IDHwt Glioblastoma Research Articles

P08.01.A FAMILY SUPPORT NEEDS IN GLIOBLASTOMA - FROM RELATIVES’ PERSPECTIVES

Abstract BACKGROUND Glioblastoma is the most common and aggressive brain tumour in adults. In spite of standard treatment by surgery followed by radiation and chemotherapy, the average life expectancy is only 10-15 months. As such, glioblastoma affects the family situation and relatives around the patient need support. There is a lack of knowledge about their experiences of support during the illness. The aim of this study was to investigate support needs during the illness trajectory in glioblastoma from the relatives’ perspectives. MATERIAL AND METHODS In this study, 12 relatives (7 partners, 3 children, 1 sister, 1 friend) to patients with a histologically-confirmed diagnosis of IDH-wt glioblastoma since at least 2 months and age ≥ 18 years at the time of diagnosis, were interviewed individually based on a semi-structured interview guide. Interviews took place on one occasion by physical or digital meeting or via telephone, based on the participant's preferences. The interviews were audio recorded and transcribed verbatim. Data collection occurred from March 2022 until December 2022. The analysis using inductive qualitative content analysis is in progress. RESULTS Preliminary results show that relatives were mostly satisfied with the support from the health care. However, they lived in a very stressful situation and parts of the support could be better organized. It was important for them with clear and correct information regardless of the outcome. The waiting time before and after MRI, living in limbo with anxiety of what would be found, was particularly stressful and they desired processing with faster answers. Availability to the health care was an important factor and because of the difficult situation they preferred digital meetings instead of physical meetings. Further, they found it difficult to manage the contacts with different health care instances and thought it would be helpful with support to navigate them to correct instance. CONCLUSION The preliminary results indicate that support to the families should be more structured och focused on clearness, high availability and openness for the family’s needs, and guidance in the health care system.

Isocitrate dehydrogenase wt and IDHmut adult-type diffuse gliomas display distinct alterations in ribosome biogenesis and 2'O-methylation of ribosomal RNA.

High-grade adult-type diffuse gliomas (HGGs) constitute a heterogeneous group of aggressive tumors that are mostly incurable. Recent advances highlighting the contribution of ribosomes to cancer development have offered new clinical perspectives. Here, we uncovered that isocitrate dehydrogenase (IDH)wt and IDHmut HGGs display distinct alterations of ribosome biology, in terms of rRNA epitranscriptomics and ribosome biogenesis, which could constitute novel hallmarks that can be exploited for the management of these pathologies. We analyzed (1) the ribosomal RNA 2'O-ribose methylation (rRNA 2'Ome) using RiboMethSeq and in-house developed bioinformatics tools (https://github.com/RibosomeCRCL/ribomethseq-nfandrRMSAnalyzer) on 3 independent cohorts compiling 71 HGGs (IDHwt n = 30, IDHmut n = 41) and 9 non-neoplastic samples, (2) the expression of ribosome biogenesis factors using medium throughput RT-qPCR as a readout of ribosome biogenesis, and (3) the sensitivity of 5 HGG cell lines to RNA Pol I inhibitors (CX5461, BMH-21). Unsupervised analysis demonstrated that HGGs could be distinguished based on their rRNA 2'Ome epitranscriptomic profile, with IDHwt glioblastomas displaying the most significant alterations of rRNA 2'Ome at specific sites. In contrast, IDHmut HGGs are largely characterized by an overexpression of ribosome biogenesis factors compared to non-neoplastic tissues or IDHwt glioblastomas. Finally, IDHmut HGG-derived spheroids display higher cytotoxicity to CX5461 than IDHwt glioblastoma, while all HGG spheroids display a similar cytotoxicity to BMH-21. In HGGs, IDH mutational status is associated with specific alterations of the ribosome biology and with distinct sensitivities to RNA Pol I inhibitors.

Spatially resolved transcriptomic profiles reveal unique defining molecular features of infiltrative 5ALA-metabolizing cells associated with glioblastoma recurrence

BackgroundSpatiotemporal heterogeneity originating from genomic and transcriptional variation was found to contribute to subtype switching in isocitrate dehydrogenase-1 wild-type glioblastoma (GBM) prior to and upon recurrence. Fluorescence-guided neurosurgical resection utilizing 5-aminolevulinic acid (5ALA) enables intraoperative visualization of infiltrative tumors outside the magnetic resonance imaging contrast-enhanced regions. The cell population and functional status of tumor responsible for enhancing 5ALA-metabolism to fluorescence-active PpIX remain elusive. The close spatial proximity of 5ALA-metabolizing (5ALA +) cells to residual disease remaining post-surgery renders 5ALA + biology an early a priori proxy of GBM recurrence, which is poorly understood.MethodsWe performed spatially resolved bulk RNA profiling (SPRP) analysis of unsorted Core, Rim, Invasive margin tissue, and FACS-isolated 5ALA + /5ALA − cells from the invasive margin across IDH-wt GBM patients (N = 10) coupled with histological, radiographic, and two-photon excitation fluorescence microscopic analyses. Deconvolution of SPRP followed by functional analyses was performed using CIBERSORTx and UCell enrichment algorithms, respectively. We further investigated the spatial architecture of 5ALA + enriched regions by analyzing spatial transcriptomics from an independent IDH-wt GBM cohort (N = 16). Lastly, we performed survival analysis using Cox Proportinal-Hazards model on large GBM cohorts.ResultsSPRP analysis integrated with single-cell and spatial transcriptomics uncovered that the GBM molecular subtype heterogeneity is likely to manifest regionally in a cell-type-specific manner. Infiltrative 5ALA + cell population(s) harboring transcriptionally concordant GBM and myeloid cells with mesenchymal subtype, -active wound response, and glycolytic metabolic signature, was shown to reside within the invasive margin spatially distinct from the tumor core. The spatial co-localization of the infiltrating MES GBM and myeloid cells within the 5ALA + region indicates PpIX fluorescence can effectively be utilized to resect the immune reactive zone beyond the tumor core. Finally, 5ALA + gene signatures were associated with poor survival and recurrence in GBM, signifying that the transition from primary to recurrent GBM is not discrete but rather a continuum whereby primary infiltrative 5ALA + remnant tumor cells more closely resemble the eventual recurrent GBM.ConclusionsElucidating the unique molecular and cellular features of the 5ALA + population within tumor invasive margin opens up unique possibilities to develop more effective treatments to delay or block GBM recurrence, and warrants commencement of such treatments as early as possible post-surgical resection of the primary neoplasm.

