bluuebunny/crossref_metadata_2025 huggingface dataset:
Introduction
Authors: Hobér Kaj
Published: December 2020

This introductory chapter provides a background of the Energy Charter Treaty, which entered into force on April 16, 1998. The ECT is a unique international instrument which covers the promotion and protection of investments, trade in energy, transit in the energy sector, environmental aspects, as well as the settlement of disputes under the Treaty. It was negotiated and drafted under considerable time pressure by a large number of States and what is now the European Union. Nevertheless, the ECT was not negotiated and drafted in a legal vacuum. Other relevant international instruments were there for the negotiators to take account of and to be guided by as they deemed appropriate. As far as investment protection is concerned, there were in place several thousands of bilateral investment protection treaties (BITs) providing for the protection of foreign investment in a manner very similar to the corresponding provisions which eventually found their way into the ECT. With respect to international trade, the General Agreement on Tariffs and Trade (GATT) was in force when the ECT negotiations commenced. It was eventually replaced by the World Trade Organization (WTO) in 1995. The present legal commentary on the ECT will not discuss general aspects of these two fields—international investment law and international trade law—in detail. Rather, an attempt has been made to limit the discussion of such general aspects—and of arbitral awards relating thereto—which are relevant for the ECT-provisions in question.
The Energy Charter Treaty
Authors: Hobér Kaj
Published: December 2020

This chapter traces the negotiating history of the Energy Charter Treaty. The genesis of the Treaty goes back to a European Council meeting held in Dublin in June of 1990. At that meeting, the Dutch Prime Minister at that time, Mr. Ruud Lubbers, presented a proposal for the creation of a European Energy Community. During 1991, the text of the ECT was negotiated and drafted by the Conference on the European Energy Charter. The Charter recognized the need for a legally binding agreement for co-operation in the energy sector. Delegations from more than fifty States were involved in the negotiations, including the USA, Russia, and most members of the Organisation for Economic Co-operation and Development (OECD), including its non-European members—Australia, Canada, Japan, and New Zealand. The various drafts of the ECT are divided into three categories: Basic Protocols; Basic Agreements; and ECT Drafts. The first ECT draft was presented on March 15, 1993. It must be kept in mind, however, that the ECT was negotiated under time pressure, as a consequence of which the language is sometimes ambiguous and unclear, bearing the hallmarks of the ambition to create a comprehensive treaty covering the entire energy sector to be agreed by a large number of participants.
Part III: Investment Promotion and Protection
Authors: Hobér Kaj
Published: December 2020

This chapter explores Part III of the Energy Charter Treaty, which sets forth the provisions on substantive protection of investments. Promotion and protection of investments are different things. Promotion of investment is concerned with attracting and permitting foreign investments. Protection of investment deals with the way in which investments must be treated, once they have been made. As a matter of policy, however, promotion and protection of investments are closely linked. That explains why the two concepts are addressed in one and the same article of the ECT: Article 10, which is entitled ‘Promotion, Protection and Treatment of Investments’. The chapter then describes the concept of fair and equitable treatment (FET). Article 11 of the ECT obliges Contracting Parties to treat key personnel of Investors in a fair way. Article 12 deals with loss of and damage to the property of Investors in situations where Article 13, concerning expropriation, is not applicable. Article 14 in essence creates a right for Investors to repatriate capital and earnings in a prompt and effective way. Article 15, a so-called subrogation clause, provides for the transfer of rights that a foreign investor may have in relation to the host State, if it has received compensation from its home state under an investment insurance or guarantee. Meanwhile, Article 16 addresses the situation when the ECT overlaps with other treaties. Lastly, Article 17 restricts the benefits of Part III of the ECT to certain categories of legal entities or Investments of Investors.
Tissue sodium stores in peritoneal dialysis and hemodialysis patients determined by sodium-23 magnetic resonance imaging
Authors: Sahinoz Melis , Tintara Supisara , Deger Serpil Muge , Alsouqi Aseel , Crescenzi Rachelle L , Mambungu Cindy , Vincz Andrew , Mason Olivia J , Prigmore Heather L , Guide Andrew , Stewart Thomas G , Harrison David G , Luft Friedrich C , Titze Jens , Ikizler Talat Alp
Published: December 2020

Background

Tissue sodium (Na+) content in patients on maintenance hemodialysis (MHD) and peritoneal dialysis (PD) was previously explored using 23Na+ magnetic resonance imaging (23NaMRI). Larger studies would provide a better understanding of Na+ stores in patients on dialysis as well as the factors influencing this Na+ accumulation.

Methods

In this cross-sectional study, we quantified the calf muscle and skin Na+ content in 162 subjects (10 PD, 33 MHD patients and 119 controls) using 23NaMRI. Plasma levels of interleukin-6 (IL-6) and high-sensitivity C-reactive protein (hsCRP) were measured to assess systemic inflammation. Sixty-four subjects had repeat 23NaMRI scans that were analyzed to assess the repeatability of the 23NaMRI measurements.

Results

Patients on MHD and PD exhibited significantly higher muscle and skin Na+ accumulation compared with controls. African American patients on dialysis exhibited greater muscle and skin Na+ content compared with non–African Americans. Multivariable analysis showed that older age was associated with both higher muscle and skin Na+ and male sex was associated with increased skin Na+ deposition. Greater ultrafiltration was associated with lower skin Na+ in patients on PD (Spearman’s ρ = −0.68, P = 0.035). Higher plasma IL-6 and hsCRP levels correlated with increased muscle and skin Na+ content in the overall study population. Patients with higher baseline tissue Na+ content exhibited greater variability in tissue Na+ stores on repeat measurements.

