Challenging anatomical paradigms: unexpected supratentorial localization of posterior fossa B ependymomas identified by DNA methylation

Abstract

Molecular subgroups of ependymomas are strongly associated with their primary anatomical compartments, namely the supratentorial region, posterior fossa and spine locations. Although it is generally accepted that posterior fossa ependymomas may arise from radial glia-like progenitor cells, the precise cellular origin of the distinct posterior fossa molecular subgroups remains to be determined. Given that DNA methylation profile strongly recapitulates developmental lineage and the cell of origin, the characterization of unexpectedly located molecular subgroups of ependymomas may provide insights into their cells of origin and potential migratory dynamics during embryogenesis and brain development. Herein, we describe two male school-aged pediatric patients with supratentorial tumors located in the third ventricle/pineal region that were classified as posterior fossa group B ependymomas based on DNA methylation profiling. To our knowledge, this represents the first reports of posterior fossa group B ependymomas identified outside the posterior fossa compartment.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s40478-026-02366-z.

Introduction

During the last decade, the study of cancer epigenetics has reshaped the diagnosis and treatment approach of central nervous system tumors. The World Health Organization (WHO) 2021 Classification of Tumors of the Central Nervous System (CNS) recognizes nine molecular subtypes of ependymal tumors that are primarily stratified by their anatomical location (posterior fossa, supratentorial and spine). Around 90% of ependymomas (EPN) occur intracranially, of which 60% are located in the posterior fossa, including the ependymoma group B (PF-EPN-B) [1]. This class of EPN usually has favorable outcomes and occurs in adolescents and adults (age range from 10 to 65 years) with a slight prevalence in females [2]. They primarily arise from the floor of the fourth ventricle or the lateral recesses (Foramen of Luschka), present uniform small cells arranged in perivascular pseudorosettes by histology, and generally retain H3K27me3 immunohistochemical expression. Mitotic activity and microvascular proliferation can be used to distinguish between grades 2 and 3 [1].

PF-EPN-B is characterized by chromosomal instability and aneuploidy [3] resulting in high heterogeneity between tumors [2]. There are no known oncogenic drivers and the most common genomic alterations include chr6 loss (63%), chr18 gain (52%), 22q loss (48%) and other rare alterations [2, 3].

Genome-wide DNA methylation profiling allows for the capture of signatures of cellular lineages and their origin, overcoming the limitations of histopathology itself which strongly relied on histomorphology and immunohistochemical markers [4, 5]. Furthermore, conventional neuroimaging modalities are constrained by current anatomical structures and remain insensitive to the dynamic migratory patterns of progenitor cells during embryogenesis. Knowingly, the location of medulloblastoma subtypes is related to the cells of origin during embryogenesis. While medulloblastoma subtypes WNT and Sonic Hedgehog (SHH) derive from lower rhombic lip/dorsal brainstem and upper rhombic lip/early cerebellum, respectively [6], the transcriptional and methylation profiles of medulloblastomas groups 3/4 mirror a continuum of the transcriptome of cell types from early fetal cerebellar development [7].

Given the high molecular heterogeneity of ependymal tumors - identified by methylation patterns and further characterized by genomic alterations, gene expression, location, demographics and outcomes [2], we herein raise the possibility that ependymomas arise from progenitor cells at different stages of lineage commitment, by reporting two definitive PF-EPN-B DNA methylation profiles located in the third ventricle / pineal region, challenging the strict anatomical-molecular relation of posterior-fossa ependymal subgroups [3].