Innovative Approach to Isolate and Characterize Glioblastoma Circulating Tumor Cells and Correlation with Tumor Mutational Status.

Circulating tumor cells (CTCs) are one of the most important causes of tumor recurrence and distant metastases. Glioblastoma (GBM) has been considered restricted to the brain for many years. Nevertheless, in the past years, several pieces of evidence indicate that hematogenous dissemination is a reality, and this is also in the caseof GBM. Our aim was to optimize CTCs' detection in GBM and define the genetic background of single CTCs compared to the primary GBM tumor and its recurrence to demonstrate that CTCs are indeed derived from the parental tumor. We collected blood samples from a recurrent IDH wt GBM patient. We genotyped the parental recurrent tumor tissue and the respective primary GBM tissue. CTCs were analyzed using the DEPArray system. CTCs Copy Number Alterations (CNAs) and sequencing analyses were performed to compare CTCs' genetic background with the same patient's primary and recurrent GBM tissues. We identified 210 common mutations in the primary and recurrent tumors. Among these, three somatic high-frequency mutations (in PRKCB, TBX1, and COG5 genes) were selected to investigate their presence in CTCs. Almost all sorted CTCs (9/13) had at least one of the mutations tested. The presence of TERT promoter mutations was also investigated and C228T variation was found in parental tumors and CTCs (C228T heterozygous and homozygous, respectively). We were able to isolate and genotype CTCs from a patient with GBM. We found common mutations but also exclusive molecular characteristics.

The efficacy and safety of anlotinib combined with bevacizumab in treatment of recurrent isocitrate dehydrogenase wildtype glioblastoma: A retrospective analysis.

e14028 Background: Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor with a 5-year survival rate of 5%. There is no standard and effective treatment for recurrent GBM. Angiogenesis is recognized as a key event in the progression of GBM. Bevacizumab as an anti-angiogenic monoclonal antibody has been recommended by NCCN guidelines for recurrent GBM. Anlotinib is a small-molecule multi-target tyrosine kinase inhibitor (TKI) which has different function mechanisms and targets with bevacizumab. Therefore, we investigated the clinical efficacy and safety of anlotinib combined with bevacizumab in patients with recurrent IDH-wide type (wt) GBM on the basis of TMZ treatment. Methods: We retrospectively analyzed the clinical data of 18 patients with IDH-wt GBM in Shandong Cancer Hospital and Institute from 2019 to 2022. The primary endpoint was progression-free survival (PFS) and overall survival (OS) for combination treatment. The secondary endpoint was 6-months PFS rate, 1-year OS rate, objective response rate (ORR), disease control rate (DCR), OS and safety and toxicity profile. Results: All patients were diagnosed as WHO grade IV IDH-wt GBM and the median age of all patients was 55 years (range 39-76). Patients with recurrent GBM were first treated with anlotinib, followed by bevacizumab when patients developed progression or cerebral edema. In the overall population, the median OS for overall patients is 27 months, and the median survival time after first recurrence is 13.3 months. The median survival time of anlotinib treatment and combination treatment is 15.6 months and 12 months, respectively and the median PFS is 14.3 months and 9.1 months, respectively. The ORR for combination treatment was 27.8%, and 6-months DCR was 77.8%. The last follow-up date was October 15, 2022. By the last follow-up date, 3 patients were continuing anlotinib combined with bevacizumab treatment with no disease progression. The 6-months PFS rate was 78.9%, and the 1-year OS rate was 44.4%. The treatment-related adverse events (AEs) were shown in Table 3. The main AEs was bone marrow suppression (83.3%, 15/18), most of which were grade I and II (50%, 9). The AEs were relieved by adjusting the drug dose and symptomatic treatment. There were no treatment related deaths. Conclusions: Our study reported the clinical effect of anlotinib combined with bevacizumab treatment in recurrent GBM patients on the basis of TMZ treatment in a real-world setting. This is the first attempt to conduct dual antiangiogenic treatment in GBM patients. We demonstrate a promising result and suggest that this combination treatment may be a novel and safety treatment option for recurrent IDH-wt GBM patients.

