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Uni-and multivariate Analysis of IL1B and SASP in PA cohort

Supplemental Table Legend

Primers and antibodies

Clinical annotations of the PA patient cohort

Clinical annotations of the multiplex assay PA cohort and normal brain

GEP results of SASP in DKFZ-BT66 in OIS vs. proliferation

Figure S1: Markers of OIS in murine PA model Figure S2: Viability of DKFZ-BT66 cells under rIL1B treatment Figure S3: SASP factor protein levels in primary PAs Figure S4: Treatments of astrocytes with senolytic agents and combinations treatments of ABT-737 in DKFZ-BT66 cells

Background Plexiform neurofibromas can transform into atypical neurofibromas (ANF) and then further progress to aggressive malignant peripheral nerve sheath tumors (MPNST). ANF have been described to harbor distinct histological features and frequent loss of CDKN2A/B. However, histological evaluation may be rater-dependent, and detailed knowledge about the molecular mechanisms of malignant transformation is scarce. In general, malignant transformation can be accompanied by significant epigenetic changes, and global DNA methylation profiling is able to differentiate relevant tumor subgroups. Therefore, epigenetic profiling might provide a valuable tool to distinguish and characterize ANF with differing extent of histopathological atypia from neurofibromas and MPNST. Methods We investigated 40 tumors histologically diagnosed as ANF and compared their global methylation profile to other peripheral nerve sheath tumors. Results Unsupervised class discovery and t-SNE analysis indicated that 36/40 ANF cluster with benign peripheral nerve sheath tumors with clear separation from MPNST. 21 ANF formed a molecularly distinct cluster in proximity to schwannomas. Tumors in this cluster had a frequent heterozygous or homozygous loss of CDKN2A/B and significantly more lymphocyte infiltration than MPNST, schwannomas, and NF. Few ANF clustered closely with neurofibromas, schwannomas, or MPNST, raising the question, whether diagnosis based on histological features alone might pose a risk to both over- and underestimate the aggressiveness of these lesions. Conclusions Our data suggest that ANF with varying histological morphology show distinct epigenetic similarities and cluster in proximity to benign peripheral nerve sheath tumor entities. Future investigations should pay special respect to correlating this methylation pattern to clinical outcomes.

Background The prognostic impact of clinical risk factors and DNA methylation patterns in sonic hedgehog (SHH)-activated early childhood desmoplastic/nodular medulloblastoma (DMB) or medulloblastoma with extensive nodularity (MBEN) were evaluated to better identify patients at risk for relapse. Methods One hundred and forty-four patients with DMB (n = 99) or MBEN (n = 45) aged Results Patients with DMB had less favorable 5-year progression-free survival than MBEN (5y-PFS, 71% [DMB] vs. 93% [MBEN]). Patients aged >3 years were associated with more unfavorable 5y-PFS (47% [>3 years] vs. 85% [ Conclusions These data suggest further heterogeneity within early childhood SHH-DMB/MBEN: SHH-2 splits into a very low-risk group SHH-2a enriched for MBEN histology and SMO mutations, and SHH-2b comprising older DMB patients with a higher risk of relapse.

PURPOSE Meningiomas are the most frequent primary intracranial tumors. Patient outcome varies widely from benign to highly aggressive, ultimately fatal courses. Reliable identification of risk of progression for individual patients is of pivotal importance. However, only biomarkers for highly aggressive tumors are established ( CDKN2A/B and TERT), whereas no molecularly based stratification exists for the broad spectrum of patients with low- and intermediate-risk meningioma. METHODS DNA methylation data and copy-number information were generated for 3,031 meningiomas (2,868 patients), and mutation data for 858 samples. DNA methylation subgroups, copy-number variations (CNVs), mutations, and WHO grading were analyzed. Prediction power for outcome was assessed in a retrospective cohort of 514 patients, validated on a retrospective cohort of 184, and on a prospective cohort of 287 multicenter cases. RESULTS Both CNV- and methylation family–based subgrouping independently resulted in increased prediction accuracy of risk of recurrence compared with the WHO classification (c-indexes WHO 2016, CNV, and methylation family 0.699, 0.706, and 0.721, respectively). Merging all risk stratification approaches into an integrated molecular-morphologic score resulted in further substantial increase in accuracy (c-index 0.744). This integrated score consistently provided superior accuracy in all three cohorts, significantly outperforming WHO grading (c-index difference P = .005). Besides the overall stratification advantage, the integrated score separates more precisely for risk of progression at the diagnostically challenging interface of WHO grade 1 and grade 2 tumors (hazard ratio 4.34 [2.48-7.57] and 3.34 [1.28-8.72] retrospective and prospective validation cohorts, respectively). CONCLUSION Merging these layers of histologic and molecular data into an integrated, three-tiered score significantly improves the precision in meningioma stratification. Implementation into diagnostic routine informs clinical decision making for patients with meningioma on the basis of robust outcome prediction.

We report two pituitary neuroendocrine tumors (PitNETs) with very high Ki67 labeling indices, many mitoses and TP53 mutation (nearly all tumor cell nuclei were positive for p53). One of the tumors had bone and liver metastases. One was a corticotroph cell tumor; the other was a lactotroph tumor. The classification of these tumors is the subject of this discussion. Traditionally, pituitary carcinomas are only diagnosed by demonstration of metastases according to the 2017 WHO classification. In contrast, neuroendocrine neoplasms of the gastrointestinal tract and pancreas are classified as either well differentiated NETs that are graded as G1, G2, and G3 based on proliferation as determined by Ki67 indices of ≤ 3, 3–20 and > 20%, and/or 20 mitoses per 10 high-power field respectively, or as neuroendocrine carcinomas (NECs) that are poorly differentiated neoplasms with mitoses > 20/HPF and/or a Ki67 index > 20%. With the reclassificiation of PitNETs, in our opinion, the adequate term for the well-differentiated corticotroph tumor that we report is a PitNET G3, whereas the undifferentiated prolactin tumor should be classified as PitNEC. This report expands the spectrum of pituitary neuroendocrine neoplasms.

