bluuebunny/crossref_metadata_2025 huggingface dataset:
P11.13 Radiotherapy combined with a multimodal imaging approach in a glioblastoma preclinical model
Authors: Salvatore D , Shaw G , Wright J , Teh I , Koch-Paszkowski J , Murray L , Scarsbrook A , Schneider J , Ottobrini L , Short S
Published: September 2019

BACKGROUND

Glioblastoma multiforme (GBM) carries a poor prognosis, partly due to biological and anatomical heterogeneity. Although radiotherapy (RT) is effective, high doses damage surrounding healthy tissues. Multimodal imaging with Magnetic Resonance (MRI) and Positron Emission Tomography (PET) may represent a useful approach for identifying GBM heterogeneity and visualising metabolic tumour properties. PET radiotracer [18F]-fluciclovine is preferentially accumulated in gliomas compared to healthy brain tissue via the cellular transport systems, LAT1 and ASCT2. In this study the effect of fractionated RT using multimodal imaging including [18F]-fluciclovine uptake and immunohistochemistry (IHC) in a GBM preclinical model will be validated.

MATERIAL AND METHODS

Two C57BL/6J mice cohorts were injected intracranially (i.c.) with murine CT2A-luc cells and subsequently submitted to multiparametric MRI and [18F]-fluciclovine PET imaging during hemi-brain RT (3Gy on 2 days/each week) for maximum 25 days after i.c. injection. Brains were collected for IHC characterization including LAT1 and ASCT2 staining.

RESULTS

Preliminary data showed that both MRI and PET were effective modalities to track tumour growth in this model. PET data revealed up to greater than 3-fold increase in SUVmax from regions of interest around the tumour site compared to healthy brain tissue. Time activity curves showed a steady increase in tumour uptake over 90 minutes. MRI showed a 25% increase in T2 values in tumours relative to unaffected contralateral regions. Confirmation of treatment response through matched imaging and IHC are ongoing, from which changes in glioma cell biology as well as amino acid transporter protein levels will be analysed.

CONCLUSION

These preliminary results show that multimodal imaging presents novel data in the assessment of treatment response in this model and will permit parallel IHC analyses to better define GBM tumour heterogeneity aligned with imaging changes. These data will also inform an on-going clinical study using the same imaging modalities.

Work at authors’ labs are supported by an Investigator initiated project from Blue Earth Diagnostics (AS, SCS) and a University of Leeds Biswas studentship (SCS, DS). Daniela Salvatore is also supported by a Scholarship provided by Molecular and Translational Medicine Doctorate School of University of Milan (Italy).
P11.30 Special VEGF90kDa form in exosome from GBM cells activates TAZ in endothelial cells to promote angiogenesis and contributes bevacizumab resistance
Authors: Wang Z , Yuan Y , Xu C , Liu Y
Published: September 2019

BACKGROUND

Glioblastoma (GBM) has obvious blood vessels proliferation, which is one of the important histological diagnostic criteria. Bevacizumab, VEGF targeted inhibitor, is found inefficient in 2/3 cases after the clinical trial. Here, we found a specific 90kDa form of VEGF (VEGF90K) in exosomes of GBM could activate TAZ expression in endothelial cells to promote angiogenesis, which might also contribute to Bevacizumab treatment resistance.

MATERIAL AND METHODS

Exosomes were isolated from U87, LN229 GBM cell culture medium and characterized using transmission electron microscopy, nanoparticle analysis, and western blotting. VEGF expression in GBM and TAZ expression in human umbilical vein endothelial cells (HUVEC) are inhibited by shRNA. In vitro migration, proliferation, and tube formation assays were used to assess the angiogenic potential of HUVEC cells. Western blotting was used to analysis TAZ expression in HUVEC. Bevacizumab or exosome inhibitor GW4869 were used separately or together to evaluation their blocking effects on angiogenic functions of GBM cells.

RESULTS

we found a unique 90kDa form of VEGF (VEGF90K) from exosome of GBM cells could activate TAZ expression in HUVEC cells which correlated with higher levels of cellular migration, proliferation, and tube formation. Knockdown VEGF expression in GBM exosomes or TAZ expression in HUVEC cells could decrease the angiogenic function of GBM cells. GW4869 combine Bevacizumab had significantly inhibited the angiogenic ability of GBM cells in vivo and in vitro.