Incidence of non-canonical IDH mutations and IDH co-mutations in a multicenter prospective study of patients with glioma.

e14033 Background: IDH-mutant tumors are a distinct disease entity accounting for one third of gliomas with a more favourable prognosis despite still harboring high morbidity and mortality rates. Most IDH mutations (muts) in gliomas group at R132/R172 hotspots in IDH1/IDH2, respectively. Our aim was to study IDH non-canonical muts and IDH co-mutations (co-muts) since the evidence is still limited. Methods: Prospective study of patients (pts) with gliomas at 4 third-level hospitals in Spain (Hospital Clinico Universitario San Carlos, Hospital Universitario La Paz, Hospital Universitario de Canarias, Hospital Universitario Nuestra Señora de la Candelaria). High throughput next generation sequencing (NGS) using a customized gene panel ((Illumina, Inc) including IDH1 and IDH2 was used in FFPE and/or fresh tumor samples, and run in an Illumina MiSeq instrument (Illumina, Inc). Only pathogenic mutations were considered as valid. Clonal IDH (cIDH) muts were defined as those with a MAF ≥ 1% and subclonal IDH (scIDH) muts as those with a MAF < 1%. Results: Between February 2017 and February 2021, 49 glioma pts enrolled and sequenced for IDH1/IDH2 tumor muts. Median age 51 (Min-max: 20-78). WHO 5th Ed (n = 42): IDH-MUT astrocytoma (n = 14), IDH-MUT oligodendroglioma (n = 7), IDH-WT glioblastoma (n = 21). 37/49 pts (75.5%) presented cIDH and/or scIDH muts, of which 21 pts (43%) harbored cIDH muts in IDH1 (R132H (n = 17), R132C (n = 1), R132G (n = 1), R100Q (n = 1), G161R (n = 1)) and in IDH2 (R140W (n = 1), R140Q (n = 1)). In 17/21 pts (81%) cIDH-mut MAF ≥ 10%. Of 21 cIDH-mutant pts, there were IDH co-muts in 11 pts (52%). Of these 11 pts, IDH co-muts were scIDH in 91%: #2 (IDH1 R100Q/G161R), #3 (IDH1 R132H/R119Q/G161R), #4 (IDH1 R132H/R132C; IDH2 R172G/R140Q), #11 (IDH1 R132H/R132C), #21 (IDH1 R132H/G161R; IDH2 R140Q), #23 (IDH1 R132H/R132C), #31 (IDH1 R132H/G161R; IDH2 R140Q/P162L), #32 (IDH1 R132H/R100Q), #33 (IDH1 R132H/R100Afs*29/I130V), #37 (IDH1 R132H; IDH2 R159H), #40 (IDH1 R132H; IDH2 R172G). Median overall survival (OS) longer in cIDH-mut vs IDH-wt (159 vs 16 months, P = 0.000). No difference in OS in: cIDH vs cIDH + scIDH (NR vs 67 m, P = 0.437), in scIDH vs cIDH (79 vs 159 m, P = 0.111), and scIDH vs IDH-wt (79 vs 12 m, P = 0.229). Conclusions: Subclonal (defined as MAF < 1%) canonical and non-canonical IDH-mutations and co-mutations occur frequently in gliomas. This may have implications for the design of future clinical trials and in the development of IDH inhibitors including the anticipation of potential resistance mechanisms.

Low expression of Ki-67/MIB-1 labeling index in IDH wild type glioblastoma predicts prolonged survival independently by MGMT methylation status

PurposeThe Ki-67/MIB-1 labeling index (LI) is clinically used to differentiate between high and low-grade gliomas, while its prognostic value remains questionable. Glioblastoma (GBM) expressing wild-type isocitrate dehydrogenase IDHwt, a relatively common malignant brain tumor in adults, is characterized by a dismal prognosis. Herein, we have retrospectively investigated the prognostic role of Ki-67/MIB-1-LI in a large group of IDHwt GBM.MethodsOne hundred nineteen IDHwt GBM patients treated with surgery followed by Stupp’s protocol in our Institution between January 2016 and December 2021 were selected. A cut-off value for Ki-67/MIB-1-LI was used with minimal p-value based approach.ResultsA multivariate analysis showed that Ki-67/MIB-1-LI expression < 15% significantly correlated with a longer overall survival (OS), independently from the age of the patients, Karnofsky performance status scale, extent of surgery and O6-methylguanine (O6-MeG)-DNA methyltransferase promoter methylation status.ConclusionsAmong other studies focused on Ki-67/MIB-1-LI, this is the first observational study showing a positive correlation between OS of IDHwt GBM patients and Ki-67/MIB-1-LI that we propose as a new predictive marker in this subtype of GBM.

Risk Estimation in Non-Enhancing Glioma: Introducing a Clinical Score.

The preoperative grading of non-enhancing glioma (NEG) remains challenging. Herein, we analyzed clinical and magnetic resonance imaging (MRI) features to predict malignancy in NEG according to the 2021 WHO classification and developed a clinical score, facilitating risk estimation. A discovery cohort (2012-2017, n = 72) was analyzed for MRI and clinical features (T2/FLAIR mismatch sign, subventricular zone (SVZ) involvement, tumor volume, growth rate, age, Pignatti score, and symptoms). Despite a "low-grade" appearance on MRI, 81% of patients were classified as WHO grade 3 or 4. Malignancy was then stratified by: (1) WHO grade (WHO grade 2 vs. WHO grade 3 + 4) and (2) molecular criteria (IDHmut WHO grade 2 + 3 vs. IDHwt glioblastoma + IDHmut astrocytoma WHO grade 4). Age, Pignatti score, SVZ involvement, and T2/FLAIR mismatch sign predicted malignancy only when considering molecular criteria, including IDH mutation and CDKN2A/B deletion status. A multivariate regression confirmed age and T2/FLAIR mismatch sign as independent predictors (p = 0.0009; p = 0.011). A "risk estimation in non-enhancing glioma" (RENEG) score was derived and tested in a validation cohort (2018-2019, n = 40), yielding a higher predictive value than the Pignatti score or the T2/FLAIR mismatch sign (AUC of receiver operating characteristics = 0.89). The prevalence of malignant glioma was high in this series of NEGs, supporting an upfront diagnosis and treatment approach. A clinical score with robust test performance was developed that identifies patients at risk for malignancy.