Interstitial lung diseases (ILDs) comprise over 200 parenchymal lung disorders. Among them, fibrosing ILDs, especially idiopathic pulmonary fibrosis, are associated with a poor prognosis, whereas some other ILDs, such as sarcoidosis, have a much better prognosis. A high proportion manifests as fibrotic ILD (fILD). Lung cancer (LC) is a frequent complication of fILD. Activated fibroblasts are crucial for fibrotic processes in fILD. The aim of this exploratory study was to evaluate the imaging properties of static and dynamic fibroblast activation protein (FAP) inhibitor (FAPI) PET/CT in various types of fILD and to confirm FAP expression in fILD lesions by FAP immunohistochemistry of human fILD biopsy samples and of lung sections of genetically engineered (Nedd4-2(−/−)) mice with an idiopathic pulmonary fibrosislike lung disease. Methods: PET scans of 15 patients with fILD and suspected LC were acquired 10, 60, and 180 min after the administration of 150–250 MBq of a (68)Ga-labeled FAPI tracer (FAPI-46). In 3 patients, dynamic scans over 40 min were performed instead of imaging after 10 min. The SUV(max) and SUV(mean) of fibrotic lesions and LC were measured and CT-density–corrected. Target-to-background ratios (TBRs) were calculated. PET imaging was correlated with CT-based fibrosis scores. Time–activity curves derived from dynamic imaging were analyzed. FAP immunohistochemistry of 4 human fILD biopsy samples and of fibrotic lungs of Nedd4-2(−/−) mice was performed. Results: fILD lesions as well as LC showed markedly elevated (68)Ga-FAPI uptake (density-corrected SUV(max) and SUV(mean) 60 min after injection: 11.12 ± 6.71 and 4.29 ± 1.61, respectively, for fILD lesions and 16.69 ± 9.35 and 6.44 ± 3.29, respectively, for LC) and high TBR (TBR of density-corrected SUV(max) and SUV(mean) 60 min after injection: 2.30 ± 1.47 and 1.67 ± 0.79, respectively, for fILD and 3.90 ± 2.36 and 2.37 ± 1.14, respectively, for LC). SUV(max) and SUV(mean) decreased over time, with a stable TBR for fILD and a trend toward an increasing TBR in LC. Dynamic imaging showed differing time–activity curves for fILD and LC. (68)Ga-FAPI uptake showed a positive correlation with the CT-based fibrosis index. Immunohistochemistry of human biopsy samples and the lungs of Nedd4-2(−/−) mice showed a patchy expression of FAP in fibrotic lesions, preferentially in the transition zone to healthy lung parenchyma. Conclusion: (68)Ga-FAPI PET/CT imaging is a promising new imaging modality for fILD and LC. Its potential clinical value for monitoring and therapy evaluation of fILD should be investigated in future studies.

BACKGROUND: Adenoid cystic carcinomas (ACCs) are rare epithelial tumors mostly situated in the head and neck region and characterized by infiltrative growth. The tumor stroma of ACCs includes cancer-associated fibroblasts (CAFs) expressing Fibroblast Activation Protein (FAP), a new target for positron emission tomography (PET) imaging. Here we describe the value of PET/ computed tomography (PET/CT) imaging using (68)Ga-labelled FAP-Inhibitors ((68)Ga-FAPI-PET/CT) and their clinical potential for staging and radiotherapy planning in 12 ACC patients (7 primary, 5 recurrent). PATIENTS AND METHODS: Patients underwent contrast enhanced staging CT (ceCT) and magnetic resonance imaging (ceMRI) before (68)Ga-FAPI - PET/CT. PET-scans were acquired 10, 60 and 180 minutes after administration of 150–250 MBq of (68)Ga-labelled FAPI tracers. SUV(max) and SUV(mean) values of ACCs and healthy organs were obtained using a 60% of maximum i so-contour. FAP and alpha smooth muscle actin (a-SMA) immunohistochemistry was performed in 13 cases (3 with and 10 without (68)Ga FAPI-PET/CT). Staging and radiotherapy planning based on (68)Ga-FAPI-PET/CT versus ceCT/MRI alone were compared. RESULTS: We observed elevated tracer uptake in all ACCs. lmmunohistochemistry showed FAP-expressing CAFs in the tumor. Compared to conventional staging, (68)Ga-FAPI-PET/CT led to upstaging in 2/12 patients and to detection of additional metastases in 3 patients, thus in total 42% of patients had their staging altered. Moreover, (68)Ga-FAPI PET improved the accuracy of target volume delineation for radiotherapy, as compared to CT and MRI. CONCLUSION: (68)Ga-FAPI-PET/CT is a promising imaging modality for ACC. Increasing the accuracy of staging exams and radiotherapy planning volumes, as compared conventional to CT and MRI.