CONCLUSION

A specific exosome-associated VEGF from GBM can promote angiogenesis by activating TAZ expression in endothelial cells. Targeted inhibition of exosome secretion can significantly inhibit tumor angiogenesis and restore bevacizumab effect. Our study highlights the unique properties of VEGF in exosome which might help to explore new treatment strategies of GBM.
P11.38 The Galectin-1 inhibitor OTX-008 has antiproliferative effects on human glioblastoma cells independent of MGMT-promotor status
Authors: Goett H , Kolodziej M , Pons-Kühnemann J , Schwarm F , Koch C , Uhl E , Stein M
Published: September 2019

BACKGROUND

The protein Galectin-1 (gal-1) is known to be involved in proliferation and progression of several malignancies. The allosteric gal-1 inhibitor OTX-008 has an antiproliferative effect on various tumors in vitro and in vivo. However, the effect of OTX-008 on patient derived GBM cultures is unknown. The aim of our study was to investigate the antiproliferative effects of OTX-008 on U87-MG and A172 cells, patient derived GBM cultures, and cultured human astrocytes (HCA).

MATERIAL AND METHODS

GBM specimens from 10 patients were intraoperatively retrieved out of the 5-ALA-positive areas and were cultured in DMEM. In these cell cultures, U87-MG and A172 cell lines, and in HCA the gal-1 expression was determined by quantitative RT-PCR. Gal-1 expression below the median was defined as low-gal-1 expression (LGE), above as high-gal-1 expression (HGE). OTX-008 was administered and cell viability was assessed after 72h with MTT assay. Dose-response curves and influence of gal-1 expression were calculated by probit analysis in a logit model with logarithmic transformation of x-axis. Comparison between MGMT promotor methylated and unmethylated cells was performed using the Mann-Whitney U test.

RESULTS

OTX-008 inhibited cell proliferation of all 10 patient derived GBM cell cultures with a mean ED50 of 34.28 (95% CI: 20.29- 61.57) μM and a mean ED95 of 100.90 (59.30- 199.33) μM. Antiproliferative effects of OTX-008 for U87-MG and A172 cells were found with an ED50 of 17.44 (95% CI: 13.24- 22.52) μM and 33.36 (95% CI: 24.30- 45.86) μM. In 5 cell cultures of the patient derived cells LGE and in the other 5 HGE was determined. Probit analysis showed an ED50 of 20.43 (95% CI: 16.03- 24.89) μM for the LGE group and an ED50 of 33.98 (95% CI: 28.15- 40.89) μM for the HGE-group. Between MGMT-promotor methylated and unmethylated cells no statistically significant difference for the ED50 was observed (38.36 vs. 31.56; P=0.454). Probit analysis of OTX-008 administration in HCA showed no dose-response-relation.

CONCLUSION

OTX-008 has antiproliferative effects on human glioblastoma cells independent of MGMT-promotor methylation status. The sensitivity of GBM cells to OTX-008 depends on the gal-1 expression level. In HCA no effect on cell viability was observed. OTX-008 seems a promising novel substance in glioblastoma therapy.
P11.46 Discovery of a new HDAC6 inhibitor for the treatment of glioblastoma
Authors: Auzmendi-Iriarte J , Saenz-Antoñanzas A , Andermatten J , Elua-Pinin A , Aldaba E , Vara Y , Matheu A
Published: September 2019

BACKGROUND

Glioblastoma’s origin and development is not only associated to genetic alterations, but also to epigenetic changes. Indeed, an altered expression or activity of epigenetic enzymes such as histone deacetylases (HDAC) has been associated to cancer stem cell activity, which has been widely described as a major feature for therapy resistance and tumor recurrence. In particular, inhibition of HDAC6 is an increasingly attractive pharmacological strategy, due to its association with low toxicity. Thus, the aim of the present study was to determine the impact of a new HDAC6-selective-inhibitor in glioblastoma and glioma stem cells.

MATERIAL AND METHODS

To test the effect of QTX compound in glioblastoma and glioma stem cell lines, cell viability after 72h of treatment was studied by MTT assay. After evaluation of IC50, QTX in vitro activity was analyzed, focusing on proliferation, apoptosis and stemness of U87-MG cell line and confirmed in a patient-derived glioma stem cell line. In vivo antitumor effect was evaluated using U87-MG cells xenografted in immunocompromised mice; after tumor formation, 5 mice were randomly selected as control group and another 5 for QTX treatment (intraperitoneal administration of 50 mg/kg; 5 days of dosing / 2 days off for 2 weeks). Mice weight was measured daily and tumor volume every two days.

