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Supplementary Figures S1 to S5

Purpose:Report relevance of molecular groups to clinicopathologic features, germline SMARCB1/SMARCA4 alterations (GLA), and survival of children with atypical teratoid rhabdoid tumor (ATRT) treated in two multi-institutional clinical trials.Materials and Methods:Seventy-four participants with newly diagnosed ATRT were treated in two trials: infants (SJYC07: age < 3 years; n = 52) and children (SJMB03: age 3–21 years; n = 22), using surgery, conventional chemotherapy (infants), or dose-dense chemotherapy with autologous stem cell rescue (children), and age- and risk-adapted radiotherapy [focal (infants) and craniospinal (CSI; children)]. Molecular groups ATRT-MYC (MYC), ATRT-SHH (SHH), and ATRT-TYR (TYR) were determined from tumor DNA methylation profiles.Results:Twenty-four participants (32%) were alive at time of analysis at a median follow-up of 8.4 years (range, 3.1–14.1 years). Methylation profiling classified 64 ATRTs as TYR (n = 21), SHH (n = 30), and MYC (n = 13), SHH group being associated with metastatic disease. Among infants, TYR group had the best overall survival (OS; P = 0.02). However, outcomes did not differ by molecular groups among infants with nonmetastatic (M0) disease. Children with M0 disease and 2 residual tumor had a 5-year progression-free survival (PFS) of 72.7 ± 12.7% and OS of 81.8 ± 11%. Infants with M0 disease had a 5-year PFS of 39.1 ± 11.5% and OS of 51.8 ± 12%. Those with metastases fared poorly [5-year OS 25 ± 12.5% (children) and 0% (infants)]. SMARCB1 GLAs were not associated with PFS.Conclusions:Among infants, those with ATRT-TYR had the best OS. ATRT-SHH was associated with metastases and consequently with inferior outcomes. Children with nonmetastatic ATRT benefit from postoperative CSI and adjuvant chemotherapy.

Supplementary Table 1 SampleSheet Supplementary Table 2 GEO_IDs Supplementary Table 3 EpiWNT-SHH_data Supplementary Table 4 EpiWNT-SHH_test Supplementary Table 5 EpiG3-G4_data Supplementary Table 6 EpiG3-G4_test

Germline methodology

Supplementary Methods Supplementary Figure 1 Unsupervised DNA methylation analysis of the training cohort. Supplementary Figure 2 Comparison between the Illumina Infinium Human Methylation450K BeadChip and the Methylation EPIC BeadChip 850K array platforms. Supplementary Figure 3 Specificity of the epigenetic biomarkers for medulloblastoma subgroups. Supplementary Figure 4 Assay performance for low-input titration series. Supplementary Figure 5 Validation of the epigenetic classifier Panel EpiWNT-SHH using DNA methylation microarray data of medulloblastoma samples. Supplementary Figure 6 Validation of the methylation pattern of Panel EpiWNT-SHH using pyrosequencing and direct bisulfite sequencing. Supplementary Figure 7 Schematic overview of the experimental strategy applied for identification of the epigenetic biomarkers and the development of the classifier EpiG3-G4. Supplementary Figure 8 Validation of the epigenetic classifier Panel EpiG3-G4 using DNA methylation microarray data.

Purpose: The classification of medulloblastoma into WNT, SHH, group 3, and group 4 subgroups has become of critical importance for patient risk stratification and subgroup-tailored clinical trials. Here, we aimed to develop a simplified, clinically applicable classification approach that can be implemented in the majority of centers treating patients with medulloblastoma.Experimental Design: We analyzed 1,577 samples comprising previously published DNA methylation microarray data (913 medulloblastomas, 457 non-medulloblastoma tumors, 85 normal tissues), and 122 frozen and formalin-fixed paraffin-embedded medulloblastoma samples. Biomarkers were identified applying stringent selection filters and Linear Discriminant Analysis (LDA) method, and validated using DNA methylation microarray data, bisulfite pyrosequencing, and direct-bisulfite sequencing.Results: Using a LDA-based approach, we developed and validated a prediction method (EpiWNT-SHH classifier) based on six epigenetic biomarkers that allowed for rapid classification of medulloblastoma into the clinically relevant subgroups WNT, SHH, and non-WNT/non-SHH with excellent concordance (>99%) with current gold-standard methods, DNA methylation microarray, and gene signature profiling analysis. The EpiWNT-SHH classifier showed high prediction capacity using both frozen and formalin-fixed material, as well as diverse DNA methylation detection methods. Similarly, we developed a classifier specific for group 3 and group 4 tumors, based on five biomarkers (EpiG3-G4) with good discriminatory capacity, allowing for correct assignment of more than 92% of tumors. EpiWNT-SHH and EpiG3-G4 methylation profiles remained stable across tumor primary, metastasis, and relapse samples.Conclusions: The EpiWNT-SHH and EpiG3-G4 classifiers represent a new simplified approach for accurate, rapid, and cost-effective molecular classification of single medulloblastoma DNA samples, using clinically applicable DNA methylation detection methods. Clin Cancer Res; 24(6); 1355–63. ©2018 AACR.

IDH2 R172 mutations occur in sinonasal undifferentiated carcinoma (SNUC), large-cell neuroendocrine carcinoma (LCNEC), sinonasal adenocarcinomas, and olfactory neuroblastoma (ONB). We performed a clinical, pathologic, and genetic/epigenetic analysis of a large IDH2-mutated sinonasal tumor cohort to explore their distinct features. A total 165 sinonasal/skull base tumors included 40 IDH2 mutants studied by light microscopy, immunohistochemistry, and genome-wide DNA methylation, and 125 IDH2 wild-type tumors used for comparison. Methylation profiles were analyzed by unsupervised hierarchical clustering, t-distributed stochastic neighbor embedding dimensionality reduction and assessed for copy number alterations (CNA). Thirty-nine histologically assessable cases included 25 (64.1%) SNUC, 8 (20.5%) LCNEC, 2 (5.1%) poorly differentiated adenocarcinomas, 1 (2.7%) ONB, and 3 (7.7%) IDH2-mutated tumors with ONB features. All cases were high-grade showing necrosis (82.4%), prominent nucleoli (88.9%), and median 21 mitoses/10 HPFs. AE1/AE3 and/or CAM 5.2 were positive in all and insulinoma-associated protein 1 (INSM1) in 80% cases. All IDH2 mutants formed one distinct group by t-distributed stochastic neighbor embedding dimensionality reduction separating from all IDH2 wild-type tumors. There was no correlation between methylation clusters and histopathologic diagnoses. Recurrent CNA included 1q gain (79.3%), 17p loss (75.9%), and 17q gain (58.6%). No CNA differences were observed between SNUC and LCNEC. IDH2 mutants showed better disease-specific survival than SMARCB1-deficient (P=0.027) and IDH2 wild-type carcinomas overall (P=0.042). IDH2-mutated sinonasal tumors are remarkably homogeneous at the molecular level and distinct from IDH2 wild-type sinonasal malignancies. Biology of IDH2-mutated sinonasal tumors might be primarily defined by their unique molecular fingerprint rather than by their respective histopathologic diagnoses.

