Glioblastoma hijacks neuronal mechanisms for brain invasion.

Glioblastomas are incurable tumors infiltrating the brain. A subpopulation of glioblastoma cells forms a functional and therapy-resistant tumor cell network interconnected by tumor microtubes (TMs). Other subpopulations appear unconnected, and their biological role remains unclear. Here, we demonstrate that whole-brain colonization is fueled by glioblastoma cells that lack connections with other tumor cells and astrocytes yet receive synaptic input from neurons. This subpopulation corresponds to neuronal and neural-progenitor-like tumor cell states, as defined by single-cell transcriptomics, both in mouse models and in the human disease. Tumor cell invasion resembled neuronal migration mechanisms and adopted a Lévy-like movement pattern of probing the environment. Neuronal activity induced complex calcium signals in glioblastoma cells followed by the de novo formation of TMs and increased invasion speed. Collectively, superimposing molecular and functional single-cell data revealed that neuronal mechanisms govern glioblastoma cell invasion on multiple levels. This explains how glioblastoma's dissemination and cellular heterogeneity are closely interlinked.

New Brain Tumor Entities Emerge from Molecular Classification of CNS-PNETs.

Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are highly aggressive, poorly differentiated embryonal tumors occurring predominantly in young children but also affecting adolescents and adults. Herein, we demonstrate that a significant proportion of institutionally diagnosed CNS-PNETs display molecular profiles indistinguishable from those of various other well-defined CNS tumor entities, facilitating diagnosis and appropriate therapy for patients with these tumors. From the remaining fraction of CNS-PNETs, we identify four new CNS tumor entities, each associated with a recurrent genetic alteration and distinct histopathological and clinical features. These new molecular entities, designated "CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2)," "CNS Ewing sarcoma family tumor with CIC alteration (CNS EFT-CIC)," "CNS high-grade neuroepithelial tumor with MN1 alteration (CNS HGNET-MN1)," and "CNS high-grade neuroepithelial tumor with BCOR alteration (CNS HGNET-BCOR)," will enable meaningful clinical trials and the development of therapeutic strategies for patients affected by poorly differentiated CNS tumors.

Both YAP1-MAML2 and constitutively active YAP1 drive the formation of tumors that resemble NF2 mutant meningiomas in mice.

YAP1 is a transcriptional coactivator regulated by the Hippo signaling pathway, including NF2. Meningiomas are the most common primary brain tumors; a large percentage exhibit heterozygous loss of chromosome 22 (harboring the NF2 gene) and functional inactivation of the remaining NF2 copy, implicating oncogenic YAP activity in these tumors. Recently, fusions between YAP1 and MAML2 have been identified in a subset of pediatric NF2 wild-type meningiomas. Here, we show that human YAP1-MAML2-positive meningiomas resemble NF2 mutant meningiomas by global and YAP-related gene expression signatures. We then show that expression of YAP1-MAML2 in mice induces tumors that resemble human YAP1 fusion-positive and NF2 mutant meningiomas by gene expression. We demonstrate that YAP1-MAML2 primarily functions by exerting TEAD-dependent YAP activity that is resistant to Hippo signaling. Treatment with YAP-TEAD inhibitors is sufficient to inhibit the viability of YAP1-MAML2-driven mouse tumors ex vivo. Finally, we show that expression of constitutively active YAP1 (S127/397A-YAP1) is sufficient to induce similar tumors, suggesting that the YAP component of the gene fusion is the critical driver of these tumors. In summary, our results implicate YAP1-MAML2 as a causal oncogenic driver and highlight TEAD-dependent YAP activity as an oncogenic driver in YAP1-MAML2 fusion meningioma as well as NF2 mutant meningioma in general.

Autonomous rhythmic activity in glioma networks drives brain tumour growth.

