression compared to Mensechymal, Classic and Neural subtypes. We performed functional assays knocking down CD99 with two different shRNAs in the human GBM cell line U87MG. The CD99 silencing efficiency was 80 and 97%, for shRNA_1 and shRNA_2, respectively, confirmed at transcriptional level by quantitative real time PCR and at protein level by Western blot. Significant reduction of migration and invasion of CD99 knocked-down U87MG cells was observed. Additionally, cell shrinkage with relevant reduction of cell area was detected after the silencing of this target, with decreased CD99 localization at lamellipodia by immunocytochemistry. These findings pointed out the cytoskeleton rearrangement with reduction in adhesion sites. Further expression profile analysis after CD99 knockdown by a cancer panel RNAseq confirmed its involvement in cell adhesion, regulation of actin cytoskeleton and ECM-receptor interaction in the KEGG pathways analysis. Interestingly, CD99 knockdown impacted in the reduction of cell adhesion in a distinct quantitative pattern, suggesting that the result is dose dependent. Refinement of this aspect is ongoing in further in vivo assays which will better clarify the CD99 role in tumor cell migration/invasion. Altogether, our results suggest CD99 as a potential new therapeutic target to control GBM infiltrative process, allowing a more efficient treatment outcome. Citation Format: Lais Cavalca Cardoso, Suely Kazue Marie, Roseli Silva Soares, Sueli M. Oba-Shinjo. CD99 expression in astrocytomas and functional analysis in glioblastoma cell line [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 1075.

Abstract 3615: Analysis of microRNAs in cerebrospinal fluid of brain tumor patients

Abstract Introduction: Cerebrospinal fluid (CSF) is a body fluid with many important functions that is in direct contact with the extracellular environment of the central nervous system (CSN). Therefore, CSF serves inter alia as a both communication channel allowing the distribution of various substances among CNS cells as well as a reservoir of waste products that these cells released. For these reasons, CSF is a potential source of diagnostic biomarkers of many neurological diseases including brain tumors. Recent studies have revealed that CSF contains also circulating microRNAs (miRNAs), short non-coding RNAs, which have been described as biofluid diagnostic markers in many cancers. The analysis of CSF miRNAs in patients affected by brain tumors could enable more precise diagnosis and patient stratification.Material and methods: We performed next-generation sequencing analysis of small RNAs (small RNAseq) in 89 CSF samples taken from 35 GBM, 14 low-grade glioma (LGG), 14 meningioma, and 8 brain metastasis patients as well as 18 non-tumor donors. Informed consent approved by the local Ethical Commission was obtained from each patient before the lumbar puncture. cDNA libraries were prepared using CleanTaq Small RNA Library Prep Kit (TriLink BioTechnologies) and, subsequently, purified by Agencourt AMPure XP (Beckman Coulter). The final sequencing analysis was performed by Next 500/550 High Output v2 Kit - 75 cycles using the NextSeq 500 instrument (both Illumina). For miRNA mapping and analysis, an online tool Chimira was used. Obtained data were subsequently statistically evaluated in the environment of statistical language R using the Bioconductor edgeR and DESeq2 package.Results: We observed 211 miRNAs to be expressed in more than 60 CSF samples with 1 395 normalized counts in average (min 3, max 77 284; miR-486-5p was excluded from analysis due to its artificial expression). From these miRNAs, 63 showed different levels in CSF of GBM, 16 in LGG, 24 in meningioma, and 31 in brain metastases patients in comparison with CSF of non-tumor donors. MiR-10a-5p and miR-196a-5p have significantly highest levels in CSF of GBM samples. Moreover, 6 miRNAs (miR-30c-5p, miR-30e-5p, miR-145-5p, miR-320c, miR-3960, and miR-6131) indicated significantly different levels between GBM and LGG (P<0.01).Conclusion: We have demonstrated that CSF of various brain tumors (GBM, LGG, meningioma, and brain metastasis) is characterized by specific miRNA signature. Moreover, we found miRNA signature with ability to differentiate LGG and GBM from CSF. Our results suggest, that after independent validations, CSF miRNAs could serve as promising biomarkers in brain tumors. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-34553A, 15-33158A, 15-31627A, 15-34678A, 16-31314A, 16-31765A and by grant of Czech Grant Agency nr. 16-18257S, 17-17636S. Citation Format: Jiri Sana, Alena Kopkova, Marek Vecera, Jaroslav Juracek, Tana Machackova, Parwez Ahmad, Natalia Anna Gablo, Pavel Fadrus, Marek Svoboda, Ondrej Slaby. Analysis of microRNAs in cerebrospinal fluid of brain tumor patients [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 3615.

