Search

Aim: Analysis of cerebrospinal fluid (CSF) is essential for diagnostic workup of patients with neurological diseases and includes differential cell typing. The current gold standard is based on microscopic examination by specialised technicians and neuropathologists, which is time‐consuming, labour‐intensive and subjective. Methods: We, therefore, developed an image analysis approach based on expert annotations of 123,181 digitised CSF objects from 78 patients corresponding to 15 clinically relevant categories and trained a multiclass convolutional neural network (CNN). Results: The CNN classified the 15 categories with high accuracy (mean AUC 97.3%). By using explainable artificial intelligence (XAI), we demonstrate that the CNN identified meaningful cellular substructures in CSF cells recapitulating human pattern recognition. Based on the evaluation of 511 cells selected from 12 different CSF samples, we validated the CNN by comparing it with seven board‐certified neuropathologists blinded for clinical information. Inter‐rater agreement between the CNN and the ground truth was non‐inferior (Krippendorff's alpha 0.79) compared with the agreement of seven human raters and the ground truth (mean Krippendorff's alpha 0.72, range 0.56–0.81). The CNN assigned the correct diagnostic label (inflammatory, haemorrhagic or neoplastic) in 10 out of 11 clinical samples, compared with 7–11 out of 11 by human raters. Conclusions: Our approach provides the basis to overcome current limitations in automated cell classification for routine diagnostics and demonstrates how a visual explanation framework can connect machine decision‐making with cell properties and thus provide a novel versatile and quantitative method for investigating CSF manifestations of various neurological diseases.

Background Myxopapillary ependymoma (MPE) is a heterogeneous disease regarding histopathology and outcome. The underlying molecular biology is poorly understood, and markers that reliably predict the patients’ clinical course are unknown. Methods We assembled a cohort of 185 tumors classified as MPE based on DNA methylation. Methylation patterns, copy number profiles, and MGMT promoter methylation were analyzed for all tumors, 106 tumors were evaluated histomorphologically, and RNA sequencing was performed for 37 cases. Based on methylation profiling, we defined two subtypes MPE-A and MPE-B, and explored associations with epidemiological, clinical, pathological, and molecular characteristics of these tumors. Results MPE-A occurred at a median age of 27 years and were enriched with tumors demonstrating papillary morphology and MGMT promoter hypermethylation. Half of these tumors could not be totally resected, and 85% relapsed within 10 years. Copy number alterations were more common in MPE-A. RNA sequencing revealed an enrichment for extracellular matrix and immune system-related signatures in MPE-A. MPE-B occurred at a median age of 45 years and included many tumors with a histological diagnosis of WHO grade II and tanycytic morphology. Patients within this subtype had a significantly better outcome with a relapse rate of 33% in 10 years (P = 3.4e-06). Conclusions We unraveled the morphological and clinical heterogeneity of MPE by identifying two molecularly distinct subtypes. These subtypes significantly differed in progression-free survival and will likely need different protocols for surveillance and treatment.

Rosette-forming glioneuronal tumor (RGNT) is a rare brain neoplasm that primarily affects young adults. Although alterations affecting the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling pathway have been associated with this low-grade entity, comprehensive molecular investigations of RGNT in larger series have not been performed to date, and an integrated view of their genetic and epigenetic profiles is still lacking. Here we describe a genome-wide DNA methylation and targeted sequencing-based characterization of a molecularly distinct class of tumors (n = 30), initially identified through genome-wide DNA methylation screening among a cohort of > 30,000 tumors, of which most were diagnosed histologically as RGNT. FGFR1 hotspot mutations were observed in all tumors analyzed, with co-occurrence of PIK3CA mutations in about two-thirds of the cases (63%). Additional loss-of-function mutations in the tumor suppressor gene NF1 were detected in a subset of cases (33%). Notably, in contrast to most other low-grade gliomas, these tumors often displayed co-occurrence of two or even all three of these mutations. Our data highlight that molecularly defined RGNTs are characterized by highly recurrent combined genetic alterations affecting both MAPK and PI3K signaling pathways. Thus, these two pathways appear to synergistically interact in the formation of RGNT, and offer potential therapeutic targets for this disease.

