bluuebunny/crossref_metadata_2025 huggingface dataset:
DRES-07. TMZ-LEV- IFN COCKTAIL REGIMEN SIGNIFICANTLY INHIBITED THE GROWTH OF GLIOMA IN VIVO
Authors: Ni Xiang-rong , Wang Jing , Chen Fu-rong , Cai Hai-ping , Yu Yan-jiao , Yang Qun-ying , Guo Chengcheng , Chen Zhong-ping , Qu Yue
Published: November 2019

OBJECTIVE

Temozolomide (TMZ), is the first line chemotherapeutic drug for glioma. Previous studies have suggested that interferon (IFN) and levetiracetam (LEV) could respectively reverse the resistance of TMZ by down-regulating MGMT expression. This study, we aim to investigate the therapeutic effect of a cocktail chemotherapy regimen combining TMZ, LEV, IFN in vivo.

METHODS

Glioma cell lines U251 and SKMG-4 (MGMT protein expression positive), U138 and GSC-1(MGMT protein expression negative) were used for producing xenograft tumors. The xenograft tumors were established by subcutaneously injecting 1×106 glioma cells into female BALB/C nude mice and divided into 5 treatment groups: Control, TMZ, TMZ+IFN, TMZ+LEV, TMZ+LEV+IFN. The treatment with TMZ (50 mg/kg, i.p.), IFN (2×105 IU, s.c.), LEV (150 mg/kg, i.p.) once a day for five consecutive days and xenograft tumors were measured every two days.

RESULTS

We identified that U138, U251, SKMG-4 tumor growth among TMZ, TMZ+IFN, TMZ+LEV, TMZ+LEV+IFN were all significantly inhibited (P< 0.05), compared with the control. As for U251 and SKMG-4, tumor killing effect of all 4 treatment groups were not different (P > 0.05). In the treatment of mice bearing U138 glioma, the tumor weight of TMZ+LEV+IFN (0.2688±0.1169 g) group was the lowest and significantly lower than that of TMZ+LEV (0.6574±0.08174g, P=0.0261), TMZ+IFN(0.6108±0.07317 g, P=0.0381), and TMZ (0.9054±0.07154 g, P=0.0017) group. Glioma stem cells GSC-1 was highly resistant to TMZ, tumor volume of TMZ group was not different from control group (P >0.05). While compared with TMZ (1.993±0.1274 g) group, in TMZ+IFN (1.506±0.1223g, P=0.0203), TMZ+LEV (1.178±0.1807g, P=0.0042), and TMZ+LEV+IFN (1.049±0.2171 g, P=0.0038) groups, GSC-1 tumor growth were significantly inhibited(P< 0.05).

CONCLUSION

Our data demonstrate that both IFN and LEV can sensitize TMZ effect on glioma in vivo, even for MGMT(+) tumors, and TMZ-LEV-IFN cocktail regimen seems the best. Key words: glioma, TMZ, LEV, IFN
DRES-08. CLINICAL SIGNIFICANCE OF HYPERMUTATION IN GLIOMAS
Authors: Touat Mehdi , Li Yvonne , Boynton Adam , Spurr Liam , Iorgulescu Bryan , Birzu Christina , Pal Sangita , Ferrer-Luna Ruben , Geduldig Jack , Bellamy Charlotte , Younan Nadia , Baldini Capucine , Verreault Maite , Guillerm Erell , Ammari Samy , Beuvon Frederic , Mokhtari Karima , Alentorn Agusti , Dehais Caroline , Houiller Caroline , Laigle-Donadey Florence , Lee Eudocia , Nayak Lakshmi , Carpentier Alexandre , Cornu Philippe , mathon Bertrand , Bi Wenya , Chiocca Ennio , Alexandrescu Sanda , Chi Susan , Haas-Kogan Daphne , Alexander Brian , Huang Raymond , Ligon Azra , Coulet Florence , Delattre Jean-Yves , Hoang-Xuan Khe , Meredith David , Santagata Sandro , Duval Alex , Sanson Marc , Cherniack Andrew , Wen Patrick , Reardon David , Marabelle Aurelien , Idbaih Ahmed , Beroukhim Rameen , Bandopadhayay Pratiti , Bielle Franck , Ligon Keith
Published: November 2019

BACKGROUND

Hypermutation is an emerging biomarker for predicting response to immunotherapy in cancer patients, however its clinical value in gliomas is not established. We sought to assess the determinants of hypermutation in gliomas, and its value for predicting response to standard of care and immune checkpoint blockade (ICB).

METHODS

We performed comprehensive genomic characterization of 2,420 clinically annotated gliomas. We assessed the clinical and molecular characteristics associated with hypermutation and relationships between hypermutation and response to cancer treatments.

RESULTS

Hypermutation occurred predominantly as an adaptive resistance mechanism to temozolomide in gliomas and was most prevalent in recurrent gliomas with MGMTpromoter methylation (33.8%), IDH1/2mutation (41.0%) or 1p/19q co-deletion (59.5%). Hypermutation was almost always associated with molecular defects in DNA mismatch repair (MMR), and was associated with shorter survival after its appearance based on multivariate analysis (hazard ratio 1.91; 95% CI 1.24–2.94; P=0.004). The molecular mechanisms whereby gliomas undergo hypermutation during therapy with alkylating agents were dissected using patient-derived glioma models in vitro and in vivo. Outcomes of hypermutated gliomas treated with immune checkpoint blockade or with standard of care agents will be presented at the conference.

