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Current therapies for medulloblastoma, a highly malignant childhood brain tumour, impose debilitating effects on the developing child, and highlight the need for molecularly targeted treatments with reduced toxicity. Previous studies have been unable to identify the full spectrum of driver genes and molecular processes that operate in medulloblastoma subgroups. Here we analyse the somatic landscape across 491 sequenced medulloblastoma samples and the molecular heterogeneity among 1,256 epigenetically analysed cases, and identify subgroup-specific driver alterations that include previously undiscovered actionable targets. Driver mutations were confidently assigned to most patients belonging to Group 3 and Group 4 medulloblastoma subgroups, greatly enhancing previous knowledge. New molecular subtypes were differentially enriched for specific driver events, including hotspot in-frame insertions that target KBTBD4 and 'enhancer hijacking' events that activate PRDM6. Thus, the application of integrative genomics to an extensive cohort of clinical samples derived from a single childhood cancer entity revealed a series of cancer genes and biologically relevant subtype diversity that represent attractive therapeutic targets for the treatment of patients with medulloblastoma.

Objective: Patients with probable dementia with Lewy bodies often have Alzheimer's disease-related pathology. Our objective was to determine the pattern of positron emission tomography tau tracer AV-1451 uptake in patients with probable dementia with Lewy bodies, compared to Alzheimer's disease, and its relationship to β-amyloid deposition on positron emission tomography. Methods: Consecutive patients with clinically probable dementia with Lewy bodies (n=19) from the Mayo Clinic Alzheimer's Disease Research Center underwent MRI, AV-1451 and Pittsburgh compound-B positron emission tomography examinations. Age and sex matched groups of Alzheimer's disease dementia (n=19) patients and clinically normal controls (n=95) from an epidemiological cohort served as a comparison groups. Atlas-based and voxel-based analyses were performed. Results: The Alzheimer's disease dementia group had significantly higher AV-1451 uptake than the probable dementia with Lewy bodies group, and medial temporal uptake completely distinguished Alzheimer's disease dementia from probable dementia with Lewy bodies. Patients with probable dementia with Lewy bodies had greater AV-1451 uptake in the posterior temporoparietal and occipital cortex compared to clinically normal controls, and in probable dementia with Lewy bodies, the uptake in these regions correlated with global Pittsburgh compound-B uptake (Spearman rho= 0.63; p=0.006). Interpretation: Medial temporal lobe AV-1451 uptake distinguishes Alzheimer's disease dementia from probable dementia with Lewy bodies, which may be useful for differential diagnosis. Elevated posterior temporoparietal and occipital AV-1451 uptake in probable dementia with Lewy bodies and its association with global Pittsburgh compound-B uptake suggest an atypical pattern of tau deposition in dementia with Lewy bodies. This article is protected by copyright. All rights reserved.

Purpose To evaluate the association of multiparametric and multiregional magnetic resonance (MR) imaging features with key molecular characteristics in patients with newly diagnosed glioblastoma. Materials and Methods Retrospective data evaluation was approved by the local ethics committee, and the requirement to obtain informed consent was waived. Preoperative MR imaging features were correlated with key molecular characteristics within a single-institution cohort of 152 patients with newly diagnosed glioblastoma. Preoperative MR imaging features (n = 31) included multiparametric (anatomic and diffusion-, perfusion-, and susceptibility-weighted images) and multiregional (contrast-enhancing regions and hyperintense regions at nonenhanced fluid-attenuated inversion recovery imaging) information with histogram quantification of tumor volumes, volume ratios, apparent diffusion coefficients, cerebral blood flow, cerebral blood volume, and intratumoral susceptibility signals. Molecular characteristics determined included global DNA methylation subgroups (eg, mesenchymal, RTK I "PGFRA," RTK II "classic"), MGMT promoter methylation status, and hallmark copy number variations (EGFR, PDGFRA, MDM4, and CDK4 amplification; PTEN, CDKN2A, NF1, and RB1 loss). Univariate analyses (voxel-lesion symptom mapping for tumor location, Wilcoxon test for all other MR imaging features) and machine learning models were applied to study the strength of association and discriminative value of MR imaging features for predicting underlying molecular characteristics. Results There was no tumor location predilection for any of the assessed molecular parameters (permutation-adjusted P > .05). Univariate imaging parameter associations were noted for EGFR amplification and CDKN2A loss, with both demonstrating increased Gaussian-normalized relative cerebral blood volume and Gaussian-normalized relative cerebral blood flow values (area under the receiver operating characteristics curve: 63%-69%, false discovery rate-adjusted P < .05). Subjecting all MR imaging features to machine learning-based classification enabled prediction of EGFR amplification status and the RTK II glioblastoma subgroup with a moderate, yet significantly greater, accuracy (63% for EGFR [P < .01], 61% for RTK II [P = .01]) than prediction by chance; prediction accuracy for all other molecular parameters was not significant. Conclusion The authors found associations between established MR imaging features and molecular characteristics, although not of sufficient strength to enable generation of machine learning classification models for reliable and clinically meaningful prediction of molecular characteristics in patients with glioblastoma. © RSNA, 2016 Online supplemental material is available for this article.

// Florian Selt 1, 2 , Juliane Hohloch 1 , Thomas Hielscher 3 , Felix Sahm 4, 5 , David Capper 4, 5 , Andrey Korshunov 4, 5 Diren Usta 1 , Sebastian Brabetz 6 , Johannes Ridinger 1 , Jonas Ecker 1, 2 , Ina Oehme 1 , Jan Gronych 7, 8 , Viktoria Marquardt 9 , David Pauck 9 , Heidi Bachli 10 , Charles D. Stiles 11 , Andreas von Deimling 4, 5 , Marc Remke 9 , Martin U. Schuhmann 12 , Stefan M. Pfister 2, 6 , Tilman Brummer 13 , David T.W. Jones 6 , Olaf Witt 1, 2, * , Till Milde 1, 2, * 1 Clinical Cooperation Unit Pediatric Oncology (G340), German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Heidelberg, Germany 2 Center for Individualized Pediatric Oncology (ZIPO) and Section of Pediatric Brain Tumors, Department of Pediatric Oncology, Hematology and Immunology, University Hospital Heidelberg, Heidelberg, Germany 3 Division of Biostatistics (C060), German Cancer Research Center (DKFZ), Heidelberg, Germany 4 Department of Neuropathology, University Hospital Heidelberg, Heidelberg, Germany 5 Clinical Cooperation Unit Neuropathology (G380), German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Heidelberg, Germany 6 Division of Pediatric Neurooncology (B062), German Cancer Research Center (DKFZ), Heidelberg, Germany, and German Cancer Consortium (DKTK), Heidelberg, Germany 7 Division of Molecular Genetics (B060), German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Heidelberg, Germany 8 AbbVie Deutschland GmbH & Co. KG, Medical Immunology, Wiesbaden, Germany (current affiliation) 9 Department of Pediatric Oncology, Hematology, and Clinical Immunology, Medical Faculty, University Hospital Dusseldorf, Germany, and Department of Pediatric Neuro-Oncogenomics, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany 10 Department of Neurosurgery, University Hospital Heidelberg, Heidelberg, Germany 11 Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA 12 Department of Neurosurgery, University Hospital Tubingen, Tubingen, Germany 13 Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University and University Medical Centre, Freiburg, Germany * These authors have contributed equally to this work Correspondence to: Florian Selt, email: f. selt@dkfz.de Keywords: pediatric low grade glioma, pilocytic astrocytoma, KIAA1549:BRAF-fusion, oncogene-induced senescence (OIS), MAPK-inhibitors Received: November 18, 2016 Accepted: November 23, 2016 Published: December 17, 2016 ABSTRACT Pilocytic astrocytoma (PA) is the most frequent pediatric brain tumor. Activation of the MAPK pathway is well established as the oncogenic driver of the disease. It is most frequently caused by KIAA1549:BRAF fusions, and leads to oncogene induced senescence (OIS). OIS is thought to be a major reason for growth arrest of PA cells in vitro and in vivo , preventing establishment of PA cultures. Hence, valid preclinical models are currently very limited, but preclinical testing of new compounds is urgently needed. We transduced the PA short-term culture DKFZ-BT66 derived from the PA of a 2-year old patient with a doxycycline-inducible system coding for Simian Vacuolating Virus 40 Large T Antigen (SV40-TAg). SV40-TAg inhibits TP53/CDKN1A and CDKN2A/RB1, two pathways critical for OIS induction and maintenance. DNA methylation array and KIAA1549:BRAF fusion analysis confirmed pilocytic astrocytoma identity of DKFZ-BT66 cells after establishment. Readouts were analyzed in proliferating as well as senescent states, including cell counts, viability, cell cycle analysis, expression of SV40-Tag, CDKN2A (p16), CDKN1A (p21), and TP53 (p53) protein, and gene-expression profiling. Selected MAPK inhibitors (MAPKi) including clinically available MEK inhibitors (MEKi) were tested in vitro . Expression of SV40-TAg enabled the cells to bypass OIS and to resume proliferation with a mean doubling time of 45h allowing for propagation and long-term culture. Withdrawal of doxycycline led to an immediate decrease of SV40-TAg expression, appearance of senescent morphology, upregulation of CDKI proteins and a subsequent G1 growth arrest in line with the re-induction of senescence. DKFZ-BT66 cells still underwent replicative senescence that was overcome by TERT expression. Testing of a set of MAPKi revealed differential responses in DKFZ-BT66. MEKi efficiently inhibited MAPK signaling at clinically achievable concentrations, while BRAF V600E- and RAF Type II inhibitors showed paradoxical activation. Taken together, we have established the first patient-derived long term expandable PA cell line expressing the KIAA1549:BRAF-fusion suitable for preclinical drug testing.

