bluuebunny/crossref_metadata_2025_split huggingface dataset:
P11.61 Development of a novel preclinical GBM model and therapeutic impact of IRE1 inhibition
Authors: Le Reste P , Pineau R , Jouan F , Samal J , Jegou G , Patterson J , Vauleon E , Avril T , Pandit A , Chevet E 
Published: September 2019

Abstract
Glioblastoma Multiforme (GBM) is the most severe primary brain tumor and represents more than 15% of all brain tumors. Despite an aggressive treatment comprising surgical resection and radio/chemotherapy, patient’s survival post diagnosis remains short with a median overall survival of 15 months. The lack of efficacy of the current treatments is mostly due to the tumor heterogeneity with different tumor cell types that exhibit various sensitivity to anti-cancer agents and to the diffuse feature of GBM that complicates the efficacy of complete resection. Another limitation for finding new valuable therapy approaches is the lack of relevant animal models that extensively recapitulate the current GBM patients’ standard of care. In the past couple of years, it has been demonstrated that the Unfolded Protein Response (UPR) plays an instrumental role in GBM development. It has been shown that IRE1, the most conserved UPR sensor, signals in tumor cells to reshape the tumor microenvironment to favor tumor growth and most likely to alter the response to treatment. The IRE1/XBP1s signaling axis exhibits pro-oncogenic properties and has a direct impact on patients’ survival. These observations point toward the IRE1/XBP1s axis as a potentially relevant therapeutic target. To further test the potential impact of the pharmacological targeting IRE1/XBP1 signaling, we proposed to use MKC8866, an inhibitors of IRE1, in preclinical models of GBM. As such we developed a novel GBM animal model that recapitulates the different steps engaged in GBM patient clinical handling (including surgical resection, irradiation and chemotherapy), and use this to demonstrate the relevance of IRE1 inhibition in GBM. Considering that IRE1 inhibitors do not cross the blood-brain barrier, we proposed an intraoperative delivery of the drugs though fibrin glue plugs applicated within the resection cavity, and then studied the impact on survival and tumor microenvironment. We showed that local delivery of IRE1 inhibitors combined with radio/chemotherapy had a positive effect on survival and induced tumoral and microenvironment remodeling, pointing at its high therapeutic potential and the need for an early clinical trial.
P12.02 Glioblastoma Multiforme immunological blockades and the implications of glioma cancer stem cells
Authors: Robilliard L D , MacDonald C , Angel C E , Finlay G J , Joseph W , Graham E S 
Published: September 2019

Abstract

BACKGROUND
Glioblastoma Multiforme (GBM) is classified as a WHO grade IV astrocytoma that continues to circumvent classical and novel chemo-, radio- and immuno-therapies. The recent FDA approvals for the use of targeted immunotherapies against inhibitory checkpoint ligands (for melanoma; ipilimumab and nivolumab) have brought the use of monoclonal antibody therapies to the forefront of GBM research. However, poor immunological responses, exemplified by down-regulation of anti-tumour T-cell activity, and up-regulation of immunosuppressive cells and secreted factors within the tumour micro-environment, have limited the effectiveness of immunotherapy in GBM to date. Therefore, understanding how GBM modulates an extensive repertoire of immune checkpoint ligands and the functional consequence on immune evasion is necessary to develop more targeted immuno-therapeutics.

MATERIAL AND METHODS
Patient derived glioblastoma cell lines were cultured using established serum-based or glioma cancer stem cell (gCSC) conditions. The phenotypes of resultant GBM cells and gCSC’s were characterised using flow cytometry and immunocytochemistry, to assess expression of neural lineage and stem cell-associated markers. Thereafter, cells were screened for the expression of an extensive range of inhibitory checkpoint ligands by flow cytometry. Finally, the secretion of immune modulating factors by GBM cells and gCSC’s were evaluated by using XL cytokine proteome arrays. Cytokines that appeared to be differentially expressed were subsequently measured using Cytometric Bead Arrays.

RESULTS
Adherent gCSC’s and gCSC derived glioma-spheres express nestin, CD44, A2B5 and vimentin, consistent with a stem cell phenotype. Furthermore, the gCSC’s exhibited reduced expression of the neural lineage markers NeuN and OSP. Flow cytometry analyses revealed that glioblastoma cells expressed all 11 checkpoint ligands investigated. Interestingly, gCSC’s showed higher levels of PD-L1, B7-H3, CD155 and HVEM expression than GBM cells.

