IDH-mutant Gliomas Research Articles

P08.08.A EXPLORATION OF SURVIVORSHIP ISSUES AND QUALITY OF LIFE IN A COHORT OF IDH MUTATED HIGH GRADE GLIOMA 5 YEARS FOLLOWING DEFINITIVE TREATMENT

Abstract BACKGROUND Approximately 2000 people are diagnosed with brain cancer in Australia each year. Compared to glioblastoma patients, the IDH-mutated high-grade glioma (IDHmHGG) is a more favourable diagnosis; better prognosis and survival outcomes. Coupled with improved treatments, the IDHmHGG cohort is living longer which means treatment side effects (eg. fatigue, cognitive deficits, physical limitations etc.) can significantly impact their quality of life in the survivorship years. Hence there is a need to identify and determine the survivorship issues most pertinent for the IDHmHGG cohort. MATERIAL AND METHODS A mixed- METHODS cross-sectional study was conducted to investigate the relationship between socio-demographic, clinical factors and quality of life in the IDHmHGG cohort. Qualitative data collection consisted of a neuro-oncology clinician focus group (n=11) regarding survivorship issues and was contrasted with semi-structured patient interviews (n=27). Quantitative data collection (n=41) involved socio-demographic questions, the Functional Assessment of Cancer Therapy - Brain (FACT-Br) tool and The Hospital Anxiety and Depression Scale (HADS). RESULTS The results indicate that marital status, employment status, and treatment complications are strongly associated with quality of life in IDHmHGG patients. Being single and unemployed is associated with lower quality of life (p<0.05). Similarly, our findings show that psychological distress, specifically the presence of anxiety and depressive symptoms, is a major factor that can negatively impact the QoL in an IDHmHGG cohort. The order of ranking between clinician and patient identified issues only matched for physical limitations (ranked 3rd). Although not matched in ranking, both groups identified cognitive function, employment, social function, isolation and mental health as commonly experienced in the survivorship years. CONCLUSION This study highlights the importance of considering socio-demographic and clinical factors in the care and support of brain cancer patients. The most important finding of this study was that being single and unemployed negatively predicted QoL. Additionally, it is notable that our results show a discrepancy between clinician identified issues and patient identified issues. This finding suggests that in order to deliver patient-centred and meaningful care, there is a need to incorporate a more patient focused perspective. Future survivorship models of care for the IDHmHGG cohort should be co-designed with patients.

P12.05.B THE ROLE OF ONCO-METABOLITE IN THE IDH MUTANT GLIOBLASTOMA MICROENVIRONMENT

Abstract BACKGROUND Recently, we found that the reactive astrocytes in the IDH wild type glioblastoma contribute to anti-tumor immunity and support pro-oncogenic signaling. Here, we characterized the transcriptomic signature of IDH1/2-mutant glioma associated astrocytes and determined a unique inflammatory transformation, profoundly different to astrocytes in IDH wildtype glioma patients due to an onco-metabolite R-2-hydroxyglutarate using Next generation sequencing and human ex-vivo slice model. MATERIAL AND METHODS We purified and transcriptionally profiled astrocytes from 9 patients with confirmed IDH1-R132H mutation, by means of RNA-sequencing and the data were analyzed using the established pipelines. We also used spatial transcriptomics to evidently show the spatial distribution of astrocytes in IDH-mutated/wildtype glioma samples. We validated our findings using human organotypic slice model inoculated with IDH-mutant cell line or treated with oncometabolite 2-hydroxy glutarate. Additionally, LC-MS was further used to give us a chart of neurotransmitters due to altered microenvironment. RESULTS Our results from RNA sequencing showed a transcriptional transformation of reactive astrocytes within the microenvironment of IDH-mutated tumors compared to wildtype glioma by means of RNA-sequencing of purified astrocytes. And, using our established human neocortical GBM model inoculated with IDH mutant tumor and R-2HG treatment, we showed that we were able to activate inflammatory transcriptional programs in astrocytes, mediated by the presence of microglia. Further, by spatially mapping the transcriptomic profiles of purified microglia, we were able to confirm that microglia also demonstrate inflammatory activation in IDH mutated glioma. This inflammatory astrocyte transformation is associated with a loss of neurotransmitter homeostasis (disrupted levels of glutamate) in the treated sections, as has been previously reported in IDH mutated tumors. Additionally, R-H2G increased neuronal spiking rate in, pointing to potential excitotoxicity. CONCLUSION Our work provides a crucial contribution towards understanding the role of R-2HG in the IDH mutant glioma microenvironment and sheds light on the significant microenvironmental differences to IDH wild-type glioma.