Diffuse gliomas, particularly glioblastomas, are incurable brain tumours1. They are characterized by networks of interconnected brain tumour cells that communicate via Ca2+ transients2-6. However, the networks' architecture and communication strategy and how these influence tumour biology remain unknown. Here we describe how glioblastoma cell networks include a small, plastic population of highly active glioblastoma cells that display rhythmic Ca2+ oscillations and are particularly connected to others. Their autonomous periodic Ca2+ transients preceded Ca2+ transients of other network-connected cells, activating the frequency-dependent MAPK and NF-κB pathways. Mathematical network analysis revealed that glioblastoma network topology follows scale-free and small-world properties, with periodic tumour cells frequently located in network hubs. This network design enabled resistance against random damage but was vulnerable to losing its key hubs. Targeting of autonomous rhythmic activity by selective physical ablation of periodic tumour cells or by genetic or pharmacological interference with the potassium channel KCa3.1 (also known as IK1, SK4 or KCNN4) strongly compromised global network communication. This led to a marked reduction of tumour cell viability within the entire network, reduced tumour growth in mice and extended animal survival. The dependency of glioblastoma networks on periodic Ca2+ activity generates a vulnerability7 that can be exploited for the development of novel therapies, such as with KCa3.1-inhibiting drugs.

A vaccine targeting mutant IDH1 in newly diagnosed glioma.

Mutated isocitrate dehydrogenase 1 (IDH1) defines a molecularly distinct subtype of diffuse glioma1-3. The most common IDH1 mutation in gliomas affects codon 132 and encodes IDH1(R132H), which harbours a shared clonal neoepitope that is presented on major histocompatibility complex (MHC) class II4,5. An IDH1(R132H)-specific peptide vaccine (IDH1-vac) induces specific therapeutic T helper cell responses that are effective against IDH1(R132H)+ tumours in syngeneic MHC-humanized mice4,6-8. Here we describe a multicentre, single-arm, open-label, first-in-humans phase I trial that we carried out in 33 patients with newly diagnosed World Health Organization grade 3 and 4 IDH1(R132H)+ astrocytomas (Neurooncology Working Group of the German Cancer Society trial 16 (NOA16), ClinicalTrials.gov identifier NCT02454634). The trial met its primary safety endpoint, with vaccine-related adverse events restricted to grade 1. Vaccine-induced immune responses were observed in 93.3% of patients across multiple MHC alleles. Three-year progression-free and death-free rates were 0.63 and 0.84, respectively. Patients with immune responses showed a two-year progression-free rate of 0.82. Two patients without an immune response showed tumour progression within two years of first diagnosis. A mutation-specificity score that incorporates the duration and level of vaccine-induced IDH1(R132H)-specific T cell responses was associated with intratumoral presentation of the IDH1(R132H) neoantigen in pre-treatment tumour tissue. There was a high frequency of pseudoprogression, which indicates intratumoral inflammatory reactions. Pseudoprogression was associated with increased vaccine-induced peripheral T cell responses. Combined single-cell RNA and T cell receptor sequencing showed that tumour-infiltrating CD40LG+ and CXCL13+ T helper cell clusters in a patient with pseudoprogression were dominated by a single IDH1(R132H)-reactive T cell receptor.

Dabrafenib plus Trametinib in Pediatric Glioma with BRAF V600 Mutations.

BACKGROUND: Detection of the BRAF V600E mutation in pediatric low-grade glioma has been associated with a lower response to standard chemotherapy. In previous trials, dabrafenib (both as monotherapy and in combination with trametinib) has shown efficacy in recurrent pediatric low-grade glioma with BRAF V600 mutations, findings that warrant further evaluation of this combination as first-line therapy. METHODS: In this phase 2 trial, patients with pediatric low-grade glioma with BRAF V600 mutations who were scheduled to receive first-line therapy were randomly assigned in a 2:1 ratio to receive dabrafenib plus trametinib or standard chemotherapy (carboplatin plus vincristine). The primary outcome was the independently assessed overall response (complete or partial response) according to the Response Assessment in Neuro-Oncology criteria. Also assessed were the clinical benefit (complete or partial response or stable disease for ≥24 weeks) and progression-free survival. RESULTS: A total of 110 patients underwent randomization (73 to receive dabrafenib plus trametinib and 37 to receive standard chemotherapy). At a median follow-up of 18.9 months, an overall response occurred in 47% of the patients treated with dabrafenib plus trametinib and in 11% of those treated with chemotherapy (risk ratio, 4.31; 95% confidence interval [CI], 1.7 to 11.2; P<0.001). Clinical benefit was observed in 86% of the patients receiving dabrafenib plus trametinib and in 46% receiving chemotherapy (risk ratio, 1.88; 95% CI, 1.3 to 2.7). The median progression-free survival was significantly longer with dabrafenib plus trametinib than with chemotherapy (20.1 months vs. 7.4 months; hazard ratio, 0.31; 95% CI, 0.17 to 0.55; P<0.001). Grade 3 or higher adverse events occurred in 47% of the patients receiving dabrafenib plus trametinib and in 94% of those receiving chemotherapy. CONCLUSIONS: Among pediatric patients with low-grade glioma with BRAF V600 mutations, dabrafenib plus trametinib resulted in significantly more responses, longer progression-free survival, and a better safety profile than standard chemotherapy as first-line therapy. (Funded by Novartis; ClinicalTrials.gov number, NCT02684058.).

