Low-grade epilepsy-associated tumors (LEATs) are a distinct group of tumors commonly encountered in pediatric drug-resistant epilepsy that necessitate surgical intervention. Like tumors elsewhere in the central nervous system, molecular characterization is becoming an increasingly important consideration in pediatric neuro-oncology prognostication and management for LEATs. Thus, familiarity with relevant tumor mutations and radiogenomic features of LEATs is important for radiologists caring for affected patients. This article will review the genetic alterations and imaging characteristics of LEATs, formatted according to the three categories defined by the World Health Organization (WHO): glioneuronal and neuronal tumors (ganglioglioma, dysembryoplastic neuroepithelial tumor, papillary glioneuronal tumor, multinodular and vacuolating neuronal tumor); circumscribed astrocytic gliomas (pilocytic astrocytoma, pleomorphic xanthoastrocytoma); and pediatric-type diffuse low-grade gliomas (diffuse astrocytoma MYB or MYBL1-altered, angiocentric glioma, diffuse low-grade glioma MAPK pathway-altered, polymorphous low-grade neuroepithelial tumor of the young).

Currently, there is limited information available regarding the clinical features, pathological findings, and detailed molecular characteristics of pleomorphic xan-thoastrocytoma (PXA) and epithelioid glioblastoma (eGBM). In this study, we examined 11 PXA cases [9 grade 2 PXA (PXA G2) and 2 grade 3 PXA (PXA G3)] as well as 15 eGBM cases to investigate their histopathological and molecular associations. Morphologically, PXA and eGBM exhibited distinct histological features. However, immunohistochemical analysis revealed no consistent differences between these glioma sub-types, with the exception of the Ki-67 labeling index. BRAF V600E mutation was detected in 60.0% of PXA cases and 85.7% of eGBM cases through immunohistochemistry (IHC) and/or sequencing, with complete concordance between the two methods. Molecular analysis further revealed that TERT promoter (TERT-p) mutation and copy number abnormalities were more prevalent in eGBM than in PXA. In conclusion, PXA and eGBM share similar clinical characteristics but exhibit different histological features. From a molecular perspective, PXA and eGBM belong to the same category and progress through the accumulation of genetic abnormalities, including TERT-p mutations, CDKN2A/B deletions, and TP53 mutations, based on the presence of BRAF mutation; however, larger sample sizes are required for validation.

Mixed tumor comprising atypical teratoid/rhabdoid tumor and pleomorphic xanthoastrocytoma: A rare case with molecular evidence of transformation

Multi-omic landscape of human gliomas from diagnosis to treatment and recurrence.

MTAP and p16 as immunohistochemical surrogates of CDKN2A/B homozygous deletion in central nervous system tumors: A multicentre Italian experience.

BackgroundPleomorphic xanthoastrocytoma (PXA) is a WHO grade 2 or 3 astrocytic tumor typically seen in children and young adults, with generally favorable outcomes. However, cases harboring BRAFV600E mutation, CDKN2A/B homozygous deletion, and TERT promoter mutations may show aggressive behavior with early recurrence and leptomeningeal dissemination. We report a rare case of PXA with all three alterations, showing rapid leptomeningeal spread and pulmonary metastasis.CaseA man in his 40s presented with right-hand sensory disturbance and fine motor dysfunction. MRI revealed a tumor in the left parietal lobe. Awake craniotomy with gross total resection was performed. Histology showed spindle cell fascicles, rhabdoid cell sheets, calcification, and necrosis. Molecular testing revealed BRAFV600E mutation, TERT C228T mutation, and CDKN2A homozygous deletion. Methylation profiling classified the tumor as MC PXA (score 0. 99). The patient received radiotherapy with temozolomide and was discharged. Three months later, he developed neuropathic pain, motor impairment, and dysphagia. CSF cytology and MRI confirmed carcinomatous meningitis. He developed aspiration pneumonia and required ventilatory support. MRI showed multiple infarctions in the left hemisphere and right temporal lobe. He died four months postoperatively. Autopsy revealed BRAFV600E-positive atypical glial cell clusters with vascular invasion in both lungs and pleura, consistent with distant metastases.DiscussionAlthough PXAs are generally indolent, this case exhibited rapid progression with leptomeningeal and systemic dissemination, likely due to the high-risk molecular profile. For such cases, early detection of dissemination and more intensive therapeutic strategies are warranted.ConclusionPXA with BRAFV600E, CDKN2A deletion, and TERT promoter mutation can follow an exceptionally aggressive course. This case underscores the importance of molecular risk stratification and individualized treatment planning.