Anaplastic ganglioglioma is a rare tumour, and diagnosis has been based on histological criteria. The 5th edition of the World Health Organization Classification of Tumours of the Central Nervous System (CNS WHO) does not list anaplastic ganglioglioma as a distinct diagnosis due to lack of molecular data in previous publications. We retrospectively compiled a cohort of 54 histologically diagnosed anaplastic gangliogliomas to explore whether the molecular profiles of these tumours represent a separate type or resolve into other entities.Samples were subjected to histological review, desoxyribonucleic acid (DNA) methylation profiling and next-generation sequencing. Morphological and molecular data were summarised to an integrated diagnosis.The majority of tumours designated as anaplastic gangliogliomas resolved into other CNS WHO diagnoses, most commonly pleomorphic xanthoastrocytoma (16/54), glioblastoma, isocitrate dehydrogenase protein (IDH) wild type and diffuse paediatric-type high-grade glioma, H3 wild type and IDH wild type (11 and 2/54), followed by low-grade glial or glioneuronal tumours including pilocytic astrocytoma, dysembryoplastic neuroepithelial tumour and diffuse leptomeningeal glioneuronal tumour (5/54), IDH mutant astrocytoma (4/54) and others (6/54). A subset of tumours (10/54) was not assignable to a CNS WHO diagnosis, and common molecular profiles pointing to a separate entity were not evident.In summary, we show that tumours histologically diagnosed as anaplastic ganglioglioma comprise a wide spectrum of CNS WHO tumour types with different prognostic and therapeutic implications. We therefore suggest assigning this designation with caution and recommend comprehensive molecular workup.

Pancreatic ductal carcinoma (PDAC) is a highly lethal cancer, and early detection and accurate staging are critical to prolonging survival. PDAC typically has a prominent stroma including cancer-associated fibroblasts that express fibroblast activation protein (FAP). FAP is a new target molecule for PET imaging of various tumors. In this retrospective study, we describe the clinical impact of PET/CT imaging using 68Ga-labeled FAP-inhibitors (68Ga-FAPI PET/CT) in 19 patients with PDAC (7 primary, 12 progressive/recurrent). Methods: All patients underwent contrast-enhanced CT (ceCT) for TNM staging before 68Ga-FAPI PET/CT imaging. PET scans were acquired 60 min after administration of 150–250 MBq of 68Ga-labeled FAP-specific tracers. To characterize 68Ga-FAPI uptake over time, additional scans after 10 or 180 min were acquired in 6 patients. SUVmax and SUVmean values of PDAC manifestations and healthy organs were analyzed. The tumor burden according to 68Ga-FAPI PET/CT was compared with TNM staging based on ceCT and changes in oncologic management were recorded. Results: Compared with ceCT, 68Ga-FAPI PET/CT results led to changes in TNM staging in 10 of 19 patients. Eight of 12 patients with recurrent/progressive disease were upstaged, 1 was downstaged, and 3 had no change. In newly diagnosed PDAC, 1 of 7 patients was upstaged, and the staging of 6 patients did not change. Changes in oncologic management occurred in 7 patients. Markedly elevated uptake of 68Ga-FAPI in PDAC manifestations after 1 h was seen in most cases. Differentiation from pancreatitis based on static imaging 1 h after injection was challenging. With respect to imaging after multiple time points, PDAC and pancreatitis showed a trend for differential uptake kinetics. Conclusion:68Ga-FAPI PET/CT led to restaging in half of the patients with PDAC and most patients with recurrent disease compared with standard of care imaging. The clinical value of 68Ga-FAPI PET/CT should be further investigated.

Medulloblastomas are malignant embryonal brain tumours that may harbour mutations in histone-modifying genes, while mutations in histone genes have not been detected to date. We here describe the first SHH medulloblastoma with H3 K27M mutation. This may have diagnostic implications as H3 K27M mutations are the hallmark of diffuse midline gliomas, H3 K27M mutant, WHO grade IV. Medulloblastomas arise in midline structures and thus must not be mistaken for DMG when using an antibody detecting the H3 K27M mutation.

According to the WHO classification, ependymal tumors are classified as subependymomas, myxopapillary ependymomas, classic ependymomas, anaplastic ependymomas, and RELA-fusion-positive ependymomas (RELA-EPN). Among classic ependymomas, the WHO defines rare histological variants, i.e., the clear cell, papillary, and tanycytic ependymoma. In parallel, global DNA methylation patterns distinguish nine molecular groups, some of which tightly overlap with histopathological subgroups. However, the match of the aforementioned histological variants to DNA methylation classes remains unclear. We analyzed histomorphology, clinical parameters, and global DNA methylation of tumors with the initial histological diagnoses of tanycytic (n = 12), clear cell (n = 14), or papillary ependymoma (n = 19). Forty percent of these tumors did not match to the epigenetic profile of ependymomas, using a previously published DNA methylation-based classifier for brain tumors. Instead, they were classified as low-grade glioma (n = 3), plexus tumor (n = 2), CNS high-grade neuroepithelial tumor with MN1 alteration (n = 2), papillary tumor of the pineal region (n = 2), neurocytoma (n = 1), or did not match to any known brain tumor methylation class (n = 8). Overall, integrated diagnosis had to be changed in 35.6% of cases as compared to the initial diagnosis. Among the tumors molecularly classified as ependymoma (27/45 cases), tanycytic ependymomas were mostly located in the spine (5/7 cases) and matched to spinal or myxopapillary ependymoma. 6/8 clear cell ependymomas were found supratentorially and fell into the methylation class of RELA-EPN. Papillary ependymomas with a positive ependymoma match (12/19 cases) showed either a 'papillary' (n = 5), a 'trabecular' (n = 1), or a 'pseudo-papillary' (n = 6) growth pattern. The papillary growth pattern was strongly associated with the methylation class B of posterior fossa ependymoma (PFB, 5/5 cases) and tumors displayed DNA methylation sites that were significantly different when compared to PFB ependymomas without papillary growth. Tumors with pseudo-papillary histology matched to the methylation class of myxopapillary ependymoma (4/6 cases), whereas the trabecular case was anatomically and molecularly a spinal ependymoma. Our results show that the diagnosis of histological ependymoma variants is challenging and epigenetic profiles may improve diagnostic accuracy of these cases. Whereas clear cell and papillary ependymomas display correlations between localization, histology, and methylation, tanycytic ependymoma does not represent a molecularly distinct subgroup.

