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Glioneuronal tumors are a heterogenous group of CNS neoplasms that can be challenging to accurately diagnose. Molecular methods are highly useful in classifying these tumors—distinguishing precise classes from their histological mimics and identifying previously unrecognized types of tumors. Using an unsupervised visualization approach of DNA methylation data, we identified a novel group of tumors (n = 20) that formed a cluster separate from all established CNS tumor types. Molecular analyses revealed ATRX alterations (in 16/16 cases by DNA sequencing and/or immunohistochemistry) as well as potentially targetable gene fusions involving receptor tyrosine-kinases (RTK; mostly NTRK1-3) in all of these tumors (16/16; 100%). In addition, copy number profiling showed homozygous deletions of CDKN2A/B in 55% of cases. Histological and immunohistochemical investigations revealed glioneuronal tumors with isomorphic, round and often condensed nuclei, perinuclear clearing, high mitotic activity and microvascular proliferation. Tumors were mainly located supratentorially (84%) and occurred in patients with a median age of 19 years. Survival data were limited (n = 18) but point towards a more aggressive biology as compared to other glioneuronal tumors (median progression-free survival 12.5 months). Given their molecular characteristics in addition to anaplastic features, we suggest the term glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA) to describe these tumors. In summary, our findings highlight a novel type of glioneuronal tumor driven by different RTK fusions accompanied by recurrent alterations in ATRX and homozygous deletions of CDKN2A/B. Targeted approaches such as NTRK inhibition might represent a therapeutic option for patients suffering from these tumors.

Glioblastoma is the most common primary malignant brain tumor in adults with an overall survival of only 14.6 months. Hypoxia is known to play a role in tumor aggressiveness but the influence of hypoxia on the immune microenvironment is not fully understood. The aim of this study was to investigate the expression of immune-related proteins in normoxic and hypoxic tumor areas by digital spatial profiling. Tissue samples from 10 glioblastomas were stained with a panel of 40 antibodies conjugated to photo-cleavable oligonucleotides. The free oligo-tags from normoxic and hypoxic areas were hybridized to barcodes for digital counting. Differential expression patterns were validated by Ivy Glioblastoma Atlas Project (GAP) data and an independent patient cohort. We found that CD44, Beta-catenin and B7-H3 were upregulated in hypoxia, whereas VISTA, CD56, KI-67, CD68 and CD11c were downregulated. PD-L1 and PD-1 were not affected by hypoxia. Focusing on the checkpoint-related markers CD44, B7-H3 and VISTA, our findings for CD44 and VISTA could be confirmed with Ivy GAP RNA sequencing data. Immunohistochemical staining and digital quantification of CD44, B7-H3 and VISTA in an independent cohort confirmed our findings for all three markers. Additional stainings revealed fewer T cells and high but equal amounts of tumor-associated microglia and macrophages in both hypoxic and normoxic regions. In conclusion, we found that CD44 and B7-H3 were upregulated in areas with hypoxia whereas VISTA was downregulated together with the presence of fewer T cells. This heterogeneous expression should be taken into consideration when developing novel therapeutic strategies.

Ependymoma is the second most common malignant brain tumor in children. The etiology is largely unknown and germline DNA sequencing studies focusing on childhood ependymoma are limited. We therefore performed germline whole-genome sequencing on a population-based cohort of children diagnosed with ependymoma in Denmark over the past 20 years (n = 43). Single nucleotide and structural germline variants in 457 cancer related genes and 2986 highly evolutionarily constrained genes were assessed in 37 children with normal tissue available for sequencing. Molecular ependymoma classification was performed using DNA methylation profiling for 39 children with available tumor tissue. Pathogenic germline variants in known cancer predisposition genes were detected in 11% (4/37; NF2, LZTR1, NF1 & TP53). However, DNA methylation profiling resulted in revision of the histopathological ependymoma diagnosis to non-ependymoma tumor types in 8% (3/39). This included the two children with pathogenic germline variants in TP53 and NF1 whose tumors were reclassified to a diffuse midline glioma and a rosette-forming glioneuronal tumor, respectively. Consequently, 50% (2/4) of children with pathogenic germline variants in fact had other tumor types. A meta-analysis combining our findings with pediatric pan-cancer germline sequencing studies showed an overall frequency of pathogenic germline variants of 3.4% (7/207) in children with ependymoma. In summary, less than 4% of childhood ependymoma is explained by genetic predisposition, virtually restricted to pathogenic variants in NF2 and NF1. For children with other cancer predisposition syndromes, diagnostic reconsideration is recommended for ependymomas without molecular classification. Additionally, LZTR1 is suggested as a novel putative ependymoma predisposition gene.

Treatment-refractory meningiomas have a dismal prognosis and limited treatment options. Meningiomas express high-densities of somatostatin receptors (SSTR), thus potentially susceptible to antitumorigenic effects of somatostatin analogues (SSA). Evidence for SSA in meningiomas is scarce, and it is unclear if published literature would either (1) support wider use of SSA, if (2) more evidence is desirable, or if (3) available evidence is sufficient to discard SSA. We addressed the need for more evidence with a systematic review and meta-analysis. We performed an individual patient data (IPD) meta-analysis. Main outcomes were toxicity, best radiological response, progression-free survival, and overall survival. We applied multivariable logistic regression models to estimate the effect of SSA on the probability of obtaining radiological disease control. The predictive performance was evaluated using area under the curve and Brier scores. We included 16 studies and compiled IPD from 8/9 of all previous cohorts. Quality of evidence was overall ranked “very low.” Stable disease was reported in 58% of patients as best radiological response. Per 100 mg increase in total SSA dosage, the odds ratios for obtaining radiological disease control was 1.42 (1.11 to 1.81, P = 0.005) and 1.44 (1.00 to 2.08, P = 0.05) for patients treated with SSA as monodrug therapy vs SSA in combination with everolimus, respectively. Low quality of evidence impeded exact quantification of treatment efficacy, and the association between response and treatment may represent reverse causality. Yet, the SSA treatment was well tolerated, and beneficial effect cannot be disqualified. A prospective trial without bias from inconsistent study designs is warranted to assess SSA therapy for well-defined meningioma subgroups.

