bluuebunny/crossref_metadata_2025 huggingface dataset:
P11.42 Magnetic targeting of the granzyme B functionalized nanoparticles for therapy of glioblastoma
Authors: Shevtsov M , Stangl S , Marchenko Y , Yakovleva L , Ryzhov V , Nikolaev B , Galibin O , Pitkin E , Multhoff G
Published: September 2019

BACKGROUND

Magnetic drug targeting by employing external magnetic fields represents a promising approach for treatment of glioblastoma. To increase the anti-tumor therapeutic effect magnetic nanocarriers could be functionalized with tumor-targeting bioligands such as granzyme B (GrB). The serine protease GrB that is produced as an effector molecule by activated NK and T cells can induce specific tumor cell apoptosis.

MATERIAL AND METHODS

The targeting potential of the dextran-coated superparamagnetic iron oxide nanoparticles conjugated with GrB (GrB-SPIONs) was assessed in glioblastoma cells (U87, C6, GL261) by flow cytometry, confocal and electron microscopies. Magnetic field strength (320 mT) in the models of U87 glioblastoma in NMRI nu/nu mice and C6 glioma in Wistar rats was achieved by employing NdFeB cylindrical ferromagnet. The irradiation of the implanted tumors was performed using the CT-image guided Small Animal Radiation Research Platform (SARRP). Accumulation of the nanoparticles was assessed in paraffine-embedded specimens employing Prussian blues staining. Sections were additionally analyzed by IHC for apoptosis (caspase 3).

RESULTS

Synthesized GrB-SPIONs incorporated into the cytoplasm of tumor cells via the endolysosomal pathway and induced apoptosis in a dose-dependent manner. Intravenous injection of GrB-SPIONs resulted in the glioma-specific retention of the nanoparticles as was shown by high-field (11 T) magnetic resonance imaging and biodistribution studies (NLR-M2 measurements). Magnetic targeting of the nanoparticles in vivo drastically enhanced the accumulation of nanoparticles to the location of the magnet. Thus the local retention of nanoparticles was 12.3-fold higher as compared to application of GrB-SPIONs without external magnetic field. The presence of granzyme B on SPIONs has been shown to promote tumor cells death (IHC staining for caspase 3) and significantly increased overall survival. A combination of nanoparticle treatment with a single radiation dose (10 Gy) significantly prolonged the survival of rats as compared to animals treated only with magnetic targeting of GrB-SPIONs or radiotherapy alone (P<0.001).

CONCLUSION

Single-agent therapy with GrB-SPIONs demonstrated an impressive increase in overall survival of tumor-bearing animals. Combinatorial regimen employing magnetic targeting and stereotactic radiotherapy further enhanced the therapeutic potential of magnetic conjugates.
P11.45 Synergistic growth inhibition mediated by dual PI3K/mTOR pathway targeting and genetic or direct pharmacological AKT inhibition in human glioblastoma models
Authors: von Achenbach C , Weller M , Kaulich K , Gramatzki D , Zacher A , Fabbro D , Reifenberger G , Szabó E
Published: September 2019

BACKGROUND

Molecular genetic aberrations in the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway are common in human cancers including glioblastoma, yet, novel therapeutic approaches targeting this pathway in glioblastoma have had limited success to date.

MATERIAL AND METHODS

Here we analyzed the molecular mechanisms determining sensitivity to PI3K/mTOR inhibition using gene silencing or pharmacological target inhibition, and effects on proliferation, and clonogenicity or spherogenicity as readouts, in an extended panel of human long-term glioma cell (LTC) lines and glioma-initiating cells (GIC).

RESULTS

Glioma cells, including GIC in particular, were universally sensitive to growth inhibition induced by PQR309, a novel, dual pan-PI3K/mTOR antagonist in vitro. Cells exhibited profound growth arrest, but little apoptotic or necrotic cell death as confirmed by electron microscopy; yet, there was evidence of senescence. In vivo studies confirmed the anti-glioma activity of PQR309 in the orthotopic LN-229 glioma model. Cell lines with high basal levels of phosphorylated (active) AKT, low levels of phosphorylated (inactive) protein translation repressor eukaryotic initiation factor (eIF) 4E-binding protein 1 (p4E-BP1), and high levels of Ser9-phosphorylated (inactive) glycogen synthase kinase 3 beta (pGSK3ß) were more sensitive to PQR309. Accordingly, the activity of PQR309 was synergistically enhanced by AKT gene silencing or direct pharmacological AKT inhibition.

CONCLUSION

These data justify to explore combined targeted therapy approaches in glioblastoma that aim at down-regulating AKT function to enhance the therapeutic potential of dual PI3K/mTOR inhibitors.
P11.51 Repurposing the antipsychotic chlorpromazine for the treatment of glioblastoma multiforme
Authors: Paggi M G , Abbruzzese C , Matteoni S , Matarrese P , Giannarelli D , Sperduti I , Villani V , Pace A
Published: September 2019

BACKGROUND

Background Glioblastoma multiforme (GBM) is characterized by short survival rates, thus generating a compelling need for more effective therapeutic schemes. Therapeutic approaches involving novel drugs must travel a long and expensive way in order to reach the bedside; drug repurposing and repositioning are viable strategies to develop therapies, involving shorter timelines and lower costs. Here we analyzed the activity of the antipsychotic drug chlorpromazine (CPZ) in hindering GBM growth in vitro and in vivo. The safety of this drug springs from six decades of clinical use as an antipsychotic. CPZ has been very recently identified as an inhibitor of the mitotic kinesin KSP/Eg5 and the AKT/mTOR pathway in human glioma cells.