RESULTS

We demonstrated that QTX reduces viability of all tested glioblastoma cells, even more greatly than normal astrocytes. Indeed, QTX diminishes proliferation and induces apoptosis in both conventional and patient-derived glioma cell lines. In particular, this effect was accompanied by a reduction of self-renewal properties of glioma stem cells. Interestingly, QTX in vitro activity was more effective comparing to the pan-inhibitor SAHA or the HDAC6-selective inhibitor Tubastatin A. Furthermore, QTX delayed tumor initiation and progression in vivo, without presenting significant side effects.

CONCLUSION

QTX compound presents a promising anti-tumor effect both in vitro and in vivo in glioblastoma, at least in part, inhibiting glioma stem cell activity.
P11.48 Dose-dependent effects of radiation exposure on the cell culture of glioblastoma G01, obtained from the patient with a long-term survival
Authors: Belyashova A , Golanov A , Pavlova G , Savchenko E , Antipina N , Panteleev D , Shamadykova D , Smirnov G , Nikolaeva A
Published: September 2019

Background: The predictors of a favorable prognosis regarding radiation exposure in patients with glioblastoma have not been studied enough, therefore, the search for factors determining a tumor response to the combined chemoradiation treatment is an actual task at present. DNA repair protein RAD51 homolog 1 (RAD51) is a crucial protein for homologous recombination and its inhibition has been shown to sensitize glioma stem cells to irradiation. Materials and Methods: G01 primary glioblastoma cell culture was obtained from tumor tissues from patient with long term survival (42 month).An irradiation with the energy of 6 MeV was carried out in 1 fraction 20,40,60,80,100,125,150,200,250 Gy with Linac. mRNA expression of the RAD51 gene, XRCC6, XRCC5 genes, relative amount of RAD51 protein and Ku70, Ku80 proteins, proliferative activity of cell culture was evaluated by RT-PCR, Western blotting, MTT-test, respectively.Results: When G01 cells were irradiated with doses from 5 to 180 Gy there was a uniform linear decrease in the population of proliferating cells compared to the control, but at a dose of 200 Gy a rise to 24% was observed. The mRNA expression of the RAD51 gene increased 2 times after irradiation at a dose of 20 Gy compared with control and then increased, reaching a peak at the level of 100,250 Gy. Minimal expression was observed after irradiation at a dose of 20 and also decreased at 200 Gy. The mRNA expression of the XRCC5 gene increased 2-fold after irradiation at a dose of 20 Gy compared with control and then decreased, remaining at the same level. Minimal expression was observed after irradiation at a dose of 200Gy. The relative amount of Ku80 protein reached a peak at a dose of 100 Gy, followed by a decrease. Maximum expression level of mRNA of the XRCC6 gene was observed after irradiation at a dose of 250Gy. The relative amount of Ku70 protein also decreased at 200 Gy and reached a peak at a dose of 250 Gy. Conclusions: After irradiation transplantable G01 cell culture from patient with long term survival a linear decrease in proliferative activity depending on the dose was observed with an decrease in mRNA expression of Rad51 gene and the relative amount of RAD51,Ku70 in dose level of 200Gy that could potentially indicate sensitivity point to radiation exposure in these cell culture. Further studies using cell cultures from patients may help in understanding the mechanisms of radioresistance and radiosensitivity in glioblastoma.The research was supported financially by RFBR (Project No. 18-29-01061).
P11.54 Identification of PDGFRA and MYC(N) as somatic driver genes in Glioblastoma
Authors: Berezovsky A D , Transou A , Irtenkauf S , Poisson L , Hank Wu K , Mikkelsen T , deCarvalho A
Published: September 2019

BACKGROUND

Somatic oncogene amplification happens frequently in glioblastoma (GBM). The second most frequently amplified gene encoding receptor tyrosine kinases in GBMs is platelet derived growth factor alpha (PDGFRA) (15%). In contrast, MYC and MYCN amplification occurs in 1.6% and 2.9%, respectively. Our goal was to characterize the role of PDGFRɑ and Myc in GBM.

