bluuebunny/crossref_metadata_2025 huggingface dataset:
Stenosis of the glomerulotubular neck in progressive chronic kidney disease
Authors: Cohen Eric P , Denic Aleksandar , Aperna Fnu , Mullan Aidan F , Barisoni Laura , Sharma Vidit , Gibson Ian W , Rule Andrew D
Published: October 2024

Background

Morphology and morphometric evaluation of lesions beyond conventional parameters can inform the pathophysiology of chronic kidney disease (CKD). We sought to determine whether the occurrence of glomerulotubular neck stenoses associates with progressive CKD.

Methods

We evaluated the normal parenchyma from radical nephrectomies removed for tumor between 2000 and 2021 and analyzed cortex for stenoses of the glomerulotubular neck. Stenosis of the glomerulotubular neck is defined a focal narrowing for which the draining tubule has a greater diameter than at the neck. Progressive CKD was defined as dialysis, kidney transplantation, sustained estimated glomerular filtration rate (eGFR) <10 mL/min/1.73 m2 or sustained 40% decline from the post-nephrectomy eGFR. Each case of progressive CKD was age- and sex-matched to two controls without progressive CKD. Logistic regression models assessed the risk of progressive CKD with stenotic necks adjusting for other histological features, kidney function and CKD risk factors.

Results

There were 65 cases with a mean of 255 glomeruli and 130 controls with a mean of 329 glomeruli. Among both cases and controls, 5% of glomeruli showed visible glomerulotubular necks. The proportion of necks that were stenotic was higher in cases than controls (35% vs 11%, P < .0001). Stenotic necks associated with progressive CKD independent of other histologic and clinical characteristics.

Conclusion

Glomerulotubular neck stenosis is associated with development of progressive CKD.
KS01.4.A CHARACTERIZATION AND THERAPEUTIC TARGETING OF ASTROCYTE REACTIVITY STATES DURING PROGRESSION FROM PRIMARY TO POST-RADIOTHERAPY RECURRENT GLIOBLASTOMA
Authors: Jeannot P , Rosberg R , Lindgren D , Smolag K I , Manou D , Berg T J , Dawson J , Elliott R , Ceberg C , Carragher N , Pietras A
Published: October 2024

BACKGROUND

Despite early response to radiotherapy, glioblastomas invariably recur, frequently within the field receiving high-dose irradiation. Stromal radiation responses remain poorly characterized, but recent evidence suggest that cells of the tumor microenvironment generate tumor-supportive conditions in the post-radiotherapy brain. Here, we explored the character of astrocyte reactivity states induced during tumor progression and treatment, identified compounds to target such states, and evaluated their suitability as therapeutic targets for glioblastoma.

MATERIAL AND METHODS

Murine glioblastomas were generated using the RCAS/tv-a system and treated (or not) with 10 Gy radiation upon symptoms. Healthy brain and tumor tissues were subjected to single cell RNA sequencing and spatial transcriptomics, and further analyzed using highly multiplexed immunohistochemistry. An image-based approach was developed and validated to quantify astrocyte reactivity, and this approach was utilized in high-throughput screens to identify compounds able to block astrocyte reactivity.

RESULTS

Single cell RNA sequencing revealed distinct astrocyte states in healthy brains and tumor tissues, and relative changes in state abundance as tumors progressed from primary to recurrent glioblastomas. Astrocyte states were spatially separated, with some enriched in peri-tumoral regions and others within the tumor core. Using an image-based method to quantify astrocyte reactivity in vitro, we screened 1600 compounds and identified 31 drugs able to inhibit astrocyte reactivity induced by radiotherapy. Among them, Flunarizine, a calcium-entry blocker approved to treat migraine, was confirmed to inhibit radiation-induced astrocyte reactivity and reactive astrocytes’ effect on glioblastoma cells in vitro. In a mouse model of glioblastoma, Flunarizine significantly extended the survival of mice in combination with radiotherapy while not affecting the outcome for non-irradiated mice, suggesting that Flunarizine targets a radiation-induced phenotype. Flunarizine treatment had no effect on primary glioblastoma cells, supporting the idea of microenvironmental targeting.

CONCLUSION

Astrocytes react to the tumor microenvironment by adopting cell states distinct from those present in healthy brain, and further adapt their phenotypes as tumors undergo radiotherapy. Screening for compounds able to inhibit astrocyte reactivity revealed Flunarizine as a promising candidate for glioblastoma treatment.
KS02.6.A SINGLE-CELL MULTIMODAL ANALYSIS OF IDH-MUTANT GLIOMAS UNCOVERS SHIFT FROM EPIGENETIC TO GENETIC DRIVERS WITH TUMOR PROGRESSION
Authors: Gonzalez Castro L , Wu J , Battaglia S , El Farran C , D’Antonio J , Miller T , Suva M , Bernstein B
Published: October 2024

BACKGROUND

Isocitrate dehydrogenase (IDH) mutant gliomas are progressive, invariably fatal brain tumors affecting young and middle-age adult patients. Previous single-cell transcriptomic characterization of IDH-mutant gliomas has uncovered a heterogeneity of cell states, with a developmental hierarchy that starts with neural progenitor-like stem cells that transition to more differentiated malignant cell states. Despite this understanding, the dynamics of these cell state transitions as tumors undergo grade progression, and the factors that facilitate or restrict cell state transitions remain poorly understood.

MATERIAL AND METHODS

Here, we characterize the cell states of IDH-mutant gliomas via single-cell gene expression (scRNA-seq) and single-cell analyses of accessible chromatin (scATAC-seq) in tumors spanning all grades using fresh patient samples and compare them with transcriptional programs and chromatin accessibility profiles of normal brain cells. We perform mechanism validation experiments using IDH-mutant glioma organoids.