Loss of nuclear SMARCB1 (INI1/hSNF5/BAF47) protein expression due to biallelic mutations of the SMARCB1 tumor suppressor gene is a hallmark of atypical teratoid/rhabdoid tumors (ATRT), but the presence of cytoplasmic SMARCB1 protein in these tumors has not yet been described. In a series of 102 primary ATRT, distinct cytoplasmic SMARCB1 staining on immunohistochemistry was encountered in 19 cases (19%) and was highly over-represented in cases showing pathogenic sequence variants leading to truncation or mutation of the C-terminal part of SMARCB1 (15/19 vs. 4/83; Chi-square: 56.04, p = 1.0E−10) and, related to this, in tumors of the molecular subgroup ATRT-TYR (16/36 vs. 3/66; Chi-square: 24.47, p = 7.6E−7). Previous reports have indicated that while SMARCB1 lacks a bona fide nuclear localization signal, it harbors a masked nuclear export signal (NES) and that truncation of the C-terminal region results in unmasking of this NES leading to cytoplasmic localization. To determine if cytoplasmic localization found in ATRT is due to unmasking of NES, we generated GFP fusions of one of the SMARCB1 truncating mutations (p.Q318X) found in the tumors along with a p.L266A mutation, which was shown to disrupt the interaction of SMARCB1-NES with exportin-1. We found that while the GFP-SMARCB1(Q318X) mutant localized to the cytoplasm, the double mutant GFP-SMARCB1(Q318X;L266A) localized to the nucleus, confirming NES requirement for cytoplasmic localization. Furthermore, cytoplasmic SMARCB1(Q318X) was unable to cause senescence as determined by morphological observations and by senescence-associated β-galactosidase assay, while nuclear SMARCB1(Q318X;L266A) mutant regained this function. Selinexor, a selective exportin-1 inhibitor, was effective in inhibiting the nuclear export of SMARCB1(Q318X) and caused rapid cell death in rhabdoid tumor cells. In conclusion, inhibition of nuclear export restores nuclear localization and residual tumor suppressor function of truncated SMARCB1. Therapies aimed at preventing nuclear export of mutant SMARCB1 protein may represent a promising targeted therapy in ATRT harboring truncating C-terminal SMARCB1 mutations.

Purpose: Report relevance of molecular groups to clinicopathologic features, germline SMARCB1/SMARCA4 alterations (GLA), and survival of children with atypical teratoid rhabdoid tumor (ATRT) treated in two multi-institutional clinical trials. Materials and Methods: Seventy-four participants with newly diagnosed ATRT were treated in two trials: infants (SJYC07: age < 3 years; n = 52) and children (SJMB03: age 3–21 years; n = 22), using surgery, conventional chemotherapy (infants), or dose-dense chemotherapy with autologous stem cell rescue (children), and age- and risk-adapted radiotherapy [focal (infants) and craniospinal (CSI; children)]. Molecular groups ATRT-MYC (MYC), ATRT-SHH (SHH), and ATRT-TYR (TYR) were determined from tumor DNA methylation profiles. Results: Twenty-four participants (32%) were alive at time of analysis at a median follow-up of 8.4 years (range, 3.1–14.1 years). Methylation profiling classified 64 ATRTs as TYR (n = 21), SHH (n = 30), and MYC (n = 13), SHH group being associated with metastatic disease. Among infants, TYR group had the best overall survival (OS; P = 0.02). However, outcomes did not differ by molecular groups among infants with nonmetastatic (M0) disease. Children with M0 disease and Conclusions: Among infants, those with ATRT-TYR had the best OS. ATRT-SHH was associated with metastases and consequently with inferior outcomes. Children with nonmetastatic ATRT benefit from postoperative CSI and adjuvant chemotherapy.

Atypical teratoid/rhabdoid tumors (ATRTs) are very aggressive childhood malignancies of the central nervous system. The underlying genetic cause are inactivating bi-allelic mutations in SMARCB1 or (rarely) in SMARCA4. ATRT-SMARCA4 have been associated with a higher frequency of germline mutations, younger age, and an inferior prognosis in comparison to SMARCB1 mutated cases. Based on their DNA methylation profiles and transcriptomics, SMARCB1 mutated ATRTs have been divided into three distinct molecular subgroups: ATRT-TYR, ATRT-SHH, and ATRT-MYC. These subgroups differ in terms of age at diagnosis, tumor location, type of SMARCB1 alterations, and overall survival. ATRT-SMARCA4 are, however, less well understood, and it remains unknown, whether they belong to one of the described ATRT subgroups. Here, we examined 14 ATRT-SMARCA4 by global DNA methylation analyses. We show that they form a separate group segregating from SMARCB1 mutated ATRTs and from other SMARCA4-deficient tumors like small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) or SMARCA4 mutated extra-cranial malignant rhabdoid tumors. In contrast, medulloblastoma (MB) samples with heterozygous SMARCA4 mutations do not group separately, but with established MB subgroups. RNA sequencing of ATRT-SMARCA4 confirmed the clustering results based on DNA methylation profiling and displayed an absence of typical signature genes upregulated in SMARCB1 deleted ATRT. In summary, our results suggest that, in line with previous clinical observations, ATRT-SMARCA4 should be regarded as a distinct molecular subgroup.

Background A prospective 2002–2014 study stratified 160 patients by resection extent and histological grade, reporting results in 2016. We re-analyzed the series after a median of 119 months, adding retrospectively patients’ molecular features. Methods Follow-up of all patients was updated. DNA copy number analysis and gene-fusion detection could be completed for 94/160 patients, methylation classification for 68. Results Progression-free survival (PFS) and overall survival (OS) at 5/10 years were 66/58%, and 80/73%. Ten patients had late relapses (range 66–126 mo), surviving after relapse no longer than those relapsing earlier (0–5 y). On multivariable analysis a better PFS was associated with grade II tumor and complete surgery at diagnosis and/or at radiotherapy; female sex and complete resection showed a positive association with OS. Posterior fossa (PF) tumors scoring ≥0.80 on DNA methylation analysis were classified as PFA (n = 41) and PFB (n = 9). PFB patients had better PFS and OS. Eighteen/32 supratentorial tumors were classified as RELA, and 3 as other molecular entities (anaplastic PXA, LGG MYB, HGNET). RELA had no prognostic impact. Patients with 1q gain or cyclin-dependent kinase inhibitor 2A (CDKN2A) loss had worse outcomes, included significantly more patients >3 years old (P = 0.050) and cases of dissemination at relapse (P = 0.007). Conclusions Previously described prognostic factors were confirmed at 10-year follow-up. Late relapses occurred in 6.2% of patients. Specific molecular features may affect outcome: PFB patients had a very good prognosis; 1q gain and CDKN2A loss were associated with dissemination. To draw reliable conclusions, modern ependymoma trials need to combine diagnostics with molecular risk stratification and long-term follow-up.