Glutamatergic synaptic input to glioma cells drives brain tumour progression.

A network of communicating tumour cells that is connected by tumour microtubes mediates the progression of incurable gliomas. Moreover, neuronal activity can foster malignant behaviour of glioma cells by non-synaptic paracrine and autocrine mechanisms. Here we report a direct communication channel between neurons and glioma cells in different disease models and human tumours: functional bona fide chemical synapses between presynaptic neurons and postsynaptic glioma cells. These neurogliomal synapses show a typical synaptic ultrastructure, are located on tumour microtubes, and produce postsynaptic currents that are mediated by glutamate receptors of the AMPA subtype. Neuronal activity including epileptic conditions generates synchronised calcium transients in tumour-microtube-connected glioma networks. Glioma-cell-specific genetic perturbation of AMPA receptors reduces calcium-related invasiveness of tumour-microtube-positive tumour cells and glioma growth. Invasion and growth are also reduced by anaesthesia and the AMPA receptor antagonist perampanel, respectively. These findings reveal a biologically relevant direct synaptic communication between neurons and glioma cells with potential clinical implications.

The molecular landscape of ETMR at diagnosis and relapse.

Embryonal tumours with multilayered rosettes (ETMRs) are aggressive paediatric embryonal brain tumours with a universally poor prognosis1. Here we collected 193 primary ETMRs and 23 matched relapse samples to investigate the genomic landscape of this distinct tumour type. We found that patients with tumours in which the proposed driver C19MC2-4 was not amplified frequently had germline mutations in DICER1 or other microRNA-related aberrations such as somatic amplification of miR-17-92 (also known as MIR17HG). Whole-genome sequencing revealed that tumours had an overall low recurrence of single-nucleotide variants (SNVs), but showed prevalent genomic instability caused by widespread occurrence of R-loop structures. We show that R-loop-associated chromosomal instability can be induced by the loss of DICER1 function. Comparison of primary tumours and matched relapse samples showed a strong conservation of structural variants, but low conservation of SNVs. Moreover, many newly acquired SNVs are associated with a mutational signature related to cisplatin treatment. Finally, we show that targeting R-loops with topoisomerase and PARP inhibitors might be an effective treatment strategy for this deadly disease.

Accurate and comprehensive evaluation of O6-methylguanine-DNA methyltransferase promoter methylation by nanopore sequencing.

AIMS: The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter region is essential in evaluating the prognosis and predicting the drug response in patients with glioblastoma. In this study, we evaluated the utility of using nanopore long-read sequencing as a method for assessing methylation levels throughout the MGMT CpG-island, compared its performance to established techniques and demonstrated its clinical applicability. METHODS: We analysed 165 samples from CNS tumours, focusing on the MGMT CpG-island using nanopore sequencing. Oxford Nanopore Technologies (ONT) MinION and PromethION flow cells were employed for single sample or barcoded assays, guided by a CRISPR/Cas9 protocol, adaptive sampling or as part of a whole genome sequencing assay. Methylation data obtained through nanopore sequencing were compared to results obtained via pyrosequencing and methylation bead arrays. Hierarchical clustering was applied to nanopore sequencing data for patient stratification. RESULTS: Nanopore sequencing displayed a strong correlation (R2 = 0.91) with pyrosequencing results for the four CpGs of MGMT analysed by both methods. The MGMT-STP27 algorithm's classification was effectively reproduced using nanopore data. Unsupervised hierarchical clustering revealed distinct patterns in methylated and unmethylated samples, providing comparable survival prediction capabilities. Nanopore sequencing yielded high-confidence results in a rapid timeframe, typically within hours of sequencing, and extended the analysis to all 98 CpGs of the MGMT CpG-island. CONCLUSIONS: This study presents nanopore sequencing as a valid and efficient method for determining MGMT promotor methylation status. It offers a comprehensive view of the MGMT promoter methylation landscape, which enables the identification of potentially clinically relevant subgroups of patients. Further exploration of the clinical implications of patient stratification using nanopore sequencing of MGMT is warranted.