Epileptogenic tumors affecting children and young adults are a morphologically diverse collection of neuroepithelial neoplasms that, as a group, exhibit varying levels of glial and/or neuronal differentiation. Recent advances in molecular profiling technology, including comprehensive DNA sequencing and methylation analysis, have enabled the application of more precise and biologically relevant classification schemes to these tumors. In this report, we describe a morphologically and molecularly distinct epileptogenic neoplasm, the polymorphous low-grade neuroepithelial tumor of the young (PLNTY), which likely accounts for a sizable portion of oligodendroglioma-like tumors affecting the pediatric population. Characteristic microscopic findings most notably include infiltrative growth, the invariable presence of oligodendroglioma-like cellular components, and intense immunolabeling for cluster of differentiation 34 (CD34). Moreover, integrative molecular profiling reveals a distinct DNA methylation signature for PLNTYs, along with frequent genetic abnormalities involving either B-Raf proto-oncogene (BRAF) or fibroblast growth factor receptors 2 and 3 (FGFR2, FGFR3). These findings suggest that PLNTY represents a distinct biological entity within the larger spectrum of pediatric, low-grade neuroepithelial tumors. Electronic supplementary material The online version of this article (doi:10.1007/s00401-016-1639-9) contains supplementary material, which is available to authorized users.

The mediodorsal nucleus of the thalamus (MDT) is one of the largest thalamic nuclei and is a typical association nucleus, participating in several cortico-subcortical networks, primarily those involving the prefrontal cortex.1–3 Pathologic involvement of the MDT or its cortical connections contributes to the cognitive, emotional, and behavioral manifestations of stroke4–6; metabolic disorders, such as Wernicke encephalopathy7; inflammatory disorders, such as multiple sclerosis8; prion disorders, such as fatal familial insomnia9 and Creutzfeldt-Jakob disease10; and neurodegenerative diseases, such as Alzheimer disease,11 dementia with Lewy bodies,12 and Parkinson disease.13 The MDT also participates in the pathophysiology of epilepsy14,15 and schizophrenia.16 The following case shows that its involvement affects wide cortical areas and produces disabling symptoms that may not be associated with objective abnormalities in neuropsychometric testing.

Introduction: The leucine-rich repeat kinase 2 (LRRK2) gene contains several variants that cause Parkinson's disease (PD) and others that modify PD risk. However, little is known about the role of LRRK2 in dementia with Lewy bodies (DLB). Aims of this study were to screen DLB patients for pathogenic LRRK2 variants and to evaluate associations between common LRRK2 variants and risk of DLB. Methods: 417 clinical DLB patients and 1790 controls were included in the primary analysis. Additionally, 355 Lewy body disease patients assessed as having a high likelihood of clinical DLB based on neuropathological findings were included in secondary analysis. Seven pathogenic LRRK2 variants were assessed in patients, while 17 common LRRK2 exonic variants and 1 GWAS-nominated common LRRK2 PD-risk variant were evaluated for association with DLB. Results: We identified carriers of 2 different pathogenic LRRK2 variants. One clinical DLB patient was a p.G2019S carrier, while in the pathological high likelihood DLB series there was one carrier of the p.R1441C mutation. However, examination of clinical records revealed the p.R1441C carrier to have PD with dementia. Evaluation of common variants did not reveal any associations with DLB risk after multiple testing adjustment. However, a non-significant trend similar to that previously reported for PD was observed for the protective p.N551K-R1398H-K1423K haplotype in the clinical DLB series (OR: 0.76, P = 0.061). Conclusion: LRRK2 does not appear to play a major role in DLB, however further study of p.G2019S and the p.N551K-R1398H-K1423K haplotype is warranted to better understand their involvement in determining DLB risk. (C) 2016 Elsevier Ltd. All rights reserved.

// Theophilos Tzaridis 1 , Till Milde 2, 3, 4 , Kristian W. Pajtler 1, 2, 4 , Sebastian Bender 1 , David T. W. Jones 1 , Simone Muller 5 , Andrea Wittmann 1 , Magdalena Schlotter 6 , Andreas E. Kulozik 2 , Peter Lichter 6 , V. Peter Collins 7 , Olaf Witt 2, 3, 4 , Marcel Kool 1 , Andrey Korshunov 8, 9 , Stefan M. Pfister 1, 2, 4 , Hendrik Witt 1, 2, 4 1 Division of Pediatric Neurooncology (B062), German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany 2 Department of Pediatric Oncology, Hematology and Immunology, Children’s Hospital, University of Heidelberg, Heidelberg, Germany 3 Clinical Cooperation Unit Pediatric Oncology (G340), German Cancer Research Center (DKFZ), Heidelberg, Germany 4 National Center for Tumor diseases (NCT), Clinical Trial Center, Heidelberg, Germany 5 Department of Cardiology, Angiology and Pulmology, University of Heidelberg, Heidelberg, Germany 6 Division of Molecular Genetics (B060), German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, Germany 7 Division of Molecular Histopathology, Department of Pathology, University of Cambridge, Cambridge UK 8 Clinical Cooperation Unit Neuropathology (G380), German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, Germany 9 Department of Neuropathology, Heidelberg University Hospital, Heidelberg, Germany Correspondence to: Hendrik Witt, email: h.witt@dkfz.dew Stefan M. Pfister, email: s.pfister@dkfz.de Keywords: ependymoma, p53, RELA, Actinomycin-D Received: February 02, 2016 Accepted: July 27, 2016 Published: August 20, 2016 ABSTRACT Ependymomas in children can arise throughout all compartments of the central nervous system (CNS). Highly malignant paediatric ependymoma subtypes are Group A tumours of the posterior fossa (PF-EPN-A) and RELA -fusion positive (ST-EPN-RELA) tumours in the supratentorial compartment. It was repeatedly reported in smaller series that accumulation of p53 is frequently observed in ependymomas and that immunohistochemical staining correlates with poor clinical outcome, while TP53 mutations are rare. Our TP53 mutation analysis of 130 primary ependymomas identified a mutation rate of only 3%. Immunohistochemical analysis of 398 ependymomas confirmed previous results correlating the accumulation of p53 with inferior outcome. Among the p53-positive ependymomas, the vast majority exhibited a RELA fusion leading to the hypothesis that p53 inactivation might be linked to RELA positivity. In order to assess the potential of p53 reactivation through MDM2 inhibition in ependymoma, we evaluated the effects of Actinomycin-D and Nutlin-3 treatment in two preclinical ependymoma models representing the high-risk subtypes PF-EPN-A and ST-EPN-RELA. The IC-50 of the agent as determined by metabolic activity assays was in the lower nano-molar range (0.2–0.7 nM). Transcriptome analyses of high-dose (100 nM), low-dose (5 nM) and non-treated cells revealed re-expression of p53 dependent genes including p53 upregulated modulator of apoptosis ( PUMA ) after low-dose treatment. At the protein level, we validated the Actinomycin-D induced upregulation of PUMA, and of p53 interaction partners MDM2 and p21. Proapoptotic effects of low-dose application of the agent were confirmed by flow cytometry. Thus, Actinomycin-D could constitute a promising therapeutic option for ST-EPN-RELA ependymoma patients, whose tumours frequently exhibit p53 inactivation.

Schwannomatosis is a genetic disorder characterized by the occurrence of multiple peripheral schwannomas. Segmental schwannomatosis is diagnosed when schwannomas are restricted to 1 extremity and is thought to be caused by genetic mosaicism. We studied 5 patients with segmental schwannomatosis through microstructural magnetic resonance neurography and mutation analysis of NF2, SMARCB1, and LZTR1. In 4 of 5 patients, subtle fascicular nerve lesions were detected in clinically unaffected extremities. Two patients exhibited LZTR1 germline mutations. This appears contrary to a simple concept of genetic mosaicism and suggests more complex and heterogeneous mechanisms underlying the phenotype of segmental schwannomatosis than previously thought. Ann Neurol 2016;80:625-628.

Rhabdoid phenotype and loss of SMARCB1 expression in a brain tumor are characteristic features of atypical teratoid/rhabdoid tumors (ATRT). Rare non-rhabdoid brain tumors showing cribriform growth pattern and SMARCB1 loss have been designated cribriform neuroepithelial tumor (CRINET). Small case series suggest that CRINETs may have a relatively favorable prognosis. However, the long-term outcome is unclear and it remains uncertain whether CRINET represents a distinct entity or a variant of ATRT. Therefore, 10 CRINETs were clinically and molecularly characterized and compared with 10 ATRTs of each of three recently described molecular subgroups (i.e. ATRT-TYR, ATRT-SHH and ATRT-MYC) using Illumina Infinium HumanMethylation450 arrays, FISH, MLPA, and sequencing. Furthermore, outcome was compared to a larger cohort of 27 children with ATRT-TYR. Median age of the 6 boys and 4 girls harboring a CRINET was 20 months (range 10-129 months). On histopathological examination, all CRINETs demonstrated a cribriform growth pattern and distinct tyrosinase staining. On unsupervised cluster analysis of methylation data, all CRINETs examined exclusively clustered within the ATRT-TYR molecular subgroup. As ATRT-TYR, CRINETs mainly showed large heterozygous 22q deletions (9/10) and SMARCB1 mutations of the other allele. In two patients, SMARCB1 mutations were also present in the germline. Estimated mean overall survival in patients with CRINETs was 125 months (95% confidence interval 100-151 months) as compared to only 53 (33-74) months in patients with ATRTs of the ATRT-TYR subgroup (Log-Rank P