Frequency and Prognostic Relevance of Volumetric MRI Changes in Contrast- and Non-Contrast-Enhancing Tumor Compartments between Surgery and Radiotherapy of IDHwt Glioblastoma

In newly diagnosed IDH-wildtype glioblastoma, the frequency and prognostic relevance of tumor regrowth between resection and the initiation of adjuvant radiochemotherapy are unclear. In this retrospective single-center study we included 64 consecutive cases, for whom magnetic resonance imaging (MRI) was available for both the volumetric assessment of the extent of resection immediately after surgery as well as the volumetric target delineation before the initiation of adjuvant radiochemotherapy (time interval: 15.5 ± 1.9 days). Overall, a median new contrast-enhancement volume was seen in 21/64 individuals (33%, 1.5 ± 1.5 cm3), and new non-contrast lesion volume in 18/64 patients (28%, 5.0 ± 2.3 cm3). A multidisciplinary in-depth review revealed that new contrast-enhancement was either due to (I) the progression of contrast-enhancing tumor remnants in 6/21 patients or (II) distant contrast-enhancing foci or breakdown of the blood-brain barrier in previously non-contrast-enhancing tumor remnants in 5/21 patients, whereas it was unspecific or due to ischemia in 10/21 patients. For non-contrast-enhancing lesions, three of eighteen had progression of non-contrast-enhancing tumor remnants and fifteen of eighteen had unspecific changes or changes due to ischemia. There was no significant association between findings consistent with tumor regrowth and a less favorable outcome (overall survival: 14 vs. 19 months; p = 0.423). These findings support the rationale that analysis of the postsurgical remaining tumor-volume for prognostic stratification should be carried out on immediate postoperative MRI (<72 h), as unspecific changes are common. However, tumor regrowth including distant foci may occur in a subset of IDH-wildtype glioblastoma patients diagnosed per WHO 2021 classification. Thus, MRI imaging prior to radiotherapy should be obtained to adjust radiotherapy planning accordingly.

Differential P-Glycoprotein/CD31 Expression as Markers of Vascular Co-Option in Primary Central Nervous System Tumors.

Vascular co-option is one of the main features of brain tumor progression. It is identified using histopathological analysis, but no antibody-specific markers were found, and no universally accepted histological features were defined. We employed double immunohistochemical stainings for CD31, P-gp, S100A10, and mitochondria on formalin-fixed, paraffin-embedded human samples of IDH-WT glioblastoma, IDH-mutant astrocytoma, and meningioma to study vascular co-option across different brain tumors and across normal, peritumoral, and intratumoral areas using the Aperio colocalization algorithm, which is a valid and robust method to handle and investigate large data sets. The results have shown that (i) co-opted vessels could be recognized by the presence of metabolically overactive (evaluated as mitochondria expression) and P-gp+ or S100A10+ tumor cells surrounding CD31+ endothelial cells; (ii) vascular co-option occurs in the intratumoral area of meningioma and astrocytoma; and (iii) vascular co-option is prevalent in peritumoral glioblastoma area. The described approach identifies new markers for cellular components of the vessel wall and techniques that uncover the order and localization of vascularization mechanisms, which may contribute to developing new and possibly more effective therapeutic strategies.

DDDR-24. INTEGRATED ANALYSIS OF SINGLE CELL CHROMATIN ACCESSIBILITY AND RNA EXPRESSION IDENTIFIED COMMON VULNERABILITY DESPITE GLIOBLASTOMA HETEROGENEITY

Abstract INTRODUCTION In 2021, the World Health Organization (WHO) reclassified glioblastoma, the most common form of adult brain cancer, into isocitrate dehydrogenase (IDH) wild-type glioblastomas and grade IV IDH mutant (G4 IDHm) astrocytomas. For both tumor types, intra-tumoral heterogeneity is a key contributor to therapeutic failure. METHODS we applied integrated genome-wide chromatin accessibility (snATACseq) and transcription (snRNAseq) profiles to clinical specimens derived IDHwt glioblastomas and G4 IDHm) astrocytomas, with goal of therapeutic target discovery. RESULTS The integrated analysis achieved resolution of intra-tumoral heterogeneity not previously possible, providing a molecular landscape of extensive regional and cellular variability. snATACseq delineated focal amplification down to an ~40 KB resolution. The snRNA analysis elucidated distinct cell types and cell states (neural progenitor/oligodendrocyte cell-like or astrocyte/mesenchymal cell-like) that were superimposable onto the snATACseq landscape. Paired-seq (parallel snATACseq and snRNAseq using the same clinical sample) provided high resolution delineation of extrachromosomal circular DNA (ecDNA), harboring oncogenes including CCND1 and EGFR. Importantly, the copy number of ecDNA genes correlated closely with the level of RNA expression. Integrated analysis across all specimens profiled suggests that IDHm grade 4 astrocytoma and IDHwt glioblastoma cells shared a common chromatin structure defined by open regions enriched for Nuclear Factor 1 transcription factors (NFIA and NFIB). Silencing of NF1A or NF1B suppressed in vitro and in vivo growth of patient-derived IDHwt glioblastomas and G4 IDHm astrocytoma models that mimic distinct glioblastoma cell states. CONCLUSION Our findings suggest despite distinct genotypes and cell states, glablastoma/G4 astrocytoma cells share dependency on core transcriptional programs, yielding an attractive platform for addressing therapeutic challenges associated with intra-tumoral heterogeneity.