P19.06.B T2/FLAIR MISMATCH, SPECIFIC RADIOGENOMIC MARKER FOR A DIFFUSE LOW GRADE ASTROCYTOMA

Abstract A 36-year-old woman presented with recurring episodes of generalized tonic clonic seizures. An MRI from an outside institution demonstrated an intra-axial brain mass suspicious for a primary brain tumor. Repeat conventional and advanced MRI imaging revealed a primary brain mass centred on the left postcentral gyrus involving cortex and subcortical white matter. The lesion was homogenously hyperintense on T2-weighted imaging, and centrally iso-to hypointense on FLAIR with a rim of hyperintense signal on the FLAIR sequence. There was no significant peritumoral edema or minimal effacement of the central sulcus. No abnormal enhancement was seen post-contrast. The lesion demonstrated no reduced diffusivity on diffusion-weighted imaging. MR perfusion showed no elevation in regional cerebral blood volume or flow. Findings are most compatible with a low grade glioma. Patient underwent total surgical resection and biopsy for histopathology. Genetic sequencing revealed an IDH mutant, non 1P,19q codeleted grade II astrocytoma. The diagnosis of adult infiltrating gliomas is predicted using imaging, and confirmed by histopathology, and molecular diagnostic methods. Regardless of grade the 1st phase in glioma diagnosis and management is IDH status. A multidisciplinary approach to the diagnosis of low grade and high grade glioma including genetic and molecular analysis is essential, as different genetic and molecular subtypes have different management and prognostic implications. Conventional MRI still represents a mandatory and extremely important step in the diagnostic workflow of neuro oncological patients for preoperative diagnosis, treatment planning, and subsequent follow up. The T2/FLAIR mismatch sign describes the MRI appearance of an IDH-mutant astrocytoma that is non 1p19q codeleted, and it is considered a preoperative novel specific MRI marker for this type of diffuse astrocytoma. In this case, the MRI findings were used preoperatively as a noninvasive tool to diagnose the patient`s low grade glioma which was confirmed by subsequent histological and genetic analysis. TEACHING POINTS1. T2/FLAIR mismatch allows for pre-operative diagnosis of IDH-mutant astrocytomas with a high level of specificity.2. It is particularly helpful in distinguishing diffuse astrocytoma from an oligodendroglioma3. Conventional MR sequences can suggest the diagnosis of IDH mutant gliomas which can assist neurosurgeons in presurgical planning and can help neuro-oncologists with treatment planning including potential clinical trials.

P14.04.B CIRCULATING TUMOR CELLS AS A NON-INVASIVE GLIOBLASTOMA DIAGNOSTIC AND PROGNOSTIC TOOL

Abstract BACKGROUND Circulating tumor cells (CTCs) detection in the blood of glioblastoma (GBM) patients can facilitate the assessment of tumoral information without the need for invasive approaches. Our aim is to use GBM CTCs to obtain knowledge about the tumor and improve the current GBM diagnosis and prognosis. MATERIAL AND METHODS We collected primary, matched recurrent GBM and healthy controls blood samples. CTCs were enriched from whole blood controls with Parsortix Cell Separation System and analysed on DEPArray system. CTCs were recognized by immunofluorescence with GFAP.The CTCs have been counted for all the samples. Copy Number Aberrations (CNAs) analysis was performed on CTCs. To compare CTCs genetic background with the same patient’s primary and recurrence tissues, whole exome sequencing (WES) was realized on tumor tissues. Recurrence of GBM was diagnosed according to the RANO criteria +/-path proven second surgery +/- FET PET imaging proven. We assessed tumor volume of contrast enhanced (CE) (as a surrogate marker of BBB damage) and nCE at the time of the CTCs collection by seminautomatic segmentation on ITKsnap software. RESULTS We found CTCs in GBM patients newly diagnosed at the time of first surgery (86%) but also at recurrence (100 %). Cell counting demonstrated in both primary and recurrent GBM patients’ blood a statistically significantly higher number of CTCs compared to healthy controls (average of cells number 15.8 vs 2.3). Interestingly, recurrent GBM patients showed more CTCs than primary GBM patients (average 9 vs 22.7). CNAs analysis confirmed the tumor status of the selected CTCs. Some of GBM classical mutations (in HTERT promoter, BRAF, PTEN, PI3KCA and EGFR genes) have been searched both in the tumor tissues and in the CTCs. HTERT was found mutated in CTCs of one patient’s tumor carrying mutant HTERT and wild type IDH1/2. We also included three IDH1 mutant glioma patients and one BRAFV600E mutant GBM patient in order to track these in CTCs. We did not find BRAF mutations in CTCs isolated from one recurrent BRAFV600E GBM patient with a very low allele frequency in the recurrent tumor (<1%). CONCLUSION We found CTCs in GBM patients newly diagnosed and at recurrence. Several factors may affect CTCs release such as tumor volume, CE and the CTCs genetic BACKGROUND , which seems to be heterogeneous. CTCs detection by our methodology could be helpful in the clinical setting for diagnostic, prognostic and theranostic purposes and for patients monitoring. Prospective study is ongoing in order to assess CTCs variations on and after therapy. This study, first investigated the genetic heterogeneity of CTCs released in the blood flow from the parental tumor.