MATERIAL AND METHODS

To explore CPZ pharmacodynamic properties, we assayed the effects of CPZ on several GBM cell lines growing in an anchorage-dependent manner or as neurospheres (cancer stem cells). Further experimentation using high-throughput proteomic and metabolomic platforms is underway.

RESULTS

In our hands CPZ, in the low micromolar range, strongly reduced GBM cell viability after 48 h in both anchorage-dependent cells (IC50=10 microM) and neurospheres (IC50=16 microM), cloning efficiency after 15 d (IC 50=4 and 9 microM, respectively), inducing also, at the respective IC50 concentrations for viability, spindle aberrations up to mitotic catastrophe in almost all cells, as highlighted by means of alpha-tubulin immunofluorescent staining. A Phase II clinical trial involving CPZ, in addition to the adjuvant phase of the Stupp protocol, in GBM patients carrying a hypo-methylated MGMT gene is now ongoing.

CONCLUSION

We proved the effectiveness of CPZ in potently hindering GBM cell growth, without sparing cancer stem cells. This last characteristic is important in order to overcome disease relapse and drug resistance. Recently, many drugs have been repositioned as potentially useful in GBM and for many of these, such as metformin, disulfiram, chloroquine, sirolimus and others, there are clinical trials that challenge them individually combined with the Stupp protocol. On the basis on our results, we consider the choice to undertake a Phase II clinical trial adding CPZ to TMZ in the adjuvant phase of the Stupp protocol as a rational and ethical option to be pursued
P11.52 Reduced expression of proteolipid protein 2 increases ER-stress-induced apoptosis and autophagy in glioblastoma
Authors: Feng Z , Zhou W , Wang J , Huang B , Chen A , Zhang D , Bjerkvig R , Wang J , Thorsen F , Li X
Published: September 2019

BACKGROUND

The PLP2 gene encodes for the Proteolipid protein 2 (PLP2) which is an integral ion channel membrane protein of the endoplasmic reticulum. The protein has been shown to be involved in several human cancers, but the importance of PLP2 in gliomas is poorly understood. In the present study, we therefore investigated the role of PLP2 in human glioma development.

MATERIAL AND METHODS

Immunohistochemistry was carried out for paraffin-embedded glioma samples, and changes in protein level were detected by western blot analysis. Small interfering RNA transfections were used for knockdown of specific genes. Cell viability and proliferation was then assessed by CCK-8 assay and EdU assay. Transmission electron microscopy was used to observe the ultrastructure of the cells and flow cytometry for the evaluation of apoptosis. Finally, U87 and U251 cells were treated with lentiviral transduction to obtain stably PLP2-knockdown cell lines, which were used for an in vivo study.

RESULTS

Data from publicly available datasets (Rembrandt, TCGA and CGGA) showed a correlation between up-regulation of PLP2 levels and increased malignancy. This was confirmed by IHC staining of sections from our own clinical glioma samples. Mechanistically, down-regulation of PLP2 in U87 and U251 glioma cell lines decreased the proliferation and increased apoptosis and autophagy, mediated by ER-stress. In PLP2 knockdown U87 and U251 cells, autophagy inhibition by chloroquine (CQ) augmented apoptotic cell death. Finally, orthotopic U87-shPLP2 and U251-shPLP2 intracranial xenograft models revealed that down-regulating of PLP2 inhibited glioma development in vivo.

CONCLUSION

In conclusion, the results indicate that PLP2 expression is related to glioma progression, and could be a potential target for future treatment strategies.
P11.53 Genotype trumps phenotype: Oligodendroglioma and oligodendroglioma like tumors- A 1p19q fluorescence in situ hybridization based study at a tertiary referral centre in north India
Authors: Dewan A , Kini L , Sharma S , Bhardwaj M
Published: September 2019

BACKGROUND

The 2016 WHO update of CNS tumours defines oligodendroglioma as a diffusely infiltrating glioma with IDH 1 or IDH 2 mutation and co-deletion of chromosomal arms 1p and 19q. We evaluated the role of 1p19q testing by Fluorescence in situ hybridization (FISH) in the diagnosis and correct classification of CNS tumours in accordance with WHO 2016 classification and exclusion of morphological mimickers of oligodendrogliomas.

MATERIAL AND METHODS

A retrospective analysis of CNS tumours was carried out that were tested for 1p19q co-deletion by FISH over a period of fourteen months at our institute. Correlation with tumour morphology as well as other molecular tests (IDH mutation and MGMT) where available was then done. A tumour was considered to have 1p or 19q deletion when the 1p probe to 1q probe ratio (1p/1q) or the 19q probe to 19p probe ratio (19q/19p) was <0.80.

RESULTS

A total of 125 cases underwent FISH testing for 1p19q co-deletion over a period of last fourteen months at our institute with 74 male patients and 51 women. Thirty-eight of the 125 evaluated patients demonstrated 1p19q co-deletion on FISH. These included 25 oligodendrogliomas, 9 astrocytomas (Grade II/III) and 4 cases of oligoastrocytomas by morphology. Out of these cases, 21 cases had a co-existent IDH mutation, while two cases were IDH wild type and for 15 cases the status was unknown. Rest of the 87 patients were negative for 1p19q co-deletion and included cases of oligodendroglioma, other gliomas (pilocytic astrocytoma, oligoastrocytoma, glioblastoma) and few glioneuronal tumours (ganglioglioma, dysembryoplastic neuroepithelial tumour and central neurocytoma).