Conclusions

Our findings highlight greater muscle and skin Na+ content in dialysis patients compared with controls without kidney disease. Tissue Na+ deposition and systemic inflammation seen in dialysis patients might influence one another bidirectionally.
BIOM-22. EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) AS A MOLECULAR DETERMINANT OF GLIOBLASTOMA RESPONSE TO DOPAMINE RECEPTOR 2 (DRD2) INHIBITORS
Authors: He Yuyu , Li Jie , Koga Tomoyuki , Ma Jun , Dhawan Sanjay , Suzuki Yuta , Furnari Frank , Prabhu Varun , Allen Joshua , Chen Clark
Published: December 2020

BACKGROUND

There are ongoing clinical trials exploring the efficacy of dopamine receptor 2 (DRD2) inhibition against glioblastomas, the most common primary brain tumor. Here we examine potential molecular determinants of this efficacy.

METHODS

The Cancer Genome Atlas (TCGA) glioblastoma database and other published mRNA profiles were used to analyze the DRD2 and EGFR expression pattern. In vitro and in vivo responses to DRD2 inhibitors were determined using patient derived xenograft (PDX) glioblastoma models. Immunohistochemical studies were performed on clinically annotated glioblastoma samples derived from patients treated with ONC201, a DRD2 inhibitor.

RESULTS

Analysis of clinical glioblastoma specimens derived from independent patient cohorts revealed an inverse correlation between EGFR and DRD mRNA expression, with implication that signaling mediated by these proteins shares overlapping functions. In independent panels of PDX glioblastoma lines, high EGFR expression was associated with poor in vitro and in vivo response to DRD2 inhibitors, including haloperidol and ONC201. Moreover, ectopic expression of a constitutively active EGFR, EGFRvIII, suppressed glioblastoma sensitivity to ONC201. DRD2 expression positively correlated with expression of rate-limiting enzymes for dopamine synthesis as well as dopamine secretion, suggesting contribution of autocrine DRD2 signaling. Analysis of specimens from patients treated with ONC201 (n = 15) showed an inverse correlation between the intensity of EGFR staining and clinical response. The median overall survival for patients with high and low EGFR staining was 162 and 373 days, respectively (p = 0.037).

CONCLUSIONS

High EGFR expression is a determinant of poor glioblastoma response to DRD2. This finding should inform future clinical trial designs.
BIOM-23. MOLECULAR PROFILING IDENTIFIES NOVEL BIOMARKERS IN A RURAL COHORT OF PATIENTS WITH GLIOBLASTOMA
Authors: Bystrom Rebecca , Craig Wendy , Rueter Jens , Lu-Emerson Christine
Published: December 2020

BACKGROUND

The rural state of Maine has one of the highest incidences of primary malignant brain cancer in the United States. We sought to investigate the genomic landscape of glioblastoma (GBM) in Maine.

METHODS

Targeted next generation sequencing of 209 cancer related genes was performed on tumor samples from 60 patients diagnosed with GBM at our institution between 2014 and 2019. We compared mutation frequencies between patients grouped according to the published median overall survival for GBM: < 14 months (n = 14) and ≥ 14 months (n = 43, including 23 patients still alive). We excluded patients who were alive but diagnosed less than 14 months prior to analysis (n = 3).

RESULTS

Across all groups frequent molecular markers included EGFR (49%), TP53 (38%), CDKN2A (17.5%), PIK3R1 (15.8%), PDGFRA (12.2%), PIK3CA (10.5%), PTEN (19%), NF1 (10.5%) and IDH1/2 (10%); frequencies did not differ significantly between groups. MGMT methylation was similar between groups (6/14 (42.9%) in shorter survivors and 18/41 (43.9%) in longer survivors). FANC group mutations were more frequent in longer survivors (13/43 [30%] versus 0/14 [0%], p = 0.025). Among shorter survivors CDKN2A deletions were slightly more frequent (5/14 (45%) versus 5/43 (11%), p = 0.099). FES, KIAA1524, FLT (all 5/43,11.6%) and EPH (6/43, 14.0%) were only observed in longer survivors. These mutations co-occurred with 4/5 CDKN2A deletions in longer survivors. After excluding those 4 cases, CDKN2A deletions were significantly associated with shorter survival (5/14 [35.7%] versus 1/39 [2.6%]), p=0.004.

CONCLUSION

FANC group mutations may be a novel prognostic biomarker for longer survival in a rural cohort with GBM. CDKN2A deletions may be associated with shorter survival, but this may be ameliorated by co-occurring FES, KIAA1524, FLT, and EPH mutations.
BIOM-33. LOW EXPRESSION OF DYNLT3 PREDICTS BETTER PROGNOSIS FOR FEMALE GLIOBLASTOMA PATIENTS
Authors: Hu Yuanjun , Wang Jing , Chen Zhong-ping
Published: December 2020

BACKGROUND

Glioblastoma (GBM) is the most malignant tumor in the central nervous system. It has been confirmed that the prognosis of GBM has obvious gender predilection. As female patients have an advantage than males, it is increasingly accepted to take gender into account while treating GBM. Therefore, it is important to figure out molecular mechanisms related to gender predilection.

METHODS

We first analyzed the protein expression of GBM (Male=24, Female=16) based on the dataset from Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute’s（NCI）and mainly focused on those differential expression proteins between males and females on X chromosome. Then the differentially expressed genes from CPTAC was further confirmed in TCGA data and their role in the overall survival of GBM patients were analyzed.