BIOM-43. THE GENOMIC, TRANSCRIPTOMIC, AND EPIGENOMIC LANDSCAPE OF ISOCITRATE DEHYDROGENASE WILD TYPE GLIOBLASTOMA ACROSS THE AGE CONTINUUM

Abstract BACKGROUND Older age is a poor prognostic factor for glioblastoma (GBM) patients. We tested whether the intrinsic molecular landscape of the tumor may contribute to this poor prognosis. METHODS In accordance with the 2021 WHO classification scheme, we included only isocitrate dehydrogenase (IDH) wild type GBM. Based on published literature, we defined older as age &amp;gt; 65. RNA expression, gene amplification, tumor mutational burden (TMB) and mutational profiles were analyzed in three unique datasets: Tempus (n = 1,410), Caris (n = 1,432), and TCGA (n = 557). Comparison were made between &amp;lt; 65 and ³ 65 year olds using Pearson’s Chi-squared tests, Fisher’s exact tests, or Wilcoxon rank-sum where appropriate. RESULTS From our evaluable gene sets, TERT promoter mutations were more prevalent in patients ³ 65 years old (Caris 82.64 vs 77.27%, p = 0.016; Tempus 58.0 vs 49.0%, p = 0.002). There were no significant differences in PDCD1, CD274, CD3E, TNFRSF18, CD40, CD8A, TNFRSF4, CTLA4, HAVCR2, TNFSF9, CD274, or CDKN2A; PDL-1 (by IHC); dMMR/MSI-H, TMB; CDK6 amplification, EGFR amplification, EGFR, EGFRvIII, EGFR fusions, MET fusions, PTEN, TP53, or NF-1. MGMT promoter methylation (Caris data) was more common in the older group (49.73 v 34.14%, p &amp;lt; 0.001). TGCA data demonstrated that gene expression, TMB, and methylation did not change significantly with age. Additionally, PCOLCE2 and SLC10A4 were differentially methylated, and missense mutations, of any type, were more common in the older group (p=0.006). CONCLUSION Despite worse survival outcomes for older patients with IDHwt GBM as compared to younger counterparts, the molecular landscape is similar at the genomic, transcriptomic and epigenomic levels. The key exception is TERT promoter mutations that are more common in older GBM patients. Poorer survival is therefore not likely to be attributable solely to intratumoral factors.

IMMU-20. MODULATION OF THE PERIPHERAL T CELL BIOLOGY IN GLIOBLASTOMA

Abstract Cancer is a systemic disease. Due to the exceedingly rare occurrence of metastasis of cerebral glioma, systemic alterations have, however, not been considered to play a major role in disease progression of glioma. T cells orchestrate the adaptive immune response in an antigen-specific, cytokine mediated manner. The aim of our study was to investigate how cerebral glioma impacts systemic T cell immunobiology. We performed gene expression profiling of peripheral blood T cells in patients with IDHwt glioblastomas as well as in a murine glioma model. In addition, we analyzed the levels of soluble immune biomarkers in patient blood and performed flow-cytometric phenotyping of human peripheral blood CD3+ T cells. We discovered a significant skewing of peripheral T cell phenotypes in IDHwt glioblastoma patients compared to healthy donors (HD), showing CD4+ TH1 expansion and reduced numbers of T follicular helper cells (TFH), TH1* and mucosa associated invariant T (MAIT) cells, while TH2 and TH17 percentages remained stable. Interestingly, peripheral memory CD4+ T cells exhibited reduced Fas and PD-1 expression, while CD8 T cells were primarily affected in the non-memory CD45RA+ compartment, displaying reduced numbers of CD8+ CD127+ CD27+ cells. Compared to healthy individuals, GBM patients had significantly increased levels of soluble CD27 while levels of soluble CD25 were reduced (p &amp;lt; 0.05). GSEA and ORA analysis of differentially expressed genes in murine gliomas showed alterations in RNA binding and processing, as well as ribosomal activity in both cell types, indicating systemic modulation in translational- and cell cycle pathways in glioblastoma. Taken together, our results demonstrate a significant skewing of the peripheral T immunobiology in patients with IDHwt gliomas. Our data highlights the importance of considering malignant glioma as a systemic disease that fundamentally alters the immune repertoire in affected patients.