CONCLUSIONS

Using the largest set of hypermutated gliomas described to date, this study establishes that mutational burden and mutation signatures are clinically and biologically significant biomarkers that can be used to predict therapy response and guide treatment decisions in gliomas
DRES-09. REGULATORY EFFECTS OF THE CILIARY GTPASE ARL13B ON PURINE METABOLISM IN GBM
Authors: Saathoff Miranda , Shireman Jack , Ali Eunus , Park Cheol , Ben-Sahra Issam , Ahmed Atique
Published: November 2019

Glioblastoma (GBM) is the most common form of adult primary brain cancer. Despite an aggressive treatment regimen – surgical resection, irradiation, and temozolomide (TMZ) chemotherapy – patients’ prognosis is still grim. TMZ acts by methylating purines, specifically at the O6 and N7 positions of guanine, to induce cytotoxic DNA double-strand breaks. We thus wanted to explore how purine metabolism may contribute to TMZ-resistance. In mammalian cells, purine nucleotides can be recycled by the salvage pathway or generated via de novo synthesis. The salvage pathway is energetically inexpensive relative to de novo thus, highly proliferative GBM cells preferentially utilize the salvage pathway. We have shown that salvage synthesis is reduced in response to TMZ (p-value=0.0021), hinting that the cells may utilize de novo to evade therapy induced alkylation of purines. Using immunoprecipitation-mass spectroscopy analysis, we found a novel interaction between the ciliary GTPase ARL13B and IMPDH2, the rate-limiting enzyme in de novo synthesis. We have shown that this interaction, occurring at the C-terminal domain of ARL13B, plays a significant role in the regulation of purine biosynthesis as abolishing it through ARL13B knockdown reduced flux through de novo (p-value< 0.0001) synthesis as measured by the specific activity of IMPDH2. Further, the lentiviral-mediated rescue of ARL13B brings IMPDH2 activity back to basal levels (p< 0.0001). Given its canonical function as a GTPase, we hypothesize that ARL13B acts as a novel regulator of de novo synthesis by sequestering GDP, allowing IMPDH2 to sense and respond to the cytosolic levels of guanine nucleotides. Without ARL13B the de novo pathway is halted, forcing the cells to rely on salvage to replenish nucleotide pools. Reliance on this pathway in the presence of TMZ causes cells to incorporate damaged nucleotides as a result of the drug’s alkylating action leading to the increased therapeutic efficacy of TMZ.
EPID-20. TRENDS IN GLIOBLASTOMA OUTCOMES OVER TIME, GEOGRAPHIC LOCATION AND TYPE OF INTERVENTION
Authors: Marenco-Hillembrand Lina , Wijesekera Olindi , Suarez-Meade Paola , Mampre David , Jackson Christina , Ortiz Kyle , Lesser Elizabeth , Hawayek Maria , Vivas-Buitrago Tito , Quinones-Hinojosa Alfredo , Chaichana Kaisorn
Published: November 2019

INTRODUCTION

The management of glioblastoma (GBM) has changed over time and varies based on geographic location. Despite its universally fatal prognoses, it is unclear if GBM outcomes have changed over time, if they vary by geographic location, and differ based on type of intervention. As a result, we conducted a systematic review of the literature to identify the average survival differences of GBM patients across time, between different continents, and among various treatments.

METHODS

The systematic review of PubMed included glioblastoma, GBM, and survival. Inclusion criteria consisted of full-text titles with human subjects, GBM/Grade IV astrocytoma, age >18 years old, and with available survival data.

RESULTS

9,162 articles were screened for survival data, 1728 were eligible, 405 complied with the inclusion criteria. Of the 405 studies, 179 (44.2%) were conducted in Europe, 151 (37.3%) in the Americas, 61 (15.1%) in Asia, 9 (2.2%) in Oceania, and 1 (0.2%) in Africa. Leading countries are the United States (33.1%), Germany (12.3%), and Italy (11.1%). Asia had the highest median survival (14.9 months), followed by Europe (13.2 months), and the United States (12.9 months) (P=0.003). Regarding treatment, survival was significantly higher after the introduction of the Stupp protocol (P< 0.001). Post-Stupp Studies had longer survival estimates (15.3 months, range: 3.8–29.6) than Pre-Stupp Studies (12.2 months, range: 2.3–28). In the included clinical trials (182), radiation (43.4%), temozolomide (25.3%), or other chemotherapies (24.2%) were the most common interventions. Clinical trial median survival estimates were significantly higher than all other studies (P< 0.017). Bevacizumab (19.3 months) and Temozolomide (15.9 months) had the longest recorded median survival estimates; while radiation (14 months), other chemotherapies, and carmustine (13.2 months) had the shortest (P=0.009).

CONCLUSION

This epidemiological study describes the current global state of GBM. Important differences regarding survival and treatment of patients with GBM were found.
EPID-25. HOW MANY PATIENTS IN A REAL WORLD GLIOBLASTOMA POPULATION MEET ELIGIBILITY CRITERIA IN CLINICAL TRIALS?
Authors: Skaga Erlend , Andrea Skretteberg Marthe , Børge Johannesen Tom , Brandal Petter , Osland Vik-Mo Einar , Helseth Eirik , Arne Langmoen Iver
Published: November 2019

In population series of glioblastoma (GBM) the median survival is < 1 year. For patients eligible for combined oncological treatment (surgery, radiotherapy (RT) and temozolomide (TMZ)) the median survival raises to 15 months. These patients are younger, have higher performance status and less comorbidity compared to the total GBM population. The extent of selection bias of GBM patients enrolled in clinical trials is, however, unclear. Using the Cancer Registry of Norway and the Brain Tumor Database of Oslo University Hospital, all patients diagnosed with GBM between 2012 and 2016 within a defined geographical area that received all oncological treatment at Oslo University Hospital were identified. Their hospital charts were retrospectively investigated and all patients were evaluated for the possibility to be included in clinical trials according to standard criteria from recent published phase 3 studies. We defined three time points for possible enrollment in trials: before-, halfway through-, and after concomitant RT/TMZ. We identified 424 patients of whom 26 (6.1%) did not undergo surgery. The median survival in the population was 11.7 months. Of the patients that underwent surgery (biopsy or resection), the median survival progressively increased with additional oncological treatment; no RT/TMZ 1.1 months, only RT 7.9 months, RT/TMZ 13.9 months. A potential study patient group≥18 years undergoing surgery for a supratentorial GBM with ECOG ≤2, with normal bone marrow, hepatic and renal function and no severe comorbidity or oncological disease constituted 54% of the population before start of RT/TMZ. Patients adhering to RT/TMZ-treatment and without tumor progression represented 46% and 43% of the population halfway through- and after concomitant RT/TMZ, respectively. The survival of patients that could be included in clinical trials was significantly higher (p< 0.001) than patients not fulfilling standard criteria. Our data suggest a significant patient selection in clinical trials from a real-world GBM population.
EXTH-05. THERAPEUTIC IMPLICATIONS OF TTFIELDS INDUCED DNA DAMAGE AND REPLICATION STRESS IN NOVEL COMBINATIONS FOR CANCER TREATMENT
Authors: Kumar Karanam Narasimha , Ding Lianghao , Aroumougame Asaithamby , Story Michael
Published: November 2019

