bluuebunny/crossref_metadata_2025 huggingface dataset:
CNSC-04. CEREBROSPINAL FLUID-DERIVED CIRCULATING TUMOR DNA IS SUPERIOR TO PLASMA IN NSCLC PATIENTS WITH CNS PROGRESSION
Authors: Yang Hainan , Wen Lei , Zhou Caicun
Published: November 2022

Introduction

Central nervous system (CNS) metastases are devastating neurologic complications. However, the clinical value of cerebrospinal fluid (CSF) as liquid biopsy medium in EGFR-mutated or ALK-rearranged patients with brain metastases (BM) or leptomeningeal metastases (LM) remains unclear.

Patients and Methods

We retrospectively analyzed the gene map of 14/48 NSCLC-BM patients and 34/48 NSCLC-LM patients. Compare the clinical predictive value of CSF and matched plasma (PLA) for patients with single CNS progression (cohort 1) or intracranial progression simultaneously with extracranial disease progression (cohort 2). Circulating cell-free tumor DNA (ctDNA) in CSF and matched PLA underwent next-generation sequencing targeting 168 cancer-relevant genes.

Results

For patients with single CNS progression, the mutation rate of major driver genes is much higher than the rate in matched PLA. Besides, CSF also demonstrated higher levels of potential prognostic markers in comparison with matched PLA samples, showing EGFR-amp presented more than two times the rate compared to matched PLA, and the detection rate of TBX3, PTEN were almost double the rate in CSF than paired PLA samples. In addition, poor outcomes associated with makers including CDKN2A/B, PIK3CA/G, CDK4/6, and MET showed exclusively in CSF samples.Almost all mutations identified in the PLA could detect in matched CSF samples in NSCLC-LM patients with both intracranial and extracranial disease progression. In addition, detection rates of driver mutations, consisting of EGFR or ALK are much higher in CSF compared to matched PLA, and these potential survival marker genes like CDK4/6, CDKN2A/B, EGFR-amp, MET, and PIK3CA identified uniquely in CSF samples.

Conclusion

CSF is more comprehensive in genomic mutation profile than matched PLA in NSCLC -LM regardless of single intracranial progression or intracranial progression simultaneously with extracranial evolution.
SURG-38. ASSESSING THE ROLE OF FRAILTY IN CRANIAL NEUROSURGERY FOR PRIMARY CNS NEOPLASMS IN THE GERIATRIC POPULATION: SEVEN YEAR RETROSPECTIVE ANALYSIS USING THE NATIONWIDE READMISSION DATABASE
Authors: Shahrestani Shane , Brown Nolan , Singh Rohin , Gendreau Julian
Published: November 2022

Frailty is a clinical state of increased vulnerability due to age-associated decline and has been well established as a perioperative risk factor. Yet, literature describing the effects of frailty on short- and long-term complications in geriatric patients is limited. In this study, the authors evaluate the effects of frailty in geriatric patients receiving cranial neurosurgery for a primary CNS neoplasm. For this purpose, we conducted a retrospective cohort study between 2010 and 2017 by using the Nationwide Readmission Database. Demographics and frailty were queried at primary admission, and readmissions were analyzed at 30-, 90-, and 180-day intervals. Nearest-neighbor propensity score matching for demographics was implemented to identify nonfrail control patients with similar diagnoses and procedures. The analysis used Welch two-sample t-tests for continuous variables and chi-square test with odds ratios. A total of 6713 frail patients and 6629 nonfrail patients were identified at primary admission. At primary admission, frail geriatric patients undergoing cranial neurosurgery had increased odds of developing acute posthemorrhagic anemia (OR 1.56, 95% CI 1.23-1.98; p = 0.00020); acute infection (OR 3.16, 95% CI 1.70-6.36; p = 0.00022); acute kidney injury (OR 1.32, 95% CI 1.07-1.62; p = 0.0088); urinary tract infection prior to discharge (OR 1.97, 95% CI 1.71-2.29; p < 0.0001); acute postoperative cerebral infarction (OR 1.57, 95% CI 1.17-2.11; p = 0.0026); and mortality (OR 1.64, 95% CI 1.22-2.24; p = 0.0012) compared to nonfrail geriatric patients receiving the same procedure. In addition, frail patients had a significantly increased inpatient length of stay (p < 0.0001) and all-payer hospital cost (p < 0.0001) compared to nonfrail patients at primary admission. Frailty may significantly increase the risks of short-term acute complications in geriatric patients receiving cranial neurosurgery for a primary CNS neoplasm. Long-term analysis revealed no significant difference in complications between frail and nonfrail patients.
TMET-03. STEAROYL-COA DESATURASE INHIBITOR SUPPRESSES IDH MUTANT GLIOMA GROWTH VIA ENHANCING LIPOLYSIS
Authors: Yamasaki Tomohiro , Lita Adrian , Zhang Lumin , Dowdy Tyrone , Koizumi Shinichiro , Kurozumi Kazuhiko , Gilbert Mark , Larion Mioara
Published: November 2022

BACKGROUND

Mutant isocitrate dehydrogenase (IDH) produces 2-hydroxyglutarate (D2HG) and causes metabolic reprograming, but so far, little is known about the role of mutant IDH and D2HG in de novo lipogenesis and fatty acids synthesis. In this study, to develop a feasible drug for IDH mutant glioma, we targeted Stearoyl-CoA desaturase 1 (SCD1) catalyzing the biosynthesis of monosaturated fatty acids (MUFA) to suppress IDH mutant glioma progression. Materials and

METHODS

We prepared genetically engineered glioma cell lines (U251 wild type: U251WT and U251 IDHR132H mutant: U251RH), normal human astrocytes (empty vector induced-NHA: NHAEV and IDHR132H mutant: NHARH) and patient derived cell lines. Lipid metabolic analysis was conducted by using LC-MS, and functional analysis for the role of SCD1 expression was investigated by RNA sequence and Western-blotting.

