Posterior fossa ependymoma comprises two distinct molecular variants termed EPN_PFA and EPN_PFB that have a distinct biology and natural history. The therapeutic value of cytoreductive surgery and radiation therapy for posterior fossa ependymoma after accounting for molecular subgroup is not known.

We report a new family with autosomal dominant inheritance of a late onset rapidly progressive leukodystrophy in which exome sequencing has revealed a novel mutation p.R782G in the Colony-Stimulating Factor 1 Receptor gene (CSF1R). Neuropathology of two affected family members showed cerebral white matter degeneration with axonal swellings and pigmented macrophages. The few recently reported families with CSF1R mutations had been previously labelled "hereditary diffuse leukencephalopathy with axonal spheroids" (HDLS) and "pigmentary orthochromatic leukodystrophy" (POLD), disorders which now appear to form a disease continuum. The term "adult-onset leukoencephalopathy with axonal spheroids and pigmented glia" (ALSP) has been proposed to encompass this spectrum. As CSF1R regulates microglia this mutation implies that dysregulation of microglia is the primary cause of the disease.

Malignant glioblastoma (GBM) is a highly aggressive brain tumor with a dismal prognosis and limited therapeutic options. Genomic profiling of GBM samples has identified four molecular subtypes (Proneural, Neural, Classical and Mesenchymal), which may arise from different glioblastoma stem-like cell (GSC) populations. We previously showed that adherent cultures of GSCs grown on laminin-coated plates (Ad-GSCs) and spheroid cultures of GSCs (Sp-GSCs) had high expression of stem cell markers (CD133, Sox2 and Nestin), but low expression of differentiation markers (βIII-tubulin and glial fibrillary acid protein). In the present study, we characterized GBM tumors produced by subcutaneous and intracranial injection of Ad-GSCs and Sp-GSCs isolated from a patient-derived xenoline. Although they formed tumors with identical histological features, gene expression analysis revealed that xenografts of Sp-GSCs had a Classical molecular subtype similar to that of bulk tumor cells. In contrast xenografts of Ad-GSCs expressed a Mesenchymal gene signature. Adherent GSC-derived xenografts had high STAT3 and ANGPTL4 expression, and enrichment for stem cell markers, transcriptional networks and pro-angiogenic markers characteristic of the Mesenchymal subtype. Examination of clinical samples from GBM patients showed that STAT3 expression was directly correlated with ANGPTL4 expression, and that increased expression of these genes correlated with poor patient survival and performance. A pharmacological STAT3 inhibitor abrogated STAT3 binding to the ANGPTL4 promoter and exhibited anticancer activity in vivo. Therefore, Ad-GSCs and Sp-GSCs produced histologically identical tumors with different gene expression patterns, and a STAT3/ANGPTL4 pathway is identified in glioblastoma that may serve as a target for therapeutic intervention.

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

Glioblastoma (GB) is the most frequent primary brain tumor in adults with a dismal prognosis despite aggressive treatment including surgical resection, radiotherapy and chemotherapy with the alkylating agent temozolomide. Thus far, the successful implementation of the concept of targeted therapy where a drug targets a selective alteration in cancer cells was mainly limited to model diseases with identified genetic drivers. One of the most commonly altered oncogenic drivers of GB and therefore plausible therapeutic target is the epidermal growth factor receptor (EGFR). Trials targeting this signaling cascade, however, have been negative, including the phase III OSAG 101-BSA-05 trial. This highlights the need for further patient selection to identify subgroups of GB with true EGFR-dependency. In this retrospective analysis of treatment-naïve samples of the OSAG 101-BSA-05 trial cohort, we identify the EGFR signaling activity markers phosphorylated PRAS40 and phosphorylated ribosomal protein S6 as predictive markers for treatment efficacy of the EGFR-blocking antibody nimotuzumab in MGMT promoter unmethylated GBs. Considering the total trial population irrespective of MGMT status, a clear trend towards a survival benefit from nimotuzumab was already detectable when tumors had above median levels of phosphorylated ribosomal protein S6. These results could constitute a basis for further investigations of nimotuzumab or other EGFR- and downstream signaling inhibitors in selected patient cohorts using the reported criteria as candidate predictive biomarkers.