CONCLUSION

1. Analysis of 1p19q co-deletion by FISH plays an important role in correctly identifying cases of oligodendroglioma as shown in our study. This is important as it is both a predictive as well as prognostic biomarker 2. In cases with co-deletion but IDH wild type status, the 1p19q status should be confirmed by other methods such as comparative genomic hybridization to confirm the presence of whole chromosome loss.3. FISH analysis also helps in excluding morphologic mimics of oligodendroglioma such as glioneuronal tumours and pilocytic astrocytomas in young patients. 4. However, in a resource limited country like ours, a good morphological diagnosis should still form the basis for FISH and other molecular tests to avoid unnecessary testing.
P11.60 Neurosurgical delivery of the poly ADP ribose polymerase-1 inhibitor olaparib from a thermo-responsive biodegradable paste potentiates radiotherapy and prolongs survival
Authors: Smith S , Serra R , Rowlinson J , Gorelick N , Veal G , Shakesheff K , Brem H , Grundy R , Tyler B , Rahman R
Published: September 2019

BACKGROUND

There has been considerable interest in repurposing the poly ADP ribose polymerase inhibitor and purported radiosensitiser olaparib (Lynparza), with a recent dose escalation study of olaparib plus temozolomide in recurrent GBM showing good tolerance. Due to systemic therapy-associated caveats such as dose-limiting toxicities and blood-brain-barrier penetration, here we assess localised post-surgical delivery of olaparib from our previously developed PLGA/PEG thermo-sensitive biodegradable paste.

MATERIAL AND METHODS

Metabolic and clonogenic assays were used to assess effects on proliferation and clonal growth upon in vitro glioma exposure to olaparib. Flow cytometry and Annexin V/Propidium iodide were used to determine apoptosis. The 9L high-grade glioma orthotopic allograft model was utilised to assess survival upon intra-cavity olaparib delivery.

RESULTS

Metabolic and clonogenic assays revealed impaired proliferation and clonal growth respectively, upon acute exposure of high-grade glioma cells to olaparib (3–5µM), an effect dramatically potentiated with 3Gy radiation. Flow cytometry of Annexin V+/Propidium iodide+ rodent and human high-grade glioma cells, revealed a significant cell proportion increase at late stage apoptosis when exposed to 2–3µM olaparib and 3Gy radiation (relative to untreated, olaparib alone or radiation alone). A high-grade glioma orthotopic allograft study revealed a significant overall survival benefit of locally-delivered 10% and 20% w/w (drug:polymer ratio) olaparib via PLGA/PEG paste post-surgery with adjuvant radiotherapy, compared to surgery/oral temozolomide/radiotherapy (GBM standard-of-care) and surgery/systemic olaparib (95 vs. 44 vs. 30 days respectively). A more pronounced survival benefit, as measured by number of animals surviving long-term, was observed with combined PLGA/PEG/olaparib/temozolomide/radiotherapy or PLGA/PEG/olaparib/etoposide/radiotherapy, relative to standard-of-care (95 vs. 44 days). Clinical correlation was determined using RNAseq data from 10 GBM patients, showing significantly elevated levels of apoptosis-inducing factor-1 in 5-aminolevulinic acid (5ALA)+ fluorescence-activated cell sorted populations (i.e. purified tumour cells from the invasive margin), relative to 5ALA- cells, confirming PARP-1 activity in infiltrative tumour cells.

CONCLUSION

Collectively our data supports a clinical rationale for localised olaparib delivery with adjuvant radiotherapy.
P11.63 PI3K inhibition in conjunction with the ketogenic diet reduces growth and neuroinflammation in pediatric high-grade glioma
Authors: Noch E , Yim I , Cantley L
Published: September 2019

Background: Pediatric high-grade glioma remains a poorly treatable disease with high mortality.Therapeutic advances have lagged behind that of adult glioblastoma, due to small patient numbers, inappropriate generalization from adult tumor types, and unique biology. The use of targeted therapy has recently gained interest in this disease, but efficacy is limited by therapeutic resistance, often as a result of tumor heterogeneity. In the case of phosphatidylinositol 3-kinase (PI3K) inhibition, clinically relevant PI3K inhibitors represent a strong class of drugs for pediatric high-grade glioma, but their use is associated with insulin feedback that reactivates the PI3K pathway and drives therapeutic resistance. Here, we target insulin feedback that is the primary mechanism of PI3K inhibitor-related therapeutic resistance in pediatric high-grade glioma using the ketogenic diet.

Materials and Methods: Patient-derived pediatric high-grade glioma stem cells were treated with vehicle or the pan-PI3K inhibitor, BKM-120, with glucose deprivation or phenformin to decrease glucose utilization. NSG mice containing patient-derived pediatric high-grade glioma xenografts were treated with vehicle or BKM-120 on a regular or ketogenic diet to determine whether reducing insulin feedback increases BKM-120 efficacy. To determine the effect of glioma cells on neuro-inflammation, we measured pro-inflammatory cytokines in glioma cells treated with BKM-120 and phenformin in comparison to vehicle-treated cells. We then applied conditioned medium from glioma cells treated with BKM-120 and phenformin to cortical neuronal cultures to measure oxidative stress and neuro-inflammation. Results: Pediatric high-grade glioma cells exhibited increased toxicity when exposed to BKM-120 with glucose deprivation or phenformin. Furthermore, mice with intracranial high-grade glioma xenografts survived longer when treated with BKM-120 on the ketogenic diet than with BKM-120 or the ketogenic diet alone. Phenformin reduced the production of pro-inflammatory cytokines by BKM-120-treated glioma cells. Cortical neurons treated with conditioned medium from BKM-120- and phenformin-treated glioma cells exhibited less oxidative stress than those treated with BKM-120 alone. Our results demonstrate that strategies to lower glucose utilization and insulin feedback increase efficacy of BKM-120 and decrease neuro-inflammation.