Molecular markers have become pivotal in brain tumor diagnostics. Mutational analyses by targeted next-generation sequencing of DNA and array-based DNA methylation assessment with copy number analyses are increasingly being used in routine diagnostics. However, the broad variety of gene fusions occurring in brain tumors is marginally covered by these technologies and often only assessed by targeted assays. Here, we assessed the feasibility and clinical value of investigating gene fusions in formalin-fixed paraffin-embedded (FFPE) tumor tissues by next-generation mRNA sequencing in a routine diagnostic setting. After establishment and optimization of a workflow applicable in a routine setting, prospective diagnostic application in a neuropathology department for 26 months yielded relevant fusions in 66 out of 101 (65%) analyzed cases. In 43 (43%) cases, the fusions were of decisive diagnostic relevance and in 40 (40%) cases the fusion genes rendered a druggable target. A major strength of this approach was its ability to detect fusions beyond the canonical alterations for a given entity, and the unbiased search for any fusion event in cases with uncertain diagnosis and, thus, uncertain spectrum of expected fusions. This included both rare variants of established fusions which had evaded prior targeted analyses as well as the detection of previously unreported fusion events. While the impact of fusion detection on diagnostics is highly relevant, it is especially the detection of "druggable" fusions which will most likely provide direct benefit to the patients. The wider application of this approach for unbiased fusion identification therefore promises to be a major advance in identifying alterations with immediate impact on patient care.

Purpose: Pilocytic astrocytoma is the most common childhood brain tumor, characterized by constitutive MAPK activation. MAPK signaling induces oncogene-induced senescence (OIS), which may cause unpredictable growth behavior of pilocytic astrocytomas. The senescence-associated secretory phenotype (SASP) has been shown to regulate OIS, but its role in pilocytic astrocytoma remains unknown. Experimental Design: The patient-derived pilocytic astrocytoma cell culture model, DKFZ-BT66, was used to demonstrate presence of the SASP and analyze its impact on OIS in pilocytic astrocytoma. The model allows for doxycycline-inducible switching between proliferation and OIS. Both states were studied using gene expression profiling (GEP), Western blot, ELISA, and cell viability testing. Primary pilocytic astrocytoma tumors were analyzed by GEP and multiplex assay. Results: SASP factors were upregulated in primary human and murine pilocytic astrocytoma and during OIS in DKFZ-BT66 cells. Conditioned medium induced growth arrest of proliferating pilocytic astrocytoma cells. The SASP factors IL1B and IL6 were upregulated in primary pilocytic astrocytoma, and both pathways were regulated during OIS in DKFZ-BT66. Stimulation with rIL1B but not rIL6 reduced growth of DKFZ-BT66 cells and induced the SASP. Anti-inflammatory treatment with dexamethasone induced regrowth of senescent cells and inhibited the SASP. Senescent DKFZ-BT66 cells responded to senolytic BCL2 inhibitors. High IL1B and SASP expression in pilocytic astrocytoma tumors was associated with favorable progression-free survival. Conclusions: We provide evidence for the SASP regulating OIS in pediatric pilocytic astrocytoma, with IL1B as a relevant mediator. SASP expression could enable prediction of progression in patients with pilocytic astrocytoma. Further investigation of the SASP driving the unpredictable growth of pilocytic astrocytomas, and its possible therapeutic application, is warranted.

Pediatric central nervous system (CNS) tumors differ substantially from their adult counterparts, are marked by considerable molecular and clinical heterogeneity, and diagnosis through histopathology alone can be challenging. Using DNA methylation-based CNS tumor classification in combination with copy number and RNAseq analysis, we identify a rare, novel pediatric CNS tumor type (n=32) which is characterized by focal high-level amplification and consecutive overexpression of one of the PLAG family genes – PLAGL1 or PLAGL2. It is epigenetically divergent from other known tumor types such as high-grade gliomas, medulloblastomas, embryonal tumors, or CNS sarcomas. The wide range of original histopathologic diagnosis rendered attests to their polyphenotypic nature in terms of morphology. We suggest that these tumors may arise from early to intermediate neural progenitor cells with some neuronal commitment. Using ChIPseq data, we show that both PLAGL1 and PLAGL2 act as transcription factors for: i) the oncogenic kinase RET, a potential drug target, that was overexpressed in our cohort; ii) components of the Wnt/β-Catenin pathway; iii) a set of imprinted genes, reported to regulate the imprinted gene network in mouse models, that was deregulated in the PLAGL-amplified tumors. Consequently, a 250-gene expression PLAGL-signature indicated dysregulation of imprinting control and differentiation/development as a prominent feature. We report differences regarding age and sex distribution between PLAGL1- and PLAGL2-amplified tumors and shed light on differences in clinical behavior and outcomes between these subtypes in male and female patients. PLAGL1-amplified tumors were more prevalent in school-age children and teenagers, while PLAGL2-amplified cases occurred in very young patients. Kaplan-Meier analysis showed a trend towards a more favorable outcome in patients with PLAGL1-amplified tumors and in female patients. Survival rates remained constant after 5 years with a five-/ten-year overall survival of 75% for PLAGL1, 24% for PLAGL2, 18% for male patients, and 88% for female patients.