RESULTS

LC-MS analysis of extracted Endoplasmic Reticulum revealed that there was significantly higher amount of MUFA in IDH mutant than wild type. SCD1 expression was increased in IDH mutant compared to wild type due to D2HG-mediated upregulation of SCD1 in IDH mutant. Therefore, IDH mutant in which SCD1 expression level was higher than wild type indicated high sensitivity to SCD inhibitor, and apoptosis was highly induced in IDH mutant compared to wild type. RNA sequencing was performed in U251RH treated with SCD inhibitor compared to U251RH treated with DMSO, and lipid droplet metabolism-associated RNA expression was significantly changed in SCD inhibitor-treated U251RH. Based on the RNA sequence data, we checked lipid droplet in U251RH with presence or absence of SCD inhibitor, and lipolysis was induced by SCD inhibitor treatment, suggesting that SCD inhibition is associated with the apoptosis in IDH mutant via enhanced lipolysis mechanism.

CONCLUSIONS

D2HG produced in IDH mutant glioma directly induces SCD1 expression and enhances sensitivity to SCD inhibitor, which suggests that SCD inhibitor would be IDH mutant glioma-specific treatment strategy.
TMET-08. METABOLOMIC CHANGES IN GLIOBLASTOMA PATIENTS UNDERGOING CONCURRENT CHEMORADIATION THERAPY
Authors: Aboud Orwa , Liu Yin Allison , Fiehn Oliver , Bridges Christopher , Fragoso Ruben , Hodeify Rawad , Bloch Orin
Published: November 2022

OBJECTIVE

To illustrate changes of untargeted metabolomics in patients with glioblastoma IDH wildtype undergoing concurrent radiation therapy (RT) with temozolomide (TMZ). This study implemented machine learning (ML) algorithms to predict treatment phase: pre-surgery, post-surgery, pre-radiation, and post radiation based on untargeted metabolomics data.

METHODS

Thirty-six patients with glioblastoma IDH wildtype (18 methylguanine methyltransferase [MGMT] methylated, 16 MGMT unmethylated, 2 MGMT status unknown) were enrolled into this study. Serum samples obtained from patients on the same day before surgery, 2 days after surgery, before starting their concurrent chemoradiation, and after concluding this phase of treatment. Blood samples were obtained via antecubital phlebotomy without regard for time of the day, diet, or fasting status. Untargeted metabolomics by GC-TOF mass spectrometry were obtained and compared. The proposed ML models analyzed 105 samples from 36 patients utilizing 157 structurally identified blood metabolites. Multinomial Logistic Regression (MLR) and GradientBoostingClassifier (GB Classifier) were used to classify patient samples based on detected changes in blood metabolites. The classification performance of these models was evaluated using performance metrics and AUC scores.

RESULTS

Post radiation; significant increase in the following metabolites: glycine, serine, threonine, oxoproline, 6-deoxyglucose, gluconic acid, glycerol-alpha-phosphate, ethanolamine, propyleneglycol, triethanolamine, xylitol, and fumaric acid were noted while significant decrease in 3-aminopiperidine 2,6-dione was noted post radiation. MLR produced 78% accuracy, 75% precision, and AUC = 0.89, and GB Classifier produced 75% accuracy, 77% precision and AUC = 0.91. Finally, we presented a pattern of metabolites changes per clinical stage based on pairwise correlations.

CONCLUSIONS

This study represent the first serum metabolic signature associated with RT in patients with glioblastoma. The results from the classification algorithms and pairwise correlations showed that blood metabolites have the potential to predict phase of treatment and potentially enable to evaluate response to treatment in patients with glioblastoma in a relatively small cohort.
TMET-10. FATTY ACID METABOLISM REGULATES IMMUNE REACTIVITY OF IRRADIATED GLIOBLASTOMA
Authors: De Martino Mara , Daviaud Camille , Laiakis Evagelia , Vanpouille-Box Claire
Published: November 2022

Radiation therapy (RT) is the standard-of-care for glioblastoma (GBM) and is the only treatment for inoperable brain tumors. In multiple cancer, RT stimulates anti-tumor immunity by at least activating cancer cell-intrinsic type I interferon (IFN-I) responses. However, GBM inevitably recur, suggesting that RT promotes immune evasion mechanisms. Altered fatty acid (FA) metabolism is an emerging mechanism that can account for treatment resistance and immune escape of GBM. Therefore, we hypothesize that RT induces a metabolic shift toward the production of FAs to foster immunosuppression of GBM. Supporting this hypothesis, our data show that RT upregulates the expression of the fatty acid synthase (FASN) and the activity of the fatty acid desaturase 2 (FADS2) to generate FAs in murine GBM models, GL261 and CT2A. To determine whether FA metabolism was preventing RT-induced IFN-I, we impaired FA synthesis by inhibiting either FASN or FADS2 using genetic (CRISPR-Cas9) or pharmacological inhibitors (FASNi or FADS2i). As expected, protein quantification (ELISA) as well as gene expression (RT-qPCR) revealed that irradiated GBM cells released greater levels of IFN-beta 24hrs post RT; an effect that was more pronounced when FASN or FADS2 were blocked. Reinforcing the role of FA metabolism in regulating immune activation, in situ immunofluorescence of irradiated intracranial GL261 tumors showed that mice treated with FASNi improved the recruitment of CD8+ T cells into GBM. Along similar lines, flow cytometry of digested GL261 tumors revealed an increase infiltration of CD11c+ CD103+ dendritic cells as well as activated CD86+ CD11b+ cells when FAs synthesis was blocked with FADS2i. Altogether, our data suggest that RT-induced FA synthesis represents a mechanism of resistance by preventing IFN-I and promoting immunological evasion of GBM. Targeting FA metabolism is a promising strategy to promote anti-tumor immunity and sensitize irradiated GBM to immunotherapies.
TMET-13. SEX DIFFERENCES IN GLUCOSE METABOLISM AND MITOCHONDRIAL FUNCTION IN GLIOBLASTOMA IMPLICATE HYPOXIA-INDUCIBLE FACTOR 1 ALPHA (HIF1A) ACTIVITY
Authors: Lu Jianyun , Sponagel Jasmin , Jones Jill , Gass Hannah , Nunez Elena , Frankfater Cheryl , Williams-McLeod Sierra , Marquez Camryn , Chinnaiyan Prakash , Rubin Joshua , Ippolito Joseph
Published: November 2022