Background/aim Malignant rhabdoid tumor (MRT) is a rare, aggressive neoplasm found in young children, caused by inactivation of a single gene, SNF5 (INI1, SMARCB1). MRT cases with multifocal tumors at diagnosis are categorized as synchronous MRT, often with a germline mutation of SNF5. The aim of this study was to establish new models useful in clarifying the biological basis of synchronous MRT. Materials and methods We established two novel MRT cell lines, designated as KP-MRT-KS and KP-MRT-KSa, derived from different lesions and at a different time from a synchronous multifocal 7-month-old female MRT patient. Results Both cells showed typical morphology of MRT, with a compound genomic mutation in exons 2 and 5 of the SNF5 gene. The exon 2 mutation was found in the germline. Conclusion These cell lines could serve as powerful tools for unveiling the molecular mechanism of refractory synchronous MRT.

INTRODUCTION Granulocyte transfusions have long been used to bridge the time to neutrophil recovery in patients with neutropenia and severe infection. Recent randomized controlled trials did not prove a beneficial effect of granulocyte transfusions, but were likely underpowered and suffered from very heterogeneous study populations. METHODS We retrospectively reviewed data of all patients treated with granulocyte transfusions at our pediatric center from 2004 to 2019. In order to identify parameters that predict the success of granulocyte transfusions, we stratified patients in 3 groups. Patients in group 1 cleared their infection, whereas patients in group 2 succumbed to an infection in neutropenia despite granulocyte transfusions. A third group included all patients who died of causes that were not related to infection. RESULTS We demonstrate that patients without respiratory or cardiocirculatory insufficiency are enriched in group 1 and more likely to benefit from granulocyte transfusions than patients who already require these intensive care measures. The effect of granulocyte transfusions correlates with the cell dose per body weight applied per time. With our standard twice weekly dosing, patients with a body weight below 40 kg are more likely to achieve a sufficient leukocyte increment and clear their infection in comparison to patients with a higher body weight. DISCUSSION/CONCLUSIONS We suggest that future studies on the benefits of granulocyte transfusions stratify patients according to clinical risk factors that include the need for respiratory or cardiocirculatory support and strive for a sufficient dose density of granulocyte transfusions.

Background More than 40% of patients with intracranial ependymoma need a salvage treatment within 5 years after diagnosis, and no standard treatment is available as yet. We report the outcome after first relapse of 64 patients treated within the 2nd AIEOP protocol. Methods We considered relapse sites and treatments, that is, various combinations of complete/incomplete surgery, if followed by standard or hypofractionated radiotherapy (RT) ± chemotherapy (CT). Molecular analyses were available for 38/64 samples obtained at first diagnosis. Of the 64 cases, 55 were suitable for subsequent analyses. Results The median follow-up was 147 months after diagnosis, 84 months after first relapse, 5-year EFS/OS were 26.2%/30.8% (median EFS/OS 13/32 months) after relapse. For patients with a local relapse (LR), the 5-year cumulative incidence of second LRs was 51.6%, with a 5-year event-specific probability of being LR-free of 40.0%. Tumor site/grade, need for shunting, age above/below 3 years, molecular subgroup at diagnosis, had no influence on outcomes. Due to variation in the RT dose/fractionation used and the subgroup sizes, it was not possible to assess the impact of the different RT modalities. Multivariable analyses identified completion of surgery, the absence of symptoms at relapse, and female sex as prognostically favorable. Tumors with a 1q gain carried a higher cumulative incidence of dissemination after first relapse. Conclusions Survival after recurrence was significantly influenced by symptoms and completeness of surgery. Only a homogeneous protocol with well-posed, randomized questions could clarify the numerous issues, orient salvage treatment, and ameliorate prognosis for this group of patients.

OBJECTIVE: Embryonal tumors with multi-layered rosettes (ETMR) (CNS WHO grade 4) comprise a rare and malignant tumor type affecting predominantly infants below 3 years of age. The treatment consists of maximal safe surgical resection, irradiation, and intensive medulloblastoma type chemotherapy. Despite aggressive treatment, the prognosis of these patients remains poor, especially for brainstem tumors. We present the case of a male infant diagnosed with a brainstem ETMR, successfully treated with an interdisciplinary multimodal approach, including stereotactic interstitial brachytherapy. RESULTS: A 19 month old boy first presented with hemiparesis, intermittent bradycardia and reduced consciousness. Initial imaging showed a brainstem lesion with characteristic features of a diffuse intrinsic pontine glioma (DIPG). We performed stereotactic biopsy to confirm the diagnosis and initiated temozolomide treatment. While the pathology result was still pending, the boy’s clinical condition deteriorated to a soporic state with stretch synergisms. By emergency open surgery, partial resection was achieved. Eventually, the patient recovered rapidly. After the diagnosis of ETMR was established, medulloblastoma type chemotherapy (systemic carboplatin/etoposide; intrathecal methotrexate) was administered. After two cycles, the patient showed only residual right-sided hemiparesis. However, imaging demonstrated only a minimal reduction of the tumor size. Therefore, stereotactic interstitial brachytherapy using 125iodine seeds and subsequent high-dose chemotherapy (HDCT) with autologous stem cell transplantation (ASCT) was performed and overall tolerated well. After two months of brachytherapy and two additional cycles of chemotherapy, the MRI showed ≥ 50% reduction in tumor volume and no neurological deficit can be clinically detected. CONCLUSION: This case indicates that stereotactic interstitial brachytherapy during intensive systemic chemotherapy is feasible. It may provide a suitable treatment for malignant infant brain tumors. Furthermore, it shows that paediatric patients are capable of recovery even after devastating neurological symptoms. Lastly, it emphasizes the importance of multidisciplinary and multimodal treatment for rare diseases.