RESULTS

18 proteins, differentially expressed between male and female were selected. 12 out of them expressed higher in female while the other 6 were higher in male (P< 0.05). We found one protein, DYNLT3 might be a predictor for GBM patients’ survival through analyzing the transcriptome profiling database of TCGA. The low expression of DYNLT3 was related to a longer survival of female group. The median survival was 556 days (95% CI, 446 to 665) with lower expression level of DYNLT3, compared with 375 days (95% CI, 291 to 459) in higher expression group (P=0.011 by log-rank test) in all cases including both male and female. For female group, the median survival was 653 days (95% CI, 464 to 842) with lower expression of DYNLT3, which was much better than 305 days (95% CI, 379 to 642) (P=0.008) with higher expression of DYNLT3. No significant predicting value of DYNLT3 expression was found in male group.

CONCLUSIONS

The expression of DYNLT3 might be a positive predictor for survival of GBM patients, especially for female cases.
BIOM-38. PI3K/AKT/mTOR SIGNALING PATHWAY ACTIVITY IN IDH-MUTANT DIFFUSE GLIOMA
Authors: Mohamed Esraa , Kumar Anupam , Hilz Stephanie , Wang Albert , Shai Anny , Taylor Jennie , Clarke Jennifer , Molinaro Annette , Lee Julieann , Costello Joseph , Solomon David , Phillips Joanna
Published: December 2020

PI3K/AKT/mTOR signaling pathway activation is a common mechanism of tumor progression in diffuse lower grade glioma. Robust and accurate biomarkers are needed to stratify patients for therapies targeting this pathway. To investigate the potential of phosphoprotein quantification to provide a direct and functional pathway readout, we analyzed 90 tumors from 83 patients with IDH-mutant diffuse glioma. The cohort comprised 50 IDH-mutant astrocytomas, 40 IDH-mutant and 1p/19q-codeleted oligodendrogliomas, 7 of whom had paired samples from initial diagnosis and recurrence. We developed and validated a pipeline using multiplex immunofluorescence to quantify tumor cell-specific phospho-protein expression of 3 pathway nodes, ribosomal protein S6 (RPS6), PRAS40, and 4E-BP1. In oligodendroglioma the fraction of tumor cells expressing each of the three phosphorylated proteins increased with tumor grade (p< 0.05). Comparing tumors at initial diagnosis (n=48) and at recurrence (n=42), p-RPS6 and p-PRAS40 increased in tumor cells (p< 0.05) and there was an overall increase in intertumoral heterogeneity of signaling activity at recurrence (p< 0.04). Analysis of paired samples demonstrated increased signaling pathway activity in a subset at recurrence. Robust signaling activity, defined as a phospho-positive tumor cell fraction ≥ median for all three phosphoproteins, was identified in 71.4% of grade 3 IDH-mutant astrocytoma(5/7) and 45.4% of grade 3 IDH-mutant, 1p/19q-codeleted oligodendroglioma(5/11). In a subset of cases analyzed by targeted NGS, robust signaling pathway activity was identified in 38%(11/29) at the protein level while genetic alterations predicted to activate the pathway were present in only 17.2% (5/29). Our results demonstrate robust PI3K/AKT/mTOR signaling activity in a significant fraction of IDH-mutant diffuse glioma, an association with increasing tumor grade in oligodendroglioma, and an increase at recurrence in both oligodendroglioma and astrocytoma. Overall, our data suggest that quantitative evaluation of phosphoproteins may be a sensitive method to detect PI3K/AKT/mTOR pathway activity and may be useful for patient stratification.
BIOM-47. LONGITUDINAL MONITORING OF PLASMA H3K27M IN DIFFUSE MIDLINE GLIOMA PATIENTS TREATED WITH ONC201
Authors: Tarapore Rohinton S , Field Amanda , Hill D Ashley , Allen Joshua
Published: December 2020

Diffuse midline glioma, H3 K27M-mutant (DMG) is a 2016 WHO Grade IV glioma that has no established treatment beyond first-line radiation. ONC201 is an investigational small molecule that has been shown to be clinically active in recurrent DMG clinical trials. While biopsies of DMG are sometimes feasible, many patients defer secondary to complication risk. MR scans have many limitations in monitoring DMG progression, including distinguishing pseudoprogression and pseudoresponse and measuring diffuse lesions that often do not contrast enhance. Digital droplet PCR (ddPCR) is capable of sensitively detecting and quantifying the allelic frequency of circulating-tumor DNA (ctDNA) fragments against a backdrop of non-tumor DNA. Using sequence-specific probes for H3F3A (H3.3 K27M) and HIST1H3B (H3.1 K27M) ddPCR detects very low frequency variants and provides an assessment of mutational burden. A pilot cohort of 5 patients treated with ONC201 who had a range of outcomes were assessed with serial ctDNA analyses. Two patients with immediately progressive disease had a concordant H3 K27M ctDNA increase that precedes radiographic detection by 4 weeks. Two patients with >50% tumor regressions while on ONC201 had concordant H3 K27M ctDNA burden at the onset of response and subsequent radiographic progression was preceded by increases in ctDNA 8–16 weeks prior. One patient who had prolonged stable disease had decreased H3 K27M ctDNA burden over time. Upon radiographic progression, the addition of bevacizumab with ONC201 caused a radiographic pseudoresponse, however H3 K27M ctDNA remained stable. These pilot results suggest H3 K27M ctDNA may be a sensitive and accurate biomarker of disease burden. Longitudinal evaluation of H3 K27M ctDNA in a cohort of 34 recurrent contrast-enhancing H3 K27M-mutant glioma patients while on ONC201 will be reported. Primary tumor locations range across the thalamus, cerebellum, basal ganglia, temporal lobe, and midbrain; median age is 31 years old (range 20–70).
BIOM-49. SERUM microRNA IS A PROGNOSTIC AND POST-OPERATIVE MONITORING BIOMARKER IN GLIOMA
Authors: Jones Jordan , Morokoff Andrew , Drummond Kate
Published: December 2020

