bluuebunny/crossref_metadata_2025 huggingface dataset:
Principles of International Criminal Law
Authors: Werle Gerhard , Jeßberger Florian
Published: December 2020

This book is one of the most influential textbooks in the field of international criminal justice. It offers a systematic and comprehensive analysis of the foundations and general principles of substantive international criminal law, including thorough discussion of its core crimes. It provides a detailed understanding of the general principles, sources, and evolution of international criminal law, demonstrating how it has developed, and how its application has changed. After establishing the general principles, the book assesses the four key international crimes as defined by the statute of the International Criminal Court: genocide, crimes against humanity, war crimes, and the crime of aggression. This new edition revises and updates the work with developments in international criminal justice since 2014. The book retains its systematic approach and consistent methodology, making it essential reading for both students and scholars of international criminal law, as well as for practitioners and judges working in the field.
BIOM-02. IDENTIFYING MGMT ALTERATIONS AS BIOMARKERS OF SURVIVAL IN LUNG ADENOCARCINOMA WITH BRAIN METASTASES
Authors: Mamatjan Yasin , Zuccato Jeffrey , Moraes Fabio , Cabanero Michael , Shali Wumairehan , Weiss Jessica , Tsao Ming , Aldape Kenneth , Shepherd Frances , Zadeh Gelareh
Published: December 2020

EGFR-mutant lung adenocarcinomas (EGFRm-LUAD) have a higher risk of developing brain metastases (BM) compared to non-EGFR-mutant tumors. BM development has significant prognostic impact and leads to poorer patient survival. MGMT promoter methylation is known to determine response to therapy in other cancer types including intracranial gliomas but has not been investigated in EGFRm-LUAD BM. This work aims to assess whether MGMT promoter methylation predicts patient survival or BM development in EGFRm-LUAD patients. A large cohort of 90 primary EGFRm-LUAD tumors, of which 33 (37%) developed BM, were profiled using the Illumina Infinium MethylationEPIC Bead chip. Using the previously reported MGMT-STP27 approach that uses two CpG sites to predict MGMT methylation status, Cox modeling was performed to assess whether MGMT methylation status correlates with overall survival independent of other clinical factors. MGMT methylation significantly predicted poorer survival in EGFRm-LUAD patients that developed BM (p=0.0003) and did not develop BM (p=0.003). A multivariate cox analysis, adjusting for cancer stage and smoking status as potential confounders, showed that MGMT methylation (HR=6.2, 95%CI:2.2–17.4, p=0.0005) and BM development (HR=2.6, 95%CI:1.3–5.3, p=0.007) were both independently predictive of worse overall survival in EGFRm-LUAD patients. This finding of poorer survival in MGMT methylated EGFRm-LUAD is validated in an independent LUAD patient cohort. Total mutation burden, calculated by the number of mutations per megabase of DNA, was substantially higher in MGMT methylated tumours with an interquartile range (IQR) of 58 (30–71) compared to MGMT unmethylated tumours with the IQR of 5.5 (4.3–6.1) resulting p-value of 0.01 for this comparison. Overall, this work shows that MGMT promoter methylation status is an important prognostic biomarker in LUAD patients. MGMT promoter methylation status in EGFRm–LUAD patients with BM may be used to guide patient treatment with potentially a greater extent of treatment for high-risk patients.
BIOM-04. BRAIN METASTASIS AMONG FEMALE PATIENTS DIAGNOSED WITH STAGE IV BREAST CANCER
Authors: Konduri Santhi , Singh Maharaj , Bobustuc George , Rovin Richard
Published: December 2020

Among the patients diagnosed with advanced stage of breast cancer there is a possibility of developing distant metastases, brain is one of the predominant sites. The objective of this study is to identify patient characteristics and biomarkers for brain metastasis. From National Cancer Database, 2010 through 2015, we identified 52,753 female patients diagnosed with stage IV breast cancer. Patient demographics, tumor characteristics and biomarkers were analyzed using descriptive statistics. Odds ratios (OR) were computed using logistics regression. Kaplan-Meier estimate was used to compute survival. Of 52,753 female patients diagnosed with stage IV breast cancer, there were 73% non-Hispanic white (NHW), 17% non-Hispanic black (NHB), 6% Hispanic and 4% from ‘other’ race category. The median age of the patients at the time of diagnosis was 62 years. Of the total patients only 8.2% had brain metastatic spread. Brain metastasis was less likely among NHB vs NHW (OR=0.85, p =0.01), among others vs NHW (OR=0.69, p =0.01), with insurance status, government vs no insurance, (OR=0.71, p< 0.01), with private vs no insurance (OR=0.66, p< 0.01), with laterality (left vs bilateral, OR=0.61, p=0.01), (right vs bilateral OR=0.61, p=0.01), ER positive (OR=0.69, p < 0.01), PR positive (OR=0.75, p < 0.01), tumor grade moderately or well differentiated vs poorly or undifferentiated (OR=0.54, p =0.02; OR=0.45, p =0.01) and with 10-year increment in age at the time of diagnosis (OR=0.89, p < 0.001). In this study population five-year survival with and without brain metastasis was 11% and 25% respectively. The protective factors for brain metastatic spread includes NHB or other race category, government or private insurance, cancer either left or right side, moderately or well differentiated tumor, the patients with ER and PR expression. The chance of brain metastatic spread decreases with increasing age. These finding might provide insights for clinicians in treating these patients.
BIOM-05. CIRCULATING TUMOR CELLS (CTC) IN CEREBROSPINAL FLUID (CSF) AS A PREDICTOR OF SURVIVAL IN CNS METASTASES
Authors: Diaz Maria , Singh Priya , Kotchetkov Ivan , Skakodub Anna , Reiner Anne , Panageas Katherine , Ramanathan Lakshmi , Pentsova Elena
Published: December 2020

BACKGROUND

CSF-CTC testing using the CellSearch® platform is a validated diagnostic tool for leptomeningeal metastases (LM) from solid tumors. CSF-CTCs can also be detected in patients with brain metastases (BM), but their significance is unclear. Our objective was to evaluate the utility of CSF-CTC quantification in predicting outcomes in CNS metastases.