Atypical Teratoid/Rhabdoid Tumors (ATRTs) are known for exhibiting high inter-tumor heterogeneity, even though they are almost all characterized by a common loss of SMARCB1 (or rarely SMARCA4). Three subgroups have been identified at bulk methylome and transcriptome level: ATRT-TYR, ATRT-SHH, and ATRT-MYC. To better understand the biology underlying each subgroup and potentially unveil their (different) cell(s) of origin, we performed single-cell transcriptomic analyses in 22 ATRTs using fresh frozen samples and both 10X and Smartseq technology. All data, grouped by technology, underwent quality control and normalization, regressing out the biases introduced by each sample. Tumor microenvironment (TME) and tumor bulk (TB) clusters were characterized by a combination of copy number variant analyses, enrichment in literature lists of marker genes for specific cell populations, and in-depth analysis of differentially enriched (DE) genes. Non-negative Matrix Factorization (NMF) was applied to TB to reveal major transcriptional profiles, which were grouped into meta-signatures. A total of 71 gene lists were retrieved from NMF (TB) and DE analyses (TME + TB), that gathered into 11 signature groups by Jaccard similarity, with one extra group accounting for unique signatures. Three groups targeted TME, accounting for either microglia, fibroblasts and endothelial cells, or OPCs, oligodendrocytes, astrocytes and neurons. These signatures are enriched in specific clusters across technologies. The remaining eight groups divide into two types, either enriched in clusters predominantly formed by cells of one or two ATRT subgroups or signatures enriched for a particular phenotype, such as cilial, cycling, axonogenesis or EM transition. While the first type is enriched across clusters in a gradient fashion, the second shows enrichment for selected clusters across technologies. Further analyses on the integrated dataset and additional samples are ongoing to validate and refine these 11 signature groups in ATRTs to see how this may lead to new treatment approaches.

BackgroundAtypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive CNS tumors of infancy and early childhood. Hallmark is the surprisingly simple genome with inactivating mutations or deletions in the SMARCB1 gene as the oncogenic driver. Nevertheless, AT/RTs are infiltrated by immune cells and even clonally expanded T cells. However, it is unclear which epitopes T cells might recognize on AT/RT cells.MethodsHere, we report a comprehensive mass spectrometry (MS)-based analysis of naturally presented human leukocyte antigen (HLA) class I and class II ligands on 23 AT/RTs. MS data were validated by matching with a human proteome dataset and exclusion of peptides that are part of the human benignome. Cryptic peptide ligands were identified using Peptide-PRISM.ResultsComparative HLA ligandome analysis of the HLA ligandome revealed 55 class I and 139 class II tumor-exclusive peptides. No peptide originated from the SMARCB1 region. In addition, 61 HLA class I tumor-exclusive peptide sequences derived from non-canonically translated proteins. Combination of peptides from natural and cryptic class I and class II origin gave optimal representation of tumor cell compartments. Substantial overlap existed with the cryptic immunopeptidome of glioblastomas, but no concordance was found with extracranial tumors. More than 80% of AT/RT exclusive peptides were able to successfully prime CD8+T cells, whereas naturally occurring memory responses in AT/RT patients could only be detected for class II epitopes. Interestingly, >50% of AT/RT exclusive class II ligands were also recognized by T cells from glioblastoma patients but not from healthy donors.ConclusionsThese findings highlight that AT/RTs, potentially paradigmatic for other pediatric tumors with a low mutational load, present a variety of highly immunogenic HLA class I and class II peptides from canonical as well as non-canonical protein sources. Inclusion of such cryptic peptides into therapeutic vaccines would enable an optimized mapping of the tumor cell surface, thereby reducing the likelihood of immune evasion.

Background Refined therapy has helped to improve survival rates in rhabdoid tumors (RT). Prognosis for patients with chemoresistant, recurrent, or progressive RT remains dismal. Although decitabine, an epigenetically active agent, has mainly been evaluated in the management of hematologic malignancies in adults, safety in children has also been demonstrated repeatedly. Materials and methods A retrospective series of patients who received decitabine upon relapse or progression following therapy according to the EU‐RHAB regimen is presented. Due to the retrospective nature of analyses, response was defined as measurable regression of at least one lesion on imaging. 850k methylation profiling was done whenever tumor tissue was available. Results A total of 22 patients with RT of any anatomical localization were included. Most patients (19/22) presented with metastases. All received low‐dose decitabine with or preceding conventional chemotherapy. Patients received a median of two (1‐6) courses of decitabine; 27.3% (6/22) demonstrated a radiological response. Molecular analyses revealed increased methylation levels in tumors from responders. No excessive toxicity was observed. Clinical benefits for responders included eligibility for early phase trials or local therapy. Responders showed prolonged time to progression and overall survival. Due to small sample size, statistical correction for survivorship bias demonstrated no significant effect on survival for responders. Conclusions Patients with RT demonstrate promising signs of antitumor activity after multiagent relapse therapy including decitabine. Analyses of methylation data suggest a specific effect on an epigenetic level. We propose to consider decitabine and other epigenetic drugs as candidates for further clinical investigations in RT., publishedVersion

Background With the advent of array-based techniques to measure methylation levels in primary tumor samples, systematic investigations of methylomes have widely been performed on a large number of tumor entities. Most of these approaches are not based on measuring individual cell methylation but rather the bulk tumor sample DNA, which contains a mixture of tumor cells, infiltrating immune cells and other stromal components. This raises questions about the purity of a certain tumor sample, given the varying degrees of stromal infiltration in different entities. Previous methods to infer tumor purity require or are based on the use of matching control samples which are rarely available. Here we present a novel, reference free method to quantify tumor purity, based on two Random Forest classifiers, which were trained on ABSOLUTE as well as ESTIMATE purity values from TCGA tumor samples. We subsequently apply this method to a previously published, large dataset of brain tumors, proving that these models perform well in datasets that have not been characterized with respect to tumor purity . Results Using two gold standard methods to infer purity – the ABSOLUTE score based on whole genome sequencing data and the ESTIMATE score based on gene expression data- we have optimized Random Forest classifiers to predict tumor purity in entities that were contained in the TCGA project. We validated these classifiers using an independent test data set and cross-compared it to other methods which have been applied to the TCGA datasets (such as ESTIMATE and LUMP). Using Illumina methylation array data of brain tumor entities (as published in Capper et al. (Nature 555:469-474,2018)) we applied this model to estimate tumor purity and find that subgroups of brain tumors display substantial differences in tumor purity. Conclusions Random forest- based tumor purity prediction is a well suited tool to extrapolate gold standard measures of purity to novel methylation array datasets. In contrast to other available methylation based tumor purity estimation methods, our classifiers do not need a priori knowledge about the tumor entity or matching control tissue to predict tumor purity. Electronic supplementary material The online version of this article (10.1186/s12859-019-3014-z) contains supplementary material, which is available to authorized users.