Although magnetic resonance imaging (MRI) provides accurate anatomical and spatial details regarding gliomas, it is not reliable at predicting biological behaviour and is confounded by problems such as pseudo-progression. A circulating biomarker has potential to improve predictions of glioma outcomes and identify tumor progression post treatment, however no such biomarker is currently available. We aimed to discover a microRNA (miRNA) serum biomarker for longitudinal monitoring of glioma patients as well as identify miRNAs that are predictive of survival outcomes. To investigate this a prospectively collected cohort of 91 gliomas and 17 healthy controls underwent pre- and post-operative serum miRNA profiling using the next-generation sequencing platform Nanostring®. Differentially expressed miRNAs were discovered using a machine learning random forest analysis. Candidate miRNAs were then assessed by droplet digital PCR in 11 patients with multiple follow-up samples and compared to tumor volume based on MRI. A lasso-regression model was used to identify candidate miRNAs from pre-operative serum that were associated with both progression-free and overall survival. We identified a 9-gene miRNA signature that could distinguish between glioma and healthy controls with 99.8% accuracy. From this signature, we identified two miRNAs that demonstrated dynamic changes which correlated closely with tumor volume in both LGG and GBM. Importantly, miRNA levels did not increase in two cases of pseudo-progression, indicating the potential utility of this test in guiding treatment decisions. Additionally, altered expressions of 6 miRNAs were found to be associated with overall and progression-free survival and were incorporated in a predictive model with already known survival factors. In conclusion, we have identified a highly accurate 9-miRNA signature associated with glioma serum and observed specific miRNAs that both correlated with tumor volume over long-term follow up and were predictive of survival. These results support a large prospective validation study of serum miRNA biomarkers in glioma.
BIOM-50. GENETIC PREDISPOSITION TO LONGER TELOMERE LENGTH AND RISK OF CHILDHOOD, ADOLESCENT AND ADULT-ONSET EPENDYMOMA
Authors: Zhang Chenan , Ostrom Quinn , Semmes Eleanor , Ramaswamy Vijay , Hansen Helen , Morimoto Libby , de Smith Adam , Pekmezci Melike , Vaksman Zalman , Hakonarson Hakon , Diskin Sharon , Metayer Catherine ,   The Glioma International Case Control Consortium, Taylor Michael , Wiemels Joe , Bondy Melissa , Walsh Kyle
Published: December 2020

INTRODUCTION

Ependymoma is the third most common brain tumor in children, with well-described molecular characterization but poorly understood underlying germline risk factors. Telomerase reactivation in somatic cells has been linked to ependymoma progression, recurrence, and survival, and has been implicated as an important prognostic marker and potential therapeutic target.

METHODS

To investigate whether inherited predisposition to longer telomere length influences ependymoma risk, we utilized case-control data from three studies: 1) a population-based pediatric and adolescent ependymoma case-control sample from California (153 cases, 696 controls), 2) a hospital-based pediatric posterior fossa type A ependymoma (EPN-PF-A) case-control study from Toronto’s Hospital for Sick Children and the Children’s Hospital of Philadelphia (83 cases, 332 controls), and 3) a multicenter adult-onset ependymoma case-control dataset nested within the Glioma International Case-Control Consortium (GICC) (103 cases, 3287 controls). We investigated the individual effect of telomere-length associated SNPs on ependymoma risk, as well as the combined effect of these SNPs through polygenic score and Mendelian randomization analyses.

RESULTS

We observed an association between genetic predisposition to longer LTL and increased risk of adolescent-onset (P= 3.97x10-3) and adult-onset (P =0.042) ependymoma, but not ependymoma diagnosed in children < 12 years old (P=0.21), or among the pediatric EPN-PF-A sample (P=0.59). Comparing ependymoma patients ages 12–19 to those under 12 years of age demonstrated that age significantly modified the association between longer telomere length and ependymoma risk (P=0.021).

CONCLUSIONS

These findings complement emerging literature suggesting that dysregulated telomere maintenance is important for ependymoma pathogenesis and that longer telomere length is a risk factor for various neoplasms of the peripheral and central nervous system.
BIOM-51. FACTORS ASSOCIATED WITH DECLINING SEX DISCREPANCY IN MENINGIOMA WITH INCREASING TUMOR AGGRESSIVENESS: RETROSPECTIVE REVIEW OF A RURAL PENNSYLVANIA COHORT
Authors: Sofoluke Richard , Chu Sally , Bross Shane , Gatson Na Tosha
Published: December 2020

INTRODUCTION

Meningioma is the most common benign primary brain tumor in adults comprising 37% of all primary CNS tumors. Approximately 81.1% of meningiomas are grade I, 16.9% - grade II, and 1.7% - grade III. While females are 2.5 times more likely to have meningiomas, males more commonly have aggressive grades. Loss of progesterone receptor (PR) and presence of androgen receptor (AR) are associated with tumor aggressiveness. We observed a declining sex-discrepancy with increasing tumor grade. We present an ongoing assessment of a rural population to define potential factors contributing to this observation.

METHODS

We completed a single institutional retrospective review of 406 pathology confirmed meningiomas in rural Northeast Pennsylvania. We compared WHO grade, hormone receptors, and proliferative indices between males and females. We further evaluated tumor location and comorbid endocrine disease for possible association.

RESULTS

Our patient cohort had a high frequency of aggressive tumors 73.5% (n= 297) grade I, 24% (n= 99) grade II, and 2.5% (n= 10) grade III. The ratio of females versus males was 3.1:1, 1.3:1, and 1:1 in grades I, II, and III respectively. Factors contributing to closing the sex-gap were: (1) grade II: males expressed less PR than females (76% vs 94%); (2) grade III: females expressed less AR than males (0% vs 100%), while PR expression remained equivalent; and (3) males had higher tumor proliferative indices compared to females (7% and 22% vs 2% and 14%), grades I and II respectively. There was no relationship determined for tumor location or comorbid endocrine disease between the sexes.