Medulloblastomas, the most frequent malignant brain tumours affecting children, comprise at least 4 distinct clinicogenetic subgroups. Aberrant sonic hedgehog (SHH) signalling is observed in approximately 25% of tumours and defines one subgroup. Although alterations in SHH pathway genes (e.g. PTCH1, SUFU) are observed in many of these tumours, high throughput genomic analyses have identified few other recurring mutations. Here, we have mutagenised the Ptch+/- murine tumour model using the Sleeping Beauty transposon system to identify additional genes and pathways involved in SHH subgroup medulloblastoma development.

Amplification of the C19MC oncogenic miRNA cluster and high LIN28 expression has been linked to a distinctly aggressive group of cerebral CNS-PNETs (group 1 CNS-PNETs) arising in young children. In this study, we sought to evaluate the diagnostic specificity of C19MC and LIN28, and the clinical and biological spectra of C19MC amplified and/or LIN28+ CNS-PNETs. We interrogated 450 pediatric brain tumors using FISH and IHC analyses and demonstrate that C19MC alteration is restricted to a sub-group of CNS-PNETs with high LIN28 expression; however, LIN28 immunopositivity was not exclusive to CNS-PNETs but was also detected in a proportion of other malignant pediatric brain tumors including rhabdoid brain tumors and malignant gliomas. C19MC amplified/LIN28+ group 1 CNS-PNETs arose predominantly in children <4 years old; a majority arose in the cerebrum but 24 % (13/54) of tumors had extra-cerebral origins. Notably, group 1 CNS-PNETs encompassed several histologic classes including embryonal tumor with abundant neuropil and true rosettes (ETANTR), medulloepithelioma, ependymoblastoma and CNS-PNETs with variable differentiation. Strikingly, gene expression and methylation profiling analyses revealed a common molecular signature enriched for primitive neural features, high LIN28/LIN28B and DNMT3B expression for all group 1 CNS-PNETs regardless of location or tumor histology. Our collective findings suggest that current known histologic categories of CNS-PNETs which include ETANTRs, medulloepitheliomas, ependymoblastomas in various CNS locations, comprise a common molecular and diagnostic entity and identify inhibitors of the LIN28/let7/PI3K/mTOR axis and DNMT3B as promising therapeutics for this distinct histogenetic entity.

Telomeres are the protective arrays of tandem TTAGGG sequence and associated proteins at the termini of chromosomes. Telomeres shorten at each cell division due to the end-replication problem and are maintained above a critical threshold in malignant cancer cells to prevent cellular senescence or apoptosis. With the recent advances in massive parallel sequencing, assessing telomere content in the context of other cancer genomic aberrations becomes an attractive possibility. We present the first comprehensive analysis of telomeric DNA content change in tumors using whole-genome sequencing data from 235 pediatric cancers.

Medulloblastoma is the most common malignant brain tumour of childhood. The identification of critical genes involved in its pathogenesis will be central to advances in our understanding of its molecular basis, and the development of improved therapeutic approaches.

Although Temozolomide is effective against glioblastoma, the prognosis remains dismal and new regimens with synergistic activity are sought for.

The implementation of personalised medicine in childhood cancers has been limited by a lack of clinically validated multi-target sequencing approaches specific for paediatric solid tumours. In order to support innovative clinical trials in high-risk patients with unmet need, we have developed a clinically relevant targeted sequencing panel spanning 311 kb and comprising 78 genes involved in childhood cancers. A total of 132 samples were used for the validation of the panel, including Horizon Discovery cell blends (n=4), cell lines (n=15), formalin-fixed paraffin embedded (FFPE, n=83) and fresh frozen tissue (FF, n=30) patient samples. Cell blends containing known single nucleotide variants (SNVs, n=528) and small insertion-deletions (indels n=108) were used to define panel sensitivities of ≥98% for SNVs and ≥83% for indels [95% CI] and panel specificity of ≥98% [95% CI] for SNVs. FFPE samples performed comparably to FF samples (n=15 paired). Of 95 well-characterised genetic abnormalities in 33 clinical specimens and 13 cell lines (including SNVs, indels, amplifications, rearrangements and chromosome losses), 94 (98.9%) were detected by our approach. We have validated a robust and practical methodology to guide clinical management of children with solid tumours based on their molecular profiles. Our work demonstrates the value of targeted gene sequencing in the development of precision medicine strategies in paediatric oncology.