KS01.5. A LATE BREAKING ABSTRACT: LONG-TERM FOLLOW-UP OF PATIENTS WITH NEWLY DIAGNOSED IDH-MUTANT ASTROCYTOMA AFTER IDH1-VAC TREATMENT

Abstract BACKGROUND The Neurooncology Working Group of the German Cancer Society trial 16 (NOA16), a multicenter, single-arm, open-label, first-in-human phase I trial, met its primary endpoints in demonstrating safety and immunogenicity of a long peptide vaccine targeting IDH1(R132H) (IDH1-vac) added to standard of care in 32 patients with newly diagnosed World Health Organization (WHO) Grade 3 (n=21) and 4 (n=11) IDH1(R132H)+ astrocytomas (ClinicalTrials.gov identifier NCT02454634). MATERIAL AND METHODS Here, we report on the clinical and immunological long-term follow-up of NOA16. Data cut-off was May 2023. RESULTS Six-year progression-free and survival rates of patients in the safety data set were 0.47 (CI: 0.29 - 0.63) and 0.66 (CI: 0.46 - 0.79), respectively. Six-year progression-free and survival rates in the Grade 3 population were 0.52 (CI: 0.30 - 0.71) and 0.67 (CI: 0.43 - 0.83), respectively. Six-year progression-free and survival rates in the Grade 4 population were 0.55 (CI: 0.23 - 0.78) and 0.62 (CI: 0.28 - 0.84), respectively. Six-year progression-free and survival rates in patients with complete resection (n=17) were 0.82 (CI: 0.55 - 0.94) and 0.88 (CI: 0.61 - 0.97), respectively. Calculated via the Simon-Makuch method, patients in the immunogenicity dataset (n=30) with immune responses (IR) had six-year progression-free and survival rates of 0.46 (0.26 - 0.64) and 0.75 (CI: 0.55 - 0.87), respectively. Two patients without IR showed progressive disease (PD) within two years of first diagnosis and died within 3 years of first diagnosis. In one-year landmark analyses, best mutation-specific B cell response at the time point of last vaccination or later was associated with a favorable clinical course, suggesting a relevant but differential response to repetitive therapeutic vaccination regimes. CONCLUSION This data supports a randomized clinical trial of IDH-vac in patients with newly diagnosed IDH-mutant gliomas. A booster treatment regime should be considered as continuous source of IDH1R132H-reactive T cells.

P02.15.A PRONEURAL, CLASSICAL AND MESENCHYMAL PROTEIN SIGNATURES LACK PROGNOSTIC VALUE IN WHO 2021 ADULT TYPE DIFFUSE LOWER-GRADE GLIOMAS

Abstract BACKGROUND Mesenchymal transformation in glioblastomas is associated with therapy resistance and increased aggressiveness. In WHO 2021-defined adult-type diffuse gliomas of lower grade (dLGG), the transition between phenotypic states, has not been studied. Most reports correlating proneural, classical or mesenchymal signatures with outcome in dLGG were made prior to the WHO 2021 classification. Here, we set out to investigate if phenotype as well as the transition of phenotype predicted survival in a clinical cohort of WHO 2021 defined dLGGs. MATERIAL AND METHODS We used a TMA-based approach and five immunohistochemical markers (EGFR, p53, MERTK, CD44 and OLIG2) to examine 183 primary and 49 recurrent tumors from patients with previously diagnosed dLGG. Of the 49 recurrent tumors, nine tumors relapsed a second time, and one a third time. RESULTS In total, 71.0% of all tumors could be subtyped into proneural, classical or mesenchymal. Proneural was the predominant subtype among IDH-mut tumors (78.5%), while in IDH-wt tumors mesenchymal was more common (63.6%). A significant difference in survival was found between subtypes (proneural, classical and mesenchymal) in the total cohort (p<0.001), but not when molecularly stratified (IDH-mut: p=0.220, IDH-wt: p=0.623). Upon relapse, proneural signature was retained in 66.7% of the proneural IDH-mut dLGGs (n=21), whereas IDH-wt tumors (n=10) mainly retained or gained mesenchymal phenotype. There was no significant difference in survival between IDH-mut gliomas remaining proneural and those switching to mesenchymal signature (p=0.347). CONCLUSION Subtyping into proneural, classical and mesenchymal phenotypes by five immunohistochemical markers, was possible for the majority of tumors, but statistical significance between phenotypes and survival was lost after molecular stratification according to the WHO 2021 criteria. IDH-mut tumors mainly remained proneural at recurrence, while IDH-wt tumors mostly retained or gained mesenchymal phenotype. This phenotypic shift, which in glioblastoma is associated with increased aggressiveness, did not affect survival. Sample sizes were, however, too small to draw any firm conclusions on whether this negative finding is due to lack of statistical power or reflects a true biological difference from glioblastomas.