METHODS

We retrospectively reviewed charts of patients with solid tumors who underwent CSF-CTC measurement between 2016–2019 at Memorial Sloan Kettering Cancer Center. Information on neuroaxis imaging, CSF results, systemic cancer status, tumor molecular profile and survival was collected. LM was diagnosed by MRI and/or CSF cytology. Survival analyses were performed using Cox proportional hazards modeling, and CSF-CTC splits associated with survival were identified through recursive partitioning analysis (RPA).

RESULTS

A total of 407 patients (38% lung primary, 34% breast, 28% other tumor types) were included; of these, 144 had LM and 233 had BM diagnosed before or around the time of CSF tests (97 had both). For a subgroup of 101 patients with LM diagnosed within 30 days of CSF sampling, mean CSF-CTCs were 127.3/3ml, compared to 44.6/3ml in the overall cohort. CSF-CTCs predicted survival in these patients, with optimal cutoff at 61 CSF-CTCs/3ml, above which the risk of death doubled (HR=2.09, 95% CI: 1.13–3.87, p=0.02). For 53 patients with BM diagnosed 1–6 months prior to CSF tests, CSF-CTCs (mean 54.1/3ml) also determined higher risk of death when above the optimal cutoff of 94 (HR=5.15, 95% CI: 2.08–12.78, p=0.004). For both groups, positive/suspicious cytology was associated with higher risk of death as well, but these results were not statistically significant (LM group: HR=1.79, 95% CI: 0.95–3.35, p=0.07; BM group: HR=1.86, 95%CI: 0.82–4.22, p=0.14).

CONCLUSION

In newly diagnosed LM and BM, quantification of CSF-CTCs predicts survival. CSF-CTC analysis can be used as a prognostic tool in patients with CNS metastases.
BIOM-06. CLINICAL AND PROGNOSTIC SIGNIFICANCE OF KRAS MUTATIONS FOR BRAIN METASTASES FROM COLORECTAL ADENOCARCINOMA IN THE UNITED STATES
Authors: Lamba Nayan , Smith Timothy , Iorgulescu Bryan
Published: December 2020

BACKGROUND

KRAS mutations in colorectal adenocarcinomas have been associated with metastatic involvement of the brain (BM); and predict a lack of benefit to EGFR-targeted antibodies. Herein we leverage national data to evaluate the prevalence and impact of KRAS mutations on colorectal BMs.

METHODS

Newly-diagnosed patients with stage 4 colorectal adenocarcinoma were identified from the National Cancer Database, comprising >70% of newly-diagnosed cases in the U.S. from 2010–2016. Multivariable logistic regression was used to evaluate predictors of brain involvement, including KRAS mutation status. Overall survival (OS) was estimated with Kaplan-Meier techniques, and compared by logrank test and multivariable Cox regression.

RESULTS

Of 86,719 patients newly presenting with stage 4 colorectal adenocarcinoma, 1.5% (n=1,318) had BMs. 39.7% of BM cases had KRAS mutation testing, in which 57.9% (n=361) of BM cases were KRAS-mutant, as opposed to only 45.6% (n=13,259) of non-BM cases (p< 0.001). Mutant KRAS persisted as independently associated with BMs (OR 1.37, 95%CI: 1.09–1.72, p=0.006) following adjustment for age at diagnosis, sex, AJCC cT and cN status, and metastatic lung, liver, or bone involvement. Overall, BM patients displayed a median OS of 5.3 months (95%CI: 4.6–6.1): 7.5 months (95%CI: 5.7–10.6) if KRAS-wildtype vs. 12.0 months (95%CI: 10.4–15.4) if KRAS-mutant (p=0.01). The improved OS associated with KRAS-mutant BMs also persisted (HR 0.63, 95%CI: 0.47–0.84, p=0.001) after adjusting for the aforementioned clinical variables, in addition to comorbidity index, chemoradiotherapy, metastasectomy, and treating hospital type.

CONCLUSIONS

Nationally, testing colorectal BM patients for KRAS mutation status—a predictive biomarker for EGFR-mab therapy—remains underutilized. KRAS-mutant colorectal adenocarcinomas were more likely to have metastatic brain involvement, with KRAS-wildtype BMs demonstrating significantly worse OS than KRAS-mutant BMs. Our data suggest that KRAS mutation testing is underutilized in colorectal BM patients and provides useful prognostic information.
BIOM-08. TARGETSELECTORTM CEREBROSPINAL FLUID (CSF) CIRCULATING TUMOR CELLS AND BIOMARKER ANALYSIS: IMPROVING SENSITIVITY AND TARGETED TREATMENT OPTIONS IN BREAST AND NSCLC CANCER PATIENTS WITH CNS INVOLVEMENT
Authors: Singh Veena , Fisher Deanna , Schultz Robbie , Mayer Julie , Boorgula Smitha , Gill Jaya , Nguyen Minhdan , Troung Judy , Dobrawa Lucia , Carrillo Jose , Wagle Naveed , Kesari Santosh
Published: December 2020

BACKGROUND

Liquid biopsy has emerged as a minimally invasive and cost-effective strategy to assess cancer biomarkers without the risk of complications associated with biopsies. Once a tumor has metastasized to the brain, circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) can be found in the cerebrospinal fluid (CSF). We analyzed CSF samples from patients(pts) with primary lung or breast cancer with either brain (BM) or leptomeningeal disease (LMD). Here we report the analytical and clinical validation of Target Selector™ CSF assays.

METHODS

CSF was collected prospectively from pts with a prior solid tumor diagnosis and confirmed or clinical/ radiological suspicion of BM or LMD. CTCs were captured in microfluidic channel and classified as either CK+ or CK-. Cell-free total nucleic acids (cfTNA) was extracted from CSF supernatant and underwent both Target Selector™ single gene and next-generation sequencing (NGS) NSCLC and breast multi-gene testing. For NGS, data analysis was performed using Torrent Suite with annotation and curation by Ion Reporter and Oncomine Knowledgebase Reporter software.