RESULTS

We find that tumor transcriptional cell states experience a marked shift as tumors undergo progression. In low-grade tumors, predominant cell states mimic slow-cycling oligodendrocyte progenitor-like cells (OPCs). With grade progression, predominant cell states transition to resemble highly proliferative neural progenitor-like cells (NPCs). Additionally, scATAC-seq analyses revealed novel permissive chromatin states, which could facilitate this transition. Genetic analyses identified several molecular alterations in high-grade tumors, including loss of DNA methylation, oncogene amplification, and the deletion of genes associated with the interferon (IFN) response. Those IFN genes, as well as tumor suppressors, are initially repressed by DNA hypermethylation in low-grade tumors. Lastly, we validate the role of DNA hypomethylation in favoring immune evasion with grade progression using an IDH-mutant glioma organoid model.

CONCLUSION

Overall, our findings provide evidence that tumor progression in IDH-mutant gliomas is driven by an interplay of epigenetic and genetic mechanisms, with the former being determinant in low-grade tumors and the latter in high-grade tumors. The emergence of genetically-driven mechanisms promotes more mitotically active cell states as well as decreased anti-tumoral immune activation.
JS06.4.A RADIOTHERAPY QUALITY ASSURANCE IN THE ROAM/EORTC1308 ATYPICAL MENINGIOMA RANDOMISED CONTROLLED TRIAL: PROSPECTIVE REVIEW IMPROVES RADIOTHERAPY QUALITY AND CONSISTENCY
Authors: Jenkinson M D , Mayles H , Weber D C , Haylock B J ,   ROAM/EORTC1308 Trial Management Group
Published: October 2024

BACKGROUND

ROAM/1308 is an international randomised controlled phase III trial comparing radiotherapy (60Gy in 30 fractions) to observation following complete surgical resection of atypical meningioma. We embedded prospective radiotherapy quality assurance (RTQA) within the trial with the aim of ensuring consistent radiotherapy planning and delivery.

MATERIAL AND METHODS

The radiotherapy protocol described the consensus contouring/planning guidelines. Pre-trial assessment included benchmark cases (outlining/planning) and a technology ‘Dummy Run’. Individual case reviews were undertaken. Non-conformities were classed as ‘major’ (undertreatment of dural margins/over treatment of brain or organs at risk) or ‘minor’ (for education). Resubmissions were mandated for all major non-conformities. All plans were approved before radiotherapy delivery.

RESULTS

The UK RTQA group coordinated European sites. The Australian RTQA group (TROG) reviewed their sites and provided all data to the UK for review. Four radiation oncologists reviewed contours and physicists/dosimetrists reviewed plans. All sites (20 UK, 25 EORTC, 11 TROG) undertook the benchmark/’Dummy Run’ cases prior to opening. Resubmissions were: UK (1 contour, 0 plans), EORTC (2 contours, 6 plans) and TROG (1 contour, 3 plans). Trial communications and outlining workshops were used to provide feedback. Radiotherapy was delivered to 30, 26 and 7 patients at 11 UK, 12 EORTC and 5 TROG sites, respectively. Overall, 25 (40%) patient cases needed resubmitting: 23 (37%) sets of outlines (2 cases twice) and 9 (14%) treatment plans (1 plan twice).

CONCLUSION

Prospective quality assurance and case review are essential to ensure consistent high-quality radiotherapy and benchmark/’Dummy Run’ alone is not sufficient. RTQA should be used for future trials in rare tumours where centres may only treat a few patients. The consistent high-quality radiotherapy in ROAM/EORTC1308 ensures the primary outcome (progression free survival: to report 2026) will be a robust assessment and any difference between treatment arms cannot be attributed to variation in radiotherapy planning or delivery.
OS03.5.A A PHASE I CLINICAL TRIAL ON THE INTRACRANIAL ADMINISTRATION OF AUTOLOGOUS CD1C(BDCA-1)+ /CD141(BDCA-3)+ MYELOID DENDRITIC CELLS IN COMBINATION WITH IPILIMUMAB AND NIVOLUMAB IN PATIENTS WITH RECURRENT HIGH-GRADE GLIOMA
Authors: Duerinck J , Lescrauwaet L , Dirven I , Del’haye J , Cammaert M , Geens W , Geeraerts X , Stevens L , Brock S , Kockx M , Everaert H , Vanbinst a , Tuyaerts S , Neyns B
Published: October 2024

BACKGROUND

Intracranial (iCran) administration of the PD-1 and CTLA-4 blocking monoclonal antibodies nivolumab (NIVO) and ipilimumab (IPI) at the time of resection of recurrent high-grade glioma (rHGG) is safe and resulted in encouraging survival (NCT03233152). CD1c(BDCA-1)+ /CD141(BDCA-3)+ myeloid dendritic cells (myDC) play a pivotal role in initiating an adaptive anti-tumor immune response and licensing immune anti-tumor effector cells within the tumor microenvironment. iCran injection of autologous myDC in pts with rHGG was found to be safe but the potential added benefit for survival has not been established.

MATERIAL AND METHODS

rHGG pts (after prior RT and TMZ, not in need of supraphysiological doses of corticotherapy), underwent leukapheresis followed by isolation/cryopreservation of myDC. NIVO (10 mg IV) was administered preoperatively. Autologous myDC (5-, 10-, or 20.106 cells) were injected into the brain tissue lining the resection cavity (iCer) following a maximal safe resection (MSR) or intratumorally (iTum) following a stereotactic core needle biopsy (SCNB). IPI (5 mg) plus NIVO (10 mg) were co-injected iCer or iTum with the myDC. Postoperative NIVO was administrated intracavitary via an Ommaya reservoir (iCav, 10 mg) and intravenously (IV, 10 mg) Q2w (max 11x).

