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Background and ObjectivesD-2-hydroxyglutarate (2HG) characterizesIDH-mutant gliomas and can be detected and quantified with edited MRS (MEGA-PRESS). In this study, we investigated the clinical, radiologic, and molecular parameters affecting 2HG levels.MethodsMEGA-PRESS data were acquired in 71 patients with glioma (24 untreated, 47 treated) on a 3 T system. Eighteen patients were followed during cytotoxic (n = 12) or targeted (n = 6) therapy. 2HG was measured in tumor samples using gas chromatography coupled to mass spectrometry (GCMS).ResultsMEGA-PRESS detected 2HG with a sensitivity of 95% in untreated patients and 62% in treated patients. Sensitivity depended on tumor volume (>27 cm3;p= 0.02), voxel coverage (>75%;p= 0.002), and expansive presentation (defined by equal size of T1and FLAIR abnormalities,p= 0.04). 2HG levels were positively correlated withIDH-mutant allelic fraction (p= 0.03) and total choline levels (p< 0.001) and were higher inIDH2-mutant compared withIDH1R132H-mutant and non–R132HIDH1-mutant patients (p= 0.002). In patients receiving IDH inhibitors, 2HG levels decreased within a few days, demonstrating the on-target effect of the drug, but 2HG level decrease did not predict tumor response. Patients receiving cytotoxic treatments showed a slower decrease in 2HG levels, consistent with tumor response and occurring before any tumor volume change on conventional MRI. At progression, 1p/19q codeleted gliomas, but not the non-codeleted, showed detectable in vivo 2HG levels, pointing out to different modes of progression characterizing these 2 entities.DiscussionMEGA-PRESS edited MRS allows in vivo monitoring of 2-hydroxyglutarate, confirming efficacy ofIDHinhibition and suggests different patterns of tumor progression in astrocytomas compared with oligodendrogliomas.

Rosette-forming glioneuronal tumors (RGNT) are rare low-grade primary central nervous system (CNS) tumors. The methylation class (MC) RGNT (MC-RGNT) delineates RGNT from other neurocytic CNS tumors with similar histological features. We performed a comprehensive molecular analysis including whole-exome sequencing, RNAseq, and methylome on 9 tumors with similar histology, focusing on the immune microenvironment and cell of origin of RGNT. Three RGNT in this cohort were plotted within the MC-RGNT and characterized by FGFR1 mutation plus PIK3CA or NF1 mutations. RNAseq analysis, validated by immunohistochemistry, identified 2 transcriptomic groups with distinct immune microenvironments. The "cold" group was distinguishable by a low immune infiltration and included the 3 MC-RGNT and 1 MC-pilocytic astrocytoma; the "hot" group included other tumors with a rich immune infiltration. Gene set enrichment analysis showed that the "cold" group had upregulated NOTCH pathway and mainly oligodendrocyte precursor cell and neuronal phenotypes, while the "hot" group exhibited predominantly astrocytic and neural stem cell phenotypes. In silico deconvolution identified the cerebellar granule cell lineage as a putative cell of origin of RGNT. Our study identified distinct tumor biology and immune microenvironments as key features relevant to the pathogenesis and management of RGNT.

Background:Strategies to modulate the tumor microenvironment (TME) have opened new therapeutic avenues with dramatic yet heterogeneous intertumoral efficacy in multiple cancers, including glioblastomas (GBMs). Therefore, investigating molecular actors of TME may help understand the interactions between tumor cells and TME. Immune checkpoint proteins such as a Cluster of Differentiation 80 (CD80) and CD86 are expressed on the surface of tumor cells and infiltrative tumor lymphocytes. However, their expression and prognostic value in GBM microenvironment are still unclear.Methods:In this study, we investigated, in a retrospective local discovery cohort and a validation TCGA dataset, expression of CD80 and CD86 at mRNA level and their prognostic significance in response to standard of care. Furthermore, CD80 and CD86 at the protein level were investigated in the discovery cohort.Results:Both CD80 and CD86 are expressed heterogeneously in the TME at mRNA and protein levels. In a univariate analysis, the mRNA expression of CD80 and CD86 was not significantly correlated with OS in both local OncoNeuroTek dataset and TCGA datasets. CD80 and CD86 mRNA high expression was significantly associated with shorter progression free survival (PFS) (p < 0.05). These findings were validated using the TCGA cohort; higher CD80 and CD86 expressions were correlated with shorter PFS (p < 0.05). In multivariate analysis, CD86 mRNA expression was an independent prognostic factor for PFS in the TCGA dataset only (p < 0.05).Conclusion:CD86 could be used as a potential biomarker for the prognosis of GBM patients treated with immunotherapy; however, additional studies are needed to validate these findings.