Diffuse midline gliomas (DMGs) are malignant gliomas that arise in the midline structures of the central nervous system. Due to their aggressive and diffuse growth and a two-year survival rate of less than 10%, DMGs are assigned to CNS WHO grade 4. Depending on the localization, median age of patients is about 11‒20 years. Genetically, most tumors are defined by a K28M-mutation in one of the highly homologous genes encoding histone protein H3. Since DMGs most frequently occur in pons and thalamus, comparatively little is known about spinal DMGs. Therefore, we histologically, molecularly, and clinically characterized spinal DMGs and analyzed, in which aspects they differ from DMGs of other localizations. Our cohort currently consists of 25 spinal DMGs and 40 pontine/thalamic reference cases. Histological, immunohistochemical and molecular analyses (DNA methylation, DNA panel sequencing) were done from FFPE tissue. Spinal DMGs were histologically very heterogeneous, both regarding different areas of single tumors as well as in comparison to other spinal and reference cases. First cluster analyses of DNA methylation data indicated a separation into three main clusters enriched for pontine, thalamic or spinal cases. The cluster enriched for spinal cases contained many tumors from elderly patients. Overall, mean age of patients with spinal DMGs was 28 years. Patients were significantly older than those with pontine DMGs. 19/20 spinal DMGs were H3-3A K28M-mutant, while one tumor had an H3-2B mutation. 4/19 (21%) spinal DMGs had mutations in FGFR1, and 6/10 (60%) in NF1. Three tumors had KRAS or BRAF mutations. In summary, first analyses suggest slight histological differences of spinal DMGs compared to DMGs of other localizations. Preliminary cluster analyses of DNA methylation data showed an enrichment of clusters for different localizations. About one third of spinal DMGs had mutations in a gene associated with the MAPK-signaling pathway.

Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications., This work was funded by Deutsche Krebshilfe grant 70112499, the NCT Heidelberg and an Illumina Medical Research Grant. Part of this work was funded by the National Institute of Health Research (to S.B. and Z.J.) and to UCLH Biomedical research centre (BRC399/NS/RB/101410). Human tissues were obtained from University College London NHS Foundation Trust as part of the UK Brain Archive Information Network (BRAIN UK, Ref: 18/004) which is funded by the Medical Research Council and Brain Tumour Research UK. The methylation profiling at NYU is supported by a grant from the Friedberg Charitable Foundation (to M.Sn.). M.Mi. would like to thank the Luxembourg National Research Fond (FNR) for the support (FNR PEARL P16/BM/11192868 grant). Open Access funding enabled and organized by Projekt DEAL.

Aims KIAA1549-BRAF fusions occur in certain brain tumours and provide druggable targets due to a constitutive activation of the MAP-kinase pathway. We introduce workflows for calling the KIAA1549-BRAF fusion from DNA methylation-array-derived copy number as well as DNA panel sequencing data. Methods Copy number profiles were analysed by automated screening and visual verification of a tandem duplication on chromosome 7q34, indicative of the KIAA1549-BRAF fusion. Pilocytic astrocytomas of the ICGC cohort with known fusion status were used for validation. KIAA1549-BRAF fusions were called from DNA panel sequencing data using the fusion callers Manta, Arriba with modified filtering criteria and deFuse. We screened DNA methylation and panel sequencing data of 7790 specimens from brain tumour and sarcoma entities. Results We identified the fusion in 337 brain tumours with both DNA methylation and panel sequencing data. Among these, we detected the fusion from copy number data in 84% and from DNA panel sequencing data in more than 90% using Arriba with modified filters. While in 74% the KIAA1549-BRAF fusion was detected from both methylation-array-derived copy number and panel sequencing data, in 9% it was detected from copy number data only and in 16% from panel data only. The fusion was almost exclusively found in pilocytic astrocytomas, diffuse leptomeningeal glioneuronal tumours and high-grade astrocytomas with piloid features. Conclusions The KIAA1549-BRAF fusion can be reliably detected from either DNA methylation array or DNA panel data. The use of both methods is recommended for the most sensitive detection of this diagnostically and therapeutically important marker.

Recently, we described a machine learning approach for classification of central nervous system tumors based on the analysis of genome-wide DNA methylation patterns [6]. Here, we report on DNA methylation-based central nervous system (CNS) tumor diagnostics conducted in our institution between the years 2015 and 2018. In this period, more than 1000 tumors from the neurosurgical departments in Heidelberg and Mannheim and more than 1000 tumors referred from external institutions were subjected to DNA methylation analysis for diagnostic purposes. We describe our current approach to the integrated diagnosis of CNS tumors with a focus on constellations with conflicts between morphological and molecular genetic findings. We further describe the benefit of integrating DNA copy-number alterations into diagnostic considerations and provide a catalog of copy-number changes for individual DNA methylation classes. We also point to several pitfalls accompanying the diagnostic implementation of DNA methylation profiling and give practical suggestions for recurring diagnostic scenarios. Electronic supplementary material The online version of this article (10.1007/s00401-018-1879-y) contains supplementary material, which is available to authorized users.