SURG-42. MOLECULAR PROFILE CONSENSUS CLUSTERING DEFINES CLINICALLY DISTINCT GROUPS IN IDH WILD-TYPE GLIOBLASTOMA

Abstract OBJECTIVES Although the impact of surgery in glioblastoma (GBM) is well-established, there is insufficient data to determine how surgical resection affects overall survival (OS). The impact of other GBM genomic alterations on EOR-induced survival remains poorly understood. Here, we leverage a large cohort of GBM patients with genomic data to establish the impact of genomic alterations on resection-dependent overall survival in GBM. METHODS 537 IDH WT GBMs from UPMC were analyzed using Glioseq next generation sequencing panel. Using molecular profiles derived from Glioseq data, semi-supervised Monte Carlo reference-based consensus clustering was used to define 5 distinct groups. Cox Proportional Hazards models were used to identify predictors of OS within molecular groups. RESULTS Clustering of 537 IDH wild-type GBM identified 5 distinct groups: 1: TERT mutated/MGMT methylated/EGFR mutated or gain; 2: TERT mutated /MGMT methylated/p53 mutated; 3: TERT WT; 4: TERT mutated/MGMT methylated/CDKN2A loss; 5: TERT mutated/MGMT unmethylated. Thresholds in postoperative residual contrast enhancing volume (rCEV) and residual non-contrast enhancing volume (rNCEV) were identified which marked significant differences in median overall survival (MOS) between molecular groups in rCEV -Group 1: 11.7cc, Group 2: 14.4cc, Group 3: 1.1cc, Group 4: 0.1cc, and Group 5 1.1cc. Sex, age, KPS &amp;gt;70, temozolomide, and radiation along with thresholded rCEV and rNCEV were used in multivariate Cox analyses. Groups demonstrated distinct predictors of OS: radiation only in Group 1 tumors, low rCEV in Group 2 tumors, younger age, temodar, low CEV, and KPS &amp;gt;70 in Group 3 tumors, female sex, younger age, radiation, KPS &amp;gt;70 low rCEV in Group 4 tumors, and KPS &amp;gt;70, temodar, and low rCEV in Group 5 tumors. CONCLUSIONS Clustering results suggest that there are clinically distinct molecular groups of IDH wild-type glioblastomas. Age, sex, KPS &amp;gt;70, temozolomide treatment, radiation therapy, and EOR have varying affects on these molecular groups.

CNSC-07. MECHANISMS OF GLIOBLASTOMA-INDUCED CORTICAL REMODELING IDENTIFY THERAPEUTIC VULNERABILITIES

Abstract Prior work demonstrated synaptic integration of malignant gliomas into neural circuits induces local hyperexcitability and tumor proliferation. However, prognostication and therapeutic vulnerabilities are lacking from preclinical models. Here, we integrate in vivo and in vitro neurophysiology spatially matched with gene expression programs and protein signaling mechanisms across 66 IDH WT glioblastoma patients to identify thrombospondin-1 (TSP-1) as a molecular driver of glioma-induced network remodeling. Bulk and single cell RNA-sequencing of 11 intratumoral regions maintaining functional connectivity (13,730 cells analyzed) revealed a distinct neurogenic signature enriched for the synaptogenic factor TSP-1. Mechanistic and functional studies validating therapeutic vulnerabilities to TSP-1 silencing by shRNA knockdown and FDA-approved inhibitors (Gabapentin and LSKL) of excitatory synapse formation through the gabapentin a2d-1 receptor was performed in vitro and in vivo. Glioma-neuron co-culture of TSP-1 overexpressing cells demonstrated increased Ki67 proliferation and tumor microtube (TMT) formation when cultured in the presence of neurons. Pharmacological inhibition of TSP-1 using gabapentin or TSP-1 shRNA inhibited the proliferation and TMT-mediated expansion. Hippocampal xenografted mice with TSP-1 over expressing primary patient cultures demonstrated shorter survival and gabapentin treatment of xenografted mice significantly reduced the proliferation of TSP-1 overexpressing cells in vivo. Electrophysiological properties of glioma-neuron co-cultures analyzed using multi-electrode array (MEA) demonstrated increased neuronal spiking activity and network burst synchrony in the presence of TSP-1 over expressing cells. Strikingly, these increases were eliminated in the presence of gabapentin. We modeled survival risk in patients incorporating the effects of glioma intrinsic neuronal activity, molecular, therapeutic, and clinical factors on overall survival by recursive partitioning. Three risk groups were identified based on tumor intrinsic neuronal activity, pre- and post-operative tumor volume with shortest overall survival in patients with glioma intrinsic neuronal activity. These data identify glioma-induced secretion of TSP-1 as a key contributor of tumor proliferation shedding light on new therapies.

TMIC-27. SPATIALLY RESOLVED T CELL RECEPTOR SEQUENCING (SPTCR-SEQ) UNCOVERS REGIONAL DIVERSITY OF ANTI-TUMOR IMMUNE RESPONSES