Clinically unfavorable transcriptome subtypes of non-WNT/non-SHH medulloblastomas are associated with a predominance in proliferating and progenitor-like cell subpopulations.

The non-WNT/non-SHH (Grp3/Grp4) medulloblastomas (MBs) include eight second-generation subgroups (SGS; I-VIII) each with distinct molecular and clinical characteristics. Recently, we also identified two prognostically relevant transcriptome subtypes within each SGS MB, which are associated with unique gene expression signatures and signaling pathways. These prognostic subsets may be in connection to the intra-tumoral cell landscape that underlies SGS MB clinical-molecular diversity. Here, we performed a deconvolution analysis of the Grp3/Grp4 MB bulk RNA profiles using the previously identified single-cell RNA-seq reference dataset and focusing on variability in the cellular composition of SGS MB. RNA deconvolution analysis of the Grp3/Grp4 MB disclosed the subgroup-specific neoplastic cell subpopulations. Neuronally differentiated axodendritic GP3-C1 and glutamatergic GP4-C1 subpopulations were distributed within Grp3- and Grp4-associated SGS MB, respectively. Progenitor GP3-B2 subpopulation was prominent in aggressive SGS II MB, whereas photoreceptor/visual perception GP3/4-C2 cell content was typical for SGS III/IV MB. The current study also revealed significant variability in the proportions of cell subpopulations between clinically relevant SGS MB transcriptome subtypes, where unfavorable cohorts were enriched with cell cycle and progenitor-like cell subpopulations and, vice versa, favorable subtypes were composed of neuronally differentiated cell fractions predominantly. A higher than median proportion of proliferating and progenitor cell subpopulations conferred the shortest survival of the Grp3 and Grp 4 MB, and similar survival associations were identified for all SGS MB except SGS IV MB. In summary, the recently identified clinically relevant Grp3/Grp4 MB transcriptome subtypes are composed of different cell populations. Future studies should aim to validate the prognostic and therapeutic role of the identified Grp3/Grp4 MB inter-tumoral cellular heterogeneity. The application of the single-cell techniques on each SGS MB separately could help to clarify the clinical significance of subgroup-specific variability in tumor cell content and its relation with prognostic transcriptome signatures identified before.

Plasma extracellular vesicles in meningioma patients following radiotherapy as liquid biopsy- a prospective explorative biomarker study (ARO 2023-05/AG-NRO-07).

BACKGROUND: While surgical resection remains the primary treatment approach for symptomatic or growing meningiomas, radiotherapy represents an auspicious alternative in patients with meningiomas not safely amenable to surgery. Biopsies are often omitted in light of potential postoperative neurological deficits, resulting in a lack of histological grading and (molecular) risk stratification. In this prospective explorative biomarker study, extracellular vesicles in the bloodstream will be investigated in patients with macroscopic meningiomas to identify a biomarker for molecular risk stratification and disease monitoring. METHODS: In total, 60 patients with meningiomas and an indication of radiotherapy (RT) and macroscopic tumor on the planning MRI will be enrolled. Blood samples will be obtained before the start, during, and after radiotherapy, as well as during clinical follow-up every 6 months. Extracellular vesicles will be isolated from the blood samples, quantified and correlated with the clinical treatment response or progression. Further, nanopore sequencing-based DNA methylation profiles of plasma EV-DNA will be generated for methylation-based meningioma classification. DISCUSSION: This study will explore the dynamic of plasma EVs in meningioma patients under/after radiotherapy, with the objective of identifying potential biomarkers of (early) tumor progression. DNA methylation profiling of plasma EVs in meningioma patients may enable molecular risk stratification, facilitating a molecularly-guided target volume delineation and adjusted dose prescription during RT treatment planning.

PerSurge (NOA-30) phase II trial of perampanel treatment around surgery in patients with progressive glioblastoma.

