bluuebunny/crossref_metadata_2025 huggingface dataset:
What is the real value of omics data? Enhancing research outcomes and securing long-term data excellence
Authors: Price Eva , Feyertag Felix , Evans Thomas , Miskin James , Mitrophanous Kyriacos , Dikicioglu Duygu
Published: October 2024

A wealth of high-throughput biological data, of which omics constitute a significant fraction, has been made publicly available in repositories over the past decades. These data come in various formats and cover a range of species and research areas providing insights into the complexities of biological systems; the public repositories hosting these data serve as multifaceted resources. The potentially greater value of these data lies in their secondary utilization as the deployment of data science and artificial intelligence in biology advances. Here, we critically evaluate challenges in secondary data use, focusing on omics data of human embryonic kidney cell lines available in public repositories. The emerging issues are obstacles faced by secondary data users across diverse domains as they concern platforms and repositories, which accept deposition of data irrespective of their species type. The evolving landscape of data-driven research in biology prompts re-evaluation of open access data curation and submission procedures to ensure that these challenges do not impede novel research opportunities through data exploitation. This paper aims to draw attention to widespread issues with data reporting and encourages data owners to meticulously curate submissions to maximize not only their immediate research impact but also the long-term legacy of datasets.
KEGG: biological systems database as a model of the real world
Authors: Kanehisa Minoru , Furumichi Miho , Sato Yoko , Matsuura Yuriko , Ishiguro-Watanabe Mari
Published: October 2024

KEGG (https://www.kegg.jp/) is a database resource for representation and analysis of biological systems. Pathway maps are the primary dataset in KEGG representing systemic functions of the cell and the organism in terms of molecular interaction and reaction networks. The KEGG Orthology (KO) system is a mechanism for linking genes and proteins to pathway maps and other molecular networks. Each KO is a generic gene identifier and each pathway map is created as a network of KO nodes. This architecture enables KEGG pathway mapping to uncover systemic features from KO assigned genomes and metagenomes. Additional roles of KOs include characterization of conserved genes and conserved units of genes in organism groups, which can be done by taxonomy mapping. A new tool has been developed for identifying conserved gene orders in chromosomes, in which gene orders are treated as sequences of KOs. Furthermore, a new dataset called VOG (virus ortholog group) is computationally generated from virus proteins and expanded to proteins of cellular organisms, allowing gene orders to be compared as VOG sequences as well. Together with these datasets and analysis tools, new types of pathway maps are being developed to present a global view of biological processes involving multiple organism groups.
Reference values for handgrip strength, five times sit-to-stand and gait speed in patients on hemodialysis
Authors: Duarte Marvery P , Nóbrega Otávio T , Vogt Barbara P , Pereira Marina S , Silva Maryanne Z C , Mondini Dario R , Disessa Henrique S , Adamoli Angélica N , Bündchen Daiana C , Sant'Helena Bruna R M , Krug Rodrigo R , Bohlke Maristela , Inda-Filho Antônio J , Lima Ricardo M , Avesani Carla M , Nakamura Luiz R , Reboredo Maycon M , Ribeiro Heitor S ,   the SARC-HD Study Group, Baião Victor M , Rocha João M , Silva João P , Varela Priscila M , Monteiro Jacqueline S , Bainha Ana C , Gravina Emanuele P , Castro Abner R , Costa Fabiana L , Rosa Clara C , Monteiro Henrique L , Medina Luiz R , Nishimaru Flávio I , Rosa Maria G , Picinato Ana C , Uchida Marco C , Moreira Paulo R , Vignali Camila R , de Oliveira Luane B , Mendez Samy , Mansur Augusto H , Colling Catiussa , Thomé Fernando S , Ferreira Aparecido P
Published: October 2024

Background

Low levels of muscle strength and physical performance predict adverse clinical outcomes in patients on hemodialysis; however, reference values remain lacking. We described reference values for handgrip strength, five times sit-to-stand (STS-5) and 4-m gait speed in a large-scale sample of patients on hemodialysis.

Methods

Baseline data from the SARCopenia trajectories and associations with adverse clinical outcomes in patients on HemoDialysis (SARC-HD) study were analyzed. Muscle strength was evaluated using handgrip strength and the STS-5, whereas physical performance was evaluated using usual 4-m gait speed before a midweek dialysis session. Sex- and age-specific smoothed reference curves for each test at the 3rd, 15th, 50th, 85th and 97th percentiles were constructed using generalized additive models for location shape and scale. Comparisons between sex and age were also performed.

Results

Data from 1004 patients (39% female; 19–96 years; 49% ≥60 years) were analyzed. Declines in muscle strength and physical performance were observed with advancing age in both sexes. However, among males, muscle strength and performance were similar between 18 and 49 years of age. Males exhibited substantially greater performance in handgrip strength [10.3 kg, 95% confidence interval (CI) 9.1 to 11.4] and 4-m gait speed (0.10 s, 95% CI 0.05 to 0.14) compared with females. Older patients, independent of sex, exhibited poorer performance on most tests. Lower handgrip strength in the arm with arteriovenous fistula was observed in both sexes (males –2.3 kg, 95% CI –2.8 to –1.7; and females –2.1 kg, 95% CI –2.6 to –1.6).