PO-281 SIRT1 inhibition exhibits decreased pluripotency in glioma cancer stem cells

IntroductionSIRT1, a class III histone deacetylase has cancer relevance because it regulates lifespan in multiple organisms and down-regulates p53 function through deacetylation. Cancer Stem Cells are a small subset of cells that are responsible for initiation, development, and recurrence of tumours. Therefore, it is important to understand the molecular mechanism of Cancer Stem Cells for translational applications in the treatment of patients with cancer. Role of SIRT1 in enhancing tumorigenesis has been well documented in many cancers. However, its functional role in glioma cancer stem cells is largely unknown.Material and methodsIn order to comprehend the role of SIRT1 in glioma cancer stem cells, we used EX-527, a nanomolar inhibitor of SIRT1 in three glioma Grade IV cell lines –U87MG, A172 and LN229. This was followed by expression analysis by Q-PCR, Immunofluorescence and Tumour sphere assay in glioma and cancer stem cells of glioma. Cell proliferation was assessed by XTT. Expression of genes in 20 grade IV glioma patient samples was compared to normal brain sample.Results and discussionsU87MG, A172 and LN229 cells cultured in Stem Media exhibited an increase in SIRT1 expression compared to Normal media. XTT assay after EX527 treatment (10 nM to 100 µM) did not exhibit any change in cell proliferation compared to control cells in all the three glioma cell lines. SIRT1 inhibition exhibited a decreased expression of stemness markers –Sox-2 and Oct-4 and a decreased capacity to form gliomaspheres compared to the Control cells. Finally, we have correlated our in-vitro results with human GBM samples and found that SIRT1 and stemness markers correlated well in native tumour samples.ConclusionThe results indicate a role of SIRT1 as a pluripotent marker in cancer stem cells of glioma.SIRT1 deacetylates p53 rendering it inactive and thus initiating a cascade of events with oncogenic potential. Cancer stem cells are the cells responsible for drug resistance and tumour relapse. Thus knowing the mechanism of SIRT1 action in cancer stem cells would open doors for more target specific therapy. Our study provides a functional interaction of SIRT1 with the stemness markers in cancer stem cells of glioma and glioma patient samples thus aiming for target specific approaches in glioma.

Identification of a Novel Splice Variant of Neural Cell Adhesion Molecule in Glioblastoma Through Proteogenomics Analysis.

Splice variants are known to be important in the pathophysiology of tumors, including the brain cancers. We applied a proteogenomics pipeline to identify splice variants in glioblastoma (GBM, grade IV glioma), a highly malignant brain tumor, using in-house generated mass spectrometric proteomic data and public domain RNASeq dataset. Our analysis led to the identification of a novel exon that maps to the long isoform of Neural cell adhesion molecule 1 (NCAM1), expressed on the surface of glial cells and neurons, important for cell adhesion and cell signaling. The presence of the novel exon is supported with the identification of five peptides spanning it. Additional peptides were also detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel separated proteins from GBM patient tissue, underscoring the presence of the novel peptides in the intact brain protein. The novel exon was detected in the RNASeq dataset in 18 of 25 GBM samples and separately validated in additional 10 GBM tumor tissues using quantitative real-time-polymerase chain reaction (qRT-PCR). Both transcriptomic and proteomic data indicate downregulation of NCAM1, including the novel variant, in GBM. Domain analysis of the novel NCAM1 sequence indicates that the insertion of the novel exon contributes extra low-complexity region in the protein that may be important for protein-protein interactions and hence for cell signaling associated with tumor development. Taken together, the novel NCAM1 variant reported in this study exemplifies the importance of future multiomics research and systems biology applications in GBM.