// Samantha Mascelli 1 , Paolo Nozza 2 , David T.W. Jones 3 , Carole Colin 4 , Angela Pistorio 5 , Claudia Milanaccio 1 , Marcello Ravegnani 1 , Alessandro Consales 1 , Olaf Witt 6 , Giovanni Morana 7 , Armando Cama 1 , Valeria Capra 1 , Roberto Biassoni 8 , Stefan M. Pfister 3, 6 , Dominique Figarella-Branger 4, 9 , Maria Luisa Garre 10 , Alessandro Raso 1 1 Dipartimento Testa, Collo e Neuroscienze, Istituto Giannina Gaslini, 16147 Genoa, Italy 2 UOC Anatomia Patologica, Istituto Giannina Gaslini, 16147 Genoa, Italy 3 Division of Paediatric Neurooncology, German Cancer Research Center (DKFZ), Heidelberg 69120, Germany 4 CRO2 UMR_S911, Inserm, Aix-Marseille Universite, 13385 Marseille, France 5 Epidemiologia, Biostatistica e Comitati, Istituto Giannina Gaslini, 16147 Genoa, Italy 6 Department of Paediatric Oncology, Haematology and Immunology, University of Heidelberg, Heidelberg, 69120 Heidelberg, Germany 7 Neuroradiologia Istituto Giannina Gaslini, 16147 Genoa, Italy 8 Laboratory Molecular Medicine, Translational Medicine Department, Istituto Giannina Gaslini, 16147 Genoa, Italy 9 APHM, Hopital de la Timone, Service d’Anatomie Pathologique et de Neuropathologie, 13385 Marseille, France 10 Centro di Neuro-Oncologia, Istituto Giannina Gaslini, 16147 Genoa, Italy Correspondence to: Samantha Mascelli, email: SamanthaMascelli@gaslini.org Keywords: low-grade gliomas, polymorphism, pilocytic astrocytoma, paediatric, TP53 Received: March 25, 2016 Accepted: May 10, 2016 Published: June 25, 2016 ABSTRACT Pilocytic astrocytoma and ganglioglioma may occur in inaccessible or surgically difficult areas. In case of incomplete resection, the availability of biological predictors of tumour progression could be particularly important. To this end, an analysis of p53 codon 72 polymorphism and assessment of its role as prognostic marker were performed. The status of the p53 Arg72Pro polymorphism was evaluated by pyrosequencing method in a multicenter cohort of 170 paediatric patients. Genotype/phenotype associations were investigated either by means of bivariate or multivariate analyses. In the partially resected pilocytic astrocytomas, the Arg/Arg variant predicts early tumour progression (median survival time: 23.1 months) and is associated with poor event-free survival ( p value = 0.0009). This finding remains true also in case of adjuvant therapies, with a 5-year event-free survival of 30.6% for cases with Arg/Arg variant vs. 78.7% for those with other genotypes. There is no association between ganglioglioma and the polymorphism. The assessment of Arg/Arg variant could improve the management of pilocytic astrocytoma. TP53 codon 72 analysis could distinguish low-risk cases, in which surgery could be conservative, from high-risk cases needing an aggressive surgery plan.

Background The clinical course of dementia with Lewy bodies patients is heterogeneous. The ability to more accurately prognosticate survival is important. Objective The objective of this study was to investigate hippocampal volume as a predictor of survival in dementia with Lewy bodies patients. Methods Survival analysis for time from onset of cognitive symptoms to death was carried out using Cox proportional hazards models. Given their age and total intracranial volume, patients were dichotomized into low/medium (0%-66.7%) and high (66. 7%-100%) hippocampal volume categories. The models using these categories to predict survival were adjusted for field strength, APOE e4 status, and estimated onset age of cognitive problems. Results We investigated 167 consecutive patients with dementia with Lewy bodies. The median age at MRI was 72 years (interquartile range 67-76), and 80% were male. The median time from estimated first cognitive symptom to death was 7.4 years (interquartile range:5.7-10.2). Lower hippocampal volumes were significantly associated with higher risk of death (hazard ratio 1.28; 95% confidence interval 1.04-1.58; P = .024). The predicted median survival for participants with onset of cognitive symptoms at age 68 was 10.63 years (95% confidence interval 8.66-14.54) for APOE e4 negative, high hippocampal volume participants; 8.89 years (95% confidence interval 7.56-12.36) for APOE e4 positive, high hippocampal volume participants; 8.10 years (95% confidence interval 7.34-11.08) for APOE e4 negative, low/medium hippocampal volume participants; and 7.38 (95% confidence interval 6.74-9.29) years for APOE e4 positive, low/medium hippocampal volume participants. Conclusions Among patients with clinically diagnosed dementia with Lewy bodies, those with neuroimaging evidence of hippocampal atrophy have shorter survival times. © 2016 International Parkinson and Movement Disorder Society

Altered iron metabolism has been hypothesized to be associated with Alzheimer’s disease pathology, and prior work has shown associations between iron load and beta amyloid plaques. Quantitative susceptibility mapping (QSM) is a recently popularized MR technique to infer local tissue susceptibility secondary to the presence of iron as well as other minerals. Greater QSM values imply greater iron concentration in tissue. QSM has been used to study relationships between cerebral iron load and established markers of Alzheimer’s disease, however relationships remain unclear. In this work we study QSM signal characteristics and associations between susceptibility measured on QSM and established clinical and imaging markers of Alzheimer’s disease. The study included 421 participants (234 male, median age 70 years, range 34–97 years) from the Mayo Clinic Study of Aging and Alzheimer’s Disease Research Center; 296 (70%) had a diagnosis of cognitively unimpaired, 69 (16%) mild cognitive impairment, and 56 (13%) amnestic dementia. All participants had multi-echo gradient recalled echo imaging, PiB amyloid PET, and Tauvid tau PET. Variance components analysis showed that variation in cortical susceptibility across participants was low. Linear regression models were fit to assess associations with regional susceptibility. Expected increases in susceptibility were found with older age and cognitive impairment in the deep and inferior gray nuclei (pallidum, putamen, substantia nigra, subthalamic nucleus) (betas: 0.0017 to 0.0053 ppm for a 10 year increase in age, p = 0.03 to < 0.001; betas: 0.0021 to 0.0058 ppm for a 5 point decrease in Short Test of Mental Status, p = 0.003 to p < 0.001). Effect sizes in cortical regions were smaller, and the age associations were generally negative. Higher susceptibility was significantly associated with higher amyloid PET SUVR in the pallidum and putamen (betas: 0.0029 and 0.0012 ppm for a 20% increase in amyloid PET, p = 0.05 and 0.02, respectively), higher tau PET in the basal ganglia with the largest effect size in the pallidum (0.0082 ppm for a 20% increase in tau PET, p < 0.001), and with lower cortical gray matter volume in the medial temporal lobe (0.0006 ppm for a 20% decrease in volume, p = 0.03). Overall, these findings suggest that susceptibility in the deep and inferior gray nuclei, particularly the pallidum and putamen, may be a marker of cognitive decline, amyloid deposition, and off-target binding of the tau ligand. Although iron has been demonstrated in amyloid plaques and in association with neurodegeneration, it is of insufficient quantity to be reliably detected in the cortex using this implementation of QSM.

Histone H3.3 glycine 34 to arginine/valine (G34R/V) mutations drive deadly gliomas and show exquisite regional and temporal specificity, suggesting a developmental context permissive to their effects. Here, we show that 50% of G34R/V-tumours (n=95) bear activating PDGFRA mutations that display strong selection pressure at recurrence. While considered gliomas, G34R/V-tumours actually arise in GSX2/DLX-expressing interneuron progenitors, where G34R/V-mutations impair neuronal differentiation. The lineage-of-origin may facilitate PDGFRA co-option through a chromatin loop connecting PDGFRA to GSX2 regulatory elements, promoting PDGFRA overexpression and mutation. At the single-cell level, G34R/V-tumours harbour dual neuronal/astroglial identity and lack oligodendroglial programs, actively repressed by GSX2/DLX-mediated cell-fate specification. G34R/V may become dispensable for tumour maintenance, while mutant-PDGFRA is potently oncogenic. Collectively, our results open novel research avenues in deadly tumours. G34R/V-gliomas are neuronal malignancies, where interneuron progenitors are stalled in differentiation by G34R/V-mutations, and malignant gliogenesis is promoted by co-option of a potentially targetable pathway, PDGFRA signalling.

Tumors of the CNS represent one of the most complex groups of human cancer, with a vast number of different entities occurring across a spectrum of ages and anatomic locations. This heterogeneity makes accurate diagnosis challenging, with the current gold standard relying on multiple subjective elements. We recently proposed a classification algorithm based on tumor DNA methylation profiling as an objective way to assign samples to over 80 distinct molecular classes. Here we present a substantial update to our machine learning-based algorithm, with more than 170 molecular classes now being represented amongst the 5,915 samples in our reference cohort. These new classes include further subclassification of known groups such as medulloblastoma and ependymoma, as well as multiple new molecular entities described here for the first time. A further improvement is the introduction of a more rationally layered output, making use of ‘families’ of closely-related molecular classes to improve the compatibility with the current WHO classification of CNS tumors. This approach is designed to increase the clinical relevance of the primary output, while also retaining the full information content from the random forest-driven classification. Benchmarking our new algorithm by cross-validation and on an independent validation cohort indicates a retention of the excellent accuracy of diagnosis (error-rate < 4%), with a significant improvement in the proportion of confidently classifiable tumors compared with our previous tool. We believe that this approach, freely accessible through an online web portal, has the potential to enhance diagnostic precision and thereby support clinical care for brain tumor patients.

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.