Non-Invasive Assessment of Isocitrate Dehydrogenase-Mutant Gliomas Using Optimized Proton Magnetic Resonance Spectroscopy on a Routine Clinical 3-Tesla MRI.

The isocitrate dehydrogenase (IDH) mutation has become one of the most important prognostic biomarkers in glioma management, indicating better treatment response and prognosis. IDH mutations confer neomorphic activity leading to the conversion of alpha-ketoglutarate (α-KG) to 2-hydroxyglutarate (2HG). The purpose of this study was to investigate the clinical potential of proton MR spectroscopy (1H-MRS) in identifying IDH-mutant gliomas by detecting characteristic resonances of 2HG and its complex interplay with other clinically relevant metabolites. Thirty-two patients with suspected infiltrative glioma underwent a single-voxel (SVS, n = 17) and/or single-slice-multivoxel (1H-MRSI, n = 15) proton MR spectroscopy (1H-MRS) sequence with an optimized echo-time (97 ms) on 3T-MRI. Spectroscopy data were analyzed using the linear combination (LC) model. Cramér-Rao lower bound (CRLB) values of <40% were considered acceptable for detecting 2HG and <20% for other metabolites. Immunohistochemical analyses for determining IDH mutational status were subsequently performed from resected tumor specimens and findings were compared with the results from spectral data. Mann-Whitney and chi-squared tests were performed to ascertain differences in metabolite levels between IDH-mutant and IDH-wild-type gliomas. Receiver operating characteristic (ROC) curve analyses were also performed. Data from eight cases were excluded due to poor spectral quality or non-tumor-related etiology, and final data analyses were performed from 24 cases. Of these cases, 9/12 (75%) were correctly identified as IDH-mutant or IDH-wildtype gliomas through SVS and 10/12 (83%) through 1H-MRSI with an overall concordance rate of 79% (19/24). The sensitivity, specificity, positive predictive value, and negative predictive value were 80%, 77%, 86%, and 70%, respectively. The metabolite 2HG was found to be significant in predicting IDH-mutant gliomas through the chi-squared test (p < 0.01). The IDH-mutant gliomas also had a significantly higher NAA/Cr ratio (1.20 ± 0.09 vs. 0.75 ± 0.12 p = 0.016) and lower Glx/Cr ratio (0.86 ± 0.078 vs. 1.88 ± 0.66; p = 0.029) than those with IDH wild-type gliomas. The areas under the ROC curves for NAA/Cr and Glx/Cr were 0.808 and 0.786, respectively. Noninvasive optimized 1H-MRS may be useful in predicting IDH mutational status and 2HG may serve as a valuable diagnostic and prognostic biomarker in patients with gliomas.

Oncological and functional outcomes support early resection of incidental IDH-mutated glioma.

The main objective was to assess the neuropsychological, epileptical, and oncological outcomes in a series of patients operated on for a IDH-mutated diffuse low-grade glioma (DLGG) of incidental discovery (iDLGG). We retrospectively reviewed a consecutive series of surgically treated adults with DLGG and selected cases incidentally discovered. Tumor volumes, growth rates, and extents of resection (EOR) were assessed by volumetric measures of fluid-attenuated inversion recovery magnetic resonance imaging. The data on oncological, functional, and epileptical results were retrieved from the patients' digital files. Among all patients with DLGG resected at our center between June 2011 and April 2022, we found eleven cases with an incidental discovery. Resection was supratotal, gross total, and subtotal in 45.5%, 26.4%, and 18.1% of cases, respectively. The rate of epileptic seizures after the surgery was 9.1%. There were 45.4% of patients that had tumor progressions and the overall mean time to tumor progression was 42months. After the surgery, 3 (27.3%) patients had mild neurocognitive deteriorations, which impeded the return to work in one patient (9.1%). There were no differences with previous series regarding clinical, radiological, and molecular characteristics. Similar results were also found for functional, surgical, epileptical, and oncological outcomes. Although the right approach for iDLGG is still a matter of debate, our data support the safety and effectiveness of early surgical resection. More studies are needed to firmly ground this early "preventive" surgery approach.

Decision system for extent of resection in WHO grade 3 gliomas: a Chinese Glioma Genome Atlas database analysis.

