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Abstract Background Pituitary neuroendocrine tumors (PitNET) are among the most common intracranial tumors. Despite a frequent benign course, aggressive behavior can occur. Tumor behavior is known to be under the influence of the tumor microenvironment (TME). However, the relationship between TME cells and aggressive tumor behavior has not been adequately explored in PitNET. Methods We performed differential expression analysis as well as gene expression program identification based on single-cell RNA sequencing to comparatively characterize the transcriptome of seven gonadotroph and three lactotroph PitNET and correlate it with clinical features using bulk RNA-seq data from an independent cohort of 134 PitNET. Tumor immune infiltration was quantified via immunostaining on tissue sections of gonadotroph and lactotroph PitNET. Results In lactotroph PitNET, we detect a highly proliferative gene profile with significantly increased expression levels in aggressively growing tumors within bulk RNA-seq data of an independent cohort of 134 PitNET samples. We also report high intratumoral heterogeneity in gonadotroph PitNET (GoPN) and lactotroph PitNET (LaPN) and identify signatures of epithelial, endocrine, and immunological gene networks in both subtypes. A comparison of their TME composition shows enrichment of SPP1+ macrophages and CD4+ T cells in GoPN, as well as enrichment of CD4/CD8 double-negative T cells (DN) and natural killer cells (NK) in LaPN. Also notable is the presence of proliferative lymphocytes, the occurrence of which positively correlates with more aggressive tumor behavior in the bulk RNA-seq cohort. However, increased CD8+ T and NK cell abundances correlate significantly with reduced aggressiveness indicating potential anti-tumoral effects. Conclusions Our study expands the knowledge of the differences in cellular composition of gonadotroph and lactotroph PitNET subtypes. It lays the foundation for further studies on the influence of lymphoid cells on the variable aggressive behavior of PitNET. Regarding the treatment of drug-resistant lactotroph PitNET, proliferative lymphocytes, CD8+ T, and NK cells could represent potentially valuable targets for developing new cancer immunotherapies.

Abstract Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is a heterogeneous group of malignancies with poor outcome. Here, we identify a subgroup, PTCL-NOS SMARCB1- , which is characterized by the lack of the SMARCB1 protein and occurs more frequently in young patients. Human and murine PTCL-NOS SMARCB1- show similar DNA methylation profiles, with hypermethylation of T-cell-related genes and hypomethylation of genes involved in myeloid development. Single-cell analyses of human and murine tumors revealed a rich and complex network of interactions between tumor cells and an immunosuppressive and exhausted tumor microenvironment (TME). In a drug screen, we identified histone deacetylase inhibitors (HDACi) as a class of drugs effective against PTCL-NOS Smarcb1- . In vivo treatment of mouse tumors with SAHA, a pan-HDACi, triggered remodeling of the TME, promoting replenishment of lymphoid compartments and reversal of the exhaustion phenotype. These results provide a rationale for further exploration of HDACi combination therapies targeting PTCL-NOS SMARCB1- within the TME.

Abstract Medulloblastomas (MBs) are malignant pediatric brain tumors that are molecularly and clinically heterogenous. The application of omics technologies—mainly studying nucleic acids—has significantly improved MB classification and stratification, but treatment options are still unsatisfactory. The proteome and their N-glycans hold the potential to discover clinically relevant phenotypes and targetable pathways. We compile a harmonized proteome dataset of 167 MBs and integrate findings with DNA methylome, transcriptome and N-glycome data. We show six proteome MB subtypes, that can be assigned to two main molecular programs: transcription/translation (pSHHt, pWNT and pG3myc), and synapses/immunological processes (pSHHs, pG3 and pG4). Multiomic analysis reveals different conservation levels of proteome features across MB subtypes at the DNA methylome level. Aggressive pGroup3myc MBs and favorable pWNT MBs are most similar in cluster hierarchies concerning overall proteome patterns but show different protein abundances of the vincristine resistance-associated multiprotein complex TriC/CCT and of N-glycan turnover-associated factors. The N-glycome reflects proteome subtypes and complex-bisecting N-glycans characterize pGroup3myc tumors. Our results shed light on targetable alterations in MB and set a foundation for potential immunotherapies targeting glycan structures.

