Update on cancer predisposition syndromes and surveillance guidelines for childhood brain tumors.

Tumors of the central nervous system (CNS) comprise the second most common group of neoplasms in childhood. The incidence of germline predisposition among children with brain tumors continues to grow as our knowledge on disease aetiology increases. Some children with brain tumors may present with non-malignant phenotypic features of specific syndromes (e.g. nevoid basal cell carcinoma syndrome, neurofibromatosis type 1 and type 2, DICER1 syndrome, and constitutional mismatch repair deficiency), while others may present with a strong family history of cancer (e.g. Li-Fraumeni syndrome), or with a rare tumor commonly found in the context of germline predisposition (e.g. rhabdoid tumor predisposition syndrome). Approximately 50% of patients with a brain tumor may be the first in a family identified to have a predisposition. The past decade has witnessed a rapid expansion in our molecular understanding of CNS tumors. A significant proportion of CNS tumors are now well characterized and known to harbor specific genetic changes that can be found in the germline. Additional novel predisposition syndromes are also being described. Identification of these germline syndromes in individual patients has not only enabled cascade testing of family members and early tumor surveillance but increasingly has also impacted cancer management in those patients. Therefore, the AACR Cancer Predisposition Working Group chose to highlight these advances in CNS tumor predisposition and summarize and/or generate surveillance recommendations for established and more recently emerging pediatric brain tumor predisposition syndromes.

Rebound growth of BRAF mutant pediatric glioma cells after MAPKi withdrawal is associated with MAPK reactivation and secretion of microglia-recruiting cytokines.

INTRODUCTION: Patients with pediatric low-grade gliomas (pLGGs), the most common primary brain tumors in children, can often benefit from MAPK inhibitor (MAPKi) treatment. However, rapid tumor regrowth, also referred to as rebound growth, may occur once treatment is stopped, constituting a significant clinical challenge. METHODS: Four patient-derived pediatric glioma models were investigated to model rebound growth in vitro based on viable cell counts in response to MAPKi treatment and withdrawal. A multi-omics dataset (RNA sequencing and LC-MS/MS based phospho-/proteomics) was generated to investigate possible rebound-driving mechanisms. Following in vitro validation, putative rebound-driving mechanisms were validated in vivo using the BT-40 orthotopic xenograft model. RESULTS: Of the tested models, only a BRAFV600E-driven model (BT-40, with additional CDKN2A/Bdel) showed rebound growth upon MAPKi withdrawal. Using this model, we identified a rapid reactivation of the MAPK pathway upon MAPKi withdrawal in vitro, also confirmed in vivo. Furthermore, transient overactivation of key MAPK molecules at transcriptional (e.g. FOS) and phosphorylation (e.g. pMEK) levels, was observed in vitro. Additionally, we detected increased expression and secretion of cytokines (CCL2, CX3CL1, CXCL10 and CCL7) upon MAPKi treatment, maintained during early withdrawal. While increased cytokine expression did not have tumor cell intrinsic effects, presence of these cytokines in conditioned media led to increased attraction of microglia cells in vitro. CONCLUSION: Taken together, these data indicate rapid MAPK reactivation upon MAPKi withdrawal as a tumor cell intrinsic rebound-driving mechanism. Furthermore, increased secretion of microglia-recruiting cytokines may play a role in treatment response and rebound growth upon withdrawal, warranting further evaluation.

Developmental basis of SHH medulloblastoma heterogeneity.

Many genes that drive normal cellular development also contribute to oncogenesis. Medulloblastoma (MB) tumors likely arise from neuronal progenitors in the cerebellum, and we hypothesized that the heterogeneity observed in MBs with sonic hedgehog (SHH) activation could be due to differences in developmental pathways. To investigate this question, here we perform single-nucleus RNA sequencing on highly differentiated SHH MBs with extensively nodular histology and observed malignant cells resembling each stage of canonical granule neuron development. Through innovative computational approaches, we connect these results to published datasets and find that some established molecular subtypes of SHH MB appear arrested at different developmental stages. Additionally, using multiplexed proteomic imaging and MALDI imaging mass spectrometry, we identify distinct histological and metabolic profiles for highly differentiated tumors. Our approaches are applicable to understanding the interplay between heterogeneity and differentiation in other cancers and can provide important insights for the design of targeted therapies.