TTFields are low-intensity, intermediate frequency, alternating electric fields which are applied to tumor regions using non-invasive arrays. TTFields is approved for the treatment of glioblastoma and mesothelioma with clinical trials ongoing in other cancer types. The mechanism of action for TTFields includes interference with mitosis, reduced DNA double strand break (DSB) repair capacity and the frank induction of DNA DSBs. The mechanism by which TTFields induces DNA DSBs appears to be through the enhancement of DNA replication stress with continued TTFields exposure. The induction of DNA DSBs appears to be as a result of significantly reduced expression of the DNA replication complex genes MCM6 and MCM10 as well as the Fanconi’s Anemia (FA) pathway genes. TTFields treatment increases the number of RPA foci, decreases nascent DNA length and increases R-loop formation which are markers of DNA replication stress. These results suggest that TTFields-induced replication stress is the underlying mechanism and cellular endogenous source of DNA DSB generation via replication fork collapse. The current study suggests that TTFields exposure causes a conditional vulnerability environment that renders cells more susceptible to chemotherapeutic agents that induce DNA damage and/or cause replication stress. Supporting this is the synergistic cell killing seen with TTFields exposure concomitant with cisplatin, TTFields plus concomitant PARP inhibition with or without subsequent radiation, or radiation given at the completion of a TTFields exposure. Finally, TTFields-induced mitotic aberrations and DNA damage/replication stress events, although intimately linked to one another as one can expose the other, are likely initiated independently of one another as suggested by the gene expression analysis of 47 key mitosis regulator genes. These results establish that enhanced replication stress and reduced DNA repair capacity are also major mechanisms of TTFields effects, effects for which there are therapeutic implications.
EXTH-29. GALECTIN-3 AS A POTENTIAL THERAPEUTIC TARGET IN RECURRENT ATYPICAL AND MALIGNANT MENINGIOMA
Authors: Das Arabinda , Martinez Santos Jaime , Kanginakudru Indira , McDonald Daniel G , Kosnik Infinger Libby , Vandergrift III William , Varma Abhay , Vanek Kenneth N , Lindhorst Scott , Patel Sunil , Cachia David
Published: November 2019

Atypical and malignant meningiomas are rare tumors that unlike WHO I meningiomas are characteristically more aggressive in nature and are associated with higher recurrence risks of recurrence. In fact despite aggressive treatment of malignant meningiomas, the average 5-year survival rates are in the range of 30% to 60%. Still the standard of care for atypical and malignant meningiomas (AM and MM) has yet to be established. Our laboratory data demonstrated that galectin-3 (Gal-3), a multifunctional β-galactoside-binding protein, is highly expressed in AM and MM as compared to normal tissue. However, the biological functions of Gal-3 in meningioma cells are not fully understood. To address this, we used either small interfering RNA (siRNA) to knock down Gal-3 expression or Gal-3 inhibitor, TD139 to suppress Gal-3 expression in in vitro cell culture model. Silencing or inhibiting of Gal-3 expression significantly decreased the protein levels of urokinase-type plasminogen activator receptor (uPAR) as well as uPAR’s downstream signaling transduction pathway, including phosphorylation of AKT. In both cases, we found that silencing of Gal-3 or inhibiting Gal-3 expression decreased the proliferative activity, and migratory potential of AM and MM cells. Furthermore, we demonstrated that TD139 inhibits MM growth in an in vivo xenograft MM model. Taken together, our results suggest that Gal-3 modulates uPAR expression and that Gal-3 may be a potential therapeutic target for the treatment of atypical and malignant meningiomas.
EXTH-42. QUANTITATIVE BIODISTRIBUTION OF ADOPTIVELY TRANSFERRED T CELLS IN BRAIN TUMOR-BEARING MICE
Authors: Hoang-Minh Lan , Pohl-Guimarães Fernanda , Rivera-Rodriguez Angelie , Currlin Seth , Otto Kevin , Rinaldi Carlos , Mitchell Duane
Published: November 2019

SIGNIFICANCE

Adoptive T cell therapy (ACT) has emerged as the most effective treatment strategy against advanced malignant melanoma, eliciting remarkable objective clinical responses in up to 75% of patients with refractory metastatic disease, including those with lesions within the central nervous system. Importantly, immunologic surrogate endpoints that correlate with treatment outcome have been identified in these patients, with clinical responses being dependent on the migration of transferred T cells to sites of tumor growth.

OBJECTIVE

We investigated the biodistribution of exogenously administered T cells in a murine model of glioblastoma at whole body, organ, and cellular levels.

METHODS

T cells were isolated from the spleens of DsRed transgenic C57BL/6 mice and injected intravenously, after in vitro expansion and activation, in murine KR158B glioma-bearing mice. To determine transferred T cell spatial distribution, brains, lymph nodes, hearts, lungs, spleens, livers, kidneys and stomachs were isolated for active clearing, immunostaining, and 3D imaging using light sheet microscopy, or processed for fluorescent immunohistochemistry and confocal imaging. Transferred T cell quantification in various organs was performed using flow cytometry, 2D optical imaging (IVIS), and magnetic particle imaging (MPI) after ferucarbotran nanoparticle labeling. T cell distribution was also assessed in vivo using IVIS and MPI.