Immunologic treatment options are uncommon in low-grade gliomas, although such therapies might be beneficial for inoperable and aggressive cases. Knowledge of the immune and stromal cells in low-grade gliomas is highly relevant for such approaches but still needs to be improved. Published gene-expression data from 400 low-grade gliomas and 193 high-grade gliomas were gathered to quantify 10 microenvironment cell populations with a deconvolution method designed explicitly for brain tumors. First, we investigated general differences in the microenvironment of low- and high-grade gliomas. Lower-grade and high-grade tumors cluster together, respectively, and show a general similarity within and distinct differences between these groups, the main difference being a higher infiltration of fibroblasts and T cells in high-grade gliomas. Among the analyzed entities, gangliogliomas and pleomorphic xanthoastrocytomas presented the highest overall immune cell infiltration. Further analyses of the low-grade gliomas presented three distinct microenvironmental signatures of immune cell infiltration, which can be divided into T-cell/dendritic/natural killer cell-, neutrophilic/B lineage/natural killer cell-, and monocytic/vascular/stromal-cell-dominated immune clusters. These clusters correlated with tumor location, age, and histological diagnosis but not with sex or progression-free survival. A survival analysis showed that the prognosis can be predicted from gene expression, clinical data, and a combination of both with a support vector machine and revealed the negative prognostic relevance of vascular markers. Overall, our work shows that low- and high-grade gliomas can be characterized and differentiated by their immune cell infiltration. Low-grade gliomas cluster into three distinct immunologic tumor microenvironments, which may be of further interest for upcoming immunotherapeutic research.

ABSTRACT Purpose This study describes oncological outcomes and investigates prognostic factors for patients with gliosarcomas (GSM). Methods Histopathologically confirmed GSM patients who underwent treatment at five European institutions were retrospectively analyzed. Results We analyzed 170 patients with a median clinical follow‐up time of 9.2 months. The majority received surgery (94.1%), postoperative radiotherapy (pRT, 81.8%), and temozolomide (TMZ)‐based postoperative chemotherapy (66.5%). The median overall survival (OS) and progression‐free survival (PFS) were 12.3 and 6.6 months, respectively. In the multivariable Cox regression analysis (MVA), the following factors were significantly associated with OS: age per year (hazard ratio (HR): 1.03, p

Abstract DNA methylation analysis based on supervised machine learning algorithms with static reference data, allowing diagnostic tumour typing with unprecedented precision, has quickly become a new standard of care. Whereas genome-wide diagnostic methylation profiling is mostly performed on microarrays, an increasing number of institutions additionally employ nanopore sequencing as a faster alternative. In addition, methylation-specific parallel sequencing can generate methylation and genomic copy number data. Given these diverse approaches to methylation profiling, to date, there is no single tool that allows (1) classification and interpretation of microarray, nanopore and parallel sequencing data, (2) direct control of nanopore sequencers, and (3) the integration of microarray-based methylation reference data. Furthermore, no software capable of entirely running in routine diagnostic laboratory environments lacking high-performance computing and network infrastructure exists. To overcome these shortcomings, we present EpiDiP/NanoDiP as an open-source DNA methylation and copy number profiling suite, which has been benchmarked against an established supervised machine learning approach using in-house routine diagnostics data obtained between 2019 and 2021. Running locally on portable, cost- and energy-saving system-on-chip as well as gpGPU-augmented edge computing devices, NanoDiP works in offline mode, ensuring data privacy. It does not require the rigid training data annotation of supervised approaches. Furthermore, NanoDiP is the core of our public, free-of-charge EpiDiP web service which enables comparative methylation data analysis against an extensive reference data collection. We envision this versatile platform as a useful resource not only for neuropathologists and surgical pathologists but also for the tumour epigenetics research community. In daily diagnostic routine, analysis of native, unfixed biopsies by NanoDiP delivers molecular tumour classification in an intraoperative time frame.

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.

Abstract DNA methylation analysis has become a powerful tool in neuropathology. Although DNA methylation-based classification usually shows high accuracy, certain samples cannot be classified and remain clinically challenging. We aimed to gain insight into these cases from a clinical perspective. To address, central nervous system (CNS) tumors were subjected to DNA methylation profiling and classified according to their calibrated score using the DKFZ brain tumor classifier (V11.4) as “≥ 0.84” (score ≥ 0.84), “0.3–0.84” (score 0.3–0.84), or “