Despite producing a panoply of potential cancer-specific targets, the proteogenomic characterization of human tumors has yet to demonstrate value for precision cancer medicine. Integrative multi-omics using a machine-learning network identified master kinases responsible for effecting phenotypic hallmarks of functional glioblastoma subtypes. In subtype-matched patient-derived models, we validated PKCδ and DNA-PK as master kinases of glycolytic/plurimetabolic and proliferative/progenitor subtypes, respectively, and qualified the kinases as potent and actionable glioblastoma subtype-specific therapeutic targets. Glioblastoma subtypes were associated with clinical and radiomics features, orthogonally validated by proteomics, phospho-proteomics, metabolomics, lipidomics and acetylomics analyses, and recapitulated in pediatric glioma, breast and lung squamous cell carcinoma, including subtype specificity of PKCδ and DNA-PK activity. We developed a probabilistic classification tool that performs optimally with RNA from frozen and paraffin-embedded tissues, which can be used to evaluate the association of therapeutic response with glioblastoma subtypes and to inform patient selection in prospective clinical trials.

D-2-hydroxyglutarate (2HG) characterizesMEGA-PRESS data were acquired in 71 patients with glioma (24 untreated, 47 treated) on a 3 T system. Eighteen patients were followed during cytotoxic (n = 12) or targeted (n = 6) therapy. 2HG was measured in tumor samples using gas chromatography coupled to mass spectrometry (GCMS).MEGA-PRESS detected 2HG with a sensitivity of 95% in untreated patients and 62% in treated patients. Sensitivity depended on tumor volume (27 cmMEGA-PRESS edited MRS allows in vivo monitoring of 2-hydroxyglutarate, confirming efficacy of

Current transcriptomic classification of Glioblastoma Multiforme (GBM) has been ineffective to predict survival and therapeutic vulnerabilities. Recently, we proposed a four-group functional classification of GBM that included proliferative/progenitor, neuronal, mitochondrial and glycolytic/plurimetabolic subtypes with prognostic and therapeutic implications as the mitochondrial subtype carries the best survival and exhibits distinct sensitivity to mitochondrial OXPHOS inhibitors. To uncover novel therapeutic targets for each functional GBM subtype, we focused on protein kinases for their attractive features as both drivers and drug targets, with current availability of 62 FDA-approved inhibitors available for cancer precision therapeutics. We designed an unbiased integrative, machine learning-based proteomics/phosphoproteomics network for the identification of Master Kinases (MKs) responsible for effecting key phenotypic hallmarks of each of the four GBM subtypes. Here we report the identification and validation of protein kinase C delta (PRKCd) and DNA-PKcs as MKs that sustain the glycolytic/plurimetabolic and proliferative/progenitor GBM subtypes, respectively. Genetic and pharmacologic inactivation of PKCd in GBM patient-derived organoids of the glycolytic/plurimetabolic subtype blocked glucose uptake and lipid accumulation, resulting in marked anti-tumor effects. We also validated the role of PKCd in oncometabolic processes at the intersection between insulin, IGF, and lipid metabolism. DNA-PKcs was qualified as MK for the proliferative/progenitor GBM subtype, which is characterized by high basal level of replication stress. Biochemical experiments showed activation of DNA-PK in GBM patient-derived organoids of the proliferative/progenitor subgroup. Targeting DNA-PK in proliferative/progenitor GBM organoids with the clinically tested DNA-PKcs inhibitor nedisertib in association with gamma irradiation, the essential component of the standard of care for patients with GBM, led to an unsustainable level of DNA damage and massive GBM cell death selectively in this GBM subtype. As DNA-PKcs inhibitors have been introduced into clinical trials, our findings suggest that pre-selection of patient with PPR tumors is likely to enhance therapeutic success.