RESULTS

The Target Selector™ CTC platform assays performed on clinical samples (n = 89) resulted in clinical sensitivity = 80.0%, clinical specificity = 96.6%, positive predictive value (PPV) = 98%, negative predictive value (NPV) = 70.0% at a limit of detection of 2 CTCs. For molecular analyses, Target Selector™ platform assays resulted in clinical sensitivity = 85.2%, clinical specificity = 88.3%, PPV = 76.7%, and NPV = 93.0%.

CONCLUSIONS

Target Selector™ is a viable and sensitive platform for CTC detection and molecular analysis of CSF samples from patients with NSCLC or breast cancer with CNS metastases compared to the current standard of care (CSF cytology) and when imaging findings are equivocal. Identifying CTCs and actionable biomarkers can help to confirm CNS involvement when clinically suspected, guide targeted therapy selection and potentially monitor treatment response.
BIOM-10. PREVALENCE OF NF1 MISSENSE MUTATIONS AND CANDIDATE MODIFIER GENES IN SPINAL NEUROFIBROMATOSIS PATIENTS
Authors: Riva Paola , Bianchessi Donata , Mangano Eleonora , Cesaretti Claudia , Bettinaglio Paola , Bordoni Roberta , Tritto Viviana , Battaglia Cristina , Cagnoli Giulia , Saletti Veronica , Melone Marina , Schettino Carla , Natacci Federica , Finocchiaro Gaetano , Eoli Marica
Published: December 2020

INTRODUCTION

Spinal Neurofibromatosis (SNF), a distinct clinical entity of NF1, characterized by bilateral neurofibromas involving all spinal roots and a few, if any, cutaneous manifestations, entails greater morbidity than the classical form of disease. Nevertheless, there are no reliable patterns to sort out patients at risk for developing SNF.

MATERIALS AND METHODS

We investigated 19 NF1 families with at least one SNF member, 37 sporadic SNF patient and 100 NF1 patients with classical form of disease. We applied Targeted NGS using a panel consisting of 139 genes encoding RAS pathway effectors, neurofibromin interactors and genes mapping at 17q11.2 region.

RESULTS

In SNF patients we found a higher percentage of missense (21% versus 8%, p=0 0.016, OR 3.13 (95% CI 01.1 -8.2) and a lower percentage of nonsense NF1 mutations (12.5% versus 28%,, p= 0.026, OR 0.36 (95% CI 0.14–0.9) than in classical NF1 cases. Furthermore, we evaluated rare variants with damaging potential predictors in genes of the RAS pathway and in neurofibromin interactors. In more than one sporadic case possible pathogenic variants were found in LIMK2 (neurofibromin interactor), RASAL1, RASAL3, SOS1, A2ML1, MAP3K1 (RAS pathway effectors), while in more than one SNF family were detected RASAL1, RASAL3, MAP3K1 genes variations.

CONCLUSIONS

Our results confirm the correlation between NF1 genotype and SNF phenotype as previously reported (Ruggieri, 2015), suggesting that neurofibromin gain-of-functions mutations are associated to SNF. In some patients, the co-occurrence of potential pathogenic variants in NF1 related genes with severe phenotypes was detected supporting their role as modifier genes and promising therapeutic targets.
BIOM-14. METHODS FOR SCREENING AND MONITORING BY GBM MASTER REGULATORY GENE MARKERS IN LIQUID BIOPSY
Authors: Liu Tianyi , Le Son , Tran David
Published: December 2020

BACKGROUND

Current liquid-based cancer screening relies on massive deep NGS to detect rare cancer cell-derived genetic materials - a costly method fraught with high false-negative and false-positive rates. We aim to develop a non-NGS-centered, AI-directed liquid-based detection of GBM stem-like cells (GSC).

METHODS

Utilized a robust AI suite, NETZEN, we defined a common master regulatory gene network (MRGN) in GSC. Since master regulators (MR) in MRGN are developmentally restricted, their chromosomal loci are accessible in GSC but not in normal cells. Downstream factors in MRGN are massively overexpressed in GSC compared to normal cells. Thus, we measured 1) accessibility of MR genes using transposase/transposons carrying unique barcodes that can be detected after insertion into the MR’s predetermined accessible locations, and 2) expression of downstream factors using nested qRT-PCR, in PBMC from healthy controls spiked with known quantities of GSC or patients with GBM.

RESULTS

We characterized 10 MR genes in GSC with ≥1 GC-rich promoter region that is hypomethylated and accessible (ATACseq) in GBM/GSC per GSE70175/92460/52271 (19 samples) and GSE67633/96088 (14 samples), and hypermethylated and inaccessible in lymphocytes/PBMC per GSE98837 (6) and GSE74912 (13), respectively. Using barcoded transposons, we specifically disrupted 4 MR’s accessible regions only in GSC, not in PBMC. We also characterized 50 upregulated downstream factors with the top 20 having 3 to 5-orders-of-magnitude higher mean expression in GSC compared to PBMC (GSE79362/86884, 451 samples). Currently our method has a detection limit of 0.2–1 GSC in 106 PBMC. Using the first iteration, we detected GSC’s MRGN in blood samples of 14/14 GBM patients before resection, compared to in none of 15 healthy donors.

CONCLUSIONS

Chromosomal accessibility of MR and signal amplification in MRGN of GSC provide powerful substrates for a non-NGS, low-cost, liquid-based GBM detection system with potentially high sensitivity and specificity. Further testing and optimization are ongoing.
BIOM-16. IMMUNOMIC ANALYSIS OF GLIOBLASTOMA (GBM) USING GENE EXPRESSION PROFILING
Authors: Castro Michael , Badra-Azar Nilofar , Kessler Thomas , Schütte Moritz , Lange Bodo , Yaspo Marie-Laure
Published: December 2020

BACKGROUND

Despite the success of immunotherapy across the spectrum of human cancer, a successful strategy has not emerged for GBM. While PD-L1 IHC and TMB have demonstrated some utility as predictors of immunotherapy benefit, responsiveness is complexly determined by factors affecting T cell trafficking, antigen presentation, other immune checkpoints, and mediators of immune exhaustion. Thus, we set out to to characterize mediators of immune resistance and their diversity in a population of GBM patients utilizing quantitative gene expression.