Diffuse IDH-mutant astrocytoma mostly occurs in adults and carries a favorable prognosis compared to IDH-wildtype malignant gliomas. Acquired mismatch repair deficiency is known to occur in recurrent IDH-mutant gliomas as resistance mechanism towards alkylating chemotherapy. In this multi-institutional study, we report a novel epigenetic group of 32 IDH-mutant gliomas with proven or suspected hereditary mismatch repair deficiency. None of the tumors exhibited a combined 1p/19q deletion. These primary mismatch repair-deficient IDH-mutant astrocytomas (PMMRDIA) were histologically high-grade and were mainly found in children, adolescents and young adults (median age 14 years). Mismatch repair deficiency syndromes (Lynch or Constitutional Mismatch Repair Deficiency Syndrom (CMMRD)) were clinically diagnosed and/or germline mutations in DNA mismatch repair genes (MLH1, MSH6, MSH2) were found in all cases, except one case with a family and personal history of colon cancer and another case with MSH6-deficiency available only as recurrent tumor. Loss of at least one of the mismatch repair proteins was detected via immunohistochemistry in all, but one case analyzed. Tumors displayed a hypermutant genotype and microsatellite instability was present in more than half of the sequenced cases. Integrated somatic mutational and chromosomal copy number analyses showed frequent inactivation of TP53, RB1 and activation of RTK/PI3K/AKT pathways. In contrast to the majority of IDH-mutant gliomas, more than 60% of the samples in our cohort presented with an unmethylated MGMT promoter. While the rate of immuno-histochemical ATRX loss was reduced, variants of unknown significance were more frequently detected possibly indicating a higher frequency of ATRX inactivation by protein malfunction. Compared to reference cohorts of other IDH-mutant gliomas, primary mismatch repair-deficient IDH-mutant astrocytomas have by far the worst clinical outcome with a median survival of only 15 months irrespective of histological or molecular features. The findings reveal a so far unknown entity of IDH-mutant astrocytoma with high prognostic relevance. Diagnosis can be established by aligning with the characteristic DNA methylation profile, by DNA-sequencing-based proof of mismatch repair deficiency or immunohistochemically demonstrating loss-of-mismatch repair proteins.

Atypical teratoid/rhabdoid tumor (ATRT) is a highly malignant brain tumor predominantly occurring in infants. Mutations of the SMARCB1 gene are the characteristic genetic lesion. SMARCB1-mutant tumors in adolescents and adults are rare and may show uncommon histopathological and clinical features. Here we report seven SMARCB1-deficient intracranial tumors sharing distinct clinical, histopathological and molecular features. Median age of the four females and three males was 40years (range 15–61years). All tumors were located in the pineal region. Histopathologically, these tumors displayed spindled and epithelioid cells embedded in a desmoplastic stroma alternating with a variable extent of a loose myxoid matrix. All cases showed loss of nuclear SMARCB1/INI1 protein expression, expression of EMA and CD34 was frequent and the Ki67/MIB1 proliferation index was low in the majority of cases (median 3%). Three cases displayed heterozygous SMARCB1 deletions and two cases a homozygous SMARCB1 deletion. On sequencing, one tumor showed a 2bp deletion in exon 4 (c.369_370del) and one a short duplication in exon 3 (c.237_276dup) both resulting in frameshift mutations. Most DNA methylation profiles were not classifiable using the Heidelberg Brain Tumor Classifier (version v11b4). By unsupervised t-SNE analysis and hierarchical clustering analysis, however, all tumors grouped closely together and showed similarities with ATRT-MYC. After a median observation period of 48months, three patients were alive with stable disease, whereas one patient experienced tumor progression and three patients had succumbed to disease. In conclusion, our series represents an entity with distinct clinical, histopathological and molecular features showing epigenetic similarities with ATRT-MYC. We propose the designation desmoplastic myxoid tumor (DMT), SMARCB1-mutant, for these tumors.