BACKGROUND: Glioblastoma is the most frequent and a particularly malignant primary brain tumor with no efficacy-proven standard therapy for recurrence. It has recently been discovered that excitatory synapses of the AMPA-receptor subtype form between non-malignant brain neurons and tumor cells. This neuron-tumor network connectivity contributed to glioma progression and could be efficiently targeted with the EMA/FDA approved antiepileptic AMPA receptor inhibitor perampanel in preclinical studies. The PerSurge trial was designed to test the clinical potential of perampanel to reduce tumor cell network connectivity and tumor growth with an extended window-of-opportunity concept. METHODS: PerSurge is a phase IIa clinical and translational treatment study around surgical resection of progressive or recurrent glioblastoma. In this multicenter, 2-arm parallel-group, double-blind superiority trial, patients are 1:1 randomized to either receive placebo or perampanel (n = 66 in total). It consists of a treatment and observation period of 60 days per patient, starting 30 days before a planned surgical resection, which itself is not part of the study interventions. Only patients with an expected safe waiting interval are included, and a safety MRI is performed. Tumor cell network connectivity from resected tumor tissue on single cell transcriptome level as well as AI-based assessment of tumor growth dynamics in T2/FLAIR MRI scans before resection will be analyzed as the co-primary endpoints. Secondary endpoints will include further imaging parameters such as pre- and postsurgical contrast enhanced MRI scans, postsurgical T2/FLAIR MRI scans, quality of life, cognitive testing, overall and progression-free survival as well as frequency of epileptic seizures. Further translational research will focus on additional biological aspects of neuron-tumor connectivity. DISCUSSION: This trial is set up to assess first indications of clinical efficacy and tolerability of perampanel in recurrent glioblastoma, a repurposed drug which inhibits neuron-glioma synapses and thereby glioblastoma growth in preclinical models. If perampanel proved to be successful in the clinical setting, it would provide the first evidence that interference with neuron-cancer interactions may indeed lead to a benefit for patients, which would lay the foundation for a larger confirmatory trial in the future. TRIAL REGISTRATION: EU-CT number: 2023-503938-52-00 30.11.2023.

LOGGIC/FIREFLY-2: a phase 3, randomized trial of tovorafenib vs. chemotherapy in pediatric and young adult patients with newly diagnosed low-grade glioma harboring an activating RAF alteration.

BACKGROUND: Pediatric low-grade glioma (pLGG) is essentially a single pathway disease, with most tumors driven by genomic alterations affecting the mitogen-activated protein kinase/ERK (MAPK) pathway, predominantly KIAA1549::BRAF fusions and BRAF V600E mutations. This makes pLGG an ideal candidate for MAPK pathway-targeted treatments. The type I BRAF inhibitor, dabrafenib, in combination with the MEK inhibitor, trametinib, has been approved by the United States Food and Drug Administration for the systemic treatment of BRAF V600E-mutated pLGG. However, this combination is not approved for the treatment of patients with tumors harboring BRAF fusions as type I RAF inhibitors are ineffective in this setting and may paradoxically enhance tumor growth. The type II RAF inhibitor, tovorafenib (formerly DAY101, TAK-580, MLN2480), has shown promising activity and good tolerability in patients with BRAF-altered pLGG in the phase 2 FIREFLY-1 study, with an objective response rate (ORR) per Response Assessment in Neuro-Oncology high-grade glioma (RANO-HGG) criteria of 67%. Tumor response was independent of histologic subtype, BRAF alteration type (fusion vs. mutation), number of prior lines of therapy, and prior MAPK-pathway inhibitor use. METHODS: LOGGIC/FIREFLY-2 is a two-arm, randomized, open-label, multicenter, global, phase 3 trial to evaluate the efficacy, safety, and tolerability of tovorafenib monotherapy vs. current standard of care (SoC) chemotherapy in patients < 25 years of age with pLGG harboring an activating RAF alteration who require first-line systemic therapy. Patients are randomized 1:1 to either tovorafenib, administered once weekly at 420 mg/m2 (not to exceed 600 mg), or investigator's choice of prespecified SoC chemotherapy regimens. The primary objective is to compare ORR between the two treatment arms, as assessed by independent review per RANO-LGG criteria. Secondary objectives include comparisons of progression-free survival, duration of response, safety, neurologic function, and clinical benefit rate. DISCUSSION: The promising tovorafenib activity data, CNS-penetration properties, strong scientific rationale combined with the manageable tolerability and safety profile seen in patients with pLGG led to the SIOPe-BTG-LGG working group to nominate tovorafenib for comparison with SoC chemotherapy in this first-line phase 3 trial. The efficacy, safety, and functional response data generated from the trial may define a new SoC treatment for newly diagnosed pLGG. TRIAL REGISTRATION: ClinicalTrials.gov: NCT05566795. Registered on October 4, 2022.