INTRODUCTION The next generation of clinical trials for relapsed medulloblastoma demands a thorough understanding of the clinical behavior of relapsed tumors as well as the molecular relationship to their diagnostic counterparts. METHODS A multi-institutional molecular cohort of patient-matched (n=126 patients) diagnostic MBs and relapses/subsequent malignancies was profiled by DNA methylation array. Entity, subgroup classification, and genome-wide copy-number aberrations were assigned while parallel next-generation (whole-exome or targeted panel) sequencing on the majority of the cohort facilitated inference of somatic driver mutations. RESULTS Comprised of WNT (2%), SHH (41%), Group 3 (18%), Group 4 (39%), primary tumors retained subgroup affiliation at relapse with the notable exception of 10% of cases. The majority (8/13) of discrepant classifications were determined to be secondary glioblastomas. Additionally, rare (n=3) subgroup-switching events of Group 4 primary tumors to Group 3 relapses were identified coincident with MYC/MYCN pathway alterations. Amongst truly relapsing MBs, copy-number analyses suggest somatic clonal divergence between primary MBs and their respective relapses with Group 3 (55% of alterations shared) and Group 4 tumors (63% alterations shared) sharing a larger proportion of cytogenetic alterations compared to SHH tumors (42% alterations shared; Chi-square p-value < 0.001). Subgroup- and gene-specific patterns of conservation and divergence amongst putative driver genes were also observed. CONCLUSION Integrated molecular analysis of relapsed MB discloses potential mechanisms underlying treatment failure and disease recurrence while motivating rational implementation of relapse-specific therapies. The degree of genetic divergence between primary and relapsed MBs varied by subgroup but suggested considerably higher conservation than prior estimates.

Pilocytic astrocytomas (PA) are low-grade gliomas (pLGG) and are the most frequent childhood brain tumors. They are characterized by oncogene-induced senescence (OIS) initiated and sustained by senescence-associated secretory phenotype (SASP) factors. OIS and SASP in PA are thought to be driven by aberrations of the mitogen-activated protein kinase (MAPK) pathway (e.g. KIAA1549:BRAF fusion, BRAFV600E mutation, for the most common MAPK alterations occuring in PA), leading to its sustained activation. The MAPK pathway cascade is activated in a sequential manner: 1) ERK activation, which phosphorylates downstream partners in both cytoplasm and nucleus. 2) ERK-mediated induction of immediate early genes encoding transcription factors. 3) Induction of MAPK target genes expression. 4) Activation of downstream pathways. Our aim is to unravel the molecular partners involved at each level of the sustained MAPK pathway activation in pLGG with different genetic backgrounds (KIAA1549:BRAF fusion and BRAFV600E mutation), and leading to the induction of OIS and SASP factors expression. pLGG cell lines DKFZ-BT66 (KIAA1549:BRAF) and BT-40 (BRAFV600E) were treated with the MEK inhibitor trametinib at key time points, and gene expression profile analysis was performed, allowing transcriptome analysis at each step of the MAPK cascade. This will be combined with a whole proteomic and phospho-proteomic analysis. Combination of the transcriptome and proteome data layers will allow the identification of a) downstream targetable partners activated by the MAPK pathway involved in PA senescence, b) new putative targets that might bring benefit in combination with MAPK inhibitors.

BACKGROUND Impact of isolated spread into the cerebrospinal fluid (CSF) is still not investigated comprehensively for childhood medulloblastoma and the best therapeutic strategy is currently unclear. MATERIAL AND METHODS Sixty-six patients with isolated M1-MB registered to the HIT-MED-database from 2000–2018 were identified. CSF and MRI were centrally reviewed for all patients. Patients were stratified by age and either treated with upfront craniospinal irradiation (CSI) followed by maintenance chemotherapy (CT) or with postoperative CT and delayed CSI. RESULTS Forty-nine patients were non-infants ≥4 years and seventeen were infants CONCLUSION Isolated M1-MB is rare. Patients without contraindication for CSI appear to benefit from treatment by upfront CSI followed by maintenance CT, while cumulative CT-doses would be reduced compared to sandwich strategies.

Medulloblastoma (MB) is a highly malignant pediatric brain tumor originating from the cerebellum and brainstem. Identification of molecular subgroups forming this heterogeneous tumor entity was initially achieved from transcriptome characterization and further strengthened using DNA methylation profiling. While subgroup classification improved clinical diagnosis and treatment options, the lack of knowledge of the cell-of-origin for some of the subgroups hinders further treatment improvements. In addition identification of the precise cells of origin for each subgroup could help to understand tumor cell biology. Single cell sequencing is the optimal way to solve this task; recently, there were attempts to uncover putative MB cell-of-origin by using such information obtained from mouse embryonic cerebellum. However, such a comparative strategy can miss important results due to the differences between mouse and human. To solve this issue, we performed global single nucleus sequencing on human cerebellum pre- and postnatal materials across several developmental time points and generated transcriptome profiles from ~200k single cells. We identified known cell types forming the human cerebellum and performed detailed comparison of normal cells to RNA-seq bulk data from MB brain tumors across all subgroups. By selecting an optimal analysis strategy, we verified granule neuron precursors as cells of origin for the SHH MB subgroup. Additionally, we also found other cell types in conjunction with the remaining MB subgroups, suggesting new potential targets for investigation. Notably, this strategy can be further applied to the examination of other brain tumors and has perspectives in medical application.

AIM Advances in molecular classification of paediatric ependymoma have been pivotal in improving risk stratification and understanding of this disease. C11orf95-RELA fused supratentorial ependymoma (ST-EPN) have been reported to have a poor outcome, with 10-year overall survival (OS) of 49% and progression free survival (PFS) of 19%. A cohort of patients from multiple international institutions with molecularly confirmed C11orf95-RELA fused ST-EPN were reviewed to assess their disease behaviour. METHOD: We reviewed patients with molecularly determined C11orf95-RELA supratentorial ependymoma diagnosed between 1999 – 2019. Demographic information, extent of surgical resection, use of radiotherapy and/or chemotherapy, disease recurrence, treatment at recurrence and clinical outcome data was collected. PFS and OS of all patients were estimated using Kaplan-Meier method. RESULTS A total of 76 ST-EPN patients with C11orf95-RELA fusion were identified (median age: 7 years3 months, range: 5 months – 18 years7 months). 58 patients (76.3%) had complete surgical resection. 70 patients(92.1%) received radiotherapy. 55 patients(72.3%) received chemotherapy. The 10-year OS of C11orf95-RELA fused ST-EPN was 72.4% and PFS was 63.8%. In contrast, ST-EPN at a single institution with unconfirmed molecular status had an OS of 61.1% and PFS of 34.9%. CONCLUSION Detailed molecular analysis identified distinct subgroups of patients with ST-EPN. Patients from this cohort with C11orf95-RELA methylation profiles had a significantly higher OS compared to previous reports and those with unconfirmed fusion status, emphasising the critical importance of complete molecular profiling to assist in treatment decision making. Complete molecular analysis in future prospective cohorts is essential for accurate risk stratification and treatment selection.

Atypical teratoid/rhabdoid tumors (ATRT) represents a genomically homogeneous disease characterized by loss of SMARCB1 protein in the vast majority of cases. In recent years, it has become clear that these tumors display a high degree of intertumoral heterogeneity with three molecularly distinct subgroups. However, the degree of intratumoral heterogeneity and the information on cellular subpopulations currently remains largely an unchartered territory. To explore the transcriptomic composition of ATRTs, we performed single nuclei RNA sequencing for 16 ATRTs representing all three molecular subgroups (5 ATRT-TYR, 7 ATRT-SHH, 4 ATRT-MYC). By performing tSNE cluster analyses of all the single cell data (~50.000 cells have been sequenced), we were able to gain unprecedented insights into the phenotypic composition of ATRTs and unravelled substantial differences between the three subgroups. Integrating transcriptomic information from non-neoplastic brain cells and the data derived from single nuclei sequencing, we found an OPC like gene signature in ATRT-SHH. In contrast, ATRT-TYR subpopulations overexpressed more markers of neuronal stem cells suggesting a larger fraction of undifferentiated cells in this subgroup. We also identified a subpopulation of cells with a clear overexpression of cell cycle associated genes (CDK4, CDKN3), predominantly present in ATRT-MYC samples, a finding which may harbour important consequences for a targeted therapy with e.g. CDK inhibitors. In summary, our analyses reveal different cellular compartments in ATRT and provide important insights into the cellular differentiation of the three ATRT-subgroups. Further analyses to achieve a specific mapping of ATRT to its physiological cell of origin are currently being pursued.

OBJECTIVES Reduced neuropsychological outcomes are a major concern in pediatric patients with malignant brain tumors. We aimed to estimate decline in cognitive function according to treatment regimens. METHODS Cross-sectional analysis of cognitive functions tested with the Neuropsychological Basic Diagnostic tool (NBD) in 279 patients >4 years at diagnosis (median: 8.66; range: 4.01–18.98) with medulloblastoma (n=110, 23.7–25.0Gy CSI; n=131, >30Gy CSI) or posterior fossa ependymoma (n=38 local radiotherapy) who participated in the HIT-2000 trial. Multivariable regression analysis was conducted to adjust for postoperative cerebellar mutism syndrome, preoperative hydrocephalus, postoperative shunt placement, the interval between diagnosis and assessment, sex and age. RESULTS Mean time from diagnosis to assessment was 5.1 years. Increasing CSI-dose was significantly associated with a deterioration in performance of most subtests, particularly in areas of fluid intelligence (mean z-values per test for no CSI/23.4Gy/>30Gy respectively: matrix reasoning:-0.40/-0.52/-0.98, p30Gy CSI, selective attention, but no other function was reduced in patients treated with pre-radiotherapy chemotherapy including intraventricular MTX (selective attention (with chemotherapy/without chemotherapy mean z-values: -0.66/0.00, p=.006)). Patients with SHH-activated medulloblastoma did significantly better than WNT or Group3/Group4 medulloblastoma patients in fluid intelligence and fine motor skills. CONCLUSION CSI dose among other highly relevant factors had significant effects on neuropsychological outcome. Pre-radiotherapy intraventricular MTX had only minor effects. Patients with SHH-activated medulloblastomas showed a more favorable outcome when compared to patients in the other subgroups.