Extensive surgical resection has been found to be associated with longer survival in patients with gliomas, but the interactive prognostic value of molecular pathology of the surgical resection is unclear. This study evaluated the impact of molecular pathology and clinical characteristics on the surgical benefit in WHO grade 3 IDH-mutant gliomas. Clinical and pathological information of 246 patients with WHO grade 3 IDH-mutant gliomas were collected from the Chinese Glioma Genome Atlas database (2006-2020). The role of the extent of resection on overall survival, stratified by molecular pathology and clinical characteristics, was investigated. We then assessed prognostic factors using a univariate log-rank test and multivariate Cox proportional hazards model in the subgroups. The extent of resection was an independent prognostic factor in the entire cohort, even when adjusted for molecular pathology. Gross total resection was found to be associated with longer survival in all patients and in the astrocytoma group but not in the oligodendroglioma group. Compared with subtotal resections, gross total resections resulted in a longer survival time for astrocytoma patients aged ≤ 45 years. However, there was no survival benefit from total resection in patients with astrocytoma aged > 45 years. Extensive resection benefits only a proportion of patients with WHO grade 3 IDH-mutant gliomas. Younger patients with astrocytomas had survival benefits from extensive resection. In addition to clinical characteristics (especially age), molecular pathology impacted prognosis in patients with gliomas. Our findings provide guiding information to neurosurgeons while planning surgeries.

Neurochemical Differences between 1p/19q Codeleted and Noncodeleted IDH-mutant Gliomas by in Vivo MR Spectroscopy.

Background Noninvasive identification of glioma subtypes is important for optimizing treatment strategies. Purpose To compare the in vivo neurochemical profiles between isocitrate dehydrogenase (IDH) 1-mutant 1p/19q codeleted gliomas and their noncodeleted counterparts measured by MR spectroscopy at 3.0 T with a point-resolved spectroscopy (PRESS) sequence optimized for D-2-hydroxyglutarate (2HG) detection. Materials and Methods Adults with IDH1-mutant gliomas were retrospectively included for this study from two university hospitals (inclusion period: January 2015 to July 2016 and September 2019 to June 2021, respectively) based on availability of 1p/19q codeletion status and a PRESS acquisition optimized for 2HG detection (echo time, 97 msec) at 3.0 T before any treatment. Spectral analysis was performed using LCModel and a simulated basis set. Metabolite quantification was performed using the water signal as a reference and correcting for water and metabolite longitudinal and transverse relaxation time constants. Concentration ratios were computed using total creatine (tCr) and total choline. A two-tailed unpaired t test was used to compare metabolite concentrations obtained in codeleted versus noncodeleted gliomas, accounting for multiple comparisons. Results Thirty-one adults (mean age, 39 years ± 8 [SD]; 19 male) were included, and 19 metabolites were quantified. Cystathionine concentration was higher in codeleted (n = 13) than noncodeleted (n = 18) gliomas when quantification was performed using the water signal or tCr as references (2.33 mM ± 0.98 vs 0.93 mM ± 0.94, and 0.34 mM ± 0.14 vs 0.14 mM ± 0.14, respectively; both P < .001). The sensitivity and specificity of PRESS to detect codeletion by means of cystathionine quantification were 92% and 61%, respectively. Other metabolites did not show evidence of a difference between groups (P > .05). Conclusion Higher cystathionine levels were detected in IDH1-mutant 1p/19q codeleted gliomas than in their noncodeleted counterparts with use of a PRESS sequence optimized for 2HG detection. Of 19 metabolites quantified, only cystathionine showed evidence of a difference in concentration between groups. Clinical trial registry no. NCT01703962 © RSNA, 2023 See also the editorial by Lin in this issue.

Prognostic value of integrative genomic approaches for IDH-mutant gliomas.

Prognostic value of integrative genomic approaches for IDH-mutant gliomas.

Exploiting nanopore sequencing for characterization and grading of IDH-mutant gliomas.

The 2021 WHO Classification of Central Nervous System Tumors recommended evaluation of cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) deletion in addition to codeletion of 1p/19q to characterize IDH-mutant gliomas. Here, we demonstrated the use of a nanopore-based copy-number variation sequencing (nCNV-seq) approach to simultaneously identify deletions of CDKN2A/B and 1p/19q. The nCNV-seq approach was initially evaluated on three distinct glioma cell lines and then applied to 19 IDH-mutant gliomas (8 astrocytomas and 11 oligodendrogliomas) from patients. The whole-arm 1p/19q codeletion was detected in all oligodendrogliomas with high concordance among nCNV-seq, FISH, DNA methylation profiling, and whole-genome sequencing. For the CDKN2A/B deletion, nCNV-seq detected the loss in both astrocytoma and oligodendroglioma, with strong correlation with the CNV profiles derived from whole-genome sequencing (Pearson correlation (r) = 0.95, P < 2.2 × 10-16 to r = 0.99, P < 2.2 × 10-16 ) and methylome profiling. Furthermore, nCNV-seq can differentiate between homozygous and hemizygous deletions of CDKN2A/B. Taken together, nCNV-seq holds promise as a new, alternative approach for a rapid and simultaneous detection of the molecular signatures of IDH-mutant gliomas without capital expenditure for a sequencer.