According to the current WHO classification, ependymal tumors are classified as subependymomas, myxopapillary ependymomas, classic ependymomas, anaplastic ependymomas and RELA-fusion positive ependymoma (RELA-EPN). Among classic ependymomas, the WHO defines rare histological variants, i.e. the clear-cell, papillary, and tanycytic ependymoma. In parallel to this WHO classification scheme, DNA methylation patterns can distinguish nine distinct molecular ependymoma subgroups, some of which tightly overlap with certain histopathological subgroups, e.g. subependyomas or myxopapillary ependymomas. Since very little is known about the molecular background of histological classic ependymoma variants, we analyzed histomorphology, clinical parameters and global DNA methylation patterns of diagnosed tanycytic ependymomas (n=12), clear-cell ependymomas (n=14) and papillary ependymomas (n=19). Surprisingly, up to 42% of these variants did not match to ependymomas using a previously published DNA methylation-based classifier for brain tumors. Among the tumors with a match to one of the nine known ependymoma methylation classes, tanycytic ependymomas were predominantly located in the spine, but showed diverse molecular methylation patterns. Most clear-cell ependymomas showed a common histomorphology, were found supratentorially and fell into the methylation class of RELA-EPN. Papillary ependymomas showed a “papillary”, “trabecular” or “pseudo-papillary” growth pattern. Interestingly, a true papillary growth pattern was strongly associated with the molecular class B of posterior fossa ependymoma (PFB), but tumors displayed DNA methylation sites that were significantly different when compared to PFB ependymomas without papillary growth. Our results show that the diagnosis of classic histological ependymoma variants can be challenging. While clear-cell and papillary ependymomas harbor common molecular features, tanycytic ependymoma may not represent a molecularly distinct subgroup.

All isocitrate dehydrogenase (IDH) mutant solid neoplasms exhibit highly elevated levels of D-2-hydroxyglutarate (D-2HG). Detection of 2HG in tumor tissues currently is performed by gas or liquid chromatography-mass spectrometry (GC- or LC-MS) or biochemical detection. While these methods are highly accurate, a considerable amount of time for tissue preparation and a relatively high amount of tissue is required for testing. We here present a rapid approach to detect 2HG in brain tumor tissue based on matrix-assisted laser desorption ionization - time of flight mass spectrometry (MALDI-TOF). We analyzed 26 brain tumor samples with known IDH1 or IDH2 mutation and compared readouts to those from 28 brain tumor samples of wildtype IDH status. IDH mutant samples exhibited a clear positive signal for 2HG which was not observed in any of the IDH wildtype tumors. Our analytical pipeline allowed for 2HG detection in less than 5 min. Data were validated by determining 2HG levels in all tissues with a biochemical assay. In conclusion, we developed a protocol for rapid detection of 2HG levels and illustrate the possibility to use MALDI-TOF for the detection of metabolites on frozen tissue sections in a diagnostic setting. Electronic supplementary material The online version of this article (10.1186/s40478-018-0523-3) contains supplementary material, which is available to authorized users.

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.

Isomorphic diffuse glioma (IDG) was first described in 2004 as an epilepsy-associated supratentorial diffuse glioma with low cellularity, low proliferation and very monomorphic tumour cells. Most patients had seizures since childhood but were operated on as adults. To study the position of these lesions among brain tumours we histologically, molecularly and clinically analysed 26 histologically typical IDGs. Tumour cells were GFAP-positive, MAP2-, OLIG2- and CD34-negative and the nuclear ATRX-expression was retained. Proliferation was very low. Sequencing of 24 cases revealed an IDH-wildtype status. Cluster analyses of DNA methylation data showed that IDG has a DNA methylation profile distinct from those of different glial/glio-neuronal brain tumours and normal hemispheric tissue. About half of IDGs had copy number alterations of MYBL1 or MYB (13/25) and half of the cases analysed by RNA-sequencing had gene fusions of MYBL1 or MYB with various gene partners (11/22), often associated with an increased RNA-expression of the respective MYB-family gene. Integrating all data available, 77% of IDGs had either MYBL1 (54%) or MYB (23%) alterations. All patients had a good outcome and most were seizure-free after surgery. In summary, we show that isomorphic diffuse glioma is a distinct benign tumour in the family of MYB/MYBL1-altered gliomas. DNA methylation analysis is very helpful for their identification. More recent analyses of a large cohort of MYB/MYBL1-altered brain tumours suggest the presence of a third methylation group that primarily contains paediatric cases and seems to be distinct from IDG and angiocentric gliomas. Further histological, molecular and clinical analyses are ongoing.