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. It is more prevalent in males and female patients have better survival. Investigating the molecular mechanisms underlying this disparity is imperative for understanding its development and progression as well as developing novel treatment paradigms. Carbohydrate (namely glucose) metabolism is a critical GBM nutrient source for biosynthesis, energetics, and reducing equivalents. Previously, our group discovered that elevated glycolytic activity uniquely predicted the outcomes of male, but not female, lower grade glioma patients. Our goal was to characterize sex differences in GBM carbohydrate metabolism and their effects on cellular phenotype. First, we discovered that male transformed murine astrocytes were more susceptible to glucose deprivation than females. We confirmed this phenotype with irreversible inhibition of hexokinase with 2-deoxyglucose as well as a GLUT1-selective inhibitor. Time-resolved stable isotope tracing of cell metabolism with carbon-13 glucose in transformed astrocytes further supported these findings; male cells had significantly higher rates of glucose uptake and metabolism than female cells. These results were validated with stable isotope metabolomics datasets from human cancer cell lines. Using additional assays of cellular metabolism, we discovered that male transformed astrocytes had a higher glycolytic rate, higher pyruvate kinase activity, higher mitochondrial respiration, and higher mitochondrial mass compared to females. This was validated by a TCGA pancancer analysis that revealed significantly higher expression of nuclear genes involved in mitochondrial regulation in males than in females. This prompted us to identify possible regulators of this metabolic phenotype. We discovered that HIF1A had robust hypoxia-inducible expression that was significantly higher in male transformed astrocytes. Moreover, HIF1A expression as well as its target transcripts were significantly higher in TCGA pancancer tumor datasets. Together, our data underscore the potential for developing sex-specific metabolic targeting approaches for patients with GBM.
TMET-16. DIRECT DETECTION OF 2HG AND GLUTAMATE PRODUCTION USING HYPERPOLARIZED [1-13C-5-12C]-Α-KETOGLUTARATE IN CELL AND IN VIVO GLIOMA MODELS
Authors: Hong Donghyun , Kim Yaewon , Mushti Chandrasekhar , Minami Noriaki , Gillepspie Anne Marie , Batsios Georgios , Taglang Celine , Cherukuri Murali , Wu Jing , Viswanath Pavithra , Swenson Rolf , Vigneron Daniel , Ronen Sabrina
Published: November 2022

Mutant IDH leads to 2HG production, which drives glioma development. 13C MRS monitoring of hyperpolarized [1-13C]α-ketoglutarate (αKG) metabolism to 2HG and glutamate provides a non-invasive assessment of the IDH mutation and normal metabolism, respectively. However, monitoring 2HG production in vivo has been challenging because its resonance is within 0.1 ppm of the natural abundance [5-13C]αKG signal of the [1-13C]αKG substrate. Here, we utilized [1-13C -5-12C]αKG, which eliminated the [5-13C]αKG peak. This new approach, combined with an optimized sequence, made it possible to readily monitor the production of both 2HG and glutamate in cells and in vivo in healthy rats or rats with orthotopic patient-derived glioma. Hyperpolarized [1-13C -5-12C]αKG was injected into genetically engineered NHAIDHmut cell lysates, healthy rats, and rats implanted orthotopically with BT257 cells intravenously. 1-D dynamic 13C MRS spectra from cells and in vivo slab spectroscopy data were then acquired using an 11.7 T NMR system and a 3 T pre-clinical scanner, respectively. Injection of the hyperpolarized [1-13C -5-12C]αKG into cell lysates showed clearly detectable dynamic conversion of hyperpolarized [1-13C-5-12C]αKG to 2HG and glutamate. The normal brain showed clear production of glutamate but no 2HG was detected. In tumor-bearing rats, we were able to clearly detect the dynamic production of both 2HG and glutamate. This study demonstrated the utility of hyperpolarized [1-13C-5-12C]αKG as a substrate to clearly assess 2HG production without the confounding presence of the natural abundance peak which cannot be distinguished from 2HG in vivo. Importantly, the detection of 2HG provides a clear indicator of the IDH mutation within the tumor.
TMET-21. ENHANCING THE METABOLIC FITNESS OF T CELLS TO TREAT BRAIN TUMORS
Authors: Ghosh Tanya , Jin Linchun , Feier Diana , Silver Aryeh , Tian Guimei , Dajac Mariana , Rahman Maryam , Flores Catherine , Sarkisian Matthew , Huang Jianping , Mitchell Duane , Deleyrolle Loic
Published: November 2022

INTRODUCTION

Glioblastoma is a challenge for neuro-oncologists and current therapies are minimally effective. Standard-of- care treatment is almost inevitably followed by disease recurrence. Adoptive T cell transfer has emerged as a viable therapeutic for brain malignancies. While promising, the efficacy of this approach is often limited by a complex immunosuppressive tumor microenvironment. These complexities mean that more sophisticated T cell products are required. Objectives: The brain tumor microenvironment provides local restraints via metabolic competition suppressing antitumor immunity, specifically inhibiting infiltration and tumoricidal functions of host and adoptively transferred tumor-reactive T cells. The overall goal of this project is to test new treatments to reverse immune dysfunction in cancer through the regulation of T cell metabolic signaling. We propose that modulating the glucose pathway in T cells can potentiate their anti-tumor activity once adoptively transferred.

METHODS

The glucose metabolic pathway of T cells was modulated via overexpression of glucose transporters. The functionality of metabolically modified T cells was investigated in murine and human models.