NEIM-11 DEUTERIUM METABOLIC IMAGING (DMI) DETECTS A LARGER WARBURG EFFECT IN HIGH-GRADE BRAIN TUMORS AND IN IDH WILD TYPE GLIOMAS

Abstract BACKGROUND The Warburg Effect (WE) is a metabolic change in which tumors favor glycolysis over oxidative phosphorylation and has been linked to cancer aggressiveness. Mutations in the isocitrate dehydrogenase (IDH) genes in gliomas are associated with improved outcomes. We deployed a magnetic resonance (MR) spectroscopic imaging technique coined Deuterium Metabolic Imaging (DMI) to measure the WE in multiple brain tumor types. METHODS Twenty-one patients underwent DMI, acquired on a Bruker 4T MR scanner after oral administration of 0.75g/kg of deuterated glucose. The WE was defined as the ratio of deuterated lactate, representing glycolysis, over deuterated glutamate and glutamine, representing oxidative phosphorylation. We devised an interval scale from 0 to 2 for the WE RESULTS The Wilcoxon rank sum test was performed to detect differences between groups. RESULTS The WE measurements for the largest groups, by diagnosis, showed: glioblastoma (n=9, mean=1.67); astrocytoma, IDH-mutant, WHO grade 3 (n=2, mean=0.25); oligodendroglioma, WHO grade 3 (n=3, mean=0.5); low grade glioma, IDH-mutant (n=2, mean=0); and meningioma, WHO grade 2 (n=2, mean=0). The WE for high-grade tumors (n=15, mean=1.27) was significantly different from the WE for low-grade tumors (n=5, mean=0), with a p=0.010. The WE for IDH wild type gliomas (n=9, mean=1.67) was significantly different from the WE for IDH-mutant gliomas (n=9, mean=0.44), with a p=0.007. Analysis is ongoing. CONCLUSIONS DMI successfully and non-invasively detects the WE in many types of brain tumors. The WE was larger in high-grade tumors as compared to low-grade tumors, and the WE was larger in IDH wild type gliomas as compared to IDH-mutant gliomas. If validated with future studies, our results suggest that DMI is a useful tool to measure differences in the metabolism of brain tumors with different clinical behaviors.

SDPS-27 P16 IMMUNOHISTOCHEMISTRY AS A SCREENING TOOL FOR HOMOZYGOUS CDKN2A DELETIONS IN GLIAL TUMORS

Abstract The 2021 WHO classification of tumors of the central nervous system emphasizes the significance of molecular parameters for an integrated diagnosis. Homozygous deletion of CDKN2A has been associated with an adverse prognosis in IDH-mutant gliomas, supratentorial ependymomas, meningiomas, and MPNST. In this study, we examined the value of p16 protein immunohistochemistry as a screening tool for predicting a homozygous CDKN2A deletion. Genetic analyses for CDKN2A deletion in 30 pleomorphic xanthoastrocytomas, 32 IDH-wildtype high-grade gliomas and 40 supratentorial ependymomas with ZFTA-RELA gene fusion, 10 IDH-mutant astrocytomas, and 10 meningiomas were performed either by a molecular inversion probe assay (MIP), a high-resolution, quantitative technology for the accurate, genome-wide assessment of chromosomal copy number alterations, or by 850k methylation profile-deduced copy number analysis or multiplex ligation-dependent probe amplification analysis. In pleomorphic xanthoastrocytomas and IDH-wildtype high-grade gliomas, p16 immunohistochemistry showed high specificity (100% both), moderate sensitivity (76% and 56%), and a high positive predictive value (PPV; 100% both) for the presence of a homozygous CDKN2A deletion. In supratentorial ependymoma with ZFTA-RELA gene fusion, immunohistochemistry for p16 protein was highly sensitive and specific (90% and 97%) for a homozygous CDKN2A deletion and exhibited a high PPV and NPV (90% and 97%, respectively). For lower-grade meningioma, no correlation between the p16 immunohistochemical status and the CDKN2A deletion status was possible. Therefore, p16 protein expression detected by immunohistochemistry is a useful prescreening tool for detecting a homozygous CDKN2A deletion in glial tumors.