MATERIAL AND METHODS

Neurosphere cultures were implanted in cohorts of 10–15 nude mice. 5 PDX lines, presenting median survival of 29–59 days were classified as short survivors, and 5 lines with median survival between 104–134 days classified as long survivors. Total RNA was extracted from PDX terminal tumors (3 biological replicates) and sequenced in a paired-end read format. Mouse reads were filtered out using Xenome. MYC and PDGFRA expression patterns were analyzed in tissue microarrays representing duplicated samples from 40 glioma neurosphere-derived PDX lines by IHC (1 anaplastic oligodendroglioma, 8 recurrent GBM with 2 newly diagnosed/recurrent pairs). Normalized staining intensity (MI) and area (A) were quantified using Fiji/ImageJ.

RESULTS

PDGFRA, MYC, MYCN gene amplifications were represented in a molecularly diverse panel of GBM patient-derived cancer stem-like cells (CSC) and orthotopic mouse xenografts (PDX). Transforming to a normal distribution (log10), 4/13 of cell lines had a PDGFRA mRNA expression (RPKM) higher than 1.5. Similarly, one PDX line had a staining index of greater than 10, 11 (27.5%) had an index between 5–10. The range of intra-tumoral variance, represented by standard deviation, was 0.09–24.25 highlighting the heterogeneity of PDGFRɑ expression. PDGFRɑ phosphorylation (Y754) did not differ between 8 cell lines cultured in NMGF, but deviated in alternate medias without growth factors, supplemented with FBS. In comparison, MYC(N) mRNA expression is only elevated in the context of a known amplification. Furthermore, a a MYC activity signature consisting of 18 target genes was only evident in the 5 amplified CSC lines. Taking advantage of genomic heterogeneity, we have isolated subclones lacking PDGFRA amplification from a PDGFRA amplified GBM CSC. The absence of PDGFRA amplification reduced the self-renewal potential to 37% of the PDGFRA amplified cell population (p=0.001) in clone 1 and 57% in clone 2 (p=0.013). Pertaining to determinants of in vivo survival, MYC was altered in 80% of short survivors (2/5 MYC, 2/5 MYCN amplification) and in 0% of long survivors. Myc signature was highly correlated with in vivo survival (Pearsons’ corr. = -0.77) and MYC gene expression was correlated with in vivo TMZ resistance (corr. = 0.7).

CONCLUSION

These results suggest that PDGFRɑ expression and activity can occur in the absence of gene amplification, while Myc activity is dependent on gene amplification. Both oncogenes drive oncogenic pathways that should be explored as therapeutic targets.
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

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

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

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

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

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

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

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

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.
P14.20 Anxiety level in a prospective cohort of newly diagnosed glioblastoma patients: preliminary results from the IMAGE study
Authors: Maillet D , Narme P , Menard V , Larrieu M , Sahel K , Belin C , Carpentier A
Published: September 2019

BACKGROUND

Quality of life (QoL) is an important area of clinical neuro-oncology, especially in glioblastoma patients. Although previous studies showed an association between QoL and anxiety, few studies have focused on the anxiety level throughout the disease progression (Bunevicius et al., 2017; Kilbride et al., 2007). Underestimating anxiety may have several consequences: a low treatment compliance, exacerbation of somatic symptoms or side effects of treatment, difficulties with understanding medical information and lower cooperation with the medical staff (Spencer, 2010). In the present study, we aimed assessing the current state of anxiety in a prospective cohort of patients treated for glioblastoma. We also investigated potential correlations with other clinical and psychological variables to better understand determinants of anxiety in this population.

MATERIAL AND METHODS

At the beginning of their cycle of temozolomide cure and after radio-chemotherapy, 30 patients with glioblastoma were included. Inclusion criteria were: Karnofsky index (IK) ≥ 70% and absence of cognitive disorder that could interfere with the completion of questionnaires. The characteristics of patients were as follows: mean age of 56.6 years ± 12.5 (70% were more than 50 years old); 20% were women; 50% had a university degree and IK of 87% ± 5. Anxiety level was assessed using the State-Trait Anxiety Inventory. This scale consists of 40 items on a self-report basis measuring how patients feel right now (“state”) and how they generally feel (“trait”). We also assessed (i) QoL using the Cancer Quality of Life Questionnaire 30 and the Brain Cancer Module-20; (ii) the presence of depressive symptoms using the Hospital Anxiety and Depression Scale; (iii) the self-esteem using the Rosenberg self-esteem scale and (iv) memory complains using the Prospective and Retrospective Memory Questionnaire.