Conclusion

Reference values obtained in this study may be used in clinical and research settings to identify patients on hemodialysis with low physical function according to sex and age. Future studies should test these reference values as potential predictors of adverse clinical outcomes.
PL01.5.A ASSOCIATION BETWEEN RESTING-STATE-NETWORK FUNCTIONAL CONNECTIVITY AND SURVIVAL IN RECURRENT GLIOMAS DIAGNOSED USING FET PET
Authors: Friedrich M , Werner J M , Steinbach J P , Sabel M , Herrlinger U , Piroth M , Stoffels G , Filss C P , Lohmann P , Shah N J , Ruge M I , Mottaghy F M , Goldbrunner R , Langen K J , Fink G R , Kocher M , Galldiks N
Published: October 2024

BACKGROUND

Functional connectivity between glioma-infiltrated brain tissue and remote cortical regions has been linked to overall survival (OS) in patients with newly diagnosed glioblastoma. Here, we studied the association of functional connectivity between tumor and resting-state networks and OS in patients with recurrent gliomas.

PATIENTS AND METHODS

The study included 82 patients (age range, 26-81 years; median ECOG performance score, 0) with recurrent gliomas (CNS WHO grade 4 glioblastoma, n=57; CNS WHO grade 3 and 4 astrocytoma, n=12; CNS WHO grade 2 and 3 oligodendroglioma, n=13) who had undergone first-line therapy by current guidelines. In addition to the RANO criteria for anatomical MRI, diagnosis of glioma recurrence was confirmed by amino acid PET using the tracer O-(2-[18F]-fluoroethyl)-L-tyrosine (FET). FET PET and resting-state functional MRI were simultaneously acquired on a 3T hybrid PET/MR scanner. A seed-based dual regression analysis was performed to determine the functional connectivity between metabolically active recurrent tumors and a set of seven canonical resting state networks.

RESULTS

Average functional connectivity was significantly higher (p=0.028) in oligodendrogliomas (z-score, 6.9±1.9) and astrocytomas (z-score, 5.5±1.3) than in glioblastomas (z-score, 4.9±2.6) and was inversely related to the tumor grade (p=0.031). Univariate Cox regression analysis revealed a significant positive association between OS and tumor connectivity with the visual, somatomotor, and dorsal attention network (all p<0.05). Subsequent stepwise multivariate Cox regression analysis refined this association to the dorsal attention network alone (HR, 0.88; 95% CI, 0.80-0.97; p=0.007). Patients with the highest functional connectivity (z-score >8.3, n=21) had a median OS of 31.4 months, while those with the lowest functional connectivity (z-score <4.3, n=21) had a median OS of 10.6 months. Functional connectivity between tumors and the dorsal attention network showed an independent association with OS (p=0.002) in a multivariate model, including the metabolic tumor volume (p=0.003).

CONCLUSION

Recurrent gliomas exhibit significant functional connectivity to most of the major known resting-state networks, which was generally higher in astrocytomas and oligodendrogliomas. The data suggest that high functional connectivity between the tumor and the dorsal attention network is an independent prognostic factor for improved OS.
PL02.1.A LOCOREGIONAL BI-SPECIFIC CAR-T CELLS TARGETING CD44 AND CD133 IN RECURRENT GLIOBLASTOMA: THE INTERIM RESULTS OF THE FIRST IN-HUMAN CLINICAL TRIAL
Authors: Zhai Y , Li G , Zhang W , Jiang T
Published: October 2024

BACKGROUND

Recurrent glioblastoma (rGBM) remains no standard of care exists, with a median overall survival of less than 1 year. Here, we report the pre-clinical data and the first four patients with rGBM in a phase I study of locoregionally administration of newly developed tandem CAR-T cell, targeting CD44 and CD133, armored with a truncated IL7Ra intracellular domain (Tris-CAR-T).

MATERIAL AND METHODS

The primary outcomes were safety and any adverse events associated with Tris-CAR-T administration. Secondary outcomes include the distribution and persistence of Tris-CAR-T cells, cytokine concentration, and the therapeutic effect according to immunotherapy Response Assessment in Neuro-Oncology (iRANO) criteria. At present, six patients of newly diagnosed rGBM underwent different doses of Tris-CAR-T administration via Ommaya reservoir of intratumoral or ventricular implantation. The first two patients had single or multi-doses of 107 Tris-CAR-T cells, the third and fourth patients had multiple doses of 108 Tris-CAR-T cells, and the last 2 patients had weekly doses of 0.5x108 Tris-CAR-T cells administration. Cytokine release syndrome and other side effects were assessed via cytokine quantification (serum and cerebrospinal fluid), K-CRP, and body temperature. Specimens of peripheral blood, cerebrospinal fluid, CAR-T products were collected for further bioinformatics analysis.

RESULTS

No neurotoxicity or adverse event greater than grade 1 was observed until now. Radiographic response and improvement in symptoms and signs were observed within days after the first infusion, and continued in the follow-up. Some patients underwent a remarkable tumor regression.

CONCLUSION

The newly constructed Tris-CAR-T cells are capable of GBM suppression with limited side effects. Long-term tumor control is considered to be related to dosage, product quality, and intervention frequency. A weekly or biweekly administration might be considered. The inspiring clinical response needs to be strengthened by follow-up and investigations in biological specimens (ClinicalTrials.gov identifier: NCT05577091)
KS03.6.A TUMOR-SPECIFIC B CELL RESPONSE IN THE CEREBROSPINAL FLUID OF MELANOMA PATIENTS: RECOMBINANT ANTIBODIES SPECIFICALLY BIND TO THE TUMOR-ASSOCIATED ANTIGENS DDX53, AKR1A AND KIFC3
Authors: Schembecker S , Vasilenko N , Ruschil C , Kowarik M C
Published: October 2024

BACKGROUND

Meningeosis carcinomatosa is a diffuse dissemination of tumor cells in the cerebrospinal fluid (CSF) and/or meninges and occurs in around 5% of patients with malignant melanoma. In previous studies on CSF immune cell subsets an elevated CSF B cell fraction was found in a subset of melanoma patients with leptomeningeal spread. Elevated CSF B cells are mostly associated with neuro-inflammatory diseases and are largely absent during physiological conditions. Hence this study aims to assess if these CSF B cells resemble an intrathecal tumor-specific immune reaction and to test CSF B cell derived recombinant antibodies for tumor specificity from these melanoma patients.