RESULTS

The spleen, liver, and lungs accounted for more than 90% of transferred T cells in the body. The proportion of DsRed T cells in tumor-bearing brains was found to be very low, hovering below 1% (and representing ~15% of total tumor-infiltrating lymphocytes). Transferred T cells mostly concentrated at the periphery of the tumor mass and in proximity to blood vessels.

CONCLUSIONS

The success of ACT immunotherapy for brain tumors likely requires optimization of delivery route, dosing regimen, and modification of tumor-specific lymphocyte trafficking and effector functions in order to achieve maximal penetration and persistence at sites of invasive tumor growth.
EXTH-43. DYNAMIC MULTI-OMICS ANALYSIS OF MLN4924-TREATED GLIOBLASTOMA CELLS REVEALS ABSENCE OF PTEN AS A CANDIDATE DRIVER OF NEDDYLATION INHIBITION RESISTANCE
Authors: Taylor Brett , Ferdosi Shayesteh , Bybee Rita , Tang Nanyun , Peng Sen , Garcia-Mansfield Krystine , David-Dirgo Victoria , Sharma Ritin , Pirrotte Patrick , Berens Michael , Dhruv Harshil
Published: November 2019

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor and calls for novel therapeutic development. The neddylation pathway may be a therapeutic target, as global over activation of neddylation has been found in GBM patients and has correlated with shorter patient survival. Analogous to ubiquitination in reaction scheme and enzyme classes used, neddylation is a post-translational modification that is essential to many protein regulation and biological processes. Although non-cullin NEDD8 substrates have been investigated in recent years, the most well-characterized substrates of neddylation are the cullin subunits of Cullin-RING ligases (CRLs), which act as a scaffold for substrates to be ubiquitinated and degraded. The neddylation inhibitor MLN4924 targets NEDD8 Activating Enzyme (NAE), an upstream activator of neddylation, and induces cell cycle arrest, apoptosis and senescence in cancer cells. In this work, we investigated the context of vulnerability to Pevonedistat (MLN4924) in GBM. Efficacy of MLN4924 in glioma cell models was evaluated by measuring cell viability (CellTiterGlo®), colony formation efficiency, and cell cycle progression (flow cytometry with propidium iodide staining). GB1 (IC50= 0.28 μM), LN18 (IC50 = 0.19 μM), and GBM43 (IC50= 0.45 μM) were established as sensitive and M059K (IC50 = 5.5 μM), SNU1105 (IC50 = 20.9 μM), and GBM39 (IC50= 10.3 μM) as non-sensitive cell lines based on IC50 values. To discover genomic and/or proteomic markers of differential response, we collected RNA and protein from sensitive and non-sensitive cell lines after 0, 2, 8 and 24 hour treatment with MLN4924 for RNA sequencing and mass spectroscopy analyses. Multi-OMICS data point towards the absence of functional PTEN as a driver of MLN4924 resistance. Uncovering the mechanism underlying GBM’s vulnerability to MLN4924 will expand the knowledge of neddylation’s role in cancer and may arm physicians with an understanding of whether a GBM patient would respond to MLN4924 treatment or not.
EXTH-50. THIOREDOXIN REDUCTASE1 AND MGMT SYNTHETIC LETHALITY ENHANCES CYTOTOXICITY OF PRIMA-1MET(APR-246) AND AURANOFIN IN GLIOBLASTOMA
Authors: Raveendrakumar Rehka , Abdulkarim Bassam , Bagchi Monisha , Daniel Paul , Deri Aymane , Jean-Claude Bertrand , Sabri Siham
Published: November 2019

Glioblastoma multiforme (GBM), the most common and advanced primary brain malignancy in adults remains an incurable disease, despite aggressive treatment with surgery, radiation therapy and chemoradiation using the alkylating agent, Temozolomide (TMZ). PRIMA-1MET (APR-246), a small molecule designed to restore mutant (mut)p53 function has been shown to affect cellular redox status through targeting thioredoxin reductase 1 (TrxR1) in wild-type (wt)p53 cancer cells. We have recently shown that PRIMA-1MET exerts cytotoxic effects status preferentially in GBM cell lines expressing low levels of the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT), known for its role in resistance to TMZ. We hypothesized that PRIMA-1MET mediates its growth inhibitory effects by modulating the redox balance and investigated the potential relationship between MGMT, redox balance and TrxR1. We show that PRIMA-1MET decreased TrxR1 expression levels preferentially in MGMT-low expressing isogenic GBM cell lines. Using pharmacological agents that modulate reactive oxygen species (ROS) levels i.e., ROS scavenger, N-acetylcysteine and ROS inducer, L-Buthionine-Sulfoximine, we show that PRIMA-1MET exerts its growth-inhibitory effects through increased ROS. Strikingly, we identified a novel positive relationship between MGMT and TrxR1, wherein high MGMT expression is associated with high expression of TrxR1 and low levels of ROS. Treatment with the MGMT inhibitor, O6-Benzylguanine, or the TrxR1-targeting FDA-approved drug Auranofin validated our findings. Interestingly, the latter exerted significantly more pronounced cytotoxic effects compared to PRIMA-1MET in GBM cell lines. Additional studies are warranted to assess PRIMA-1MET in combination with TrxR1-targeting therapies and propose repurposing of Auranofin as a novel strategy to improve the dismal outcome of patients with GBM.
GENE-12. ANALYSIS OF FAILURE PATTERNS IN MALIGNANT GLIOMA: EXPLORING THE GENETIC LANDSCAPE OF PATTERN OF FAILURE
Authors: Won Choi Seung , Nam Do-Hyun , Sa Jason , Koo Harim
Published: November 2019

