Analysis of safety and efficacy of proton radiotherapy for optic nerve sheath meningioma.

Background: Primary optic nerve sheath meningiomas (ONSMs) represent a group of benign tumors originating from the optic nerve sheath, typically causing painless, gradual onset monocular visual loss, which can result in blindness if left untreated. Radiation therapy represents an important treatment option for patients with ONSM, allowing for preservation and potential improvement in visual function. In particular, proton radiotherapy may enable a reduction of the side effects due to its physical advantage of an inverted dose profile with a steep dose gradient. The study investigates the visual acuity, local tumor control, and treatment-related toxicities following proton beam radiotherapy with a single institutional cohort comprising 32 patients treated for ONSM. Methods: Patients with primary ONSM, either histologically (16/32) or radiologically confirmed (16/32), which were treated at the Department of Radiation Oncology at the University Hospital Heidelberg (Germany) were assessed in regard to their visual outcomes, treatment toxicity, and local tumor control following radiotherapy according to response assessment in neuro-oncology criteria. Results: After a median follow-up time of 39.5 months, the 5-year local progression-free survival was estimated at 100%, with 84.4% of patients reporting improvement or stability in visual acuity during their last follow-up. Radiation-induced optic neuropathy (RION) was encountered in 9.4%. Conclusions: Our study demonstrates proton beam therapy as a safe and effective treatment alternative in the therapeutic management of ONSMs. RION represents a rare but dreaded complication after treatment. Future head-to-head comparisons with photon radiotherapy in a prospective setting are required to demonstrate a potential, additional clinical benefit.

Toward standardized brain tumor tissue processing protocols in neuro-oncology: a perspective for gliomas and beyond.

Implementation of standardized protocols in neurooncology during the surgical resection of brain tumors is needed to advance the clinical treatment paradigms that use tissue for diagnosis, prognosis, bio-banking, and treatment. Currently recommendations on intraoperative tissue procurement only exist for diffuse gliomas but management of other brain tumor subtypes can also benefit from these protocols. Fresh tissue from surgical resection can now be used for intraoperative diagnostics and functional precision medicine assays. A multidisciplinary neuro-oncology perspective is critical to develop the best avenues for practical standardization. This perspective from the multidisciplinary Oncology Tissue Advisory Board (OTAB) discusses current advances, future directions, and the imperative of adopting standardized protocols for diverse brain tumor entities. There is a growing need for consistent operating room practices to enhance patient care, streamline research efforts, and optimize outcomes.

Uterine mesenchymal tumours harboring the KAT6B/A::KANSL1 gene fusion represent a distinct type of uterine sarcoma based on DNA methylation profiles.

Uterine mesenchymal tumours harboring KAT6B/A::KANSL1 gene fusions typically exhibit histological and immunophenotypic overlap with endometrial stromal and smooth muscle tumours. To date it remains uncertain whether such neoplasms should be regarded as variants of smooth muscle or endometrial stromal neoplasm, or if they constitute a distinct tumour type. In this study we investigated DNA methylation patterns and copy number variations (CNVs) in a series of uterine tumours harboring KAT6B/A::KANSL1 gene fusions in comparison to other mesenchymal neoplasms of the gynecological tract. Unsupervised hierarchical clustering and t-SNE analysis of DNA methylation data (Illumina EPIC array) identified a distinct cluster for 8/13 KAT6B/A::KANSL1 tumours (herein referred to as core cluster). The other 5 tumours (herein referred to as outliers) did not assign to the core cluster but clustered near various other tumour types. CNV analysis did not identify significant alterations in the core cluster. In contrast, various alterations, including deletions at the CDKN2A/B and NF1 loci were identified in the outlier group. Analysis of the DNA methylation clusters in relation to histological features revealed that while tumours in the core KAT6B/A::KANSL1 cluster were histologically bland, outlier tumours frequently exhibited "high-grade" histologic features in the form of significant nuclear atypia, increased mitotic activity and necrosis. Three of the five patients with outlier tumours died from their disease while clinical follow-up in the remaining two patients was limited (less than 12 months). In comparison, none of the 7 out of 8 patients with tumors in the core KAT6B/A::KANSL1 sarcoma cluster, where follow-up was available, died from disease. Furthermore, only 1 out of 7 patients recurred (mean follow-up of 30 months). In conclusion, KAT6B/A::KANSL1 uterine sarcoma is a molecularly unique type of uterine tumour that should be recognized as a distinct entity. These tumors typically exhibit low-grade histologic features but are occasionally morphologically high-grade; the latter have a DNA methylation profile different from the typical low-grade neoplasms and may be associated with aggressive behaviour.