RESULTS

We demonstrated the existence of a competition for glucose between T cells and tumor cells, with tumor cells imposing glucose restriction mediating T cell hyporesponsiveness. Overexpression of glucose transporters such as Glut1 and Glut3 increased T cell glucose utilization and provided a survival/growth advantage and enhanced T cell activation in glucose-restricted conditions. We also established that glucose transporter overexpression improves intratumoral infiltration and expansion of adoptively transferred T cells, resulting in improved survival.

CONCLUSION

This project integrates fundamental concepts of tumor and immune metabolism in the design of immunotherapy and confirms that immunometabolism represents a viable target for new cancer therapy to treat brain tumors.
TMET-22. ENHANCED LIPID METABOLISM IN BRAIN TUMORS: AN EXPLOITABLE METABOLIC VULNERABILITY THROUGH DIET-INDUCED KETOSIS
Authors: Kant Shiva , Kesarwani Pravin , Zhao Yi , Chinnaiyan Prakash
Published: November 2022

Enhanced lipid metabolism contributes towards carcinogenesis at multiple levels, serving as a "gain of function" in several cancers, including glioblastoma. Seemingly paradoxical to these findings, pre-clinical investigations have demonstrated anti-tumor activity in mice fed a high fat/low carbohydrate ketogenic diet (KD), both alone and in combination with conventional therapy. Therefore, we sought to define mechanistic underpinnings contributing towards this apparent disconnect. Consistent with previous reports, mice fed a KD demonstrated independent anti-tumor activity and potent synergy with RT when tested in an aggressive glioblastoma orthotopic model. To provide a window into metabolic consequences of a KD in glioblastoma, we performed global metabolomic profiling on tumors and serum from mice fed a standard diet and KD. Although ketosis was confirmed, no change in glucose was observed in serum of KD mice, suggesting presence/absence of carbohydrates might not be contributing to the observed anti-tumor activity. Interestingly, profound intra-tumoral metabolic changes were observed in intracranial tumors from mice fed a KD, with an accumulation of unsaturated fatty acids emerging as a central metabolic node. Intriguingly, recapitulating these findings in culture conditions in vitro, polyunsaturated fatty acid linoleic acid demonstrated anti-proliferative activity, decreased clonogenic capacity, and synergy with RT in GBM cells, recapitulating in vivo findings. This was not observed when cultured with the monounsaturated fatty acid oleic acid. Through a series of investigations, we went on to identify lipid storage homeostasis and lipotoxicity associated with free fatty acid accumulation played a contributory role in the differential activity of these different classes of fatty acids. Importantly, PUFA-rich modified diet showed potent antitumor activity in combination with RT in our preliminary investigation. These studies collectively reaffirm and provide mechanistic underpinnings of the anti-tumor activity of a KD, suggesting that enhanced lipid metabolism in brain tumors may serve as an exploitable metabolic vulnerability through diet modification.
TMET-23. GLIOBLASTOMA EXHIBIT SEX DIFFERENCES IN BRANCHED-CHAIN AMINO ACID METABOLISM
Authors: Sponagel Jasmin , Gass Hannah , Cho Kevin , Chinnaiyan Prakash , Patti Gary , Rubin Joshua , Ippolito Joseph
Published: November 2022

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. It is more prevalent in males and female patients exhibit a survival advantage. Understanding the molecular mechanisms that underlie those sex differences could support novel treatment strategies. Amino acids, such as glutamine and the branched-chain amino acids (leucine, isoleucine, valine; BCAAs), play important roles in GBM as they are involved in biosynthesis, energetics, redox homeostasis, and mTOR signaling. Using murine transformed astrocytes, we recently discovered that male cells are more dependent on glutamine and utilize more glutamine to replenish biosynthetic pathways, including BCAA regeneration. BCAA regeneration (i.e., re-amination of branched-chain ketoacids to BCAA) is catalyzed by branched-chain aminotransferases (BCATs) and requires glutamate as a nitrogen donor. Importantly, BCAA regeneration has been linked to cancer progression and poor outcome. Using stable isotope glutamine labeling we found that male transformed astrocytes exhibit greater nitrogen transfer from glutamate onto branched-chain ketoacids to regenerate BCAAs. In line with increased glutamate utilization for BCAA regeneration via BCAT, we found that male transformed astrocytes exhibit greater BCAT1 protein expression levels. Concordantly, male transformed astrocytes are more sensitive to BCAA deprivation compared to female cells, indicating that BCAA metabolism may play a sex-specific role in GBM. Using human GBM surgical specimens, we found greater BCAA metabolite abundance in male GBM. Importantly, our findings on sex differences in BCAA abundance and dependency in GBM are in line with previous reports that show that BCAA abundance and utilization differ between males and females under normal physiological conditions. Together, these data suggest that male GBM are more dependent on BCAAs and that BCAT1 may play unique roles in male and female GBM metabolism. Our data highlight that GBM metabolism differs between males and females and underline the importance of considering sex in metabolic targeting approaches.
CNSC-23. NEURONAL ORIGIN INFLUENCES SPONTANEOUS NETWORK ACTIVITY IN GLIOBLASTOMA-NEURON COCULTURES
Authors: Daniel Andy , Krishna Saritha , Popova Galina , Kaur Jasleen , Hervey-Jumper Shawn L
Published: November 2022