Papillary glioneuronal tumor (PGNT) is a WHO-defined brain tumor entity that poses a major diagnostic challenge. Recently, SLC44A1-PRKCA fusions have been described in PGNT. We subjected 28 brain tumors from different institutions histologically diagnosed as PGNT to molecular and morphological analysis. Array-based methylation analysis revealed that 17/28 tumors exhibited methylation profiles typical for other tumor entities, mostly dysembryoplastic neuroepithelial tumor and hemispheric pilocytic astrocytoma. Conversely, 11/28 tumors exhibited a unique profile, thus constituting a distinct methylation class PGNT. By screening the extended Heidelberg cohort containing over 25,000 CNS tumors, we identified three additional tumors belonging to this methylation cluster but originally histologically diagnosed otherwise. RNA sequencing for the detection of SLC44A1-PRKCA fusions could be performed on 19 of the tumors, 10 of them belonging to the methylation class PGNT. In two additional cases, SLC44A1-PRKCA fusions were confirmed by FISH. We detected fusions involving PRKCA in all cases of this methylation class with material available for analyses: the canonical SLC44A1-PRKCA fusion was observed in 11/12 tumors, while the remaining case exhibited a NOTCH1-PRKCA fusion. Neither of the fusions was found in the tumors belonging to other methylation classes. Our results point towards a high misclassification rate of the morphological diagnosis PGNT and clearly demonstrate the necessity of molecular analyses. PRKCA fusions are highly diagnostic for PGNT, and detection by RNA sequencing enables the identification of rare fusion partners. Methylation analysis recognizes a unique methylation class PGNT irrespective of the nature of the PRKCA fusion.

© Springer-Verlag GmbH Germany, part of Springer Nature 2019., The "isomorphic subtype of diffuse astrocytoma" was identified histologically in 2004 as a supratentorial, highly differentiated glioma with low cellularity, low proliferation and focal diffuse brain infiltration. Patients typically had seizures since childhood and all were operated on as adults. To define the position of these lesions among brain tumours, we histologically, molecularly and clinically analysed 26 histologically prototypical isomorphic diffuse gliomas. Immunohistochemically, they were GFAP-positive, MAP2-, OLIG2- and CD34-negative, nuclear ATRX-expression was retained and proliferation was low. All 24 cases sequenced were IDH-wildtype. In cluster analyses of DNA methylation data, isomorphic diffuse gliomas formed a group clearly distinct from other glial/glio-neuronal brain tumours and normal hemispheric tissue, most closely related to paediatric MYB/MYBL1-altered diffuse astrocytomas and angiocentric gliomas. Half of the isomorphic diffuse gliomas had copy number alterations of MYBL1 or MYB (13/25, 52%). Gene fusions of MYBL1 or MYB with various gene partners were identified in 11/22 (50%) and were associated with an increased RNA-expression of the respective MYB-family gene. Integrating copy number alterations and available RNA sequencing data, 20/26 (77%) of isomorphic diffuse gliomas demonstrated MYBL1 (54%) or MYB (23%) alterations. Clinically, 89% of patients were seizure-free after surgery and all had a good outcome. In summary, we here define a distinct benign tumour class belonging to the family of MYB/MYBL1-altered gliomas. Isomorphic diffuse glioma occurs both in children and adults, has a concise morphology, frequent MYBL1 and MYB alterations and a specific DNA methylation profile. As an exclusively histological diagnosis may be very challenging and as paediatric MYB/MYBL1-altered diffuse astrocytomas may have the same gene fusions, we consider DNA methylation profiling very helpful for their identification., This study was supported by an International League against Epilepsy (ILAE) Grant to D. Capper. I. Blumcke was supported by the European Union (FP6 DESIRE Grant Agreement #602531). M. Mittelbronn would like to thank the Luxembourg National Research Fund (FNR) for the support (FNR PEARL P16/BM/11192868 grant). K. Ligon is funded by the Paediatric Low Grade Astrocytoma Foundation and NCI R01CA215489. T. Jacques is funded by The Brain Tumour Charity, Children with Cancer UK, Great Ormond Street Hospital Children’s Charity, Cancer Research UK, the Olivia Hodson Cancer Fund and the National Institute of Health Research. T. Jacques’s work is partly funded by the NIHR GOSH BRC. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. We thank the biomedical scientists in the Division of Neuropathology, the National Hospital for Neurology and Neurosurgery (NHNN) for excellent technical assistance. We also thank clinicians and neuropathologists for referring cases for molecular analysis. Part of the study was funded by the National Institute for Health Research to UCLH Biomedical research centre (BRC399/NS/RB/101410). S. Brandner and Z. Jaunmuktane are also supported by the Department of Health’s NIHR Biomedical Research Centre’s funding scheme. Additional funding was provided by the Brain Tumour Charity (UK) for the Everest Centre for Paediatric Low-Grade Brain Tumour Research