Medulloblastoma (MB) with extensive nodularity (MBEN) represent a rare subtype of cerebellar tumors of infancy which comprise two histologically distinct components, nodular reticulin-free zones and inter-nodular reticulin-rich regions. We applied single nucleus RNA-sequencing (snRNA-seq) using the 10X Genomics and the SMARTseq V2 protocols, bulk RNA-sequencing, DNA-methylation profiling and DNA-panel sequencing to ten histologically confirmed MBEN specimens. All tumors were classified as sonic hedgehog (SHH) MB based on DNA methylation. Somatic mutations within the SHH-pathway were detected in seven samples (3x SUFU, 2x PTCH1, 2x SMO) by DNA panel sequencing. The combined snRNAseq approach resulted in data on ~30.000 single cells. Several non-malignant cell types were identified, e.g. endothelial cells, astrocytes, and microglia. Amongst malignant cell populations SHH-pathway activation and mitotic activity differed revealing actively cycling embryonic stem (ES) cell-like and more differentiated neuronal-like cell types. In addition, distinct histological components of these tumours were subjected to bulk RNA sequencing following microdissection. This approach was repeated for DNA methylation profiling in an independent paraffin embedded MBEN cohort. However, these analyses did not reveal significant transcriptomic differences or differential methylation patterns between the two histological components. In summary, snRNA-seq identified a strongly proliferating, ES-like subset of cells in MBEN, which might represent the driving cell population in these malignancies, while direct analyses of nodular and inter-nodular regions did not reveal any significant differences. These findings suggest that both components originate from the same cell of origin but represent different cellular developmental stages.

The majority of supratentorial ependymomas (ST-EPN) are driven by fusions between RELA and a zinc finger containing gene, C11orf95. Apart from fusions to the Hippo effector YAP1, which affects a small group of infant patients, the oncogenic mechanism of remaining ST-EPNs is unclear. Aiming at refining the molecular classification of ST-EPNs, we analyzed methylation profiles, RNA and DNA sequencing results as well as clinical data in a cohort of 617 ST-EPNs. Unsupervised clustering analysis of DNA methylation data revealed four distinct clusters that formed in addition to the known molecular groups ST-EPN-RELA and –YAP1. Tumors within these additional clusters were characterized by fusions of C11orf95 to numerous fusion partners different from RELA, e.g. MAML2, MAML3, NCOA2 and SS18, suggesting a general role of C11orf95 in tumorigenesis of ST-EPN. Transforming capacity of newly identified fusion genes was validated using an electroporation-based in vivo gene transfer technology. All fusion genes were sufficient to drive malignant transformation in the cerebral cortex of mice and resulting tumors faithfully recapitulated molecular characteristics of their human counterparts. We found that both, the partner gene and the zinc finger DNA binding domain of C11orf95, were essential to exert tumorigenesis. When exploring genes commonly upregulated in C11orf95 fusion-expressing tumors of human and murine origin, the Sonic Hedgehog effector gene Gli2 was identified as a promising downstream target. Subsequent co-expression of C11orf95:RELA and a dominant negative form of Gli2 indeed hampered tumorigenesis. We thus propose GLI2 as a potential therapeutic downstream target of C11orf95 fusion-dependent oncogenic signaling in ST-EPN.

Diffuse midline gliomas (DMG) H3 K27M-mutant were introduced in the 2016 WHO Classification unifying diffuse intrinsic pontine gliomas (DIPG) and gliomas from the thalamus and spinal cord harboring a histone H3-K27M mutation leading to Polycomb Repressor Complex 2 (PRC2) inhibition. However, few cases of DMG tumors presenting a H3K27 trimethylation loss, but lacking an H3-K27M mutation were reported. To address this question, we combined a retrospective cohort of 10 patients biopsied for a DIPG at the Necker Hospital or included in the BIOMEDE trial (NCT02233049) and extended our analysis to H3-wildtype (WT) diffuse gliomas from other midline locations presenting either H3K27 trimethylation loss or ACVR1 mutation from Necker, ICR, the HERBY trial, the INFORM registry study and the St. Jude PCGP representing 9 additional cases. Genomic profiling identified alterations frequently found in DMG, but none could explain the observed loss of H3K27 trimethylation. Similar observations were previously made in the PF-A subgroup of ependymoma, where the H3K27me3 loss resulted from EZHIP/CXorf67 overexpression rather than H3-K27M mutations. We thus analyzed EZHIP expression and observed its overexpression in all but one H3-WT DMGs compared to H3-K27M mutated tumors (EZHIP negative). Strikingly, based on their DNA methylation profiles, all H3-WT DMG samples analyzed clustered close to H3-K27M DIPG, rather than EZHIP overexpressing PF-A ependymomas. To conclude, we described a new subgroup of DMG lacking H3-K27M mutation, defined by H3K27 trimethylation loss and EZHIP overexpression that can be detected by IHC. We propose that these EZHIP/H3-WT DMGs extend the spectrum of DMG with PRC2 inhibition beyond H3-K27M mutation.

Pineoblastomas (PB) are rare, aggressive pineal gland tumours with poor global OS of 50–70% and only 15–49% OS for patients

The large variety of CNS tumor entities affecting children and adolescents, some of which are exceedingly rare, results in very diverging patient outcomes and renders accurate diagnosis challenging. To assess the diagnostic utility of routine DNA methylation-based CNS tumor classification and gene panel sequencing, the Molecular Neuropathology 2.0 study prospectively integrated these (epi-)genetic analyses with reference neuropathological diagnostics as an international trial for newly-diagnosed pediatric patients. In a four-year period, 1,215 patients with sufficient tissue were enrolled from 65 centers, receiving a reference neuropathological diagnosis according to the WHO classification in >97%. Using 10 FFPE sections as input, DNA methylation analysis was successfully performed in 95% of cases, of which 78% with sufficient tumor cell content were assigned to a distinct epigenetic tumor class. The remaining 22% did not match any of 82 represented classes, indicating novel rare tumor entities. Targeted gene panel sequencing of >130 genes performed for 96% of patients with matched blood samples detected diagnostically, prognostically, or therapeutically relevant somatic alterations in 48%. Germline DNA sequencing data indicated potential predisposition syndromes in ~10% of patients. Discrepant results by neuropathological and epigenetic classification (29%) were enriched in histological high-grade gliomas and implicated clinical relevance in 5% of all cases. Clinical follow-up suggests improved survival for some patients with high-grade glioma histology and lower-grade molecular profiles. Routine (epi-)genetic profiling at the time of primary diagnosis adds a valuable layer of information to neuropathological diagnostics and will improve clinical management of CNS tumors.

About half of all pediatric high-grade gliomas (HGG) harbor mutations in histone 3 or IDH genes. The remaining HGG are currently broadly classified as H3-/IDH-wild-type. Since the introduction of a uniform approach to DNA methylation-based classification of CNS tumors in 2018, DNA methylation data from over 45,000 CNS tumor samples have been generated. From this large cohort, a number of smaller yet distinct subgroups start to emerge within H3-/IDH-wild-type HGG. Three such subgroups are enriched for focal gene amplifications and have been provisionally termed pedGBM_MYCN, pedGBM_RTK1 and pedGBM_RTK2. Since a significant subset of samples in each subgroup is lacking characteristic alterations, we further investigated the molecular and transcriptional composition of H3-/IDH-wild-type HGG. We evaluated DNA methylation and copy-number profiles in >1000 tumors classified as H3-/IDH-wild-type HGG. Tumors classified pedGBM_MYCN showed a focal MYCN amplification in 25%, with a similar fraction showing amplification of EGFR (8% of samples harbored both alterations) compared to 4% and 4% in pedGBM_RTK1 and 14% and 22% in pedGBM_RTK2. Deletion of CDKN2A/B was much more prevalent in the pedGBM_RTK2 subgroup (~50% compared to 27% in pedGBM_RTK1 and

OBJECTIVE Molecular subtypes of Group 3/4 medulloblastoma have been identified by unsupervised clustering methods in different studies. We hypothesized that risk stratification using these subtypes I-VIII improves outcome prediction. PATIENTS AND METHODS n=340 patients with Group 3 or Group 4 medulloblastoma defined by DNA methylation array profiling enrolled into the HIT2000 study and HIT-MED registries were subtyped by the Heidelberg Medulloblastoma Classifier. The discovery cohort consisted of n=162 previously published samples, the validation cohort of n=178 newly analyzed samples. RESULTS AND DISCUSSION: n=300/340 (88%) MBs could be assigned to one of the subtypes with confidence (score >0.8; Heidelberg Medulloblastoma classifier). Subtype II,III and V showed a poor PFS and OS and were classified as HR (discovery:5y-PFS 45%[95%-CI:33–62], 5y-OS 50%[37–67]; validation:5y-PFS 32%[20–50], 5y-OS 40%[27–61]). Subtypes I, IV, VI-VIII fared better (discovery:5y-PFS 67%[58–77], 5y_OS 84%[77–91]; Validation:5y-PFS 70%[58–83], 5y-OS 89%[81–99]). Survival prediction by subtype-based risk assessment was improved compared to Group 3 versus 4 differentiation in both cohorts in univariate and multivariable Cox regression models (PFS:Hazard ratio HR versus LR 2.474, p CONCLUSION We showed that molecular subtypes I-VIII improved risk stratification of Group 3/4 medulloblastomas. Group 3 subtype IV MB treated with RT had very high cure rates.