Neuronal activity is emerging as a driver of cancer initiation and proliferation. Several models have been developed to recapitulate cancer-neuron interactions within the tumor microenvironment; however, there is substantial diversity of neuronal subtypes within the murine and human cortex. Differences in the neuronal microenvironment may influence glioma cell behavior and fail to recapitulate in vivo mechanisms. Here, we investigate the electrophysiological properties of cortical neurons and activity-regulated paracrine signaling across co-culture conditions using microelectrode arrays (MEA). Cortical neurons were obtained from three conditions, (1) human prenatal tissues, (2) murine embyronic tissue (E18), and (3) murine postnatal tissue (P1.5), then co-cultured with primary patient-derived glioblastoma cultures. Neuronal activity was assessed using weighted mean firing rate (WMFR; spike rate multiplied by number of active electrodes) and network burst synchrony index. Characterization of the activity-dependent paracrine signaling was performed using proteomics. Initiation of spontaneous firing activity differed across neuronal conditions, with activity occurring earlier in postnatal P1.5 cultures (day in vitro [DIV] 7), whereas E18 and human prenatal cultures demonstrated delayed patterns of activity (DIV 14 and DIV 21, respectively). Glioblastoma cells were added on the first day of consistent spiking. All neuronal co-culture conditions demonstrated glioma-induced hyper-activity (increased WMFR), however in different patterns. E18 co-cultures exhibited glioma-induced hyperexcitability within 24 hours with a subsequent decrease in activity over 14 days with minimal synchrony. P1.5 demonstrated no glioma-induced hyper-excitability at 24 hours; however, WMFR peaked by 7 days and remained elevated through co-culture day 14 (including concurrent increase in synchrony). Human postnatal neurons demonstrated a steady and consistent increase in WMFR as early as 24 hours post co-culture and remained active through day 14 with no synchrony change. Our data suggest that careful consideration should go into selecting neuronal co-culture conditions for mechanistic studies.
TMIC-03. TUMOR INFILTRATING LYMPHOCYTES IN BRAIN METASTASIS: CLINICOPATHOLOGICAL EVALUATION AND COMPARISON WITH PAIRED PRIMARY
Authors: Sahay Ayushi , Indave Mayur , Chatterjee Abhishek , Moiyadi Aliasgar , Patil Vijay , Gupta Tejpal , Shetty Prakash , Singh Vikas , Menon Nandini , Sahu Arpita , Chaudhari Amit , Sridhar Epari
Published: November 2022

INTRODUCTION

Little is known about tumour-infiltrating lymphocytes (TILs) in brain metastasis (BM) and their utility as predictive biomarker for immunotherapy. AIM: To analyze TILs in BM, and correlate with corresponding paired primary (PP).

METHODS

Inclusion: Histologically confirmed BM and PP (2015-2020). Exclusion: Hematological malignancies, spinal cord/meningeal metastasis, pediatric age, slides/blocks not available. Final Nf 277 BM; 64 PP. One H&E slide from both PP &BM assessed for stromal TILs (sTILs) and intratumoral TILs (iTILs) as per International TIL Working Group guidelines.

RESULTS

Median sTILs (10%) were significantly higher than iTILs (1%) in primary and BM (p-value < 0.001). sTILs were mild (<10%) in majority of primary (57.8%) and BM (59.9%), while iTILs were nearly always mild (primary 97.1%; BM 96.9%). Between PP and BM, no change in sTIL category in 48.43%, while conversion in 51.56%, with higher to lower conversion (66.67%) > lower to higher (33.33%). Conversion rate higher for breast (54.3%) &GIT (62.5%) than lung (45.5%) &GUT (40%). In BM, preoperative steroid therapy (PST) showed lower sTILs (≤10%) (p-value= 0.041), and adenocarcinoma histology higher sTILs ( >10%) (p-value= 0.001), while precocious metastasis (p-value= 0.051) and absence of extracranial metastasis (p-value= 0.083) trended towards higher sTILs. Median survival of whole cohort was 20 months (95% CI 14.4-25.68 months). Presence of extracranial metastasis (p-value= 0.01); precocious BM (p-value= 0.007) showed lower overall survival (OS), while adenocarcinoma histology better OS (p-value= 0.029), in both univariate and multivariate analysis. Absence of PST showed longer median survival, though not significant (24.1 vs 19.8months; p-value= 0.19).

CONCLUSIONS

sTILs predominantly show high >low conversion between PP and BM, signifying lowered immune response in BM. Thus, evaluation of sTILs in BM, wherever tissue available, may be indicated if immunotherapy is considered. Parameters with higher sTILs showed longer survival, possibly indicating prognostic role of tumor microenvironment in BM.
TMIC-06. ANTAGONISM OF DRD2 USING ONC201 INCREASED EXPRESSION OF ANTIGEN PRESENTATION PATHWAY PROTEINS IN DIFFUSE MIDLINE GLIOMA, RECRUITING TUMOR INFILTRATING LYMPHOCYTES IN VIVO
Authors: Persson Mika , Jackson Evangeline , Duchatel Ryan , Bramberger Liesl , McEwen Holly , Kearney Padraic , Findlay Izac , Douglas Alicia , Kobbe Birgit , Wagener Raimund , Larsen Martin , Faridi Pouya , Holst Jeff , Mayall Jemma , Gedye Craig , Hondermarck Hubert , Horvat Jay , Nixon Brett , Cartaxo Rodrigo , Koschmann Carl , Valdes-Mora Fatima , Ortega David Gallego , Nazarian Javad , Alonso Marta M , Hulleman Esther , Van der Lugt Jasper , Vitanza Nicholas , Mueller Sabine , Dun Matthew
Published: November 2022