CONCLUSION
Glioblastoma Multiforme is highly immuno-suppressive, which is reinforced by this study. Glioblastoma cells express all the inhibitory checkpoint ligands investigated and glioma cancer stem cell cultures up-regulate expression levels further. This implies that GBM cells are heavily equipped to inhibit infiltrating T-cells, exemplifying the need to find suitable therapeutics that target multiple immuno-suppressive mechanisms simultaneously.
P12.03 Bi-specific T cell engagers targeting IL13Rá2 activate tumor-infiltrating lymphocytes and improve survival in pre-clinical models of glioblastoma
Authors: Zannikou M , Pituch K C , Ilut L , Balyasnikova I V 
Published: September 2019

Abstract

BACKGROUND
The outstanding efficacy of bi-specific T-cell engagers (BiTE against hematological malignancies offers hope that they can similarly target solid tumors like GBM. In this study, we have designed a BiTE protein with specificity to the tumor-associated antigen, IL13Rα2, and investigated how BiTE protein engages a host’s T cell immune response to promote anti-glioma activity in pre-clinical models of GBM.

MATERIAL AND METHODS
BiTE molecule consisting of two single chain variable regions (scFv) of antibodies against either murine or human CD3ε and scFv47 against human IL13Rα2 connected through a flexible linker (BiTEIL13Rα2) were sub-cloned in a lentiviral expression cassette. The BiTE molecule (BiTEIL13Rα2off) modified to abrogate the interaction of scFv47 with IL13Rα2 served as a negative control. The BiTE proteins were isolated from the supernatants of HEK293T cells using His-Tag affinity chromatography and validated in SDS-PAGE, Western Blot, and ELISA assays. The BiTEIL13Rα2-induced T cells activation was measured in (i) cytotoxicity assay against IL13Rα2+ glioma cells, (ii) flow cytometry measuring for CD69 and CD25 T cells’ activation markers, and (iii) the production of cytokines, IFNγ and TNFα. For in vivo analysis, VmDk and C57Bl/6 mice bearing established intracranial glioma were treated systemically with BiTE proteins. The survival of the mice was recorded and analyzed using the log-rank test.

RESULTS
Here we show that BiTEIL13Rα2 specifically binds to IL13Rα2 but not to IL13Rα1, whereas BiTEIL13Rα2off has no binding activity to both IL13 receptors. The co-culture of naïve murine or donor’s human CD3+ T cells with IL13Rα2+ glioma cells in the presence of BiTEIL13Rα2 but not with BiTEIL13Rα2off (i) activates CD3+CD8+ T cells as judged by upregulation of CD69, CD25, and production of IFNγ and TNFα and (ii) results in concentration- and antigen-dependent cytotoxicity in glioma cells. Furthermore, a direct comparison of CD3+ T cells obtained from the peripheral blood and tumor tissue of GBM patients revealed that BiTEIL13Rα2 induces a potent cytotoxic activity of CD3+ T cells against IL13Rα2+ glioma cells. Finally, treatment of immunocompetent mice bearing IL13Rα2+ murine glioma with BiTEIL13Rα2 resulted in a higher frequency of intratumoral CD8+ T cells, and significant (p<0.05) improvement of survival over a negative control, BiTEIL13Rα2off group of mice.

CONCLUSION
Our data demonstrate that BiTEIL13Rα2 protein activates CD3+ T cells in an antigen-specific fashion. Furthermore, systemic treatment with BiTEIL13Rα2 protein confers a significant survival benefit in pre-clinical syngeneic glioma models, warranting investigations in other IL13Rα2-expressing cancers and translation to clinical settings.
P12.05 Evaluating the compatibility of tumor treating electric fields with key antitumoral immune functions
Authors: Diamant G , Simchony H , Shiloach T , Globerson-Levin A , Gasri Plotnitsky L , Eshhar Z , Pencovich N , Grossman R , Ram Z , Volovitz I 
Published: September 2019

Abstract

BACKGROUND
TTFields has the ability to induce immunogenic cell death (ICD). As immunotherapy and TTFields have different mechanisms of action (MOA), combining these therapies is a rational approach. Contrarily, TTFields may interfere with immune functions critical for effective T cell function.

MATERIAL AND METHODS
We cultured T cells from healthy donors’ peripheral blood or from viably dissociated glioblastoma samples under normal or TTFields conditions, with or without superantigen-stimulation. In order to assess T cell responses we used eight-color flow cytometry by monitoring select pivotal antitumoral functions: proliferation (CFSE), IFNγ secretion, cytotoxic degranulation (CD107a), activation/exhaustion (PD1) and viability. Evaluation of direct cytotoxicity was done by using chimeric antigen receptor (CAR) T cells.

RESULTS
TTFields did not change T cell activation rates for all evaluated functions with the exception of reduced proliferation - in line with TTFields’ MOA. TTFields substantially reduced the viability of activated proliferating T cells, moderately affected activated nonproliferating T cells and had almost no effect on the viability of non-activated cells. Polyfunctionality analysis of T-cells, associated with effective antitumoral responses, demonstrated that under TTFields, the activated non-proliferating T cells retained polyfunctional capabilities. 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).