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.

Pilocytic astrocytomas (PAs) and other pediatric low-grade gliomas (pLGGs) exhibit aberrant activation of the MAPK signaling pathway caused by genetic alterations, most commonly KIAA1549:BRAF fusions, BRAF V600E and NF1 mutations. In such a single-pathway disease, novel drugs targeting the MAPK pathway (MAPKi) are prime candidates for treatment. We developed an assay suitable for pre-clinical testing of MAPKi in pLGGs, aiming at the identification of novel MAPK pathway suppressing synergistic drug combinations. We generated a reporter plasmid (pDIPZ) expressing destabilized firefly luciferase driven by a MAPK-responsive ELK-1-binding element, packaged in a lentiviral vector system. We stably transfected pediatric glioma cell lines with a BRAF fusion (DKFZ-BT66) and a BRAFV600E mutation (BT-40) background, respectively. Measurement of MAPK pathway activity was performed using the luciferase reporter. pERK protein levels were detected for validation. We performed a screen of a MAPKi library and calculated Combination Indices of selected combinations. The MAPKi library screen revealed MEK inhibitors as the class inhibiting the pathway with the lowest IC50s, followed by ERK and second generation RAF inhibitors. Synergistic effects in both BRAF-fusion and BRAFV600E mutation backgrounds were observed following combination treatments with different MAPKi classes (RAFi/MEKi, > RAFi/ERKi > MEKi/ERKi). We have generated a novel reporter assay for medium- to high-throughput pre-clinical drug testing of MAPKi in pLGG cell lines. MEK, ERK and next-generation RAF inhibitors were confirmed as potential treatment approaches for KIAA1549:BRAF and BRAFV600E mutated pLGGs. Synergistic suppression of MAPK pathway activity upon combination treatments was revealed using our assay in addition.

BRCAness is a phenotypical trait resembling tumors with germline BRCA1/BRCA2 mutation and homologous recombination deficiency (HRD). Recently, osteosarcoma (OS), which is the most common primary malignant bone tumor, was shown to frequently present molecular features resembling BRCAness. The inability of the HR pathway to keep the genome integrity leads to its instability, resulting in copy number variations and loss of heterozygosity. Tumors characterized by BRCAness might have an increased sensitivity to poly ADP ribose polymerase inhibitors, which leads to synthetic lethality through tumor cell apoptosis. There are several genomic methods to assess BRCAness such as calculating the HRD score based on large genomic rearrangements. We developed a new approach based on DNA-methylation patterns and gene expression signatures. Low-coverage whole-genome (lcWGS) data, DNA-methylation, and RNA-Seq data of 41 patients with relapsed OS were obtained within the scope of the INdividualized Therapy FOr Relapsed Malignancies in Childhood (INFORM) project. Percentage of genome changed (PGC) by large state transitions and telomeric allele imbalance was computed with Nexus CN (BioDiscovery, Inc) and open source Control-FREEC/HRDtools from lcWGS and used for BRCAness scoring. llumina Human Methylation450k and EPIC arrays were used to determine DNA methylation profiles. We selected 18 BRCA-positive and 12 BRCA-negative samples based on the PGC to train the classifier algorithm Random Forest (RF) (R-project) and set aside 11 samples as a test set. Probes were ranked according to minimum variability within groups and maximum between groups. The top 1,000 probes were selected for RF training. The importance of probes was assessed as a mean decrease in accuracy (MDA). Probes with zero MDA were removed from training set. RNA-seq differential expression analysis was performed with DESeq2. Expression signatures were extracted by gene set enrichment analysis (GSEA). Training samples were clustered unambiguously into BRCA-positive and BRCA-negative groups with unsupervised hierarchical clustering of 1,000 probes. All 11 test samples set aside before RF training were accurately classified. Excluding noninformative probes reduced the number of informative probes to 628 and correctly clustered them into two groups. Differential expression analysis of BRCA positive and negative RNA-Seq samples resulted in 1,458 differentially expressed genes. Only upregulated genes in BRCA-positive samples showed highly significant homologous recombination, nucleotide excision, and mismatch repair signatures in GSEA. We established and validated a DNA methylation-based classifier that differentiates BRCA-negative and BRCA-positive OS samples with high accuracy. The differential expression and enrichment analyses identified genome instability signatures. Our method complements existing genomic BRCAness scoring and brings new epigenomic/trancriptomic dimensions into the computational prediction of BRCAness. Citation Format: Maxim Barenboim, Michal Kovac, Baptiste Ameline, David T.W. Jones, Olaf Witt, Stefan Bielack, Stefan Burdach, Daniel Baumhoer, Michaela Nathrath. Identifying BRCAness in osteosarcoma with DNA-methylation profiling and gene expression signatures [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A24.

Introduction: Immune checkpoint inhibition is a novel approach of restimulating immune cells to overcome immunosuppressive microenvironment in tumors and has shown significant clinical benefit in 20-40% of adult patients. Although this success in adult cancers has resulted in a plethora of immunogenomic efforts to delineate mechanisms of response, such studies in pediatric cancers are lacking. The main objective of this study is to uncover the overall characteristics of immune microenvironment in pediatric solid tumors. Methods: We leveraged transcriptomes of 1,268 pediatric primary solid tumors acquired from multiple international initiatives, including Therapeutically Applicable Research to Generate Effective Treatments (TARGET, n = 269), International Cancer Genome Consortium (ICGC, n = 216) and Children Brain Tumor Tissue Consortium (CBTTC, n = 803). We compared our results with those of 7 adult cancer types from The Cancer Genome Atlas (TCGA, n = 2720). We applied methods of immune deconvolution, repurposed RNAseq data to recover infiltrating T- and B-cell clonotypes and studied checkpoint gene expression in pediatric tumors. In collaboration with Gabriela Miller’s Kids First Data Resource Centre, all analyses were performed and shared on CAVATICA computational platform (cavatica.sbgenomics.com). Results: Embryonal tumors with multilayered rosettes (ETMR) and medulloblastomas (MB) harbored the lowest levels of immune infiltration in our pediatric cohort. Immune deconvolution analysis revealed that neuroblastomas (NBL) had the highest T-cell infiltration amongst pediatric cancers. We found atypical teratoid/rhabdoid tumors (ATRT) had highest levels of CD8+ T cells among pediatric CNS tumors. While tumor mutational burden (TMB) was associated with immune cell infiltration in adult lung cancers and melanomas, we found no significant association in pediatric cancers. We found specific genetic alterations such as BRAF, H3F3A, and CTNNB1 had significant impact on the composition of the immune microenvironment. Analysis of T-cell repertoire revealed an inverse correlation between clonal diversity and TMB, suggesting a T-cell clonal expansion in high TMB samples. Finally, while the majority of NBL samples expressed LAG3, ~10% of samples had elevated levels of TIM3 gene, suggesting a distinct mode of immunosuppression in this subset. 2% of pediatric CNS tumors, mostly consisted of ATRTs, harbored high expression of PRF1 and GZMA genes suggestive of functionally capable immune cells in these cases. Conclusions: We report characteristics of the tumor microenvironment in pediatric tumors at primary diagnosis. We found that specific gene mutations, rather than mutational load, shape the composition of immune cell types. Furthermore, hypermutant samples exhibit evidence of clonal T-cell expansion. Our results suggest immune activity in 2% of CNS tumors and uncover a subtype of NBL expressing TIM3 checkpoint gene, calling for investigations to evaluate the effectiveness of immunotherapy. This abstract is also being presented as Poster A74. Citation Format: Arash Nabbi, Pengbo Sun, Sudhaman Sumedha, Kelsey Zhu, S.Y. Cindy Yang, Joseph N. Paulson, Marcel Kool, Komal Rathi, Karthik Kalletla, Pichai Raman, Yuankun Zhu, Adam C. Resnick, David T.W. Jones, Natalie Jäger, Stefan M. Pfister, Trevor J. Pugh. The immunogenomic landscape of pediatric primary solid tumors [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR06.

LBA10503 Background: Several pediatric precision oncology programs have identified molecular actionable variants. However, the clinical benefit is largely unknown. We here report a target prioritization algorithm and associated clinical outcome. Methods: INFORM is a prospective, non-interventional, multi-center, multi-national, and feasibility registry collecting clinical and molecular data. Patients with refractory/relapsed/progressive malignant disease, including primary diagnosis high-risk entities, can be enrolled. Fresh frozen tumor material (incl. germline DNA) was subjected to WES, lcWGS, RNA-Seq, RNA expression array and DNA-methylation. A weekly interdisciplinary molecular board reviewed and prioritized alterations based on a 7- step scale from ‘very high’ to ‘very low’, depending on the type of alteration and its entity specific relevance (described by Worst et al. Eur J Cancer 2016). Results: To date, more than 1300 patients were enrolled. 525 patients finished follow-up and were included in this analysis. They were enrolled in 72 centers in 8 countries. The median age was 12.0 (range 0 - 40) years. Average turnaround time from submission to report was 25.4 days. Median PFS and OS were 116 (95% CI 105 – 135) and 289 (95% CI 250 – 335) days. The distribution of the highest priority target per patient was: very high 8.0%, high 14.8%, moderate 20.3%, intermediate 23.6%, borderline 14.4%, low 2.5%, very low 1.0% and no actionable target 15.4%. 149 patients received targeted treatment on the basis of identified targets, of which 20 had a very high priority target (mostly ALK, BRAF and NRAS mutations and MET and NTRK-fusions) with a median PFS of 204.5 (95% CI 91.0 – 628.0) compared to 114 (95% CI 103 – 133) days in all other 505 patients (p = 0.0095). OS did not show clinically relevant differences. Explorative analysis of the time to progression (TTP) ratio (before compared to after enrollment) showed that patients treated according to a very high priority target had a higher TTP ratio (1.0) compared to all other patients (0.7). Possible predisposition syndromes were identified in 7.8% of patients, half of which were newly diagnosed. Methylation analysis provided a diagnosis refinement in 8% of CNS tumors. Conclusions: Pediatric precision oncology in a real world, multi-national setting is feasible. The prioritization algorithm identifies subgroups benefitting from molecularly matched targeted treatment. Still, for the patients without a very high priority target further layers of molecular and functional data should be incorporated in future programs.