CONCLUSION

The association between declining sex discrepancy in meningioma with increasing tumor aggressiveness is potentially related to the expression PR, AR, and increased tumor proliferation indices with increasing meningioma grade.
BIOM-53. DIFFUSION MR IMAGING DERIVED APPARENT DIFFUSION COEFFICIENT AS A POTENTIAL BIOMARKER OF CLINICAL OUTCOME IN RECURRENT CENTRAL NERVOUS SYSTEM LYMPHOMA
Authors: Ho Ka-wai , Nosov Anton , Young Robert , Grommes Christian
Published: December 2020

BACKGROUND

Studies have shown that diffusion-weighted imaging (DWI) derived apparent diffusion coefficient (ADC) measurements inversely correlate with tumor cell density and may have prognostic value in newly-diagnosed CNS lymphoma patients. This study characterizes ADC measurements in a relapsed/refractory CNS lymphoma population and describes the potential prognostic value of ADC measurements in these patients.

METHODS

ADC measurements were assessed in immunocompetent patients enrolled on a prospective clinical trial studying ibrutinib-based treatment in recurrent/refractory CNS lymphoma (NCT02315326; 42 treated with single agent ibrutinib and 15 with an ibrutinib containing combination). Measurements were normalized to values contralateral to the lesion to generate an ADC measurement ratio. Imaging and clinical variables were correlated with treatment response. High-quality lesions were defined as areas with clear delineation from preserved brain parenchyma ≥ 10mm in maximal transverse dimension. Cerebral spinal fluid (CSF) was collected at time of MR imaging in all patients.

RESULTS

Thirty-nine out of 57 patients had lesions with measurable ADC of which 17 (44%) were high-quality ADC lesions. The median ADCmean ratio was 0.909 (range 0.497–1.444). There was no association between ADC ratios and tumor volume, CSF cell count, or CSF protein. The high-quality lesions had a median ADCmean ratio of 0.774 (range 0.497–1.051). These patients with a high ADCmean ratio (≥ 0.774) had increased rates of partial and complete responses (p=0.057) as well as a longer progression free survival (PFS; median not reached) compared to those with low ADCmean ratios (< 0.774; median PFS=1.5 months, p=0.016).

CONCLUSION

ADC ratios in the recurrent CNS population do not correlate with tumor size, cell count, or protein in the CSF. In high-quality DWI measurements, ADC might be a prognostic marker in relapsed/refractory CNS lymphoma patients treated with ibrutinib.
CBIO-07. CELL DEATH INDUCED BY AN OSCILLATING MAGNETIC FIELD IN PATIENT DERIVED GLIOBLASTOMA CELLS IS MEDIATED BY REACTIVE OXYGEN SPECIES
Authors: Hambarde Shashank , Sharpe Martyn , Baskin David , Helekar Santosh
Published: December 2020

Noninvasive cancer therapy with minimal side effects would be ideal for improving patient outcome in the clinic. We have developed a novel therapy using strong rotating magnets mounted on a helmet. They generate oscillating magnetic fields (OMF) that penetrate through the skull and cover the entire brain. We have demonstrated that OMF can effectively kill patient derived glioblastoma (GBM) cells in cell culture without having cytotoxic effects on cortical neurons and normal human astrocytes (NHA). Exposure of GBM cells to OMF reduced the cell viability by 33% in comparison to sham-treated cells (p< 0.001), while not affecting NHA cell viability. Time lapse video-microscopy for 16 h after OMF exposure showed a marked elevation of mitochondrial reactive oxygen species (ROS), and rapid apoptosis of GBM cells due to activation of caspase 3. Addition of a potent antioxidant vitamin E analog Trolox effectively blocked OMF-induced GBM cell death. Furthermore, OMF significantly potentiated the cytotoxic effect of the pro-oxidant Benzylamine. The results of our studies demonstrate that OMF-induced cell death is mediated by ROS generation. These results demonstrate a potent oncolytic effect on GBM cells that is novel and unrelated to any previously described therapy, including a very different mechanism of action and different technology compared to Optune therapy. The effect is very powerful, and unlike Optune, can be seen within hours after initiation of treatment. We believe that this technology holds great promise for new, effective and nontoxic treatment of glioblastoma.
CBIO-09. INTRATUMORAL HETEROGENEITY OF DIELECTRIC PROPERTIES IN GLIOBLASTOMA
Authors: Proescholdt Martin , Haj Amer , Doenitz Christian , Schmidt Nils Ole , Bomzon Zeev
Published: December 2020

INTRODUCTION

Recently, tumor treating fields (TTFields) were established for the treatment of newly diagnosed glioblastoma (GBM). One of the most crucial parameters defining the treatment efficacy of TTFields is the electric field intensity, which depends on the dielectric properties of the tumor tissue. In this study we determined the dielectric properties of GBM by analyzing resected tissue following a fast acquisition protocol. To account for the intratumoral heterogeneity, different regions of the tumor were analyzed separately.

METHODS

A cohort of 38 patients with newly diagnosed GBM were analyzed. Tissue probes were acquired from the vital tumor area and perinecrotic compartment. The tissue was measured immediately to avoid artifacts. A fragment was dissected from each tissue sample and was placed into a cylindrical cell with a known diameter. The impedance was recorded at frequencies 20Hz-1MHz using a software specifically developed for this study, which controls the LCR meter. The measured impedance was translated into dielectric properties of the sample (conductivity and relative permittivity) based on the parallel plate model, the recorded complex impedance and the geometry of the samples. Each tissue probe was fixed, and stained with H&E to visualize cellularity, luxol fast blue to analyze the myelinated fiber content and against factor VIII related antigen to assess tumor vascularity.