Odontogenic Myxomas Harbor Recurrent Copy Number Alterations and a Distinct Methylation Signature.

Odontogenic myxoma is a rare, benign, and locally aggressive tumor that develops in the tooth-bearing areas of the jaw. The molecular mechanisms underlying odontogenic myxomas are unknown and no diagnostic markers are available to date. The aim of this study was to analyze DNA methylation and copy number variations in odontogenic myxomas to identify new molecular signatures for diagnostic decision-making. We collected a cohort of 16 odontogenic myxomas from 2006 to 2021 located in the mandible (n = 10) and maxilla (n = 6) with available formalin-fixed paraffin-embedded or fresh frozen tumor tissue from a biopsy or resection material. Genome-wide DNA methylation and copy number variation data were generated from 12 odontogenic myxomas using the Illumina Infinium Methylation EPIC array, interrogating >850,000 CpG sites. Unsupervised clustering and dimensionality reduction (Uniform Manifold Approximation and Projection) revealed that odontogenic myxomas formed a distinct DNA methylation class. Copy number profiling showed recurrent whole-chromosome gains (trisomies) of chromosomes 5, 8, and 20 in all cases, and of chromosomes 10, 12, and 17 in all except one case. In conclusion, odontogenic myxomas harbor recurrent copy number patterns and a distinct DNA methylation profile, which can be used as an additional diagnostic tool in the appropriate clinical and radiologic context. Further research is needed to explain the genetic mechanisms caused by these alterations that drive these locally aggressive neoplasms.

Recurrent CLTC::SYK fusions and CSF1R mutations in juvenile xanthogranuloma of soft tissue.

Juvenile xanthogranuloma (JXG) is a histiocytic neoplasm that usually presents in the skin. Rarely, extracutaneous localizations occur; the genetic drivers of this clinical variant of JXG remain incompletely characterized. We present detailed clinicopathologic and molecular data of 16 children with extracutaneous JXG and 5 adults with xanthogranulomas confined to the central nervous system (CNS) or soft tissue. Tissue samples were obtained through the Dutch Nationwide Pathology Databank and analyzed with an innovative sequencing technique capable of detecting both small genomic variants and gene rearrangements. Targetable kinase alterations were detected in 16/16 children and 1/5 adults. Alterations included CLTC::SYK fusions in 6 children and CSF1R mutations in 7 others - all below 2 years old with soft tissue tumors. One child had a CSF1R mutation and MRC1::PDGFRB fusion. Most were treated surgically, although spontaneous regression occurred in 1/6 with CLTC::SYK and 2/7 with CSF1R mutations - underscoring that treatment is not always necessary. Tumors with CLTC::SYK fusions generally lacked Touton giant cells, but exhibited many other histologic features of JXG and concordant methylation profiles. Using multispectral immunofluorescence, phosphorylated-SYK expression was localized to CD163+ histiocytes; tumors with CLTC::SYK fusions also demonstrated mTOR activation, Cyclin D1 expression, and variable phosphorylated-ERK expression. BRAFV600E was detected in 1 child and 1 adult with CNS xanthogranulomas; both responded to BRAF inhibition. Finally, a TPM3::NTRK1 fusion or MAP2K1 deletion were detected in 2 children with systemic JXG who experienced spontaneous disease regression. This study advances the molecular understanding of histiocytic neoplasms and may guide diagnostics and clinical management.