The introduction of the classification of brain tumours based on their DNA methylation profile has significantly changed the diagnostic approach for cases with ambiguous histology, non-informative or contradictory molecular profiles or for entities where methylation profiling provides useful information for patient risk stratification, for example in medulloblastoma and ependymoma. We present our experience that combines a conventional molecular diagnostic approach with the complementary use of a DNA methylation-based classification tool, for adult brain tumours originating from local as well as national referrals. We report the frequency of IDH mutations in a large cohort of nearly 1550 patients, EGFR amplifications in almost 1900 IDH-wildtype glioblastomas, and histone mutations in 70 adult gliomas. We demonstrate how additional methylation-based classification has changed and improved our diagnostic approach. Of the 325 cases referred for methylome testing, 179 (56%) had a calibrated score of 0.84 and higher and were included in the evaluation. In these 179 samples, the diagnosis was changed in 45 (25%), refined in 86 (48%) and confirmed in 44 cases (25%). In addition, the methylation arrays contain copy number information that usefully complements the methylation profile. For example, EGFR amplification which is 95% concordant with our Real-Time PCR-based copy number assays. We propose here a diagnostic algorithm that integrates histology, conventional molecular tests and methylation arrays.

As more biomarkers for Alzheimer’s disease and age-related brain conditions become available, more sophisticated analytic approaches are needed to take full advantage of the information they convey. Most work has been done using categorical approaches but the joint relationships of tau PET, amyloid PET and cortical thickness in their continuous distributions to cognition have been under-explored. We evaluated non-demented subjects over age 50 years in the Mayo Clinic Study of Aging, 2037 of whom had undergone 3 T MRI scan, 985 amyloid PET scan with 11C-Pittsburgh compound B (PIB) and MRI, and 577 PIB-PET, 18F-AV1451 flortaucipir PET and MRI. Participants received a nine-test cognitive battery. Three test scores (logical memory delayed recall, visual reproduction delayed recall and auditory verbal learning test delayed recall) were used to generate a memory composite z-score. We used Gradient Boosting Machine models to analyse the relationship between regional cortical thickness, flortaucipir PET signal, PIB-PET signal and memory z-scores. Age, education, sex and number of test exposures were included in the model as covariates. In this population-based study of non-demented subjects, most of the associations between biomarkers and memory z-scores accrued after 70 years of age. Entorhinal cortex exhibited the strongest associations between biomarkers and memory z-scores. Other temporal regions showed similar but attenuated associations, and non-temporal regions had negligible associations between memory z-scores and biomarkers. Entorhinal flortaucipir PET signal, PIB-PET signal and entorhinal cortical thickness were independently and additively associated with declining memory z-scores. In contrast to global PIB-PET signal where only very high amyloid-β levels were associated low memory z-scores, entorhinal flortaucipir PET signal just above background levels was associated with low memory z-scores. The lowest memory z-scores occurred with the confluence of elevated entorhinal flortaucipir PET signal and lower entorhinal cortical thickness.

There has been no comparison of excessive daytime sleepiness (EDS) in patients with Alzheimer’s disease dementia (AD), dementia with Lewy bodies (DLB), and behavioral variant frontotemporal dementia (bvFTD). We identified patients with mild dementia who met criteria for these disorders who also had the Epworth Sleepiness Scale (ESS) completed. The sample included 17 bvFTD, 111 AD, and 31 DLB. An ESS score ≥10 was considered abnormal and consistent with EDS. Analyses with age and sex as covariates revealed higher mean ESS scores for DLB compared to the other groups (DLB 13.9 [5], bvFTD 9.6 [8], AD 8.8 [5], P < .05). An ESS score ≥10 was significantly more likely to occur in DLB compared to bvFTD or AD (DLB 81% vs bvFTD 47% vs AD 45%, P < .01). In patients with mild dementia, EDS is greatest in DLB and comparably lower in bvFTD and AD.

BACKGROUND: Pediatric low-grade glioma (pLGG) is the most common central nervous system tumor of childhood. Although overall survival is very good, many children suffer from multiple progressions and functional morbidities. There is no one universally accepted therapy for children with recurrent disease, however, standard cytotoxic chemotherapies are often utilized by most practitioners. The Pediatric Brain Tumor Consortium conducted a multi-institutional phase II study evaluating selumetinib (AZD6244, ARRY-142886), a MAP/ERK Kinase I/II inhibitor, in patients with recurrent, refractory or progressive pLGG assigned to numerous strata. The aim of the study was to evaluate the efficacy of selumetinib in these patients. METHODS: Eligibility required age 3–21 y/o, a Lansky or Karnofsky performance score greater than 60 and the presence of recurrent, refractory or progressive pLGG after at least one standard therapy. Stratum 1 included children with World Health Organization (WHO) grade I pilocytic astrocytoma (PA) harboring either one of the two most common BRAF aberrations (KIAA1549-BRAF fusion or the BRAF(V600E) mutation). Stratum 3 included children with any neurofibromatosis type 1 (NF1)-associated pLGG (WHO grades I and II). Selumetinib was provided as capsules given orally at the recommended phase II dose of 25 mg/m(2) twice daily. The primary endpoint was stratum-specific objective response rate assessd by the local site and sustained for at least 8 weeks. All responses were reviewed centrally and statistical analyses were done as per protocol. Although the trial (NCT01089101) is still ongoing in other strata, enrollment and planned follow-up is compete on both strata 1 and 3. FINDINGS: Between July 25, 2013, and June 12, 2015, 25 eligible and evaluable children were accrued to stratum 1, and between August 28, 2013, and June 25, 2015, 25 eligible and evaluable children were accrued to stratum 3. On stratum 1, 9/25 (36%) patients achieved a partial response (PR). The median follow-up for the 11 patients who have not yet experienced an event is 36.4 months (4.4–50.5; IQR=23.9). On stratum 3, 10/25 (40%) patients achieved a PR with a median follow-up of 48.6 months (8.6–59.1; IQR=12.2) for the 17 subjects without progressions. All patients evaluable for visual acuity had improved or stable vision. The most common attributable toxicities on both strata were grade 1 and 2 CPK elevation, hypoalbuminemia, dyspnea, rash, duodenal ulcer, anemia, dry skin, fatigue and diarrhea. Rare grade 3 toxicities included elevated CPK (n=5), maculopapular rash (n=5), neutropenia (n=3), nausea (n=3), paronychia (n=3), acneiform rash (n=2), diarrhea (n=2), elevated ALT (n=1), decreased ejection fraction (n=1), gastric hemorrhage (n=1), headache (n=1), skin infection (n=1), tooth infection (n=1) and weight gain (n=1). There was only one grade 4 toxicity, lymphopenia. There were no treatment-realted deaths. Patient reported outcomes and quality of life assessments were not part of the current study. INTERPRETATION: Selumetinib is active against recurrent, refractory or progressive PA harboring common BRAF aberrations and NF1-associated pLGG. To our knowledge, this is one of the first prospectively tested and successful molecularly-targeted agents in pLGG. These data not only provide an alternative to standard chemotherapy for these subgroups of patients, but this success has led to an interest in exploring efficacy in patients as a first-line therapy. In fact, these data have directly led to the development of two Children’s Oncology Group phase III studies in newly diagnosed pLGG patients both with and without NF1 comparing standard chemotherapy to selumetinib. The current trial was funded by a National Cancer Institute (NCI) Cancer Therapy Evaluation Program (CTEP) PBTC U01 Grant: 2UM1CA081457 (UM1) and by the American Lebanese Syrian Associated Charities.

Since it was first described in 1965, normal pressure hydrocephalus (NPH) has been a controversial subject. New studies have shed light on its epidemiology and pathogenesis and provided objective ways to measure outcome in patients with NPH. Neuroimaging has improved and allows better recognition of both NPH and the presence of overlapping diseases RECENT FINDINGS: Several recent epidemiologic studies confirm that NPH is a rare disease, but the presence of large ventricles is a common finding with aging. NPH may be multifactorial, including congenital causes, vascular disease, and impaired CSF absorption. MRI features of NPH include enlarged ventricular size and CSF fluid collection outside the ventricles not due to atrophy. The term disproportionately enlarged subarachnoid space hydrocephalus (DESH) has been used to describe prognostic MRI features in NPH, including a "tight high convexity" and enlargement of CSF spaces in the sylvian fissure. DESH has been included in the Japanese guideline for the diagnosis and treatment of NPH. A new NPH scale has been published that provides an objective framework for evaluating patients with NPH before and after shunt placement. Programmable shunts can noninvasively manage overdrainage complications. Surgical outcome has been improving over time. Recent studies have led to improved recognition of overlapping diseases such as Alzheimer pathology, which co-occurs in about 30% of NPH cases. Fludeoxyglucose positron emission tomography (FDG-PET) is a promising imaging modality for diagnosing NPH and detecting concomitant degenerative disease.A systematic approach to patients with possible NPH allows recognition of the subset of patients who will respond to shunt surgery and identification of those with alternative diagnoses.