METHODS

Three melanoma patients with leptomeningeal spread and a presumable CSF B cell reaction were selected by analyzing CSF parameters including intrathecal immunoglobulin (Ig) synthesis. Single cell sorting of CSF B cells was performed followed by conventional sequencing of Ig heavy and light chain transcripts. 13 recombinant antibodies were generated from CSF B cells and were tested for antigen specificity using antigen microarrays, FACS, ELISA and immunocytochemistry as well as bioinformatic tools for binding predictions.

RESULTS

By applying single cell analysis on CSF B cells, representative Ig transcriptome repertoires were obtained. CSF Ig repertoires showed features of a clonal expansion and multiple mutations to germline. These mutations were specifically found in the complementary determining regions suggesting an antigen stimulated affinity maturation within the CSF compartment. By systematic microarrays, the number of potential antigen targets was narrowed down and further evaluated by binding properties of each individual antibody to a well characterized melanoma cell line. Using FACS and immunocytochemistry on the melanoma cell line, antibody binding was confirmed for a subset of antibodies. In custom made ELISA tests, the specific binding of three antibodies to the individual proteins AKR1A1, KIFC3 and DDX53, a known cancer-testis antigen, was detected. Furthermore, potential binding epitopes for these three antibodies were characterized.

CONCLUSION

In summary, this study shows evidence for a targeted intrathecal B cell reaction with features of a clonally expanded CSF B cell population in melanoma patients with meningeal carcinomatosis. Specific antibody binding against the targets AKR1A1, KIFC3 and DDX53 was confirmed for three antibodies indicating that these antibodies are indeed tumor-specific and might represent a B cell response against the tumor cells. These antibodies and their corresponding tumor antigens may be extremely useful as they appear to be sufficiently immunogenic to elicit an immune response and thus may be utilized for tumor treatments.

GRANTS AND FELLOWSHIPS

This study was funded by the DFG (GZ: KO 4367/5-1; AOBJ: 692835; Projektnummer: 517964704).
JS04.5.A THE ROLE OF SURGERY IN IDH WILDTYPE DIFFUSE ASTROCYTOMA
Authors: Krenzlin H , Lange F , Dauth A , Kalasauskas D , Freyschlag C , Schnell O , Misch M , von der Brelie C , Wagner A , Krigers A , Rohde V , Meyer B , Vajkoczy P , Beck J , Thomé C , Ringel F , Keric N
Published: October 2024

BACKGROUND

Isocitrate dehydrogenase (IDH)1/2 wildtype astrocytomas, classified as CNS WHO grade 4 tumors in the latest WHO classification of 2021, have high recurrence rates. The role of surgery in these tumors remains uncertain.. In this study the impact of the extent of resection (EOR) on progression free (PFS) and overall survival (OS) in astrocytomas IDH1/2 wt (CNS WHO Grad 4) is compared to glioblastoma IDH1/2 wt (CNS WHO Grad 4).

METHODS

Data acquisition was conducted as a multi-center retrospective analysis at 6 University Hospitals (2016-2020). Patients with IDH 1/2 wildtype diffuse astrocytoma (CNS WHO grade 2-4) were included in our study. Patients presenting with IDH wildtype glioblastoma from one institution served as controls. Primary outcome parameters were EOR (GTR; <5% residual tumor (RT), subtotal resection (STR; 5-75% RT), and biopsy (> 75% RT) and OS.

RESULTS

160 patients with IDH-wildtype glioblastoma (CNS WHO grade 4) (45.6 % females) and 138 patients with IDH-wildtype diffuse astrocytoma (CNS WHO grade 2-4), (37.7 % females) were enrolled. The median age was 68 years (37-86 years) in the classic glioblastoma cohort and 55 years (20-87 years) in those with astrocytomas. GTR was achieved more often in patients with classic glioblastoma: 58.8% compared to those with diffuse astrocytoma 39.1% (p = 0.0001); STR (32.6%, p = 0.0048) and biopsy (28.3%, p = 0.0032) were more often in astrocytomas. The overall survival was 12.7±10.4 months in classic glioblastoma compared to 27.3±2.9 months in astrocytoma. GTR significantly increased OS in both entities (glioblastoma: 15.3±10.4 months, 95% CI 13.13 - 17.40, p = <0.0001; diffuse astrocytoma: 26.7±17.9 months, 95% CI 20.13 - 33.25, p = <0.0001). Further, also STR significantly increased OS in glioblastoma (11.46±11.03 months, 95% CI 7.55 - 15.37, p = 0.0257) and astrocytoma (18.8±13.54 months, 95% CI 14.47- 23.13, p = 0.0035). ROC analysis identified a residual cut-off tumor volume of >20% in the glioblastoma cohort and >60% in the difuse astrocytoma cohort to be associated with impaired OS. Multivariate logistic regression analysis identified age and EOR, not radio-chemotherapy, as independent predictors of OS in diffuse astrocytoma. MGMT methylation had no impact on OS (p = 0.11).