Conclusions: We show that strategies to lower glucose utilization and insulin feedback increase efficacy of BKM-120. Furthermore, reducing insulin feedback decreases the production of pro-inflammatory cytokines in tumor cells and reduces oxidative stress in neurons treated with conditioned medium from BKM-120-treated glioma cells. By using the ketogenic diet to reduce glucose levels, this strategy may enhance efficacy of PI3K inhibitors in this patient population.
P12.01 Local implantation of thermoresponsive interferon alpha-polypeptide conjugate combined with temozolomide for post-surgical glioblastoma chemoimmunotherapy
Authors: Liang P , Zhuoran W , Weiping G , Guihuai W
Published: September 2019

BACKGROUND

Glioblastoma(GBM) is associated with gloomy prognosis despite maximal safe resection and following chemoradiation. In recent years, significant progress has been made in cancer immunotherapy except for GBM, which is largerly due to its local immunosuppressive microenvironment. Some research has demonstrated that local immunotherapy may be more efficient than systematic administrators. IFNα has been investigated as antitumor agent for some cancers, including glioma. Nevertheless, the short circulating half-life greatly limits its clinical application. Hence, a thermoresponsive IFNα-elastin-like polypeptide (IFNα-ELP) was genetically engineered for the earliest local intervention post-surgery.

MATERIAL AND METHODS

Firstly, IFNα-ELP(V) was constructed, expressed and purified, then, its physicochemical characterization was verified. The tumor was resected 10 days after U87MG-mCherry-luc cells orthotopic implantation, and IFNα-ELP(V) was injected into the resection cavity. Two days later, temozolomide(TMZ) was intraperitoneally injected. Using in vivo imaging technique, we could monitor the trends in tumor size. The survival time of mice was counted. Biosafety was evaluated by peripheral blood biochemistry analysis and pathology of the organs.

RESULTS

In this study, the bioconjugate not only in situ deposited in the resection cavity because of the thermoresponsive characteristic, but also showed zero-order release kinetics from the depot and dramatically improved pharmacokinetics and biodistribution of IFNα. Consequently, it showed the inhibition of tumor relapse in GBM orthotopic resection mice models. When followed by TMZ intraperitoneal injection, IFNα-ELP(V) could significantly prevent the tumor recurrence than itself or TMZ alone. Biosafety results indicated that the systemic toxicity of IFNα-ELP(V) in mice can be reduced to safe levels.

CONCLUSION

The results reveal that local implantation of thermoresponsive IFNα-ELP(V) combined with TMZ exhibits the synergy of post-surgical GBM chemoimmunotherapy.
P12.07 Minimal bimodular G-quadruplex nano-construct as cryptic therapeutic DNA oligonucleotide to suppress glioblastoma cell functioning
Authors: Kopylov A , Antipova O , Legatova V , Samoylenkova N , Savchenko E , Golovin A , Zavyalova E , Pavlova G
Published: September 2019

BACKGROUND

G-quadruplex folding oligodeoxyribonucleotides (G4 ODNs) are considers to be potential anticancer agents, because G4 interferes with some functions of cell, like survival, proliferation, ets, by interacting with G4 binding regulatory proteins. There are some experimental data on anti-proliferative activities of G4: for telomerase complex, aptamers for STAT3, nucleolin, TOP1, SP1, VEGF, and SHP-2, but there is no molecular mechanism yet. Because ODNs are supposed to block regulatory proteins, like cryptic aptamers do, they could be potentially therapeutics, like monoclonal antibodies. The only current approach to find proper model for G4 plus reference cell line, is to screen sets of G4 for sets of cancer cell lines. To avoid selection from large number of G4 structures, we have initiated anti-proliferative study of minimal DNA nano-construct biHD1, built from two minimal active G4 15-mer, by testing 7 human cancer cell lines, including glioblastoma (GBM): U87 and primary cultures from GBM surgery samples.

MATERIAL AND METHODS

ODN were from Evrogen (Russia). ODN were folded: heated to 950C, slowly cooled to 250C. biHD1, GGTTGGTGTGGTTGG TGGTTGGTGTGGTTGG, and derivatives with G4 structure variations. CD in saline phosphate, pH 7.5; CHIRASCAN (UK) and MARK-5 (France); melting curves at 294 nm. 7 human cancer cell lines: colon, HCT116; prostate, PC3; lung, RL-67; breast adenocarcinoma, MCF7; melanoma, mS; HeLa; GBM, U87 and primary cultures from surgery samples; and human embryonic fibroblast, hEF, as control. MTT for cell viability by colorimetric measurements of metabolic activity (Tecan plate reader, Switzerland). ODN: 0.10 μM, 1.0 μM, 10 μM for 3 days.

RESULTS

biHD1 is dose-dependent toxic for several human cancer cell lines, most active for lung cancer RL-67 and GBM U87, survival at 10 μM ODN 35% and 45%, respectively. According to data for biHD1 derivatives with deviations from G4 conformation: length of linker joining modules, light distortions of loops, strong distortions of G4, freezing of G4 conformation, it is G4 fold which is responsible for toxicity for U87 cell line.

CONCLUSION

Cell survival after treatment with G4 minimal bimodular DNA oligonucleotide biHD1 and its derivatives have been tested for 7 human cancer cell lines, including CNS, U87. biHD1 is toxic for cancer cells, but not for embryonic fibroblast, as control; activity depends on G4 fold. Therefore, it is possible to manipulate with minimal G4 as module, and build G4 nano-construct being toxic for cancer cells, including GBM, U87.