METHODS

A set of 54 immunotherapy and checkpoint relevant genes and seven genes related to immune failure were selected from the literature. RNA gene counts for TCGA glioblastoma multiforme samples (N=163) were downloaded from https://portal.gdc.cancer.gov/. Annotation on subtypes and PFS values were obtained from PMID: 24120142. Gene expression normalization as FPKM, hierarchical clustering and box-plots were performed using R-3.6.0. Statistical differences of gene expression between subtypes were quantified using a TurkeyHSD test.

RESULTS

A heatmap with hierarchical clustering for immune related genes for the TCGA GBM cohort was generated including colored annotation for the subtype and progression free survival. The graph shows a rough separation into two groups, where one group of the genes is tentatively associated with mesenchymal subtype and shorter survival and showing higher expression for most immune evasion genes. However, a heterogeneity of immune evasion signatures was identified within and across subtypes. Transcripts related to antigen presentation, EZH2, and LDHA varied significantly between GBM subtypes (p < 0.05).

CONCLUSION

Gene expression analysis has utility to identify specific mediators of immune evasion and to inform the selection of combination therapies for discrete subsets of patients. A Bayesian approach to patient selection for specific immunotherapy strategies may enhance the likelihood of successful implementation of immunotherapy in the clinic.
BIOM-19. METABOLIC ALTERATION INDUCED BY SELECTIVE KNOCK DOWN OF GABPB1L IN U251 CELLS
Authors: Minami Noriaki , Ayyappan Vinay , Stevers Nick , Molloy Abigail , Batsios Georgios , Hong Donghyun , Gillespie Anne Marie , Subramani Elavarasan , Radoul Marina , Costello Joseph , Viswanath Pavithra , Ronen Sabrina
Published: December 2020

BACKGROUND

TERT promoter mutations that result in TERT expression are observed in over 80% of GBM and upstream inhibition of TERT expression by targeting GABPB1L is currently under investigation. In that context, non-invasive reliable biomarkers that can help detect TERT expression are needed. The aim of this research was to assess the value of magnetic resonance spectroscopy (MRS)-detectable metabolic changes as biomarkers of TERT expression in GBM.

METHODS

GABPB1L knock down clones (GABPB1LKD) were established by introducing Crispr Cas9 plasmid vector targeting GABPB1L into U251 cells. Two representative clones with different knock down efficiency were chosen and compared to control cells. Tumor forming capacity was evaluated by colony formation assay and magnetic resonance imaging of orthotopically implanted tumors in mice. Cells were extracted using the dual phase extraction method and 1H-MRS data of cell extracts acquired using a Bruker 500 scanner. The data was analyzed using Mnova software. Multivariate analysis was performed using the SIMCA software.

RESULTS

TERT expression was significantly reduced in GABPB1LKD compared to control cells depending on the GABPB1L knock down efficiency. Colony forming capacity was impaired in GABPB1LKD compared to control cells. In vivo MRI data showed significantly smaller tumor volumes in GABPB1LKD compared to control. Unbiased PCA analysis of 1H-MRS data showed separation of GABPB1LKD and control extracts and VIP scores derived from the OPLS-DA analysis, demonstrated that the common metabolites leading to separation of GABPB1LKD and control cells were aspartate, glutathione, glycerophosphocholine, glutamine, NAD(P)+, AXP. This data was confirmed by univariate analysis that revealed that aspartate, glutathione, glutamine, NAD(P)+, AXP level was significantly reduced in GABPB1LKD.

CONCLUSIONS

GABPB1L knock down cells that show reduced TERT expression demonstrate MRS-detectable metabolic changes. These could be translated into clinical applications, improve the monitoring of GBM patients and advance precision medicine.
BIOM-25. IDENTIFYING EXTRACELLULAR VESICLES FROM GLIOBLASTOMA OR NON-NEOPLASTIC CELLS VIA IMAGING FLOW CYTOMETRY
Authors: Milbeth Cumba Garcia Luz , Aibaidula Abudumijiti , Kazemi Nazanin Yeganeh , Jung Miyeon , Lucien-Matteoni Fabrice , Markovic Svetomir , Parney Ian
Published: December 2020

Patients with glioblastoma (GBM) have a median survival of 15 months despite aggressive treatment. Immunosuppressive monocytes are heavily infiltrated in these tumors and in patients’ circulation. Treatment-related pseudo-progression confounds outcome assessment by MRI. Thus, there is a need for additional non-invasive methods to assess treatment response. Extracellular vesicles (EVs) contain tumor-specific microRNA (miRNA) cargo that could serve as a liquid biopsy to distinguish true progression from treatment-related pseudo-progression. We had found significant differences in plasma EVs molecular profile (i.e. miRNA signatures) between GBM patients and healthy donors. Our overall hypothesis is that these differences reflect the EVs cell of origin. Our goal in this project was to develop a fluorescent staining paradigm by flow cytometry to distinguish EVs from different cells in vitro and determine differences in EV miRNA expression profile between GBM and monocytic cell-derived EVs. Gleolan (5-ALA) is an FDA-approved orally available agent for fluorescence-guided resection of GBM tumors. It is metabolized to protoporphyrin IX (PpIX) in GBM cells but not in non-neoplastic cells and has been reported to aid in the detection of GBM-derived EVs by flow cytometry. However, distinguishing between GBM-derived EVs and EVs from other cells of origin has not been described. We co-cultured human GBM cells (dBT114 or dBT116) and CD14+ monocytes for 72 hours in the presence or absence of 5-ALA. EVs were isolated by ultracentrifugation and stained for CD11b (myeloid cell marker). ImageStream Imaging Flow Cytometry was performed showing clear differentiation between PpIX+ EVs from GBM cells and CD11b+ EVs from monocytes. Interestingly, a small number of double-positive EVs (presumably representing monocyte-derived EVs that had taken up PpIX after phagocytizing GBM cells) were also present. Taken together, we were able to optimize a technique to distinguish EVs originating from GBM and monocytes for further characterization by short non-coding RNA sequencing.
BIOM-28. IDENTIFICATION OF SOLUBLE AXL AS A POTENTIAL NON-INVASIVE SERUM BIOMARKER FOR GLIOBLASTOMA
Authors: Akhtar Parvez , Raymond Daniel , Fukui Melanie , Zwernik Samuel , Winn Robert , LaCrosse Amber , Last Brittany , Polinske Natalie , Rovin Richard
Published: December 2020