Pineal anlage tumor: clinical and diagnostic features, and rationales for treatment.

PURPOSE: To provide a treatment-focused review and develop basic treatment guidelines for patients diagnosed with pineal anlage tumor (PAT). METHODS: Prospectively collected data of three patients with pineal anlage tumor from Germany was combined with clinical details and treatment information from 17 published cases. RESULTS: Overall, 20 cases of PAT were identified (3 not previously reported German cases, 17 cases from published reports). Age at diagnosis ranged from 0.3 to 35.0 (median: 3.2 ± 7.8) years. All but three cases were diagnosed before the age of three years. For three cases, metastatic disease at initial staging was described. All patients underwent tumor surgery (gross-total resection: 9, subtotal resection/biopsy: 9, extent of resection unknown: 2). 15/20 patients were alive at last follow-up. Median follow-up for 10/15 surviving patients with available follow-up and treatment data was 2.4 years (0.3-6.5). Relapse was reported for 3 patients within 0.8 years after diagnosis. Five patients died, 3 after relapse and 2 from early postoperative complications. Two-year-progression-free- and -overall survival were 65.2 ± 12.7% and 49.2 ± 18.2%, respectively. All 4 patients who received intensive chemotherapy including high-dose chemotherapy combined with radiotherapy (2 focal, 2 craniospinal [CSI]) had no recurrence. Focal radiotherapy- and CSI-free survival rates in 13 evaluable patients were 46.2% (6/13) and 61.5% (8/13), respectively. CONCLUSION: PAT is an aggressive disease mostly affecting young children. Therefore, adjuvant therapy using intensive chemotherapy and considering radiotherapy appears to comprise an appropriate treatment strategy. Reporting further cases is crucial to evaluate distinct treatment strategies.

Repeat surgery of recurrent glioma for molecularly informed treatment in the age of precision oncology: A risk-benefit analysis.

PURPOSE: Surgery for recurrent glioma provides cytoreduction and tissue for molecularly informed treatment. With mostly heavily pretreated patients involved, it is unclear whether the benefits of repeat surgery outweigh its potential risks. METHODS: Patients receiving surgery for recurrent glioma WHO grade 2-4 with the goal of tissue sampling for targeted therapies were analyzed retrospectively. Complication rates (surgical, neurological) were compared to our institutional glioma surgery cohort. Tissue molecular diagnostic yield, targeted therapies and post-surgical survival rates were analyzed. RESULTS: Between 2017 and 2022, tumor board recommendation for targeted therapy through molecular diagnostics was made for 180 patients. Of these, 70 patients (38%) underwent repeat surgery. IDH-wildtype glioblastoma was diagnosed in 48 patients (69%), followed by IDH-mutant astrocytoma (n = 13; 19%) and oligodendroglioma (n = 9; 13%). Gross total resection (GTR) was achieved in 50 patients (71%). Tissue was processed for next-generation sequencing in 64 cases (91%), and for DNA methylation analysis in 58 cases (83%), while immunohistochemistry for mTOR phosphorylation was performed in 24 cases (34%). Targeted therapy was recommended in 35 (50%) and commenced in 21 (30%) cases. Postoperatively, 7 patients (11%) required revision surgery, compared to 7% (p = 0.519) and 6% (p = 0.359) of our reference cohorts of patients undergoing first and second craniotomy, respectively. Non-resolving neurological deterioration was documented in 6 cases (10% vs. 8%, p = 0.612, after first and 4%, p = 0.519, after second craniotomy). Median survival after repeat surgery was 399 days in all patients and 348 days in GBM patients after repeat GTR. CONCLUSION: Surgery for recurrent glioma provides relevant molecular diagnostic information with a direct consequence for targeted therapy under a reasonable risk of postoperative complications. With satisfactory postoperative survival it can therefore complement a multi-modal glioma therapy approach.