Ependymomas encompass multiple clinically relevant tumor types based on localization, genetic alterations, as well as epigenetic and transcriptomic profiles. Distinct global DNA methylation signatures serve as the most powerful diagnostic tool to distinguish these types. The methylation class of spinal ependymomas (SP-EPN) comprises mostly WHO°II tumors with slow progression and incomplete surgical resection rate. Molecular data of SP-EPN are scarce and clear treatment recommendations are lacking although these neoplasms represent the most common intramedullary tumors in children and adults. The only known recurrent genetic events in SP-EPN are the loss of chromosome 22q and mutations of the NF2 gene. However, data on the frequency of NF2 mutations range from 16 % to 71 % and originate from small series that lack epigenetic or transcriptomic characterization. Furthermore, it remains unclear whether SP-EPN with germline or sporadic NF2 mutation or with NF2 wild type status display clinical and other molecular differences. Finally, the underlying genomic and transcriptomic changes of SP-EPN without NF2 mutations are fully unclear. To provide a comprehensive molecular profile of SP-EPN, we integrated genomic and epigenetic analyses and clinical data of 170 cases. Unsupervised hierarchical clustering and t-SNE analyses of methylation data revealed three distinct molecular SP-EPN subtypes. Of the three subtypes, only subtype 1 and subtype 2 contained tumors with NF2 mutations, either as previously known germline mutations or as sporadic mutations without evidence for a syndromic disease (p< 0.0001). Besides the lack of NF2 mutations, subtype 3 tumors showed a higher frequency of MGMT promoter methylation (p= 0.0015) and occurred in significantly older patients compared to tumors of subtypes 1 and 2 (p= 0.0038). Further investigations such as whole-exome sequencing, copy number variation profiling, gene expression analysis, and histological evaluation are ongoing and will add to the picture of molecular and clinical heterogeneity within SP-EPN.

The cerebellum arguably constitutes one of the best characterized central nervous circuits, and its structure, cellular function, and histogenesis have been described in exceptional quantitative detail. A notable exception to this is the development of its inhibitory interneurons, and in particular the extensive migrations of future basket and stellate cells. Here, we used acute slices from 8-day-old mice to assess the migration of Pax2-EGFP-tagged precursors of these cells en route to the molecular layer during their transit through the nascent cerebellar cortex. We document that movement of these cells is highly directed. Their speed and directional persistence are larger in the nascent granule cell layer than in the molecular layer. And they migrate periodically, with periods of effective, directed translocation separated by bouts of rather local movement. Finally, we document that the arrangement of these cells in the adult molecular layer is characterized by clustering. These data are discussed with a focus on potential generative mechanisms for the developmental pattern observed.

Embryonal tumors with multilayered rosettes (ETMRs) have recently been described as a new entity of rare pediatric brain tumors with a fatal outcome. We show here that ETMRs are characterized by a parallel activation of Shh and Wnt signaling. Co-activation of these pathways in mouse neural precursors is sufficient to induce ETMR-like tumors in vivo that resemble their human counterparts on the basis of histology and global gene-expression analyses, and that point to apical radial glia cells as the possible tumor cell of origin. Overexpression of LIN28A, which is a hallmark of human ETMRs, augments Sonic-hedgehog (Shh) and Wnt signaling in these precursor cells through the downregulation of let7-miRNA, and LIN28A/let7a interaction with the Shh pathway was detected at the level of Gli mRNA. Finally, human ETMR cells that were transplanted into immunocompromised host mice were responsive to the SHH inhibitor arsenic trioxide (ATO). Our work provides a novel mouse model in which to study this tumor type, demonstrates the driving role of Wnt and Shh activation in the growth of ETMRs and proposes downstream inhibition of Shh signaling as a therapeutic option for patients with ETMRs.

Summary Background The WHO classification of brain tumours describes 15 subtypes of meningioma. Nine of these subtypes are allotted to WHO grade I, and three each to grade II and grade III. Grading is based solely on histology, with an absence of molecular markers. Although the existing classification and grading approach is of prognostic value, it harbours shortcomings such as ill-defined parameters for subtypes and grading criteria prone to arbitrary judgment. In this study, we aimed for a comprehensive characterisation of the entire molecular genetic landscape of meningioma to identify biologically and clinically relevant subgroups. Methods In this multicentre, retrospective analysis, we investigated genome-wide DNA methylation patterns of meningiomas from ten European academic neuro-oncology centres to identify distinct methylation classes of meningiomas. The methylation classes were further characterised by DNA copy number analysis, mutational profiling, and RNA sequencing. Methylation classes were analysed for progression-free survival outcomes by the Kaplan-Meier method. The DNA methylation-based and WHO classification schema were compared using the Brier prediction score, analysed in an independent cohort with WHO grading, progression-free survival, and disease-specific survival data available, collected at the Medical University Vienna (Vienna, Austria), assessing methylation patterns with an alternative methylation chip. Findings We retrospectively collected 497 meningiomas along with 309 samples of other extra-axial skull tumours that might histologically mimic meningioma variants. Unsupervised clustering of DNA methylation data clearly segregated all meningiomas from other skull tumours. We generated genome-wide DNA methylation profiles from all 497 meningioma samples. DNA methylation profiling distinguished six distinct clinically relevant methylation classes associated with typical mutational, cytogenetic, and gene expression patterns. Compared with WHO grading, classification by individual and combined methylation classes more accurately identifies patients at high risk of disease progression in tumours with WHO grade I histology, and patients at lower risk of recurrence among WHO grade II tumours (p=0·0096) from the Brier prediction test). We validated this finding in our independent cohort of 140 patients with meningioma. Interpretation DNA methylation-based meningioma classification captures clinically more homogenous groups and has a higher power for predicting tumour recurrence and prognosis than the WHO classification. The approach presented here is potentially very useful for stratifying meningioma patients to observation-only or adjuvant treatment groups. We consider methylation-based tumour classification highly relevant for the future diagnosis and treatment of meningioma. Funding German Cancer Aid, Else Kroner-Fresenius Foundation, and DKFZ/Heidelberg Institute of Personalized Oncology/Precision Oncology Program.