RESULTS

21 pts (13 M; med 49 y [range 20 -78]; IDHwt 17 pts, ECOG PS 0 or 1: 18-, 3 pts) underwent procurement of myDC; intraoperative administration of myDC was preceded by MSR in 19 pts, and SCNB in 2 pts. Respectively 6-, 3-, and 12 pts were treated at the 3 myDC dose levels. All pts received the peri-op iTum/iCer/IV-administration of IPI and NIVO as planned. The median postoperative iCav and IV NIVO-administration was 7 (range 0-11) and 8 (0-11). Study treatment was discontinued early at first progression in 9- and AE in 3 pts. Most important TRAEs: bacterial colonization of the Ommaya reservoir (n=4), craniotomy wound dehiscence (n=2), and bacterial meningitis (n=1). At DBL (01MAY2024), 6 pts (29%) remain progression-free (incl. 3 pts with >2y PFS). PFS (med. 24w [95% CI 8- 39]) is superior when compared to the pts (n=70) with resectable rHGG treated in 4 other cohorts of the GlITIpNi trial (p=.003). When including a durable benefit from bevacizumab at first PD in 3 pts (out of 10 pts who were treated with a low-dose bevacizumab regimen at first progression), PFS compared favorably to a historical pooled cohort (n: 469) of rHGG treated with VEGF(R)-inhibitors (p=.007). The 1-year OS-rate was 50% [95% CI 24-76].

CONCLUSION

Intracranial administration of autologous myDC combined with IPI/NIVO is feasible, safe and associated with encouraging survival, deserving further investigation.
OS05.6.A RADIOLOGICAL TREATMENT-ASSOCIATED CHANGES IN GLIOBLASTOMAS WITH OR WITHOUT AN MGMT PROMOTER-METHYLATION TREATED WITH CILENGITIDE: REVIEW OF THE EORTC CENTRIC AND CORE STUDIES
Authors: Flies C M , Friedrich M , Lohmann P , van Garderen K , Smits M , Tonn J C , Weller M , Galldiks N , Snijders T J
Published: October 2024

BACKGROUND

Radiological progression during follow-up of patients with glioblastoma after treatment can result from progressive disease (PD) or treatment-associated changes such as pseudoprogression. Previous studies reported higher rates of treatment-associated changes in patients with an O6-methylguanine DNA methyltransferase (MGMT) promoter-methylated glioblastoma. We reviewed patients from two prospective, randomised EORTC studies with an MGMT promoter-methylated (CENTRIC) or -unmethylated (CORE) glioblastoma who were treated with standard temozolomide chemoradiation with or without the integrin inhibitor cilengitide. The addition of cilengitide did not improve survival. We previously reported on treatment-associated changes in the CENTRIC study. We now assessed the rate of treatment-associated changes for all patients in relation to the MGMT status, cilengitide exposure, and timing after radiotherapy.

MATERIAL AND METHODS

Patients with three or more MRI follow-up scans were included. We applied the modified RANO criteria of 2017. Preliminary PD (PPD) was defined as a 25% or more increase of the sum of perpendicular diameter (SPD) or a new or increasing contrast-enhancing lesion compared to baseline or nadir. Confirmed PD was defined as a further 25%- or more increase of the SPD or as a new lesion outside the radiation field. According to the RANO criteria 2.0 of 2023, we classified patients with PPD and no further follow-up due to clinical progression as PD. Treatment-associated changes were defined as stabilisation or partial/complete regression after PPD. Survival of patients with treatment-associated changes was compared to those with PD.

RESULTS

Follow-up was available for 424 patients from the CENTRIC and for 181 patients from the CORE study. PPD rates were 65% (274 of 424) in methylated tumours, and 86% (155 of 181) in unmethylated tumours. This resulted in treatment-associated changes in 32% (88 of 274) and 17% (27 of 155). Treatment-associated changes were more common in the first three months after radiotherapy (28% (CORE) and 54% (CENTRIC) of all PPD), in patients with tumours with MGMT promoter methylation (relative risk (RR), 1.8; 95%-confidence interval, 1.3-2.7; P=0.002), and after cilengitide (RR, 1.4; 95%-CI, 1.02-1.8; P=0.038). In addition, late treatment-associated changes after six months after radiotherapy were also observed (9% (CORE) and 13% (CENTRIC)). In both studies, patients with treatment-associated changes lived significantly longer than patients with PD (P=0.044 (CORE); P=0.003 (CENTRIC)).

CONCLUSION

The rate of treatment-associated changes varied with timing after radiotherapy, MGMT status and the addition of cilengitide. The role of integrins in the pathophysiology of treatment-associated changes needs to be elucidated further.
OS06.3.A EFFECT OF EARLY INTEGRATION OF PALLIATIVE CARE ON THE QUALITY OF LIFE IN GLIOBLASTOMA PATIENTS
Authors: Golla H , Nettekoven C , Hellmich M , Bausewein C , Thon N , Becker G , Schnell O , Rolke R , Clusmann H , Radbruch L , Herrlinger U , Vatter H , Neukirchen M , Sabel M , Civello D , Müller D , Brumbarova T , Voltz R , Goldbrunner R ,   EPCOG study group
Published: October 2024

BACKGROUND

The disease course of glioblastoma (GB) patients is usually characterized by high symptom burden and short median survival (approx. 14 months). Nevertheless, they rarely receive specialized palliative care, especially in the early stage of the disease (early integration of palliative care, EIPC). The “Early Palliative Care for Patients with Glioblastoma” (EPCOG) study therefore aimed at investigating the impact of EIPC on quality of life (QoL) in this cohort of patients.

