AOSNP‐ADAPTR resource level‐based recommendations on practical diagnostic strategies for ependymomas

Abstract

Ependymomas are uncommon primary tumors of the central nervous system (CNS) that affect both children and adults. In recent years, molecular analyses have identified multiple subtypes of ependymal tumors based on location, patterns of CpG island methylation, and gene fusion events. While this knowledge is highly important in progressing diagnostic precision, prognostication, and effective treatments, an unwanted side effect is that best practice diagnostics are often inaccessible because of cost and technical barriers for many low‐ or lower middle‐income countries. The ADAPTR group (Adapting Diagnostic Approaches for Practical Taxonomy in Resource‐Restrained Regions) is an initiative of the Asian Oceanian Society of Neuropathology and aims to provide practical guidelines for CNS tumor diagnostics at various levels of pathology resource restrictions. In this manuscript, we propose a practical approach to the diagnosis of ependymal tumors based on ADAPTR resource levels. These recommendations provide a practical adaptation of World Health Organisation Classification of Tumours of the Central Nervous System guidelines suited to routine diagnosis in resource‐restrained regions.

Adapting Diagnostic Approaches for Practical Taxonomy in Resource‐Restrained Regions (ADAPTR) recommendations for ependymomas in resource‐restrained settings (RL = resource level; created in BioRender).

1. INTRODUCTION

Solid tumor classification increasingly relies on integration of complex molecular information with classical histological assessments. This requirement is most prevalent and longstanding in central nervous system (CNS) tumors, where integration of molecular features in some tumor types has been mandated for over a decade [1]. The grading of ependymoma has historically suffered from poor reproducibility and clinical utility, with a reliance on histological grading parameters [2]. Since the publication of the 2021 WHO Classification of CNS tumors [3], the histological subtyping and grading of ependymoma has been improved by the introduction of molecular subclassification, with a schema based on anatomical location, genomic alterations, and methylation profiling [4, 5]. Within this schema, the definition of some ependymal tumor subtypes has moved to one based solely on complex molecular profiles—for example, posterior fossa group B (PFB) ependymomas are now defined by their methylation profile [3]. While the improved precision in both diagnosis and prognosis is much needed, delivering the necessary molecular testing methodologies to low‐ and lower middle‐income countries (LICs and LMICs) incurs costs that are prohibitive for routine use [4, 6].

The AOSNP‐ADAPTR initiative (Asian Oceanian Society of Neuropathology recommendations for Adapting Diagnostic Approaches for Practical Taxonomy in Resource‐Restrained Regions) proposes practical, resource‐stratified pathways that emphasize morphology and immunohistochemistry (IHC) in CNS tumor diagnostics, with recommended targeted molecular tests when essential or highly informative [7, 8]. The AOSNP‐ADAPTR recommendations are a practical adaptation of the World Health Organization Classification of Tumors of the Central Nervous System 5th edition [3], intended to suit routine diagnostic practices in resource‐limited settings without altering established diagnostic categories [6, 7]. As previously reported by our group and others, LICs and LMICs face significant barriers to the implementation of molecular techniques required for the diagnosis of many (CNS) tumors [6, 7, 8, 9].

The classic morphologic features of ependymal tumors are well described, with the current World Health Organisation Classification of Tumours of the Central Nervous System (WHO CNS5) diagnosis of some tumor types possible on morphology alone (e.g., subependymoma) [3]. However, the accurate diagnosis of other ependymal tumors necessitates advanced molecular testing. When genetic testing is not feasible or does not categorize the tumor in the current WHO Classification [6], then the designation of NOS (not otherwise specified) or NEC (not elsewhere classified), respectively, is assigned to the diagnosis as per current WHO guidelines [10].

In this paper, the AOSNP‐ADAPTR Ependymoma Working Group outlines a histology‐oriented, resource‐level approach for ependymoma diagnosis that leverages surrogate immunohistochemical markers and clinical–radiological correlation, and escalates to molecular testing when it is essential for WHO CNS5 entities and may alter management. Referral pathways to higher‐resource centers are suggested or recommended in specific circumstances.