Impact of signal intensity normalization of MRI on the generalizability of radiomic-based prediction of molecular glioma subtypes.

OBJECTIVES: Radiomic features have demonstrated encouraging results for non-invasive detection of molecular biomarkers, but the lack of guidelines for pre-processing MRI-data has led to poor generalizability. Here, we assessed the influence of different MRI-intensity normalization techniques on the performance of radiomics-based models for predicting molecular glioma subtypes. METHODS: Preoperative MRI-data from n = 615 patients with newly diagnosed glioma and known isocitrate dehydrogenase (IDH) and 1p/19q status were pre-processed using four different methods: no normalization (naive), N4 bias field correction (N4), N4 followed by either WhiteStripe (N4/WS), or z-score normalization (N4/z-score). A total of 377 Image-Biomarker-Standardisation-Initiative-compliant radiomic features were extracted from each normalized data, and 9 different machine-learning algorithms were trained for multiclass prediction of molecular glioma subtypes (IDH-mutant 1p/19q codeleted vs. IDH-mutant 1p/19q non-codeleted vs. IDH wild type). External testing was performed in public glioma datasets from UCSF (n = 410) and TCGA (n = 160). RESULTS: Support vector machine yielded the best performance with macro-average AUCs of 0.84 (naive), 0.84 (N4), 0.87 (N4/WS), and 0.87 (N4/z-score) in the internal test set. Both N4/WS and z-score outperformed the other approaches in the external UCSF and TCGA test sets with macro-average AUCs ranging from 0.85 to 0.87, replicating the performance of the internal test set, in contrast to macro-average AUCs ranging from 0.19 to 0.45 for naive and 0.26 to 0.52 for N4 alone. CONCLUSION: Intensity normalization of MRI data is essential for the generalizability of radiomic-based machine-learning models. Specifically, both N4/WS and N4/z-score approaches allow to preserve the high model performance, yielding generalizable performance when applying the developed radiomic-based machine-learning model in an external heterogeneous, multi-institutional setting. CLINICAL RELEVANCE STATEMENT: Intensity normalization such as N4/WS or N4/z-score can be used to develop reliable radiomics-based machine learning models from heterogeneous multicentre MRI datasets and provide non-invasive prediction of glioma subtypes. KEY POINTS: • MRI-intensity normalization increases the stability of radiomics-based models and leads to better generalizability. • Intensity normalization did not appear relevant when the developed model was applied to homogeneous data from the same institution. • Radiomic-based machine learning algorithms are a promising approach for simultaneous classification of IDH and 1p/19q status of glioma.

DNA methylation-based classification of central nervous system tumours.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

Simultaneous Nbs1 and p53 inactivation in neural progenitors triggers high-grade gliomas.

AIMS: Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder caused by hypomorphic mutations of NBS1. NBS1 is a member of the MRE11-RAD50-NBS1 (MRN) complex that binds to DNA double-strand breaks and activates the DNA damage response (DDR). Nbs1 inactivation in neural progenitor cells leads to microcephaly and premature death. Interestingly, p53 homozygous deletion rescues the NBS1-deficient phenotype allowing long-term survival. The objective of this work was to determine whether simultaneous inactivation of Nbs1 and p53 in neural progenitors triggered brain tumorigenesis and if so in which category this tumour could be classified. METHODS: We generated a mouse model with simultaneous genetic inactivation of Nbs1 and p53 in embryonic neural stem cells and analysed the arising tumours with in-depth molecular analyses including immunohistochemistry, array comparative genomic hybridisation (aCGH), whole exome-sequencing and RNA-sequencing. RESULTS: NBS1/P53-deficient mice develop high-grade gliomas (HGG) arising in the olfactory bulbs and in the cortex along the rostral migratory stream. In-depth molecular analyses using immunohistochemistry, aCGH, whole exome-sequencing and RNA-sequencing revealed striking similarities to paediatric human HGG with shared features with radiation-induced gliomas (RIGs). CONCLUSIONS: Our findings show that concomitant inactivation of Nbs1 and p53 in mice promotes HGG with RIG features. This model could be useful for preclinical studies to improve the prognosis of these deadly tumours, but it also highlights the singularity of NBS1 among the other DNA damage response proteins in the aetiology of brain tumours.