Failure patterns in malignant gliomas have been described in previous literatures, however, studies were limited to analyze clinical features to account for predisposition to distinct failure patterns. Present study aimed to describe the landscape of failure patterns in malignant glioma from large cohort by integrating multi-omics data and investigate the genetic backgrounds of distinct failure patterns. A total of 423 cases from 325 patients who enrolled at the registry of IRCR at SMC were reviewed for their pattern of failure. Failure patterns were categorized into local, distant recurrence and leptomeningeal seeding regarding recurrent tumors’ spatial relation to primary location. Genomic data was available for 327 (DNAseq) and 259 samples (RNAseq), respectively. Glioblastoma was the most prevalent histologic type in study cohort (81.2%)) and majority of cases experienced the recurrence (79.0%). None of clinical parameters (e.g. age, sex, extent of operation and history of prior therapy) failed to show any significant association with failure patterns. Although local recurrence was most prevalent (63.8%) among failure patterns in malignant gliomas, considerable portion of patients (37.8%) demonstrated other types of failure patterns even in their initial relapse. Multivariate analysis demonstrated that failure pattern was significant prognostic factor to overall survival (remote recurrence, HR=1.59, p-value=0.009; leptomeningeal seeding, HR=2.17, p-value< 0.001). Genomic analysis including mutational profile revealed distinct molecular landscape of malignant gliomas according to failure patterns, which suggested that innate biologic characteristics of tumors might contribute to develop distinct failure patterns upon recurrence. PTEN mutation was significantly enriched in tumors of distant recurrence (p-value=0.026). We described the landscape of failure patterns in malignant gliomas by integrating clinical and genomic data. Considerable amount of malignant glioma patients experienced distinct failure patterns other than local recurrence and their clinical outcome as well as genetic background demonstrated invasive characteristic of these tumors.
GENE-22. GENOME-WIDE METHYLATION PROFILING OF GLIOBLASTOMA EXTRACELLULAR VESICLE DNA ALLOWS TUMOR CLASSIFICATION
Authors: Ricklefs Franz , Maire Cecile , Kolbe Katharina , Holz Mareike , Westphal Manfred , Schüller Ulrich , Lamszus Katrin
Published: November 2019

BACKGROUND

Genome-wide methylation profiling has recently been developed into a tool that allows subtype tumor classification in central nervous system (CNS) tumors. Extracellular vesicles (EVs) are released by CNS tumor cells 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 glioma-derived EVs reflects genome-wide tumor methylation profiles and allows tumor subtype classification.

METHODS

DNA was isolated from EVs secreted by cultured glioma stem-like cells (GSC) as well as from the cells of origin and from the original tumor samples (n=3 patients). EVs were classified by nanoparticle analysis (NTA), immunoblotting, imaging flow cytometry (IFCM), multiplex EV assays 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.

RESULTS

The size range of GSC-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 and multiplex EV assays. Genome-wide methylation profiling of GSC-derived EVs correctly identified the methylation class of the original tumor, including information on the IDH mutation status and subclass classification (RTK1, RTK2). In addition, copy number alterations and the MGMT metyhlation status matched the pattern of the parental GSCs and original tumor samples.

CONCLUSION

EV DNA faithfully reflects the tumor methylation class as well as the MGMT methylation status and copy number variations 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 CNS tumors.
GENE-37. VESTIBULAR SCHWANNOMA IS COMPRISED OF NEURAL CREST AND IMMUNE SUBGROUPS
Authors: John Liu S , Vasudevan Harish , Pekmezci Melike , Choudhury Abrar , Lien Brian , Breshears Jonathan , Swaney Danielle , Magill Stephen , Lang Ursula , Chen William , Gopinath Chetna , Castro Maria , Villanueva-Meyer Javier , Braunstein Steve , Sneed Patricia , Lim Daniel , Perry Arie , Krogran Nevan , McDermott Mike , Berger Mitchel , Theodosopoulos Philip , Raleigh David
Published: November 2019

BACKGROUND

Vestibular schwannomas (VS) are tumors arising from cranial nerve Schwann cells and show variable outcomes after treatment, including oscillation in size for many years after radiosurgery. To understand the unique biology of VS, we performed multiplatform molecular profiling to develop a single cell atlas of VS and reveal that VS exists on a molecular axis defined by neural crest and immune genes.

METHODS

Sixty-six sporadic VS with available tissue for molecular profiling from 59 consecutive patients at a single institution were included. 850K DNA methylation arrays and RNA sequencing were used to profile both primary (76%) and recurrent (24%) tumors. Single nuclei RNA sequencing of 7 tumors and single cell RNA sequencing of 3 tumors and cell lines were used to define the cellular composition of VS and heterogeneous changes in molecular programs following irradiation. Molecular subtyping was performed by hierarchical clustering of differentially-methylated DNA probes and validated using transcriptomic data. Mechanistic experiments were performed using cultured human schwann cells and human vestibular schwannoma cells, confocal microscopy, CRISPR interference, proteomic mass spectrometry and lymphocyte migration assays.

RESULTS

Multiplatform genomic profiling and machine learning revealed that VS is comprised of two distinct molecular subtypes characterized by heterogeneous cell populations. Neural crest enriched VS express primary cilia and are associated with misactivation of the Hedgehog pathway. Consistently, we find that the Hedgehog pathway antagonist vismodegib blocks the growth of human Schwann cells. Irradiation epigenetically reprograms tumors and cell lines to reduce ciliary length, attenuate Hedgehog signaling, activate senescence pathways, and express cytokines and apolipoproteins that recruit lymphocytes and macrophages to immune enriched VS.

CONCLUSIONS

Our data reveal novel molecular subtypes of VS and establish a framework for understanding how irradiation modifies the epigenome and tumor microenvironment.
GENE-39. CRISPRi-BASED RADIATION MODIFIER SCREEN IDENTIFIES LONG NON-CODING RNA THERAPEUTIC TARGETS IN GLIOMA
Authors: John Liu S , Malatesta Martina , Seo Kyounghee , Lien Brian , Hong Sung , Koontz Mark , Raleigh David , Ullian Erik , Lim Daniel
Published: November 2019