Resource levels (RLs) have been previously defined [6, 8] and are reproduced in Supporting Information S1. Imaging, morphology, and relevant surrogate immunohistochemical markers for ependymal tumors are detailed below, as well as ADAPTR recommendations based on local RLs with diagnostic flowcharts.

2. CHALLENGES IN DIAGNOSIS AND MANAGEMENT OF EPENDYMOMAS IN RESOURCE‐RESTRAINED SETTINGS

The diagnostic classification of an ependymoma is dictated by its anatomical location, with each site typified by different molecular profiles requiring different molecular testing methodologies [3]. For example, supratentorial (ST) ependymoma requires gene fusion testing, whereas methylation profiling is an essential diagnostic criterion for PFB ependymoma [3]. Therefore, it was difficult to synthesize molecular testing of ependymal tumors as a whole, and our ADAPTR recommendations have followed the WHO Classification's anatomical site‐by‐site approach for ependymoma.

The standard of care for ependymal tumors remains primarily surgical, with gross total resection (GTR) and timely adjuvant radiotherapy being an important prognostic factor [11, 12]. Significant challenges exist in the delivery of the expert neurosurgical care required for GTR, with an international survey of neurosurgical and pediatric surgical societies estimating one pediatric neurosurgeon for every 3.6 million children in LICs/LMICs [13]. In a study of GTR rates in posterior fossa ependymoma from Hospital for Sick Children, Toronto, GTR rates increased from 35% in 1985 to 77% in 2014, correlating with an improvement in both progression‐free (PFS) and overall survival (OS), alongside upfront radiotherapy [14]. By contrast, studies from institutions in LICs/LMICs report GTR rates of 17% (Egypt) [15], 31% (Iran) [16], 32% (Peru) [17], and 38% (Brazil) [18], all of which attribute higher mortality rates to lower rates of GTR [19]. Furthermore, equitable access to radiotherapy for the treatment of ependymomas may be limited in LICs/LMICs, with socio‐economic factors such as travel costs and loss of income affecting treatment adherence [16]. A multicohort study of centers from high‐income and upper‐middle income countries found that a considerable proportion of PFB ependymoma could be treated with surgical intervention alone [20]. Therefore, molecular profiling, while expensive, may play a role in identifying subtypes of ependymoma that can be spared post‐operative radiotherapy, saving patients in LICs/LMICs the financial burden of adjuvant treatment [16, 20].

2.1. Imaging features of ependymoma

The AOSNP‐ADAPTR survey showed that over half of centers in LICs/LMICs have access to magnetic resonance imaging (MRI) with contrast, highlighting the key supporting role of radiological data in CNS tumor diagnosis in this setting [6]. Following the ADAPTR adult‐type diffuse glioma framework [8], imaging features provide a first‐line diagnostic context for ependymal tumors at all RLs, based on tumor location (ST, posterior fossa [PF], and spinal [SP]). Supratentorial tumors are often well‐demarcated masses with cystic change and calcification, showing strong but heterogeneous enhancement in solid portions [21]. Posterior fossa group A (PFA) ependymoma are usually calcified lateral or median tumors, show incomplete enhancement, invasion into posterior fossa foramina (particularly the foramina of Luschka), resulting in significant hydrocephalus and are seen in young children [22]. In contrast, PFB ependymoma are minimally invasive and non‐calcified median tumors, generally seen in adults and adolescents, arising from the floor of the fourth ventricle, and predominantly cystic with complete homogeneous enhancement in the solid parts [22]. Machine learning algorithms based on MRI features may also assist in distinguishing PFA from PFB ependymomas [23]. Spinal ependymomas are usually intramedullary and frequently cervical–cervicothoracic; syringomyelia is common [24, 25]. Aggressive appearances such as extramedullary extension or leptomeningeal spread should raise suspicion for SP ependymoma, MYCN‐amplified [26]. Incorporating imaging features in diagnostic workflows requires close collaboration with local expert (neuro‐)radiologists.