RESULTS

The preliminary results showed that - at baseline - 21% of our sample reported high levels of anxiety. Correlation analyses showed that state anxiety was correlated with trait anxiety (rho=0.799, p< .001), QoL (rho=0.678, p< .001), level of self-esteem (rho=-0.514, p=0.004) and memory complains (rho=0.618, p< .001). Any correlation was found with age, education level, lesional lateralization or depressive symptoms. Women had higher state anxiety scores than men (t(27)=-2.4, p=0.02).

CONCLUSION

These preliminary results suggest that higher current state of anxiety is associated with lower QoL and lower self-esteem, regardless the presence of depressive symptoms, age, education level or lesional lateralization. Anxiety level at the follow-up (4 and 6 months after the baseline) and determinants of its progression will also be presented and might help health professionals to understand patients’ experience and better meet their needs.
P14.34 Priority of comet tail sign in the planning of stereotactic irradiation for the treatment of brain metastases
Authors: Mitsuya K
Published: September 2019

BACKGROUND

A highly enhanced cap attached to the surface of metastatic tumours in the brain parenchyma is occasionally encountered on magnetic resonance (MR) images. This atypical cap termed “comet tail sign (CTS)” is attributed to leakage of contrast medium from the tumour body into the oedema surrounding it and is containing no neoplastic tissue (Mitsuya, et al. 2016. J Neurooncol). The purpose of this study was to demonstrate our clinical results of excluding CTS in the planning of stereotactic irradiation (STI) for brain metastases to avoid radiation necrosis.

METHODS

We selected 16 consecutive cases of newly diagnosed brain metastases with CTS treated with STI between May 2007 and January 2019. Patients underwent STI with target fields including CTS until December 2014 (Group A, n=4), while STI with fields excluding CTS from January 2015 (Group B, n=12). CTS was carefully diagnosed using contrasted T1-weighted images and T2-weighted images by radiation oncologists and neurosurgeons. Treatment outcome and toxicity were compared among two groups.

RESULTS

Primary cancers were lung in 13 and colon in 3 patients. Ten (63%) were male, and the median age was 68 years (range, 47 to 78). The median follow-up time was 13.5 months from STI. There was no significant difference of the mean tumour volume between two groups (4.28ml vs. 4.68ml). Disease control rates were 25% in Group A and 92% in Group B. Symptomatic radiation necrosis was observed in two (50%) patients of Group A but none (0%) of Group B.

CONCLUSION

CTS could be excluded from STI target planning to increase local control and to decrease radiation toxicity. Prospective study is needed to clarify optimal STI planning for patients with brain metastasis accompanied CTS in the future.
P14.40 Incidence and risk factors identification for lung cancer brain metastasis
Authors: Fariña H , Rodríguez-Salazar M , Rodríguez-Yanes M , Plata-Bello J
Published: September 2019

BACKGROUND

Lung Cancer (LC) is the most frequent malignant neoplasm around the world and it is one of the most frequent brain metastasis origins. The aim of the present work is to measure the incidence of brain metastasis in new-diagnosed LC patients and to identify the risk factors associated with its development.

MATERIAL AND METHODS

A retrospective observational study has been performed. Patients with new diagnosis of LC between January 2015 and December 2016 in our Center were included. Demographic, clinical and molecular variables were studied. Statistical analysis included a uni- and multivariate COX regression, survival analysis (log-rank test) and non-parametric two-independent sample tests.

RESULTS

Three-hundred and thirty-nine patients were diagnosed with LC in our Center between 2015 and 2016 (99 female; mean age: 66.1 years, SD=10.89). The incidence of brain metastasis was 16.25 cases each year since the initial diagnosis (19.2% of all patients). The risk of brain metastasis was increased in the first year after the diagnosis of LC (83.07% of cases). Patients with metastasis were younger than non-metastatic patients (64.0 vs. 66.6 years of age; p=.041). Small-cell lung carcinoma was the most frequent histological subtype associated with the development of metastasis (HR=2.267; p=.011), followed by the adenocarcinoma (HR=1.131; p=.624) and epidermoid (HR=.546; p=.067). No molecular factor (EGFR, ALK or p63 expression) was identified as risk factor. The overall survival of brain-metastatic patients was significantly lower than non-metastatic patients (167.0 vs. 273.0 days; p=.0002).