INTRODUCTION: Anecdotal case reports suggest an association between assisted reproduction technologies (ART) and malignant rhabdoid tumors (MRT). We performed a multi-institutional retrospective analysis of the EU-RHAB database, complemented by additional cases outside of EU-RHAB to compile clinical, (epi)genetic characteristics and outcome data of children with MRT following ART. METHODS: Data of 14 patients (from 311 patients with MRT) from 9 countries were analyzed (2010-2018). Tumors and matching blood samples were examined for SMARCB1 mutations using FISH, MLPA and sequencing. Molecular subgroups were determined using DNA methylation arrays and correlated with a validation cohort (n=22, tumor samples of MRT; n=39 blood samples of patients small for gestational age). RESULTS: The median age at diagnosis of the 13 girls and 1 boy was 9 months (0 – 66). 8 patients with ATRT, 3 with extracranial, extrarenal-, 1 with renal rhabdoid tumor and 2 with synchronous tumors were identified. Distant metastases at diagnosis were present in 6 patients. A germline mutation (GLM) was detected in 5 patients. In 11 tumors complete data on SMARCB1 mutational status were available. DNA methylation subgrouping was available in 10 tumors and 6 blood samples. A female predominance was noted as compared to the EU-RHAB cohort with MRT born without ART (n=213, p=0.009). A total of 8 patients received gross total resection, n=12 patients received conventional chemotherapy (EU-RHAB=9, Head Start II=2, IRS III=1). Radiotherapy was applied to 6 patients. 10 patients achieved CR, and 5 remain in continuing CR. Significant genome-wide DNA methylation differences (including imprinted genes) between patients born after ART and patients born without ART could not be demonstrated. CONCLUSIONS: Long-term survival is achievable in patients who develop MRT after ART, even in cases with GLM, metastatic disease at diagnosis, or relapse. Larger epidemiological studies are needed to confirm a potential association between MRT and ART.

Rhabdoid tumors (RT) are embryonal neoplasms occurring most frequently in the central nervous system where they are termed atypical teratoid rhabdoid tumor (ATRT). A common hallmark of RT is homozygous loss of the BAF complex subunit SMARCB1. RT patients have a poor prognosis with an overall survival time of 17 months and >60% of patients suffer from relapses. The lack of an optimal treatment strategy could be attributed to the heterogeneity within and between different subgroups of ATRT. Despite the recent advancements in characterizing RT at a molecular level, the cellular origin of RT remains elusive. Thus, this study focused on the identification of the cellular origin of MYC-RT and underlying epigenetic deregulations which account for the cellular heterogeneity in these tumors. We showed that Smarcb1 abrogation in Sox2-positive progenitor cells at E6.5 give rise to RT of the MYC and SHH subgroup in genetically engineered mouse models (GEMM). To uncover distinct cells of origin (COO) for the SHH and MYC subgroups, unbiased computational approaches were used to compare single-cell transcriptomes of GEMMs with single-cell reference maps of murine early embryogenesis. While SHH tumors arise from mid/hindbrain progenitor cells, primordial germ cells (PGCs) emerge as COO of both intracranial and extracranial MYC tumors. PGCs as COO of MYC-RT were validated in vivo by using PGC-specific Smarcb1 knockout mouse model. We further characterized a deregulated transcriptome in MYC-RT compared to PGCs, which is sustained by a subset of epigenetically driven tumor cells. Deregulated expression of genes driving methylation/demethylation processes in MYC tumors and regression of these tumors upon treatment with decitabine in vitro and in vivo, indicates that DNA methylation plays a key role in cellular transformation and development of MYC-RT.

INTRODUCTION: Atypical teratoid/rhabdoid tumors (ATRT) represent frequent brain tumors in infants. In recent years, large-scale landscaping efforts on the epigenome and transcriptome of these tumors have unravelled a high degree of heterogeneity and three major molecular subgroups, termed ATRT-TYR, ATRT-SHH,ATRT-MYC, have been identified. The ATRT-proteome, in turn, still represents a largely unchartered territory. METHODS: We have performed a peptide-based screening approach to characterize the proteome of 40 ATRTs and six ATRT cell-lines. All of these samples had matching methylation data available and 28 also corresponding gene expression data. RESULTS: Unsupervised clustering recapitulated the previously described ATRT groups, revealing also a clear split of the SHH-subgroup in a supratentorial (SHH_1) and an infratentorial subgroup (SHH_2). Overall, we identified 7265 proteins, of which 1320 were differentially expressed between the groups, with an enrichment of spliceosome associated terms in SHH_1 and integrins/cell adhesions molecules in SHH_2. ATRT-MYC displayed an overrepresentation of immune cell markers and the TYR subgroup an enrichment of PI3K- as well as mTOR-signaling. Particularly, genes that have previously been described as signature genes for the ATRT-groups such as FABP7 in ATRT-SHH and OTX2 and MITF in ATRT-TYR were among the highly correlating genes that were both expressed in the proteome and the gene expression datasets. On top of this, our analysis revealed highly differentially expressed drug targets such as the tyrosine-kinase MARCKS (overexpressed in ATRT-TYR) not previously identified in ATRT transcriptome data, which warrant investigation by in vitro drug tests. CONCLUSION: Our data reveal the importance of previously described regulatory hubs in the ATRT subgroups, but additionally highlight novel drug targets that merit further exploration. Currently, drug treatment experiments in ATRT cell lines are ongoing to validate these proteins as drug targets, ultimately aiming to establish new therapeutic strategies in this deadly disease.

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant tumor that may not only contain rhabdoid tumor cells but also poorly differentiated small‐round‐blue cells as well as areas with mesenchymal or epithelial differentiation. Little is known on factors associated with histopathological diversity. Recent studies demonstrated three molecular subgroups of AT/RT, namely ATRT‐TYR, ATRT‐SHH, and ATRT‐MYC. We thus aimed to investigate if morphological patterns might be related to molecular subgroup status. Hematoxylin‐eosin stained sections of 114 AT/RT with known molecular subgroup status were digitalized and independently categorized by nine blinded observers into four morphological categories, that is, “rhabdoid,” “small‐round‐blue,” “epithelial,” and “mesenchymal.” The series comprised 48 ATRT‐SHH, 40 ATRT‐TYR, and 26 ATRT‐MYC tumors. Inter‐observer agreement was moderate but significant (Fleiss’ kappa = 0.47; 95% C.I. 0.41‐0.53; p