In this multi-institutional study we compiled a retrospective cohort of 86 posterior fossa tumors having received the diagnosis of cerebellar glioblastoma (cGBM). All tumors were reviewed histologically and subjected to array-based methylation analysis followed by algorithm-based classification into distinct methylation classes (MCs). The single MC containing the largest proportion of 25 tumors diagnosed as cGBM was MC anaplastic astrocytoma with piloid features representing a recently-described molecular tumor entity not yet included in the WHO Classification of Tumours of the Central Nervous System (WHO classification). Twenty-nine tumors molecularly corresponded to either of 6 methylation subclasses subsumed in the MC family GBM IDH wildtype. Further we identified 6 tumors belonging to the MC diffuse midline glioma H3 K27 M mutant and 6 tumors allotted to the MC IDH mutant glioma subclass astrocytoma. Two tumors were classified as MC pilocytic astrocytoma of the posterior fossa, one as MC CNS high grade neuroepithelial tumor with BCOR alteration and one as MC control tissue, inflammatory tumor microenvironment. The methylation profiles of 16 tumors could not clearly be assigned to one distinct MC. In comparison to supratentorial localization, the MC GBM IDH wildtype subclass midline was overrepresented, whereas the MCs GBM IDH wildtype subclass mesenchymal and subclass RTK II were underrepresented in the cerebellum. Based on the integration of molecular and histological findings all tumors received an integrated diagnosis in line with the WHO classification 2016. In conclusion, cGBM does not represent a molecularly uniform tumor entity, but rather comprises different brain tumor entities with diverse prognosis and therapeutic options. Distinction of these molecular tumor classes requires molecular analysis. More than 30% of tumors diagnosed as cGBM belong to the recently described molecular entity of anaplastic astrocytoma with piloid features. Electronic supplementary material The online version of this article (10.1186/s40478-019-0801-8) contains supplementary material, which is available to authorized users.

AIM Recurrent structural variants involving MYB and MYBL1 transcription factors were recently identified in pediatric low-grade gliomas (pLGGs), such as the MYB-QKI rearrangement in Angiocentric Gliomas and truncations of MYBL1 (MYBL1tr) in Diffuse Astrocytomas. However, therapeutic dependencies induced by these alterations remain unexplored. METHODOLOGY We have generated in vitro pLGG mouse neural stem cell (NSCs) models engineered to harbor distinct MYB/MYBL1 genomic alterations. We used single cell RNA sequencing approaches to determine the transcriptional profile and dissect the central regulatory networks of our in vitro pLGG models over time. To identify specific genetic dependencies associated with MYB/MYBL1 mutations, we employed the Brie genome-wide mouse CRISPR lentiviral knockout pooled library, consisting of 78,637 single guide RNAs (sgRNAs) targeting 19,674 mouse genes. RESULTS MYB/MYBL1 expression in neural stem cells induced activation of cell-cycle related, glioma-related and senescence-related pathways that are involved in normal development, including activation of MAPK and mTOR signaling which are also activated in human pLGG samples. Genome-scale CRISPR-cas9 screens in isogenic NSCs expressing MYB-QKI or MYBL1tr identified differential genetic dependencies relative to GFP controls. These included regulators of cell-cycle progression and several modulators of the ubiquitin-proteasome degradation pathway. Analysis of RNA-sequencing data from human tumors revealed several of these dependencies identified in the cell line model to be differentially expressed in MYB-altered pLGG tumors relative to normal brain. CONCLUSION Expression of MYB family alterations induces expression of key developmental and oncogenic pathways and genetic dependencies that represent potential therapeutic targets for MYB or MYBL1 rearranged pLGGs.