AXL, a member of the TAM family of tyrosine kinases, engages cellular pathways that promote a cancer phenotype. In glioblastoma, increasing AXL expression is associated with worse prognosis. The extracellular domain of AXL is cleaved by the sheddases ADAM10 and ADAM17, releasing soluble AXL (sAXL), which is detectable in blood and tumor related fluid collections. In this study we use ELISA to measure serum sAXL levels in 23 patients with newly diagnosed GBM pre- and post- operatively and every 3 months during treatment. Relative AXL expression was assessed in tumor tissue obtained from 13 patients via western blotting. Additionally, we used T1-weighted MRI scans to interpolate pre-operative tumor volume for all patients enrolled in the study. We found that serum sAXL concentrations were elevated in 84 GBM serum samples (35.77 ±1.25 ng/mL) compared to 40 healthy volunteer samples (30.16 ±1.88 ng/mL). When normalized to total serum albumin the difference between GBM and healthy controls was statistically significant (t(df)=5.647(122), p= 0.0132). In the 19 patients with paired samples, the pre- and post- operative sAXL levels were not significantly different. Using the Pearson method, we did not find a significant correlation between tumor volume and sAXL level. However, in patients with sAXL levels above the healthy population average, there was a moderate negative correlation between sAXL and pre-operative tumor volume (r= -0.58; p= 0.23). Interestingly, there was a strong correlation between sAXL level and tumor expression of AXL in patients with high preoperative sAXL levels (r= 0.85; p= 0.03). Though sAXL shed from brain tumors is detectable in the serum of GBM patients, in this small series of patients, it does not correlate with tumor volume. That said, we feel a larger study correlating sAXL level with improved volumetric MRI determination of viable GBM is warranted.
BIOM-42. ASSOCIATION OF NEUTROPHIL-LYMPHOCYTE RATIO WITH GLIOMA GRADING AND SURVIVAL
Authors: Ghazali Malueka Rusdy , Dwianingsih Ery Kus , Alethea Maria , Argo Ibnu Widya , Ramadhani Aulia Fitri , Saleh Sabillal , Wicaksono Adiguno Suryo , Dananjoyo Kusumo , Asmedi Ahmad , Hartanto Rachmat Andi
Published: December 2020

INTRODUCTION

Gliomas are the most common primary central nervous system tumor. Inflammatory responses are thought to play an important role in cancer progression. Neutrophil-lymphocyte ratio (NLR) is a simple, low cost and easy to measure inflammation marker that has been shown to be a poor prognostic factor in many solid tumors. The preoperative NLR has been shown to be associated with histopathological grading of glioblastoma. This study aimed to find the association of NLR with glioma grading and overall survival in Indonesian patients.

METHODS

The patients were enrolled from Dr. Sardjito General Hospital, Yogyakarta Province, Indonesia. Neutrophil and lymphocyte counts were extracted from the complete blood count test before surgery. NLR was calculated as neutrophil count divided by lymphocyte count using standard units. Detailed demographic and clinical data were collected from medical records.

RESULT

A total of 79 patients were included in this study, consisting of 48 (60.8%) males and 31(39.2%) females. Most patients were diagnosed as WHO grade IV gliomas (43%), followed by grade II (26%), grade III (13%), and grade I (7.6%). In multivariate analysis, NLR ≥ 5.01 was significantly associated with high grade glioma (HR 5.50, 95%CI 1.96–15.44, p=0.001). We also found that patients with NLR ≥ 10.5 have significantly shorter median overall survival compared with patients with NLR < 10.5 (4.6 months and 27.2 months respectively, HR 2.13, 95%CI 1.03–4.40, p=0.041). Multivariate analysis using Cox Regression method showed that higher NLR (≥ 10.5) and IDH1 wild type were independent predictors for worse outcome.

CONCLUSION

In conclusion, NLR is associated with grading and overall survival of glioma patients in Indonesia.
BIOM-55. DGKζ-TARGETED REGULATION OF MIR-34A IN THE PROLIFERATION AND TUMORIGENICITY OF HUMAN GLIOBLASTOMA
Authors: Gu Wangxian , Wan Guoqing , Zheng Yanjun , Yang Xintong , Zhang Peng , Lu Changlian , Gu Xuefeng
Published: December 2020

Diacylglycerol kinase (DGK) is a lipid kinase that catalyzes the phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA), which uses ATP as a phosphate donor. Diacylglycerol kinases ζ(DGKζ) is characterized as specific type IV due to its myristoylated alanine-rich C-kinase substrate (MARCKS), ankyrin, and PDZ binding domain. Similar to other DGKs, DGKζ is also reported to be abnormally expressed in human colorectal cancer cells, and it is indispensable for the proliferation of cancer cells. However, its implications in human glioblastoma (GBM) is largely unknown. Both the mRNA and protein levels of DGKζ were significantly higher in GBM tissues than in precancerous lesions. Knockdown of DGKζ inhibited GBM cell proliferation, cell cycle and promoted apoptosis of GBM cells. Moreover, down-regulation of DGKζ markedly reduced in vitro colony formation and in vivo tumorigenic capability. Furthermore, we confirmed that DGKζ was the downstream target of miR-34a. The expression level of DGKζ was negatively correlated with miR-34a in GBM tissues. Overexpression of DGKζ reversed the tumor suppressive roles of miR-34a in GBM cells. Taken together, DGKζ can act as a potential prognostic biomarker for GBM patients and promote the growth of GBM cells was regulated by miR-34a, and it may represent a promising therapeutic target for patients with GBM.
BIOM-57. PEDIATRIC TUMOR CLASSIFICATION THROUGH GENOME-WIDE METHYLATION PROFILING OF EXTRACELLULAR VESICLE DNA
Authors: Ricklefs Franz , Maire Cecile , Fita Krys , Fritzsche Friederike , Kammler Gertrud , Westphal Manfred , Lamszus Katrin , Schüller Ulrich
Published: December 2020

BACKGROUND

Genome-wide methylation profiling reliably classifies pediatric central nervous system (CNS) tumors. Extracellular vesicles (EVs) are released by pediatric CNS tumor cells (pCC) and contain high molecular weight tumor DNA, rendering EVs a potential biomarker source to identify tumor subgroups, stratify patients and monitor therapy by liquid biopsy. We investigated whether the DNA in pCC-derived EVs reflects genome-wide tumor methylation profiles and allows tumor subtype classification.