DNA Methylation Profiling of Salivary Gland Tumors Supports and Expands Conventional Classification.

Tumors of the major and minor salivary glands histologically encompass a diverse and partly overlapping spectrum of frequent diagnostically challenging neoplasms. Despite recent advances in molecular testing and the identification of tumor-specific mutations or gene fusions, there is an unmet need to identify additional diagnostic biomarkers for entities lacking specific alterations. In this study, we collected a comprehensive cohort of 363 cases encompassing 20 different salivary gland tumor entities and explored the potential of DNA methylation to classify these tumors. We were able to show that most entities show specific epigenetic signatures and present a machine learning algorithm that achieved a mean balanced accuracy of 0.991. Of note, we showed that cribriform adenocarcinoma is epigenetically distinct from classical polymorphous adenocarcinoma, which could support risk stratification of these tumors. Myoepithelioma and pleomorphic adenoma form a uniform epigenetic class, supporting the theory of a single entity with a broad but continuous morphologic spectrum. Furthermore, we identified a histomorphologically heterogeneous but epigenetically distinct class that could represent a novel tumor entity. In conclusion, our study provides a comprehensive resource of the DNA methylation landscape of salivary gland tumors. Our data provide novel insight into disputed entities and show the potential of DNA methylation to identify new tumor classes. Furthermore, in future, our machine learning classifier could support the histopathologic diagnosis of salivary gland tumors.

Wnt/ß-Catenin-Activated Nonpilomatrical Carcinoma of the Skin: A Case Series.

Among skin epithelial tumors, recurrent mutations in the APC/CTNNB1 genes resulting in activation of the Wnt/ß-catenin pathway have been reported predominantly in neoplasms with matrical differentiation. In the present study, we describe the morphologic, immunohistochemical, and genetic features of 16 primary cutaneous carcinomas harboring mutations activating the Wnt/ß-catenin pathway without evidence of matrical differentiation, as well as 4 combined tumors in which a similar Wnt/ß-catenin-activated carcinoma component was associated with Merkel cell carcinoma (MCC) or pilomatrical carcinoma. Among the pure tumor cases, 6 of 16 patients were women with a median age of 80 years (range, 58-98 years). Tumors were located on the head and neck (n = 7, 44%), upper limb (n = 4, 25%), trunk (n = 3, 18%), and leg (n = 2, 13%). Metastatic spread was observed in 4 cases resulting in death from disease in 1 patient. Microscopically, all cases were poorly differentiated neoplasms infiltrating the dermis and/or subcutaneous tissue. In 13 cases, solid "squamoid" areas were associated with a basophilic component characterized by rosette/pseudoglandular formation resulting in a biphasic appearance. Three specimens consisted only of poorly differentiated carcinoma lacking rosette formation. Immunohistochemical studies showed frequent expression of EMA (100%), BerEP4 (100%), cytokeratin 7 (94%), chromogranin A (44%), synaptophysin (82%), and cytokeratin 20 (69%). Complete loss of Rb expression was observed in all but 1 case. Nuclear ß-catenin and CDX2 expressions were detected in all cases. Recurrent pathogenic somatic mutations were observed in APC (60%), CTNNB1 (40%), and RB1 (n = 47%). Global methylation analysis confirmed that cases with rosette formation constituted a homogeneous tumor group distinct from established skin tumor entities (pilomatrical carcinoma, MCC, and squamous cell carcinoma), although the 3 other cases lacking such morphologic features did not. In addition, we identified 4 combined neoplasms in which there was a component showing a similar poorly differentiated rosette-forming carcinoma demonstrating Rb loss and ß-catenin activation associated with either MCC (n = 3) or pilomatrical carcinoma (n = 1). In conclusion, we describe a distinctive neoplasm, for which we propose the term "Wnt/ß-catenin-activated rosette-forming carcinoma," morphologically characterized by the association of rosette formation, squamous and/or neuroendocrine differentiation, diffuse CDX2 expression, Rb loss, and mutations in CTNNB1/APC genes.