MATERIAL AND METHODS

Confirmatory, multicenter, single blinded, randomized, controlled clinical study. Patients with newly diagnosed/recurrent GB were randomly assigned to the control or interventional group. Patients in the interventional arm received proactive specialized palliative care (PC physician and PC social worker) on a monthly basis for 12 months. The control group received standard care. Data were collected by a blinded researcher every three months for 24 months. The primary outcome was QoL assessed by the sum of three subscales (trial outcome index, TOI) of the Functional Assessment of Cancer Therapy for brain tumor (FACT-Br) from baseline to six months of treatment.

RESULTS

556 patients were screened for eligibility, 231 patients were included in the study. 109 patients were treated in the intervention and 108 patients in the control group. Groups did not differ significantly in terms of age, gender or first/recurrent diagnosis. 34 patients were still alive after 24 months (intervention group: n=15, control group: n=19). 131 patients died during the study (intervention group: n=71, control group: n=60); without a statistically significant difference between groups. Within the intervention period, the TOI relative to the baseline tended to be better in the intervention group than in the control group, especially after three months. However, the difference was not statistically significant.

CONCLUSION

This is the first multicenter, randomized, confirmatory clinical trial to investigate the impact of EIPC on GB patients with an observation period long enough to realistically reflect the course of the disease. Despite the very vulnerable patient group, the study was even feasible with a higher number of patients than originally planned. The selected primary outcome parameter (TOI) included physical and functional, but not emotional and social factors, which may play a more important role in assessing QoL of GB patients.
OS06.9.A A PHASE 0/1 ‘TRIGGER’ TRIAL OF BDTX-1535 IN RECURRENT HIGH-GRADE GLIOMA (HGG) PATIENTS WITH EGFR ALTERATIONS OR FUSIONS
Authors: Sanai N , Umemura Y , Karapetyan V , Margaryan T , Huttenlocher D , Harmon J , Hong A , Yoo W , Tien A , Tovmasyan A , Mehta S
Published: October 2024

BACKGROUND

Alterations to epidermal growth factor receptor (EGFR) occur frequently in high-grade gliomas (HGG) and are critical to its pathogenesis. This Phase 0/1 study evaluates pharmacokinetic (PK), pharmacodynamic (PD), and clinical response to an ATP-competitive, irreversible EGFR inhibitor, BDTX-1535, in recurrent HGG patients with EGFR alterations and/or fusions.

MATERIALS/METHODS

Recurrent HGG patients with EGFR alterations (Arm A) or EGFR fusions (Arm B) received 5 days of BDTX-1535 at 200 mg/daily (cohort 1) or 400 mg/every other day (cohort 2) prior to planned resection at 2-4 hours following the final dose. In the Phase 0 component of the study, total and unbound drug concentrations were measured in tumor tissue (gadolinium (Gd) enhancing and non-enhancing regions), cerebrospinal fluid (CSF), and plasma using validated LC-MS/MS methods. A PK ‘trigger’, defined as unbound drug concentration above 1 nM and 4.1 nM in Gd non-enhancing tumor in patients with EGFR mutations and amplification, respectively, determined eligibility for Phase 1 therapeutic dosing. PD response was assessed by quantification of percentage of positive pEGFR, pERK and MIB-1 cells in the surgical tumor tissue compared to baseline pre-treatment tissue.

RESULTS

Eleven recurrent glioblastoma (GBM) patients were enrolled (Arm A, n=11; Arm B, n=0) and two were excluded from PK/PD analysis due to pseudoprogression. The mean unbound concentrations of BDTX-1535 in Gd non-enhancing tumor regions were 11.9 nM and 18.8 nM in cohorts 1 and 2, respectively. Eight of nine (88.9%) evaluable patients exceeded the PK threshold and qualified for Phase 1. Suppression of pEGFR and MIB1 was observed in 55% and 67% of patients, respectively. No serious adverse events related to BDTX-1535 were observed. Median progression-free and overall survival have not yet been reached.

CONCLUSIONS

BDTX-1535 is well-tolerated in recurrent GBM patients, achieves pharmacologically relevant concentrations in Gd non-enhancing tumor tissue and is associated with suppression of EGFR signaling. Clinical efficacy data accompanying this PK/PD response is maturing.
OS11.7.A DELETION OF MONOCARBOXYLATE TRANSPORTER 1 IN MYELOID CELLS FAVOURS GLIOBLASTOMA DEVELOPMENT
Authors: LILLO S , DERIEPPE M , BOMONT D , BURBAN A , MARTIN O , SALEH M , DAUBON T
Published: October 2024

BACKGROUND

Glioblastoma (GB) is the most aggressive tumor of the central nervous system, with a median survival of less than 15 months after diagnosis. The standard of care consists in surgical resection followed by radio-chemotherapy. However, patients are subject to a high rate of tumor recurrence, explained by an enriched immune environment in myeloid cells, which represent up to 70% of tumor weight and have immunosuppressive and/or pro-tumoral properties. GB possesses highly invasive properties due to its hypoxic and acidic core. Acidity is mainly mediated by the released of protons with lactate coming from tumor cells. Lactate is known to be responsible for immuno-metabolic modifications of immune cells in other cancers. Altogether, immunosuppressive/pro-tumoral macrophages abundancy and lactate concentration may impact tumor development and patient’s outcome. However, the specific effects of lactate on the phenotypes and functions of myeloid cells in the GB tumor microenvironment is poorly understood.

MATERIAL AND METHODS

The brain’s immune environment is unique. To study effects of microglia, which are resident macrophages, and of macrophages, that infiltrated brain from the bloodstream, on GB development, mouse models deficient for the lactate transporter MonoCarboxylate Transporter 1 (MCT1) either in all myeloid cells (Mct1Δmye), or specifically depleted in microglia (Mct1ΔMG) as well as constitutively depleted in lactate signalling, for the receptors GPR81 or GPR132 have been generated in both sexes.