Abstract BACKGROUND Pleomorphic xanthoastrocytoma (PXA) represents a rare glial brain tumor with variable prognosis. The majority of cases in adulthood harbor BRAFV600E mutation and CDKN2A homozygous deletion, while pediatric PXA remained poorly investigated. METHODS 42 pediatric patients with morphologically verified PXA (28 grade 2 and 14 grade 3) with median age of 9.9 years were investigated. Molecular studied included BRAF codon 600 allele-specific PCR, targeted RNA sequencing, DNA methylation assay. First-line management was conducted as per SIOP-LGG or HIT-HGG protocols depending on tumor grade. Targeted therapy was applied mostly after disease progression and first-line in two cases. Median of follow-up time achieved 4.0 years. RESULTS Molecular genetic markers included BRAF V600E mutation in 25 cases (59.5%), CDKN2A deletion either homo or heterozygous in 23 (54.8%) and rearrangements of receptor tyrosine-kinase (RTK) genes (CCDC88A::ALK, SFPQ::ALK, NOS1AP::NTRK1, TPM3::NTRK1, NACC2::NTRK2, ETV6::NTRK2, VIM::NTRK3, GOPC::ROS1) in 8 patients (19.0%). Remarkably, four patients with RTK fusions aged >10 years did not matched any known DNA methylation class (DKFZ v.12.8) while others were perfectly consistent with MC PXA (>0.90). CDKN2A deletions were uniformly distributed between BRAF-mutated and RTK-fused cases. Patients with PXA grade 2 were observed after surgery and extent of tumor resection predicted survival (p=0.01). Radiation therapy of PXA grade 3 was not able to control the disease: 2-year progression-free survival (PFS) – 31% (95%CI14-70%). Targeted therapy was used in 15 cases (9 PXA grade 3 and 6 PXA grade 2) with combination of dabrafenib and trametinib in 12 and entrectinib in 3. This resulted in 2-year PFS 100% and 50.0%(95%CI27-93%) for PXA grade 2 and 3, respectively. CONCLUSION PXA is a molecularly heterogenic tumor with high frequency of actionable genetic alterations. RTK fusions should be investigated in all BRAF-negative cases. Targeted therapy demonstrated promising results.

Abstract BACKGROUND Consistent data support the efficacy of BRAF and MEK inhibitors in paediatric patients with low-grade gliomas, while data in adults are more limited, and the clinical benefit is to be defined. PATIENTS AND METHODS We retrospectively collected data of adult patients with BRAF-mutant gliomas treated between 2004 and 2024 in our Institution. Twelve patients received dabrafenib plus trametinib at recurrence after standard radiotherapy and chemotherapy. Histo-molecular diagnoses were reviewed according to WHO 2021. Radiological response was re-evaluated according to RANO criteria. RESULTS We identified 12 patients (5 males and 7 females), with age ranging from 21 to 53 years. At baseline, all patients had contrast-enhancing tumours and were neurologically symptomatic. Three out of 12 patients (25%) displayed a complete response: 1 pilocytic astrocytoma (PA), 1 anaplastic pleomorphic xanthoastrocytoma (PXA), and 1 glioblastoma (GBM). Two patients achieved either seizure reduction >50% or seizure freedom. A F-DOPA PET confirmed a metabolic response in the GBM patient. Four out of 12 patients (33%) displayed a partial response (2 PA, 1 ganglioglioma - GG, 1 GBM), together with neurological improvement. Two (1 PXA, 1 GBM) out of 12 patients (17%) displayed a stable disease, with seizure freedom in 1. Three patients (25%) (1 PXA, 2 GBM) displayed an early tumour progression. PFS ranged from 7 to 24 months and OS from 8 to 46 months. Five patients are still on treatment. Adverse effects included headache (7/12) fever (4/12) and cutaneous reactions (3/12), leading to dose reduction in 2 patients. CONCLUSIONS This cohort of recurrent and/or disseminated BRAF-mutant gliomas displayed a high response rate on MRI following dabrafenib plus trametinib (CR+PR 58%), with improvement of seizures (3/8, 37%). More data are needed to assess the long-term duration of response, and to investigate molecular factors influencing the probability of response.