Molecular characteristics and improved survival prediction in a cohort of 2023 ependymomas.

The diagnosis of ependymoma has moved from a purely histopathological review with limited prognostic value to an integrated diagnosis, relying heavily on molecular information. However, as the integrated approach is still novel and some molecular ependymoma subtypes are quite rare, few studies have correlated integrated pathology and clinical outcome, often focusing on small series of single molecular types. We collected data from 2023 ependymomas as classified by DNA methylation profiling, consisting of 1736 previously published and 287 unpublished methylation profiles. Methylation data and clinical information were correlated, and an integrated model was developed to predict progression-free survival. Patients with EPN-PFA, EPN-ZFTA, and EPN-MYCN tumors showed the worst outcome with 10-year overall survival rates of 56%, 62%, and 32%, respectively. EPN-PFA harbored chromosome 1q gains and/or 6q losses as markers for worse survival. In supratentorial EPN-ZFTA, a combined loss of CDKN2A and B indicated worse survival, whereas a single loss did not. Twelve out of 200 EPN-ZFTA (6%) were located in the posterior fossa, and these tumors relapsed or progressed even earlier than supratentorial tumors with a combined loss of CDKN2A/B. Patients with MPE and PF-SE, generally regarded as non-aggressive tumors, only had a 10-year progression-free survival of 59% and 65%, respectively. For the prediction of the 5-year progression-free survival, Kaplan-Meier estimators based on the molecular subtype, a Support Vector Machine based on methylation, and an integrated model based on clinical factors, CNV data, and predicted methylation scores achieved balanced accuracies of 66%, 68%, and 73%, respectively. Excluding samples with low prediction scores resulted in balanced accuracies of over 80%. In sum, our large-scale analysis of ependymomas provides robust information about molecular features and their clinical meaning. Our data are particularly relevant for rare and hardly explored tumor subtypes and seemingly benign variants that display higher recurrence rates than previously believed.

Clinically relevant molecular hallmarks of PFA ependymomas display intratumoral heterogeneity and correlate with tumor morphology.

Posterior fossa type A (PF-EPN-A, PFA) ependymoma are aggressive tumors that mainly affect children and have a poor prognosis. Histopathology shows significant intratumoral heterogeneity, ranging from loose tissue to often sharply demarcated, extremely cell-dense tumor areas. To determine molecular differences in morphologically different areas and to understand their clinical significance, we analyzed 113 PF-EPN-A samples, including 40 corresponding relapse samples. Cell-dense areas ranged from 0 to 100% of the tumor area and displayed a higher proportion of proliferating tumor cells (p < 0.01). Clinically, cell density was associated with poor progression-free and overall survival (pPFS = 0.0026, pOS < 0.01). Molecularly, tumor areas with low and high cell density showed diverging DNA methylation profiles regarding their similarity to distinct previously discovered PF-EPN-A subtypes in 9/21 cases. Prognostically relevant chromosomal changes at 1q and 6q showed spatial heterogeneity within single tumors and were significantly enriched in cell-dense tumor areas as shown by single-cell RNA (scRNA)-sequencing as well as copy number profiling and fluorescence in situ hybridization (FISH) analyses of different tumor areas. Finally, spatial transcriptomics revealed cell-dense areas of different tumors to be more similar than various different areas of the same tumor. High-density areas distinctly overexpressed genes encoding histone proteins, WNT5A, TGFB1, or IGF2. Relapsing tumors displayed a higher proportion of cell-dense areas (p = 0.036), a change in PF-EPN-A methylation subtypes (13/32 patients), and novel chromosome 1q gains and 6q losses (12/32 cases) compared to corresponding primary tumors. Our data suggest that PF-EPN-A ependymomas habor a previously unrecognized intratumoral heterogeneity with clinical implications, which has to be accounted for when selecting diagnostic material, inter alia, by histological evaluation of the proportion of cell-dense areas.

Cerebrospinal fluid cfDNA sequencing for classification of central nervous system glioma.