Although children with acute lymphoblastic leukemia (ALL) generally have a good outcome, some patients do relapse and survival following relapse is poor. Altered DNA methylation is highly prevalent in ALL and raises the possibility that DNA methylation-based biomarkers could predict patient outcome. In this study, genome-wide methylation analysis, using the Illumina Infinium HumanMethylation450 BeadChip platform, was carried out on 52 diagnostic patient samples from 4 genetic subtypes [ETV6-RUNX1, high hyperdiploidy (HeH), TCF3-PBX1 and dic(9;20)(p11-13;q11)] in a 1:1 case-control design with patients who went on to relapse (as cases) and patients achieving long-term remission (as controls). Pyrosequencing assays for selected loci were used to confirm the array-generated data. Non-negative matrix factorization consensus clustering readily clustered samples according to genetic subgroups and gene enrichment pathway analysis suggested that this is in part driven by epigenetic disruption of subtype specific signaling pathways. Multiple bioinformatics approaches (including bump hunting and individual locus analysis) were used to identify CpG sites or regions associated with outcome. However, no associations with relapse were identified. Our data revealed that ETV6-RUNX1 and dic(9;20) subtypes were mostly associated with hypermethylation; conversely, TCF3-PBX1 and HeH were associated with hypomethylation. We observed significant enrichment of the neuroactive ligand-receptor interaction pathway in TCF3-PBX1 as well as an enrichment of genes involved in immunity and infection pathways in ETV6-RUNX1 subtype. Taken together, our results suggest that altered DNA methylation may have differential impacts in distinct ALL genetic subtypes.

Ependymoma with RELA fusion has been defined as a novel entity of the revised World Health Organization 2016 classification of tumors of the central nervous system (CNS), characterized by fusion transcripts of the RELA gene and consequent pathological activation of the NFkB pathway. These tumors represent the majority of supratentorial ependymomas in children. The validation of diagnostic tools to identify this clinically relevant ependymoma entity is essential. Here, we have used interphase fluorescent in situ hybridization (FISH) for C11orf95 and RELA, immunohistochemistry (IHC) for p65-RelA and the recently developed DNA methylation-based classification besides conventional histopathology, and compared the precision of the methods in 40 supratentorial pediatric brain tumors diagnosed as ependymomas in the past years. Reverse transcription PCR (RT-PCR) and RNA sequencing were performed to explore discordant cases. Furthermore, we integrated imaging and clinical features as additional layers of information. The concordance between nuclear RelA expression by IHC and RELA FISH was 100%. Concordance between IHC and DNA methylation profiling, and between FISH and DNA methylation profiling was also high (96.4% and 95.2%, respectively). Thirty-four out of 40 (85%) cases were confirmed by integrated diagnoses as ependymal tumors, including 22 RELA-fused ependymomas (71% of ependymal tumors), two YAP1-fused ependymomas (6%), six non-RELA/non-YAP1 ependymomas (18%) and four ependymal/subependymal mixed tumors (12%). Ependymal/subependymal mixed tumors had an excellent clinical outcome despite the presence of histopathological signs of malignancy, suggesting that these tumors should not be diagnosed as classic ependymomas. DNA methylation profiling helped in the differential diagnosis of RELA-fused ependymomas. IHC and FISH, which are available in the majority of pathology laboratories, are valuable tools to identify RELA-fused ependymomas.

The co-receptor lymphocyte-activation gene-3 (LAG3, LAG-3, CD223) is a potential target for immune checkpoint inhibition immunotherapies. However, little is known about the biological and clinical significance of LAG3 DNA methylation in melanoma and its microenvironment.