Abstract Regional diversity of anti-tumor immunity in glioblastoma (GBM) has been poorly investigated due to technical limitations. To overcome these hurdles, we developed SPTCR-seq (SPatial TCell Receptor sequencing) to spatially profile T cell immune response. Here, we performed 10X-Visium spatial transcriptomics and SPTCR-seq on 9 de-novo IDH wt glioblastoma. For SPTCR-seq, we captured enriched T-cell receptor (TCR) sequences by hybridization followed by Oxford nanopore (ONT) long-read sequencing. We established a computational pipeline for spatial demultiplexing, error correction and TCR reconstruction using a Seq2Vector model, followed by a long-term memory autoencoder to deconvolute and compare CDR3 sequence similarities across patients. The on-target rate for detected TCR genes reached over 85%, of which our long-TCR algorithm could completely reconstruct TCRs in over 70%. To decipher the regional diversity of T-cell clonality, we performed spatially weighted regression to estimate the overlap of T-cell clonal expansion to the recently described regional transcriptional niches. We found that myeloid cell infiltration was highly correlated with increased T cell diversity. We integrated single cell RNA-seq data and annotated T cell subtypes through the “natural barcode” of the CDR3 sequence, revealing that CD4+ T cells interact with myeloid cells via MHCII in segregated areas, also described as the Reactive Immune niche. In contrast, CD8+ T cells exhibit a dysfunctional/exhausted phenotype at the same location. In comparison to the myeloid-infiltrated regions, the hypoxia-associated niches showed increased CD8+ T cells with greater clonal expansion, suggesting an immune response anti-cancer immunity. Global spatial vector field analysis revealed a spatial co-occurrence of metabolic vulnerability and a mutational burden with CD8+ T effector cells negatively associated with myeloid cell infiltration. Integration of CDR3 sequences in different patients revealed higher similarity of motifs of CD4+ compared with CD8+ T cells in hypoxia areas. Our data provide new insights into the regional heterogeneity of cancer immunity.

MODL-16. SOMATICALLY ENGINEERED MOUSE MODELS RECAPITULATING CELLULAR AND MOLECULAR FEATURES OF HUMAN GBM

Abstract INTRODUCTION Mouse models are instrumental in advancing our understanding of glioblastoma (GBM), however many commonly used models have features that limit their practicality or applicability. These limitations can include an incomplete immune context, a requirement for complex breeding strategies, high cost of specialized lines, and unpredictability in where tumors arise. We sought to develop and validate a mouse modeling system for some of the major human GBM subtypes that overcame these practical challenges. METHODS In vivo electroporation was used to introduce genetic alterations into periventricular cells of early postnatal C57Bl6/j mice. PiggyBac transposon/transposase and CRISPR-Cas9 systems were used to overexpress (OE) or knock out (KO) genes associated with mesenchymal (Nf1-KO/Pten-KO/p53-KO), classical (EGFRvIII-OE/Cdkn2a-KO/Pten-KO), and proneural (Pdgfra-OE/Cdk4-OE/p53-KO) IDH-WT GBM subtypes. KO or OE was confirmed by Indel Detection by Amplicon Analysis and immunofluorescent staining (IF). Tumours were analyzed for histologic, immunohistochemical, and transcriptional (RNAseq) features. RESULTS Tumors arose with near complete penetrance and median survivals of 36 to 116 days amongst models. All tumors were GFAP-expressing high-grade gliomas, with distinct phenotypes associated with different genetic combinations. Nf1-KO/Pten-KO/p53-KO tumors were composed of spindle cells. EGFRvIII-OE/Cdkn2a-KO/Pten-KO and Pdgfra-OE/Cdk4-OE/p53-KO tumors showed prevalence of small, round cells. Molecularly, Nf1-KO/Pten-KO/p53-KO tumors were enriched for human mesenchymal signature and displayed more differentiated phenotype. The Nf1-KO/Pten-KO/p53-KO model also showed increased stromal cells and macrophages. The other models had mixed proneural and classical profiles and were enriched for OPC- and NPC-like gene signatures found in human GBM. Once tumors formed, cells from each model were transplantable into C57Bl6/j mice to generate subsequent tumors. CONCLUSION We developed and validated a rapid, versatile, and reproducible system to model GBMs. These models allow for controlled study of GBM pathogenesis, progression, and treatment response, and allow for robust generation of syngeneic implantable mouse models that can serve as valuable tools for preclinical testing.

TMIC-10. IDENTIFICATION, VALIDATION AND BIOLOGICAL CHARACTERIZATION OF NOVEL GLIOBLASTOMA TUMOUR MICROENVIRONMENT SUBTYPES: IMPLICATIONS FOR PRECISION IMMUNOTHERAPY

Abstract New precision medicine therapies are urgently required for glioblastoma (GBM). However, to date, efforts to subtype patients based on molecular profiles, have failed to direct treatment strategies. We hypothesized that interrogation of the GBM tumor microenvironment (TME) and identification of novel TME-specific subtypes could inform new precision treatment strategies. To this end, a refined and validated microenvironment cell population (MCP)-counter method was applied to &amp;gt; 800 GBM patient tumours and validated by multiplex-immunohistochemistry. The MCP-counter deconvolution method interrogates the TME composition from transcriptomic data. Using this refined method, we classified the GLIOTRAIN(www.gliotrain.eu) IDHwt GBM cohort (n=123) into 3 novel clusters characterised by differences in TME composition and subsequently validated findings in the TCGA (n=69), CGGA (n=72) and DUKE (unpublished)(n=162) cohorts. TMEHigh tumours (30%) displayed elevated immune populations, functional orientation markers, immune checkpoint genes, and upregulated immunoregulatory pathways. Moreover, tertiary lymphoid structures were a feature of TMEHigh/mesenchymal+ patients. TMEMed (46%) tumours displayed heterogeneous immune populations and upregulated neuronal signalling pathways. TMELow (24%) tumours represented an ‘immune-desert’ group, high EGFR mutation frequency and upregulated EGFR signalling pathways. Longitudinal analysis of the GLASS cohort revealed TME-subtype transitions upon recurrence, influenced by TME composition changes. Finally, assessment of three GBM immunotherapy clinical trial cohorts revealed that TMEHigh patients treated with neo-adjuvant anti-PD1 have a significantly improved survival (P=0.04). Moreover, TMEHigh patients treated with anti-PD1 and an oncolytic virus (PVSRIPO) in the adjuvant setting, showed a trend towards improved survival (P=0.15 and P=0.056 respectively). Overall, we have established a novel TME-based classification system for application in intracranial malignancies. This system may be used to better inform a precision targeting approach in the brain tumour setting. For example, we hypothesise that patients bearing TMELow tumours may be amenable to neoadjuvant anti-TIM3 + EGFR inhibitor, TMEMed to anti-angiogenic immunotherapy, and TMEHigh patients to neoadjuvant anti-PD1 + anti-CTLA4.