MiR-134, epigenetically silenced in gliomas, could mitigate the malignant phenotype by targeting KRAS.

Gliomas are characterized by a malignant phenotype with proliferation, cell cycle arrest and invasion. To explore the biological consequences of epigenetically regulated miRNAs, we performed a microarray-based screening (whose expression was affected by 5-AZA treatment) followed by bisulfite sequencing validation. We found that miR-134 as an epigenetically regulated suppressor gene with prognostic value in gliomas. MicroRNA-134 was downregulated in high-grade gliomas, especially in GBM samples. Functional studies in vitro and in vivo in mouse models showed that overexpression of miR-134 was sufficient to reduce cell cycle arrest, cell proliferation and invasion. Target analysis and functional assays correlated the malignant phenotype with miR-134 target gene KRAS, an established upstream regulator of ERK and AKT pathways. Overall, our results highlighted a role for miR-134 in explaining the malignant phenotype of gliomas and suggested its relevance as a target to develop for early diagnostics and therapy.

Abstract B58: The effect of differential miRNA expression in glioblastoma multiforme molecular subtypes

Abstract Glioblastoma multiforme (GBM) is characterized by a distinct profile of specific miRNAs that regulate pathways of therapeutic importance, such as cell proliferation, apoptosis, cell cycle regulation, migration, invasion, and angiogenesis. Recently, the molecular classification of gliomas was updated, and three methylation clusters with distinct prognostic values for GBM were characterized. The aim of this research is to evaluate the expression, the functional roles, and the target genes of the miRNAs that have potential value for diagnosis, prognosis, and treatment in patients within the newest molecular subgroup classification of GBM. First, we performed an in silico analysis 606 GBM samples from the Genome Data Commons (GDC) cohort. Based on the methylation subgroups of gliomas recently published (Cecarelli et al., 2016), we performed a comparison of the miRNA expression between LGm4, LGm5, and LGm6 subgroups through Nexus Expression 3.0 (Biodiscovery). The comparison between LGm4 x LGm6 indicated that eight miRNAs were upregulated, while 17 were downregulated. The LGm5 x LGm4 comparison showed two downregulated miRNAs, and the LGm5 x LGm6 comparison showed one upregulated miRNA. By comparing the differentially expressed miRNAs found in our analysis with two other in silico studies, we found an overlap of 54% of the differentially expressed miRNAs. We will now evaluate the expression levels of the upregulated miRNAs obtained through the in silico analysis of samples of the Hospital of Clinics - Faculty of Medicine of Ribeirão Preto (HCFMRP/USP) through a customized nCounter NanoString panel of 24 miRNA. We will also evaluate the plasma exosomal miRNAs from the same patients using the same 24 miRNA nCounter NanoString panel. By identifying the most significant miRNAs regulating cell proliferation and invasive behavior in this tumor, we will be able to use expression levels of the most informative miRNA to both guide the molecular diagnosis of GBM and facilitate future targeted therapeutics for this aggressive disease. Citation Format: Livia Ferreira Silva Verzola, Thiago Vidotto, Jeremy Andrew Squire. The effect of differential miRNA expression in glioblastoma multiforme molecular subtypes [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B58.