Desmoplastic/nodular medulloblastomas (DNMB) and medulloblastomas with extensive nodularity (MBEN) were outlined in the current WHO classification of tumors of the nervous system as two distinct histological MB variants. However, they are often considered as cognate SHH MB entities, and it is a reason why some clinical MB trials do not separate the patients with DNMB or MBEN histology. In the current study, we performed an integrated DNA/RNA-based molecular analysis of 83 DNMB and 36 MBEN to assess the etiopathogenetic relationship between these SHH MB variants. Methylation profiling revealed "infant" and "children" SHH MB clusters but neither DNMB nor MBEN composed separate epigenetic cohorts, and their profiles were intermixed within the "infant" cluster. In contrast, RNA-based transcriptional profiling disclosed that expression signatures of all MBEN were clustered separately from most of DNMB and a set of differentially expressed genes was identified. MBEN transcriptomes were enriched with genes associated with synaptic transmission, neuronal differentiation and metabolism, whereas DNMB profiling signatures included sets of genes involved in phototransduction and NOTCH signaling pathways. Thus, DNMB and MBEN are distinct tumor entities within the SHH MB family whose biology is determined by different transcriptional programs. Therefore, we recommend a transcriptome analysis as an optimal molecular tool to discriminate between DNMB and MBEN, which may be of benefit for patients' risk stratification in clinical trials. Molecular events identified in DNMB by RNA sequencing could be considered in the future as potent molecular targets for novel therapeutic interventions in treatment-resistant cases.

Introduction Conventional Z-scores are generated by subtracting the mean and dividing by the standard deviation. More recent methods linearly correct for age, sex, and education, so that these “adjusted” Z-scores better represent whether an individual's cognitive performance is abnormal. Extreme negative Z-scores for individuals relative to this normative distribution are considered indicative of cognitive deficiency. Methods In this article, we consider nonlinear shape constrained additive models accounting for age, sex, and education (correcting for nonlinearity). Additional shape constrained additive models account for varying standard deviation of the cognitive scores with age (correcting for heterogeneity of variance). Results Corrected Z-scores based on nonlinear shape constrained additive models provide improved adjustment for age, sex, and education, as indicated by higher adjusted-R2. Discussion Nonlinearly corrected Z-scores with respect to age, sex, and education with age-varying residual standard deviation allow for improved detection of non-normative extreme cognitive scores.

© 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

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.

International audience; We investigated the challenging diagnostic case of a ventricular cystic glioneuronal tumor with papillary features, by RNA sequencing using the Illumina TruSight RNA Fusion panel. We did not retrieve the SLC44A1-PRKCA fusion gene specific for papillary glioneuronal tumor, but an EWSR1-PATZ1 fusion transcript. RT-PCR followed by Sanger sequencing confirmed the EWSR1-PATZ1 fusion. It matched with canonic EWSR1 fusion oncogene, juxtaposing the entire N terminal transcriptional activation domain of EWSR1 gene and the C terminal DNA binding domain of a transcription factor gene, PATZ1. PATZ1 protein belongs to the BTB-ZF (broad-complex, tramtrack and bric-à-brac -zinc finger) family. It directly regulates Pou5f1 and Nanog and is essential to maintaining stemness by inhibiting neural differentiation. EWSR1-PATZ1 fusion is a rare event in tumors: it was only reported in six round cell sarcomas and in three gliomas of three exclusively molecular studies. The first reported glioma was a BRAF[V600E] negative ganglioglioma, the second a BRAF[V600E] negative glioneuronal tumor, not otherwise specified, and the third, very recently reported, a high grade glioma, not otherwise specified. In our study, forty BRAF[V600E] negative gangliogliomas were screened by FISH using EWSR1 break-apart probes. We performed methylation profiling for the index case and for seven out of the ten FISH positive cases. The index case clustered apart from other pediatric low grade glioneuronal entities, and specifically from the well-defined ganglioglioma methylation group. An additional pediatric intraventricular ganglioglioma clustered slightly more closely with ganglioglioma, but showed differences from the main ganglioglioma group and similarities with the index case. Both cases harbored copy number variations at the PATZ1 locus. EWSR1-PATZ1 gene fusion might define a new type of glioneuronal tumors, distinct from gangliogliomas.

Background and objectives We assessed correlations between the resting state functional connectivity (RSFC) of different thalamic nuclei and seizure frequency in patients with drug-resistant medial temporal lobe epilepsy (mTLE). Methods Seventeen patients with mTLE and 17 sex-/age-/handedness-matched controls participated. A seed-based correlation method for the resting-state FMRI data was implemented to get RSFC maps of 70 thalamic nuclei seed masks. Group statistics for individual RSFC for subjects and seed masks were performed to obtain within-group characteristics and between-group differences with age covariates. A linear regression was applied to test whether seizure frequency correlated with thalamic nuclear RSFC with the whole brain in mTLE patients. Results RSFC of thalamic nuclei showed spatially distinguishable connectivity patterns that reflected principal inputs and outputs that were derived from priori anatomical knowledge. We found group differences between normal control and mTLE groups in RSFC for nuclei seeds located in various subdivisions of thalamus. The RSFCs in some of those nuclei were strongly correlated with seizure frequency. Conclusions Mediodorsal thalamic nuclei may play important roles in seizure activity or in the regulation of neuronal activity in the limbic system. The RSFC of motor- and sensory-relay nuclei may help elucidate sensory-motor deficits associated with chronic seizure activity. RSFC of the pulvinar nuclei of the thalamus could also be a key reflection of symptom-related functional deficits in mTLE., Highlights • We assessed the functional connectivity for 70 thalamic nuclei of temporal lobe epilepsy subjects. • Each nucleus showed distinguishable connectivity along its known pathological preference. • Especially, the mediodorsal thalamic nuclei may play important roles in seizure frequency.

Background Establishment of telomere maintenance mechanisms is a universal step in tumor development to achieve replicative immortality. These processes leave molecular footprints in cancer genomes in the form of altered telomere content and aberrations in telomere composition. To retrieve these telomere characteristics from high-throughput sequencing data the available computational approaches need to be extended and optimized to fully exploit the information provided by large scale cancer genome data sets. Results We here present TelomereHunter, a software for the detailed characterization of telomere maintenance mechanism footprints in the genome. The tool is implemented for the analysis of large cancer genome cohorts and provides a variety of diagnostic diagrams as well as machine-readable output for subsequent analysis. A novel key feature is the extraction of singleton telomere variant repeats, which improves the identification and subclassification of the alternative lengthening of telomeres phenotype. We find that whole genome sequencing-derived telomere content estimates strongly correlate with telomere qPCR measurements (r = 0.94). For the first time, we determine the correlation of in silico telomere content quantification from whole genome sequencing and whole genome bisulfite sequencing data derived from the same tumor sample (r = 0.78). An analogous comparison of whole exome sequencing data and whole genome sequencing data measured slightly lower correlation (r = 0.79). However, this is considerably improved by normalization with matched controls (r = 0.91). Conclusions TelomereHunter provides new functionality for the analysis of the footprints of telomere maintenance mechanisms in cancer genomes. Besides whole genome sequencing, whole exome sequencing and whole genome bisulfite sequencing are suited for in silico telomere content quantification, especially if matched control samples are available. The software runs under a GPL license and is available at https://www.dkfz.de/en/applied-bioinformatics/telomerehunter/telomerehunter.html. Electronic supplementary material The online version of this article (10.1186/s12859-019-2851-0) contains supplementary material, which is available to authorized users.

ObjectiveTo study the in vivo binding properties of 18F-AV-1451 (tau-PET) and Pittsburgh compound B (PiB-PET) in a unique kindred with a familial prion disorder known to produce amyloid plaques composed of prion protein alongside Alzheimer disease (AD)–like tau tangles.MethodsA case series of 4 symptomatic family members with the 12-octapeptide repeat insertion in the PRNP gene were imaged with 3T MRI, PiB-PET, and tau-PET in their fourth decade of life.ResultsThere was significant neocortical uptake of the tau-PET tracer in all 4 familial prion cases. However, PiB-PET images did not demonstrate abnormally elevated signal in neocortical or cerebellar regions for any of the patients.ConclusionsIn vivo detection of molecular hallmarks of neurodegenerative diseases will be a prerequisite to well-conducted therapeutic trials. Understanding the in vivo behavior of these PET biomarkers in the setting of various neurodegenerative processes is imperative to their proper use in such trials and for research studies focused on the basic neurobiology of neurodegeneration. This study supports the high specificity of neocortical 18F-AV-1451 binding to AD-like tau and the lack of PiB binding to PrP plaques. It is uncertain how early in the disease course tau pathology appears in the brains of individuals who carry this PRNP gene mutation or how it evolves throughout the disease course, but future longitudinal 18F-AV-1451 imaging of symptomatic and asymptomatic individuals in this kindred will help address these uncertainties.

One key advantage of the CRISPR/Cas9 system in comparison with other gene editing approaches lies in its potential for multiplexing. Here, we describe an elaborate procedure that allows the assembly of multiple gRNA expression cassettes into a vector of choice within a single step, termed ASAP(Adaptable System for Assembly of multiplexed Plasmids)-cloning. We demonstrate the utility of ASAP-cloning for multiple CRISPR-mediated applications, including efficient multiplex gene editing, robust transcription activation and convenient analysis of Cas9 activity in the presence of multiple gRNAs.