Long noncoding RNAs (lncRNAs) exhibit highly cell type-specific expression and function, making this class of transcript attractive for targeted cancer therapy. However, the vast majority of lncRNAs have not been tested as potential therapeutic targets, particularly in the context of currently used cancer therapy. Malignant glioma is rapidly fatal, and ionizing radiation is part of the current standard-of-care used to slow tumor growth in both adult and pediatric patients. Here, we used a CRISPR interference (CRISPRi) platform to systematically screen 5689 lncRNA loci in human glioblastoma (GBM) cells, identifying 467 hits that modified cell growth in the presence of clinically relevant doses of fractionated radiation. 34 of these lncRNA hits sensitized cells to radiation, and based on their expression in adult and pediatric glioma, nine of these hits were prioritized as lncRNA Glioma Radiation Sensitizers (lncGRS). Knockdown of lncGRS-1 – a primate-conserved, nuclear-enriched lncRNA – inhibited the growth and proliferation of primary adult and pediatric glioma cells, but not the viability of normal brain cells. Using human brain organoids comprised of mature neural cell types as a 3-dimensional tissue substrate to model the invasive growth of glioma, we found that antisense oligonucleotides (ASOs) targeting lncGRS-1 selectively decreased tumor growth synergistically with radiation therapy. These studies identify lncGRS-1 as a glioma-specific therapeutic target and establish a generalizable approach to rapidly identify novel therapeutic targets in the vast non-coding genome that can enhance the standard-of-care for cancer therapy.
GENE-50. SWI/SNF COMPLEX HETEROGENEITY RELATES WITH POLYPHENOTYPIC DIFFERENTIATION, PROGNOSIS AND IMMUNE RESPONSE IN RHABDOID TUMORS
Authors: Venneti Sriram , Panwalkar Pooja , Pratt Drew , Chung Chan , Dang Derek , Lee Paul , Martinez Daniel , Bayliss Jill , Smith Kyle , Adam Mike , Potter Steve , Northcott Paul , Mascarenhas Leo , Shows Jared , Pawel Bruce , Margol Ashley , Huang Annie , Judkins Alexander
Published: November 2019

PURPOSE

Rhabdoid tumors (RTs) arise within (Atypical Teratoid/Rhabdoid Tumor-AT/RT) or outside the brain (extraCNS-RT-eCNS-RT) and are driven mainly by inactivation of the SWI/SNF complex subunit SMARCB1. A pathognomonic hallmark of RTs is heterogeneity characterized by multilineage differentiation of tumor cells including anomalous neuronal differentiation in a subset of eCNS-RT. The mechanisms that regulate heterogeneity in RTs are unknown. Because remodeling of the SWI/SNF complex regulates differentiation, we hypothesized that SWI/SNF-BAF and PBAF complex heterogeneity correlates with both multilineage differentiation and clinical outcome. EXPERIMENTAL DESIGN: We performed an integrated analysis of SWI/SNF complex subunit alterations in the developing kidney and cerebellum (most common regions of origin for RT) in comparison to eCNS-RT (n=14) and AT/RT (n=25) tumors. RT samples were interrogated using immunohistochemistry, DNA methylation and gene expression analyses.

RESULTS

(A) The SWI/SNF-BAF paralogs ACTL6A/ACTL6B were expressed in a mutually exclusive manner in the developing cerebellum and kidney. In contrast, a subset of eCNS-RTs lost mutual exclusivity and co-expressed both subunits. These tumors showed aberrant DNA methylation of genes that regulate neuronal and renal development and demonstrated immunohistochemical evidence of neuronal differentiation. (B) Low expression of the PBAF subunit-PBRM1 identified a group of AT/RTs in younger children with better overall prognosis. PBRM1-low AT/RT and eCNS-RTs showed altered DNA methylation and gene expression in immune-related genes accompanied by increased CD8 cytotoxic T-cell infiltration.

CONCLUSIONS

Heterogeneity in SWI/SNF BAF (ACTL6A/ACTL6B) and PBAF (PBRM1) subunits correlates with histogenesis, contributes to the immune microenvironment and prognosis in RTs and may inform opportunities to develop immunotherapies.
GENE-60. THE EPITRANSCRIPTOMIC CODE IN LGG: METABOLICALLY REPROGRAMMED IDH-MUTANT GLIOMAS ALTER tRNA MODIFICATION LANDSCAPE
Authors: Scheepbouwer Chantal , Borland Kayla , Aparicio Ernesto , Verschueren Heleen , Wedekind Laurine , Ramaker Jip , Misovic Branko , CM Kouwenhoven Mathilde , Noske David , Vandertop Peter , Wesseling Pieter , Wurdinger Tom , Hackenberg Michael , Kellner Stefanie , Koppers-Lalic Danijela
Published: November 2019

BACKGROUND

Diffuse lower grade gliomas (LGGs) are generally slow growing primary central nervous system tumors that occur in early adult life. The prevalence of isocitrate dehydrogenase (IDH) mutations is high in LGG, and induces excess production of the oncometabolite 2-hydroxyglutarate (2-HG). These gain-of-function mutations play a key role in promoting metabolic reprogramming of the cancer cell that affects activity of α-KG dependent demethylases. Inhibition of DNA demethylase activity leads to glioma with a CpG island methylator phenotype (G-CIMP). Whether the activity of RNA demethylases and methylation status of tRNAs in LGG are modulated by changes in IDH-status is unknown. AIM: To investigate whether IDH mutations play a role in reprogramming of tRNA modifications in adult glioma.

MATERIALS AND METHODS

We combined small RNAseq and LC-MS/MS analysis to identify distinct tRNA processing patterns and methylation signatures in LGG tissues. To address important experimental bottlenecks that limit RNAseq-based detection of tRNA and possibly other modified small noncoding RNAs, we employed a tailored small RNAseq method with validation of specific methylation sites by mass-spectrometry.

RESULTS

Our customized small RNAseq approach yielded >100 fold increase in sequencing reads per tRNA type, thereby dramatically improving tRNA detection when compared to currently used small RNAseq approaches. Moreover, LC-MS/MS analysis revealed a higher abundance of modified nucleosides in tRNA from IDH-mutant LGG compared to IDH-wildtype LGG. Analysis of tRNA from IDH-mutant and IDH-wildtype LGG using the combination of our tailored small RNAseq and LC-MS/MS methodology demonstrated strong differential tRNA expression, tRFs processing and tRNA methylation.