EZH2 suppression in glioblastoma shifts microglia toward M1 phenotype in tumor microenvironment

BackgroundGlioblastoma multiforme (GBM) induces tumor immunosuppression through interacting with tumor-infiltrating microglia or macrophages (TAMs) with an unclear pathogenesis. Enhancer of zeste homolog 2 (EZH2) is abundant in GBM samples and cell lines and is involved in GBM proliferation, cell cycle, and invasion, whereas its association with innate immune response is not yet reported. Herein, the aim of this study was to investigate the role of EZH2 in GBM immune.MethodsCo-culturing models of human/murine GBM cells with PBMC-derived macrophages/primary microglia were employed. EZH2 mRNAs and function were suppressed by siEZH2 and DZNep. Real-time PCR and flow cytometry were used to determine levels of microglia/macrophages markers. The fluorescence-labeled latex beads and flow cytometry were utilized to evaluate phagocytic abilities of microglia. CCK8 assay was performed to assess microglia proliferation.ResultsEZH2 inhibition led to significant reduction of TGFβ1-3 and IL10 and elevation of IL1β and IL6 in human and murine GBM cells. More importantly, EZH2 suppression in GBM cells resulted in significant increase of M1 markers (TNFα and iNOS) and decrease of a pool of M2 markers in murine microglia. The proportion of CD206+ cells was decreased in PBMC-derived macrophages as co-incubated with EZH2-inhibited GBM cells. Functional researches showed that phagocytic capacities of microglia were significantly ameliorated after EZH2 inhibition in co-culturing GBM cells and microglia proliferation was declined after addition of TGFβ2 antibodies to co-incubated GBM cells with EZH2 inhibition. Besides, we found that EZH2 suppression in GBM cells enhanced co-culturing microglia engulfment through activation of iNOS.ConclusionsOur data demonstrates that EZH2 participates in GBM-induced immune deficient and EZH2 suppression in GBM can remodel microglia immune functions, which is beneficial for understanding GBM pathogenesis and suggests potential targets for therapeutic approaches.

Detailed longitudinal sampling of glioma stem cells in situ reveals Chr7 gain and Chr10 loss as repeated events in primary tumor formation and recurrence.

Intratumoral heterogeneity at the genetic, epigenetic, transcriptomic, and morphologic levels is a commonly observed phenomenon in many aggressive cancer types. Clonal evolution during tumor formation and in response to therapeutic intervention can be predicted utilizing reverse engineering approaches on detailed genomic snapshots of heterogeneous patient tumor samples. In this study, we developed an extensive dataset for a GBM case via the generation of polyclonal and monoclonal glioma stem cell lines from initial diagnosis, and from multiple sections of distant tumor locations of the deceased patient's brain following tumor recurrence. Our analyses revealed the tissue-wide expansion of a new clone in the recurrent tumor and chromosome 7 gain and chromosome 10 loss as repeated genomic events in primary and recurrent disease. Moreover, chromosome 7 gain and chromosome 10 loss produced similar alterations in mRNA expression profiles in primary and recurrent tumors despite possessing other highly heterogeneous and divergent genomic alterations between the tumors. We identified ETV1 and CDK6 as putative candidate genes, and NFKB (complex), IL1B, IL6, Akt and VEGF as potential signaling regulators, as potentially central downstream effectors of chr7 gain and chr10 loss. Finally, the differences caused by the transcriptomic shift following gain of chromosome 7 and loss of chromosome 10 were consistent with those generally seen in GBM samples compared to normal brain in large-scale patient-tumor data sets.

Abstract 3348: UniD: unified and integrated diagnostic pipeline for malignant gliomas based on DNA methylation data