Research is supported by RFBR grants 17-00-00157, 18-29-01047
P12.09 Multidimensional Personalized Response Assessment to Microglia Modulators in Gioblastoma Bioreactors
Authors: Shekarian T , Wachnowicz A , Flammer J , Paganetti C , Martins T , Muraro M , Schürch C , Hutter G
Published: September 2019

BACKGROUND

Recently, strategies harnessing the non-neoplastic, immune tumor microenvironment (iTME) consisting of myeloid-derived macrophages and yolk sac derived microglia (termed TAMs) as well as adaptive immune components have been employed to treat glioblastoma (GBM). To evaluate the effect of TAM-modulating therapies in combination with T-cell checkpoint inhibitor approaches, we generated 3D GBM bioreactor cultures from patient-derived samples. Here, we report patient-tailored, tumor region specific response assessment to microglia modulators and T-cell checkpoint inhibitors using multidimensional fluorescent microscopy techniques

MATERIAL AND METHODS

GBM tissue fragments from the tumor center and periphery were placed into perfusion bioreactors shortly after resection and cultured for up to 3 weeks. Control conditions included non-perfused cultures of the same tissue. Cultures were treated with combinations of TAM and T-cell modulating, FDA approved drugs including anti-PD1, anti-CTLA4 and anti-CD47 antibodies. Tissue was harvested for histology, RNA extraction, and supernatants were processed for multiplexed cytokine analysis. Multidimensional CODEX technology analysis using a customized TAM/microglia-centric 50 marker panel was implemented, and a map of individualized response criteria to specific immunotherapies developed.

RESULTS

We were able to cultivate viable GBM tissue with intact iTME. Tumor cell proliferation and invasion capacity were preserved for up to 3 weeks. Conventional immunohistochemistry confirmed the presence of TAMs and T cells. Treatment with immunomodulators resulted in a profound polarization shift of TAMs. Furthermore, cytokine analysis confirmed proinflammatory immune responses in most assessed samples. We present preliminary data of the CODEX analysis of our combinatorial immunotherapies in a series of 8 patient-specific explant samples.

CONCLUSION

GBM tissue could be incubated in the perfused 3D bioreactor model and kept viable for up to 21 days. The proposed model allows patient-tailored testing of immunomodulatory drugs by taking into account the patients individual iTME response. GBM tissue could be incubated in the perfused 3D bioreactor model and kept viable for up to 21 days. The proposed model allows patient-tailored testing of immunomodulatory drugs by taking into account the patients individual iTME response.
P13.01 TERTpromoter methylation is significantly associated withTERTupregulation and tumor progression in pituitary adenomas
Authors: Adachi J , Miyake Y , Suzuki T , Mishima K , Araki R , Nishikawa R
Published: September 2019

BACKGROUND

Activation of telomerase plays a critical role in tumor development. Somatic alterations in the promoter of the telomerase reverse transcriptase (TERT) gene are a major mechanism of upregulating telomerase. Several mechanisms have been associated with TERT abnormalities, including TERT promoter mutations or methylation. Mutations in the TERT promoter have been observed in a subset of brain tumors, especially in adult gliomas. In pituitary adenomas (PAs), however, TERT abnormalities are not fully understood. The present study aimed to investigate not only mutational but also methylation status changes in the TERT promoter in PAs and to analyze their correlations with clinical variables.

MATERIAL AND METHODS

We retrospectively studied 70 PAs consisting of 53 primary and 17 recurrent samples. Clinical data, including age at surgery, sex, tumor size, tumor subtype, resection rate, presence or absence of postoperative irradiation, and progression-free survival (PFS), were obtained from medical records. First, we investigated TERT promoter hotspot mutations via Sanger sequencing. Next, we quantified the methylation status of the TERT promoter using methylation-sensitive high-resolution melting analysis (MS-HRM). Finally, we investigated TERT mRNA expression levels using real-time quantitative PCR. Fisher’s exact test was applied to evaluate the statistical significance between TERT promoter methylation status and tumor recurrence. PFS was calculated using Kaplan-Meier estimates and compared between methylated with ummethylated PAs with the log-rank test. The correlation between TERT promoter methylation status and mRNA levels was analyzed with the Mann-Whitney U-test. PFS was analyzed using multivariate analysis with the Cox proportional hazards model and included the following variables: age, sex, tumor size, tumor subtype, resection rate, radiation therapy, and methylation status.

RESULTS

TERT promoter hotspot mutations were not observed in any PA sample. Nineteen percent of PAs exhibitedTERT promoter methylation, which was significantly predominant in recurrent PA samples. PFS was significantly shorter in the methylated cases than in the unmethylated cases. Higher TERT expression levels were correlated with methylation status.

CONCLUSION

We found that TERT promoter methylation upregulated TERT expression and was associated with shorter PFS in PAs. Our results suggest thatTERT promoter methylation may be a potential biomarker for predicting tumor recurrence in PAs.
P13.03 Identification of novel gene fusions in glioblastomas with chromothripsis
Authors: Ah-Pine F , Casas D , Boisselier B , Guardiola P , Menei P , Garcion E , Rousseau A
Published: September 2019

BACKGROUND

Genomic rearrangements are thought to occur in a stepwise manner during cancer development. Recent findings, however, suggest an alternative mechanism, involving massive chromosome (chr) rearrangements in a one-step catastrophic event termed chromothripsis (CT). In this scenario, the chr shatters into tens to hundreds of pieces, some of which are then stitched together in a mosaic patchwork of genomic fragments. Such random stitching may give rise to fusion genes with oncogenic potential. CT has not been thoroughly studied in diffuse gliomas, such as glioblastomas (GB). The aim of the project was to detect CT in GB and identify novel fusion genes in CT regions. Those genes are potential therapeutic targets.

MATERIAL AND METHODS

170 de novo IDH wild-type GB were screened for CT patterns using whole genome SNP arrays. RNA sequencing was performed in 52 cases with CT features to identify novel fusion genes within the rearranged genomic regions. Expression levels of transcripts were measured using FPKM (fragments per kilobase of exon per million fragments mapped) normalization method. Candidate fusion genes were validated by RT-PCR followed by Sanger sequencing.