CONCLUSION

We described an approach that makes use of tailored small RNA sequencing combined with mass-spectrometry that enables insights into cancer driven alterations in tRNA methylation patterns and differential tRNA processing signatures. Our data implies that tumor metabolic reprogramming deregulates tRNA methylation, contributing to an altered epitranscriptomic code in IDH-mutant LGG.
HOUT-03. CLINICAL OUTCOMES OF PATIENTS WITH VESTIBULAR SCHWANNOMAS: THE RELEVANCE OF TUMOR SIZE AND RECURRENCE
Authors: Zhao Amy , Fomchenko Elena , Tyrtova Evgeniya , Huttner Anita , Zhang Yawei , Fulbright Robert , Zeynep Erson-Omay E , Jin Lan , Moliterno Jennifer
Published: November 2019

INTRODUCTION

Vestibular schwannomas (VS) are known to be slow-growing lesions that can present significant issues for patients. Here, we sought to better understand their outcomes and risks for recurrence.

METHODS

We retrospectively reviewed clinical outcomes data for patients who had VS tumor that underwent whole-exome sequencing after surgical resection between 2015 and 2018. The association between any two categorical variables was investigated using Fisher’s exact test. The mean of a continuous variable was compared between groups using either a two-tailed t-test or ANOVA.

RESULTS

Forty patients met our inclusion criteria. Interestingly, men were more likely than women to experience recurrence (p = 0.05). In accordance with previous research demonstrating a significant association between NF2 and bilateral vestibular schwannomas, most VS were NF2-mutants, with only four being were non-NF2. Pre-operative tumor size significantly correlated with progression-free survival time from surgical resection to recurrence, such that larger tumors were more likely to recur slower post-operatively (p = 0.043). There appeared to be no significant association between the extent of surgical resection (EOR) and recurrence (p = 0.653). In addition, there was no significant difference in post-operative complications or clinical outcomes, including EOR and facial nerve function, between different surgical approaches (p > 0.30). Patients with pre-operative intra-tumoral hemorrhage were more likely to have smaller tumors, suggesting they present more acutely (p = 0.119). Finally, patients with NF2-mutated VS had a longer time from original diagnosis to surgery than patients who had non-NF2 VS mutants (p = 0.041).

CONCLUSIONS

Surgery for VS offers good clinical outcomes overall with low recurrence rates, regardless of prior treatment. Though not related to Ki67, larger tumors appear to confer a slower growth potential and lower risk of recurrence over time.
HOUT-05. COMPARING OUTCOMES OF ADULT DIFFUSE MIDLINE GLIOMAS TO GLIOBLASTOMA (GBM) IN YOUNG PATIENTS
Authors: Sarangi Sasmit , Wong Eric
Published: November 2019

Diffuse midline gliomas are a diagnostic entity with astrocytic tumors in a midline location and in the pediatric age group they have been associated with poor outcomes. There is ongoing debate if adults with diffuse midline gliomas also have a poor prognosis particularly as complete surgical resection is often not achievable in these patients. One of the limitations of looking at case-series based outcomes with these patients is that the incidence of these tumors has a skew towards younger patients and recent case-series with observed better than expected outcomes, might be subject to influence from this parameter. To better compare outcomes of these patients we reviewed our institutional tumor registry to identify 31 patients with diffuse midline gliomas from January 2010 to December 2017. We also identified a contemporaneous consecutive series of 46 patients of age ≤ 55 with a diagnosis of GBM, as the control group for comparison. Both groups were comparable in median age (53 vs 50), gender distribution and median Karnofsky Performance Status (80 vs 90). But fewer resections were performed on diffuse midline gliomas (35.5% vs 80%). Our results indicate that patients with a diffuse midline glioma have a higher likelihood of a poorer outcome with a median overall survival of 52 vs 91 weeks in the control. The hazard ratio for death in the diffuse midline glioma cohort as compared to the control was 1.68 (95% CI – 0.97–2.91, log-rank P value = 0.038). The 2-year survival rate was also lower in the diffuse midline glioma with 22.6% vs 39.1% in the control. This poorer outcome could be related to the slightly lower median performance status or the lack of complete resection in diffuse midline glioma patients. Additional analysis is being planned to genetically differentiate these patients and identify possible prognostic indicators.
HOUT-19. THE SHORT-TERM OVERALL SURVIVAL ASSOCIATED WITH SINGLE- VS. MULTI-AGENT CHEMOTHERAPEUTIC REGIMENS FOR 1p/19q-CODELETED WHO GRADE III ANAPLASTIC OLIGODENDROGLIOMAS: A NATIONAL EVALUATION
Authors: Touat Mehdi , Harary Maya , Kavouridis Vasileios , Smith Timothy , Iorgulescu Bryan
Published: November 2019

INTRODUCTION

Although diffuse gliomas of oligodendrocytic lineage demonstrate chemosensitivity, the survival outcomes associated with single- (i.e. temozolomide; TMZ) or multi-agent (i.e. PCV) chemotherapy regimens remain uncertain for anaplastic oligodendrogliomas (AO).

METHODS

Patients presenting between 2010–2016 with 1p/19q-codeleted WHO grade III AO were identified by ICD-O3 and site-specific factors from the National Cancer Database, which comprises >70% of cancers newly-diagnosed in the U.S. Predictors of receiving first-line single- vs. multi-agent chemotherapy were assessed by multivariable logistic regression. Overall survival (OS) was estimated by Kaplan-Meyer approaches and evaluated by multivariable Cox regression.