Abstract Li-Fraumeni syndrome increases the risk for multiple cancer types, including glioma. The behavior of gliomas associated with Li-Fraumeni syndrome is not well characterized in the literature. We performed a retrospective review at MD Anderson Cancer Center using the terms “glioma” and “Li-Fraumeni” from January 2013 through January 2025. Patients were followed until June 2025. A total of 25 patients were included. The median age of diagnosis was 25 years and 52% were females. Tumor type included 44% astrocytomas, 32% glioblastoma, and 8% ependymoma. Less common tumors included anaplastic astroblastoma, diffuse high grade glioma histone 3 wildtype and IDH wildtype, and pleomorphic xanthoastrocytoma, each with one patient. In the astrocytoma group, 50% were graded as WHO grade II and 30% as WHO grade III. In the ependymoma group, one patient was graded as WHO grade II and one patient as WHO grade III. In the glioblastoma group, 87.5% were females, compared to 20% in the astrocytoma group. Initial treatment varied by tumor type, and included a combination of resection, radiation, and chemotherapy. Eleven patients had other primary cancer sites, including multiple tumors with the higher frequency of breast cancer (6 patients), thyroid cancer (2 patients), and sarcoma (2 patients). Median overall survival was not reached given that only 7 patients died. Progression free survival after initial diagnosis to first recurrence was 57 months for the entire group, and was 33.8 months for astrocytomas, and not reached for glioblastoma (p=0.925 comparing astrocytomas and glioblastomas). The spectrum of gliomas among Li-Fraumeni syndrome patients is diverse, favoring high-grade glioma as initial presentation in young adults/pediatric patients. However, survival can be longer to what is expected per tumor type.

Abstract Pediatric brain tumors are a leading cause of cancer-related mortality in children, yet many subtypes remain poorly characterized at the cellular, developmental, and molecular levels. To address this gap, we performed a comprehensive single-cell RNA sequencing analysis across a diverse cohort of primary pediatric brain tumors, including ganglioglioma, DNET, pilocytic astrocytoma, pleomorphic xanthoastrocytoma (PXA), oligodendroglioma, diffuse astrocytoma, IDH mutant glioma, IDH wild-type glioblastoma, G34 diffuse hemispheric glioma, ependymoma, and atypical teratoid/rhabdoid tumor (ATRT). Tumor-derived transcriptomes were mapped to our previously published high-resolution reference atlas of the developing human brain to identify cell type signatures, specifically shared progenitor signatures. This revealed widespread enrichment of radial glia–like and early neurogenic populations in aggressive tumor types. PTPRZ1, a canonical radial glia marker, was among the most consistently expressed genes across tumor types. Its diffuse expression was confirmed by immunostaining in patient samples across multiple pediatric tumor pathologies. Notably, tumors with high PTPRZ1 expression, including G34 glioma, high-grade glioma, ATRT, and ependymoma, demonstrated robust enrichment for radial glia–like populations, suggesting a potential developmental dependency on this progenitor program. To functionally model these dependencies, we optimized a human tumor organoid co-culture platform to support the growth of freshly resected pediatric ependymoma tissue. Using single-cell RNA sequencing, we confirmed that this platform preserved the tumor’s cellular heterogeneity, including radial glia-like states. The ability to maintain and study these progenitor populations in vitro creates a powerful system for dissecting pediatric tumor progression and cellular states and allows for testing of therapeutic options. Our findings highlight conserved radial glial signatures as shared developmental programs across aggressive pediatric brain tumors and establish a patient-derived organoid model to interrogate their functional relevance in tumor biology.