RESULTS AND CONCLUSION

Disruption of lactate signalling through Gpr81 deficiency did not affect GB growth in our model. In contrast, male Gpr132 knockout mice (Gpr132-/-) displayed significantly reduced tumor volume compared to WT. ScRNA seq showed that Gpr132 is highly expressed in immune cells. Surprisingly, flow cytometry analysis of the immune infiltrate revealed no significant difference in in bone-marrow-derived or microglia tumor-associated macrophages (BM-TAMs vs MG-TAMs), suggesting that lactate signalling through Gpr132 regulate tumor growth with minimal influence of immune system. ScRNAseq also showed that Mct1 is expressed in myeloid cells. Then, we investigated the role of Mct1 by using the myeloid-specific deficiency model (Mct1Δmye). In vivo experiments revealed a sex-dependent effect in Mct1Δmye mice compared to control, with males exhibiting significantly larger tumors than WT mice. Interestingly, in vitro respiration assays demonstrated that Mct1Δmye BMDMs have a lower respiration rate on lactate compared to controls. For Mct1Δmye, a detailed analysis of the metabolic differences between WT and Mct1Δmye BMDMs is currently under investigation. These findings suggest intriguing regulatory roles of lactate signalling and transport on GB tumor growth, with a potential sex-specific responses mediated by Mct1 and lactate flux.
P01.09.B IDENTIFICATION OF TIME-DEPENDENT PHENOTYPIC SHIFTS IN PATIENT-DERIVED GLIOMA STEM-LIKE CELLS AFTER CHEMORADIOTHERAPY
Authors: Van Eupen L , De Visser Y , Bravo Gonzalez-Blas C , Cole B , Loers J , Derweduwe M , Claeys A , Tabury K , Primac I , Vermeirssen V , De Smet F , Quintens R
Published: October 2024

BACKGROUND

Glioblastoma remains among the deadliest tumors without a suitable cure. The failure of virtually all clinical trials from the past 20 years can be attributed to the extensive (epi)genetic and phenotypic heterogeneity within and across GBM patients but also to the extensive plasticity of cancer stem-like cells that can easily adapt and drive resistance to treatment. A fundamental understanding of this plasticity and its underlying mechanisms is still largely missing, but it remains pivotal to identify suitable targets that can prevent cells from surviving treatment.

METHODS

In this project, we start by defining the landscape of standard-of-care-induced plasticity using multi-omic profiling of patient-derived glioma stem-like cells (PDGSCLs) after radiation therapy (RT, 2 Gy) and temozolomide (TMZ, 150 μM). In a pilot experiment, bulk RNA sequencing was performed on six PDGSCLs at different time points after RT+TMZ (16 hours, 3 days, 10 days). A single-cell RNA sequencing (scRNAseq) experiment has been set up in a similar fashion, including the 16-h and 3-d time points.

RESULTS

Analysis of the bulk transcriptomic data, including module scoring based on transcriptional cell states defined by Neftel et al. (2019), revealed a time-dependent shift in phenotype after treatment. Two of the six PDGSCLs did not show clear indications of plasticity, while two others showed minor shifts in phenotype and two PDGSCLs displayed clear shifts towards the mesenchymal (MES) phenotype, with one of them reverting back to its original state after 10 days. Pathway analysis additionally indicated an upregulation of MES-associated processes including ameboidal-type cell migration, cell-substrate adhesion and wound healing, with differentially upregulated pathways between sensitive and resistant PDGSCLs that both displayed MES-shifts.

CONCLUSIONS

Our results confirm that treatment can induce phenotypic changes in PDGSCLs in a cell line-specific manner, suggesting different mechanisms can lead to the enrichment of the same phenotype. Moreover, our time course data highlights that the timing of read-out is important as well, and comparing tumors at diagnosis vs. at recurrence might not suffice to fully understand this plastic behavior, for which these intermediate time points are more relevant. The scRNAseq experiment is currently ongoing and will inform on the gene regulatory networks responsible for the observed shifts while also highlighting the heterogeneity within these PDGSCLs with greater resolution, as seen in the patient population. This project is funded by FWO (11B2323N) and the Foundation against Cancer (F/2022/2026).
P01.14.B SYNGN: A NOVEL EPIGENETIC PLATFORM TO DISCOVER PATIENT-SPECIFIC DISEASE MECHANISMS AND DRUGGABLE TARGETS IN GLIOBLASTOMA
Authors: Marino S , Vinel C , Constantinou M
Published: October 2024

BACKGROUND

The alteration of the glioblastoma genome by epigenetic mechanisms that share functions with normal developmental processes, such as self-renewal and fate specification of neural stem cells (NSC), is a key piece of evidence that links brain cancer pathogenesis with dysregulated stem cell functions. A patient-specific comparison of glioblastoma stem cells (GSC) with NSC, a putative cell of origin of at least a proportion of these tumours, is not feasible as patient-matched endogenous NSC are not surgically accessible and all epigenetic studies in glioblastoma have so far compared epigenetic changes of different tumours with each other or to comparators obtained from foetal brains or an unrelated donor. We reasoned that availability of syngeneic GSC and NSC pairs would allow to identify crucial epigenetic differences on a patient-specific basis and would provide essential therapeutic contrast to define disease-and patient-intrinsic biomarkers of drug response that are less confounded by germline variation.