BSBM-07 HIGH-DIMENSIONAL HISTOPATHOLOGIC EVALUATION OF THE HYPOXIC MICROENVIRONMENT IN GLIOBLASTOMA

Abstract Rapidly growing solid tumors such as glioblastoma (GBM) are characteristically hypoxic, displaying large areas of necrosis surrounded by hyperproliferative pseudopalisading cells. Intra-tumoral hypoxia develops over time in the three-dimensional space and the degree of tissue oxygenation is a dynamic process that varies continuously. Combined with the extensive inter- and intra-tumoral heterogeneity associated with GBM at the bulk and single cell level, hypoxia contributes to a gradient of molecular alterations that are specific to the different cell populations that make up the bulk of the tumor and reside in specific niches. To date, high dimensional histopathologic analyses of the hypoxic regions within GBM tissue have not been performed. Here, we took a combined spatial and single-cell proteomic profiling approach to investigate the histopathologic features of hypoxia by leveraging a unique clinical study where the exogenous hypoxia marker pimonidazole (PIMO) is administered to patients with GBM prior to surgery. Tissue specimens were subjected to imaging mass cytometry and serial immunohistochemistry using a panel of 27 markers associated with cellular hallmarks of hypoxia, metabolism, proliferation, stemness, angiogenesis, and immune cell types. We took high-resolution imaging and statistical approaches to explore the interplay of the different markers within hypoxic regions of primary and recurrent GBMs, in addition to IDH-mutant gliomas. Our findings elucidate the expression pattern of key biological markers relative to one another, altered composition of different cell types, along with differential proliferative, transcriptional, and translational activation states associated with each cell type within the hypoxic regions of GBM.

Molecular and clonal evolution in vivo reveal a common pathway of distant relapse gliomas

The evolutionary trajectories of genomic alterations underlying distant recurrence in glioma remain largely unknown. To elucidate glioma evolution, we analyzed the evolutionary trajectories of matched pairs of primary tumors and relapse tumors or tumor in situ fluid (TISF) based on deep whole-genome sequencing data (ctDNA). We found that MMR gene mutations occurred in the late stage in IDH-mutant glioma during gene evolution, which activates multiple signaling pathways and significantly increases distant recurrence potential. The proneural subtype characterized by PDGFRA amplification was likely prone to hypermutation and distant recurrence following treatment. The classical and mesenchymal subtypes tended to progress locally through subclonal reconstruction, trunk genes transformation, and convergence evolution. EGFR and NOTCH signaling pathways and CDNK2A mutation play an important role in promoting tumor local progression. Glioma subtypes displayed distinct preferred evolutionary patterns. ClinicalTrials.gov, NCT05512325.

HDAC1 and HDAC6 are essential for driving growth in IDH1 mutant glioma

Low-grade and secondary high-grade gliomas frequently contain mutations in the IDH1 or IDH2 metabolic enzymes that are hypothesized to drive tumorigenesis by inhibiting many of the chromatin-regulating enzymes that regulate DNA structure. Histone deacetylase inhibitors are promising anti-cancer agents and have already been used in clinical trials. However, a clear understanding of their mechanism or gene targets is lacking. In this study, the authors genetically dissect patient-derived IDH1 mutant cultures to determine which HDAC enzymes drive growth in IDH1 mutant gliomas. A panel of patient-derived gliomasphere cell lines (2 IDH1 mutant lines, 3 IDH1 wildtype lines) were subjected to a drug-screen of epigenetic modifying drugs from different epigenetic classes. The effect of LBH (panobinostat) on gene expression and chromatin structure was tested on patient-derived IDH1 mutant lines. The role of each of the highly expressed HDAC enzymes was molecularly dissected using lentiviral RNA interference knock-down vectors and a patient-derived IDH1 mutant in vitro model of glioblastoma (HK252). These results were then confirmed in an in vivo xenotransplant model (BT-142). The IDH1 mutation leads to gene down-regulation, DNA hypermethylation, increased DNA accessibility and H3K27 hypo-acetylation in two distinct IDH1 mutant over-expression models. The drug screen identified histone deacetylase inhibitors (HDACi) and panobinostat (LBH) more specifically as the most selective compounds to inhibit growth in IDH1 mutant glioma lines. Of the eleven annotated HDAC enzymes (HDAC1-11) only six are expressed in IDH1 mutant glioma tissue samples and patient-derived gliomasphere lines (HDAC1-4, HDAC6, and HDAC9). Lentiviral knock-down experiments revealed that HDAC1 and HDAC6 are the most consistently essential for growth both in vitro and in vivo and target very different gene modules. Knock-down of HDAC1 or HDAC6 in vivo led to a more circumscribed less invasive tumor. The gene dysregulation induced by the IDH1 mutation is wide-spread and only partially reversible by direct IDH1 inhibition. This study identifies HDAC1 and HDAC6 as important and drug-targetable enzymes that are necessary for growth and invasiveness in IDH1 mutant gliomas.