CONCLUSION
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 antitumoral immune responses. Our findings support the further preclinical and clinical investigation into combining TTFields with immunotherapy.
P13.07 Multi-omics as a tool for elucidating temozolomide resistance in glioblastoma multiforme
Authors: Fabro F , Tóth E , Dekker L J M , Luider T M , Pierson T M , Leenstra S 
Published: September 2019

Abstract

BACKGROUND
Glioblastoma multiforme is the most common and aggressive brain tumor in adults, with an average overall survival of 14 months. Current standard of care consists of tumor resection followed by radiotherapy with concomitant temozolomide and adjuvant temozolomide. However, glioblastoma recurs in all patients. The causes reside in the enhanced invasiveness and resistance to treatment, giving a clear indication that recurrent and resistant glioblastoma biology must be understood better in order to achieve future treatment strategies to benefit the patients. The complex nature of recurrent glioblastoma makes its understanding still a challenging achievement in the field. Nowadays multi-omics approaching is developing further and further and it may be used to unravel, by combining different layers of biological information, a comprehensive view of the changes occurring during the treatment.

MATERIAL AND METHODS
A discovery set of 13 primary patient-derived glioblastoma stem-like cultures were analysed, comprising selected resistant, induced resistant and with pre-existing resistance conditions. A characterization of transcriptome, proteome and phosphoproteome was performed using RNAseq and liquid chromatography mass spectrometry. Additional 10 paired primary and recurrent tumor tissues were utilized as a validation set. The data obtained was visualised, explored and integrated through TIBCO Spotfire, Ingenuity Pathway Analysis, STRING and COREMINE medical software.

RESULTS
Genetic regulatory processes such as DNA repair mechanism, mRNA splicing and chromatin assembly were shown to be common over-represented trends in resistant and recurrent glioblastomas as a result of increased genomic instability and stress deriving from acute an repeated temozolomide exposure. Due to the immense heterogeneity of glioblastomas, other proteins and genes here identified as differentially expressed need a further investigation as they also may play an important role in relevant biological processes in a patient-specific way.

CONCLUSION
This study provides further understanding of glioblastoma biology revealing an association with processes of recurrence and temozolomide resistance, moreover offering potential therapeutic targets for better treatment options for glioblastoma patients.
P13.10 Glioblastoma within the subventricular zone associates with increased mesenchymal transition: an intratumoral gene expression analysis
Authors: Berendsen S , Dalemans D , Draaisma K , Robe P A , Snijders T J 
Published: September 2019

Abstract

BACKGROUND
Involvement of the subventricular zone (SVZ) in GBM is associated with poor prognosis and suggested to associate with specific tumor-biological characteristics. The SVZ microenvironment can influence gene expression and migration in GBM cells in preclinical models. We aimed to investigate whether the SVZ microenvironment has any influence on intratumoral gene expression patterns in GBM patients.

MATERIAL AND METHODS
The publicly available Ivy GBM database contains clinical, radiological and whole exome sequencing data from multiple regions from en bloc resected GBMs. SVZ involvement of the various tissue samples was evaluated on MRI scans. In the tumors that contacted the SVZ, we performed gene expression analyses and gene set enrichment analyses to compare gene (set) expression in tumor regions within the SVZ to tumor regions outside the SVZ, within the same tumors. We also compared these samples to GBMs that made no contact with the SVZ.

RESULTS
Within GBMs that contacted the SVZ, tissue samples within the SVZ showed enrichment of gene sets involved in (epithelial-)mesenchymal transition, NF-κB and STAT3 signaling, angiogenesis and hypoxia, compared to the samples outside of the SVZ region from the same tumors (p<0.05, FDR<0.25). Comparison of GBM samples within the SVZ region to samples from tumors that did not contact the SVZ yielded similar results. In contrast, we observed no difference in gene set enrichment when comparing the samples outside of the SVZ from SVZ-contacting GBMs with samples from GBMs that did not contact the SVZ at all.

CONCLUSION
GBM samples in the SVZ region associate with increased (epithelial-)mesenchymal transition and angiogenesis/hypoxia signaling, possibly mediated by the SVZ microenvironment.
P14.02 Influence of individual CpG methylation status on outcome in adult patients with glioblastoma multiforme receiving alkylating agent treatment
Authors: Siller S , Lauseker M , Giese A , Tonn J , Niyazi K , Thon N , Suchorska B , Kreth F 
Published: September 2019