Diffuse midline glioma (DMG) is a high-grade glioma with a median overall survival of 9-11 months. Radiotherapy is the only recognized treatment. The DMG tumor microenvironment (TME) contains few, if any, tumor infiltrating lymphocytes (TILs) or inflammatory cytokines, thus is distinctive of an ‘immunologically cold’ tumor/TME.1 DMG lack the expression of immunosuppressive immune checkpoint proteins, likely explaining the failure of immune checkpoint inhibitors (ICI) tested under clinical trials for DMG patients, and suggestive of an alternative mechanism underpinning the cold TME. 1 Glioblastomas also harbor a cold TME, which can be somewhat explained by T cell lymphopenia, influenced by the sequestration of T cells in the bone marrow (through Beta-arrestin-induced internalization of Sphingosine-1-phosphate receptor 1 [S1PR1]). 2 Dopaminergic activation of Beta-arrestin and hence S1PR1 internalization, is potentially regulated through dopaminergic peripheral nerves in primary and secondary lymphoid organs, regulated by the Dopamine receptor D2 (DRD2), that is highly expressed on T cells. ONC201 is a potent DRD2 antagonist, currently in phase I-III clinical trials for DMG patients, alone and in combination with radiotherapy and the PI3K/AKT inhibitor paxalisib (NCT05009992). Proteomic profiling of DMG patient-derived cells +/-ONC201 showed increased expression of several antigen presenting pathway proteins, including Beta-2-microglobulin (B2M) and HLA class I histocompatibility antigen, A alpha chain (HLA-A). This was confirmed in vivo using SU-DIPG-VI patient-derived xenograft mouse model tissues +/-ONC201 alone, and together with paxalisib. Excitingly, this combination (given orally) promoted the recruitment of TILs to the tumor, revealing novel immunomodulatory effects. In vivo, ONC201 promoted the expression of EMILIN-3, a TGF-β antagonist that is known to inhibit HLA-A/B2M expression, possibly explaining the increased MHC-I activity. This study uncovers a novel link between treatment of DMG with ONC201 and paxalisib and the role dopaminergic peripheral nerves signaling may play on the sequestration of T cells within lymphoid organs and lymphopenia.
TMIC-15. AGE-RELATED MARKERS FOR SENESCENCE INCREASE IN THE OLDER ADULT EXTRATUMORAL MOUSE BRAIN DUE TO GLIOBLASTOMA
Authors: Bell April , Johnson Margaret , Ladomersky Erik , Kim Miri , Zhao Junfei , Miska Jason , Zhai Lijie , Lauing Kristen , Asimakidou Evridiki , Bommi Prashant , Chandler James , Mi Xinlei , Kocherginski Masha , Lukas Rimas , Wainwright Derek
Published: November 2022

OBJECTIVE

The median age of onset for glioblastoma (GBM; IDHwt) is 68-70 years. Age is a strong prognostic factor for GBM patient outcomes such that overall survival in older adults is less than their younger counterparts – even after adjustment for MGMT promoter methylation status. Aging is associated with increased levels of senescence in the brain. Several age-related neurological disorders have been shown to improve with senolytic treatments. Here, we explored the effects and therapeutic neutralization of syngeneic brain tumors on increasing senescence levels in the extratumoral brain (ie. outside of the brain tumor) of young and old mice.

METHODS

General RNA-sequencing, as well as single-cell (sc) RNA-sequencing was performed on extratumoral tissue from young (8-12 weeks) and older adult (80-90 weeks) C57BL/6 mice with or without GL261 and key markers were validated with RT-PCR. The combined effects of the senolytics, dasatinib and quercetin, with radiation, anti-PD-1 mAb, and IDO enzyme inhibitor treatment, was also investigated.

RESULTS

General- and sc-RNA sequencing revealed a distinct gene expression profile in the extratumoral brain of older mice with syngeneic GL261 as compared to all other groups. RT-PCR results confirmed that the brain tumor increased gene expression for senescence levels, p53, and NFkB signaling in the older adult extratumoral brain. Expression of the senescence marker p16INK4A was primarily localized to oligodendrocyte progenitor cells in the older adult brain. The combinatorial treatment of senolytics with RT, anti-PD-1 mAb, and IDO enzyme inhibitor led to a synergistic survival benefit in older adult mice with GL261 as compared to the treatment with senolytics, or immunotherapy, alone.

CONCLUSIONS

The data suggest that the extratumoral brain may be responsible in-part for the poorer outcomes of older adults with GBM and that treatment approaches that target senescent cells may provide clinical benefit.
TMIC-21. THE ROLE OF CLEC5A ON M2-LIKE TUMOR-ASSOCIATED MACROPHAGES POLARIZATION AND DISEASE PROGRESSION IN GLIOBLASTOMA
Authors: Li Jiabo , Wang Xuya , Markwell Steven , Carneiro-Lobo Tatiana , Olson Cheryl , Shih Ling-Kai , Tong Luqing , Yang Xuejun , Brat Daniel
Published: November 2022

Glioblastoma, IDH-wildtype (GBM, WHO grade 4) is the most common primary intracranial malignant tumor of adults and is characterized by an immunosuppressive tumor micro-environment (TME) and poor clinical outcomes. CLEC5A is a protein-coding gene involved in inflammatory and infectious diseases, where its activation enhances immune response. However, its role in GBM immune regulation is unclear. Seurat analysis of scRNA-seq data and CIBERSORTx analysis of GBM RNA-seq data showed that M2-like TAMs accounted for the largest proportion of immune cells in the GBM TME. Among CD163+ cells (M2-like TAM marker) in GBM, CLEC5A expression was most highly associated with overall patient survival. IHC on tumor microarrays (TMA) and analysis of Ivy GAP data indicated that CLEC5A and M2-like TAMs were preferentially expressed in tumor peri-necrotic zone. THP-1 cells exposed to GBM conditioned media showed increased CLEC5A and CD163 expression by RT-qPCR and Western blot compared to other immune-activating factors (IL-4, IL-13). CLEC5A overexpression in THP-1 cells enhanced their migration toward glioma cells in vitro, and also led to increased M2-like TAM biomarker expression. Cytokine antibody microarray results revealed that overexpressing CLEC5A in THP-1 cells also significantly increased the expression of IL-1β, IL-4, IL-6, IL-8, IL-10, IL-13, GM-CSF, and other cytokines, which all contribute to the immunosuppressive TME. In TCGA GBM data, tumors with higher CLEC5A expression were enriched in IL6-JAK-STAT3 signaling. In vivo, silencing CLEC5A delayed glioma growth and prolonged survival of tumor-bearing mice, and analysis of tumors showed reduced expression of CLEC5A, CD163 and Ki-67. Collectively, CLEC5A is expressed by M2-like TAMs and enhances M2-like TAM polarization, likely through IL6-JAK-STAT3 signaling.
TMIC-23. EXPLORING IBRUTINIB'S EFFECTS ON BLOOD-BRAIN BARRIER INTEGRITY AND GLIOMA PROGRESSION
Authors: Dalmage Mahalia , Kundu Nabanita , Kwak Minhye , Lim Sanghee , Jackson Sadhana
Published: November 2022

BACKGROUND

The blood-brain barrier (BBB), among malignant gliomas limits certain agents from impairing tumor growth. Thus, identifying agents that can transiently increase BBB permeability may prove useful in enhancing glioma treatment response. Previous ibrutinib studies showed it independently disrupted gut epithelial integrity and hampered glioma growth through BMX (bone marrow x-linked tyrosine kinase) inhibition. We propose BMX inhibition via ibrutinib disrupts BBB integrity while impairing glioma progression.