METHODS

DNA was isolated from EVs secreted by pediatric CNS tumor cells (pCC) as well as from the shortly cultured tumor cells and from the original tumor samples (n=4 patients). Pediatric Fibroblasts and EVs derived thereof were used as a non-tumorous control. EVs were classified by nanoparticle analysis (NTA), immunoblotting, imaging flow cytometry (IFCM and electron microscopy. Genome-wide DNA methylation profiling was performed using an 850k Illumina EPIC array and results were classified according to the DKFZ brain tumor classifier and further analysed by t-SNE and Copy number alteration analysis (CNA).

RESULTS

The size range of pCC-derived EVs was 120–150 nm, as measured by NTA. The majority of secreted EVs exhibited high expression of common EV markers (i.e. CD9, CD63 and CD81), as characterized by IFCM. Genome-wide DNA methylation profiling of pCC-derived EVs correctly identified the methylation class of the original tumor (i.e. pilocytic astrocytoma, medulloblastoma). In addition, t-SNE analysis and copy number alterations matched the pattern of the parental pCC and original tumor samples.

CONCLUSION

EV DNA faithfully reflects the tumor methylation class and copy number alterations present in the parental cells and the original tumor. Methylation profiling of circulating tumor EV DNA could become a useful tool to detect and classify pediatric CNS tumors.
BIOM-58. ASSESSMENT OF ANEUPLOIDY BY REPETITIVE ELEMENT SEQUENCING TO DETECT PEDIATRIC AND ADULT BRAIN CANCERS IN PLASMA
Authors: Douville Christopher , Mattox Austin , Blair Cherie , Bettegowda Chetan
Published: December 2020

Central nervous system cancers are the tenth leading cause of death for adults and the leading cause of cancer mortality in children. Non-invasive detection methods are desperately needed for earlier diagnosis and recurrence monitoring. Aneuploidy is common to nearly all cancers and can be detected in circulating tumor DNA (ctDNA) isolated from a variety of biofluids, including plasma. We utilized a sensitive PCR-based assay called Repetitive Element AneupLoidy Sequencing System (RealSeqS) on plasma samples from 90 patients diagnosed with the most common primary brain tumors including pilocytic astrocytoma, Grade II and III astrocytoma, glioblastoma (GBM), oligodendroglioma, ependymoma, and medulloblastoma to detect aneuploidy in ctDNA. RealSeqS employs PCR amplification and sequencing of ~350,000 genome-wide loci to identify chromosomal abnormalities and then aggregate them into a single Genome Aneuploid Score via a supervised machine learning algorithm. RealSeqS identified aneuploidy in 16 of 90 (17.8%) patients, including 20.9% (9/43) of anaplastic astrocytomas and GBMs, 57.1% (4/7) ependymomas, and 18.8% (3/16) pilocytic astrocytomas. Notably, ependymomas have significant chromothripsis and aneuploidy, which was reflected in our plasma analyses. Detection of aneuploidy also correlated with higher mortality rates at the time of last follow-up (p < 0.001). RealSeqS did not detect aneuploidy in the plasma of patients with Grade II astrocytoma, medulloblastoma, or oligodendroglioma. RealSeqS detected focal amplifications of 11q in 16.7% (2/12) and a focal deletion of TERT in 8.3% (1/12) of Grade III astrocytomas, focal deletions of EXT1 (14.3%; 1/7) and focal amplifications of EGFR (14.3%; 1/7) in the plasma of patients with ependymomas, and focal deletions of EXT1 (3.2%, 1/31) and focal amplifications of CDKN2A and CDKN2B in (3.2%; 1/31) GBM. These findings suggest detection of aneuploidy by RealSeqS may be a rapid, cheaper, and more sensitive alternative to low pass whole genome sequencing for the earlier detection of certain CNS neoplasms.
BIOM-59. TERT PROMOTER MUTATION AND MGMT PROMOTER METHYLATION-MEDIATED SENSITIVITY TO TEMOZOLOMIDE IN IDH-WILDTYPE GLIOBLASTOMA: IS THERE A LINK?
Authors: Gramatzki Dorothee , Felsberg Jörg , Hentschel Bettina , Wolter Marietta , Schackert Gabriele , Westphal Manfred , Regli Luca , Thon Niklas , Tatagiba Marcos , Wick Wolfgang , Schlegel Uwe , Krex Dietmar , Matschke Jakob , Roth Patrick , Suresh Marian , Kamp Marcel , Rushing Elisabeth , Pietsch Torsten , von Deimling Andreas , Sabel Michael , Loeffler Markus , Weller Michael , Reifenberger Guido
Published: December 2020

BACKGROUND

Benefit from temozolomide (TMZ) chemotherapy in the treatment of isocitrate dehydrogenase (IDH)-wildtype glioblastoma is essentially limited to patients with O6-methylguanine DNA methyltransferase (MGMT) promoter-methylated tumors. Recent studies suggest that the impact of the MGMT status on chemosensitivity may be modulated by telomerase reverse transcriptase (TERT) promoter hotspot mutations.

METHODS

MGMT promoter methylation and TERT promoter mutation status were assessed in an exploratory prospective cohort of IDH-wildtype glioblastoma patients of the German Glioma Network (GGN) (n=298) and validated in a retrospective cohort from Düsseldorf, Germany, and Zurich, Switzerland (n=302).

RESULTS

In the prospective GGN discovery cohort of patients with MGMT promoter-unmethylated tumors, TERT promoter mutation showed inferior outcome (p=0.044). In contrast, TERT promoter mutations were not associated with improved outcome in patients with MGMT promoter-methylated tumors. Different TERT promoter hotspot mutations were not associated with distinct outcomes. The association of TERT promoter mutation in MGMT promoter-unmethylated tumors was not confirmed in the retrospective validation cohort.

