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.

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.

A hallmark of pediatric low-grade glioma (pLGG) is aberrant signaling of the mitogen activated protein kinase (MAPK) pathway. Hence, inhibition of MAPK signaling using small molecule inhibitors such as MEK inhibitors (MEKi) may be a promising strategy.

Long-term complications such as radiation-induced second malignancies occur in a subset of patients following radiation-therapy, particularly relevant in pediatric patients due to the long follow-up period in case of survival. Radiation-induced gliomas (RIGs) have been reported in patients after treatment with cranial irradiation for various primary malignancies such as acute lymphoblastic leukemia (ALL) and medulloblastoma (MB). We perform comprehensive (epi-) genetic and expression profiling of RIGs arising after cranial irradiation for MB (n = 23) and ALL (n = 9). Our study reveals a unifying molecular signature for the majority of RIGs, with recurrent PDGFRA amplification and loss of CDKN2A/B and an absence of somatic hotspot mutations in genes encoding histone 3 variants or IDH1/2, uncovering diagnostic markers and potentially actionable targets.

Large-scale molecular profiling studies in recent years have shown that central nervous system (CNS) tumors display a much greater heterogeneity in terms of molecularly distinct entities, cellular origins and genetic drivers than anticipated from histological assessment. DNA methylation profiling has emerged as a useful tool for robust tumor classification, providing new insights into these heterogeneous molecular classes. This is particularly true for rare CNS tumors with a broad morphological spectrum, which are not possible to assign as separate entities based on histological similarity alone. Here, we describe a molecularly distinct subset of predominantly pediatric CNS neoplasms (n = 60) that harbor PATZ1 fusions. The original histological diagnoses of these tumors covered a wide spectrum of tumor types and malignancy grades. While the single most common diagnosis was glioblastoma (GBM), clinical data of the PATZ1-fused tumors showed a better prognosis than typical GBM, despite frequent relapses. RNA sequencing revealed recurrent MN1:PATZ1 or EWSR1:PATZ1 fusions related to (often extensive) copy number variations on chromosome 22, where PATZ1 and the two fusion partners are located. These fusions have individually been reported in a number of glial/glioneuronal tumors, as well as extracranial sarcomas. We show here that they are more common than previously acknowledged, and together define a biologically distinct CNS tumor type with high expression of neural development markers such as PAX2, GATA2 and IGF2. Drug screening performed on the MN1:PATZ1 fusion-bearing KS-1 brain tumor cell line revealed preliminary candidates for further study. In summary, PATZ1 fusions define a molecular class of histologically polyphenotypic neuroepithelial tumors, which show an intermediate prognosis under current treatment regimens.

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.

MYC-driven Group 3 medulloblastoma (MB) (subtype II) is a highly aggressive childhood brain tumor. Sensitivity of MYC-driven MB to class I histone deacetylase inhibitors (HDACi) has been previously demonstrated in vitro and in vivo. In this study we characterize the transcriptional effects of class I HDACi in MYC-driven MB and explore beneficial drug combinations.

Patients with MYC-amplified Group 3 medulloblastoma (MB) (subtype II) show poor progression-free survival rates. Class I histone deacetylase inhibitors (HDACi) are highly effective for the treatment of MYC-amplified MB in vitro and in vivo. Drug combination regimens including class I HDACi may represent an urgently needed novel treatment approach for this high risk disease.

Pilocytic astrocytoma is the most common childhood brain tumor, characterized by constitutive MAPK activation. MAPK signaling induces oncogene-induced senescence (OIS), which may cause unpredictable growth behavior of pilocytic astrocytomas. The senescence-associated secretory phenotype (SASP) has been shown to regulate OIS, but its role in pilocytic astrocytoma remains unknown.Experimental Design: The patient-derived pilocytic astrocytoma cell culture model, DKFZ-BT66, was used to demonstrate presence of the SASP and analyze its impact on OIS in pilocytic astrocytoma. The model allows for doxycycline-inducible switching between proliferation and OIS. Both states were studied using gene expression profiling (GEP), Western blot, ELISA, and cell viability testing. Primary pilocytic astrocytoma tumors were analyzed by GEP and multiplex assay.