Abstract Group 3 medulloblastoma is one of the most aggressive types of childhood brain tumors. Roughly 30% of cases carry genetic alterations in MYC, SMARCA4, or both genes combined. While overexpression of MYC has previously been shown to drive medulloblastoma formation in mice, the functional significance of SMARCA4 mutations and their suitability as a therapeutic target remain largely unclear. To address this issue, we combined overexpression of MYC with a loss of SMARCA4 in granule cell precursors. Both alterations did not increase proliferation of granule cell precursors in vitro. However, combined MYC overexpression and SMARCA4 loss successfully induced tumor formation in vivo after orthotopic transplantation in recipient mice. Resulting tumors displayed anaplastic histology and exclusively consisted of SMARCA4-negative cells although a mixture of recombined and non-recombined cells was injected. These observations provide first evidence for a tumor-promoting role of a SMARCA4 deficiency in the development of medulloblastoma. In comparing the transcriptome of tumors to the cells of origin and an established Sonic Hedgehog medulloblastoma model, we gathered first hints on deregulated gene expression that could be specifically involved in SMARCA4/MYC driven tumorigenesis. Finally, an integration of RNA sequencing and DNA methylation data of murine tumors with human samples revealed a high resemblance to human Group 3 medulloblastoma on the molecular level. Altogether, the development of SMARCA4-deficient medulloblastomas in mice paves the way to deciphering the role of frequently occurring SMARCA4 alterations in Group 3 medulloblastoma with the perspective to explore targeted therapeutic options.

Abstract Pediatric high-grade gliomas of the subclass MYCN (HGG-MYCN) are highly aggressive tumors frequently carrying MYCN amplifications, TP53 mutations, or both alterations. Due to their rarity, such tumors have only recently been identified as a distinct entity, and biological as well as clinical characteristics have not been addressed specifically. To gain insights into tumorigenesis and molecular profiles of these tumors, and to ultimately suggest alternative treatment options, we generated a genetically engineered mouse model by breeding hGFAP-cre::Trp53 Fl/Fl ::lsl-MYCN mice. All mice developed aggressive forebrain tumors early in their lifetime that mimic human HGG-MYCN regarding histology, DNA methylation, and gene expression. Single-cell RNA sequencing revealed a high intratumoral heterogeneity with neuronal and oligodendroglial lineage signatures. High-throughput drug screening using both mouse and human tumor cells finally indicated high efficacy of Doxorubicin, Irinotecan, and Etoposide as possible therapy options that children with HGG-MYCN might benefit from.

In contrast to adults, meningiomas are uncommon tumors in childhood and adolescence. Whether adult and pediatric meningiomas differ on a molecular level is unclear. Here we report detailed genomic analyses of 37 pediatric meningiomas by sequencing and DNA methylation profiling. Histologically, the series was dominated by meningioma subtypes with aggressive behavior, with 70% of patients suffering from WHO grade II or III meningiomas. The most frequent cytogenetic aberrations were loss of chromosomes 22 (23/37 [62%]), 1 (9/37 [24%]), 18 (7/37 [19%]), and 14 (5/37 [14%]). Tumors with NF2 alterations exhibited overall increased chromosomal instability. Unsupervised clustering of DNA methylation profiles revealed separation into three groups: designated group 1 composed of clear cell and papillary meningiomas, whereas group 2A comprised predominantly atypical meningiomas and group 2B enriched for rare high-grade subtypes (rhabdoid, chordoid). Meningiomas from NF2 patients clustered exclusively within groups 1 and 2A. When compared with a dataset of 105 adult meningiomas, the pediatric meningiomas largely grouped separately. Targeted panel DNA sequencing of 34 tumors revealed frequent NF2 alterations, while other typical alterations found in adult non-NF2 tumors were absent. These data demonstrate that pediatric meningiomas are characterized by molecular features distinct from adult tumors.

Abstract To date, several studies on genomic events underlying medulloblastoma (MB) biology have expanded our understanding of this tumour entity and led to its division into four groups—WNT, SHH, group 3 (G3) and group 4 (G4). However, there is little information about the relevance of pathogenic mitochondrial DNA (mtDNA) mutations and their consequences across these. In this report, we describe the case of a female patient with MB and a mitochondriopathy, followed by a study of mtDNA variants in MB groups. After being diagnosed with G4 MB, the index patient was treated in line with the HIT 2000 protocol with no indications of relapse after five years. Long-term side effects of treatment were complemented by additional neurological symptoms and elevated lactate levels ten years later, resulting in suspected mitochondrial disease. This was confirmed by identifying a mutation in the MT-TS1 gene which appeared homoplasmic in patient tissue and heteroplasmic in the patient’s mother. Motivated by this case, we explored mtDNA mutations across 444 patients from ICGC and HIT cohorts. While there was no statistically significant enrichment of mutations in one MB group, both cohorts encompassed a small group of patients harbouring potentially deleterious mtDNA variants. The case presented here highlights the possible similarities between sequelae caused by MB treatment and neurological symptoms of mitochondrial dysfunction, which may apply to patients across all MB groups. In the context of the current advances in characterising and interpreting mtDNA aberrations, recognising affected patients could enhance our future knowledge regarding the mutations’ impact on carcinogenesis and cancer treatment.