CONCLUSION

The presented data suggests that resection prolongs OS in patients with IDH-wildtype astrocytomas and is of higher value than radio-chemotherapy. Of note, STR also positively influences OS in these patients. These results underline the importance of surgical resection even if GTR is not achievable and thus mark a potential paradigm shift in clinical decision making.
OS02.4.A CAREGIVER PROGRAM IN NEURO-ONCOLOGY: UCSF EXPERIENCE CARING FOR MORE THAN 4,000 CAREGIVERS OVER THE LAST 10 YEARS
Authors: Taylor J W , Page M , Rodriguez Almaraz E , Angelo D , Velasco C , Chang S M
Published: October 2024

BACKGROUND

Caregivers of patients with primary brain tumors are at high risk for distress and as they navigate caring for their loved one with both a cancer diagnosis and neurologic disorder. The last decade has seen growth of caregiver research in the brain tumor community, with evidence that caregiver mastery may correlate with survival in patients with glioblastoma (GBM). The UCSF Gordon Murray Neuro-Oncology Program, the first of its kind, launched in 2014 with a mission to address their unique needs. Caregiver care is now fully integrated into the clinical care of patients with brain tumors at UCSF, providing personalized care across the disease trajectory and programming to address common challenges and foster resilience. The Program includes a nurse, administrator, and social worker. Referrals occur 1) at first clinic appointment; 2) from clinical staff; and 3) caregivers of patients with GBM receive proactive outreach over the first 6 months after diagnosis. Caregivers are also contacted for bereavement support. Dedicated programming provides educational materials focusing on common concerns and building community through monthly support groups, educational webinars, annual retreats and family camp. We report our practical experience of the Program serving over 4,000 caregivers since its inception.

MATERIAL AND METHODS

Total number of individual caregivers were included from 01/2014 - 12/2023. Detailed demographic information was extrapolated during clinical interviews from a subset. Information regarding caregiver concerns and interventions was extracted from the medical records between 01/2017 - 12/2023.

RESULTS

The UCSF Gordon Murray Caregiver serves an annual average of 400 caregivers and over 800 encounters. Demographic information obtained from a subset revealed 66% females; 77% between 41-64 years of age; 60% a spouse; 89% working full time; and 30% had children in the home. The most common concerns for caregivers were informational (42%), followed by emotional (25%), and practical (23%). Examples of informational concerns included desire for informational resources, understanding the treatment plan/side effects, and understanding patient behavioral changes. Emotionally, they feel overwhelmed, uncertain, and sad. Practical concerns included physically caring for patients and issues around medical insurance, disability, and finances. Caregivers also expressed frequent concerns around talking with children/family members and feelings of isolation.

CONCLUSION

Caregivers of patients with primary brain tumors are at high risk of distress and have unique needs based on the tumor location and symptoms from treatment. Our practical 10 year experience of a dedicated, fully integrated, caregiver program highlights real world concerns of caregivers and identifies opportunities to develop tailored interventions to address these needs.
OS06.7.A PLATFORM-AGNOSTIC METHYLATION CLASSIFICATION OF 184 CLASSES OF CNS TUMOURS
Authors: Patel A , Schrimpf D , Jäger N , Loose M , Wick W , Krieg S , Schlesner M , Pfister S M , von Deimling A , Jones D T W , Sill M , Sahm F
Published: October 2024

BACKGROUND

The 2021 WHO classification underscores the importance of methylation-based Central Nervous System (CNS) tumour diagnostics. The Heidelberg MNP classifier has been widely used for methylation classification. However, current approaches for methylation classification warrant the use of methylation arrays which suffer from technical complexity, considerable equipment and reagent cost, as well as lengthy turnaround times. Recent advances in nanopore sequencing have demonstrated the feasibility of methylation classification of broad classes of CNS tumours. Here we introduce and validate MNP-Flex- a platform agnostic methylation classifier that classifies 184 classes of CNS tumours and is compatible with the version v12.8 of the Heidelberg MNP classifier. MNP-Flex is made available as a web-based service.

MATERIAL AND METHODS

We trained a gradient boosted model on a training set comprising 7,495 methylome profiles spanning 184 classes and covering multiple sparsity levels. We validated the model on a comprehensive multi-centric dataset comprising over 79,000 samples processed using diverse approaches like- methylation arrays, nanopore shallow and deep whole genome sequencing using cryopreserved or FFPE tissue, intraoperative and post-hoc nanopore adaptive sampling (Rapid-CNS2) data, whole genome bisulphite sequencing using tissue and liquid biopsy samples, as well as methylation panels.

RESULTS

We achieved an overall accuracy of 94% across a diverse, cross-platform, global cohort of 79,000 samples spanning 9 different sequencing conditions. MNP-Flex could also successfully classify 85% of samples with sparse profiles including those obtained using nanopore sequencing in an intraoperative setting and from liquid biopsies.

CONCLUSION

MNP-Flex addresses the crucial need for accessibility of methylation-based CNS tumour diagnostics. It offers a single platform-agnostic methylation classifier applicable across diverse sample types and sequencing conditions which is provided as a web-service. The ability to effectively classify samples with sparse profiles extends its clinical utility to low throughput conditions. MNP-Flex aims to streamline methylation-based classification, ultimately improving access to molecularly-informed patient care in neuro-oncology.
OS09.7.A GBM AGILE PLATFORM TRIAL FOR NEWLY DIAGNOSED AND RECURRENT GBM: RESULTS OF FIRST EXPERIMENTAL ARM, REGORAFENIB
Authors: Lassman A , Alexander B , Berry D , Buxton M , Cavenee W , Colman H , De Groot J , Ellingson B , Gordon G , Hyddmark E M V , Khasraw M , Lim M , Mellinghoff I , Mikkelsen T , Perry J , Powell A , Sulman E , Tanner K , Weller M , Yung W K A , Blondin N , Brenner A , Butt O , de la Fuente M I , Drappatz J , Iwamoto F , Kim L , Lee E , Mantica M , Nabors B , Newton H , Schiff D , Walbert T , Weathers S , Cloughesy T , Wen P Y
Published: October 2024

BACKGROUND

GBM AGILE (NCT03970447;https://www.gcaresearch.org/research/gbm-agile) is a phase 3 Bayesian adaptive platform trial that efficiently tests multiple arms against common control, with 6 arms included to date. Primary endpoint is overall survival (OS). Stage 1 experimental arms are adaptively randomized against other arms. Demonstrated efficacy in stage 1 leads to fixed randomization stage 2. Stages 1 and 2 are combined for registration. Control randomization is fixed. Regorafenib, a multikinase-inhibitor, entered into GBM AGILE as the first arm and therefore was equally randomized against control. Regorafenib showed OS benefit in recurrent disease (RD) in randomized phase 2 REGOMA trial.