METHODS

To evaluate ibrutinib’s effect on brain endothelium, we evaluated electrical cell-cell impedance, junctional/cytoskeletal expression, downstream protein expression and functional ABC transporter activity; monitoring changes over time (0 to 8h) and at varied drug concentrations (1-10μM ibrutinib). Using rat glioma cells (S635) in vitro and in models, we examined cytotoxicity, apoptosis, model survival, and drug concentrations with ibrutinib alone or combined with poorly permeable Abcb1 substrate doxorubicin.

RESULTS

Ibrutinib dose-dependently decreased brain endothelial cell-cell impedance by 60% at 2h, without affecting cell viability. We observed decreased ZO-1 junctions, actin cytoskeletal rearrangement, and downstream pErk and pMek decreased expression optimally 2h (10μM ibrutinib). Dose-dependently ibrutinib inhibited Abcb1 efflux activity, further favoring a more permeable endothelium. Synergy with doxorubicin was seen in rat glioma cells treated with ibrutinib via increased apoptosis and cytotoxicity. However, while combined treatment resulted in decreased systemic doxorubicin concentrations, no differences in brain:plasma or tumor:plasma concentrations were evident. Combination therapy also did not increase rodent glioma model survival nor decrease tumor size.

CONCLUSION

Our results suggest that while ibrutinib induces brain endothelial permeability by junctional/cytoskeletal disruption and inhibition of Abcb1 efflux, no sustained effect can be seen in rodent glioma models in combination with a cytotoxic agent. Additional studies exploring similar agents that can enhance BBB permeability while slowing glioma growth are warranted, so as to improve upon current bleak malignant glioma treatment options.
TMIC-26. USING INTEGRATED MULTIDIMENSIONAL MASS CYTOMETRY AND MULTIPLEX IMMUNOHISTOCHEMISTRY TO INFER SPATIAL RELATIONSHIPS BETWEEN PHENOTYPICALLY DISTINCT GLIOBLASTOMA INFILTRATING IMMUNE CELLS
Authors: Bartkowiak Todd , Brockman Asa , Lima Sierra , Hayes Madeline , Roe Caroline , Sinnaeve Justine , Mistry Akshitkumar , Leelatian Nalin , Mobley Bret , Chambless Lola , Thompson Reid , Weaver Kyle , Irish Jonathan , Ihrie Rebecca
Published: November 2022

Glioblastomas (GBM) account for ~60% of adult primary brain tumors. With few advances in therapeutics, median overall survival remains 15-months post-diagnosis. Immunotherapies may provide therapeutic benefit; however, no predictive immune features have informed therapeutic stratification. Radiographic tumor contact with the lateral ventricle (C-GBM) correlates with 7-months worse prognosis compared to patients with ventricle non-contacting GBM (NC-GBM), yet the influence of ventricle contact on anti-tumor immunity is unknown. This study characterized the GBM immune microenvironment and identified targetable mechanisms of immunosuppression correlating with worse outcomes in C-GBM patients.Primary glioblastoma tissue was provided with written informed consent in accordance with the Declaration of Helsinki and with approval of the Vanderbilt Institutional Review Board (IRB #131870). Seventeen patients presented with primary, IDH-wildtype C-GBM and 15 with NC-GBM. Machine learning integrated mass cytometry and matched multiplex immunohistochemistry on FFPE embedded tissue to identify phenotypic, functional, and spatial biomarkers correlating with patient outcome. C-GBM tumors were enriched in STAT3-driven CD32+CD44+HLA-DR+ monocyte-derived macrophages (MDM) compared to NC-GBM (19 ± 8% vs. 6 ± 2%; p< 0.001) and depleted in lymphocytes (2.9 ± 1% vs. 7.6 ± 2%; p< 0.001) and tissue-resident microglia (1.8 ± 0.3% vs. 7 ± 3%; p< 0.001). Exhausted T cells in C-GBM co-expressed checkpoint receptors PD-1 and TIGIT. K-means clustering identified 10 immunological niches in GBM. Macrophage-tumor niches were most common in C-GBM (17.93% of niches), followed by T cell-microglia-tumor niches (17.72%). Within NC-GBM niches, T cell-T cell interactions were more prevalent (log odds ratio = 0.90) and correlated with improved outcome.These findings suggest that factors within the periventricular space negatively influence the immune microenvironment within GBM tumors. Clinically targetable immune biomarkers were identified in C-GBM. Notably, radiologic assessment of lateral ventricle contact may guide clinical trial design for immunotherapies in neuro-oncology based on tumor proximity to the ventricle wall.
TMIC-28. COMBINING INDUCED NEURAL STEM CELL THERAPY AND IMMUNOMODULATION IN GLIOBLASTOMA
Authors: Thang Morrent , Valdivia Alain , Hingtgen Shawn
Published: November 2022