Abstract Prognostic and predictive molecular diagnostics for patients with gliomas typically rely on multiple assays, requiring large amounts of tissue and high cost. DNA methylation has been utilized to identify prognostic subsets and high-throughput platforms exist that are suitable for archival tissue. Therefore, we developed a unified and integrated diagnostic pipeline that can assess multiple prognostic and predictive biomarkers using only the Illumina Infinium Methylation array. This pipeline includes two parts: data processing and diagnostic biomarkers. Data processing starts from the raw data and followed with quantitative sample, probe, and batch quality control. The diagnostic biomarkers include a glioma methylation assay that predicts radiation response (GaMA); tumor classification enriched for TCGA expression subclasses; copy number alterations including phosphatase and tensin homolog (PTEN) loss, epidermal growth factor receptor (EGFR) amplification, and chromosomes 1p/19q co-deletion; CpG island methylation phenotype (G-CIMP); isocitrate dehydrogenase (IDH) mutation, and O6-methylguanine-DNA methyltransferase (MGMT) promoter hypermethylation. WHO grade II-IV gliomas were analyzed in both publically available and institutional datasets. A signature was identified to effectively distinguished radiation resistant from radiation sensitive glioma stem-like cells (GSCs). Signature has been applied to 272 TCGA GBM samples from patients who received standard radiotherapy (RT). The survival analysis showed that the subgroup with RT-sensitive and RT-resistant have significant difference in survival time (log-rank test p-value = 0.0016). Gene expression subclasses prediction biomarker was build by using the revised TCGA gene expression subclasses as gold standard. The prediction accuracy in test data set was 83.5% in the homogeneous subgroup, 71.0% in the semi-heterogeneous subgroup, and 62.1% in the heterogeneous subgroup. The prediction accuracy decreased as tumor heterogeneity increased. Certain copy number alteration events were predicted by developing specific signatures. Revised methylation signatures were developed for IDH mutation and G-CIMP status respectively, which can identified 99% of those samples. 230 GBM samples with 450k data available were tested with MGMT methylation-specific real-time PCR for MGMT methylation status. The methylation-based MGMT prediction accuracy reached about 90%. In summary, we have developed a single, FFPE-based pipeline for unified and integrated determination of multiple biomarkers of malignant glioma. Citation Format: Jie Yang, Qianghu Wang, Lihong Long, Ravesanker Ezhilarasan, Erik Sulman. UniD: unified and integrated diagnostic pipeline for malignant gliomas based on DNA methylation data [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 3348. doi:10.1158/1538-7445.AM2017-3348

Abstract 899: CD99 plays an important role in glioblastoma cell migration

Abstract Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults and the standard treatment consists of surgical resection of the tumor followed by radiation and chemotherapy with temozolomide. A combination of standard therapy with other biologically-based therapies is necessary to improve the survival of patients with GBM, which currently stands only to 15-17 months. In this sense many studies have been developed in pursuit of expressed membrane proteins in GBM, which are potential targets for immunotherapy. Our laboratory demonstrated increased CD99 mRNA and protein expression in GBM samples. CD99 has a key role in several biological processes, including cell adhesion, migration, apoptosis, differentiation of T cells and thymocytes, diapedesis of lymphocytes to inflamed vascular endothelium, maintenance of cellular morphology and regulation of intracellular membrane protein trafficking. In this study, the transcriptome of two GBM cell lines (U87MG and A172) transfected with siRNA for CD99 were analyzed in relation to the non-target control (NTC). A total of 728 genes (hypoexpressed with fold change ≤-1.5 relative to NTC or hyperexpressed with fold change ≥1.5 relative to NTC) were observed in both cell lines. An enrichment analysis by MetaCoreTM revealed the following processes as the most significant: (1) Cytoskeleton_Regulation of cytoskeleton rearrangement, (2) Cell adhesion_Cell-matrix interactions (3) Development_Blood vessel morphogenesis. Further, both cell lines were silenced for CD99 expression by shRNA (two clones) to perform functional assays, as wound healing (migration assay), adhesion, invasion and apoptosis. The CD99 knockdown reduced migration in both cell lines, with the highest decrease of the migration observed in the highest CD99 knockdown. Adhesion assay was performed using fibronectin as an extracellular matrix substrate. U87MG cells showed greater adhesion to fibronectin than the control (scrambled shRNA). On the other hand, A172 cells knocked down for CD99 presented lower adherence to fibronectin than the control. Apoptosis analysis with shCD99 U87MG cells showed a tendency of cells entering into late apoptosis when treated with temozolamide for 48 hours, but the same was not observed in shCD99 A172 cells. Additionally, CD99 (RPE) and phaloidin (Alexa Fluor 488) colocalized at lamellipodia, suggesting that CD99 is related to cytoskeleton rearrangement in both GBM cell lines. The different phenotypic behavior of migration and apoptosis observed in these two GBM cell lines with different somatic mutation background may be due to distinct signaling activation. Migration/invasion is the major characteristics of GBM which limits the surgical tumor resection, and consequently leads to tumor recurrence. Therefore, further analysis of CD99 activating pathways in the context of cell migration is worthwhile to unveil new therapeutic strategies to halt GBM progression. Citation Format: Lais C. Cardoso, Antonio M. Lerario, Suely K. Marie, Roseli S. Soares, Sueli M. Oba-Shinjo. CD99 plays an important role in glioblastoma cell migration [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 899. doi:10.1158/1538-7445.AM2017-899