RESULTS

We found significant differences between the conductivity and permittivity of tissue samples from each individual tumor (mean conductivity [S/m]: 0.302; range: 0.607 – 0.100; mean permittivity [Farad/m]: 3519.8; range: 11182.5 – 135.7). Consistently, the perinecrotic areas displayed lower conductivity values compared to the solid tumor compartments. Histological analysis revealed significantly higher cellularity and lower myelinated fiber content in tissue samples with high conductivity and permittivity.

CONCLUSION

The dielectric properties of GBM show a high intratumoral heterogeneity which correlate to the extent of cellularity and myelin fiber content within the tissue.
CBIO-11. NOVEL THERAPY TO TARGET PR-RECURRENT GLIOMA
Authors: Rahman Masum , Olson Ian E , Saber Rehan , Zhang Jibo , Carlstrom Lucas P , Sisi Chen , Rajani Karishma , Brown Desmond , Parney Ian , Sarkaria Jann , Burns Terry C
Published: December 2020

BACKGROUND

Glioblastoma is a fatal infiltrative primary brain tumor, and standard care includes maximal safe surgical resection followed by radiation and Temozolomide (TMZ). Therapy-resistant residual cells persist in a latent state a long time before inevitable recurrence. Conventional radiation and Temozolomide (TMZ) treatment cause oxidative stress and DNA damage resulting senescent-like state of cell-cycle arrest. However, increasing evidence demonstrates escaping senescence leads to tumor recurrence. Thus, the ablation of senescent tumor cells after chemoradiation may be an avenue to limit tumor recurrence.

METHODS

100uM TMZ for 7days or 10-20Gy radiation (cesium gamma radiator) was used for senescence induction in human glioblastoma in vitro and confirmed by SA-Beta gal staining and PCR. Replication arrest assessed by automated quantification of cellular confluence (Thermo Scientific Series 8000 WJ Incubator). We evaluated the IC50 for several senolytics targeting multiple SCAPs, including Dasatinib, Quercetin, AMG-232, Fisetin, Onalespib, Navitoclax, and A1331852, and in senescent vs. proliferating cells.

RESULTS

Among the senolytic tested, the Bcl-XL inhibitors A1331852 and Navitoclax both shown senolytic effect by selectively killing radiated, senescent tumor cells at lower concentrations as compared to 0Gy treated non-senescent cells. Across 12 GBM cell lines, IC50 for senescent cells was 6–500 times lower than non-senescent GBM(p< 0.005). Such differential sensitivity to Bcl-XL inhibition after radiation has also observed by BCL-XL knockdown in radiated glioma.

CONCLUSION

These findings suggest the potential to harness radiation-induced biology to ablate surviving quiescent cells and demonstrate Bcl-XL dependency as a potential vulnerability of surviving tumor cells after exposure to chemoradiation.
CBIO-15. MDM2 INHIBITOR SYNERGY WITH BCL-XL INHIBITION FOR p53 WILD TYPE GLIOBLASTOMA
Authors: Rahman Masum , Olson Ian E , Saber Rehan , Carlstrom Lucas P , Jhang Jibo , Brown Desmond , Rajani Karishma , Sarkaria Jann , Parney Ian , Burns Terry C
Published: December 2020

BACKGROUND

Glioblastoma (GBM) is an infiltrative, uniformly fatal brain tumor, treated with surgery, radiation, and Temozolomide (TMZ). Chemoradiation induces a senescent-like phenotype, which contributes to disease recurrence. We recently found that, radiated GBM cells can be eliminated by inhibition of BCL-XL. However, it remains unknown whether p53 is involved in this process and whether the elimination of senescent cells by BCL-XL inhibition could be augmented by MDM2 inhibition, a negative p53 regulator.

METHODS

p53-mutant (GBM6/GBM123) and p53-WT (GBM39/GBM76) human GBM cells were treated with 5Gy or TMZ following 48 hours of MDM2 inhibitor (AMG232) or vehicle treatment and maintained for seven days to establish a senescent-like phenotype. We evaluated the IC50 for BCL-XL inhibitor (A1331852) in radiated vs. non-radiated cells with or without MDM2 inhibitor pre-treatment.

RESULTS

MDM2-inhibitor treatment prior to radiation increased the expression of p21 and lead to increased cell death when combined with BCL-XL inhibition in p53-WT GBM cells. IC50 of BCL-XL inhibitor after prior MDM2 pretreatment and radiation in GBM76 was 4.5□M compared to 33.5□M, 18.1□M, and 32.3□M in vehicle without radiation, vehicle with radiation, and MDM2 inhibitor-alone treatment groups, respectively(p=0.0036). The IC50 of BCL-XL inhibitor without MDM2-inhibition in non-irradiated and radiated, as well as pre-administration of MDM2 inhibitor in non-irradiated and radiated GBM39 cells was 5618nM, 16117nM, 3926nM and 276.5nM, respectively(p=0.0003). Conversely, MDM2 co-inhibition with BCL-XL did not lead to increased rates of cell death in p53-mutant cell(p>0.05).