PURPOSE: Primary central nervous system (CNS) gliomas can be classified by characteristic genetic alterations. In addition to solid tissue obtained via surgery or biopsy, cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) is an alternative source of material for genomic analyses. EXPERIMENTAL DESIGN: We performed targeted next-generation sequencing (NGS) of CSF cfDNA in a representative cohort of 85 patients presenting at two neurooncological centers with suspicion of primary or recurrent glioma. Copy-number variation (CNV) profiles, single nucleotide variants (SNVs), and small insertions/ deletions (indels) were combined into a molecular-guided tumor classification. Comparison with the solid tumor was performed for 38 cases with matching solid tissue available. RESULTS: Cases were stratified into four groups: glioblastoma (n = 32), other glioma (n = 19), non-malignant (n = 17) and non-diagnostic (n = 17). We introduced a molecular-guided tumor classification, which enabled identification of tumor entities and/ or cancer specific alterations in 75.0 % (n = 24) of glioblastoma and 52.6 % (n = 10) of other glioma cases. The overlap between CSF and matching solid tissue was highest for CNVs (26-48 %) and SNVs at pre-defined gene loci (44 %), followed by SNVs/ indels identified via uninformed variant calling (8-14 %). A molecular-guided tumor classification was possible for 23.5 % (n = 4) of non-diagnostic cases. CONCLUSIONS: We developed a targeted sequencing workflow for CSF cfDNA as well as a strategy for interpretation and reporting of sequencing results based on a molecular-guided tumor classification in glioma.

Compartments in medulloblastoma with extensive nodularity are connected through differentiation along the granular precursor lineage.

Medulloblastomas with extensive nodularity are cerebellar tumors characterized by two distinct compartments and variable disease progression. The mechanisms governing the balance between proliferation and differentiation in MBEN remain poorly understood. Here, we employ a multi-modal single cell transcriptome analysis to dissect this process. In the internodular compartment, we identify proliferating cerebellar granular neuronal precursor-like malignant cells, along with stromal, vascular, and immune cells. In contrast, the nodular compartment comprises postmitotic, neuronally differentiated malignant cells. Both compartments are connected through an intermediate cell stage resembling actively migrating CGNPs. Notably, we also discover astrocytic-like malignant cells, found in proximity to migrating and differentiated cells at the transition zone between the two compartments. Our study sheds light on the spatial tissue organization and its link to the developmental trajectory, resulting in a more benign tumor phenotype. This integrative approach holds promise to explore intercompartmental interactions in other cancers with varying histology.

Molecular analysis of cancer genomes in children with Lynch syndrome: Exploring causal associations.

Lynch syndrome (LS) predisposes to cancer in adulthood and is caused by heterozygous germline variants in a mismatch repair (MMR) gene. Recent studies show an increased prevalence of LS among children with cancer, suggesting a causal relationship. For LS-spectrum (LSS) cancers, including high-grade gliomas and colorectal cancer, causality has been supported by typical MMR-related tumor characteristics, but for non-LSS cancers, causality is unclear. We characterized 20 malignant tumors of 18 children with LS, including 16 non-LSS tumors. We investigated second hits, tumor mutational load, mutational signatures and MMR protein expression. In all LSS tumors and three non-LSS tumors, we detected MMR deficiency caused by second hit somatic alterations. Furthermore, these MMR-deficient tumors carried driver variants that likely originated as a consequence of MMR deficiency. However, in 13 non-LSS tumors (81%), a second hit and MMR deficiency were absent, thus a causal link between LS and cancer development in these children is lacking. These findings demonstrate that causality of LS in children with cancer, which can be determined by molecular tumor characterization, seems to be restricted to specific tumor types. Large molecular and epidemiological studies are needed to further refine the tumor spectrum in children with LS.

Genomic characterization of IDH-mutant astrocytoma progression to grade 4 in the treatment setting.