Case presentation

CASE #1: An 8-year-old boy was admitted to the pediatric emergency department with a 1-week history of gait ataxia, urinary incontinence, headache, and vomiting. Initial laboratory investigations were unremarkable, while head computed tomography (CT) revealed a supratentorial mass associated with raised intracranial pressure, prompting neurosurgical evaluation. A right frontal external ventricular drain (EVD) was placed, and intravenous dexamethasone was initiated in the pediatric intensive care unit (PICU). Given the initial diagnostic suspicion of craniopharyngioma, a comprehensive pituitary hormonal axis evaluation was performed and yielded normal results. Brain magnetic resonance imaging (MRI) revealed a highly cellular neoplastic mass filling the third ventricle (Fig. 1A and C). Spinal MRI showed no evidence of tumor implants or dissemination. The patient underwent an endoscopic biopsy with ventriculoperitoneal shunt (VPS) placement, with insufficient tissue for a definitive histopathological diagnosis. The patient then underwent a left frontal transcallosal approach to the third ventricle neoplasm, achieving a subtotal resection. The anatomopathological examination revealed a neuroepithelial neoplasm composed of monomorphic cells, predominantly arranged in a solid growth pattern, with round to oval nuclei, delicate chromatin, and inconspicuous nucleoli. The cytoplasm was abundant, eosinophilic, and ill-defined, with occasional cells showing eccentric nuclei and columnar morphology. Perivascular pseudorosettes and rare papillary structures were observed (Fig. 1D). Immunohistochemistry demonstrated focal positivity for AE1/AE3, strong and diffuse positivity for S100 and partial positivity for GFAP (Fig. 1E), along with a dot-like EMA pattern in rare cells (Fig. 1F). Neurofilament, MAP2, and OLIG2 were negative, with retained INI-1 expression. Trimethylation at lysine 27 of histone H3 (H3K27me3) was also retained (Fig. 1G). The proliferative index (Ki-67) was low (around 4.5%). A provisional diagnosis of papillary tumor of the pineal region (PTPR), WHO grade 2, was suggested; however, due to the unusual features of the case, the pathologist recommended tumor DNA methylation profiling. To aid in the differential diagnosis, an in-house PCR-based panel targeting BRAF V600E, KIAA1549::BRAF fusions, and two FGFR1 hotspot mutations (N546 and K656) was performed, with all results being negative. Methylation profiling ultimately classified the tumor as a posterior fossa B ependymoma (class mean score of 0.906 in the Bethesda V2 classifier and calibrate score of 0.9956 in the Heidelberg v12.8 classifier). The Uniform Manifold Approximation and Projection (UMAP) grouped this case within the PFB ependymoma group (Fig. 1H). The tumor also had copy number alterations (CNAs) recurrently found in PF-EPN-B: loss of chr6 and 22q and a gain of chr18 in addition to other CNAs (Supplementary Fig. 1A). The patient subsequently received volumetric modulated arc (VMAT) radiotherapy with daily image-guided radiotherapy (IGRT) to the residual CNS lesion, delivering a total dose of 30 fractions of 180 cGy. Post-treatment surveillance with serial brain MRI was initiated to monitor residual disease.

Fig. 1.

A Axial, B Coronal and C Sagittal Post-gadolinium T1-weighted images demonstrating a lesion confined to the third ventricle, showing a marked contrast enhancement. D Photomicrograph of hematoxylin and eosin (200×). Photomicrograph of immunohistochemistry of E S100 (strong and diffuse) (400×), F EMA (dot-like pattern) (400×), G H3 K27me3 (strong) (400×). H UMAP analysis showing clustering of case #1 (labeled as TU2233) within the PFB ependymoma subgroup. I Axial, J Coronal and K Sagittal Post-gadolinium T1-weighted images demonstrating the tumor arising at the third ventricle/pineal region, with marked contrast enhancement. L Photomicrograph (hematoxylin and eosin, 200×) - Round cells without atypia arranged in perivascular pseudorosettes. M Photomicrograph (immunohistochemistry, S100 protein, 200×) - The neoplastic cells show diffuse positivity. N Photomicrograph (immunohistochemistry, GFAP, 400×) - The neoplastic cells show diffuse positivity. O Photomicrograph (immunohistochemistry, EMA, 400×) - The neoplastic cells are positive, exhibiting a paranuclear dot-like staining pattern. P The UMAP analysis showing clustering of case #2 (identified in the figure as mnpACC_128) within the PFB ependymoma subgroup