2.2. Morphological features of ependymoma

Classic ependymal morphology includes tumor cells with round to ovoid nuclei, arranged in perivascular pseudorosettes, true ependymal rosettes and also as simulated ependymal lining (canals) [1, 3, 27]. Ependymomas are typically well‐demarcated from the surrounding brain [1, 3, 27]. Other histological features that may be encountered include fibrillary areas, calcification, and cystic degeneration [1, 3, 27]. Although ultrastructural findings are not required for ADAPTR recommendations, ependymal features (e.g., intracytoplasmic villi, cilia, and complex intercellular zipper‐like junctions) are present in all the ependymoma subtypes [28]. The morphological patterns previously described (clear cell, tanycytic, and papillary) in the 2016 WHO classification [27] are not listed as subtypes in the 2021 WHO classification [3], because of their lack of clinicopathological usefulness. These morphological patterns are associated with certain anatomical locations and are therefore described within the anatomically defined subtypes below.

The current classification assigns CNS WHO grades 2 or 3 to ST, posterior fossa, and SP ependymoma tumor types, based solely on histologic findings [3]. However, it is recognized that the histopathologic grading of ependymoma does not consistently relate to patient outcome [2] and is often related to anatomical site [29]. Though the relevance of histological grading in a molecular classification framework can be questioned, in a resource‐limited setting, histological grading may be the only indicator of prognosis for patients and their carers, and is therefore included in these recommendations. High‐grade features in ependymomas include brisk mitotic activity and microvascular proliferation [2, 29], which have more prognostic value than other features such as nuclear pleomorphism or necrosis [30].

2.3. Lineage and surrogate immunohistochemistry for ependymoma

Lineage markers for ependymoma at Resource Level II (RL II), termed “basic IHC” by the ADAPTR Ependymoma Working Group, include immunohistochemical expression of glial fibrillary acidic protein (GFAP) and epithelial membrane antigen (EMA) (dot‐like/ring‐like luminal patterns) [31, 32, 33]. OLIG2 and SOX10 negativity assist in distinguishing ependymomas from mimics such as diffuse gliomas, as well as schwannoma and pilocytic astrocytoma [3, 34]. Ki‐67 index has not been included as an IHC marker, as it is not used in the WHO CNS5 grading of ependymomas [3]. Nevertheless, it is noteworthy that in a single institution study of 141 cases, a Ki‐67 cut‐off of 7% was found to be a useful adjunct in grading ependymomas at all anatomical locations [35].

Surrogate IHC that is practical at Resource Level III (RL III) includes: (i) L1 cell adhesion molecule (L1CAM) and p65/RELA; (ii) H3 K27me3/H3 K27M; (iii) enhancer of zeste homolog inhibitory protein (EZHIP); (iv) MYCN; and (v) homeobox B13 (HOXB13). EBP50, expressed at the luminal surface of human epithelia, has also been proposed as a potential IHC marker [36]. Surrogate IHC markers are further described in the ADAPTR recommendations below. Aligning with cIMPACT‐NOW update 11 [37], we refer to the hotspot H3 p.K28 as “H3 K27” in this manuscript.

3. ADAPTR RECOMMENDATIONS FOR THE EVALUATION OF EPENDYMOMA

The ADAPTR Ependymoma Working Group presents the three ADAPTR components in this manuscript, similar to the approach used for adult‐type diffuse gliomas [8]: (i) RLs of diagnostic pathology services with relevance to ependymal tumors; (ii) recommendations for “Histology‐oriented integrated diagnosis” format for each ependymal tumor type at different RLs, including an “ADAPTR grade” where tumor grading cannot be performed at WHO CNS5 levels; (iii) corresponding diagnostic flow charts for each ependymal tumor type at different RLs.