CONCLUSION

Brain metastasis are diagnosed in almost 20% of LC patients with, at least, three-years follow-up. Small-cell carcinoma subtype and younger patients were associated with a higher risk of brain metastasis, which is associated with a bad prognosis.
P14.46 SUBEPENDYMAL GIANT CELLS ASTROCYTOMA (SEGA) IN TUBEROUS SCLEROSIS COMPLEX (TSC): A SERIES OF 31 PATIENTS
Authors: Giordano F , Moscheo C , Lenge M , Mari F , Grandoni M , Buccoliero A , Sardi I , Genitori L , Guidi M
Published: September 2019

Background

Subependymal Giant Cells Astrocytomas (SEGAs) are characteristic of Tuberous Sclerosis Complex (TSC). They are usually benign tumors but may rapidly grow and cause hydrocephalus and raised intracranial pressure. Surgery is mandatory for large and symptomatic SEGAs

METHODS

31 patients harboring SEGAs in TSC were admitted for surgery. The main indications for surgery were tumor size and location, tumoral growth and cystization/hemorrhage, and hydrocephalus. In presence of symptomatic hydrocephalus firts surgery aimed to reduce intracranial pressure

RESULTS

Forty-four surgeries were performed in 31 patients achieving Gross Total and Subtotal Removal in 36 and 8 patients respectively. Recurrences occurred in 11 patients; nine of them were reoperated while two were administered therapy with m-TOR pathway inhibitors. Surgical morbidity and mortality accounted for 22.7% and 2.3% respectively; hydrocephalus was the main complication. After an average follow-up of 5 years, 90% of patients had no evidence of the disease and most (93,3%) had a good clinical status after surgery; 12 out of 30 patients (40%) had a VP-shunt for hydrocephalus

Conclusions

GTR is feasible and represents the treatment of choice of SEGAs in TSC. Therapy with m-TOR pathway inhibitors is to be considered in selected patients and especially in recurrences of SEGAs
P14.53 Deconstructing pathologically increased MEG network clustering in glioma patients
Authors: Kulik S D , Derks J , Numan T , Hillebrand A , de Witt Hamer P C , Klein M , Geurts J J G , Reijneveld J C , Stam C J , Schoonheim M M , Douw L
Published: September 2019

Introduction

Functional brain networks in glioma patients are characterized by higher global clustering than healthy controls, indicating stronger connectivity in triads of brain regions when averaging across the entire brain. However, this could be due to either primary increased local clustering of (peri)tumor regions or higher local clustering throughout the entire brain.

METHODS

Magnetoencephalography recordings of 71 glioma patients and 53 HCs were analyzed by calculating functional connectivity with the phase lag index between source-localized time series of 78 cortical regions of the automated anatomical labelling atlas. Per participant, we calculated (1) global average clustering, (2) local clustering of tumor and non-tumor regions, and (3) Euclidean distance between tumor centroids and of all other region centroids.

RESULTS

Glioma patients had higher global average clustering (p=0.002) than HCs. This increase was indeed global: there was no difference between tumor and non-tumor regions (p=0.154) and no association between distance and local clustering (p=0.759). When splitting patients into high (top 25%, n=18) and normal global clustering (other 75%, n=53) to more specifically pick up on the determinants of pathological global average clustering, again no localized or distance-dependent effects were found. High clustering patients were younger than patients with normal global clustering (p=0.027). Posthoc analysis into tumor localization preference for particular network regions in the entire patient cohort revealed greater tumor occurrence in regions with high clustering in HC (p<0.001), while patients with high global clustering showed tumors localized in regions with lower clustering in HC (p=0.032).

CONCLUSION

The functional brain network of a subset of (relatively young) glioma patients is disturbed on a global level, suggesting that treatment thereof might benefit patients. Moreover, our exploratory analyses suggest that gliomas occur more often in normally highly clustered regions, but that tumors occurring in less clustered regions are associated with more extensive global network alterations. These findings may speculatively indicate that patients with and without such pathologically altered global clustering represent distinct phenotypes (both in terms of age and tumor localization) and may also need to be treated as such.