Molecular groups of medulloblastoma (MB) are well established. Novel risk stratification parameters include Group 3/4 (non-WNT/non-SHH) methylation subgroups I-VIII or whole-chromosomal aberration (WCA) phenotypes. This study investigates the integration of clinical and molecular parameters to improve risk stratification of non-WNT/non-SHH MB. Non-WNT/non-SHH MB from the HIT2000 study and the HIT-MED registries were selected based on availability of DNA-methylation profiling data. MYC or MYCN amplification and WCA of chromosomes 7, 8, and 11 were inferred from methylation array-based copy number profiles. In total, 403 non-WNT/non-SHH MB were identified, 346/403 (86%) had a methylation class family Group 3/4 methylation score (classifier v11b6) ≥ 0.9, and 294/346 (73%) were included in the risk stratification modeling based on Group 3 or 4 score (v11b6) ≥ 0.8 and subgroup I-VIII score (mb_g34) ≥ 0.8. Group 3 MB (5y-PFS, survival estimation ± standard deviation: 41.4 ± 4.6%; 5y-OS: 48.8 ± 5.0%) showed poorer survival compared to Group 4 (5y-PFS: 68.2 ± 3.7%; 5y-OS: 84.8 ± 2.8%). Subgroups II (5y-PFS: 27.6 ± 8.2%) and III (5y-PFS: 37.5 ± 7.9%) showed the poorest and subgroup VI (5y-PFS: 76.6 ± 7.9%), VII (5y-PFS: 75.9 ± 7.2%), and VIII (5y-PFS: 66.6 ± 5.8%) the best survival. Multivariate analysis revealed subgroup in combination with WCA phenotype to best predict risk of progression and death. The integration of clinical (age, M and R status) and molecular (MYC/N, subgroup, WCA phenotype) variables identified a low-risk stratum with a 5y-PFS of 94 ± 5.7 and a very high-risk stratum with a 5y-PFS of 29 ± 6.1%. Validation in an international MB cohort confirmed the combined stratification scheme with 82.1 ± 6.0% 5y-PFS in the low and 47.5 ± 4.1% in very high-risk groups, and outperformed the clinical model. These newly identified clinico-molecular low-risk and very high-risk strata, accounting for 6%, and 21% of non-WNT/non-SHH MB patients, respectively, may improve future treatment stratification.

Choroid plexus carcinoma (CPC) is a rare and aggressive tumor of infancy without a clear treatment strategy. This study describes the outcomes of children with CPC treated on the multi-institutional phase 2 SJYC07 trial and reports on the significance of clinical and molecular characteristics.

Two distinct genetically defined entities of ependymoma arising in the supratentorial compartment are characterized by the presence of either a C11orf95-RELA or a YAP-MAMLD1 fusion, respectively. There is growing evidence that supratentorial ependymomas without these genetic features exist. In this study, we report on 18 pediatric non-RELA/non-YAP supratentorial ependymomas that were systematically characterized by means of their histology, immunophenotype, genetics, and epigenomics. Comprehensive molecular analyses included high-resolution copy number analysis, methylation profiling, analysis of fusion transcripts by Nanostring technology, and RNA sequencing. Based upon histological and immunohistochemical features two main patterns were identified-RELA-like (n = 9) and tanycytic ependymomas (n = 6). In the RELA-like group histologically assigned to WHO grade III and resembling RELA-fused ependymomas, tumors lacked nuclear expression of p65-RelA as a surrogate marker for a pathological activation of the NF-κB pathway. Three tumors showed alternative C11orf95 fusions to MAML2 or NCOA1. A methylation-based brain tumor classifier assigned two RELA-like tumors to the methylation class "EP, RELA-fusion"; the others demonstrated no significant similarity score. Of the tanycytic group, 5/6 tumors were assigned a WHO grade II. No gene fusions were detected. Methylation profiling did not show any association with an established methylation class. We additionally identified two astroblastoma-like tumors that both presented with chromothripsis of chromosome 22 but lacked MN1 breaks according to FISH analysis. They revealed novel fusion events involving genes in chromosome 22. One further tumor with polyploid cytogenetics was interpreted as PFB ependymoma by the brain tumor methylation classifier but had no relation to the posterior fossa. Clinical follow-up was available for 16/18 patients. Patients with tanycytic and astroblastoma-like tumors had no relapse, while 2 patients with RELA-like ependymomas died. Our data indicate that in addition to ependymomas discovered so far, at least two more supratentorial ependymoma types (RELA-like and tanycytic) exist.