As the progression of low-grade diffuse astrocytomas into grade 4 tumors significantly impacts patient prognosis, a better understanding of this process is of paramount importance for improved patient care. In this project, we analyzed matched IDH-mutant astrocytomas before and after progression to grade 4 from six patients (discovery cohort) with genome-wide sequencing, 21 additional patients with targeted sequencing, and 33 patients from Glioma Longitudinal AnalySiS cohort for validation. The Cancer Genome Atlas data from 595 diffuse gliomas provided supportive information. All patients in our discovery cohort received radiation, all but one underwent chemotherapy, and no patient received temozolomide (TMZ) before progression to grade 4 disease. One case in the discovery cohort exhibited a hypermutation signature associated with the inactivation of the MSH2 and DNMT3A genes. In other patients, the number of chromosomal rearrangements and deletions increased in grade 4 tumors. The cell cycle checkpoint gene CDKN2A, or less frequently RB1, was most commonly inactivated after receiving both chemo- and radiotherapy when compared to other treatment groups. Concomitant activating PDGFRA/MET alterations were detected in tumors that acquired a homozygous CDKN2A deletion. NRG3 gene was significantly downregulated and recurrently altered in progressed tumors. Its decreased expression was associated with poorer overall survival in both univariate and multivariate analysis. We also detected progression-related alterations in RAD51B and other DNA repair pathway genes associated with the promotion of error-prone DNA repair, potentially facilitating tumor progression. In our retrospective analysis of patient treatment and survival timelines (n = 75), the combination of postoperative radiation and chemotherapy (mainly TMZ) outperformed radiation, especially in the grade 3 tumor cohort, in which it was typically given after primary surgery. Our results provide further insight into the contribution of treatment and genetic alterations in cell cycle, growth factor signaling, and DNA repair-related genes to tumor evolution and progression.

Mapping pediatric brain tumors to their origins in the developing cerebellum.

BACKGROUND: Distinguishing the cellular origins of childhood brain tumors is key for understanding tumor initiation and identifying lineage-restricted, tumor-specific therapeutic targets. Previous strategies to map the cell-of-origin typically involved comparing human tumors to murine embryonal tissues, which is potentially limited due to species-specific differences. The aim of this study was to unravel the cellular origins of the 3 most common pediatric brain tumors, ependymoma, pilocytic astrocytoma, and medulloblastoma, using a developing human cerebellar atlas. METHODS: We used a single-nucleus atlas of the normal developing human cerebellum consisting of 176 645 cells as a reference for an in-depth comparison to 4416 bulk and single-cell transcriptome tumor datasets, using gene set variation analysis, correlation, and single-cell matching techniques. RESULTS: We find that the astroglial cerebellar lineage is potentially the origin for posterior fossa ependymomas. We propose that infratentorial pilocytic astrocytomas originate from the oligodendrocyte lineage and MHC II genes are specifically enriched in these tumors. We confirm that SHH and Group 3/4 medulloblastomas originate from the granule cell and unipolar brush cell lineages. Radiation-induced gliomas stem from cerebellar glial lineages and demonstrate distinct origins from the primary medulloblastoma. We identify tumor genes that are expressed in the cerebellar lineage of origin, and genes that are tumor specific; both gene sets represent promising therapeutic targets for future study. CONCLUSION: Based on our results, individual cells within a tumor may resemble different cell types along a restricted developmental lineage. Therefore, we suggest that tumors can arise from multiple cellular states along the cerebellar "lineage of origin."

Key Pharmacokinetic Parameters of 74 Pediatric Anticancer Drugs Providing Assistance in Preclinical Studies.

Novel drug treatments for pediatric patients with cancer are urgently needed. Success of drug development in pediatric oncology has been promising, but many drugs still fail in translation from preclinical to clinical phases. To increase the translational potential, several improvements have been implemented, including the use of clinically achievable concentrations in the drug testing phase. Although pharmacokinetic (PK) parameters of numerous investigated drugs are published, a comprehensive PK overview of the most common drugs in pediatric oncology could guide preclinical trial design and improve the translatability into clinical trials. A review of the literature was conducted for PK parameters of 74 anticancer drugs, from the drug sensitivity profiling library of the INdividualized Therapy FOr Relapsed Malignancies in Childhood (INFORM) registry. PK data in the pediatric population were reported and complemented by adult parameters when no pediatric data were available. In addition, blood-brain barrier (BBB)-penetration assessment of drugs was provided by using the BBB score. Maximum plasma concentration was available for 73 (97%), area under the plasma concentration-time curve for 69 (92%), plasma protein binding for 66 (88%), plasma half-life for 57 (76%), time to maximum concentration for 54 (72%), clearance for 52 (69%), volume of distribution for 37 (49%), lowest plasma concentration reached by the drug before the next dose administration for 21 (28%), and steady-state concentration for 4 (5%) of drugs. Pediatric PK data were available for 48 (65%) drugs. We provide a comprehensive review of PK data for 74 drugs studied in pediatric oncology. This data set can serve as a reference to design experiments more closely mimicking drug PK conditions in patients, and may thereby increase the probability of successful clinical translation.