Abstract 2454: Genetic landscape of glioma reveals defective neuroactive ligand receptor interaction pathway as a poor prognosticator in glioblastoma patients

Abstract Glioblastoma (GBM; grade IV), is highly proliferative, infiltrative and treatment refractory. Hence, understanding the complete genetic alteration profile of GBM would help us in identifying molecules or pathways that have strong implications in GBM pathogenesis, thus opening up avenues for targeted therapy. Recent large scale studies suggest that three pathways - receptor-tyrosine kinase, TP53 and RB, are significantly altered in GBM. However, even with the tremendous increase in our understanding of the tumor, advancement in therapeutics is minimal and the median survival still remains at 15 months. Hence, we need to elucidate novel altered molecules and pathways in GBM progression such that more effective therapeutic options can be explored. Here, we have carried out whole exome sequencing of grade II, grade III and GBM samples which revealed the mutation spectrum of glioma from our patient set. Further, we performed integrative analysis of mutated genes from our patient cohort as well as TCGA cohort (The Cancer Genome Atlas) to find out mutated pathways that predict survival in GBM patients. The most significant pathway - neuroactive ligand-receptor interaction pathway was explored further. Patients with mutations in one or more genes of this pathway had poor survival. The pathway comprises of G-protein coupled receptors, ion channels and ligands which functions in modulation of neural plasticity, memory processes, behavior etc. Of the enriched genes belonging to this pathway, Calcitonin Receptor (CALCR), which was highest mutated in GBM (2.75%), was taken up for further investigation. CALCR was found to be downregulated in GBM and mutation or downregulation of the gene was found to predict poor survival in patients. Functional studies through cell-line based experiments revealed CALCR is a tumor suppressor in GBM. The peptide hormone calcitonin (CT), a high affinity CALCR ligand, inhibited proliferation, migration and anchorage-independent growth of glioma cells expressing CALCR with a concomitant decrease in the phosphorylation levels of ERK, AKT and JNK signaling molecules. However, CT failed to do these functions in CALCR silenced glioma cells. Exogenous overexpression of CALCR in glioma cells expressing low levels of the receptor was found to inhibit proliferation, migration and anchorage independent growth and this effect was further augmented when CT was added. Further, introduction of tumor-derived mutations in CALCR led to the abrogation of its tumor suppressor function. Studies are ongoing to demonstrate the tumor suppressive nature of CALCR using in vitro astrocyte transformation and intracranial orthotopic mouse glioma model. Thus, our study finds CT-CALCR signaling axis is an important tumor suppressor pathway in glioma development and underscores the importance of using CT as a novel therapeutic molecule for GBM treatment. Citation Format: Jagriti Pal, Vikas Patil, Anupam Kumar, Kavneet Kaur, Chitra Sarkar, Kumaravel Somasundaram. Genetic landscape of glioma reveals defective neuroactive ligand receptor interaction pathway as a poor prognosticator in glioblastoma patients [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 2454. doi:10.1158/1538-7445.AM2017-2454