CONCLUSIONS

Analysis of two independent cohorts of glioblastoma patients, including the prospective GGN cohort, did not confirm previous data suggesting that TERT promoter mutations confer an enhanced benefit from TMZ in patients with MGMT promoter-methylated glioblastoma. Thus, diagnostic testing for TERT promoter mutations may not be required for prediction of TMZ sensitivity in IDH-wildtype glioblastoma patients.
BIOM-62 SENSITIVE DETECTION AND DISCRIMINATION OF INTRACRANIAL TUMORS BY BLOOD
Authors: Nassiri Farshad , Chakravarthy Ankur , Feng Shengrui , Shen Roxana , Nejad Romina , Zuccato Jeffrey , Voisin Mathew , Patil Vikas , Horbinski Craig , Aldape Kenneth , Zadeh Gelareh , de Carvalho Daniel
Published: December 2020

BACKGROUND

The diagnosis of intracranial tumors relies on tissue specimens obtained by invasive surgery. Non-invasive diagnostic approaches, particularly for patients with brain tumours, provide an opportunity to avoid surgery and mitigate unnecessary risk to patients. We reasoned that DNA methylation profiles of circulating tumor DNA in blood can be used as a clinically useful biomarker for patients with brain tumors, given the specificity of DNA methylation profiles for cell-of-origin.

METHODS

We generated methylation profiles on the plasma of 608 patients with cancer (219 intracranial, 388 extracranial) and 60 healthy controls using a cell-free methylated DNA immunoprecipitation combined with deep sequencing (cfMeDIP-seq) approach. Using machine-learning approaches we generated and evaluated models to distinguish brain tumors from extracranial cancers that may metastasize to the brain, as well as additional models to discriminate common brain tumors included in the differential diagnosis of solitary extra-axial and intra-axial tumors.

RESULTS

We observed high sensitivity and discriminative capacity for our models to distinguish gliomas from other cancerous and healthy patients (AUC=0.99, 95%CI 0.96–1), with similar performance in IDH mutant and wildtype gliomas as well as in lower- and high-grade gliomas. Excluding non-malignant contributors to plasma methylation did not change model performance (AUC=0.982, 95%CI 0.93–1). Models generated to discriminate intracranial tumors from each other also demonstrated high accuracy for common extra-axial tumors (AUCmeningioma=0.89, 95%CI 0.80–0.97; AUChemangiopericytoma=0.95, 95%CI 0.73–1) as well as intra-axial tumors ranging from low-grade indolent glial-neuronal tumors (AUC 0.93, 95%CI 0.80 – 1) to diffuse intra-axial gliomas with distinct molecular composition (AUCIDH-mutant glioma = 0.82, 95%CI 0.66 -0.98; AUCIDH-wildtype-glioma = 0.71, 95%CI 0.53 – 0.9). Plasma cfMeDIP-seq signals originated from corresponding tumor tissue DNA methylation signals (r=0.37, p< 2.2e-16).

CONCLUSIONS

These results demonstrate the potential for cfMeDIP-seq profiles to not only detect circulating tumor DNA, but to accurately discriminate common intracranial tumors that share cell-of-origin lineages.
CBIO-04. MUTATION-SPECIFIC NON-CANONICAL PATHWAY OF PTEN AS A DISTINCT THERAPEUTIC TARGET FOR GLIOBLASTOMA
Authors: Won Choi Seung , Lee Yeri , Shin Kayoung , Kim Donggeon , Lee Se Jeong , Kim Hyunho , Chung Seok , Shin Jihye , Lee Cheolju , Nam Do-Hyun
Published: December 2020

The dominant-negative effect of PTEN mutation has been described previously, suggesting that aberrant gain of function attributed to mutation might be more disastrous than deletion in respect to malignant potential. In present study, we explored the functional implications of hot spot mutations of PTEN in GBM tumors. Subcellular location of PTEN is important for its distinct function and spatial distribution within the cytoplasm is known to be associated with cellular locomotion. We evaluated the subcellular compartmentalization of different PTEN mutants and found that some PTEN mutants located at cellular edges of chemotaxing cells. Moreover, these PTEN mutations exhibited invasive phenotype, which was not disrupted by PI3K inhibitor, but microtubule inhibitors. This finding suggests that cytoskeletal assembly as a novel non-canonical pathway of PTEN, thus unraveling a novel therapeutic vulnerability of PTEN. Mutation-specific therapeutic options should be considered in treating GBM patients with PTEN mutations.
CBIO-05. LIPID METABOLIC REPROGRAMMING SENSITIZES A MOLECULARLY-DEFINED SUBSET OF GLIOBLASTOMAS TO FERROPTOSIS
Authors: Morrow Danielle , Nathanson David , Cloughesy Timothy , Prins Robert , Bayley Nicholas , Liau Linda , Bensinger Steven , Williams Kevin
Published: December 2020

Cancers, including the universally lethal glioblastoma (GBM), have reprogrammed lipid metabolism to fuel tumor growth. However, the molecular alterations responsible for aberrant lipid metabolism, and the potential for identifying new therapeutic opportunities are not fully understood. To systematically investigate the GBM lipidome, we performed integrated transcriptomic, genomic and shotgun lipidomic analysis of an extensive library of molecularly diverse patient-derived GBM samples. Using this comprehensive approach, we discovered two GBM sub-groups defined by their combined molecular and lipidomic profile. Triacylglycerides (TAGs) enriched in polyunsaturated fatty acids (PUFAs) were among the most significantly altered lipids between the two groups of GBM tumors. TAGs are the main components of lipid droplets, which sequester PUFA-TAGs away from membrane phospholipids where their peroxidation can lead to ferroptosis – a regulated from of PUFA-peroxidation dependent cell death. Accordingly, the GBM subgroup with a depletion of PUFA TAGs showed heightened sensitivity to ferroptosis. Our findings suggest a novel association between specific molecular signatures of GBM, lipid metabolism and ferroptosis. This relationship may present a new therapeutic opportunity to target reprogrammed lipid metabolism in a molecularly-defined subset of GBMs.
CBIO-12. THE ROLES OF lncRNAs IN GBM RADIATION RESISTANCE AND TUMOR RECURRENCE
Authors: Stackhouse Christian , Rowland James , Anderson Joshua , Wang Jelai , Nguyen Thanh , Yue Zongliang , Chen Jake , Ianov Lara , Gillespie Yancey , Willey Christopher
Published: December 2020