RESULTS

Out of 66 GB with CT patterns (66/170, 38.8%), 40 cases (out of 52 RNA-sequenced cases, 77%) harbored at least one fusion gene within CT regions, which were mostly detected on chr 7, 9, and 12. Overall, 137 candidate fusion genes were identified, 26 of which with a predicted open reading frame. Twelve out of 26 fusions were validated by RT-PCR followed by Sanger sequencing (validation of the remaining fusions is ongoing). The fusions involved either well-known key oncogenes such as EGFR, MDM2, and MET or potential oncogenes (CPM, PPM1H, SEPT14, SEC61G) with a yet-to-discover role in GB pathogenesis. GB with CT on chr 12 (11/66, 16.7%) showed a distinct transcript expression profile at the locus 12q13-15, particularly in the vicinity of MDM2 and CDK4 genes. In our cohort, this so-called “multi-aberration region” recurrently harbored gene fusions, involving, among others, CPM (12q15). CPM, encoding an extracellular membrane-bound carboxypeptidase, is recurrently amplified in liposarcoma and is a potential oncogene involved in the EGFR signaling pathway. Three cases (3/26, 11.5%) harboring an in-frame gene fusion involving CPM showed higher levels of CPM transcripts and did not exhibit any EGFR amplification. Gene fusions involving CPM might constitute an alternative mechanism of EGFR pathway activation in de novo IDH wild-type GB.

CONCLUSION

The occurrence of CT points to underlying gene fusions in IDH wild-type GB. Such fusions may drive tumorigenesis and represent promising therapeutic targets. We identified CPM as a recurrent fusion partner and a potential alternative mechanism of EGFR signaling pathway activation in de novo IDH wild-type GB.
P13.06 Transcriptome-wide Mendelian randomization study to identify brain-specific causal genes influencing glioma
Authors: Robinson J W , Zheng J , Tscavachidis S , Howell A E , Relton C L , Armstrong G N , Bondy M , Martin R M , Kurian K M
Published: September 2019

BACKGROUND

The drug treatment regimen for glioma has remained relatively static since the introduction of temozolomide, although new drugs and drug combinations are being trialled. The human transcriptome can provide promising insights into causal genes as potential drug interventions for glioma treatment and may guide drug discovery methods.

MATERIAL AND METHODS

We apply two sample Mendelian randomisation and colocalisation to explore the influence of genetically-predicted gene expression across 12 tissue types located in the brain (4,554 genes from GTEx) and whole blood (16,112 genes from eQTLGen) on glioma risk (5,739 cases, 5,501 controls from a meta-analysis of GICC and MDA glioma GWAS). We used the MR-Base R package to conduct these analyses.

RESULTS

We identify 9 genes whose genetically-predicted expression was strongly associated with glioma risk. Of these genes, 7/9 are shown to have tissue-specific expression while the other two genes showed an association with glioma across multiple brain tissues and whole blood. For example, JAK1, involved in the well-known JAK-STAT pathway, is found in the frontal cortex (OR=1.49 for glioma per standard deviation increase in gene expression; 95% CI: 1.28 to 1.73; P=1.79 × 10−7), the cerebellar hemisphere (OR=1.32; 95% CI: 1.19 to 1.47; P=2.64 × 10−7), the cerebellum (OR=1.27; 95% CI: 1.16 to 1.39; P=2.64 × 10−7) and the cortex (OR=1.38; 95% CI: 1.22 to 1.57; P=2.64 × 10−7). This pathway has been highlighted in previous research as a potential intervention target for glioma therapies. We found that 5/9 of the genes from the MR analysis are expressed in the cerebellum. However malignant cerebellar glioma is a rare tumour (~3% of all malignant gliomas). This suggests that tumourigenesis elsewhere in the brain may be affected by other tissue-specific genes, specifically in the cerebellum, though this will require further research to elucidate. We further triangulate the MR findings with evidence from the OpenTargets platform to strengthen the putative causal associations. OpenTargets aims to “generate evidence on the validity of therapeutic targets based on genome-scale experiments and analysis”. For example, JAK1 receives an overall OpenTargets score of 0.89 out of 1, with most of the evidence for this JAK1-glioma association coming from affected pathways data.

CONCLUSION

This study has combined genetic epidemiological approaches to the analysis of the human transcriptome on glioma incidence. We provide evidence that these genes may inform putative drug targets for tertiary treatment of glioma. Future research specifically towards this aim will be required to fully elucidate intervention targets.
P13.09 Genomic analysis of paired IDHwt glioblastoma (GB) reveals recurrent alterations of MPDZ at relapse after radiotherapy and temozolomide (RTCT)
Authors: Chanez B , Appay R , Guille A , Lagarde A , Bequet C , Jiguet-Jiglaire C , Graillon T , Dufour H , Figarella-Branger D , Chinot O , Tabouret E
Published: September 2019

BACKGROUND

GB are highly aggressive tumors which systematically relapse. Our objective was to identify disease progression mechanisms and genomic drivers of GB treatment resistance.

MATERIAL AND METHODS

Ten paired frozen tumors from initial and recurrent surgery after RTCT were screened by CGH Array. Next, NGS of the selected genes was performed on 19 paired tumors (38 samples). Molecular alterations were correlated with patient data. TCGA was used to characterize the molecular profile of MPDZ.