Abstract Pediatric gliomas have diverse molecular profiles that can evade detection through conventional DNA-based testing. While DNA profiling is a frontline tool, it may miss oncogenic fusions or expression patterns critical for targeted therapy. RNA sequencing has emerged as a powerful complementary modality, especially for identifying targetable gene fusions that influence treatment decisions. We present two cases where RNA analysis directly impacted clinical management. Cases were selected for the relevance of RNA sequencing to diagnosis and treatment. Clinical course, sequencing modality, therapy, and outcomes were reviewed. In one case, a patient with a hypothalamic pilocytic astrocytoma experienced multiple treatment failures with conventional chemotherapy. DNA testing on two surgical samples did not yield a definitive molecular diagnosis. RNA sequencing, performed over six years after the initial diagnosis, identified the canonical BRAF-KIAA1549 fusion. By then, the patient had already started selumetinib with a positive clinical response. The RNA finding retrospectively validated the treatment and informed future care. In the second case, the patient presented with a glioma exhibiting high-grade histologic features, while DNA methylation profiling aligned most closely with a pleomorphic xanthoastrocytoma. DNA sequencing revealed CDKN2A/B and MTAP co-deletions, adding further diagnostic uncertainty. RNA sequencing identified a KANK1: NTRK2 fusion, likely the oncogenic driver. This guided treatment with larotrectinib, which has been well tolerated and has led to sustained disease control for over two years without recurrence. These cases highlight how RNA sequencing can resolve diagnostic uncertainty, uncover therapeutic targets, and minimize exposure to ineffective therapies. RNA-based fusion detection is particularly valuable in tumors that mimic known subtypes but lack canonical mutations. Early integration of RNA testing alongside DNA profiling should be standard in pediatric neuro-oncology workflows to expand therapeutic options and enhance treatment precision. Larger cohort studies are underway to validate these findings and optimize RNA-driven decision-making in pediatric CNS tumors.

Abstract BACKGROUND Maturation is an enigmatic property of neurogenic tumors, including neuroblastoma and brain tumors in the unique cases. Multiple factors (TrkA-signaling, microenvironment, telomerase and epigenetics) have been linked to the tumor maturation. METHODS We analyzed four cases of pediatric CNS tumors (medulloblastoma SHH-activated; ETMR; unspecified CNS embryonal tumor (ET-CNS); thalamic high-grade glioma, HGG) having undergone a morphologically verified maturation to ganglioglioma, pleomorphic xanthoastrocytoma or diffuse low-grade glioma in parallel with 23 cases of maturating extracranial neuroblastoma. The paired tumor samples were analyzed using targeted NGS and gene expression profiling by NanoString. RESULTS The NanoString experiment revealed an identical shift of gene expression profile, all investigated tumors after maturation clustered together regardless of initial tumor type. MAPK (FDR=1.54×10-14), PI3K-AKT-mTOR (FDR=6.78×10-13) and TrkA signaling (FDR=2.72×10-11), cellular senescence (FDR=3.90×10-9) were predicted to be activated, while cell cycle control, DNA replication and repair (FDR respectively 1.53×10-14, 2.04×10-14 and 3.60×10-13) - repressed. Moreover, tumors completing the maturation similarly showed enhanced immunogenicity, mostly pronounced in MB and ETMR. Maturing cases of neurogenic tumors were enriched in genetic aberrations leading to MAPK cascade activation: mutations in BRAF (n=5), PTEN,PTPN11,HRAS or NF1, and a KCTD16::NTRK2 rearrangement (in neuroblastoma, ET-CNS and HGG, respectively). Genes encoding neurothophin receptors, epigenetic writers, erasers, and Kruppel-family transcription factors had similar dynamics of expression upon maturation in both CNS malignancies and neuroblastoma. NTRK1 expression was elevated at debut, whereas NTRK2 increased during maturation. Expression of negative epigenetic regulators HDAC2,HDAC10,DNMT3A and EZH2 similarly decreased in maturing tumors, while positive (SWI/SNF subunits) increased at the beginning of maturation and decreased upon the differentiation completion. KLF4, GLI1/3 expression significantly increased in the matured tumors. CONCLUSION Central and peripheral neurogenic tumors share common mechanisms of maturation, which include maintenance of MAPK signaling via neurotrophins and consistent changes in expression of epigenetic modifiers and KLF-like transcription factors.

Abstract Leptomeningeal disease (LMD) is a rare metastatic complication with a grim prognosis for most patients and limited treatment strategies. Therapy is adjusted to the primary tumor from which it arises. Targeted therapies and personalized medicine have become cornerstones in cancer treatment but its utility in LMD has been limited. In here we report a case of a female patient who developed LMD from a Pleomorphic Xanthoastrocytoma (PXA), BRAFV600-mutated, who has shown successful response to treatment with BRAF/MEKi (Encorafinib/Binimetinib) for almost four years since initial LMD diagnosis. The effectiveness of therapy in this patient was initially observed as stable disease, with radiographic progression if BRAF/MEKi were withheld, and immediate control achieved when reinstated. Despite being just one case, this hopefully could serve as proof-of-concept for use of targeted therapy for BRAF V600E-mutated tumors with LMD progression. Patient is well up to date and to our knowledge, this is the first case reported in the literature of a PXA with LMD with successful response to targeted therapy.