Abstract The tumor suppressor and chromatin modifier cAMP response element-binding protein binding protein (CREBBP) and v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN), a member of the MYC oncogene family, are critically involved in brain development. Both genes are frequently mutated in the same tumor entities, including high-grade glioma and medulloblastoma. Therefore, we hypothesized that alterations in both genes cooperate to induce brain tumor formation. For further investigation, hGFAP-cre::Crebbp Fl/Fl ::lsl-MYCN mice were generated, which combine Crebbp deletion with overexpression of MYCN in neural stem cells (NSCs). Within eight months, these animals developed aggressive forebrain tumors. The first tumors were detectable in the olfactory bulbs of seven-day-old mice. This location raises the possibility that presumptive founder cells are derived from the ventricular-subventricular zone (V-SVZ). To examine the cellular biology of these tumors, single-cell RNA sequencing was performed, which revealed high intratumoral heterogeneity. Data comparison with reference CNS cell types indicated the highest similarity of tumor cells with transit-amplifying NSCs or activated NSCs of the V-SVZ. Consequently, we analyzed V-SVZ NSCs of our mouse model aiming to confirm that the tumors originate from this stem cell niche. Mutant V-SVZ NSCs showed significantly increased cell viability and proliferation as well as reduced glial and neural differentiation in vitro compared to control cells. In summary, we demonstrate the oncogenic potential of a combined loss of function of CREBBP and overexpression of MYCN in this cell population. hGFAP-cre::Crebbp Fl/Fl ::lsl-MYCN mice thus provide a valuable tool to study tumor-driving mechanisms in a key neural stem/ progenitor cell niche.

The use of mutation analysis of homologous recombination repair (HRR) genes to estimate PARP‐inhibition response may miss a larger proportion of responding patients. Here, we provide preclinical models for castration‐resistant prostate cancer (CRPC) that can be used to functionally predict HRR defects. In vitro, CRPC LNCaP sublines revealed an HRR defect and enhanced sensitivity to olaparib and cisplatin due to impaired RAD51 expression and recruitment. Ex vivo‐induced castration‐resistant tumor slice cultures or tumor slice cultures derived directly from CRPC patients showed increased olaparib‐ or cisplatin‐associated enhancement of residual radiation‐induced γH2AX/53BP1 foci. We established patient‐derived tumor organoids (PDOs) from CRPC patients. These PDOs are morphologically similar to their primary tumors and genetically clustered with prostate cancer but not with normal prostate or other tumor entities. Using these PDOs, we functionally confirmed the enhanced sensitivity of CRPC patients to olaparib and cisplatin. Moreover, olaparib but not cisplatin significantly decreased the migration rate in CRPC cells. Collectively, we present robust patient‐derived preclinical models for CRPC that recapitulate the features of their primary tumors and enable individualized drug screening, allowing translation of treatment sensitivities into tailored clinical therapy recommendations.

Abstract Background Cancer metabolism influences multiple aspects of tumorigenesis and causes diversity across malignancies. Although comprehensive research has extended our knowledge of molecular subgroups in medulloblastoma (MB), discrete analysis of metabolic heterogeneity is currently lacking. This study seeks to improve our understanding of metabolic phenotypes in MB and their impact on patients’ outcomes. Methods Data from four independent MB cohorts encompassing 1,288 patients were analysed. We explored metabolic characteristics of 902 patients (ICGC and MAGIC cohorts) on bulk RNA level. Moreover, data from 491 patients (ICGC cohort) were searched for DNA alterations in genes regulating cell metabolism. To determine the role of intratumoral metabolic differences, we examined single-cell RNA-sequencing (scRNA-seq) data from 34 additional patients. Findings on metabolic heterogeneity were correlated to clinical data. Results Established MB groups exhibit substantial differences in metabolic gene expression. By employing unsupervised analyses, we identified three clusters of group 3 and 4 samples with distinct metabolic features in ICGC and MAGIC cohorts. Analysis of scRNA-seq data confirmed our results of intertumoral heterogeneity underlying the according differences in metabolic gene expression. On DNA level, we discovered clear associations between altered regulatory genes involved in MB development and lipid metabolism. Additionally, we determined the prognostic value of metabolic gene expression in MB and showed that expression of genes involved in metabolism of inositol phosphates and nucleotides correlates with patient survival. Conclusion Our research underlines the biological and clinical relevance of metabolic alterations in MB. Thus, distinct metabolic signatures presented here might be the first step towards future metabolism-targeted therapeutic options. Graphical Abstract