CONCLUSION

We previously identified that BCL-XL inhibition promotes cell death in senescent GBM cells. We build upon that work, demonstrating the increased rates of cell death can be augmented by MDM2 inhibition, but only in p53-WT cells. These findings highlight a novel therapeutic target for treating latent GBM tumors prior to recurrence, with the additional of MDM2 inhibition greatly increasing the efficacy of BCL-XL targeting agents.
CBIO-17. IDENTIFYING A NETWORK OF ESSENTIAL & TUMORIGENIC GENES IN GLIOBLASTOMA USING WHOLE-GENOME CRISPR Cas9 SCREENING
Authors: Baisiwala Shivani , Saathoff Miranda , Dmello Crismita , Shireman Jack , Chen Li , Park Cheol , Awah Chidibere , Goel Chirag , Budhiraja Shreya , Hardiman Clare , Sonabend Adam , Ahmed Atique
Published: December 2020

GBM is the most common primary malignant brain tumor in adults, with a 100% recurrence rate and a median survival of 21 months. As such, advances in therapy are desperately needed. Genomic approaches have shown that GBM has high intra-tumoral heterogeneity. However, a comprehensive understanding of determinants of growth is required to identify new therapeutic targets. CRISPR-Cas9 screening technology has enabled whole-genome screens that allow objective identification of genes governing specific phenotypes. Here, we performed a genome-wide CRISPR knockout screen in H4 human glioma cells to identify genes that drive proliferation. Our screen identified ~150 novel essential growth genes. From this list, we identified 5 genes that were previously unstudied, show significant elevations in expression at the RNA and protein levels (p< 0.05), and show significant survival benefit in patient datasets (p< 0.05) – PSMB3, CHCHD4, THOC1, SPDYE5, HSPA1. Our validation experiments showed that knocking out these genes resulted in cell death in multiple GBM patient-derived xenograft (PDX) lines. In addition, animals with knockout cells implanted demonstrated extended survival (p< 0.01). Furthermore, overexpression of these genes in a normal neural stem cell line resulted in transformation to a cancer phenotype, as evidenced by sphere formation in a soft agar assay (p< 0.01). Further investigation of one of these genes, PSMB3, which is a subunit of the proteasome, showed that ubiquitinated proteins were significantly increased after PSMB3 knockdown, suggesting that disruption of the proteasome system was the likely cause of cell death. We performed a ubiquitin immunoprecipitation (IP) to identify which genes were being uniquely ubiquitinated in GBM. Results showed pathways involving retinoblastoma genes, DNA repair genes, and DNA replication. Together, this data suggests that both our CRISPR screens and our ubiquitin IP have yielded promising and novel therapeutic targets for GBM, a disease desperately in need of new strategies.
COVD-22. COVID-19+ GLIOMA PATIENT CARE: LESSONS FROM A 5-PATIENT CASE SERIES
Authors: Daher Ahmad
Published: December 2020

Glioma patients, like other cancer patients, are at an increased risk of COVID-19 infections, but there are no specific guidelines on how their care should be modified during this pandemic. The challenge to develop such guidelines is largely related to the limited number of reported cases and lack of studies on this particular patient population. We present a 5-patient case series of glioma, detailing their baseline characteristics, treatment courses, lab abnormalities, and the changes made to their care after they developed COVID-19. The median age of the patient population was 66 years. All patients had IDH-wild type glioma (3 Grade IV, 1 Grade III, and 1 Grade II) and all of whom had received temozolomide chemotherapy shortly before COVID-19 diagnosis (median = 22 days). Three patients presented with mild non-respiratory symptoms requiring hospitalization to two of them, and adjuvant Temozolomide chemotherapy was held in all. One patient developed severe symptoms of shortness of breath requiring ICU-stay and expired eight days later. One patient was asymptomatic, tested positive during a routine pre-chemotherapy screening, and initiation of temozolomide was delayed by two weeks after a negative repeat test. All four symptomatic patients were rehabilitation facility residents. The most common lab abnormality was lymphopenia seen in 4/5 patients. Other abnormalities seen included elevated ferretin/total bilirubin/CRP/LDH/procalcitonin/D-dimer, thrombocytopenia/leukopenia, and low sodium/vitamin D. Chest x-ray findings were normal in 3/5 patients and showed ground glass opacities in 1 patient. COVID-19 screening during different phases of glioma therapy is recommended. Therapy interruptions or shortening duration of treatment particularly of temozolomide given its risk on lymphopenia may be needed. lymphopenia thresholds, MGMT promoter methylation status, and residence in rehabilitation facilities may help stratify glioma patient COVID-19 risks further. Patients and their family will need to be involved in therapies’ risk:benefit discussions during this pandemic.
CSIG-11. TARGETING PD-L1 IN GLIOBLASTOMA USING NANOPARTICLE-BASED GENE EDITING
Authors: Tran An , Fierro Javier , Dou Huanyu
Published: December 2020

Glioblastoma multiforme (GBM) is an astrocyte derived brain tumor with very poor prognosis, usually with a less than one year survival rate. Immunotherapy has shown promising therapeutic potentials in research and clinical application, however, GBM associated immunosuppressive microenvironment creates a significant barrier for effective anti-GBM immune responses. The immune checkpoint PD-1/PD-L1 pathways play a critical role in tumor-induced immunosupression to evade immune surveillance. Gene-editing tools targeting PD-1/PD-L1 pathway have gained increased research attention, however the major challenge is how to effectively deliver gene-editing tools without causing adverse effects for clinical translation. We have developed a nanoparticle (NP) delivery system from a low molecular weight PEI lipid shell and a PLGA core that can package PD-L1 gRNA-CRISPR/Cas9 plasmid to transfect human U87 glioma cells overexpressing PD-L1. PD-L1 gRNA-CRISPR/Cas9 plasmid is constructed by inserting single guide targeting PD-L1 sequence into GFP CRISPR/Cas9 plasmid for visualizing transfection efficacy. NP is labelled with Rhodamine 6G to monitor cellular uptake and trafficking. Fluorescence microscopy shows human U87 glioma cells can quickly uptake PD-L1 GFP-CRISPR/Cas9 plasmid-NPs within 2 hours as indicated by the Rhodamine 6G. GFP expression was obtained after 48 hours of transfection and maintained up to 7 days without causing toxicity. Western blot analysis confirmed successful knockdown of PD-L1 in U87 cells. These findings reveal that NPs made by a cationic branched PEI lipid shell and a PLGA core are non-toxic and efficient in delivering CRISPR/Cas9 system to U87 cells. Editing of pathological gene in human glioma cells with PD-L1 GFP-CRISPR/Cas9 plasmid using NPs as a delivery system may provide a novel immunotherapy platform to treat GBM.
CSIG-14. COMBINING THE GLIOBLASTOMA CELL MEMBRANE-PERMEABILIZING EFFECT OF TUMOR TREATING FIELDS (TTFIELDS) WITH WITHAFERIN A (AND OTHER) CHEMOTHERAPY
Authors: Chang Edwin , Patel Chirag , Young Caroline , Flores Thomas , Zeng Yi , Joubert Lydia , Arami Hamed , Natarajan Arutselvan , Sinclair Robert , Gambhir Sanjiv
Published: December 2020