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant central nervous system tumor predominantly occurring in infants that may also arise in older children and adults. Rare secondary AT/RT developing from other tumors such as pleomorphic xanthoastrocytoma (PXA) are on record, but AT/RT presenting with molecular features of PXA have not been described. Here, we report 3 malignant central nervous system tumors in children (10, 13, and 18 y old). All tumors were located in the temporal lobe. In 2 cases, there was no history of a low-grade precursor lesion; in 1 case anaplastic PXA had been diagnosed 3 months earlier. Histopathologically, all tumors were composed of RT cells and showed frank signs of malignancy as well as loss of nuclear SMARCB1/INI1 protein expression. Two cases displayed homozygous deletions of the SMARCB1 region while the third case showed an exon 7 mutation (c.849_850delGT; p.Met283Ilefs*77). Of note, DNA methylation profiles did not group with AT/RT or other tumor entities using the Heidelberg Brain Tumor Classifier (version v11b4). By unsupervised t-distributed stochastic neighbor embedding analysis and hierarchical clustering analysis, however, all tumors clearly grouped with PXA. Genome-wide copy number analysis revealed homozygous CDNK2A/B deletions and gains of whole chromosome 7. BRAF V600E mutations could be demonstrated in all cases. In conclusion, the possibility of AT/RT with molecular features of PXA needs to be taken into account and warrants molecular characterization of AT/RT especially in older children. Since treatments targeting mutated BRAF are available, identification of such cases may also have therapeutic consequences.

BackgroundGlioblastoma multiforme (GBM) is the most common malignant brain tumor among adult patients and represents an almost universally fatal disease. Novel therapies for GBM are being developed under the orphan drug legislation and the knowledge on the molecular makeup of this disease has been increasing rapidly. However, the clinical outcomes in GBM patients with currently available therapies are still dismal. An insight into the current drug development pipeline for GBM is therefore of particular interest.ObjectivesTo provide a quantitative clinical-regulatory insight into the status of FDA orphan drug designations for compounds intended to treat GBM.MethodsQuantitative cross-sectional analysis of the U.S. Food and Drug Administration Orphan Drug Product database between 1983 and 2020. STROBE criteria were respected.ResultsFour orphan drugs out of 161 (2,4%) orphan drug designations were approved for the treatment for GBM by the FDA between 1983 and 2020. Fourteen orphan drug designations were subsequently withdrawn for unknown reasons. The number of orphan drug designations per year shows a growing trend. In the last decade, the therapeutic mechanism of action of designated compounds intended to treat glioblastoma shifted from cytotoxic drugs (median year of designation 2008) to immunotherapeutic approaches and small molecules (median year of designation 2014 and 2015 respectively) suggesting an increased focus on precision in the therapeutic mechanism of action for compounds the development pipeline.ConclusionDespite the fact that current pharmacological treatment options in GBM are sparse, the drug development pipeline is steadily growing. In particular, the surge of designated immunotherapies detected in the last years raises the hope that elaborate combination possibilities between classical therapeutic backbones (radiotherapy) and novel, currently experimental therapeutics may help to provide better therapies for this deadly disease in the future.Article summaryStrengths and limitationsThis study provides a quantitative overview on the drug development pipeline for pediatric and adult oncology in general and specifically for the indication glioblastomaAnalyzing the therapeutic mechanisms of designated compounds in glioblastoma reveals an increased focus on personalized and targeted therapiesThe precise reasons for failure of approved drugs and for withdrawal of approved drugs in glioblastoms are unknownFor the analysis, only the databases “FDALabel” (https://nctr-crs.fda.gov/fdalabel/ui/search) and the FDA Orphan drug product designation database (https://www.accessdata.fda.gov/scripts/opdlisting/oopd/) were considered

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

Here, we present a patient with high-grade neuroepithelial tumors with mutations in the BCL6 corepressor BCOR (HGNET-BCOR), a rare, highly malignant brain tumor with poor prognosis. The patient underwent gross total tumor resection (GTR), high-dose chemotherapy, and, after local relapse, GTR, proton radiation, and chemotherapy. After a 7.5 year-long complete remission, the tumor recurred locally, was treated by GTR, and responded to temozolomide treatment. In addition to an internal tandem duplication in BCOR common to the majority of HGNET-BCOR cases, molecular analysis revealed a second BCOR mutation in this tumor: a frame shift mutation. The combination of these mutations was associated with relatively low BCOR expression compared to other HGNET-BCOR cases.

Atypical teratoid rhabdoid tumors (ATRT) are challenging pediatric brain cancers which are predominantly associated with inactivation of the gene SMARCB1, a conserved subunit of the chromatin remodeling BAF complex, which has known contributions to developmental processes. To identify potential interactions between SMARCB1 loss and the process of neural development, we introduced an inducible SMARCB1 loss of function system into human induced pluripotent stem cells (iPSCs) which were subjected to either directed neuronal differentiation or differentiation into cerebral organoids. Using this system, we have identified substantial differences in the downstream effects of SMARCB1 loss depending on differentiation state and identified an interaction between SMARCB1 loss and neural differentiation pressure which causes a resistance to terminal differentiation and a defect in maintenance of a normal cell state. Our results provide insight into how SMARCB1 loss might interact with neural development in the process of ATRT tumorigenesis.

Atypical teratoid/rhabdoid tumors (ATRTs) typically arise in the central nervous system (CNS) of children under 3 years of age. Despite intensive multimodal therapy (surgery, chemotherapy and, if age permits, radiotherapy), median survival is 17 months(1,2). We show that ATRTs robustly express B7-H3/CD276 that does not result from the inactivating mutations in SMARCB1 (refs.(3,4)), which drive oncogenesis in ATRT, but requires residual SWItch/Sucrose Non-Fermentable (SWI/SNF) activity mediated by BRG1/SMARCA4. Consistent with the embryonic origin of ATRT(5,6), B7-H3 is highly expressed on the prenatal, but not postnatal, brain. B7-H3. BB.z-chimeric antigen receptor (CAR) T cells administered intracerebroventricularly or intratumorally mediate potent antitumor effects against cerebral ATRT xenografts in mice, with faster kinetics, greater potency and reduced systemic levels of inflammatory cytokines compared to CAR T cells administered intravenously. CAR T cells administered ICV also traffic from the CNS into the periphery; following clearance of ATRT xenografts, B7-H3.BB.z-CAR T cells administered intracerebroventricularly or intravenously mediate antigen-specific protection from tumor rechallenge, both in the brain and periphery. These results identify B7-H3 as a compelling therapeutic target for this largely incurable pediatric tumor and demonstrate important advantages of locoregional compared to systemic delivery of CAR T cells for the treatment of CNS malignancies.