Transcriptome analysis stratifies second-generation non-WNT/non-SHH medulloblastoma subgroups into clinically tractable subtypes.

Medulloblastoma (MB), one of the most common malignant pediatric brain tumor, is a heterogenous disease comprised of four distinct molecular groups (WNT, SHH, Group 3, Group 4). Each of these groups can be further subdivided into second-generation MB (SGS MB) molecular subgroups, each with distinct genetic and clinical characteristics. For instance, non-WNT/non-SHH MB (Group 3/4) can be subdivided molecularly into eight distinct and clinically relevant tumor subgroups. A further molecular stratification/summarization of these SGS MB would allow for the assignment of patients to risk-associated treatment protocols. Here, we performed DNA- and RNA-based analysis of 574 non-WNT/non-SHH MB and analyzed the clinical significance of various molecular patterns within the entire cohort and the eight SGS MB, with the aim to develop an optimal risk stratification of these tumors. Multigene analysis disclosed several survival-associated genes highly specific for each molecular subgroup within this non-WNT/non-SHH MB cohort with minimal inter-subgroup overlap. These subgroup-specific and prognostically relevant genes were associated with pathways that could underlie SGS MB clinical-molecular diversity and tumor-driving mechanisms. By combining survival-associated genes within each SGS MB, distinct metagene sets being appropriate for their optimal risk stratification were identified. Defined subgroup-specific metagene sets were independent variables in the multivariate models generated for each SGS MB and their prognostic value was confirmed in a completely non-overlapping validation cohort of non-WNT/non-SHH MB (n = 377). In summary, the current results indicate that the integration of transcriptome data in risk stratification models may improve outcome prediction for each non-WNT/non-SHH SGS MB. Identified subgroup-specific gene expression signatures could be relevant for clinical implementation and survival-associated metagene sets could be adopted for further SGS MB risk stratification. Future studies should aim at validating the prognostic role of these transcriptome-based SGS MB subtypes in prospective clinical trials.

Integrated genetic analyses of immunodeficiency-associated Epstein-Barr virus- (EBV) positive primary CNS lymphomas.

Immunodeficiency-associated primary CNS lymphoma (PCNSL) represents a distinct clinicopathological entity, which is typically Epstein-Barr virus-positive (EBV+) and carries an inferior prognosis. Genetic alterations that characterize EBV-related CNS lymphomagenesis remain unclear precluding molecular classification and targeted therapies. In this study, a comprehensive genetic analysis of 22 EBV+ PCNSL, therefore, integrated clinical and pathological information with exome and RNA sequencing (RNASeq) data. EBV+ PCNSL with germline controls carried a median of 55 protein-coding single nucleotide variants (SNVs; range 24-217) and 2 insertions/deletions (range 0-22). Genetic landscape was largely shaped by aberrant somatic hypermutation with a median of 41.01% (range 31.79-53.49%) of SNVs mapping to its target motifs. Tumors lacked established SNVs (MYD88, CD79B, PIM1) and copy number variants (CDKN2A, HLA loss) driving EBV- PCNSL. Instead, EBV+ PCNSL were characterized by SOCS1 mutations (26%), predicted to disinhibit JAK/STAT signaling, and mutually exclusive gain-of-function NOTCH pathway SNVs (26%). Copy number gains were enriched on 11q23.3, a locus directly targeted for chromosomal aberrations by EBV, that includes SIK3 known to protect from cytotoxic T-cell responses. Losses covered 5q31.2 (STING), critical for sensing viral DNA, and 17q11 (NF1). Unsupervised clustering of RNASeq data revealed two distinct transcriptional groups, that shared strong expression of CD70 and IL1R2, previously linked to tolerogenic tumor microenvironments. Correspondingly, deconvolution of bulk RNASeq data revealed elevated M2-macrophage, T-regulatory cell, mast cell and monocyte fractions in EBV+ PCNSL. In addition to novel insights into the pathobiology of EBV+ PCNSL, the data provide the rationale for the exploration of targeted therapies including JAK-, NOTCH- and CD70-directed approaches.