Exomic and epigenomic analysis of pulmonary blastoma.

OBJECTIVE: Pulmonary blastoma is a rare, biphasic, adult-onset lung tumor. In this study, we investigate whether DICER1 pathogenic variants are a feature of pulmonary blastomas through in-depth analysis of the molecular events defining them. METHODS: We performed exome-wide sequencing and DNA methylation profiling of 8 pulmonary blastomas from 6 affected persons. RESULTS: We identified biallelic somatic DICER1 pathogenic variants in 7 of 8 cases. The remaining case had a solitary missense pathogenic variant in the RNase IIIb domain of DICER1. Six of 8 cases carried a CTNNB1 hotspot variant and 4 of 8 had a somatic pathogenic variant in TP53. Methylation analysis showed that the pulmonary blastomas clustered with other DICER1-mutated tumors and not with other more common types of lung cancer. CONCLUSION: We conclude somatic DICER1 pathogenic variants are the major driver of pulmonary blastoma and are likely to act in conjunction with CTNNB1 hotspot variants that are often present.

cIMPACT-NOW Update 8: Clarifications on molecular risk parameters and recommendations for WHO grading of meningiomas.

Meningiomas are the most frequent primary intracranial tumors. Hence, they constitute a major share of diagnostic specimens in neuropathology practice. The 2021 WHO Classification of Central Nervous System Tumors ("CNS5") has introduced the first molecular grading parameters for meningioma with oncogenic variants in the TERT promoter and homozygous deletion of CDKN2A/B as markers for CNS WHO grade 3. However, after publication of the new classification volume, clarifications were requested, not only on novel but also on long-standing questions in meningioma grading that were beyond the scope of the WHO "blue book". In addition, more recent research into possible new molecular grading parameters could not yet be implemented in the 2021 classification but constitute a compelling body of literature. Hence, the cIMPACT-NOW Steering Committee convened a working group to provide such clarification and assess the evidence of possible novel molecular criteria. As a result, this cIMPACT-NOW update provides guidance for more standardized morphological evaluation and interpretation, most prominently pertaining to brain invasion, identifies scenarios in which advanced molecular testing is recommended, proposes to assign CNS WHO grade 2 for cases with CNS WHO grade 1 morphology but chromosomal arm 1p deletion in combination with 22q deletion and/or NF2 oncogenic variants, and discusses areas in which the current evidence is not yet sufficient to result in new recommendations.

Analyses of combined Merkel cell carcinomas with neuroblastic components suggests that loss of T antigen expression in Merkel cell carcinoma may result in cell cycle arrest and neuroblastic transdifferentiation.

Merkel cell carcinoma (MCC) is an aggressive skin cancer frequently caused by genomic integration of the Merkel cell polyomavirus (MCPyV). MCPyV-negative cases often present as combined MCCs, which represent a distinctive subset of tumors characterized by association of an MCC with a second tumor component, mostly squamous cell carcinoma. Up to now, only exceptional cases of combined MCC with neuroblastic differentiation have been reported. Herein we describe two additional combined MCCs with neuroblastic differentiation and provide comprehensive morphologic, immunohistochemical, transcriptomic, genetic and epigenetic characterization of these tumors, which both arose in elderly men and appeared as an isolated inguinal adenopathy. Microscopic examination revealed biphasic tumors combining a poorly differentiated high-grade carcinoma with a poorly differentiated neuroblastic component lacking signs of proliferation. Immunohistochemical investigation revealed keratin 20 and MCPyV T antigen (TA) in the MCC parts, while neuroblastic differentiation was confirmed in the other component in both cases. A clonal relation of the two components can be deduced from 20 and 14 shared acquired point mutations detected by whole exome analysis in both combined tumors, respectively. Spatial transcriptomics demonstrated a lower expression of stem cell marker genes such as SOX2 and MCM2 in the neuroblastic component. Interestingly, although the neuroblastic part lacked TA expression, the same genomic MCPyV integration and the same large T-truncating mutations were observed in both tumor parts. Given that neuronal transdifferentiation upon TA repression has been reported for MCC cell lines, the most likely scenario for the two combined MCC/neuroblastic tumors is that neuroblastic transdifferentiation resulted from loss of TA expression in a subset of MCC cells. Indeed, DNA methylation profiling suggests an MCC-typical cellular origin for the combined MCC/neuroblastomas. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Non-C19MC-altered embryonal tumor with multilayered rosettes in a young woman with DICER1 syndrome: case report and review of the literature.