Abstract BACKGROUND Tovorafenib is a type II rapidly accelerated fibrosarcoma (RAF) kinase inhibitor approved by the FDA in 2024 for relapsing or chemotherapy-refractory pediatric low-grade gliomas with mutations or alterations in the gene BRAF. There is potential for the use of this drug outside the pediatric population. We present a single institution case series of adults with CNS tumors with mutations or alterations in BRAF treated (off-label) with tovorafenib. METHODS Chart review. RESULTS 8 adult (age >18) patients were treated with tovorafenib at our institution between March 2024 and June 2025. Age range was 21-66 years old (median age 42). The population included 5 males and 3 females. Indications for treatment were pilocytic astrocytoma (n = 2), high grade astrocytoma with piloid features (HGAP) (n = 2), Erdheim-Chester disease (n = 2), pleomorphic xanthoastrocytoma (n = 1), and otherwise unclassified low-grade glioma (n = 1). All patients had mutations or alterations in BRAF including KIAA1549 fusion (n = 4), V600E mutation (n = 3), or GAB2 fusion (n = 1). Radiographic response to therapy was seen in 5/8 patients with 3 cases of radiographic stability (though one patient later progressed) and 2 cases of decrease in tumor size (in a pilocytic astrocytoma and in an HGAP). Both cases of decreased tumor size after treatment were in tumors with KIAA1549 fusions. Adverse effects of tovorafenib included fatigue (n=2), headache, myalgias, diplopia, cytopenias, and transaminitis (n=1). 3/8 patients had to pause or stop treatment due to adverse effects (intolerable headache, cytopenias, and transaminitis respectively). CONCLUSION Tovorafenib is a promising new treatment for CNS tumors with BRAF mutations or alterations. Our single institution case series suggests the efficacy of this drug for multiple tumor types in adults. Certain tumor subtypes such as KIAA1549 fusions may be more likely to benefit from this treatment.

Abstract INTRODUCTION With regard to superior long-term disease control, anterior temporal lobectomies (ATL) as a supra-total resection regime have gained growing attention in surgery for temporal lobe tumors. However, aggressive temporal tumor resection might be accompanied by an impairment of a patient’s neurocognitive functioning. We therefore analyzed our database with regard to postoperative changes in neurocognitive functioning following resection of temporal-located low-grade glioma depending on the extent of tumor resection. METHODS Between 1999 and 2018, 33 patients were surgically treated for temporal-located lower-grade gliomas via ATL or lesionectomy in terms of gross-total resection (GTR) at the authors’ institution. Histopathological analysis identified 15 WHO grade I pilocytic astrocytomas, 6 grade II pleomorphic xanthoastrocytomas, 7 grade II diffuse astrocytomas, 2 polymorphous low-grade neuroepithelial tumors of the young (PLNTY), 1 ganglioglioma, and 2 astrocytomas not otherwise specified. Seventeen of these tumors were reclassified according to the 2021 WHO classification of CNS tumors. Neuropsychological examination included attention, visuoconstruction, language as well as verbal and figural memory functions. All patients underwent testing preoperatively and approximately one year postoperatively. The analyses were conducted both at the individual level and at the group level, comparing preoperative and postoperative data. RESULTS Fourteen patients (42%) underwent ATL, and 19 patients (58%) received GTR. Depending on the domain, up to 70% of patients exhibited preoperative deficits, particularly in verbal and figural memory as well as language, with varying degrees of severity (verbal/figural memory: 69.7%/72.7%). Preoperative performance did not differ between ATL and GTR group. Postoperatively, about 30% showed measurable decline, mainly in memory tasks (verbal/figural memory: 31.2%/37.5%), while more patients demonstrated stability (34.4%/25%) or improvement (34.4%/37.5%). No significant differences were observed between the GTR and ATL groups at either the individual or group level. CONCLUSIONS ATL and GTR, as differing oncosurgical approaches, did not show significant differences in postoperative neurocognitive impairment rates. ATL, as a supra-total resection strategy, thus represents a safe surgical option for temporally located lower-grade gliomas.