Genomic studies of pediatric cancer have primarily focused on specific tumor types or high-risk disease. Here, we used a three-platform sequencing approach, including whole-genome sequencing (WGS), whole-exome sequencing (WES), and RNA sequencing (RNA-seq), to examine tumor and germline genomes from 309 prospectively identified children with newly diagnosed (85%) or relapsed/refractory (15%) cancers, unselected for tumor type. Eighty-six percent of patients harbored diagnostic (53%), prognostic (57%), therapeutically relevant (25%), and/or cancer-predisposing (18%) variants. Inclusion of WGS enabled detection of activating gene fusions and enhancer hijacks (36% and 8% of tumors, respectively), small intragenic deletions (15% of tumors), and mutational signatures revealing of pathogenic variant effects. Evaluation of paired tumor-normal data revealed relevance to tumor development for 55% of pathogenic germline variants. This study demonstrates the power of a three-platform approach that incorporates WGS to interrogate and interpret the full range of genomic variants across newly diagnosed as well as relapsed/refractory pediatric cancers.

Clear cell meningioma represents an uncommon variant of meningioma that typically affects children and young adults. Although an enrichment of loss-of-function mutations in the SMARCE1 gene has been reported for this subtype, comprehensive molecular investigations are lacking. Here we describe a molecularly distinct subset of tumors (n = 31), initially identified through genome-wide DNA methylation screening among a cohort of 3093 meningiomas, of which most were diagnosed histologically as clear cell meningioma. This cohort was further supplemented by an additional 11 histologically diagnosed clear cell meningiomas for analysis (n = 42). Targeted DNA sequencing revealed SMARCE1 mutations in 33/34 analyzed samples, accompanied by a nuclear loss of expression determined via immunohistochemistry and a decreased SMARCE1 transcript expression in the tumor cells. Analysis of time to progression or recurrence of patients within the clear cell meningioma group (n = 14) in comparison to those with meningioma WHO grade 2 (n = 220) revealed a similar outcome and support the assignment of WHO grade 2 to these tumors. Our findings indicate the existence of a highly distinct epigenetic signature of clear cell meningiomas, separate from all other variants of meningiomas, with recurrent mutations in the SMARCE1 gene. This suggests that these tumors may arise from a different precursor cell population than the broad spectrum of the other meningioma subtypes.

Atypical teratoid/rhabdoid tumor (ATRT) is an aggressive central nervous system tumor characterized by loss of SMARCB1/INI1 protein expression and comprises three distinct molecular groups, ATRT-TYR, ATRT-MYC and ATRT-SHH. ATRT-SHH represents the largest molecular group and is heterogeneous with regard to age, tumor location and epigenetic profile. We, therefore, aimed to investigate if heterogeneity within ATRT-SHH might also have biological and clinical importance. Consensus clustering of DNA methylation profiles and confirmatory t-SNE analysis of 65 ATRT-SHH yielded three robust molecular subgroups, i.e., SHH-1A, SHH-1B and SHH-2. These subgroups differed by median age of onset (SHH-1A: 18 months, SHH-1B: 107 months, SHH-2: 13 months) and tumor location (SHH-1A: 88% supratentorial; SHH-1B: 85% supratentorial; SHH-2: 93% infratentorial, often extending to the pineal region). Subgroups showed comparable SMARCB1 mutational profiles, but pathogenic/likely pathogenic SMARCB1 germline variants were over-represented in SHH-2 (63%) as compared to SHH-1A (20%) and SHH-1B (0%). Protein expression of proneural marker ASCL1 (enriched in SHH-1B) and glial markers OLIG2 and GFAP (absent in SHH-2) as well as global mRNA expression patterns differed, but all subgroups were characterized by overexpression of SHH as well as Notch pathway members. In a Drosophila model, knockdown of Snr1 (the fly homologue of SMARCB1) in hedgehog activated cells not only altered hedgehog signaling, but also caused aberrant Notch signaling and formation of tumor-like structures. Finally, on survival analysis, molecular subgroup and age of onset (but not ASCL1 staining status) were independently associated with overall survival, older patients (> 3 years) harboring SHH-1B experiencing relatively favorable outcome. In conclusion, ATRT-SHH comprises three subgroups characterized by SHH and Notch pathway activation, but divergent molecular and clinical features. Our data suggest that molecular subgrouping of ATRT-SHH has prognostic relevance and might aid to stratify patients within future clinical trials.