Embryonal tumors with multilayered rosettes (ETMR) are highly aggressive and therapy-resistant pediatric central nervous system (CNS) tumors that have three histological patters: embryonal tumor with abundant neuropil and true rosettes, ependymoblastoma, and medulloepithelioma. We present a case of ETMR in an 18-year-old woman with DICER1 syndrome. This report confirms the important role of DNA-methylation analysis in the classification of CNS embryonal tumors and the importance of investigating somatic and germline DICER1 mutations in all CNS embryonal tumors.

Mesonephric-type adenocarcinomas of the ovary: prevalence, diagnostic reproducibility, outcome, and value of PAX2.

Mesonephric-type (or -like) adenocarcinomas (MAs) of the ovary are an uncommon and aggressive histotype. They appear to arise through transdifferentiation from Müllerian lesions creating diagnostic challenges. Thus, we aimed to develop a histologic and immunohistochemical (IHC) approach to optimize the identification of MA over its histologic mimics, such as ovarian endometrioid carcinoma (EC). First, we screened 1,537 ovarian epithelial neoplasms with a four-marker IHC panel of GATA3, TTF1, ER, and PR followed by a morphological review of EC to identify MA in retrospective cohorts. Interobserver reproducibility for the distinction of MA versus EC was assessed in 66 cases initially without and subsequently with IHC information (four-marker panel). Expression of PAX2, CD10, and calretinin was evaluated separately, and survival analyses were performed. We identified 23 MAs from which 22 were among 385 cases initially reported as EC (5.7%) and 1 as clear cell carcinoma. The interobserver reproducibility increased from fair to substantial (κ = 0.376-0.727) with the integration of the four-marker IHC panel. PAX2 was the single most sensitive and specific marker to distinguish MA from EC and could be used as a first-line marker together with ER/PR and GATA3/TTF1. Patients with MA had significantly increased risk of earlier death from disease (hazard ratio = 3.08; 95% CI, 1.62-5.85; p < 0.0001) compared with patients with EC, when adjusted for age, stage, and p53 status. A diagnosis of MA has prognostic implications for stage I disease, and due to the subtlety of morphological features in some tumors, a low threshold for ancillary testing is recommended.

Spindle Cell Lesions with Oncogenic EGFR Kinase Domain Aberrations: Expanding the Spectrum of Protein Kinase-Related Mesenchymal Tumors.

EGFR aberrations are reported in a subset of myofibroblastic lesions with kinase domain duplication (EGFR-KDD) and exon 20 mutations being assigned to infantile fibrosarcomas (IFS), mesoblastic nephroma, and fibrous hamartoma of infancy (FHI), respectively. In this retrospective study, we correlated molecular findings with the histomorphology of 14 myofibroblastic lesions harboring such genetic changes identified by NGS. We additionally performed DNA methylation profiling (DNAmp) and immunohistochemistry. Lesions were from 10 males and 4 females with a mean age of 3 years (range, 0.3-14) and occurred subcutaneously in the upper limbs (n = 5), lower limbs (n = 3), back/thorax (n = 5), and the nasal cavity (n = 1). Eleven were cured by surgery, including 1 relapsed case. Two patients were lost to follow-up. One case was very recent, and the patient was biopsied. Histologically, the lesions showed a wide spectrum varying from classic FHI (n = 9) to IFS (n = 1) or lipofibromatosis-like tumors (LFT-like) (n = 2) or dermatofibrosarcoma protuberans-like (DFSP-like) (n = 1) to a predominantly myxoid spindle cell lesion (n = 1). Immunohistochemically, all neoplasms stained with CD34, whereas S100 was positive in 2/14. EGFR expression was observed in 9/10 cases. Molecularly, the IFS and 1 LFT-like harbored EGFR-KDD, whereas an exon 20 mutation was identified in all FHI, 1 LFT-like, the DFSP-like, and in predominant myxoid spindle cell lesion. By DNAmp, all but 2 cases formed a well-defined cluster, demonstrating that these lesions are also epigenetically related. In conclusion, EGFR kinase domain aberrations found in FHI, IFS, LFT-like, DFSP-like, and a spindle cell lesion with a predominant myxoid stroma of children and adolescents showed that these neoplasms with a broad morphologic spectrum belong to the group of protein kinase-related lesions with a distinct epigenetic signature. Molecular analyses, including DNAmp, help to identify and characterize this emerging category and become mandatory when targeted treatment is considered.