Abstract
Background: Methylation of the O-6-methylguanine-DNA methyltransferase (MGMT) promotor causes gene silencing and has been associated with a favourable prognosis in patients with glioblastoma multiforme (GBM) receiving alkylating chemotherapy. However, analysis of MGMT promotor methylation is usually reported as a cut-off depending on the results of the correspondent CpG site testing. This approach disregards a possible heterogeneity concerning the methylation status within the individual CpG sites and its possible association with prognosis in GBM patients. The current study aimed at elucidating the association between methylation of CpG sites 74–98 within the MGMT promotor region and outcome in GBM patients receiving alkylating agents.
Material and Methods: Individual methylation status of 230 patients with histologically proven GBM following concomitant radio-chemotherapy with TMZ after stereotactic biopsy or open tumor resection (OTR) was assessed by the Sanger sequencing (Sseq) approach. Methylation of CpG sites 74–98 within the MGMT promotor region was defined according to a ratio of cytosine /thymine peak >50%. The total number of methylated CpG sites as well as clinical factors such as age, Karnofsky Performance Score (KPS) and mode of surgical procedure were correlated with outcome using proportional hazards models. In a subset of 34 patients, a correlation between individual CpG methylation and MGMT mRNA expression was performed.
Results: Median progression-free (PFS) and overall survival (OS) were 7.8 and 14.6 months, respectively. Alongside younger age, KPS> 80 and OTR, the cumulative total number of methylated loci within the CpG sites 74–98 was strongly associated with both PFS and OS and retained its prognostic influence on outcome in multivariate models (p <0.001). Furthermore, a linear coherence between the total number of methylated CpG sites 74–98 and survival parameters could be observed. Moreover, low number of methylated CpG sites was observed in tumor specimen with a high mRNA expression and vice versa (Spearman correlation coefficient: -0.62).
Conclusion: In contrast to the concept of dichotomizing the MGMT promotor status into ‘methylated’ and ‘non-methylated’, our approach shows a clear heterogeneity within the methylation status of the CpG sites 74–98 within the GBM tumor specimens. Our data suggest a strong correlation between outcome and the total number of methylated CpG sites, thus an up-front analysis of the individual GpC site methylation status prior to initiation of alkylating chemotherapy might help to improve treatment response in GBM patients.
P14.06 Phase II randomized trial of short course hypofractionated radiotherapy with or without temozolomide in elderly patients with Glioblastoma
Authors: Madan R , Thakur S , Salunke P , Ahuja C , Khosla D , Yadav B S , Kapoor R , Kumar N 
Published: September 2019

Abstract

BACKGROUND
Glioblastoma (GBM) is the most aggressive primary brain tumor. Although treatment advances and introduction of temozolomide has improved the outcome in young patients, it is still a concern in elderly patients. Elderly population is often excluded from the trials, thus established treatment guidelines are not available. Trials have shown that short course hypofractionated radiotherapy (HRT) is equally effective in terms of overall survival (OS) and progression free survival (PFS) as compared to standard RT in elderly population. While the benefit of adding Temozolomide (TMZ) to standard dose radiotherapy is well established, there is lack of evidence that addition of TMZ to HRT improves survival in elderly patients.We conducted this study to see if addition of temozolomide to HRT improves OS, PFS and quality of life (QOL) in elderly GBM patients.

MATERIAL AND METHODS
The study was conducted over a period of 21 months from January 2017 to September 2018. Histologically proven 70 newly diagnosed Glioblastoma patients (age>60 years) were enrolled and randomized into two arms (Arm I- HRT alone, Arm II- HRT and TMZ) using computer generated randomization table (1:1 ratio). The radiotherapy dose in both arms was same and consisted of a dose of 25Gy in 5 daily fractions over 1 week. In addition, arm II patients received daily concurrent TMZ 75mg/m2 1 hour prior to RT followed by adjuvant TMZ 175mg/m2 from Day1-5 for 6 cycles (q 4weeks), started after a month of RT completion. Quality of life score and global health status was assessed in both arms at baseline, 1, 3, 6 and 9 months after the treatment using QOL questionnaire EORTC QLQ-C 30 and QLQ-BN20. Data entry was done in Microsoft Excel 2016 and analysis was done in SPSS version 18. Kaplan Meier survival analysis was done for OS and PFS. To see the trend of time related observations at different point of time repeated ANOVA was used. A p value of <0.05 was taken significant.

RESULTS
The median OS in arm I and II was 121 (range 40–360 days) and 146 (range 40–450 days) days respectively (P=0.146). The median PFS in arm 1 and arm 2 were 77 (range 30–300 days) and 109.50 (range 30–300) days respectively (p-0.028). On further analysis, the prognostic factors were type of surgery (Median PFS 159 days in gross total excision and 129 days in subtotal excision or biopsy, p-0.03) and KPS (0.04). QOL score for symptom and functional domain was comparable in both arms. However score of nausea and vomiting was higher during concurrent chemoradiotherapy. A non significant improvement in QLQ BN 20 score was observed at 9 months in arm I.

CONCLUSION
Addition of Temozolomide to HRT is a compelling option in elderly Glioblastoma patients as it significantly improves the PFS and a non significant improvement in OS without compromising quality of life.