Atypical teratoid/rhabdoid tumor (ATRT) is a highly malignant brain tumor predominantly encountered in infants. Mutations of the SMARCB1 gene are the characteristic genetic lesion. A small group of ATRT stands out clinically, because these tumors are located in the sellar region of adults. To investigate if sellar region ATRT in adults represents a molecular distinct entity, we characterized molecular alterations in 7 sellar region ATRTs in adults as compared with 150 pediatric ATRTs and 47 pituitary adenomas using SMARCB1 sequencing, multiplex ligation-dependent probe amplification and fluorescence in situ hybridization as well as DNA methylation profiling. The median age of the 6 female and 1 male patients was 56 years. On histopathologic examination, all tumors were malignant rhabdoid tumors showing loss of SMARCB1/INI1 protein expression. Two cases displayed compound heterozygous SMARCB1 point mutations, 3 cases showed heterozygous SMARCB1 deletions with point mutations of the other allele and 1 case a homozygous SMARCB1 deletion; in 1 case, underlying SMARCB1 alterations could not be identified. On unsupervised hierarchical cluster analysis of DNA methylation profiles, sellar region ATRTs did not form a distinct group, but clustered with ATRT-MYC, 1 of 3 recently described molecular subgroups of ATRT. On analysis of DNA methylation array intensity data, only 1 sellar region ATRT showed characteristic features of pediatric ATRT-MYC, that is, major copy number losses affecting the SMARCB1 region. In conclusion, these results suggest that sellar region ATRTs in adults form a clinically distinct entity with a different mutational spectrum, but epigenetic similarities with pediatric ATRTs of the ATRT-MYC subgroup.

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

Mutations in chromatin remodelling genes occur in approximately 25% of all human tumours (Kadoch et al. Nat Genet 45: 592-601, 2013). The spectrum of alterations is broad and comprises single nucleotide variants, insertion/deletions and more complex structural variations. The single most often affected remodelling complex is the SWI/SNF complex (SWItch/sucrose non-fermentable). In the field of paediatric neuro-oncology, the spectrum of affected genes implicated in epigenetic remodelling is narrower with SMARCB1 and SMARCA4 being the most frequent. The low mutation frequencies in many of the SWI/SNF mutant entities underline the fact that perturbed chromatin remodelling is the most salient factor in tumourigenesis and could thus be a potential therapeutic opportunity. Here, I review the genetic basis of aberrant chromatin remodelling in paediatric brain tumours and discuss their impact on the epigenome in the respective entities, mainly medulloblastomas and rhabdoid tumours.

Background Controversy exists as to what may be defined as standard of care (including markers for stratification) for patients with atypical teratoid/rhabdoid tumors (ATRTs). The European Rhabdoid Registry (EU-RHAB) recruits uniformly treated patients and offers standardized genetic and DNA methylation analyses. Methods Clinical, genetic, and treatment data of 143 patients from 13 European countries were analyzed (2009–2017). Therapy consisted of surgery, anthracycline-based induction, and either radiotherapy or high dose chemotherapy following a consensus among European experts. Fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, and sequencing were employed for assessment of somatic and germline mutations in SWItch/sucrose nonfermentable related, matrix associated, actin dependent regulator of chromatin, subfamily B (SMARCB1). Molecular subgroups (ATRT-SHH, ATRT-TYR, and ATRT-MYC) were determined using DNA methylation arrays, resulting in profiles of 84 tumors. Results Median age at diagnosis of 67 girls and 76 boys was 29.5 months. Five-year overall survival (OS) and event-free survival (EFS) were 34.7 ± 4.5% and 30.5 ± 4.2%, respectively. Tumors displayed allelic partial/whole gene deletions (66%; 122/186 alleles) or single nucleotide variants (34%; 64/186 alleles) of SMARCB1. Germline mutations were detected in 26% of ATRTs (30/117). The patient cohort consisted of 47% ATRT-SHH (39/84), 33% ATRT-TYR (28/84), and 20% ATRT-MYC (17/84). Age Conclusions Age and molecular subgroup status are independent risk factors for survival in children with ATRT. Our model warrants validation within future clinical trials.

BACKGROUND This 2002–2014 Italian prospective study stratified 160 patients by surgical resection (complete=NED/incomplete=ED) and centrally-reviewed grade. Grade2/NED patients received focal radiotherapy (RT) up to 59.4Gy, Grade3/NED received 4 courses of VEC(vincristine,etoposide,cyclophosphamide) after RT.ED patients received 1–4 VEC courses, second-look surgery, 59.4 Gy+8Gy boost on measurable residue. METHODS We re-analyzed data at 115 months follow-up including methylation profile on available samples. RESULTS Global PFS/OS at 5/10 years were 66/59% and 80/74%, respectively. Of the 64 relapsers at median 20 months, 53 died at median 37/18 months after diagnosis/relapse, respectively.10/64 relapsed after 5 years (66–126 months); 4 died, relapse was local in 8/10, metastatic 1, combined 1;5/10 patients were below age 3, 5 females, 8 PF tumors. Their survival post-relapse was not longer than earlier relapsers’. At univariable analysis, age over 3 years, female sex, complete surgery, grade 2, no shunt confirmed better PFS/OS. 66/95 analyzed tumors received a score >0.80 through the DNA methylation-based central nervous system tumor classifier: 41/8 as PFA/PFB, respectively,14/17 ST as RELA-positive (3 scored for other molecular entities i.e. anaplastic PXA, LGG MYB, HGNET). Prognostic factors were equally distributed among PFA/PFB groups,1 only group B patient relapsed locally at 96 months. CONCLUSIONS Already published prognostic factors remained at long-term follow-up;6.2% patients had late relapses. OS after relapse was not better in late relapsers. Group B confirmed better prognosis but all patients had received «at least» adjuvant RT. Modern ependymoma trials need long follow-up to draw firm conclusions.

Atypical teratoid/rhabdoid tumor (ATRT) is a highly malignant brain tumor predominantly arising in infants. Mutations of SWI/SNF chromatin remodeling complex members SMARCB1/INI1 or (rarely) SMARCA4/Brg1 are the sole recurrent genetic lesions. Epigenetic studies revealed a large number of genes predicted to be affected by differential histone modifications in ATRT, but the role of these genes in the biology of ATRT remains uncertain. We therefore aimed at exploring the role of these genes in the detrimental effects of SMARCB1-deficiency. The functional relevance of 1083 genes predicted to be affected by epigenetic alterations in ATRT was examined in vivo using a Drosophila melanogaster model of SMARCB1-deficiency. Human orthologues of genes whose knockdown modified the phenotype in the Gal4-UAS fly model were further examined in ATRT samples and SMARCB1-deficient rhabdoid tumor cells. Knockdown of Snr1, the fly orthologue of SMARCB1, resulted in a lethal phenotype and epigenetic alterations in the fly model. The lethal phenotype was shifted to later stages of development upon additional siRNA knockdown of 89 of 1083 genes screened in vivo. These included TGF-beta receptor signaling pathway related genes, e.g. CG10348, the fly orthologue of transcriptional regulator PRDM16. Subsequently, PRDM16 was found to be over-expressed in ATRT samples and knockdown of PRDM16 in SMARCB1-deficient rhabdoid tumor cells reduced proliferation. These results suggest that a subset of genes affected by differential histone modification in ATRT is involved in the detrimental effects of SMARCB1-deficiency and also relevant in the biology of ATRT.