3.1. Resource levels of diagnostic pathology services

Resource Level I (RL I; histology) emphasizes recognition of classic ependymal morphology and provides an ADAPTR histological grade (II/III) where applicable, with management permissible on morphology alone when further testing is not feasible (Table 1). RL II adds basic IHC to confirm lineage. RL III incorporates surrogate immunomarkers—L1CAM/p65 for ST ependymoma, EZHIP/H3 K27me3 for posterior fossa ependymoma, and MYCN IHC for SP ependymoma—to approximate WHO CNS5 entities. Resource Level IV (RL IV) enables targeted molecular confirmation (e.g., FISH or reverse transcription polymerase chain reaction [RT‐PCR] for ZFTA or YAP1 fusions; FISH for MYCN amplification), and Resource Level V (RL V) uses comprehensive platforms (next generation sequencing [NGS] and DNA methylation profiling) when prior levels are inconclusive or for further risk‐stratification. While multidisciplinary discussion should take place at all RLs, it is imperative that pathologists making diagnoses at RL I and RL II discuss their findings with the treating team, to assess if referral to a higher RL center will affect treatment and prognostication. Compartment‐specific recommendations are detailed below.

TABLE 1.

Adapting Diagnostic Approaches for Practical Taxonomy in Resource‐Restrained Regions‐stratification of resource levels of diagnostic pathology services for ependymomas (ependymoma).

Resource levels	Resource Level I (RL I)	Resource Level II (RL II)	Resource Level III (RL III)		Resource Level IV (RL IV)		Resource Level V (RL V)
Available resources	Histology only	+ Basic diagnostic IHC markers	+ Surrogate IHC molecular markers		+ Basic molecular tests		+ Advanced molecular tests
Pathology services	Material FFPE sections Frozen tissue/Squash smear Techniques H&E Other histochemical stains—Alcian Blue/PAS	Material (i) and (ii) Techniques 1. and 2. 3. Basic IHC markers	Material (i) and (ii) Techniques 1. to 3. 4. Surrogate IHC molecular markers		Material (i) and (ii) (iii) Fresh tissue Techniques 1. to 4. 5. FISH 6. RT‐PCR		Material (i), (ii) and (iii) Techniques 1. to 6. 7. DNA/RNA sequencing by NGS 8. DNA methylation profiling
Basic IHC	Nil	GFAP, EMA, OLIG2, Ki‐67 Optional—SOX10	Same as RL II		Same as RL II		Same as RL II
Surrogate IHC	Nil	Nil	Markers	For diagnosis of:	Same as RL III		Same as RL III
			L1CAM/p65	ST ependymoma, ZFTA‐fusion positive
			EZHIP	PFA versus PFB ependymoma
			H3 K27M	To exclude H3 K27M‐mutant forms of diffuse midline glioma, H3 K27‐altered
			H3 K27me3	PFA versus PFB ependymoma To exclude diffuse midline glioma, H3 K27‐altered in the setting of diffuse gliomas (OLIG2+) in the midline
			MYCN	SP ependymoma, MYCN‐amplified
			HOXB13	MPE
Diagnostic molecular assays	Nil	Nil	Nil		5. FISH		Same as RL IV and 7. DNA/RNA sequencing by NGS for diagnosis
					ZFTA fusion	ST ependymoma, ZFTA‐fusion positive	ZFTA fusion	ST ependymoma, ZFTA‐fusion positive
					YAP1 fusion	ST ependymoma, YAP1‐fusion positive	YAP1 fusion	ST ependymoma, YAP1‐fusion positive
					MYCN amp	SP ependymoma, MYCN‐amplified	MYCN amp	SP ependymoma, MYCN‐amplified
					6. RT‐PCR		8. DNA methylation profiling for methylation‐based diagnosis
					ZFTA fusion	ST ependymoma, ZFTA‐fusion positive	PFB ependymoma
					YAP1 fusion	ST ependymoma, YAP1‐fusion positive	Unresolved lesions
Personnel/pathologist	General pathologist	General pathologist	Pathologist skilled in interpreting the IHC surrogate molecular markers		Neuropathologist proficient in interpretation of molecular tests and providing an integrated diagnosis		Neuropathologist proficient in interpretation of molecular tests and providing an integrated diagnosis