CASE #2: A 5-year-old male presented with worsening somnolence and ataxia that had started 2 years before admission. CNS Neuroimaging revealed hydrocephalus and a pineal/third ventricular mass (Fig. 1I and K), and a VPS was placed. Histopathological and immunohistochemical analyses initially suggested a high-grade neuroepithelial tumor demonstrating perivascular pseudorosettes positive for GFAP, paranuclear dot-like staining for EMA and positivity to S100 (Fig. 1L and O). H3K27me3 expression was retained in tumor cells. Complementary tumor DNA methylation profiling identified an epigenetic profile similar to posterior fossa ependymomas, type B (class mean score of 0.18 in the Bethesda V2 classifier and calibrate score of 0.68 in the Heidelberg v12.8 classifier). The degraded nature of FFPE sample-derived DNA typically lowers the prediction confidence score even despite accurate biological classification. Nevertheless, on UMAP dimensionality reduction analysis, the tumor clustered within the molecular landscape PFB ependymomas further supporting its classification (Fig. 1P). The tumor also had copy number alterations (CNAs) recurrently found in PF-EPN-B: loss of chr6 and 22q and a gain of chr18 in addition to other CNAs, some also described in PF-EPN-B, such as monosomy of chromosomes 6, 10 and 17 as well as trisomy of chromosomes 5 and 8 (Supplementary Fig. 1B). Three months following surgery, imaging demonstrated tumor progression with subependymal dissemination; biopsy confirmed recurrent ependymoma. He received induction and maintenance chemotherapy, complicated by severe recurrent grade 4 myelotoxicity and a chronic subdural hematoma requiring surgical drainage. Eight months later, new intraventricular lesions were identified, and second-line therapy with temozolomide and irinotecan was initiated, achieving a partial radiological response but with persistent hematologic toxicity. At 8 months after the start of maintenance therapy treatment was discontinued due to further myelosuppression and radiological progression accompanied by seizures. The latest imaging studies demonstrated multifocal intraventricular disease progression without surgical options. After multidisciplinary discussion, the disease was deemed refractory with no curative therapeutic alternatives, and the patient was transitioned to palliative care.

Discussion and conclusions

This study describes for the first time the identification of two molecularly-defined posterior fossa group B ependymomas in the unexpected third ventricle/pineal regions, stressing that the description of new subgroups or atypical presentations remains dependent on the reporting of ultra-rare tumor cases. In this context, low- and middle-income countries (LMIC) may play a key role in this scenario due to the high burden of pediatric cancer cases and their underrepresentation in public databases [8, 9].

Epigenetic classification has shown a strong correlation between molecular subgroups and anatomical sites in several CNS entities. In the particular case of EPN, this relationship is well established. Posterior fossa EPN are reproducibly segregated into three main molecular subgroups defined by methylation: posterior fossa ependymoma group A (PF-EPN-A), PF-EPN-B and posterior fossa subependymoma (PF-SE), which patients show marked differences in age. Traditionally, their classification was based on histopathological criteria, with, however, limited ability to predict clinical behavior, therapeutic response, and prognosis [10]. Methylation profile of PF-EPN-B has already suggested the existence of three or five subgroups [11, 12].

These advances in molecular characterization have revealed that EPN are biologically heterogeneous, leading to a reformulation of their diagnostic classification [10]. Transcriptomic analyses show that the posterior fossa EPN presents a gene expression profile similar to the embryonic radial glia, considered a neural progenitor cell [13,14]. In addition, gene expression patterns and copy number alterations correlate with the anatomical location of the tumour (supratentorial region, posterior fossa or spine) despite they share a consistent and similar morphology [15].

Radial glial cells located in different areas of the central nervous system are thought to acquire distinct genetic abnormalities that promote cellular transformation and underlie ependymoma development [14]. The radial glial cells come from neuroepithelial cells polarised along their apical–basal axis as a layer of pseudostratified epithelium that lines the neural plate and neural tube [16]. The radial glial cells generate intermediate progenitor cells and oligodendrocyte precursor cells, which in turn produce neurons and oligodendrocytes, respectively. They also produce intermediate progenitors that expand in number before producing astrocytes and ependymal cells [17]. These changes in morphology come with the modulation of gene expression pointing to the existence of subpopulations, specific to developmental stages and of nervous system regions [18]. Accordingly, expression of gene regulators of neural precursor cells are different according to the region of the CNS where they are located, which is recapitulated by the EPN [15].

Ependymomas with discordant anatomical and molecular features have been previously reported. Notably, a subset of tumors harboring ZFTA fusion, which are typically associated with the supratentorial location, has been identified in the posterior fossa [11, 19, 20]. These observations support the concept that methylation class reflects the cell of origin, which may occur in atypical anatomical sites in rare cases, revealing the biological complexity of these tumors and challenging the conventional practice of incorporating anatomical location into tumor nomenclature. As a result, the recently published research letter by the Consortium to Inform Molecular and Practical Approaches to Central Nervous System Tumor Taxonomy (cIMPACT-NOW) update 11 discusses the possibility of removing tumor primary location supratentorial for ZFTA-fused EPN in the next edition of the WHO CNS classification [21].