The biological significance of tumor grade, age, enhancement, and extent of resection in IDH-mutant gliomas: How should they inform treatment decisions in the era of IDH inhibitors?

The 2016 and 2021 World Health Organization 2021 Classification of central nervous system tumors have resulted in a major improvement in the classification of isocitrate dehydrogenase (IDH)-mutant gliomas. With more effective treatments many patients experience prolonged survival. However, treatment guidelines are often still based on information from historical series comprising both patients with IDH wild-type and IDH-mutant tumors. They provide recommendations for radiotherapy and chemotherapy for so-called high-risk patients, usually based on residual tumor after surgery and age over 40. More up-to-date studies give a better insight into clinical, radiological, and molecular factors associated with the outcome of patients with IDH-mutant glioma. These insights should be used today for risk stratification and for treatment decisions. In many patients with IDH-mutant grades 2 and 3 glioma, if carefully monitored postponing radiotherapy and chemotherapy is safe, and will not jeopardize the overall outcome of patients. With the INDIGO trial showing patient benefit from the IDH inhibitor vorasidenib, there is a sizable population in which it seems reasonable to try this class of agents before recommending radio-chemotherapy with its delayed adverse event profile affecting quality of survival. Ongoing trials should help to further identify the patients that are benefiting from this treatment.

Clinically unfavorable transcriptome subtypes of non-WNT/non-SHH medulloblastomas are associated with a predominance in proliferating and progenitor-like cell subpopulations.

The non-WNT/non-SHH (Grp3/Grp4) medulloblastomas (MBs) include eight second-generation subgroups (SGS; I-VIII) each with distinct molecular and clinical characteristics. Recently, we also identified two prognostically relevant transcriptome subtypes within each SGS MB, which are associated with unique gene expression signatures and signaling pathways. These prognostic subsets may be in connection to the intra-tumoral cell landscape that underlies SGS MB clinical-molecular diversity. Here, we performed a deconvolution analysis of the Grp3/Grp4 MB bulk RNA profiles using the previously identified single-cell RNA-seq reference dataset and focusing on variability in the cellular composition of SGS MB. RNA deconvolution analysis of the Grp3/Grp4 MB disclosed the subgroup-specific neoplastic cell subpopulations. Neuronally differentiated axodendritic GP3-C1 and glutamatergic GP4-C1 subpopulations were distributed within Grp3- and Grp4-associated SGS MB, respectively. Progenitor GP3-B2 subpopulation was prominent in aggressive SGS II MB, whereas photoreceptor/visual perception GP3/4-C2 cell content was typical for SGS III/IV MB. The current study also revealed significant variability in the proportions of cell subpopulations between clinically relevant SGS MB transcriptome subtypes, where unfavorable cohorts were enriched with cell cycle and progenitor-like cell subpopulations and, vice versa, favorable subtypes were composed of neuronally differentiated cell fractions predominantly. A higher than median proportion of proliferating and progenitor cell subpopulations conferred the shortest survival of the Grp3 and Grp 4 MB, and similar survival associations were identified for all SGS MB except SGS IV MB. In summary, the recently identified clinically relevant Grp3/Grp4 MB transcriptome subtypes are composed of different cell populations. Future studies should aim to validate the prognostic and therapeutic role of the identified Grp3/Grp4 MB inter-tumoral cellular heterogeneity. The application of the single-cell techniques on each SGS MB separately could help to clarify the clinical significance of subgroup-specific variability in tumor cell content and its relation with prognostic transcriptome signatures identified before.

