The mitogen-activated protein kinase (MAPK) pathway is one of the most frequently altered pathways in pediatric cancer. Activating genomic MAPK-alterations and phosphorylation of the MAPK downstream target ERK (pERK) were analyzed in the PTT2.0 registry to identify potential targets for MAPK-directed treatment in relapsed pediatric CNS tumors, sarcomas and other solid tumors. The present study investigates the association of ERK phosphorylation and genomic MAPK pathway alterations (mutations, fusions, amplifications) in the PTT2.0 dataset. PTT2.0 registry cases with available genomic and immunohistochemistry data (n = 235) were included. Samples with and without detected activating genomic MAPK alterations were compared regarding ERK phosphorylation, quantified by immunohistochemistry H-score. The association of pERK intensity and the presence of MAPK alteration was analyzed using a univariable binary logistic regression model.The mean pERK H-score was significantly higher in samples with activating genomic MAPK alterations. pERK H-score positively correlated with the presence of MAPK alterations. However, the pERK H-score predicted MAPK alterations only with a sensitivity of 58.3% and a specificity of 83.8%. The highest mean pERK H-scores were observed in low-grade gliomas, enriched for MAPK alterations, and in ependymoma, where MAPK alterations were absent. Although there is an association between pERK level and activating genetic MAPK alterations, the predictive power of pERK H-score for genetic MAPK alterations is low in pediatric tumors. Tumors/groups with absent genetic MAPK alterations but high pERK indicate a dissociation of the two parameters, as well as a possible MAPK pathway activation in the absence of genetic MAPK alterations.

DNA methylation-based classification is now central to contemporary neuro-oncology, as highlighted by the World Health Organization (WHO) classification of central nervous system (CNS) tumors. We present the Heidelberg CNS Tumor Methylation Classifier version 12.8 (v12.8), trained on 7,495 methylation profiles, which expands recognized entities from 91 classes in version 11 (v11) to 184 subclasses. This expansion is a result of newly identified tumor types discovered through our large online repository and global collaborations, underscoring CNS tumor heterogeneity. The random forest-based classifier achieves 95% subclass-level accuracy, with its well-calibrated probabilistic scores providing a reliable measure of confidence for each classification. Its hierarchical output structure enables interpretation across subclass, class, family, and superfamily levels, thereby supporting clinical decisions at multiple granularities. Comparative analyses demonstrate that v12.8 surpasses previous versions and conventional WHO-based approaches. These advances highlight the improved precision and practical utility of the updated classifier in personalized neuro-oncology.

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.A medium-throughput in vitro combination drug screen was performed in three MYC-amplified and one non-MYC-amplified MB cell line testing 75 clinically relevant drugs alone and in combination with entinostat. The drug sensitivity score (DSS) was calculated based on metabolic inhibition quantified by CellTiter-Glo. The six top synergistic combination hits were evaluated in a 5 × 5 combination matrix and a seven-ray design. Synergy was validated and characterized by cell counts, caspase-3-like-activity and poly-(ADP-ribose)-polymerase-(PARP)-cleavage. On-target activity of drugs was validated by immunoprecipitation and western blot. BCL-XL dependency of the observed effect was explored with siRNA mediated knockdown of BCL2L1, and selective inhibition with targeted compounds (A-1331852, A-1155463).20/75 drugs effectively reduced metabolic activity in combination with entinostat in all three MYC-amplified cell lines (DSS ≥ 10). The combination entinostat and navitoclax showed the strongest synergistic interaction across all MYC-amplified cell lines. siRNA mediated knockdown of BCL2L1, as well as targeted inhibition with selective inhibitors showed BCL-XL dependency of the observed effect. Increased cell death was associated with increased caspase-3-like-activity.Our study identifies the combination of class I HDACi and BCL-XL inhibitors as a potential new approach for the treatment of MYC-amplified MB cells.

Background: The recently published WHO classification of central nervous system (CNS) tumours recognizes DNA methylation profiling as a desirable and, for some diagnoses, essential diagnostic tool adjunctive to conventional histopathology. DNA methylation profiling is not routinely available in many countries, including Greece. Methods: In this collaborative study, we report the DNA methylation results in a series of children and adolescents with CNS tumours in Greece (2018-2023). In total, 130 tumour samples were analyzed using the latest applicable version of the Heidelberg brain tumour classifier. Results: Upon initial analysis, 80 % (104/130) achieved calibrated scores (Cs) >= 0.9 and matched an established methylation class family/subclass. Among them, methylation results confirmed (90/104, 86.5 %), refined (50/104, 48 %) or changed (10/104, 9.6 %) the histological diagnosis. Only four results were regarded as non-contributing (4/104, 3.9 %). Twenty-six tumour samples received Cs < 0.9. Despite low scores, methylation results supported the initial diagnosis with lower confidence in 38.5 % (10/26) and established the diagnosis in two tumours with non-conclusive histopathology. Additional t-distributed stochastic neighbour embedding (t-SNE) analysis allowed the possible classification of twelve tumours. Nine more samples reached high Cs using the newer brain tumour classifiers, since available. Samples co-tested in Greece demonstrated excellent test reproducibility, supporting the analysis' local implementation. Methylome profiling impacted the clinical management of 40 % of patients, modifying stratification, prognosis, or treatment approach. Conclusions: This study supports the need to integrate methylome analysis into routine diagnostics in our country and highlights the importance of collaboration between European pediatric oncology centres.