BACKGROUND

Efforts are underway to uncover novel, unorthodox therapies against glioblastoma (GBM). Tumor treating fields (TTFields) disrupt mitotic spindle formation and inhibits tumor growth. Combining TTFields with Withaferin A and other therapeutics, synergistically inhibited proliferation. We describe relevant mechanisms.

METHODS

Human and murine GBM cells (GBM2, GBM39, U87-MG, KR158B) were isolated from primary tumors. Cells were engineered to stably express firefly luciferase (fLuc). Proliferation was assessed by bioluminescence imaging (using D-Luciferin as a substrate for fLuc) or cell counting. Dextran-FITC binding and scanning electron microscopy (SEM) studies were used to probe effects on cellular membranes.

RESULTS

TTFields (200 kHz, 4 V/cm) significantly inhibited growth of cells (n=3/time point, p≤0.02; 2-way ANOVA, no TTFields vs. TTFields). Combination of Withaferin A or other chemotherapeutics with TTFields significantly inhibited growth of glioma cells synergistically beyond that of Withaferin A or TTFields alone (p< 0.01, at least n=3 experiments). Bioluminescence imaging suggested alterations in membrane configuration when cancer cells were exposed to TTFields. This was validated with observations of greater fluorescence of membrane-associating Dextran-FITC to U87-MG cells that were subjected to TTFields (p< 0.01, 2-way ANOVA, TTFields vs. no TTFields, n=3 experiments). SEM revealed significantly greater and larger number of holes on the membrane surface of TTFields-exposed U87-MG cancer cells (53.5±19.1 holes per field of view and mean size=240.6±91.7 nm2) compared to unexposed cells (23.9±11.0 holes per field of view and mean size=129.8±31.9 nm2, p< 0.005: TTFields exposed vs. non-exposed, n=3 samples, univariate Mann-Whitney test).

CONCLUSION

The findings suggest a novel combinatorial approach to treat GBM in a manner that is likely better than single treatment (drug or TTFields) alone and that is synergistic. Synergy is achieved through TTFields inducing increased permeability on membranes thus conferring greater accessibility to drug. Such a strategy is thus a promising candidate for future clinical translation in glioblastoma.
CSIG-20. L3MBTL3 SUPPRESSES MEDULLOBLASTOMA TUMORIGENESIS THROUGH MODULATION OF THE NOTCH/RBPJ SIGNALING PATHWAY
Authors: Zhang Honglai , Xu Tao , Peabody Claire , Fernández Ester Calvo , Budhathoki Rashmi , Huang Philip Chi-En , Hughes Elizabeth D , Saunders Thomas L , Kuick Rork , Magnuson Brian , Camelo-Piragua Sandra , Rual Jean-François
Published: December 2020

The NOTCH/RBPJ pathway governs cell proliferation in many biological contexts, including SHH and Group#3medulloblastoma (MB) tumorigenesis. Using our proteomic platform, we discovered an interaction between RBPJ, a key co-factor of NOTCH for the modulation of the NOTCH/RBPJ signaling pathway, and L3MBTL3, a methyllysine reader. L3MBTL3 is recruited by RBPJ on chromatin at the enhancers of NOTCH/RBPJ target genes to repress their expression. Deletions of the L3MBTL3 locus are observed in patients with WNT and Group#3 MB and expression of L3MBTL3 in the SHH MB-derived cell DAOY inhibits cell growth, suggesting a putative tumor suppressor role for L3MBTL3 in MB. To further investigate the putative role of L3MBTL3 as a suppressor of MB tumorigenesis, we used our novel L3mbtl3 KO mouse in combination with a genetically engineered ND2:SmoA1 mouse model of SHH MB in a survival analysis. Furthermore, to identify the biological processes regulated by L3mbtl3 in MB, we analyzed by RNA-seq the transcriptome of L3mbtl3 KO mouse cerebella. Our survival analysis validated in vivo our hypothesis that L3mbtl3 is a tumor suppressor in this disease context. Indeed, our data show that [ND2:SmoA1; L3mbtl3+/-] mice have a significantly lower survival rate than ND2:SmoA1 mice (P = 0.032; Log-rank test). Moreover, our RNA-seq studies showed that L3MBTL3 regulates cell fate in the cerebellum via modulation the NOTCH/RBPJ signaling pathway. Hence, the RBPJ-L3MBTL3 interaction is at the heart of a molecular mechanism governing the repression of NOTCH/RBPJ target genes and malfunction of this molecular mechanism contributes to L3MBTL3’s tumor suppressor role in MB through aberrant “de-repression” of NOTCH/RBPJ target genes. Our discovery provides insights into the tumor suppressor role of the L3MBTL3 in MB that could be harnessed in the future for the therapeutic benefit of patients with MB.