RESULTS

Nineteen IDHwt GB patients with a median age of 54.5 years (37.2–72.8) were included. Using CGH array, unsupervised analysis clustered the whole samples by paired of initial and recurrent tumors. However only 44% of CGH Array alterations were shared between initial and recurrent tumors (amplifications: 55%; deletions: 30%). The new alterations detected at relapse were amplifications in 25% and deletions in 23% of tumors. Two regions corresponding to 171 genes were lost at relapse (p=0.03): 19q13.33 and 19q13.41. Using DAVID genome, 3/171 genes (related to neutrophil chemotactic factors) were identified: FPR1, FPR2, FPR3. Moreover, 24 genes were lost (including MPDZ) and 2 genes were gained in 20% of recurrent tumors. Totally, 29 genes were analyzed by NGS and 4 genes showed pathogenic mutations shared by initial and recurrent tumors: FPR2, REL, TYRP1 and MPDZ. Only MPDZ showed, at relapse, an increasing rate of mutated variants and a new mutation affecting the splicing site. These alterations were independent from classical prognostic factors (age, sexe, karnofsky performans status, MMS and MGMT status) and from patient survivals. To explore MPDZ expression, we used TCGA initial dataset and observed that a lower RNA expression of MPDZ was associated with IDHwt (p<0.001) and grade IV (p<0.001) gliomas, reinforcing the potential pejorative impact of MPDZ loss.

CONCLUSION

Our results suggest that MPDZ is frequently altered at initial diagnosis with increased alterations in recurrent IDHwt GB after RTCT, suggesting that MPDZ impairment could contribute to the resistance/relapse mechanisms. Further investigations are needed to validate these results. Our results suggest that MPDZ is frequently altered at initial diagnosis with increased alterations in recurrent IDHwt GB after RTCT, suggesting that MPDZ impairment could contribute to the resistance/relapse mechanisms. Further investigations are needed to validate these results.
P14.17 Differentiation of treatment-related changes from tumor progression: A direct comparison between dynamic FET PET and ADC values obtained from DWI MRI
Authors: Werner J , Stoffels G , Lichtenstein T , Borggrefe J , Lohmann P , Ceccon G , Fink G R , Langen K , Kabbasch C , Galldiks N
Published: September 2019

BACKGROUND

Following brain cancer treatment, the capacity of anatomical MRI to differentiate neoplastic tissue from treatment-related changes (e.g., pseudoprogression) is limited. This study compared apparent diffusion coefficients (ADC) obtained by diffusion-weighted MRI (DWI) with static and dynamic parameters of O-(2-[18F]fluoroethyl)-L-tyrosine (FET) PET for the differentiation of treatment-related changes from tumor progression.

MATERIAL AND METHODS

Forty-eight pretreated high-grade glioma patients with anatomical MRI findings suspicious for progression (median time elapsed since last treatment, 16 weeks) were investigated using DWI and dynamic FET PET. Maximum and mean tumor-to-brain ratios (TBRmax, TBRmean) as well as dynamic parameters (time-to-peak and slope values) of FET uptake were calculated. For mean ADC calculation, regions-of-interest analyses were performed on ADC maps calculated from DWI co-registered with the contrast-enhanced MR image. Diagnoses were confirmed neuropathologically (21%) or clinicoradiologically. Diagnostic performance was evaluated using receiver-operating-characteristic analyses or Fisher’s exact test for a combinational approach.

RESULTS

Ten of 48 patients had treatment-related changes (21%). The diagnostic performance of FET PET was significantly higher (threshold for both TBRmax and TBRmean, 1.95; accuracy, 83%; AUC, 0.89±0.05; P<0.001) than that of ADC values (threshold ADC, 1.09x10-3 mm2/s; accuracy, 69%; AUC, 0.73±0.09; P=0.13). The addition of static FET PET parameters to ADC values increased the latter’s accuracy to 89%. The highest accuracy was achieved by combining static and dynamic FET PET parameters (93%).

CONCLUSION

Data suggest that static and dynamic FET PET provide valuable information concerning the differentiation of early treatment-related changes from tumor progression and outperform ADC measurement for this highly relevant clinical question.
P14.21 Tehila Kaisman-Elbaz MD/PhD
Authors: Kaisman-Elbaz T , Elbaz Y , Merkin V , Dym L , Noy A , Atar-Vardi M , Bari R , Turiel S , Lavrenkov K , Melamed I
Published: September 2019

BACKGROUND

Glioblastoma is known for its dismal prognosis though its dependency on patients’ readily available RBCs parameters defining the patient’s anemic status such as hemoglobin level and Red blood cells distribution Width (RDW) is not fully established. Several works demonstrated a connection between low hemoglobin level or high RDW values to overall glioblastoma patient’s survival, but in other works, a clear connection was not found. This study addresses this unclarity.

MATERIAL AND METHODS

In this work, 170 glioblastoma patients, diagnosed and treated in Soroka University Medical Center (SUMC) in the last 12 years were retrospectively inspected for their survival dependency on pre-operative RBCs parameters using multivariate analysis followed by false discovery rate procedure due to the multiple hypothesis testing. A survival stratification tree and Kaplan-Meier survival curves that indicate the patient’s prognosis according to these parameters were prepared.

RESULTS

Beside KPS>70 and tumor resection supplemented by oncological treatment, age<70 (HR=0.4, 95% CI 0.24–0.65), low hemoglobin level (HR=1.79, 95% CI 1.06–2.99) and RDW<14% (HR=0.57, 95% CI 0.37–0.88) were found to be prognostic to patients’ overall survival in multivariate analysis, accounting for false discovery rate of less than 5%.

CONCLUSION

A survival stratification highlighted a non-anemic subgroup of nearly 30% of the cohort’s patients whose median overall survival was 21.1 months (95% CI 16.2–27.2) - higher than the average Stupp protocol overall median survival of about 15 months. A discussion on the beneficial or detrimental effect of RBCs parameters on glioblastoma prognosis and its possible causes is given.
P14.25 Venous thromboembolic events in patients with brain metastases: the PICOS score
Authors: Wolpert F , Grossenbacher B , Lareida A , Roth P , Neidert M C , Andratschke N , Le Rhun E , Weller M
Published: September 2019

BACKGROUND

Venous thromboembolic events are significant complications in patients and possibly associated with an unfavorable outcome. Thrombosis risk is poorly defined for patients with brain metastasis, and available risk calculation scores are not validated for these patients.