Diagnostic Utility of Immunohistochemical Detection of MEOX2, SOX11, INSM1 and EGFR in Gliomas.

Histological identification of dispersed glioma cells in small biopsies can be challenging, especially in tumours lacking the IDH1 R132H mutation or alterations in TP53. We postulated that immunohistochemical detection of proteins expressed preferentially in gliomas (EGFR, MEOX2, CD34) or during embryonal development (SOX11, INSM1) can be used to distinguish reactive gliosis from glioma. Tissue microarrays of 46 reactive glioses, 81 glioblastomas, 34 IDH1-mutant diffuse gliomas, and 23 gliomas of other types were analysed. Glial neoplasms were significantly more often (p < 0.001, χ2) positive for EGFR (34.1% vs. 0%), MEOX2 (49.3% vs. 2.3%), SOX11 (70.5% vs. 20.4%), and INSM1 (65.4% vs. 2.3%). In 94.3% (66/70) of the glioblastomas, the expression of at least two markers was observed, while no reactive gliosis showed coexpression of any of the proteins. Compared to IDH1-mutant tumours, glioblastomas showed significantly higher expression of EGFR, MEOX2, and CD34 and significantly lower positivity for SOX11. Non-diffuse gliomas were only rarely positive for any of the five markers tested. Our results indicate that immunohistochemical detection of EGFR, MEOX2, SOX11, and INSM1 can be useful for detection of glioblastoma cells in limited histological samples, especially when used in combination.

Oncological, cognitive, and employment outcomes in a series of patients with IDH-mutated glioma resected following neoadjuvant chemotherapy.

Maximal safe tumor resection is the first line of treatment for IDH-mutated gliomas. However, when upfront surgical resection is deemed unsatisfactory due to tumor size and location, chemotherapy could represent an interesting alternative for reducing glioma extension and allowing for a safer and more efficient removal. We performed a retrospective study (June 2011 to December 2021) on patients with IDH-mutated gliomas undergoing chemotherapy with a neoadjuvant intent, followed by surgical excision in awake conditions. MRI-imaging follow-up was conducted every 3-6 months. Neuropsychological assessments (NPSA) were performed for all patients before surgery, during post-operative period, and at later follow-up, and patients were periodically interviewed about their clinical and job status. We included 6 patients who underwent awake surgery after neoadjuvant chemotherapy (temozolomide in 5 cases, PCV in 1 case) for an IDH-mutated glioma (3 oligodendrogliomas and 3 astrocytomas). Median tumor volume reduction was 47%, allowing for complete resection in one patient, subtotal resection in 4 patients, and partial resection in 1 patient. No major adverse effects were observed under chemotherapy. At the 4 months NPSA, a worsening of flexibility was observed in 2 patients (verbal fluencies in one case and trail making test in the other). Three out of the four patients working full time before procedure resumed their job full time, after a 7 to 10 months delay. Neoadjuvant chemotherapy followed by maximal safe resection can be offered to patients affected by IDH-mutated gliomas for whom upfront surgery would be inadequate. More studies are necessary given the limited size of our sample.

Integrated molecular analysis reveals hypermethylation and overexpression of HOX genes to be poor prognosticators in isocitrate dehydrogenase mutant glioma.

Diffuse gliomas represent over 80% of malignant brain tumors ranging from low-grade to aggressive high-grade lesions. Within isocitrate dehydrogenase (IDH)-mutant gliomas, there is a high variability in survival and a need to more accurately predict outcome. To identify and characterize a predictive signature of outcome in gliomas, we utilized an integrative molecular analysis (using methylation, mRNA, copy number variation (CNV), and mutation data), analyzing a total of 729 IDH-mutant samples including a test set of 99 from University Health Network (UHN) and 2 validation cohorts including the German Cancer Research Center (DKFZ) and The Cancer Genome Atlas (TCGA). Cox regression analysis of methylation data from the UHN cohort identified CpG-based signatures that split the glioma cohort into 2 prognostic groups strongly predicting survival that were validated using 2 independent cohorts from TCGA and DKFZ (all P-values < .0001). The methylation signatures that predicted poor outcomes also exhibited high CNV instability and hypermethylation of HOX gene probes. Integrated multi-platform analyses using mRNA and methylation (iRM) showed that parallel HOX gene overexpression and simultaneous hypermethylation were significantly associated with increased mutational load, high aneuploidy, and worse survival (P-value < .0001). A 7-HOX gene signature was developed and validated using the most significantly associated HOX genes with patient outcome in both 1p/19q codeleted and non-codeleted IDHmut gliomas. HOX gene methylation and expression provide important prognostic information in IDH-mutant gliomas that are not captured by current molecular diagnostics. A 7-HOX gene signature of outcome shows significant survival differences in both 1p/19q codeleted and non-codeleted IDH-mutant gliomas.