Given that ependymal tumors originate from radial glial cells, the unusual anatomical location of these tumors may highlight the biological complexity and spatial heterogeneity of their cells of origin. Of note, the radial glial cells are also precursors of the ventricular zone and pineal gland. In the 3rd ventricle, they generate the tanycytes and remain in the wall throughout adulthood [22]. In rats, the evagination of the dorsal diencephalon that gives rise to the pineal gland is populated by radially aligned precursor cells expressing Pax6 and vimentin that give rise to pinealocytes [23]. Whether the methylation classification of both tumors herein described as posterior fossa ependymoma group B might reflect the epigenetic signature of the primary progenitor cells or their precise mislocated anatomical signature remains to be explored.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

cGy

Centigray

chr

Chromosome

cIMPACT-NOW

Consortium to Inform Molecular and Practical Approaches to Central Nervous System Tumor Taxonomy

CNS

Central Nervous System

CT

Computed tomography

EVD

External ventricular drain

H3K27me3

Trimethylation of histone H3 at lysine 27

IDH

Isocitrate dehydrogenase

IGRT

Image-guided radiotherapy

LMIC

Low- and middle-income countries

MRI

Magnetic resonance imaging

PF

Posterior fossa

PFB

Ependymoma Group B

PF-EPN-A

Posterior fossa ependymoma group A

PF-EPN-B

Posterior fossa ependymoma group B

PICU

Pediatric intensive care unit

q or p

long or short arm of the chromosome

SHH

Sonic Hedgehog

UMAP

Uniform Manifold Approximation and Projection

VMAT

Volumetric modulated arc

VPS

ventriculoperitoneal shun

WHO

World Health Organization

WNT

Wingless and int-1

Author contributions

FDAC (Conceptualization; Methodology; Data Curation; Visualization; Writing–Original Draft; Writing–Review & Editing; Supervision); MM (Methodology; Data Curation; Visualization; Writing–Original Draft; Writing–Review & Editing; Supervision); MCS (Data Curation; Visualization; Writing–Original Draft; Writing–Review & Editing); CERF (Conceptualization; Methodology; Data Curation; Visualization; Writing–Original Draft; Writing–Review & Editing) JVAC (Conceptualization; Methodology; Data Curation; Visualization; Writing–Original Draft; Writing–Review & Editing); PPL (Conceptualization; Methodology; Data Curation; Visualization; Writing–Original Draft; Writing–Review & Editing); MSVD (Data Curation; Visualization; Writing–Original Draft; Writing–Review & Editing); JGRC (Data Curation; Visualization; Writing–Original Draft; Writing–Review & Editing); ACS (Visualization; Writing–Original Draft; Writing–Review & Editing); RSO (Writing–Original Draft; Writing–Review & Editing) ; MVS (Writing–Original Draft; Writing–Review & Editing); DSLF (Writing–Original Draft; Writing–Review & Editing); CAS (Writing–Original Draft; Writing–Review & Editing); ETV (Conceptualization; Methodology; Data Curation; Visualization; Writing–Original Draft; Writing–Review & Editing; Supervision). All authors made substantial contributions to the intellectual content of this study and have read and approved the final version of the manuscript.

Funding

Teaching, Research and Healthcare Support Foundation of the Hospital das Clínicas, Ribeirão Preto Medical School, University of São Paulo (FAEPA), Brazilian Ministry of Health – MoH (Programa Nacional de Genômica e Saúde de Precisão - Genomas Brasil) and National Council for Scientific and Technological Development – CNPq 406716/2022-6.

Data availability

All relevant materials are included in the article. Additional material that is not publicly available is available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Ethics approval and consent to participate: This study was approved by the Ethics Committee of the Hospital das Clínicas, Ribeirão Preto Medical School, University of São Paulo (approval number: 96342526.0.0000.5440), and by the Ethics Committee of Fundação Antônio Prudente/A.C.Camargo Cancer Center (approval number: 96171226.7.0000.5432).

Consent for publication

Written informed consent was obtained from the parents of case #1. For case #2, the requirement for informed consent was waived by the Institutional Review Board of AC Camargo Cancer Center due to the retrospective nature of this case report and because the patient was deceased at the time of data collection.

Competing interests

The authors declare no competing interests.