RESULTS

There were 952 patients with 1p/19q-codeleted WHO grade III AO and complete first-line chemotherapy data, with: 13.9% (n=132) no-, 75.0% (n=714) single-, and 11.1% (n=106) multi-agent chemotherapy. In logistic regression of chemotherapy-treated AOs, more recent diagnosis was associated with higher multi-agent (OR=1.48/year, 95%CI=1.25–1.74, p< 0.001) rates; otherwise there were no associations of single- vs. multi-agent chemotherapy with patient sex, age-at-diagnosis, race, insurance status (reference=privately insured), comorbidity index, tumor greatest dimension, tumor location (reference=frontal lobe) or crossing of midline, nor with radiotherapy or EOR (all p >0.05). Multi-agent usage rose from 3.6% in 2010 to 24.3% in 2016. Median follow-up was 34.9mos (IQR=18.8–54.8). The unadjusted 5yr-OS rate was 57.4% (95%CI=43.5–69.1) for no chemotherapy, 72.1% (95%CI=67.1–76.5) for single-agent, and 77.5% (95%CI=59.9–88.1) for multi-agent. Cox regression (adjusting for the above variables of radiotherapy and EOR, patient demographics, and tumor characteristics) demonstrated no significant OS difference between single- and multi-agent (HR=0.91, 95%CI=0.38–2.15, p=0.82) chemotherapy.

CONCLUSIONS

In a national database of AOs managed in the ‘real-world’ setting, there is increasing utilization of multi-agent (i.e. PCV) chemotherapy; but no significant difference in risk-adjusted short-term mortality (i.e. ~3-5yrs after diagnosis) between first-line multi- and single-agent (i.e. TMZ) chemotherapy. These findings provide preliminary data while we await the long-term PFS and OS results from the CODEL trial.
IMMU-25. MAGNETIC PARTICLE IMAGING FOR NON-INVASIVE TRACKING OF ADOPTIVE CELL TRANSFER IN CANCER IMMUNOTHERAPY
Authors: Rivera-Rodriguez Angelie , Hoang-Minh Lan , Marrero-Morales Leyda , Mitchell Duane , Rinaldi Carlos
Published: November 2019

BACKGROUND

Adoptive cell therapies (ACT) are strategies being explored to boost the immune response against cancer. ACT cancer immunotherapies are effective against metastatic melanoma, leukemia, and lymphoma, but face challenges in treating other solid tumors, such as in the brain. A critical step for the success of ACT in solid cancers is achieving trafficking and persistence of T-cells at tumor sites. Glioblastoma (GBM) is the most common and aggressive cancer of the central nervous system in adults, with a prognosis of 15-18-month average patient survival after diagnosis. Biomedical imaging is often used to track cell therapies. Magnetic Particle Imaging (MPI) is a novel biomedical imaging modality enabling non-invasive visualization of the distribution of biocompatible superparamagnetic iron oxide (SPIO) tracers.

OBJECTIVE

Label T-cells with SPIO to non-invasively track adoptive T cell transfer immunotherapy with MPI in the context of brain cancer.

METHODS

Murine pmel-DsRed T-cells were isolated from the spleen of a transgenic C57BL/6 mouse, and were exposed to different SPIO concentrations ex vivo. Cell viability, phenotype, and cytotoxic function were analyzed to determine if T-cells were affected by the SPIO labeling. Moreover, in vivo experiments were performed in a murine GBM model, and labeled T-cells were injected intravenously and tracked using MPI.

RESULTS

The SPIO-labeling of T-cells did not affected cell viability, phenotype, or cell cytotoxic function at all tested incubation conditions. The internalized SPIO can be quantified and spatially detected using MPI both in vitro and in vivo. In addition, MPI in vivo tracking shows T-cells accumulation in liver and lungs, as well in the spleen and brain, as showed ex vivo.

CONCLUSIONS

SPIO-labeling of T-cells did not affected its cytotoxic function and MPI allows for in vivo tracking of adoptively T-cell transfer. MPI will provide better understanding of ACT dynamics to accelerate development of novel treatments.
IMMU-27. IMMUNE CHECKPOINT BLOCKADE OF EX VIVO EXPANDED T CELLS FOR USE IN ADOPTIVE CELLULAR THERAPY (ACT) FOR GLIOBLASTOMA
Authors: Ogando-Rivas Elizabeth , Yang Changlin , Castillo Paul , Dechkovskaia Anjelika , Mitchell Duane
Published: November 2019

BACKGROUND

Despite aggressive treatments, GBM continues to have unacceptably high mortality rates. Immune-checkpoint blockade and ACT have shown excellent results in other solid tumors, especially in melanoma. Unfortunately, these results have not been extrapolated to GBM. We have developed a novel platform for ACT using tumor mRNA-pulsed dendritic cells(DCs) to in-vitro expand polyclonal populations of tumor-reactive T-cells. This platform has shown promising effects in preclinical brain tumor models (Flores et al OncoImmunology 2015, Wildes et al CCR 2018, Flores et al NatureComm 2018) and being evaluated in clinical trials at UF Health (NCT02465268,NCT03334305).

STUDY OBJECTIVE

Evaluate whether immune-checkpoint blockade during ex-vivo expansion of antigen-specific T-cells impact their use in ACT.

METHODS

CMVpp65 was used as model antigen for in-vitro activation of T-cells. Mature pp65 mRNA-pulsed DCs from CMV+ healthy donors were co-cultured with T-cells in IL2-containing medium for 15days. We tested four checkpoint inhibitor groups: PD1(n= 6), PDL1(n= 4), TIM3(n= 7) and PD1+TIM3(n= 6) that were compared with non-blockade group, respectively. Checkpoint blockade was performed every 3days. T-cell proliferation, immune-phenotyping, and IFN-g release were analyzed.

RESULTS

Cell proliferation was lower in all the blockade groups but significantly lower in the TIM3 (p= 0.03) and TIM3+PD1 (p= 0.01) blockade groups. TIM3 expression was significantly lower in the TIM3 (p= 0.006) and PD1+TIM3 blockade groups (p= 0.0001). There was a trend of reduced pp65 tetramer positive in the TIM3 and PD1+TIM3 blockade groups (PD1+TIM3 subgroup at 3mcg/mL, p= 0.02) and lower INFg release in the TIM3 and PD1+TIM3 blockade groups.

CONCLUSION

The exact role of checkpoints during expansion of T-cells for ACT is not well understood. In our study checkpoint blockade with PD-1 or TIM-3 alone or in combination did not enhance T-cell expansion or function, in fact, appeared to have an inhibitory effect on measured parameters. Our results suggest that TIM-3 may have an activating role in our system.