Glioblastoma multiforme (GBM) almost invariably recurs and tumors exhibit resistance to conventional therapies. One mechanism of resistance is enhanced DNA damage response (DDR) to alkylating chemotherapy or radiation therapy (RT). We have generated 8 novel GBM patient-derived xenograft (PDX) models of tumor recurrence following serial in vivo irradiation (6 x 2Gy fractions over 2 weeks for 6+ rounds). RNA sequencing has revealed enrichment of a number of DDR pathways in the RT selected (RTS) PDX. Differential enrichment across the RTS PDX suggests multiple molecular routes to acquired RT resistance. We have also identified differential enrichment of molecular signatures for cell cycle progression, stemness, and chromatin remodeling all suggesting decreased proliferation, increased stemness, and more compacted chromatin states associated with RTS PDX. We have identified altered kinase signaling in RTS PDX that may suggest targetable signaling pathways using small molecule inhibitors. Integrated ‘–omics’ analysis has identified SRC family kinases and altered expression of collagens related to the RTS profile. Long non-coding RNAs (lncRNA) have the potential to regulate molecular phenotypes through nucleic acid binding. We have identified 269 lncRNAs significantly differentially expressed in the RTS condition. We have determined that a number of these transcripts have DNA binding potential in gene regulatory regions proximal to kinase, DDR, cell cycle, stemness, and chromatin remodeling genes. Analysis of lncRNAs and genes proximal to their binding sites has revealed regulatory networks potentially governing cell fate and phenotype. We have observed complex correlations of some of these transcripts, such as ZFAS1, which has a positive correlation with expression of stemness-promoting genes and simultaneous inverse correlation with cell cycle genes. This suggests ZFAS1 could be a phenotypic switch between a RT-sensitive proliferating cell and a RT-resistant non-proliferating stem-like cell. LncRNAs may represent a novel therapeutic target for the treatment of therapy resistant, recurrent GBM.
CBIO-18. G-QUADRUPLEX STABILIZATION TARGETS ATRX-DEFICIENT HIGH-GRADE GLIOMA VIA INDUCTION OF p53-INDEPENDENT APOPTOSIS
Authors: Dharmaiah Sharvari , Farooqi Ahsan , Alvarez Christian , Sharin Vladislav , Irvin David , Huse Jason
Published: December 2020

Gliomas are the most common primary malignant brain tumor in adults and mutational inactivation of histone chaperone ATRX is a critical molecular marker in the classification of high-grade glioma (HGG). ATRX loss occurs with concurrent mutations in TP53 and IDH1/2, altering genome-wide accessibility of chromatin and inducing replication stress and DNA damage via accumulations of abnormal G-quadruplex (G4) DNA secondary structures. While G4 stabilizers in particular hold strong therapeutic promise, the genomic consequences and efficacy of this treatment are poorly understood. We previously showed that chemical stabilization of G4s in ATRX-deficient normal human astrocytes (NHAs) results in lethality due to induction of replication stress, but it is unknown what drives this lethality in ATRX-deficient patient-derived preclinical models. We therefore sought to evaluate the mechanisms that underlie cell death in ATRX- and p53-deficient preclinical in vitro models following G4 stabilization. We found that ATRX-deficient glioma stem cells (GSCs) demonstrated dose-dependent enhanced sensitivity to G4 stabilization, compared to ATRX-intact controls. Evaluation of cell death mechanisms following G4 stabilization revealed a significant increase in cleaved caspase 3 expression and no p21 expression in ATRX-deficient GSCs, suggesting p53-independent apoptotic activation. Cell cycle flow analysis demonstrated G2/M checkpoint arrest in ATRX-deficient GSCs upon G4 stabilization, suggesting that p53 is nonfunctional at the G1/S checkpoint. Our preliminary findings now suggest that p73, a functional and structural homologue of p53, is activated and drives apoptosis in these ATRX-deficient GSCs. Furthermore, ATRX-deficient GSCs demonstrated upregulated expression of both pATR/pChk1 and pATM/pChk2, indicating enhanced replication stress and DNA damage via double-stranded breaks, respectively. These findings indicate that G4 stabilizers could potentially synergize with ionizing radiation, the current standard of care, as both therapies are DNA-damaging. Taken together, this study elucidates mechanisms of cytotoxicity and efficacy of a novel therapeutic strategy in ATRX-deficient preclinical models.
CBIO-25. INTEGRATED MOLECULAR AND BH3 PROFILING OF THE INTRINSIC APOPTOTIC MACHINERY IDENTIFIES THERAPEUTIC VULNERABILITIES IN GLIOBLASTOMA
Authors: Fernandez Elizabeth , Mai Wilson , Bayley Nicholas , Tse Christopher , Liau Linda , Cloughesy Timothy , Letai Anthony , Nathanson David
Published: December 2020

Resistance to apoptosis is a hallmark of cancer. However, the underlying molecular mechanisms of intrinsic-apoptotic resistance in glioblastoma (GBM) are largely unknown. Here we performed integrated molecular and functional characterization (via BH3 profiling) of the intrinsic apoptotic machinery in 50 GBM patient specimens. We found that, despite significant genetic heterogeneity of our GBM samples, all GBMs have a cross compensatory reliance on BCLXL and MCL1 for basal survival. Treatment with standard of care (e.g., temozolomide or radiation) caused minimal apoptosis, yet ablated the MCL-1 block in a p53-dependent manner, thus creating an exclusive dependence on BCLXL for survival in p53 wild-type GBM tumors (65% of GBM tumors). Consequently, BCLXL inhibition caused synergistic cell death with IR/TMZ in GBM tumors with intact p53 signaling. Importantly, the degree of synergistic cell kill was best predicted by combining molecular features with BH3 profiling, providing an integrated predictive signature of response to this novel therapeutic approach. Collectively, these studies identify mechanisms of intrinsic apoptosis resistance in both basal and treatment states of GBM and demonstrate how functional and molecular data can be complementary to robustly predict therapy-induced cell death.