Glioneuronal tumors are a heterogenous group of CNS neoplasms that can be challenging to accurately diagnose. Molecular methods are highly useful in classifying these tumors-distinguishing precise classes from their histological mimics and identifying previously unrecognized types of tumors. Using an unsupervised visualization approach of DNA methylation data, we identified a novel group of tumors (n = 20) that formed a cluster separate from all established CNS tumor types. Molecular analyses revealed ATRX alterations (in 16/16 cases by DNA sequencing and/or immunohistochemistry) as well as potentially targetable gene fusions involving receptor tyrosine-kinases (RTK; mostly NTRK1-3) in all of these tumors (16/16; 100%). In addition, copy number profiling showed homozygous deletions of CDKN2A/B in 55% of cases. Histological and immunohistochemical investigations revealed glioneuronal tumors with isomorphic, round and often condensed nuclei, perinuclear clearing, high mitotic activity and microvascular proliferation. Tumors were mainly located supratentorially (84%) and occurred in patients with a median age of 19 years. Survival data were limited (n = 18) but point towards a more aggressive biology as compared to other glioneuronal tumors (median progression-free survival 12.5 months). Given their molecular characteristics in addition to anaplastic features, we suggest the term glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA) to describe these tumors. In summary, our findings highlight a novel type of glioneuronal tumor driven by different RTK fusions accompanied by recurrent alterations in ATRX and homozygous deletions of CDKN2A/B. Targeted approaches such as NTRK inhibition might represent a therapeutic option for patients suffering from these tumors.

Pediatric low-grade gliomas (pLGG) show heterogeneous responses to MAPK inhibitors (MAPKi) in clinical trials. Thus, more complex stratification biomarkers are needed to identify patients likely to benefit from MAPKi therapy. Here, we identify MAPK-related genes enriched in MAPKi-sensitive cell lines using the GDSC dataset and apply them to calculate class-specific MAPKi sensitivity scores (MSSs) via single-sample gene set enrichment analysis. The MSSs discriminate MAPKi-sensitive and non-sensitive cells in the GDSC dataset and significantly correlate with response to MAPKi in an independent PDX dataset. The MSSs discern gliomas with varying MAPK alterations and are higher in pLGG compared to other pediatric CNS tumors. Heterogenous MSSs within pLGGs with the same MAPK alteration identify proportions of potentially sensitive patients. The MEKi MSS predicts treatment response in a small set of pLGG patients treated with trametinib. High MSSs correlate with a higher immune cell infiltration, with high expression in the microglia compartment in single-cell RNA sequencing data, while low MSSs correlate with low immune infiltration and increased neuronal score. The MSSs represent predictive tools for the stratification of pLGG patients and should be prospectively validated in clinical trials. Our data supports a role for microglia in the response to MAPKi.

We and others have demonstrated that MYC-amplified medulloblastoma (MB) cells are susceptible to class I histone deacetylase inhibitor (HDACi) treatment. However, single drug treatment with HDACi has shown limited clinical efficacy. We hypothesized that addition of a second compound acting synergistically with HDACi may enhance efficacy.

Until today, adult and pediatric clinical trials investigating single-agent or combinatorial HDAC inhibitors including vorinostat in solid tumors have largely failed to demonstrate efficacy. These results may in part be explained by data from preclinical models showing significant activity only at higher concentrations compared to those achieved with current dosing regimens. In the current pediatric trial, we applied an intra-patient dose escalation design. The purpose of this trial was to determine a safe dose recommendation (SDR) of single-agent vorinostat for intra-patient dose escalation, pharmacokinetic analyses (PK), and activity evaluation in children (3-18 years) with relapsed or therapy-refractory malignancies.

Targeting oncogenic fusion-genes in pediatric high-grade gliomas (pHGG) with entrectinib has emerged as a highly promising therapeutic approach. Despite ongoing clinical studies, to date, no reports on the treatment of cerebrospinal fluid (CSF) disseminated fusion-positive pHGG exist. Moreover, clinically important information of combination with other treatment modalities such as intrathecal therapy, radiotherapy and other targeted agents is missing. We report on our clinical experience of entrectinib therapy in two CSF disseminated ROS1/NTRK-fusion-positive pHGG cases. Combination of entrectinib with radiotherapy or intrathecal chemotherapy appears to be safe and has the potential to act synergistically with entrectinib treatment. In addition, we demonstrate CSF penetrance of entrectinib for the first time in patient samples suggesting target engagement even upon CSF dissemination. Moreover, in vitro analyses of two novel cell models derived from one case with NTRK-fusion revealed that combination therapy with either a MEK (trametinib) or a CDK4/6 (abemaciclib) inhibitor synergistically enhances entrectinib anticancer effects. In summary, our comprehensive study, including clinical experience, CSF penetrance and in vitro data on entrectinib therapy of NTRK/ROS1-fusion-positive pHGG, provides essential clinical and preclinical insights into the multimodal treatment of these highly aggressive tumors. Our data suggest that combined inhibition of NTRK/ROS1 and other therapeutic vulnerabilities enhances the antitumor effect, which should be followed-up in further preclinical and clinical studies.