MATERIAL AND METHODS

Patient subtypes in GBM AGILE are newly diagnosed unmethylated (NDU), RD, and—not considered for regorafenib—ND methylated (NDM). Arm indications (signatures) are combinations of subtypes. Control is temozolomide (ND) and lomustine (RD). Efficacy is assessed by OS hazard ratio(HR), arm/control. Efficacy is demonstrated when Bayesian probability of benefit (HR< 1.00) ≥ 98% (roughly analogous P-value: 0.02). Futility occurs at any monthly analysis when Bayesian predictive power (PP) is < 25% for all signatures. Follow-up continues for 12 months after arm’s accrual stops.

RESULTS

When PP for all 3 pre-defined signatures was < 25%, regorafenib’s accrual was stopped for futility. Regorafenib/control sample sizes were 49/51, 127/128, 176/179 for signatures NDU, RD, and both. Respective PPs: 0.138, 0.030, 0.025—none close to 0.25. Respective mean HRs: 1.26, 1.25, 1.23. Probabilities of benefit (HR< 1.00): 0.35, 0.18, 0.17. At final analysis, mean HRs were 1.07, 1.08, 1.08 with final probabilities of benefit (HR< 1.00) equal to 0.421, 0.312, 0.296—none close to 0.98.

CONCLUSION

GBM AGILE efficiently and compellingly addressed regorafenib’s role in GBM, in RD and NDU. These findings are germane as they fail to confirm the REGOMA results in RD. GBM AGILE continues to efficiently assess other therapies, including utilizing concurrent and previously accrued controls. This abstract was accepted and previously presented at the 2023 SNO Annual Meeting and published in Neuro-Oncology, Volume 25, Issue Supplement_5, November 2023, Pages v97-v98.
OS11.5.A MYELOID CELLS DRIVE IMMUNOSUPPRESSION OF CHIMERIC ANTIGEN RECEPTOR T CELLS IN THE NEURONAL MICROENVIRONMENT OF GLIOBLASTOMA
Authors: Zhang J , Ehr J v , Look T , Benotmane J K , Neidert N , Kueckelhaus J , Weiss T , Heiland D H , Yabo Y A
Published: October 2024

BACKGROUND

Chimeric antigen receptor (CAR)-T cell therapy has been successful in hematologic malignancies but demonstrated limited success in solid cancers including glioblastoma. The heterogeneous nature of central nervous system malignancies and immunosuppressive microenvironment are major impediments to the efficacy of CAR-T cell therapy in glioblastoma. Here, we aim to explore the transcriptional adaptation of CAR-T cells and cellular interactions that may explain the T cell dysfunction in the glioblastoma microenvironment.

MATERIAL AND METHODS

We performed physically interacting cells sequencing (PIC-seq) to explore the transcriptional diversity of NKG2D CAR-T cells and control T cells in the glioblastoma ecosystem leveraging a human neocortical slice model. We investigated cellular interactions and receptor-ligand interactions through CellChat and integration of spatially resolved T cell receptor sequencing. CellOracle was used to reconstruct gene regulatory networks and identify key transcription factors associated with CD8 CAR-T cell dysfunction. Further, we used in-silico perturbation to identify transcriptional drivers that have the potential to enhance cytotoxic response of CAR-Ts.

RESULTS

We inoculated primary patient-derived glioma cells into human neocortical slices from surgical access tissue specimens. After 3 days of tumor growth, slices were treated with NKG2D CAR-Ts or control T cells. We observed that after an initial phase (2 days) of anti-tumor T cell response in the CAR-T group, tumor growth did not differ between conditions. After 7 days of treatment, PIC-seq was used to profile CAR-Ts (tomato+) and tumor cells (GFP+) as well as cells which were physically connected (+/+). We identified a transcriptional shift towards T cell exhaustion in CAR-T cells compared to T cells lacking the NKG2D construct. This transcriptional phenotype was driven by CAR-T cell-myeloid cell interaction. Integrative analysis identified that tumor-associated macrophages with enhanced phagocytic function in proximity to mesenchymal-like tumor cells located within hypoxic niches are the main drivers of T cell dysfunction. Gene regulatory network analysis demonstrated that MAF and BACH2 are key transcriptional regulators of CAR-T cell function.

CONCLUSION

Both myeloid-tumor and myeloid-T cell interactions collectively promote an immunosuppressive microenvironment in glioblastoma. Our data has the potential to catalyze the re-engineering and optimization of CAR-T cell therapies with sustained cytotoxic effect against glioblastoma.
P01.08.A HYOXIA-INDUCED EXPRESSION OF ARHGAP FAMILY MEMBERS PROMOTES GLIOBLASTOMA DISSEMINATION
Authors: Vaughn-Beaucaire P , Muller L , Prior S , Lawler S , Bruning-Richardson A
Published: October 2024

BACKGROUND

The Rho GTPase activating proteins (ARHGAPs) perform vital roles in the regulation of the Rho GTPases with direct effect on cellular plasticity and cell migration/invasion. We recently reported on the role of five ARHGAPs, identified by a microarray screen, in morphological plasticity in established and patient-derived Glioblastoma (GBM) cell lines and associated clinical relevance in terms of tumour recurrence after treatment. We established that the ARHGAPs either promoted or targeted cell migration/invasion by allowing cells to undergo morphological changes in preparation for dissemination. We also showed that co-expression of two ARHGAPs, 12 and 29, is associated with reduced time to recurrence after initial treatment in patients. ARHGAP activity has been also reported in metastatic cancers including breast and colorectal cancer highlighting the biological relevance of these, as yet understudied, proteins.