MATERIALS AND METHODS

We have derived GSC from IDH-wildtype glioblastoma and harnessed state-of-the-art stem cell technologies to generate patient-matched fibroblast-derived EPSC, which were induced to NSC (iNSC). We generated transcriptome, DNA methylome, ChIP-Seq (H3K4me3, H3K27ac, H3K3me3, H3K27me3) and ATACSeq datasets for 10 GIC/iNSC pairs.

RESULTS

Comparative integrated analysis has shown that redistribution of selected histone marks as well as shifts in chromatin states across the genome identifies differentially expressed genes, including novel druggable regulatory mechanisms in GSC, which are specific of the neoplastic context. In particular, we identified METTL7B as an essential regulator of lineage specification and plasticity in human glioblastoma, with impact on both tumour size and invasiveness in an in vivo xenograft model. Single cell transcriptomic analysis of these tumours and of cerebral organoids derived from expanded potential stem cells (EPSCs) overexpressing METTL7B reveal a regulatory role for the gene in the neural stem cells-to-astrocyte differentiation trajectory. Mechanistically, METTL7B downregulates the expression of key neuronal differentiation players, including SALL2, via post-translational modifications of histone marks.

CONCLUSION

SYNGN is an epigenetic platform which can identify novel disease mechanisms and druggable targets in glioblastoma.
P02.02.A AN MRI-LSM PLATFORM TO ASSESS BLOOD-BRAIN BARRIER DISRUPTION AND BIODISTRIBUTION OF IMMUNOTHERAPIES IN EXPERIMENTAL GLIOMA
Authors: Scheck J G , Boztepe B , Heinz L , Sturm V , Pan C , Fischer M , Heiland S , Winkler F , Wick W , Schregel K , Abdollahi A , Platten M , Bendszus M , Weidenfeld I , Breckwoldt M O
Published: October 2024

BACKGROUND

The inherent resistance of glioma including the recent failure of immune checkpoint blockade to prolong survival in glioma has partly been attributed to tumor heterogeneity leading to insufficient drug availability. Magnetic resonance imaging (MRI) is the main clinical modality to monitor tumor growth and treatment response but cannot assess drug availability within the tumor microenvironment (TME). To this end, we developed a platform to assess blood-brain barrier disruption utilizing high field multiparametric MRI and correlative light sheet microscopy (LSM) to investigate the underlying mechanisms of response and resistance towards immunotherapy in brain tumor models.

MATERIAL AND METHODS

Orthotopic gliomas were grown in the syngeneic GL261 or SB28 mouse glioma models. Longitudinal contrast enhanced multiparametric MRI was performed weekly after baseline. To correlate drug distribution with BBB-D and the microvasculature, mice either received a single dose of fluorescently labelled anti-PD1 or TLR7 agonist encapsulated nanoparticle CDNP-R848 at different tumor stages. After 24 hours circulation time, vasculature was labelled through lectin-FITC perfusion. Brains were cleared with fDisco and LSM was performed. Images were segmented with Ilastik and further analysis was carried out in Imaris, Fiji and VesselVio.

RESULTS

In Gl261 gliomas we achieved a response rate of 87.5% to CDNP-R848, as previously established by our group, whereas SB28 tumors were fully resistant to CDNP-R848 validating the strong immunotherapeutic resistance of SB28. In both glioma models, immunotherapies were CNS-penetrant and reached the TME without off targeting, while showing cellular specificity for myeloid cells or T cells, respectively. Gadofluorine M and Gd-DOTA allowed the assessment of different spatio-temporal patterns of BBB-D. Microbleedings as assessed by T2* imaging and BBB-D as assessed by perfusion imaging were more prominent in SB28, indicating pronounced heterogeneity in the SB28 model, likely contributing to its resistance. GfM and perfusion MRI were strongly correlated with drug distribution, indicating that these MR parameters can predict drug distribution in the TME.

CONCLUSION

Our correlated MR-LSM approach therefore allows the assessment of the 3-dimensional biodistribution of checkpoint inhibitors and immunotherapeutic NP in the TME in relation to BBB-D. By validating MR-based imaging biomarkers, we aim to non-invasively predict drug availability in the complex TME of glioma.
P02.11.A IMMUNE CELLS SUBPOPULATIONS IN PERIPHERAL BLOOD IN PATIENTS WITH GLIOBLASTOMA
Authors: Roncelli F , Boselli D , Snider S , De Domenico P , Braga M , Bisoglio A , Villa C , Gentner B , Mortini P , Gagliardi F
Published: October 2024

BACKGROUND

Glioblastoma is the deadliest type of primary brain tumors. Tumor microenvironment characterization may provide new insights in understanding disease and the balance between inflammatory and suppressor cells and represent a key factor in tumor-microenvironment interaction. This study aims at characterizing peripheral immune cells subpopulations in glioblastoma patients.

MATERIAL AND METHODS

We analyzed peripheral blood mononuclear cells (PBMC) from 57 patients with newly diagnosed glioblastoma who underwent surgery in Neurosurgery department from 2019 to 2023. Blood sample was taken before surgery and PBMC were stored in Institutional Biobank. Clinical and radiological features and steroid dosage at sample time were recorded. For flow cytometry analysis, two panels of antibodies were designed, one for myeloid and one for lymphoid cells. Acquisition was performed with BC CytoFLEX LX and CytExpert software and data were processed with FCS Express 7. Statistical analysis was performed with SPSS 28.