Plasma extracellular vesicles in meningioma patients following radiotherapy as liquid biopsy- a prospective explorative biomarker study (ARO 2023-05/AG-NRO-07).

BACKGROUND: While surgical resection remains the primary treatment approach for symptomatic or growing meningiomas, radiotherapy represents an auspicious alternative in patients with meningiomas not safely amenable to surgery. Biopsies are often omitted in light of potential postoperative neurological deficits, resulting in a lack of histological grading and (molecular) risk stratification. In this prospective explorative biomarker study, extracellular vesicles in the bloodstream will be investigated in patients with macroscopic meningiomas to identify a biomarker for molecular risk stratification and disease monitoring. METHODS: In total, 60 patients with meningiomas and an indication of radiotherapy (RT) and macroscopic tumor on the planning MRI will be enrolled. Blood samples will be obtained before the start, during, and after radiotherapy, as well as during clinical follow-up every 6 months. Extracellular vesicles will be isolated from the blood samples, quantified and correlated with the clinical treatment response or progression. Further, nanopore sequencing-based DNA methylation profiles of plasma EV-DNA will be generated for methylation-based meningioma classification. DISCUSSION: This study will explore the dynamic of plasma EVs in meningioma patients under/after radiotherapy, with the objective of identifying potential biomarkers of (early) tumor progression. DNA methylation profiling of plasma EVs in meningioma patients may enable molecular risk stratification, facilitating a molecularly-guided target volume delineation and adjusted dose prescription during RT treatment planning.

CNS tumors with PLAGL1-fusion: beyond ZFTA and YAP1 in the genetic spectrum of supratentorial ependymomas.

A novel methylation class, "neuroepithelial tumor, with PLAGL1 fusion" (NET-PLAGL1), has recently been described, based on epigenetic features, as a supratentorial pediatric brain tumor with recurrent histopathological features suggesting an ependymal differentiation. Because of the recent identification of this neoplastic entity, few histopathological, radiological and clinical data are available. Herein, we present a detailed series of nine cases of PLAGL1-fused supratentorial tumors, reclassified from a series of supratentorial ependymomas, non-ZFTA/non-YAP1 fusion-positive and subependymomas of the young. This study included extensive clinical, radiological, histopathological, ultrastructural, immunohistochemical, genetic and epigenetic (DNA methylation profiling) data for characterization. An important aim of this work was to evaluate the sensitivity and specificity of a novel fluorescent in situ hybridization (FISH) targeting the PLAGL1 gene. Using histopathology, immunohistochemistry and electron microscopy, we confirmed the ependymal differentiation of this new neoplastic entity. Indeed, the cases histopathologically presented as "mixed subependymomas-ependymomas" with well-circumscribed tumors exhibiting a diffuse immunoreactivity for GFAP, without expression of Olig2 or SOX10. Ultrastructurally, they also harbored features reminiscent of ependymal differentiation, such as cilia. Different gene partners were fused with PLAGL1: FOXO1, EWSR1 and for the first time MAML2. The PLAGL1 FISH presented a 100% sensitivity and specificity according to RNA sequencing and DNA methylation profiling results. This cohort of supratentorial PLAGL1-fused tumors highlights: 1/ the ependymal cell origin of this new neoplastic entity; 2/ benefit of looking for a PLAGL1 fusion in supratentorial cases of non-ZFTA/non-YAP1 ependymomas; and 3/ the usefulness of PLAGL1 FISH.