According to the 2016 World Health Organization Classification of Tumors of the Central Nervous System (2016 CNS WHO), IDH-mutant astrocytic gliomas comprised WHO grade II diffuse astrocytoma, IDH-mutant (AII(IDHmut)), WHO grade III anaplastic astrocytoma, IDH-mutant (AAIII(IDHmut)), and WHO grade IV glioblastoma, IDH-mutant (GBM(IDHmut)). Notably, IDH gene status has been made the major criterion for classification while the manner of grading has remained unchanged: it is based on histological criteria that arose from studies which antedated knowledge of the importance of IDH status in diffuse astrocytic tumor prognostic assessment. Several studies have now demonstrated that the anticipated differences in survival between the newly defined AII(IDHmut) and AAIII(IDHmut) have lost their significance. In contrast, GBM(IDHmut) still exhibits a significantly worse outcome than its lower grade IDH-mutant counterparts. To address the problem of establishing prognostically significant grading for IDH-mutant astrocytic gliomas in the IDH era, we undertook a comprehensive study that included assessment of histological and genetic approaches to prognosis in these tumors. A discovery cohort of 211 IDH-mutant astrocytic gliomas with an extended observation was subjected to histological review, image analysis, and DNA methylation studies. Tumor group-specific methylation profiles and copy number variation (CNV) profiles were established for all gliomas. Algorithms for automated CNV analysis were developed. All tumors exhibiting 1p/19q codeletion were excluded from the series. We developed algorithms for grading, based on molecular, morphological and clinical data. Performance of these algorithms was compared with that of WHO grading. Three independent cohorts of 108, 154 and 224 IDH-mutant astrocytic gliomas were used to validate this approach. In the discovery cohort several molecular and clinical parameters were of prognostic relevance. Most relevant for overall survival (OS) was CDKN2A/B homozygous deletion. Other parameters with major influence were necrosis and the total number of CNV. Proliferation as assessed by mitotic count, which is a key parameter in 2016 CNS WHO grading, was of only minor influence. Employing the parameters most relevant for OS in our discovery set, we developed two models for grading these tumors. These models performed significantly better than WHO grading in both the discovery and the validation sets. Our novel algorithms for grading IDH-mutant astrocytic gliomas overcome the challenges caused by introduction of IDH status into the WHO classification of diffuse astrocytic tumors. We propose that these revised approaches be used for grading of these tumors and incorporated into future WHO criteria.

Extraventricular neurocytoma (EVN) is a rare primary brain tumor occurring in brain parenchyma outside the ventricular system. Histopathological characteristics resemble those of central neurocytoma but exhibit a wider morphologic spectrum. Accurate diagnosis of these histologically heterogeneous tumors is often challenging because of the overlapping morphological features and the lack of defining molecular markers. Here, we explored the molecular landscape of 40 tumors diagnosed histologically as EVN by investigating copy number profiles and DNA methylation array data. DNA methylation profiles were compared with those of relevant differential diagnoses of EVN and with a broader spectrum of diverse brain tumor entities. Based on this, our tumor cohort segregated into different groups. While a large fraction (n = 22) formed a separate epigenetic group clearly distinct from established DNA methylation profiles of other entities, a subset (n = 14) of histologically diagnosed EVN grouped with clusters of other defined entities. Three cases formed a small group close to but separated from the epigenetically distinct EVN cases, and one sample clustered with non-neoplastic brain tissue. Four additional samples originally diagnosed otherwise were found to molecularly resemble EVN. Thus, our results highlight a distinct DNA methylation pattern for the majority of tumors diagnosed as EVN, but also indicate that approximately onethird of morphological diagnoses of EVN epigenetically correspond to other brain tumor entities. Copy number analysis and confirmation through RNA sequencing revealed FGFR1–TACC1 fusion as a distinctive, recurrent feature within the EVN methylation group (60%), in addition to a small number of other FGFR rearrangements (13%). In conclusion, our data demonstrate a specific epigenetic signature of EVN suitable for characterization of these tumors as a molecularly distinct entity, and reveal a high frequency of potentially druggable FGFR pathway activation in this tumor group.