Abbreviations: amp, amplification; EMA, epithelial membrane antigen; EZHIP; enhancer of zeste homolog inhibitory protein; FISH, fluorescent in situ hybridization; FFPE, formalin fixed paraffin embedded; GFAP, glial fibrillary acidic protein; H&E, haematoxylin and eosin; HOXB13: homeobox B13; IHC, immunohistochemistry; L1CAM, L1 cell adhesion molecule; MPE, myxopapillary ependymoma; NGS, next generation sequencing; NF‐κB p65, nuclear factor NF‐kappa‐B p65 subunit; PFA, posterior fossa group A (PFA) ependymoma; PFB, posterior fossa group B (PFB) ependymoma; RT‐PCR, reverse transcription polymerase chain reaction; SP ependymoma, MYCN‐amplified: spinal ependymoma, MYCN‐amplified; ST ependymoma, YAP1‐fusion positive, supratentorial ependymoma, YAP1 fusion‐positive; ST ependymoma, ZFTA‐fusion positive, supratentorial ependymoma, ZFTA fusion‐positive.

3.2. ADAPTR recommendations for “Histology‐oriented integrated diagnosis” format for ependymomas at different resource levels

The ADAPTR Ependymoma Working Group applied the “Histology‐oriented integrated diagnosis format” to each ependymal tumor type (Tables S1–S6 and Figures 1, 2, 3, 4, 5). We have included RL V in these recommendations, as DNA methylation profiling may be required to achieve a WHO CNS5 diagnosis or resolve ambiguous cases in ependymal tumors, which may impact clinical management. While we have included RL V, our focus is on RLs I to IV to ensure the accessibility of these recommendations. In alignment with other ADAPTR recommendations, we categorize our recommendations as “can be considered” and “highly recommended.”

FIGURE 1.

Adapting Diagnostic Approaches for Practical Taxonomy in Resource‐Restrained Regions (ADAPTR) diagnostic flow chart for supratentorial (ST) ependymoma at resource levels Resource Level I to IV (RL I to RL IV). WHO CNS5 diagnosis can be established at RL IV using basic molecular testing. If a WHO CNS5 diagnosis cannot be determined, an ADAPTR descriptive diagnosis and ADAPTR histologic grading are provided. ADAPTR recommendations—“can be considered” and “highly recommended”—are based on histopathological and immunohistochemistry (IHC) findings. FISH, fluorescent in situ hybridization; L1CAM, L1 cell adhesion molecule; NEC, not elsewhere classified; NGS, next generation sequencing; NOS, not otherwise specified; RT‐PCR, reverse transcription polymerase chain reaction; s/o, suggestive of.

FIGURE 2.

Adapting Diagnostic Approaches for Practical Taxonomy in Resource‐Restrained Regions (ADAPTR) diagnostic flow chart for posterior fossa (PF) ependymoma at resource levels Resource Level I to IV (RL I to RL IV). For posterior fossa group A (PFA) ependymoma, a WHO CNS5 diagnosis can be established at RL III using surrogate immunohistochemistry (IHC). Fluorescent in situ hybridization (FISH) for chromosome 1q gain or 6q loss can be used for prognostication at RL IV. For posterior fossa group B (PFB) ependymoma, a WHO CNS5 diagnosis can only be established at RL V. If a WHO CNS5 diagnosis cannot be determined, an ADAPTR descriptive diagnosis and ADAPTR histologic grading are provided. ADAPTR recommendations—“can be considered” and “highly recommended”—are based on histopathological and IHC findings. AOSNP, Asian Oceanian Society of Neuropathology; NOS, not otherwise specified.

FIGURE 3.

Adapting Diagnostic Approaches for Practical Taxonomy in Resource‐Restrained Regions (ADAPTR) diagnostic flow chart for spinal ependymoma (SP) ependymoma at resource levels Resource Level I to IV (RL I to RL IV). WHO CNS5 diagnosis can be established at RL II using basic immunohistochemistry (IHC). Where SP ependymoma, MYCN‐amplified, is suspected based on high‐grade histology or radiological features, referral to an RL IV center for MYCN Proto‐oncogene (MYCN) fluorescent in situ hybridization (FISH) is recommended to determine a WHO CNS5 diagnosis. If this is not feasible, referral to an RL III center for MYCN IHC can be undertaken, with an ADAPTR descriptive diagnosis then provided. ADAPTR recommendations—“can be considered” and “highly recommended”—are based on histopathological and IHC findings. AOSNP, Asian Oceanian Society of Neuropathology; NOS, not otherwise specified; PFA, posterior fossa group A ependymoma; s/o, suggestive of.