A-to-I editing in human miRNAs is enriched in seed sequence, influenced by sequence contexts and significantly hypoedited in glioblastoma multiforme

Editing in microRNAs, particularly in seed can significantly alter the choice of their target genes. We show that out of 13 different human tissues, different regions of brain showed higher adenosine to inosine (A-to-I) editing in mature miRNAs. These events were enriched in seed sequence (73.33%), which was not observed for cytosine to uracil (17.86%) editing. More than half of the edited miRNAs showed increased stability, 72.7% of which had ΔΔG values less than −6.0 Kcal/mole and for all of them the edited adenosines mis-paired with cytosines on the pre-miRNA structure. A seed-editing event in hsa-miR-411 (with A – C mismatch) lead to increased expression of the mature form compared to the unedited version in cell culture experiments. Further, small RNA sequencing of GBM patients identified significant miRNA hypoediting which correlated with downregulation of ADAR2 both in metadata and qRT-PCR based validation. Twenty-two significant (11 novel) A-to-I hypoediting events were identified in GBM samples. This study highlights the importance of specific sequence and structural requirements of pre-miRNA for editing along with a suggestive crucial role for ADAR2. Enrichment of A-to-I editing in seed sequence highlights this as an important layer for genomic regulation in health and disease, especially in human brain.

Abstract A26: Shotgun lipidomics analysis of temozolomide-treated glioblastoma

Abstract Glioblastoma (GBM) is the most common and aggressive form of primary brain tumor. Due to its extremely malignant and invasive nature, the mean survival period of GBM patients is only 12-15 months from diagnosis. GBM patients rely on current treatment options, including surgery, radiation and chemotherapy (temozolomide). However, these treatment options fail to improve quality of patients' lives. After the treatment of temozolomide (TMZ) chemotherapy, more than 80% of the GBM tumors relapse. One of the most significant issues with the standard treatment strategy is that these secondary tumors have usually developed a chemoresistance to TMZ. This indicates that the secondary tumors have different tumor characteristics than primary tumors. Our goal is to examine the TMZ-treated and non-treated tumors in order to investigate the lipid expression changes in GBM samples by shotgun lipidomics. Lipids are known to play a critical role in cancer cell initiation and progression governed by oncogenic signaling pathways. Our previous study showed that more than 90% of identified lipids from orthotopic GBM tumors were significantly under-expressed compared to the control samples, which suggests that studying changes of lipids in tumor environment might help us better develop better strategies to overcome chemoresistance. We hypothesize that temozolomide promotes tumor heterogeneity and a mutagenic microenvironment in GBM, and lipid profiles of GBM can be a potential key biomarker for chemoresistance and cancer development. Our study focuses on investigating changes in lipid expression between TMZ-treated and non-treated GBM tumors by shotgun lipidomics. Intracerebral implants of the GBM10 cell line were performed in the right cerebral hemisphere in NOD/SCID/gchainnull mice obtained from the In Vivo Therapeutics Core Indiana University School of Medicine (IUSCC/IUSM). Ten mice received three rounds of TMZ treatment, and ten mice received no treatment. Tumors were harvested approximately 25 days after the implants and stored in -80C immediately. We plan to investigate lipid biomarkers of GBM by utilizing shotgun lipidomics to examine changes in the tumor lipid composition as a result of temozolomide treatment. In order to visualize the data, our group will utilize supervised statistical analysis methods (principal component analysis - PCA), univariate statistics and receiver operating characteristics (ROC) curve to show the differences of lipid profiles and define prospective biomarkers. Our findings could contribute to the development or the monitoring of therapeutic intervention strategies for tumors that recur after standard care of treatment in glioblastoma. Citation Format: Soo Jung Ha, Lauren Bailey, Christina Ferreira, Haiyan Wang, Barbara Bailey, Jixin Ding, M Reza Saadatzadeh, Karen E. Pollok, Kari Clase. Shotgun lipidomics analysis of temozolomide-treated glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A26.