RESULTS

Survival data were available for 52 patients: the mean overall survival (OS) was 15.98 months. A Cox regression analysis was performed to identify any prognostic impact of subpopulations, on which steroids’ influence was checked. A small effect was noted for monocytes (HR 1.055, p 0.018) and CD11b+/CD33+ cells including peripheral myeloid-derived suppressor cells (MDSC, HR 1.044, p 0.055), both influenced by steroids. A stronger effect was noted for immature NK cells (HR 0.198, p 0.029), uninfluenced by steroids. A significant correlation was found between immature NK cells and ECOG performance status (PS), with higher levels in patients with lower ECOG (p 0.029). MDSC and CD8+ T cells were more represented in patients with necrosis on MRI while patients with midline shift showed higher MDSC and lower immature NK cells. FLAIR volume was positively correlated with mature NK while MDSC and mature NK showed a correlation near to significance with the difference between FLAIR and contrast-enhancing volume.

CONCLUSION

These results are promising in identifying subpopulations in peripheral blood with a putative role in patients’ OS. Higher immature NK cells correlate with higher PS at surgery and are associated with increased OS. On the other hand, monocytes and MDSC correlate with radiological parameters such as midline shift, necrosis, and FLAIR volume. Further analysis on larger sample and longer follow-up is required to confirm these promising results.
P06.04.A FIRST IN HUMAN - TARGETING ANDROGEN RECEPTOR FOR GLIOBLASTOMA THERAPY
Authors: Lavon I , Mordechai A , Lossos A
Published: October 2024

BACKGROUND

Glioblastoma represents a significant therapeutic challenge due to its aggressive nature, necessitating the development of effective targeted therapies. In a pivotal study, the amplification of the androgen receptor in 56% of glioblastomas was observed, shedding light on its therapeutic potential. Through extensive research, our team demonstrated the efficacy of the androgen receptor antagonist, enzalutamide (Xtandi), in prolonging lifespan in a mouse model of human glioblastoma. Collaborating with the Cyclotron and PET/CT teams, we established [18F]-FDHT PET/CT imaging, facilitating real-time detection of androgen receptor expression within brain tumors and enabling precise patient selection for targeted therapy.

METHODS

To proceed with a clinical trial, we conducted several steps. Initially, we examined the pharmacokinetics of enzalutamide and temozolomide, both as monotherapy and in combination, over multiple time points up to 96 hours, analyzing plasma and brain samples by LC-MS/MS. Additionally, we assessed the efficacy of PET/CT imaging for therapy response monitoring. After confirming androgen receptor expression via PET scan, patients underwent a 10-day regimen of escalating doses of the androgen antagonist Xtandi, administered daily, followed by a second PET scan.

RESULTS

In the pharmacokinetic analysis, we found that the half-life (t1/2) of enzalutamide remained consistent in both plasma and brain tissue when combined with temozolomide, compared to monotherapy. Notably, the t1/2 of temozolomide increased significantly when administered concurrently, rising from 1.8 to 4.45 in plasma and from 0.88 to 7.25 in brain tissue, respectively. These results informed the clinical trial protocol design. Regarding the PET/CT study, the outcomes were promising: two potential responders identified through imaging received either 40 or 80 mg of Xtandi. The patient who received 80 mg exhibited a significant reduction in tracer uptake after ten days of treatment, accompanied by tumor shrinkage and tangible improvements in mobility and quality of life, highlighting the transformative potential of our approach.

PLANS AND DISCUSSION

Encouraged by these results, we initiated a clinical trial to determine the safety and tolerability of Enzalutamide in conjunction with radiotherapy (RT) and temozolomide (TMZ), to assess clinical response using Response Assessment in Neuro-Oncology (RANO) criteria and to characterize the pharmacokinetics of Enzalutamide combined with TMZ. We have recently enrolled two patients for this study, and eagerly anticipate presenting the results of these participants, along with potentially others, during the forthcoming meeting. With the potential to rapidly translate these findings into clinical practice, our fast-track trial represents a beacon of hope for Glioblastoma patients worldwide.
P07.04.A THE LOSS OF THE X CHROMOSOME IN THE CONTEXT OF THE MALIGNANCY OF MENINGIOMAS
Authors: Berghaus N , Savran D , Hielscher T , Schrimpf D , Herold-Mende C , Wick W , von Deimling A , Sahm F
Published: October 2024

BACKGROUND

In recent years, molecular markers have become increasingly important in the diagnosis and risk assessment of brain tumours. Besides mutations and methylation, this also includes copy number variations (CNVs). It is well known that CNVs contribute to cancer initiation, progression and therapeutic resistance. Investigating the most common primary intracranial tumours in adults, meningiomas, several studies propose new stratification systems with a more accurate risk prediction than the WHO grading. Several of these approaches combine morphology, methylation, or both, with CNV status to derive a more precise risk assessment. However, a common shortcoming is the absence of separate analyses based on the sex of the patients or the inclusion of the X chromosome.

MATERIAL AND METHODS

A cohort of over 6000 meningiomas was stratified and analysed regarding the loss of the X chromosome. Further, a differential methylation analysis was conducted based on EPIC v1 data.

RESULTS

In general, meningiomas from females displayed a loss of chromosome X more than three times more often than tumours from males (p = <0.001) and were significantly associated with a more malignant classification, affecting both the WHO classes and the methylation classification. Calculating oncogenetic trees, females and males share the loss of chr22q as first node followed by a loss of chr1p but only in females, the loss of chromosome X directly follows the high-risk CNV event “loss of chr14q”. However, not only CNVs, but also DNA methylation is a decisive factor. Therefore, a differential methylation analysis was conducted between meningiomas from females with and without a loss of the X chromosome, stratified for methylation classes. Within the methylation class (MC) ben-1, almost no difference between the two cohorts could be detected for the autosomes. In contrast, within the MC mal, the highest and significant difference was noted for a region overlapping with the RUNX3 gene (p = <0.001) - indicating a higher methylation in the “noloss” subgroup. This is of special interest as RUNX3, a transcription factor, regulates gene expression, impacts the cell cycle, cell differentiation, apoptosis, tumor suppression, and cancer cell metastasis. Analyzing the MC mal regarding the X chromosome, for example, a significant difference was detected for a region overlapping COX7 (p = <0.001). The mitochondrial protein COX7b has previously been linked to driving metastasis of human breast cancer cells in mice.