Glioblastoma (GBM) is an aggressive malignant adult brain tumor and is characterized by poor prognosis and survival outcome. Insufficient drug accumulation to the tumor site and development of monotherapy drug resistance are critical challenges to overcome in combating GBM. Human induced neural stem cells (hiNSC) therapy is promising due to the stem cells’ innate capability to migrate to the tumors and deliver therapeutics. We previously have developed spheroidal iNSCs and engineered them to constitutively secrete pro-apoptotic protein TRAIL—hiNeuroS-TRAIL. However, investigating a combination therapy along with hiNeuroS-TRAIL therapy is needed to potentially avoid monotherapy resistance and enhance overall treatment durability. One of the GBM hallmarks is its immunosuppressed tumor microenvironment (TME). GBM-associated macrophages and microglia (GAMs) display pro-tumoral phenotypes in TME—inhibiting their ability to target the tumor cells or recruit other immune cells. In humans, higher glioma grade is associated with higher expression of Mer Tyrosine Kinase Receptor (MerTK) in both GBM cells and GAMs. Activation of MerTK in GAMs and GBM cells exacerbates immunosuppression and tumor progression respectively. Inhibiting MerTK activation via a small molecule drug, MRX-2843 and combining with hiNeuroS-TRAIL demonstrated robust synergistic tumor killing effects in vitro (CDI=0.22) and ex vivo (CDI=0.18). Additionally, the preliminary data for in vivo pilot study revealed that the median survival for the combination therapy treated group of mice extended over 24 days whereas the median survival for control, MRX-2843, and hiNeuroS-TRAIL treated groups of mice were 16, 18, and 21 days respectively. We think that combining these two therapies could significantly extend the survival in our murine model of GBM. Therefore, we will be optimizing dose concentration and cell counts for single agent therapies, and we will then repeat in vivo y synergy study with optimized therapies and a larger sample size to fully uncover the potential of this combination therapy.
TMIC-32. TUMOR CELL CILIA ASSOCIATE WITH SPECIFIC IMMUNE CELL POPULATIONS IN HUMAN AND MOUSE MODELS OF GBM
Authors: Deleyrolle Loic , Harrison Jeffrey , Takacs Gregory , Sarkisian Matthew
Published: November 2022

INTRODUCTION

Glioblastoma (GBM) typically recurs after standard of care therapies. A major obstacle is that GBMs employ various mechanisms to suppress the host immune system, preventing destruction and removal of cancer cells. A better understanding of the crosstalk mechanisms between tumor and immune cell types is needed to advance immunotherapeutic approaches against GBM. GBMs contain primary cilia, organelles likened to both cellular ‘antennae’ and transmitters, and their presence is associated with more aggressive and treatment resistant tumors.

OBJECTIVE

To explore whether ciliated GBM cells associate with infiltrating immune cells and to determine if these interactions are specific for certain immune cell populations.

METHODS

We immunostained GBM patient specimens and sections from syngeneic mouse models of GBM for markers of cilia (ARL13B and gTub) together with various immune cell markers (CD45, CD11b, Ly-6B.1, CD3). Cilia were also examined in brain tumors generated in CCR2+/rfp/CX3CR1+/gfp mice, to evaluate the proximity of glioma cell cilia to CCR2- and CX3CR1-expressing myeloid cell subpopulations (brain resident microglia as well as peripheral-derived macrophages and monocyctic- MDSCs (M-MDSCs). Proximity of tumor cell cilia to different markers or cell types was quantified using nearest neighbor analyses.

RESULTS

In patient samples, CD45+ cells extended processes that contacted the tumor cilia and cilia tip. Similar observations were made in intracranial GBM-bearing mice where we detected juxtaposition of cilia with recruited immune cells (i.e. CD45, CD11b and Ly-6B.1). Notably, cilia predominately associated with immune-suppressive M-MDSCs. Further, CD3+ T cells rarely juxtaposed tumor cilia and maintained greater distances away from ciliated tumor cells compared to M-MDSCs.

CONCLUSION

Our data suggest specific interactions between ciliated tumor cells and select immune-suppressive cell types, raising the possibility immunologically cold tumors may contain more ciliated cells. Ongoing studies are examining how the tumor immune cell landscape develops depending on the presence of tumor cilia.
TMIC-45. DISTRIBUTION AND TUMOR CELL PROXIMITY OF IMMUNE CELLS IN THE TUMOR CORE, INFILTRATION ZONE AND PERIPHERY OF GLIOBLASTOMAS
Authors: Pedersen Vilde , Knudsen Arnon Møldrup , Michaelsen Signe Regner , Kristensen Bjarne Winther
Published: November 2022

Glioblastoma is the most frequent and malignant brain tumor. Immune therapies have had limited effect, but not much is known about the frequency and type of immune cells in the infiltration zone and periphery. In these areas, tumor cells migrate into the brain parenchyma and prevent total tumor resection thereby leading to recurrence. This study aims to quantify the type and distribution of immune cells in glioblastomas. A cohort of 67 glioblastomas was established containing tissue from central tumor and/or infiltration zone and/or tumor periphery. Patients were included if most tumor cells were positive in immunohistochemical staining for P53. A multiplex staining was established, where tissue sections were stained with P53 (tumor cells), FOXP3 (FOXP3+ regulatory T cells), CD8 (CD8+ cytotoxic T cells) and IBA1 (microglia/macrophages). Cells were counted in tumor core, infiltration zone and periphery using Visiopharm software based on training of convolutional neural networks. Moreover, the number of tumor cells with immune cells in proximity (30 µm) was quantified. The number of CD8+, FOXP3+ and Iba1+ cells was significantly higher in the core than in the periphery (P < 0.001), and in the infiltration zone than in the periphery (P < 0.001). However, the CD8+, FOXP3+ and Iba1+ cells/tumor cell ratio increased from core to infiltration zone and to periphery (P < 0.001). There was a higher amount of tumor cells with CD8+ cells in proximity in the tumor core (P < 0.001), whereas the highest amount of tumor cells with IBA1+ cells and FOXP3+ cells in proximity was the infiltration zone (P < 0.001). In conclusion, although levels of immune cells are highest in the tumor core - the higher proximity and ratio of immune cells to tumor cells in the infiltration zone/periphery suggest immune-suppressive mechanisms to be active in these areas.