Abstract BACKGROUND AND AIMS Molecular diagnosis of an individual’s brain cancer is essential to guide precision therapies. NHS diagnostic turn-around-time takes weeks to months following surgery. A new sequencing technology from Oxford Nanopore Technologies, coupled with a research-based diagnostic tool ROBIN (doi.org/10.1093/neuonc/noaf103) can provide methylation-based diagnosis within hours, and molecular variant identification within 1-2 days. We independently replicate feasibility of using this diagnostic assay within the NHS, supported by a University of Birmingham-Queen Elizabeth Hospital Birmingham collaboration, and Brain Tumour Charity funding. METHOD Patients undergoing surgery with NHS whole genome sequencing for radiological presumed glioma (01/Jan/2025-01/Jun/2025) were consented for genomic research. Tumour DNA was extracted in the pathology department and NHS staff were trained by university scientists in long read sequencing using PromethION flow cells and ROBIN software. Concordance of the ROBIN methylation classifiers and glioma methylation status is reported. RESULTS 20 patients were recruited: median age 58.5 years (interquartile range 38-79); male gender 60 %, ethnicity self-reported as White (70), Asian (25%), Black/Caribbean or African (5%). The integrated histopathology-molecular diagnosis confirmed Glioblastoma IDH-wild type (13, 65%), Astrocytoma IDH-mutant (3, 15%), Diffuse hemispheric glioma (H3G34-mutant; 1, 5%), Anaplastic Pleomorphic Xanthoastrocytoma (1, 5%) Metastasis (Melanoma, Oesophageal; 2, 10%). Classifier performance demonstrated concordant diagnosis in 70 % of cases. Misclassified samples included: tumours that were outside of the classifier remit (1 oesophageal metastasis), low tumour content in recurrent glioma (1). The classifier reported inflammatory tissue in 3 high grade glioma cases with satisfactory tissue content but high necrosis content. Of 13 glioma cases with completed NHS methylation status, 9 (70%) were concordant and 2 (15%) failed. CONCLUSION This work demonstrates good concordance between a rapid diagnostic assay and current standard of care pathology diagnostics within the clinical setting. A close working partnership between surgeons, pathologists, and scientists is essential. Access to larger, more diverse samples will help refine these classifiers over time to further improve real-world accuracy and generalisability.

Pleomorphic xanthoastrocytoma with brain abscess: A case report

Published literature on epidemiological profile of paediatric brain tumours in Zambia is scarce.AimTo present a retrospective analysis of the histological spectrum of 36 paediatric age group central nervous system tumours operated in a single tertiary care hospital in Lusaka, Zambia between June 2021 and December 2024.Methods and MaterialsRetrospective analysis of the data regarding frequencies of various primary brain tumours among 36 paediatric patients (<18 years of age). The tumours were categorised according to the revised 4th edition of World Health Organization (WHO) classification of tumours of the Central Nervous system.ResultsPaediatric CNS constituted 29.2% of total intracranial tumours (35/120) operated in the study period. The mean age of the patients was 10 years, and a male predominance was noted (1.2:1). Posterior fossa tumors (24/35; 68.6%) were more common than supratentorial tumors. (12/35; 34.3%) Of the posterior fossa tumors majority were the pilocytic astrocytoma tumors (15/24; 62.5%), followed by medulloblastomas (9/24; 37.5%). Of the supratentorial tumors 6 pediatric type-high grade gliomas (17.1%) were recorded, 1 pleomorphic xanthoastrocytoma (2.9%) and 1 subependymal giant cell astrocytoma (2.9%) Our series also included 3 meningiomas (3/35; 8.6%) and one germ cell tumor (1/35; 2.9%).ConclusionsPaediatric central nervous system tumours are more common in boys and in the second decade of life. Astrocytomas are the most common paediatric brain tumours followed by medulloblastomas. Pediatric tumours affect the infratentorial compartment more often than the supratentorial compartment. The profile of paediatric brain tumours in our series is similar to that reported from other countries in sub-Saharan Africa as well as most western literature.