Tumors with histological features of pilocytic astrocytoma (PA), but with increased mitotic activity and additional high-grade features (particularly microvascular proliferation and palisading necrosis) have often been designated anaplastic pilocytic astrocytomas. The status of these tumors as a separate entity has not yet been conclusively demonstrated and molecular features have only been partially characterized. We performed DNA methylation profiling of 102 histologically defined anaplastic pilocytic astrocytomas. T-distributed stochastic neighbor-embedding(t-SNE) and hierarchical clustering analysis of these 102 cases against 158 reference cases from 12 glioma reference classes revealed that a subset of 83 of these tumors share a common DNA methylation profile that is distinct from the reference classes. These 83 tumors were thus denominated DNA methylation class anaplastic astrocytoma with piloid features (MC AAP). The 19 remaining tumors were distributed amongst the reference classes, with additional testing confirming the molecular diagnosis in most cases. Median age of patients with MC AAP was 41.5years. The most frequent localization was the posterior fossa (74%). Deletions of CDKN2A/B (66/83, 80%), MAPK pathway gene alterations (49/65, 75%, most frequently affecting NF1, followed by BRAF and FGFR1) and mutations of ATRX or loss of ATRX expression (33/74, 45%) were the most common molecular alterations. All tumors were IDH1/2 wildtype. The MGMT promoter was methylated in 38/83 tumors (45%). Outcome analysis confirmed an unfavorable clinical course in comparison to PA, but better than IDH wildtype glioblastoma. In conclusion, we show that a subset of histologically defined anaplastic pilocytic astrocytomas forms a separate DNA methylation cluster, harbors recurrent alterations in MAPK pathway genes in combination with alterations of CDKN2A/B and ATRX, affects patients who are on average older than those diagnosed with PA and has an intermediate clinical outcome.

During CNS development, interneuron precursors have to migrate extensively before they integrate in specific microcircuits. Known regulators of neuronal motility include classical neurotransmitters, yet the mechanisms that assure interneuron dispersal and interneuron/projection neuron matching during histogenesis remain largely elusive. We combined time-lapse video microscopy and electrophysiological analysis of the nascent cerebellum of transgenic Pax2-EGFP mice to address this issue. We found that cerebellar interneuronal precursors regularly show spontaneous postsynaptic currents, indicative of synaptic innervation, well before settling in the molecular layer. In keeping with the sensitivity of these cells to neurotransmitters, ablation of synaptic communication by blocking vesicular release in acute slices of developing cerebella slows migration. Significantly, abrogation of exocytosis primarily impedes the directional persistence of migratory interneuronal precursors. These results establish an unprecedented function of the early synaptic innervation of migrating neuronal precursors and demonstrate a role for synapses in the regulation of migration and pathfinding. © 2017. This is the authors’ accepted and refereed manuscript to the article. Locked until 14.11.2018 due to copyright restrictions.

Lettera no abstract

Canonical Wnt signaling is known to promote proliferation of olfactory stem cells. In order to investigate the effects of a constitutive activation of Wnt signaling in Sox2-positive precursor cells of the olfactory epithelium, we used transgenic mice that allowed an inducible deletion of exon 3 of the Ctnnb1 gene, which is responsible for the phosphorylation and degradation of Ctnnb1 protein. After induction of aberrant Wnt activation by Ctnnb1 deletion at embryonic day 14, such mice developed tumor-like lesions in upper parts of the nasal cavity. We still observed areas of epithelial hyperplasia within the olfactory epithelium following early postnatal Wnt activation, but the olfactory epithelial architecture remained unaffected in most parts when Wnt was activated at postnatal day 21 or later. In summary, our results suggest an age-dependent tumorigenic potential of aberrant Wnt signaling in the olfactory epithelium of mice.