MATERIAL AND METHODS

We identified 811 patients with brain metastasis followed at our institution and screened electronic charts retrospectively for the occurrence of venous thromboembolic events, along with candidate risk factors. Risk factors were tested in uni- and multivariate analyses and finally integrated in a score model for risk prediction.

RESULTS

Venous thromboembolic events were documented in 97 of 811 patients (12.0%). Primary tumors with high thrombogenicity (p=0.02, odds ratio 1.7, 95% CI 1.1–2.8), dexamethasone (p=0.011, odds ratio 2.27, 95% CI 1.5–4.5), chemotherapy (p=0.005, odds ratio 3.4, 95% CI 1.6–7.5), BMI > 35 kg/m2 (p=0.002, odds ratio 3.4, 95% CI 1.6–7.5) and immobilization (p=0.003, odds ratio 2.4, 95% CI 1.3–4.3) were confirmed as independent predictors of VTE. We derived a score model for venous thromboembolic event prediction, the PICOS (thrombogenic Primary, Immobilization, Chemotherapy, Obesity, Steroids) score (0–7 points). Receiver Operating Characteristic Curve Analysis demonstrated its prognostic accuracy (AUC=0.71, 95% CI 0.64–0.77), and its predictive capability was superior to that of other scores proposed for the evaluation of venous thromboembolic event risk such as the Khorana (AUC=0.51) or CONKO (AUC=0.52) scores.

CONCLUSION

We report a rate of venous thrombotic events of 12.0% in our cohort of 811 patients with brain metastasis. We define a risk model for prediction in of venous thrombotic events in patients with BM, the PICOS score. It may become a valuable tool for the identification of brain metastasis patients at high risk for venous thromboembolic events and be helpful for guidance of clinicians towards decision whether to start thrombosis prophylaxis. Further, the PICOS score might be used for stratification in controlled studies.
P14.32 Spatial discrepancies between FET PET and conventional MRI in patients with newly diagnosed glioblastoma
Authors: Lohmann P , Stavrinou P , Lipke K , Bauer E K , Ceccon G , Werner J , Fink G R , Shah N J , Langen K , Galldiks N
Published: September 2019

BACKGROUND

In patients with glioblastoma, the tissue showing contrast enhancement (CE) in MRI is usually the target for resection or radiotherapy. However, the solid tumor mass typically extends beyond the area of CE. Amino acid PET can detect tumor parts that show no CE. We systematically investigated tumor volumes delineated by amino acid PET and MRI in newly diagnosed, untreated glioblastoma patients.

MATERIAL AND METHODS

Preoperatively, 50 patients with subsequently neuropathologically confirmed glioblastoma underwent O-(2-[18F]-fluoroethyl)-L-tyrosine (FET) PET, fluid-attenuated inversion recovery (FLAIR), and CE MRI. Areas of CE were manually delineated. FET PET tumor volumes were segmented using a tumor-to-brain ratio ≥ 1.6. The percentage of overlapping volumes (OV), as well as Dice and Jaccard spatial similarity coefficients (DSC; JSC), were calculated. FLAIR images were evaluated visually.

RESULTS

In 86% of patients (n = 43), the FET PET tumor volume was significantly larger than the volume of CE (21.5 ± 14.3 mL vs. 9.4 ± 11.3 mL; P < 0.001). Forty patients (80%) showed both an increased uptake of FET and CE. In these 40 patients, the spatial similarity between FET and CE was low (mean DSC, 0.39 ± 0.21; mean JSC, 0.26 ± 0.16). Ten patients (20%) showed no CE, and one of these patients showed no FET uptake. In 10% of patients (n = 5), increased FET uptake was present outside of areas of FLAIR hyperintensity.

CONCLUSION

Our results show that the metabolically active tumor volume delineated by FET PET is significantly larger than tumor volume delineated by CE. The data strongly suggest that the information derived from FET PET should be integrated into the management of newly diagnosed glioblastoma patients.

FUNDING

This work was supported by the Wilhelm-Sander Stiftung, Germany
P14.38 Clinical experience with tumor treating fields (TTFields) treatment for posterior fossa tumors
Authors: Taliansky A , Vagner T , Jan E , Zach L
Published: September 2019

Introduction: TTFields brought a new concept in the treatment of brain tumors. It was tested and recommended for use in supra-tentorial lesions. There is a paucity of data available about TTFields safety and efficacy for infra-tentorial lesion.

Method: Patients with posterior fossa tumors were treated with the Optune® system. Initial transducer array positioning and all technical characteristic were performed with technical support from Novocure.

Results: Two patients with posterior fossa glial tumors were treated. The first is a 27 years old male with DIPG (without pathological confirmation of the tumor grade) who was treated with Stupp protocol. Treatment with TTFields commenced with TMZ adjuvant phase. The second patient is a 33 years old female with cerebello-pontine astrocytoma WHO grade III, IDH I mutated. This patient was treated with partial resection and Stupp protocol. TTFields treatment was started after completing 12 TMZ adjuvant cycles with measurable stable disease. Duration and compliance of TTFields treatment for first patient is 286 days and 86% and for second patient is 168 days and 93.6%. The skin toxicity is minimal and did not require treatment interruption. There is no other TTFields related toxicity. The MRI evaluation had showed improvement in the first patient and stable disease in the second.

Conclusion: According to our knowledge, this is the first report of clinical experience with TTFields treatment for posterior fossa tumors. In our experience, the treatment is feasible and with equal toxicity to previously reported supra-tentorial lesions. Small patient population and short follow up do not allow efficacy evaluation.