CONCLUSION

The findings indicate that the loss of the X chromosome in females is linked to tumor malignancy and can also be correlated with different methylation patterns.
P08.03.A CSF-BASED PROTEOMIC BIOMARKERS OF RESPONSE TO MULTI-AGENT INTRAVENTRICULAR CHEMOTHERAPY IN CNS LYMPHOMA
Authors: Mansouri A , Aastha A , Wilding H , Mikoljewicz N , de Macedo Filho L , Glantz M , Kislinger T
Published: October 2024

BACKGROUND

Despite promising responses to initial therapy for central nervous system lymphoma (CNSL), relapse is common and overall responses are disappointing compared to other non-CNS diffuse large B-cell lymphomas. Literature reports 1- and 3-year survival rates of 50% and 30-40% respectively, with standard treatment. Incorporation of multi-agent intrathecal chemotherapy (MAITC) alongside systemic therapy is novel but not widely adopted. Biomarkers predictive of response could help select patients most likely to benefit. Our retrospective analysis of CNSL patients treated with MAITC between 2015-2021 indicates a significant overall survival advantage. Cerebrospinal fluid (CSF) is a less invasive alternative for diagnosis and management of CNSL compared to traditional biopsies, while also reducing sampling bias. The objective of this study is to identify CSF-based proteomic biomarkers that can serve as reliable indicators of MAITC treatment response and guide CNSL management.

MATERIAL AND METHODS

Patient-matched CSF samples were banked at pre- and post-therapeutic endpoint in 22 primary and 36 secondary CNSL patients. The samples were profiled using high-throughput fluid sample protocol coupled with mass-spectrometry that only requires 30 microliters of CSF. Clinically, patients were categorized as “early responders” (objective imaging response and cytology clearance by cycle 6, both maintained until cycle 12) vs. “non responders”. Tumor burden (based on MRI and cytology) was correlated with proteomic markers to identify potential correlative markers.

RESULTS

SGCE, LCP1 and AGRN were found to reliably (AUROC 0.95, 95% CI 0.81-0.98, Sensitivity: 0.62, Specificity: 0.76) stratify patients based on MAITC response (responder vs. non-responder). Through analysis of matched pre- and post-treatment samples (>3 cycles) from the same patient cohort and correlation with brain MRI and CSF cytology, we identified H3F3A, YWHAE, LCP1, CA1 and GAPDH as candidate tumor burden biomarkers (AUROC 0.9, 95% CI 0.79-1.0). Conclusion: Our low input proteomic analyses of CSF has identified candidates potentially predictive of response to MAITC along with potential tumor burden markers. Validation studies with a larger cohort and additional timepoints are currently underway.
P10.05.B ADVANCED MACHINE LEARNING TO DETECT HETEROGENEITY OF CELL CYCLE ALTERATIONS DURING TMZ CHEMOTHERAPY AT SINGLE CELL LEVEL
Authors: Villa G , Kückelhaus J , Yabo Y , Schnell O , Delev D , Benotmane J K , Heiland H D
Published: October 2024

BACKGROUND

Glioblastoma (GBM) stands as the most aggressive tumor of the CNS. Current treatment typically involves surgical intervention followed by cycles of Temozolomide (TMZ) chemotherapy in combination with radiotherapy. However, the efficacy of these treatments remains limited due to GBM’s ability to adapt and resist therapy. TMZ treatment leads to significant alterations in the cell cycle of GBM cells, with a decrease in cells in the G0-G1 phase and an increase in cells in the S or G2 phases. This shift in cell cycle distribution suggests a dual response: re-entry of tumor cells into the cell cycle and concurrent arrest induced by TMZ, potentially contributing to tumor resistance and disease progression. Here, we leverage the variable patterns of Ki-67, a marker of cellular proliferation, which expression fluctuates throughout the cell cycle, peaking during S, G2, and M phases to evaluate cellular response to TMZ treatment across various primary cell lines and tissue samples.

MATERIAL AND METHODS

We developed an image-based approach to investigate and reconstruct the cell cycle using advanced machine learning for pattern recognition to identify the detailed cell cycle status on single cell level. To characterized primary cell lines (n=6), we performed whole-genome DNA sequencing, whole-genome methylation sequencing and RNA-sequencing. For functional read-outs, we performed large-scale TMZ treatment in a time- and concentration dependent manner in cell culture and GBM-tissue slice models.

RESULTS

Our machine learning model of cell cycle reconstructions demonstrated a robust reconstruction of detailed cell cycles stages across all cell lines with the majority of cells assigned to G0-G1 stated without treatment. TMZ treatment resulted in a significant shift towards enhanced S/G2 phase (p < 0.02) in 3 of 6 tested cell lines. Pseudo-time reconstruction revealed a dose-dependent alteration of the cell cycle showing significant changes with TMZ-doses below 50µmol. Cell lines which enhanced stem-like phenotypes based on epigenetically and transcriptionally characterisation demonstrated no cell cycle alterations even in high-dose treatment conditions (>150µmol) regardless their MGMT promotor methylation status. This heterogeneity underscores the complexity of GBM and highlights the importance of personalized treatment strategies.

CONCLUSION

Our findings provide valuable insights into GBM’s cell cycle alterations and adaptive mechanisms of resistance independent of the MGMT methylation status as a known biomarker for TMZ response. TMZ-induced cell cycle alterations can provide a novel biomarker to detect treatment response and offer a foundation for developing personalized therapeutic approaches.