MATERIAL AND METHODS

Here, we explored the GBM microenvironment and the effect of hypoxia on ARHGAP expression and localisation in glioma cell lines.

RESULTS

Using immunocytological and in vitro migration and invasion assays we were able to ascertain that ARHGAP4, 12, 22, 25 and 29 acquired subcellular distributions different to those recorded under normoxia and expression also decreased or increased under hypoxia. We also found that the effect of the migrastatic inhibitor 6-bromo-indirubin-3′-oxime (BIO) targeting GSK-3 signalling was potentiated when cells were challenged with the drug under hypoxic conditions.

CONCLUSION

Our results confirm our initial findings of biological relevance of the ARHGAPs in cellular activities and highlight important roles for the ARHGAPs for promoting migratory/invasive activity and behaviour in GBM cells. We also conclude that migrastatic inhibitors will exert enhanced activity on GBM cells under hypoxic conditions making them excellent candidates for complement treatment with cytotoxic drugs or radiotherapy for an improved management of GBM.
P01.15.A SURGICAL RESECTION-INDUCED ISCHEMIA DRIVES GLIOBLASTOMA CELL PLASTICITY AND TUMOR MICROENVIRONMENT ADAPTATION
Authors: Ballestín A , Reveilles J , Pichol-Thievend C , Bourmeau G , Anezo O , Ribecco V , Blanchard B , Pettiwala A , Leboucher S , Daubon T , van Rheenen J , Seano G
Published: October 2024

BACKGROUND

Glioblastoma IDH-wildtype (GBM) treatment involves a combination of surgical resection, radiation therapy, and chemotherapy with temozolomide. Despite the aggressive standard of care, 90% of patients experience recurrence a few months after treatment, typically marked by the regrowth of highly invasive cells spreading from the peritumoral brain resection zone. The impact of chemoradiotherapy has been studied, but the impact of microsurgical resection on the tumor microenvironment (TME) and the plasticity of GBM cells remains unknown. Hence, the objective of this work was to study histopathological and transcriptomic changes induced by microsurgical resection on residual tumor cells and TME.

MATERIAL AND METHODS

Syngeneic GBM cell lines CT2A and GL261 were orthotopically implanted in C57Bl6j mice brains meanwhile P3 xenografts were injected in immunosuppressed nude mice. We developed an optimized microsurgical resection protocol for GBM in mice, mirroring the steps performed in patients. In a size-matched study, mice were randomly assigned to four groups: control (pre-surgery) and 1, 3, and 7 days post-surgery. Tumor tissues were studied with a multilevel strategy composed of time-resolved bulk and single-cell RNA sequencing, immunohistochemistry and intravital multiphoton microscopy (IVM).

RESULTS

Our analyses showed that microsurgical resection induced pronounced proneural-to-mesenchymal transition (PMT) and stimulation of hypoxia-driven pathways in residual GB cells. Further, we detected significant changes in the TME relative to the immune response and vascular function in line with ischemia exposure, which could humper the response to treatment by altering the adequate effect and distribution of the subsequent chemoradiotherapy.

CONCLUSION

Microsurgical resection consequences play a crucial role in shaping the phenotypic landscape of GBM tumor cells and the TME, marked by a transient ischemic TME triggering PMT in GBM cells and potentially preventing the effective response to chemoradiotherapy.
P02.07.B FIBROBLAST ACTIVATION PROTEIN EXPRESSING MESENCHYMAL CELLS INFLUENCE T CELL ABUNDANCE AND FUNCTION IN GLIOBLASTOMA
Authors: Ternerova N , Houdova Megova M , Buna T , Svablova T , Vymolova B , Balaziova E , Vanickova Z , Krepela E , Hrabal P , Tomas R , Netuka D , Busek P , Sedo A
Published: October 2024

BACKGROUND

Glioblastomas (GBMs) are aggressive brain tumors with strong immunosuppressive properties. In epithelial cancers, mesenchymal cells expressing fibroblast activation protein (FAP) play an important role in modulating the T cell response. We have recently shown that FAP+ mesenchymal cells are present in human GBMs. The aim of this study was to determine their effect on T cell abundance and function.

MATERIAL AND METHODS

GBM tumors (16 samples) were mechanically and enzymatically dissociated and analyzed for immune cell subpopulations by flow cytometry. Expression of T cell related genes was analyzed by qRT-PCR in GBMs selected based on FAP protein concentration determined by ELISA (14 FAP high [upper tercile], 16 FAP low [lower tercile]). Immunohistochemistry (IHC) was used to compare the number of CD3+, CD4+, and CD8+ T cells in GBMs with high (upper tercile, n=21) and low abundance of FAP+ stroma (lower tercile, n=20). Co-localization of FAP+ mesenchymal cells and CD3, PD-L1 and PD-L2 was evaluated by immunofluorescence in frozen sections. 8 FAP+ mesenchymal and 3 glioblastoma stem-like cell cultures were derived from human GBMs and PD-L1 and PD-L2 expression was determined using ELISA. Peripheral blood mononuclear cells were isolated from healthy donors´ buffy coats. Proliferation of stimulated T cells in conditioned media from FAP+ mesenchymal cells was analyzed by flow cytometry.