Targeting fibroblast activation protein (FAP) is a new diagnostic approach allowing the visualization of tumor stroma. Here, we applied FAP-specific PET imaging to gliomas. We analyzed the target affinity and specificity of two FAP ligands (FAPI-02 and FAPI-04) in vitro, and the pharmacokinetics and biodistribution in mice in vivo. Clinically, we used 68Ga-labeled FAPI-02/04 for PET imaging in 18 glioma patients (five IDH-mutant gliomas, 13 IDH-wildtype glioblastomas). For binding studies with 177Lu-radiolabeled FAPI-02/04, we used the glioblastoma cell line U87MG, FAP-transfected fibrosarcoma cells, and CD26-transfected human embryonic kidney cells. For pharmacokinetic and biodistribution studies, U87MG-xenografted mice were injected with 68Ga-labeled compounds followed by small-animal PET imaging and 177Lu-labeled FAPI-02/04, respectively. Clinical PET/CT scans were performed 30 min post intravenous administration of 68Ga-FAPI-02/04. PET and MRI scans were co-registrated. Immunohistochemistry was done on 14 gliomas using a FAP-specific antibody. FAPI-02 and FAPI-04 showed high binding specificity to FAP. FAPI-04 demonstrated higher tumor accumulation and delayed elimination compared with FAPI-02 in preclinical studies. IDH-wildtype glioblastomas and grade III/IV, but not grade II, IDH-mutant gliomas showed elevated tracer uptake. In glioblastomas, we observed spots with increased uptake in projection on contrast-enhancing areas. Immunohistochemistry showed FAP-positive cells with mainly elongated cell bodies and perivascular FAP-positive cells in glioblastomas and an anaplastic IDH-mutant astrocytoma. Using FAP-specific PET imaging, increased tracer uptake in IDH-wildtype glioblastomas and high-grade IDH-mutant astrocytomas, but not in diffuse astrocytomas, may allow non-invasive distinction between low-grade IDH-mutant and high-grade gliomas. Therefore, FAP-specific imaging in gliomas may be useful for follow-up studies although further clinical evaluation is required.

Pediatric low-grade gliomas (pLGGs) are the most common brain tumors in children. Despite recent advances in the molecular characterization of this heterogeneous set of tumors, the separation of specific tumor types is still not fully established. Pilocytic astrocytoma (PA; WHO grade I) and pleomorphic xanthoastrocytoma (PXA; WHO grade II) are two pLGG types that can be difficult to distinguish based on histology alone. Even though their clinical course is different, they are often grouped as ‘pLGG’ in clinical trials (and therefore treated similarly). Based on a cohort of 89 human pediatric tumor samples, we show that PAs and PXAs have clearly distinct methylation and transcriptome profiles. The difference in gene expression is mainly caused by cell cycle- and development-associated genes, suggesting a key difference in the regulatory circuits involved in tumor growth. In addition to BRAF V600E, we found NTRK fusions and a previously unknown EGFR:BRAF fusion as mutually exclusive driving events in PXAs. Both tumor types show marked signs of immune cell infiltration, but with significant qualitative differences, which might represent therapeutic vulnerabilities. To pave the way for further research on PA and PXA, we developed corresponding mouse models using the virus-based RCAS system, which allows introduction of an oncogenic driver into immunocompetent mice for molecular and preclinical research. The murine tumors do not only histologically resemble their human counterparts but also show a similar growth behavior. Expression analysis revealed that the murine PXAs have a stronger gene signature of proliferation and immune cell infiltration compared to PAs.

LIN28B is a homologue of the RNA-binding protein LIN28A and regulates gene expression during development and carcinogenesis. It is strongly upregulated in a variety of brain tumors, such as medulloblastoma, embryonal tumor with multilayered rosettes (ETMR), atypical teratoid/rhabdoid tumor (AT/RT), or glioblastoma, but the effect of an in vivo overexpression of LIN28B on the developing central nervous system is unknown. We generated transgenic mice that either overexpressed Lin28b in Math1-positive cerebellar granule neuron precursors or in a broad range of Nestin-positive neural precursors. Sections of the cerebellar vermis from adult Math1-Cre::lsl-Lin28b mice had an additional subfissure in lobule IV. Vermes from p0 and p7 Nestin-Cre::lsl-Lin28b mice appeared normal, but we found a pronounced vermal hypersublobulation at p15 and p21 in these mice. Also, the external granule cell layer (EGL) was thicker at p15 than in controls, contained more proliferating cells, and persisted up to p21. Consistently, some Pax6- and NeuN-positive cells were present in the EGL of Nestin-Cre::lsl-Lin28b mice even at p21, and we detected more NeuN-positive granule neuron precursors in the molecular layer (ML) as compared to control. Finally, we found some residual Pax2-positive precursors of inhibitory interneurons in the ML of Nestin-Cre::lsl-Lin28b mice at p21, which have already disappeared in controls. We conclude that while overexpression of LIN28B in Nestin-positive cells does not lead to tumor formation, it results in a protracted development of granule cells and inhibitory interneurons and leads to a hypersublobulation of the cerebellar vermis.