Background Cerebral cavernous malformations (CCMs) are common sporadic and inherited vascular malformations of the central nervous system. Although familial CCMs are linked to loss-of-function mutations in KRIT1 (CCM1), CCM2, or PDCD10 (CCM3), the genetic cause of sporadic CCMs, representing 80% of cases, remains incompletely understood. Methods We developed two mouse models harboring mutations identified in human meningiomas with the use of the prostaglandin D2 synthase (PGDS) promoter. We performed targeted DNA sequencing of surgically resected CCMs from patients and confirmed our findings by droplet digital polymerase-chain-reaction analysis. Results We found that in mice expressing one of two common genetic drivers of meningioma - Pik3ca H1047R or AKT1 E17K - in PGDS-positive cells, a spectrum of typical CCMs develops (in 22% and 11% of the mice, respectively) instead of meningiomas, which prompted us to analyze tissue samples from sporadic CCMs from 88 patients. We detected somatic activating PIK3CA and AKT1 mutations in 39% and 1%, respectively, of lesion tissue from the patients. Only 10% of lesions harbored mutations in the CCM genes. We analyzed lesions induced by the activating mutations Pik3ca H1074R and AKT1 E17K in mice and identified the PGDS-expressing pericyte as the probable cell of origin. Conclusions In tissue samples from sporadic CCMs, mutations in PIK3CA were represented to a greater extent than mutations in any other gene. The contribution of somatic mutations in the genes that cause familial CCMs was comparatively small. (Funded by the Fondation ARC pour la Recherche contre le Cancer and others.).

International audience; AimsMutations activating the Hedgehog (Hh) signalling pathway have been described in anterior skull base meningiomas, raising hope for the use of targeted therapies. However, identification of Hh-activated tumours is hampered by the lack of a reliable immunohistochemical marker. We report the evaluation of GAB1, an immunohistochemical marker used to detect Hh pathway activation in medulloblastoma, as a potential marker of Hh-activated meningiomas.MethodsGAB1 staining was compared to SMO mutation detection with Sanger and NGS techniques as well as Hh pathway activation study through mRNA expression level analyses in a discovery set of 110 anterior skull base meningiomas and in a prospective validation set of 21 meningiomas.ResultsUsing an expression score ranging from 0 to 400, we show that a cut-off score of 250 lead to excellent detection of Hh pathway mutations (sensitivity 100%, specificity 86%). The prospective validation set confirmed the excellent negative predictive value of GAB1 to exclude Hedgehog independent meningiomas. We describe a large series of 32 SMO-mutant meningiomas and define multiple ways of Hh activation, either through somatic mutations or associated with mutually co-exclusive SHH (Sonic Hedgehog) or IHH (Indian Hedgehog) overexpression independent of the mutations.ConclusionThe assessment of GAB1 expression by an immunohistochemical score is a fast and cost-efficient tool to screen anterior skull base meningiomas for activation of the Hedgehog pathway. It could facilitate the identification of selected cases amenable to sequencing for hedgehog pathway genes as predictive markers for targeted therapy.

Purpose Strategies to modulate the tumor microenvironment (TME), including the vascular and immune components, have opened new therapeutic avenues with dramatic yet heterogeneous intertumor efficacy in multiple cancers, including brain malignancies. Therefore, investigating molecular actors of TME may help understand the interactions between tumor cells and TME. Immune checkpoint proteins such as a Cluster of Differentiation 80 (CD80) and CD86 are expressed on the surface of tumor cells and infiltrative tumor lymphocytes. However, their expression and prognostic value in glioblastoma (GBM) is still unclear. Methods In this study, we have investigated, in a retrospective local discovery cohort and a validation TCGA dataset, expression of CD80 and CD86 at mRNA level and their prognostic significance in response to standard of care. CD80 and CD86 at the protein level were also investigated in the discovery cohort. Results Both CD80 and CD86 are expressed heterogeneously in GBM at mRNA and protein levels. In a univariate analysis, the mRNA expression of CD80 and CD86 was not significantly correlated with OS in both ONT and TCGA datasets. On the other hand, CD80 and CD86 mRNA high expression was significantly associated with shorter PFS (p

International audience; The neuropathology of epilepsy aims at diagnosing the cerebral lesions underlying epilepsy that are obtained from epilepsy surgery, or rarely from biopsy or autopsy. The main histopathological and immunohistochemical characteristics of several entities are described: epilepsy-associated hippocampal sclerosis, long-term epilepsy-associated tumours, cortical malformations, vascular malformations, glial scars, encephalitides, and focal neuronal lipofuscinosis. The diagnostic approach, the differential diagnosis and the histochemical and immunohistochemical tools are detailed in order to provide the pathologist with a summarized toolkit to handle the broad range of epileptogenic lesions.