RESULTS

The percentage of T cells in CD45+ cells from GBMs positively correlated with FAP expression. Expression of the T cell inhibitory molecule PD-L2 was higher in FAP high GBMs. Immunohistochemisty revealed that the numbers of CD3+ and CD8+ T cells were higher in GMBs with a high abundance of FAP+ stroma and T cells were frequently in close proximity to FAP+ mesenchymal cells, which were often PD-L2 positive. In vitro, FAP+ mesenchymal cells expressed more PD-L2 than glioblastoma stem-like cells. T cell proliferation was decreased after exposure to conditioned media from FAP+ mesenchymal cells.

CONCLUSION

FAP+ mesenchymal cells influence T cell abundance in GBMs and may affect T cell functions by limiting their proliferation via soluble factors or cell-cell contact. Acknowledgment: This work was supported by the National Institute for Cancer Research (LX22NPO5102), the Center for Tumor Ecology (CZ.02.1.01/0.0/0.0/16_019/0000785), Charles University project GAUK 365022 and Cooperatio Program Oncology and Haematology“.
P02.10.A IDENTIFYING NOVEL HEXOKINASE-2 ASSOCIATED PROCESSES IN THE GLIOBLASTOMA TUMOUR MICROENVIRONMENT
Authors: Edwards C L , Morris M , Shivakumar S P , Richards T , Small B , Butcher K , Dawson T , Warr T , Karakoula K
Published: October 2024

BACKGROUND

Glioblastoma (GB) is the most common and aggressive type of adult brain tumour. With a median patient survival of only 16 months, new therapies for GB are desperately needed. Deregulation of cellular metabolism is a hallmark of cancers, where they upregulate glycolysis and preferentially ferment glucose to lactate (over pyruvate) in the ‘Warburg effect’. Upregulation of hexokinase 2 (HK2), which catalyses the first rate-limiting step of glycolysis, is key to Warburg metabolism. Several studies have shown HK2 to be selectively overexpressed in GB, while normal human brain tissue (NHBT) relies upon its isoform HK1. As a result, HK2-orientated therapies may selectively target GB tumours while limiting toxicity to normal metabolising healthy tissue. In this study we sought to identify HK2-associated tumour processes in GB which could serve as potential therapeutic co-targets.

MATERIAL AND METHODS

High HK2 expressing (>20-fold upregulation vs NHBT) established and patient-derived GB cell cultures (U87-MG, IN859) were identified by RT-qPCR. HK2 expression was knocked down in these cultures by transfection with targeted siRNA, while non-targeting siRNA was used as a negative control (NC). Expression profiles of HK2 knockdown (HK2KD) and NC cultures were compared using RNAseq. Differentially expressed genes (DEGs) found to be associated with HK2KD were analysed for gene ontology category enrichment (GOSeq) and protein-protein interactions (STRING). DEGs shared between HK2KD cultures were validated by RT-qPCR.

RESULTS

HK2KD was associated with significantly (padj ≤0.05) enriched GO biological processes (BPs) in both U87-MG (117) and IN859 (109). BPs related to glycolysis or glucose metabolism were not enriched. Large clusters of enriched BPs related to inflammatory and immune response were prominent in both HK2KD-U87-MG and HK2KD-IN859. Notable members of these clusters included ‘regulation of neuroinflammatory response’, ‘activation of innate immune response’, and ‘response to bacterium’. Numerous enriched BPs linked to the recruitment of immune cells, such as ‘leukocyte migration’ and ‘granulocyte chemotaxis’, were also evident. STRING analyses further revealed clusters of interacting DEG-encoded cytokines (i.e. CSF2, IL1A/1B/6, TNF) and chemokines (i.e. CCL3, CXCL1/2/3/5/6/8) associated with HK2KD. Several angiogenesis related BPs were also enriched, which corresponded with the downregulation of the major angiogenic regulators HIF2A, VEGF and FGF2 in these HK2KD cultures.

CONCLUSION

These findings highlight potential non-canonical roles for HK2 in regulating inflammatory, immune and angiogenesis related processes within the GB tumour microenvironment. Agents targeting HK2 may thus improve the efficacy of existing preclinical immuno/anti-angiogenic therapies against GB, which warrants further investigation.
P02.23.A SPATIAL PROFILING OF THE MYELOID COMPARTMENT IN MENINGIOMA REVEALS DISTINCT PHENOTYPIC HETEROGENEITY ACROSS METHYLATION CLASSES
Authors: Calafato D , Tang Y , Rukhovich G , Patel B , Suwala A , Sahm F , Gerstung M , Hartmann F
Published: October 2024

BACKGROUND

Recent advances in neuro-oncology highlighted the meninges as an important immunological hub at the crossroads between systemic immunity and the central nervous system. Meningiomas are predominantly benign tumors arising from meningeal cells. However, about 20% are high-grade cases, commonly refractory to standard treatment, showing high recurrence rates. Methylation-based classification has crucially improved meningioma patient stratification. However, the biological processes that govern differences between these classes remain unknown. Here, we investigate the role of differential immune infiltration, cellular communities, and their spatial organization. In light of previously found differential immune infiltration across methylation classes, this study aims to spatially resolve the role of myeloid cells and study their influence on tumor progression and recurrence.

MATERIAL AND METHODS

We deployed multiplexed ion beam imagining and spatial transcriptomics on a retrospective cohort with extensive clinical annotations. Analyzing single-cell transcriptomic data of a subset of patients (n=22), we delineated the expression of gene and protein markers that were used to design an antibody panel comprising 41 targets for cell-type identification, phenotypic and metabolic characterization at the protein level. Additionally, we curated a targeted gene panel for spatial transcriptomics to perform granular spatial analysis. An integration of histological, spatial transcriptomic, and protein layers provides a comprehensive multi-omics approach to studying spatial cellular interactions.