A multi-institutional retrospective pooled outcome analysis of molecularly annotated pediatric supratentorial ZFTA-fused ependymoma.

Background: ZFTA-RELA (formerly known as c11orf-RELA) fused supratentorial ependymoma (ZFTAfus ST-EPN) has been recognized as a novel entity in the 2016 WHO classification of CNS tumors and further defined in the recent 2021 edition. ZFTAfus ST-EPN was reported to portend poorer prognosis when compared to its counterpart, YAP1 ST-EPN in some previously published series. The aim of this study was to determine the treatment outcome of molecularly confirmed and conventionally treated ZFTAfus ST-EPN patients treated in multiple institutions. Methods: We conducted a retrospective analysis of all pediatric patients with molecularly confirmed ZFTAfus ST-EPN patients treated in multiple institutions in 5 different countries (Australia, Canada, Germany, Switzerland, and Czechia). Survival outcomes were analyzed and correlated with clinical characteristics and treatment approaches. Results: A total of 108 patients were collated from multiple institutions in 5 different countries across three continents. We found across the entire cohort that the 5- and 10-year PFS were 65% and 63%, respectively. The 5- and 10-year OS of this cohort of patients were 87% and 73%. The rates of gross total resection (GTR) were high with 84 out of 108 (77.8%) patients achieving GTR. The vast majority of patients also received post-operative radiotherapy, 98 out of 108 (90.7%). Chemotherapy did not appear to provide any survival benefit in our patient cohort. Conclusion: This is the largest study to date of contemporaneously treated molecularly confirmed ZFTAfus ST-EPN patients which identified markedly improved survival outcomes compared to previously published series. This study also re-emphasizes the importance of maximal surgical resection in achieving optimal outcomes in pediatric patients with supratentorial ependymoma.

Outcome of Children and Adolescents With Relapsed/Refractory/Progressive Malignancies Treated With Molecularly Informed Targeted Drugs in the Pediatric Precision Oncology Registry INFORM.

PURPOSE: INFORM is an international pediatric precision oncology registry, prospectively collecting molecular and clinical data of children with recurrent, progressive, or very high-risk malignancies. We have previously identified a subgroup of patients with improved outcomes on the basis of molecular profiling. The present analysis systematically investigates progression-free survival (PFS) and overall survival (OS) of patients receiving matching targeted treatment (MTT) with the most frequently applied drug classes and its correlation with underlying molecular alterations. METHODS: A cohort of 519 patients with relapsed or refractory high-risk malignancies who had completed a follow-up of at least 2 years or shorter in the case of death or loss to follow-up was analyzed. Survival times were compared using the log-rank test. RESULTS: MTT with anaplastic lymphoma kinase (ALK), neurotrophic tyrosine receptor kinase (NTRK), and B-RAF kinase (BRAF) inhibitors showed significantly improved PFS (P = .012) and OS (P = .036) in comparison with conventional treatment or no treatment. However, analysis of the four most commonly applied MTT groups, mitogen-activated protein kinase (MEK- n = 19), cyclin-dependent kinase (CDK- n = 23), other kinase (n = 62), and mammalian-target of rapamycin (mTOR- n = 20) inhibitors, did not reveal differences in PFS or OS compared with conventional treatment or no treatment in patients with similar molecular pathway alterations. We did not observe differences in the type of pathway alterations (eg, copy number alterations, single-nucleotide variants, InDels, gene fusions) addressed by MTT. CONCLUSION: Patients with respective molecular alterations benefit from treatment with ALK, NTRK, and BRAF inhibitors as previously described. No survival benefit was observed with MTT for mutations in the MEK, CDK, other kinase, or mTOR signaling pathways. The noninterventional character of a registry has to be taken into account when interpreting these data and underlines the need for innovative interventional biomarker-driven clinical trials in pediatric oncology.

Significant increase of high-risk chromosome 1q gain and 6q loss at recurrence in posterior fossa group A ependymoma: A multicenter study.