Abstract BACKGROUND High-grade gliomas (HGGs) are among the most aggressive CNS tumors in pediatric, adolescent, and young adult (AYA) populations, marked by rapid progression and poor survival. These tumors show considerable histopathological and molecular heterogeneity, complicating diagnosis and treatment. Although WHO CNS5 distinguishes adult and pediatric HGGs, the pediatric and AYA subgroup remains a heterogeneous group with overlapping features and considerable molecular variability. MATERIAL AND METHODS A total of 61 pediatric and AYA cases of diffuse HGG, CNS WHO Grade-4, NOS (median age: 19 years; M: F = 1.6: 1), were included in the study. Whole-genome DNA methylation profiling was performed for all cases along with eight controls and classified on the online available Epignostix brain tumor classifier (v12.8), and Methylscape. Additionally, next-generation sequencing (NGS) and RNA fusion analysis were conducted where appropriate, based on insights from methylation-based classification. RESULTS Tumors initially labeled as diffuse HGG, CNS WHO Grade 4, NOS were reclassified into distinct methylation classes. Pediatric HGGs, H3-WT/IDH-WT accounted for 20% (12/61), with 42% (5/12) showing H3K27me3 loss, deviating from WHO criteria. RTK1 subtype was enriched in MMR-deficient and radiation-induced gliomas. Glioblastoma, IDH-wildtype comprised 18% (11/61), including 45% (5/11) pediatric cases. Adult-type glioblastoma, subtype B (5%, 3/61) showed MAPK pathway alterations of therapeutic relevance. Diffuse hemispheric glioma, H3K27M-altered was identified in 6.5% (4/61) of cases, including one located in the temporal lobe—challenging conventional anatomy-based classification. Pleomorphic xanthoastrocytoma (PXA) was seen in 8.2% (5/61), all of which had poor survival outcomes. Other reclassified entities included DHG, H3G34-mutant (n=2), pilocytic astrocytoma (n=3), HGAP (n=1), MPNST (n=2), CNS-BCOR ITD (n=1), neuroepithelial tumor with PATZ1-fusion (n=1), and DLGNT (n=1). Methylation profiling refined diagnoses in 48%, changed them in 16%, confirmed in 15%, while 12% were misdiagnosed and 10% remained unclassified. CONCLUSION Methylation profiling revealed substantial molecular diversity in pediatric and AYA diffuse HGGs, enabling refined classification and identification of rare, clinically relevant subtypes. Deviations from WHO criteria highlight the need to update diagnostic frameworks to better capture subgroup variability. Expanding classifier datasets to include diagnostically challenging cases may enhance prognostic accuracy and clinical relevance. This comprehensive approach advances molecular understanding, diagnostic precision, and better informs targeted therapeutic strategies.

Abstract BACKGROUND The association between pediatric high-grade gliomas (HGG) and cancer predisposition syndromes (CPS) remains inadequately elucidated in the existing literature, while next-generation sequencing (NGS) is increasingly utilized for diagnostic support MATERIAL AND METHODS In this retrospective analysis, we evaluated a cohort of 95 consecutive pediatric patients (48 males and 47 females), with a median age at tumor diagnosis of 10.4 year, with HGG reclassified according to the latest 2021 WHO classification, diagnosed and treated at the Bambino Gesù Children’s Hospital between 2011 and 2023. All patients underwent genetic germline testing with NGS technology to detect the presence of germline variants predisposing to cancer and were included in this study. RESULTS The cohort included 23 patients (24.2%) diagnosed with pHGG H3-IDH wild-type, 46 (48.4%) with diffuse midline glioma (DMG), and 7 (7.4%) with IDH-mutant astrocytoma. Anaplastic pleomorphic xanthoastrocytoma and adult-type HGG were each identified in 4 patients (4.2%), while neuroepithelial tumor with PATZ1 fusion was found in 3 patients (3.2%). HGG H3 G34-mutant and infant-type hemispheric glioma were each diagnosed in 2 patients (2.1%). Additionally, 4 patients (4.2%) had other rare histologies. Oncological family history was positive in 43/95 (45.3%) patients. Our analysis identified 80 variants across the 95 patients, categorized as follows: 7 pathogenic (P) variants, 12 likely pathogenic (LP) variants and 61 variants of uncertain significance (VUS). Notably, 23.7% of the P/LP variants were found in genes associated with CPS. While the distribution of these variants did not show significant differences across tumor subtypes, the highest proportion of pathogenic variants was observed in DMG. In addition, functional studies led to the reclassification of one LZTR1 variant from VUS to P, highlighting the importance of functional analysis in better interpreting variants initially classified as uncertain. CONCLUSION Notably, our results revealed that 18.9% of patients had P/LP variants, a rate higher than the 10% incidence typically reported in the literature. The high rate of mutations in CPS associated genes described in our work, leads to consider performing NGS even in the absence of clinical or anamnestic criteria suggestive of it. Moreover, further research are needed to determine the role of variants of uncertain significance