RESULTS

Transcriptional programs and cell populations observed in single-nuclei data were captured in the spatial context revealing differences in the myeloid compartment across meningioma methylation classes. In specific classes, myeloid cells constitute a significant part of the tumor bulk warranting further investigation into their phenotypic differences. Accordingly, our analyses revealed a distinct phenotypic macrophage landscape along disease progression. Furthermore, spatial profiling of the metabolic regulome displayed distinct niche formation within perivascular and tumor compartments.

CONCLUSION

In this study we confirm that macrophages undergo a phenotypic switch from more tolerogenic in low-grade to inflammatory in high-grade cases of meningioma. Correspondingly, specific phenotypic and metabolic features of myeloid and tumor cells contribute to tumor progressions. Considering the limited targeted therapy options and poor responses to immunotherapy in meningioma patients, we hope that our spatial multi-omics analysis may reveal recurrent infiltration patterns of phenotypically distinct cell types that could be informative of biological processes and predictive of disease outcome.
P03.02.B DECIPHERING THE IDENTITY AND CHARACTERISTICS OF LONG-TERM LABEL RETAINING GLIOBLASTOMA CELLS AND THEIR ROLE IN TUMOUR RELAPSE
Authors: Chow H , Kourtesakis A , Hai L , Schaffranek S , Pusch S , Hoffmann D , Reibold D , Wick W , Wagener R
Published: October 2024

BACKGROUND

Glioblastoma (GB) stem cells (GSCs) are described to recapitulate the expression signatures of stem and progenitor cell classes in the neurodevelopmental hierarchy and are implicated in driving therapeutic resistance. However, the heterogeneity of both the GSC subclasses and their expression of stemness markers hinders comprehensive labelling of these cells. Cellular dormancy is a key feature of GSCs that can be captured by label retention method to track slowly dividing cells. We used CellTrace (CT) dye to identify long term label retaining GB cells (LRGC) in orthotopic xenograft model. We analysed the distribution of LRGC versus the tumour bulk in histology. Additionally, we analysed the transcriptional signature by FAC-sorting LGRC for single cell RNA sequencing. Further, we demonstrated that LRCs survived through temozolomide (TMZ) treatment in vivo and re-initiated tumour proliferation in a faster pace than other surviving glioma cells, signifying its role in therapeutic relapse.

MATERIAL AND METHODS

To label LRGC, patient derived GB cell lines were stained with CT dye before transplantation into the striatum of immunodeficient mice. After 6 weeks of growth the brains were cryosectioned, histologically stained and imaged via confocal microscopy, followed by histologic AI assisted analysis with the AIVIA software. LRGC and non-label retaining tumor cells were FAC-sorted for single-cell RNA sequencing (SMART seq2). To explore the role of LGRC in therapeutic resistance, tumors were treated with TMZ. Re-initiation of tumor growth was tracked by subcutaneous surgical implantation of BrdU pumps at different timepoints after therapy. The proliferation patterns of LRGC versus other surviving tumor cells were analysed in histology again using an AI assisted software.

RESULTS

LRGC could be identified in histology 6 weeks after tumor growth. LRGC were found near the centre of tumor but rarely at the invasion front. Single cell RNA-seq of LRGC showed differentially expressed pathways related to lipid metabolism and neurodevelopment. Re-initiation of tumor growth started 12-14 days after therapy. LRGC showed a more intense proliferation compared to other surviving tumor cells, indicating the ability of LRGC to not only survive TMZ treatment, but also drive GB relapse.

CONCLUSION

LRGC could be visualized 6-8 weeks after tumor growth in an orthotopic xenograft model. On a transcriptional level these cells share similarities with stem cells in normal brain development but also show distinct features. An important role of LRGC in re-initiation of tumor growth after therapy could be confirmed.
P06.01.A TARGETING MRNA DECAPPING ENZYME SCAVENGER (DCPS) IS A PROMISING STRATEGY AGAINST GLIOBLASTOMA VIA THE DOWN-REGULATION OF STAT5B
Authors: Duan H , Xie Y , Yu Y , Hu W , Ma W , Mou Y
Published: October 2024

BACKGROUND

Patients with glioblastoma (GBM) have poor prognosis and limited treatment options. The mRNA decapping enzyme scavenger (DCPS) is a cap-hydrolyzing enzyme. The inhibitor targeting DCPS exhibited excellent central nervous system bioavailability in vivo and was safe and well tolerated in healthy volunteers in a phase 1 clinical trial. In this study, we investigated the expression of DCPS in GBM and the anti-tumor activity of the DCPS inhibitor in various preclinical models of GBM.

MATERIAL AND METHODS

DCPS expression was examined in human GBM and paired peritumoral tissues. Its prognostic role was evaluated together with clinicopathological characteristics of patients. The anti-GBM effect of targeting DCPS was determined using GBM cell lines, patient-derived organoids,and orthotopic mouse models. The therapeutic mechanisms of DCPS inhibition were explored.

RESULTS

DCPS is overexpressed in GBMand associated with poor survival of patients with GBM. The DCPS inhibitor exhibited robust anti-GBM activities in GBM cell lines, patient-derived organoids and orthotopic mouse models, with drug exposure achievable in humans. Mechanistically, inhibition of DCPS downregulatedSTAT5Bexpression, thereby suppressing proliferation, survival and colony formation of GBM cells.

CONCLUSION

Inhibition of DCPS at doses achievable in humans downregulates STAT5B expression and reduces proliferation, survival and colony formation of GBM cells. Given the excellent anti-cancer activity and central nervous system bioavailability in vivo and good tolerance in humans, DCPS is a promising target for GBM.