Low-grade epilepsy-associated brain tumours (LEAT) are the second most common cause for drug-resistant, focal epilepsy, that is ganglioglioma (GG) and dysembryoplastic neuroepithelial tumours (DNT). However, molecular pathogenesis, risk factors for malignant progression and their frequent association with drug-resistant focal seizures remain poorly understood. This contrasts recent progress in understanding the molecular-genetic basis and targeted treatment options in diffuse gliomas. The Neuropathology Task Force of the International League Against Epilepsy examined available literature to identify common obstacles in diagnosis and research of LEAT. Analysis of 10 published tumour series from epilepsy surgery pointed to poor inter-rater agreement for the histopathology diagnosis. The Task Force tested this hypothesis using a web-based microscopy agreement study. In a series of 30 LEAT, 25 raters from 18 countries agreed in only 40% of cases. Highest discordance in microscopic diagnosis occurred between GG and DNT variants, when oligodendroglial-like cell patterns prevail, or ganglion cells were difficult to discriminate from pre-existing neurons. Suggesting new terminology or major histopathological criteria did not satisfactorily increase the yield of histopathology agreement in four consecutive trials. To this end, the Task Force applied the WHO 2016 strategy of integrating phenotype analysis with molecular-genetic data obtained from panel sequencing and 450k methylation arrays. This strategy was helpful to distinguish DNT from GG variants in all cases. The Task Force recommends, therefore, to further develop diagnostic panels for the integration of phenotype-genotype analysis in order to reliably classify the spectrum of LEAT, carefully characterize clinically meaningful entities and make better use of published literature.

Olfactory neuroblastoma/esthesioneuroblastoma (ONB) is an uncommon neuroectodermal neoplasm thought to arise from the olfactory epithelium. Little is known about its molecular pathogenesis. For this study, a retrospective cohort of n = 66 tumor samples with the institutional diagnosis of ONB was analyzed by immunohistochemistry, genome-wide DNA methylation profiling, copy number analysis, and in a subset, next-generation panel sequencing of 560 tumor-associated genes. DNA methylation profiles were compared to those of relevant differential diagnoses of ONB. Unsupervised hierarchical clustering analysis of DNA methylation data revealed four subgroups among institutionally diagnosed ONB. The largest group (n = 42, 64%, Core ONB) presented with classical ONB histology and no overlap with other classes upon methylation profiling-based t-distributed stochastic neighbor embedding (t-SNE) analysis. A second DNA methylation group (n = 7, 11%) with CpG island methylator phenotype (CIMP) consisted of cases with strong expression of cytokeratin, no or scarce chromogranin A expression and IDH2 hotspot mutation in all cases. T-SNE analysis clustered these cases together with sinonasal carcinoma with IDH2 mutation. Four cases (6%) formed a small group characterized by an overall high level of DNA methylation, but without CIMP. The fourth group consisted of 13 cases that had heterogeneous DNA methylation profiles and strong cytokeratin expression in most cases. In t-SNE analysis, these cases mostly grouped among sinonasal adenocarcinoma, squamous cell carcinoma, and undifferentiated carcinoma. Copy number analysis indicated highly recurrent chromosomal changes among Core ONB with a high frequency of combined loss of chromosome 1-4, 8-10, and 12. NGS sequencing did not reveal highly recurrent mutations in ONB, with the only recurrently mutated genes being TP53 and DNMT3A. In conclusion, we demonstrate that institutionally diagnosed ONB are a heterogeneous group of tumors. Expression of cytokeratin, chromogranin A, the mutational status of IDH2 as well as DNA methylation patterns may greatly aid in the precise classification of ONB.

We identified a rare undifferentiated cell population that is intermingled with the Bergmann glia of the adult murine cerebellar cortex, expresses the stem cell markers Sox2 and Nestin, and lacks markers of glial or neuronal differentiation. Interestingly, such Sox2+ S100− cells of the adult cerebellum expanded after adequate physiological stimuli in mice (exercise), and Sox2+ precursors acquired positivity for the neuronal marker NeuN over time and integrated into cellular networks. In human patients, SOX2+ S100− cells similarly increased in number after relevant pathological insults (infarcts), suggesting a similar expansion of cells that lack terminal glial differentiation.

Previous studies have evaluated the role of miRNAs in cancer initiation and progression. MiR-34a was found to be downregulated in several tumors, including medulloblastomas. Here we employed targeted transgenesis to analyze the function of miR-34a in vivo. We generated mice with a constitutive deletion of the miR-34a gene. These mice were devoid of mir-34a expression in all analyzed tissues, but were viable and fertile. A comprehensive standardized phenotypic analysis including more than 300 single parameters revealed no apparent phenotype. Analysis of miR-34a expression in human medulloblastomas and medulloblastoma cell lines revealed significantly lower levels than in normal human cerebellum. Re-expression of miR-34a in human medulloblastoma cells reduced cell viability and proliferation, induced apoptosis and downregulated the miR-34a target genes, MYCN and SIRT1. Activation of the Shh pathway by targeting SmoA1 transgene overexpression causes medulloblastoma in mice, which is dependent on the presence and upregulation of Mycn. Analysis of miR-34a in medulloblastomas derived from ND2:SmoA1(tg) mice revealed significant suppression of miR-34a compared to normal cerebellum. Tumor incidence was significantly increased and tumor formation was significantly accelerated in mice transgenic for SmoA1 and lacking miR-34a. Interestingly, Mycn and Sirt1 were strongly expressed in medulloblastomas derived from these mice. We here demonstrate that miR-34a is dispensable for normal development, but that its loss accelerates medulloblastomagenesis. Strategies aiming to re-express miR-34a in tumors could, therefore, represent an efficient therapeutic option.

Introduction: Tumors with histological features of pilocytic astrocytoma but with increased mitotic activity and additional high grade features (i.e. microvascular proliferation, necrosis) have been designated anaplastic pilocytic astrocytomas (APA). Patients with such tumors are thought to have an [for full text, please go to the a.m. URL]