Suppl. Table S3 shows "Screening for false positive results using the reporter assay without ELK-1 binding site in DKFZ-BT66 and BT-40 cells".

Suppl. Table S1 shows "List of cloning primers".

Purpose 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. Methods 18 pLGG tissue samples (17 (94%) histological and/or molecular diagnosis PA) were mechanically dissociated. Tumor cell positive-selection using A2B5 was perfomed in 8/18 (44%) cases. All primary cell suspensions were seeded in Neural Stem Cell Medium (NSM) and Astrocyte Basal Medium (ABM). Resulting short-term cultures were infected with SV40-TAg lentivirus. Detection of tumor specific alterations ( BRAF -duplication and BRAF V600E -mutation) by digital droplet PCR (ddPCR) at defined time points allowed for determination of tumor cell fraction (TCF) and evaluation of the workflow. DNA-methylation profiling and gene-panel sequencing were used for molecular profiling of primary samples. Results Primary cell suspensions had a mean TCF of 55% (+/− 23% (SD)). No sample in NSM (0/18) and ten samples in ABM (10/18) were successfully transduced. Three of these ten (30%) converted into long-term pLGG cell lines (TCF 100%), while TCF declined to 0% (outgrowth of microenvironmental cells) in 7/10 (70%) cultures. Young patient age was associated with successful model establishment. Conclusion A subset of primary PA cultures can be converted into long-term cell lines using SV40-TAg depending on sample intrinsic (patient age) and extrinsic workflow-related (e.g. type of medium, successful transduction) parameters. Careful monitoring of sample-intrinsic and extrinsic factors optimizes the process.

Suppl. Table S2 shows "Overview of MAPKi and other drugs used in the screen".

Suppl. Table S1 - A summary of the human cohort used for DNA methylation-profiling_DRG3003

Clinical annotations of the PA patient cohort

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.

Suppl. Table S7 - Antibody list

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.

Suppl. Table S4 - Histopathological evaluation of 25 cases

The authors regret that the acknowledgements section in the published article did not include the acknowledgement for the administrative project management provided by Martina Nesper-Brock. The full and correct acknowledgements are given here: The authors thank patients, families and submitting centers for participation in this study. The authors thank Sabine Brokmeier, Jasmin Nowy, Linda Keilbach and Christina Vasanthakumar for technical and logistic assistance, as well as Martina Nesper-Brock for administrative project management. The graphical abstract was created with BioRender.com. The authors would like to apologise for any inconvenience caused. Molecular diagnostics enables detection of actionable targets: the Pediatric Targeted Therapy 2.0 registryEuropean Journal of CancerVol. 180PreviewPrecision oncology requires diagnostic accuracy and robust detection of actionable alterations. The Pediatric Targeted Therapy (PTT) 2.0 program aims at improving diagnostic accuracy by addition of molecular analyses to the existing histological diagnosis and detection of actionable alterations for relapsed paediatric oncology patients, in cases with limited availability of tumour material. Full-Text PDF

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.MYC-amplified Group 3 MB cells (HD-MB03) were treated with class I HDACi entinostat. Changes in the gene expression profile were quantified on a microarray. Bioinformatic assessment led to the identification of pathways affected by entinostat treatment. Five drugs interfering with these pathways (olaparib, idasanutlin, ribociclib, selinexor, vinblastine) were tested for synergy with entinostat in WST-8 metabolic activity assays in a 5 × 5 combination matrix design. Synergy was validated in cell count and flow cytometry experiments. The effect of entinostat and olaparib on DNA damage was evaluated by γH2A.X quantification in immunoblotting, fluorescence microscopy and flow cytometry.Entinostat treatment changed the expression of genes involved in 22 pathways, including downregulation of DNA damage response. The PARP1 inhibitors olaparib and pamiparib showed synergy with entinostat selectively in MYC-amplified MB cells, leading to increased cell death, decreased viability and increased formation of double strand breaks, as well as increased sensitivity to additional induction of DNA damage by doxorubicin. Non-MYC-amplified MB cells and normal human fibroblasts were not susceptible to this triple treatment.Our study identifies the combination of entinostat with olaparib as a new potential therapeutic approach for MYC-driven Group 3 MB.

Suppl. Figure S1 shows "Initial MAPKi screen using metabolic activity as readout". Suppl. Figure S2 shows "Western blot validation of MAPK pathway alterations in HEK293T cells". Suppl. Figure S3 shows "Dose-response curves of MAPKi combination treatment in DKFZ-BT66 pDIPZ-CMV cells". Suppl. Figure S4 shows "Dose response curves of MAPKi combination treatment in BT 40 pDIPZ CMV cells". Suppl. Figure S5 shows "Isobolograms of MAPKi combination treatment in DKFZ-BT66 pDIPZ-CMV and BT-40 pDIPZ-CMV cells"

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