Embryonal tumours with multilayered rosettes (ETMRs) are aggressive paediatric embryonal brain tumours with a universally poor prognosis1. Here we collected 193 primary ETMRs and 23 matched relapse samples to investigate the genomic landscape of this distinct tumour type. We found that patients with tumours in which the proposed driver C19MC2-4 was not amplified frequently had germline mutations in DICER1 or other microRNA-related aberrations such as somatic amplification of miR-17-92 (also known as MIR17HG). Whole-genome sequencing revealed that tumours had an overall low recurrence of single-nucleotide variants (SNVs), but showed prevalent genomic instability caused by widespread occurrence of R-loop structures. We show that R-loop-associated chromosomal instability can be induced by the loss of DICER1 function. Comparison of primary tumours and matched relapse samples showed a strong conservation of structural variants, but low conservation of SNVs. Moreover, many newly acquired SNVs are associated with a mutational signature related to cisplatin treatment. Finally, we show that targeting R-loops with topoisomerase and PARP inhibitors might be an effective treatment strategy for this deadly disease.

Medulloblastoma (MB) is the most frequent malignant brain tumor in children. Four subgroups with distinct genetic, epigenetic and clinical characteristics have been identified. Survival remains particularly poor in patients with Group 3 tumors harbouring a MYC amplification. We herein explore the molecular mechanisms and translational implications of class I histone deacetylase (HDAC) inhibition in MYC driven MBs.

Pediatric central nervous system (CNS) tumors represent the most common cause of cancer-related death in children aged 0-14 years. They differ from their adult counterparts, showing extensive clinical and molecular heterogeneity as well as a challenging histopathological spectrum that often impairs accurate diagnosis. Here, we use DNA methylation-based CNS tumor classification in combination with copy number, RNA-seq, and ChIP-seq analysis to characterize a newly identified CNS tumor type. In addition, we report histology, patient characteristics, and survival data in this tumor type. We describe a biologically distinct pediatric CNS tumor type (n = 31 cases) that is characterized by focal high-level amplification and resultant overexpression of either PLAGL1 or PLAGL2, and an absence of recurrent genetic alterations characteristic of other pediatric CNS tumor types. Both genes act as transcription factors for a regulatory subset of imprinted genes (IGs), components of the Wnt/β-Catenin pathway, and the potential drug targets RET and CYP2W1, which are also specifically overexpressed in this tumor type. A derived PLAGL-specific gene expression signature indicates dysregulation of imprinting control and differentiation/development. These tumors occurred throughout the neuroaxis including the cerebral hemispheres, cerebellum, and brainstem, and were predominantly composed of primitive embryonal-like cells lacking robust expression of markers of glial or neuronal differentiation (e.g., GFAP, OLIG2, and synaptophysin). Tumors with PLAGL1 amplification were typically diagnosed during adolescence (median age 10.5 years), whereas those with PLAGL2 amplification were diagnosed during early childhood (median age 2 years). The 10-year overall survival was 66% for PLAGL1-amplified tumors, 25% for PLAGL2-amplified tumors, 18% for male patients, and 82% for female patients. In summary, we describe a new type of biologically distinct CNS tumor characterized by PLAGL1/2 amplification that occurs predominantly in infants and toddlers (PLAGL2) or adolescents (PLAGL1) which we consider best classified as a CNS embryonal tumor and which is associated with intermediate survival. The cell of origin and optimal treatment strategies remain to be defined.

Although pediatric low-grade gliomas (pLGG) are the most common pediatric brain tumors, patient-derived cell lines reflecting pLGG biology in culture are scarce. This also applies to the most common pLGG subtype pilocytic astrocytoma (PA). Conventional cell culture approaches adapted from higher-grade tumors fail in PA due to oncogene-induced senescence (OIS) driving tumor cells into arrest. Here, we describe a PA modeling workflow using the Simian Virus large T antigen (SV40-TAg) to circumvent OIS.