NIMG-101. COMPARISON OF PRE-SURGICAL MRI FINDINGS FROM LONG-TERM (≥ 3 YRS) VERSUS SHORT-TERM (&amp;lt; 3 YRS) GLIOBLASTOMA SURVIVORS: A BLINDED, CASE-CONTROL REVIEW

Abstract BACKGROUND The median overall survival of patients with glioblastoma (GBM) is 10-12 months and the five-year survival ranges from 5-10%. Favorable clinical and molecular prognostic markers have been described in the literature, although an individual patient’s prognosis cannot easily be predetermined on these features as shown in the NOB-LTS project by Briceno et al. (SNO 2021). Until now, no radiologic characteristics have been correlated with better patient outcomes. We reviewed eighteen MRI features to determine if these could aid the identification of long-term survivors (LTS). METHODS From the NOB-LTS cohort of our Natural History Study, 16 long-term survivals (LTS; ≥ 3 years survival post-diagnosis) were matched based on sex, age, and extent of resection to 32 control (STS, &amp;lt; 3 years survival post-diagnosis) patients with NGS-confirmed IDH-wt glioblastoma. Pre-surgical MRIs (T1, T1 post-contrast, T2, T2/FLAIR, DWI, ADC) were reviewed by three blinded reviewers and analyzed based on pre-set criteria. RESULTS T1 hypointensity occurred commonly in the LTS (71%) vs STS (29%) while central heterogeneous signal occurred commonly in the STS (73%) vs LTS (50%) patients. Restricted diffusion was seen in 82% of STS and 70% of LTS cases. No difference was seen in irregular enhancement, peripheral and grey matter enhancement on T1 post-contrast images. Evaluation of T2/FLAIR images showed no difference in the number of lobes involved, presence of mass effect, heterogeneity of the signal or the perilesional signal abnormality or involvement of grey matter. CONCLUSION Our results suggest that pre-surgical MRI might be a useful prognostic tool as T1 hypointensity more frequently, and T1 central heterogeneity less commonly seen in the LTS group. These imaging findings may provide additional insights regarding the differences in tumor biology between LTS and STS. Further validation and correlation with histological/molecular characteristics are planned to better predict patient outcomes with newly diagnosed glioblastoma.

CTNI-07. LOMUSTINE/TEMOZOLOMIDE CHEMOTHERAPY FOR NEWLY DIAGNOSED MGMT-METHYLATED IDHWT GLIOBLASTOMA ACCORDING TO CETEG/NOA-09: REAL-WORLD EXPERIENCE IN A MULTICENTER COHORT

Abstract INTRODUCTION The CeTeG/NOA-09 trial demonstrated superior median overall survival (mOS, 48.1 months) in MGMT-methylated glioblastoma treated with lomustine/temozolomide compared to temozolomide. We retrospectively analyzed an off-study cohort of patients treated with lomustine/temozolomide to gather real-world data on this new regimen. METHODS Adult patients from 20 centers in Germany, Austria and Switzerland were included. Inclusion criteria were MGMT-methylated IDHwt glioblastoma newly diagnosed prior to end of 2020, and lomustine/temozolomide treatment as part of first-line therapy. RESULTS 321 patients with a median age of 57 years (range, 21-78) and a median follow-up of 19.9 months were included. In the whole cohort, mOS was 41.0 months (95%CI, 33.0 – not reached). In patients starting lomustine/temozolomide immediately upon initiation of radiotherapy strictly following the CeTeG protocol (88%), mOS was 52.8 months (35.8 – not reached) as compared to 24.6 months (17.6 – not reached) in patients starting lomustine/temozolomide after completion of radiotherapy/concomitant temozolomide (12%, logrank test: p = 0.06). Patients with a KPS &amp;lt; 80 had a shorter mOS of 19.7 months (95%CI, 16.6 – not reached) compared to 41.0 months (33.0 – not reached, p = 0.009) in KPS 80-100. Gross total resection (GTR, 53.9%) was associated with longer mOS (52.8 months, 95%CI 24.1 – not reached) compared to partial resection/biopsy (30.5 months, 95%CI 36.8 – not reached, p=0.004). Multivariable Cox regression analysis confirmed GTR (HR 0.66, p = 0.033) and younger age ( ≤ 50 years: HR 0.42, p = 0.001), but not KPS (80-100 vs. lower: HR 0.66, p = 0.12) as independent prognostic factors. DISCUSSION In this real-world multicenter cohort, survival was similar to the promising results of CeTeG/NOA-09. Further analyses should investigate a potentially reduced benefit from lomustine/temozolomide in patients with low KPS/no GTR and a possible detrimental effect from deferred lomustine/temozolomide initiation. The median follow-up is admittedly short, updated data will be presented.