The neuropathology of epilepsy aims at diagnosing the cerebral lesions underlying epilepsy that are obtained from epilepsy surgery, or rarely from biopsy or autopsy. The main histopathological and immunohistochemical characteristics of several entities are described: epilepsy-associated hippocampal sclerosis, long-term epilepsy-associated tumours, cortical malformations, vascular malformations, glial scars, encephalitides, and focal neuronal lipofuscinosis. The diagnostic approach, the differential diagnosis and the histochemical and immunohistochemical tools are detailed in order to provide the pathologist with a summarized toolkit to handle the broad range of epileptogenic lesions.

Background Actionable fibroblast growth factor receptor 3 (FGFR3)–transforming acidic coiled-coil protein 3 fusions (F3T3) are found in approximately 3% of gliomas, but their characteristics and prognostic significance are still poorly defined. Our goal was to characterize the clinical, radiological, and molecular profile of F3T3 positive diffuse gliomas. Methods We screened F3T3 fusion by real-time (RT)-PCR and FGFR3 immunohistochemistry in a large series of gliomas, characterized for main genetic alterations, histology, and clinical evolution. We performed a radiological and radiomic case control study, using an exploratory and a validation cohort. Results We screened 1162 diffuse gliomas (951 unselected cases and 211 preselected for FGFR3 protein immunopositivity), identifying 80 F3T3 positive gliomas. F3T3 was mutually exclusive with IDH mutation (P < 0.001) and EGFR amplification (P = 0.01), defining a distinct molecular cluster associated with CDK4 (P = 0.04) and MDM2 amplification (P = 0.03). F3T3 fusion was associated with longer survival for the whole series and for glioblastomas (median overall survival was 31.1 vs 19.9 mo, P = 0.02) and was an independent predictor of better outcome on multivariate analysis. F3T3 positive gliomas had specific MRI features, affecting preferentially insula and temporal lobe, and with poorly defined tumor margins. F3T3 fusion was correctly predicted by radiomics analysis on both the exploratory (area under the curve [AUC] = 0.87) and the validation MRI (AUC = 0.75) cohort. Using Cox proportional hazards models, radiomics predicted survival with a high C-index (0.75, SD 0.04), while the model combining clinical, genetic, and radiomic data showed the highest C-index (0.81, SD 0.04). Conclusion F3T3 positive gliomas have distinct molecular and radiological features, and better outcome.

International audience; Rats with complete spinal cord transection (SCT) can recover hindlimb locomotor function under strategies combining exercise training and 5-HT agonist treatment. This recovery is expected to result from structural and functional reorganization within the spinal cord below the lesion. To begin to understand the nature of this reorganization, we examined synaptic changes to identified gastrocnemius (GS) or tibialis anterior (TA) moto-neurons (MNs) in SCT rats after a schedule of early exercise training and delayed 5-HT agonist treatment. In addition, we analyzed changes in distribution and number of lumbar interneurons (INs) presynaptic to GS MNs using retrograde transneuronal transport of rabies virus. In SCT-untrained rats, we found few changes in the density and size of inhibitory and excitatory inputs impinging on cell bodies of TA and GS MNs compared to intact rats, whereas there was a marked trend for a reduction in the number of premotor INs connected to GS MNs. In contrast, after training of SCT rats, a significant increase of the density of GABAergic and glycinergic axon terminals was observed on both GS and TA motoneuronal cell bodies, as well as of presynaptic P-boutons on VGLUT1 afferents. Despite these changes in innervation the number of premotor INs connected to GS MNs was similar to control values although some new connections to MNs were observed. These results suggest that adaptation of gait patterns in SCT-trained rats was accompanied by changes in the innervation of lumbar MNs while the distribution of the spinal premotor circuitry was relatively preserved.