Sonic hedgehog medulloblastoma encompasses a clinically and molecularly diverse group of cancers of the developing central nervous system. Here, we use unbiased sequencing of the transcriptome across a large cohort of 250 tumors to reveal differences among molecular subtypes of the disease, and demonstrate the previously unappreciated importance of non-coding RNA transcripts. We identify alterations within the cAMP dependent pathway (GNAS, PRKAR1A) which converge on GLI2 activity and show that 18% of tumors have a genetic event that directly targets the abundance and/or stability of MYCN. Furthermore, we discover an extensive network of fusions in focally amplified regions encompassing GLI2, and several loss-of-function fusions in tumor suppressor genes PTCH1, SUFU and NCOR1. Molecular convergence on a subset of genes by nucleotide variants, copy number aberrations, and gene fusions highlight the key roles of specific pathways in the pathogenesis of Sonic hedgehog medulloblastoma and open up opportunities for therapeutic intervention.

DICER1 syndrome is associated with a predisposition to multiple tumor types. Here, the authors identify and characterize 3 molecular subgroups of mesenchymal tumors with DICER1 mutations.

Abstract Background The gene of the Epidermal growth factor receptor (EGFR) is one of the most frequently altered genes in glioblastoma (GBM), with deletions of exons 2–7 (EGFRvIII) being amongst the most common genomic mutations. EGFRvIII is heterogeneously expressed in GBM. We already showed that EGFRvIII expression has an impact on chemosensitivity, replication stress, and the DNA damage response. Wee1 kinase is a major regulator of the DNA damage induced G2 checkpoint. It is highly expressed in GBM and its overexpression is associated with poor prognosis. Since Wee1 inhibition can lead to radiosensitization of EGFRvIII-negative (EGFRvIII−) GBM cells, we asked, if Wee1 inhibition is sufficient to radiosensitize also EGFRvIII-positive (EGFRvIII+) GBM cells. Methods We used the clinically relevant Wee1 inhibitor adavosertib and two pairs of isogenetic GBM cell lines with and without endogenous EGFRvIII expression exhibiting different TP53 status. Moreover, human GBM samples displaying heterogenous EGFRvIII expression were analyzed. Expression of Wee1 was assessed by Western blot and respectively immunohistochemistry. The impact of Wee1 inhibition in combination with irradiation on cell cycle and cell survival was analyzed by flow cytometry and colony formation assay. Results Analysis of GBM cells and patient samples revealed a higher expression of Wee1 in EGFRvIII+ cells compared to their EGFRvIII− counterparts. Downregulation of EGFRvIII expression by siRNA resulted in a strong decrease in Wee1 expression. Wee1 inhibition efficiently abrogated radiation-induced G2-arrest and caused radiosensitization, without obvious differences between EGFRvIII− and EGFRvIII+ GBM cells. Conclusion We conclude that the inhibition of Wee1 is an effective targeting approach for the radiosensitization of both EGFRvIII− and EGFRvIII+ GBM cells and may therefore represent a promising new therapeutic option to increase response to radiotherapy.

Sinonasal tumour diagnosis can be complicated by the heterogeneity of disease and classification systems. Here, the authors use machine learning to classify sinonasal undifferentiated carcinomas into 4 molecular classe with differences in differentiation state and clinical outcome.

Summary: The zinc-finger transcription factor GLI2 is frequently amplified in childhood medulloblastoma of the Sonic-hedgehog type (SHH-MB), with or without amplification of NMYC or deletion of TP53. Despite the aggressive tumor behavior, tumorigenesis is not well understood, and adequate mouse models are lacking. Therefore, we generated mice with a GLI2 overexpression under control of the hGFAP-promoter. These mice died within 150 days. The majority only survived until postnatal day 40. They displayed severe cerebellar hypoplasia, cortical malformations, but no brain tumors, except for one out of 23 animals with an undifferentiated hindbrain lesion. Additional loss of p53 did not result in cerebellar tumors, but partially rescued the cerebellar phenotype induced by GLI2 overexpression. Similarly, the combination of GLI2 and NMYC was neither sufficient for the development of SHH-MB. We therefore assume that the development of childhood SHH-MB in mice is either occurring in cellular origins outside the hGFAP-positive lineage or needs additional genetic drivers.