Metabolite reference standards are often not available, which results in a lack of MS/MS spectra for library matching. Consequently, the identification of suspected metabolites proves to be challenging. The present study aims at structurally elucidating the MS/MS fragmentation behavior of selected benzimidazole anthelmintics to theoretically predict characteristic product ions for rapid and systematic tentative metabolite identification. A set of common characteristic product ions was identified from accurate mass MS/MS experiments for five parent compounds. It was hypothesized that the mass shift of any metabolic transformation at the parent molecule also is observable in the mass spectrum of the corresponding metabolite. This was tested and verified with six metabolite reference standards and subsequently, formulated as a general prediction scheme. The approach was integrated into a rapid MSe QTOF workflow and tested in mouse plasma for mebendazole and its metabolites. The presented scheme allows the prediction of characteristic product ions for suspected unknown metabolites. These can be matched with measured product ions of suspected metabolites for tentative identification. The theoretically predicted spectra can contribute to the tentative identification of unknown compounds in non-target and suspect screening approaches.

Rhabdoid phenotype and biallelic mutations of the SMARCB1 gene causing loss of SMARCB1/INI1 protein expression are the hallmark of atypical teratoid/rhabdoid tumour (AT/RT), a highly malignant central nervous system tumour mainly affecting infants [1]. Loss of SMARCB1/INI1 protein expression has also been described in a variety of other rhabdoid and non-rhabdoid tumour entities including cribriform neuroepithelial tumour (CRINET) [2], poorly differentiated chordoma [3] as well as rhabdoid peripheral nerve sheath tumour [4] and familial schwannomatosis [5]. To our knowledge, however, loss of SMARCB1/INI1 protein expression has not yet been described in papillary tumours of spinal location encountered in adults such as (papillary) ependymomas [6] and spinal papillary tumour [7]. This article is protected by copyright. All rights reserved.

Rhabdoid tumors are caused by the deletion of SMARCB1, whose protein encodes the SMARCB1 subunit of the chromatin remodeling complex SWI/SNF that is involved in global chromatin organization and gene expression control. Simultaneously inhibiting the main players involved in the deregulated transcription machinery is a promising option for preventing exaggerated tumor cell proliferation and survival as it may bypass compensatory mechanisms. In support of this hypothesis, we report efficient impairment of cellular proliferation and strong induction of cell death elicited by inhibition of bromodomain protein BRD4 and transcription kinase CDK9 using small molecular compounds. Combination of both compounds efficiently represses antiapoptotic genes and the oncogene MYC. Our results provide a novel approach for the treatment of RT.

Atypical teratoid/rhabdoid tumors (ATRT) are highly malignant brain tumors arising in young children. The majority of ATRT is characterized by inactivation of the chromatin remodeling complex member SMARCB1 (INI1/hSNF5). Little is known, however, on downstream pathways involved in the detrimental effects of SMARCB1 deficiency which might also represent targets for treatment. Using Drosophila melanogaster and the Gal4-UAS system, modifier screens were performed in order to identify the role of SMAD dependent signaling in the lethal phenotype associated with knockdown of snr1, the fly homolog of SMARCB1. Expression and functional role of human homologs was next investigated in ATRT tumor samples and SMARCB1-deficient rhabdoid tumor cells. The lethal phenotype associated with snr1 knockdown in Drosophila melanogaster could be shifted to later stages of development upon additional knockdown of several decapentaplegic pathway members including Smox, and Med. Similarly, the transforming growth factor beta (TGFbeta) receptor type I kinase inhibitor SB431542 ameliorated the detrimental effect of snr1 knockdown in the fruit fly. Examination of homologs of candidate decapentaplegic pathway members in human SMARCB1-deficent ATRT samples revealed SMAD3 and SMAD6 to be over-expressed. In SMARCB1-deficent rhabdoid tumor cells, siRNA-mediated silencing of SMAD3 or SMAD6 expression reduced TGFbeta signaling activity and resulted in decreased proliferation. Similar results were obtained upon pharmacological inhibition of TGFbeta signaling using SB431542. Our data suggest that SMAD dependent signaling is involved in the detrimental effects of SMARCB1-deficiency and provide a rationale for the investigation of TGFbeta targeted treatments in ATRT.

Rhabdoid tumors (RTs) are frequent pediatric malignancies that are classified based on tumor localization: In the central nervous system, they are referred to as atypical teratoid/rhabdoid tumors (ATRTs) as compared to extra-cranial malignant RTs (MRTs). The common genetic hallmark of these tumors is the loss of SMARCB1 in 95% of all cases. Molecularly, RTs are heterogeneous. Previous studies reported molecular subgroups within RTs of the same anatomic compartment. However, biological similarities among RT subgroups of different sites, and molecular characteristics shared among them remained unknown. Investigating the similarity of RTs from different anatomic sites at a molecular level, we compared DNA methylation, gene expression, and H3K27ac profiles of 150 MRTs and 161 ATRTs generated by WGBS, 450K/850K-arrays, RNA-, and ChIP-Seq. Clustering of methylation data showed that a subset of MRTs (called Group1) clustered with the MYC-subgroup of ATRTs but not with SHH-and TYR-subgroups of ATRTs. Over-expression of MYC-ATRT representative genes, such as c-MYC and the non-coding regulatory RNA gene HOTAIR, was also observed in these MRTs. Transcription factor binding enrichment analyses displayed a high enrichment of transcription factors implicated in immune cell regulation such as GMEMB1/2, IRF5/8/9, and STAT1 in both ATRT-MYC and MRT. We therefore predicted the immune cell infiltration of RTs using the CIBERSORT and validated these in-silico analyses by immunohistochemistry (IHC) for CD3,CD8, CD68 and PD-L1. In line with the computational results, IHC demonstrated a high number of CD3+ and CD8+ in both MRT and ATRT-MYC, suggestive of abundant cytotoxic T-cell infiltration. Overall our analyses reveal a high degree of similarity between ATRT-MYC and (Group 1)- MRT not only at the epigenetic level, but also in the immune cell composition and underline the suitability of these tumors for a potential immunotherapy.