BACKGROUND: Ependymoma (EPN) posterior fossa group A (PFA) has the highest rate of recurrence and the worst prognosis of all EPN molecular groups. At relapse, it is typically incurable even with re-resection and re-irradiation. The biology of recurrent PFA remains largely unknown; however, the increasing use of surgery at first recurrence has now provided access to clinical samples to facilitate a better understanding of this. METHODS: In this large longitudinal international multicenter study, we examined matched samples of primary and recurrent disease from PFA patients to investigate the biology of recurrence. RESULTS: DNA methylome derived copy number variants (CNVs) revealed large-scale chromosome gains and losses at recurrence in PFA. CNV changes were dominated by chromosome 1q gain and/or 6q loss, both previously identified as high-risk factors in PFA, which were present in 23% at presentation but increased to 61% at first recurrence. Multivariate survival analyses of this cohort showed that cases with 1q gain or 6q loss at first recurrence were significantly more likely to recur again. Predisposition to 1q+/6q- CNV changes at recurrence correlated with hypomethylation of heterochromatin-associated DNA at presentation. Cellular and molecular analyses revealed that 1q+/6q- PFA had significantly higher proportions of proliferative neuroepithelial undifferentiated progenitors and decreased differentiated neoplastic subpopulations. CONCLUSIONS: This study provides clinically and preclinically actionable insights into the biology of PFA recurrence. The hypomethylation predisposition signature in PFA is a potential risk-classifier for trial stratification. We show that the cellular heterogeneity of PFAs evolves largely because of genetic evolution of neoplastic cells.

Second-line treatment of pediatric patients with relapsed rhabdomyosarcoma adapted to initial risk stratification: Data of the European Soft Tissue Sarcoma Registry (SoTiSaR).

BACKGROUND: Outcome of relapsed disease of localized rhabdomyosarcoma remains poor. An individual treatment approach considering the initial systemic treatment and risk group was included in the Cooperative Weichteilsarkom Studiengruppe (CWS) Guidance. METHODS: Second-line chemotherapy (sCHT) ACCTTIVE based on anthracyclines (adriamycin, carboplatin, cyclophosphamide, topotecan, vincristine, etoposide) was recommended for patients with initial low- (LR), standard- (SR), and high-risk (HR) group after initial treatment without anthracyclines. TECC (topotecan, etoposide, carboplatin, cyclophosphamide) was recommended after initial anthracycline-based regimen in the very high-risk (VHR) group. Data of patients with relapse (n = 68) registered in the European Soft Tissue Sarcoma Registry SoTiSaR (2009-2018) were retrospectively analyzed. RESULTS: Patients of initial LR (n = 2), SR (n = 16), HR (n = 41), and VHR (n = 9) group relapsed. sCHT consisted of ACCTTIVE (n = 36), TECC (n = 12), or other (n = 15). Resection was performed in 40/68 (59%) patients and/or radiotherapy in 47/68 (69%). Initial risk stratification, pattern/time to relapse, and achievement of second complete remission were significant prognostic factors. Microscopically incomplete resection with additional radiotherapy was not inferior to microscopically complete resection (p = .17). The 5-year event-free survival (EFS) and overall survival (OS) were 26% (±12%) and 31% (±14%). The 5-year OS of patients with relapse of SR, HR, and VHR groups was 80% (±21%), 20% (±16%), and 13% (±23%, p = .008), respectively. CONCLUSION: Adapted systemic treatment of relapsed disease considering the initial risk group and initial treatment is reasonable. New treatment options are needed for patients of initial HR and VHR groups.

3D genome mapping identifies subgroup-specific chromosome conformations and tumor-dependency genes in ependymoma.

Ependymoma is a tumor of the brain or spinal cord. The two most common and aggressive molecular groups of ependymoma are the supratentorial ZFTA-fusion associated and the posterior fossa ependymoma group A. In both groups, tumors occur mainly in young children and frequently recur after treatment. Although molecular mechanisms underlying these diseases have recently been uncovered, they remain difficult to target and innovative therapeutic approaches are urgently needed. Here, we use genome-wide chromosome conformation capture (Hi-C), complemented with CTCF and H3K27ac ChIP-seq, as well as gene expression and DNA methylation analysis in primary and relapsed ependymoma tumors, to identify chromosomal conformations and regulatory mechanisms associated with aberrant gene expression. In particular, we observe the formation of new topologically associating domains ('neo-TADs') caused by structural variants, group-specific 3D chromatin loops, and the replacement of CTCF insulators by DNA hyper-methylation. Through inhibition experiments, we validate that genes implicated by these 3D genome conformations are essential for the survival of patient-derived ependymoma models in a group-specific manner. Thus, this study extends our ability to reveal tumor-dependency genes by 3D genome conformations even in tumors that lack targetable genetic alterations.

Glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA): a molecularly distinct brain tumor type with recurrent NTRK gene fusions.

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.

Optimizing biomarkers for accurate ependymoma diagnosis, prognostication, and stratification within International Clinical Trials: A BIOMECA study.

BACKGROUND: Accurate identification of brain tumor molecular subgroups is increasingly important. We aimed to establish the most accurate and reproducible ependymoma subgroup biomarker detection techniques, across 147 cases from International Society of Pediatric Oncology (SIOP) Ependymoma II trial participants, enrolled in the pan-European "Biomarkers of Ependymoma in Children and Adolescents (BIOMECA)" study. METHODS: Across 6 European BIOMECA laboratories, we evaluated epigenetic profiling (DNA methylation array); immunohistochemistry (IHC) for nuclear p65-RELA, H3K27me3, and Tenascin-C; copy number analysis via fluorescent in situ hybridization (FISH) and MLPA (1q, CDKN2A), and MIP and DNA methylation array (genome-wide copy number evaluation); analysis of ZFTA- and YAP1-fusions by RT-PCR and sequencing, Nanostring and break-apart FISH. RESULTS: DNA Methylation profiling classified 65.3% (n = 96/147) of cases as EPN-PFA and 15% (n = 22/147) as ST-ZFTA fusion-positive. Immunohistochemical loss of H3K27me3 was a reproducible and accurate surrogate marker for EPN-PFA (sensitivity 99%-100% across 3 centers). IHC for p65-RELA, FISH, and RNA-based analyses effectively identified ZFTA- and YAP-fused supratentorial ependymomas. Detection of 1q gain using FISH exhibited only 57% inter-center concordance and low sensitivity and specificity while MIP, MLPA, and DNA methylation-based approaches demonstrated greater accuracy. CONCLUSIONS: We confirm, in a prospective trial cohort, that H3K27me3 immunohistochemistry is a robust EPN-PFA biomarker. Tenascin-C should be abandoned as a PFA marker. DNA methylation and MIP arrays are effective tools for copy number analysis of 1q gain, 6q, and CDKN2A loss while FISH is inadequate. Fusion detection was successful, but rare novel fusions need more extensive technologies. Finally, we propose test sets to guide future diagnostic approaches.

Pediatric-type high-grade neuroepithelial tumors with CIC gene fusion share a common DNA methylation signature.

Pediatric neoplasms in the central nervous system (CNS) show extensive clinical and molecular heterogeneity and are fundamentally different from those occurring in adults. Molecular genetic testing contributes to accurate diagnosis and enables an optimal clinical management of affected children. Here, we investigated a rare, molecularly distinct type of pediatric high-grade neuroepithelial tumor (n = 18), that was identified through unsupervised visualization of genome-wide DNA methylation array data, together with copy number profiling, targeted next-generation DNA sequencing, and RNA transcriptome sequencing. DNA and/or RNA sequencing revealed recurrent fusions involving the capicua transcriptional repressor (CIC) gene in 10/10 tumor samples analyzed, with the most common fusion being CIC::LEUTX (n = 9). In addition, a CIC::NUTM1 fusion was detected in one of the tumors. Apart from the detected fusion events, no additional oncogenic alteration was identified in these tumors. The histopathological review demonstrated a morphologically heterogeneous group of high-grade neuroepithelial tumors with positive immunostaining for markers of glial differentiation in combination with weak and focal expression of synaptophysin, CD56 and CD99. All tumors were located in the supratentorial compartment, occurred during childhood (median age 8.5 years) and typically showed early relapses. In summary, we expand the spectrum of pediatric-type tumors of the CNS by reporting a previously uncharacterized group of rare high-grade neuroepithelial tumors that share a common DNA methylation signature and recurrent gene fusions involving the transcriptional repressor CIC. Downstream functional consequences of the fusion protein CIC::LEUTX and potential therapeutic implications need to be further investigated.