Background: Glioblastomas are highly resistant to therapy, and virtually all patients experience tumor recurrence after standard-of-care treatment. Surgical tumor resection is a cornerstone in glioblastoma therapy, but its impact on cellular phenotypes in the local postsurgical microenvironment has yet to be fully elucidated. Methods: We developed a preclinical orthotopic xenograft tumor resection model in rats with integrated 18F-FET PET/CT imaging. Primary and recurrent tumors were subject to bulk and single-cell RNA sequencing. Differentially expressed genes and pathways were investigated and validated using tissue specimens from the xenograft model, 23 patients with matched primary/recurrent tumors, and a cohort including 190 glioblastoma patients. Functional investigations were performed in vitro with multiple patient-derived cell cultures. Results: Tumor resection induced microglia/macrophage infiltration, angiogenesis as well as proliferation and upregulation of several stem cell-related genes in recurrent tumor cells. Expression changes of selected genes SOX2, POU3F2, OLIG2, and NOTCH1 were validated at the protein level in xenografts and early recurrent patient tumors. Single-cell transcriptomics revealed the presence of distinct phenotypic cell clusters in recurrent tumors which deviated from clusters found in primary tumors. Recurrent tumors expressed elevated levels of pleiotrophin (PTN), secreted by both tumor cells and tumor-associated microglia/macrophages. Mechanistically, PTN could induce tumor cell proliferation, self-renewal, and the stem cell program. In glioblastoma patients, high PTN expression was associated with poor overall survival and identified as an independent prognostic factor. Conclusion: Surgical tumor resection is an iatrogenic driver of PTN-mediated self-renewal in glioblastoma tumor cells that promotes therapeutic resistance and tumor recurrence.

TERT promoter mutations have been associated with increased risk of recurrence in meningioma cohorts, thus a potential biomarker for aggressive phenotypes. A main purpose of refining tumour classification is better predictions on the patient level. We compiled data from previous published cohorts to investigate patient-level predictions of recurrence based on TERTp-mut status. Implementation of TERTp-mut into the WHO grading led to better patient prognostication by improved prediction of recurrence. Our results support implementation of TERTp-mut into diagnostics and classification of meningiomas

Introduction: The extent of meningioma resection is the most fundamental risk factor for recurrence, and exact knowledge of extent of resection is necessary for prognostication and for planning of adjuvant treatment. Currently used classifications are the EANO-grading and the Simpson grading. The former comprises radiological imaging with contrast-enhanced MRI and differentiation between “gross total removal” and “subtotal removal,” while the latter comprises a five-tiered differentiation of the surgeon’s impression of the extent of resection. The extent of resection of tumors is usually defined via analyses of resection margins but has until now not been implemented for meningiomas. PET/MRI imaging with 68Ga-DOTATOC allows more sensitive and specific imaging than MRI following surgery of meningiomas. Objective: To develop an objective grading system based on microscopic analyses of resection margins and sensitive radiological analyses to improve management of follow-up, adjuvant therapy, and prognostication of meningiomas. Based on the rationale of resection-margin analyses as gold standard and superior imaging performance of 68Ga DOTATOC PET, we propose “Copenhagen Grading” for meningiomas. Results: Copenhagen Grading was described for six pilot patients with examples of positive and negative findings on histopathology and DOTATOC PET scanning. The grading could be traceably implemented and parameters of grading appeared complementary. Copenhagen Grading is prospectively implemented as a clinical standard at Rigshospitalet, Copenhagen. Conclusion: Copenhagen Grading provided a comprehensive, logical, and reproducible definition of the extent of resection. It offers promise to be the most sensitive and specific imaging modality available for meningiomas. Clinical and cost-efficacy remain to be established during prospective implementation.

The advent of checkpoint immunotherapy, particularly with programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) inhibitors, has provided ground-breaking results in several advanced cancers. Substantial efforts are being made to extend these promising therapies to other refractory cancers such as gliomas, especially glioblastoma, which represents the most frequent and malignant glioma and carries an exceptionally grim prognosis. Thus, there is a need for new therapeutic strategies with related biomarkers. Gliomas have a profoundly immunosuppressive tumour micro-environment and evade immunological destruction by several mechanisms, one being the expression of inhibitory immune checkpoint molecules such as PD-L1. PD-L1 is recognised as an important therapeutic target and its expression has been shown to hold prognostic value in different cancers. Several clinical trials have been launched and some already completed, but PD-1/PD-L1 inhibitors have yet to show convincing clinical efficacy in gliomas. Part of the explanation may reside in the vast molecular heterogeneity of gliomas and a complex interplay within the tumour micro-environment. In parallel, critical knowledge about PD-L1 expression is beginning to accumulate including knowledge on expression levels, testing methodology, co-expression with other checkpoint molecules and prognostic and predictive value. This article reviews these aspects and points out areas where biomarker research is needed to develop more successful checkpoint-related therapeutic strategies in gliomas.