FIGURE 4.

Adapting Diagnostic Approaches for Practical Taxonomy in Resource‐Restrained Regions (ADAPTR) diagnostic flow chart for myxopapillary ependymoma (MPE) at resource levels Resource Level I to IV (RL I to RL IV). A WHO CNS5 diagnosis can be established at RL II using basic immunohistochemistry (IHC). For lesions with ambiguous morphology (i.e., morphological overlap with spinal ependymoma (SP)), RL III testing with Homeobox B13 (HOXB13) IHC is recommended, where an ADAPTR descriptive diagnosis and ADAPTR histological grade can be made. ADAPTR recommendations—“can be considered” and “highly recommended”—are based on histopathological and IHC findings. AOSNP, Asian Oceanian Society of Neuropathology; NOS, not otherwise specified; s/o, suggestive of.

FIGURE 5.

Adapting Diagnostic Approaches for Practical Taxonomy in Resource‐Restrained Regions (ADAPTR) diagnostic flow chart for subependymoma (SE) at resource levels Resource Level I to IV (RL I to RL IV). A WHO CNS5 diagnosis can be made on H&E morphology alone and therefore can be established at RL I. Basic (RL II) or surrogate immunohistochemistry (IHC) markers (RL III) are considered unnecessary in SE. Referral to an RL IV center should be considered for brainstem, spinal, or other midline locations, or mixed ependymoma‐SE morphology. ADAPTR recommendations—“can be considered” and “highly recommended”—are based on histopathological and IHC findings. AOSNP, Asian Oceanian Society of Neuropathology; FISH, fluorescent in situ hybridization.

3.2.1. Supratentorial ependymoma (ST ependymoma)

The WHO CNS5 has classified ST ependymoma tumors into three groups: ST ependymoma, ZFTA fusion‐positive; ST ependymoma, YAP1 fusion‐positive; ST ependymoma, NOS. ST ependymoma often affects children and sometimes adults [38], although epidemiological features partially depend on molecular subtypes [5, 39]. Radiologically, a well‐demarcated mass, frequently with cystic components and calcification, is observed, associated with strong but heterogenous enhancement in solid parts [40]. Despite a limited number of reports on outcome data of molecularly‐defined ST ependymoma, ZFTA fusion‐positive tumors have the poorest outcome, and YAP1 fusion‐positive tumors appear to be prognostically more favorable [4, 39], with the prognostic value of homozygous CDKN2A/B deletion uncertain [41, 42]. However, there is conflicting data regarding the relationship of ZFTA fusion with patient outcome in South Asian populations. While one study from Vellore, India (n = 61) shows no significant effect of ZFTA fusion on PFS or OS [43], another from Chandigarh, India (n = 58) shows significantly inferior PFS and OS [44]. Such regional differences, particularly in rare tumors, will require larger cohorts and longitudinal studies for accurate prognostication.

There are several histologic features described for ST ependymoma suitable for all RLs, in addition to general histological features of ependymoma (Figure 1; Table S1). A branching network of capillaries and clear cell phenotypes is frequent in ZFTA fusion‐positive tumors [45], and PAS‐positive eosinophilic granular bodies are frequently observed in YAP1 fusion‐positive tumors [46]. ST ependymoma can be assigned CNS WHO grades 2 or 3 by histological findings including brisk mitoses and microvascular proliferation [30] as per WHO criteria [3]. Referral to an RL II center for basic IHC will aid in confirming ependymoma origin and is therefore “highly recommended.”

Immunohistochemical surrogate markers for the molecular alterations of ST ependymoma are available, including L1CAM (strong diffuse membranous staining) and p65/RELA (nuclear staining) [47, 48, 49, 50]. For these markers, ZFTA fusion‐positive tumors (especially with a ZFTA::RELA fusion) should be L1CAM+/p65 (NF‐κB)+, and YAP1 fusion‐positive tumors or others (NEC) L1CAM−/p65 (NF‐κB)−. Strong and diffuse cytoplasmic L1CAM staining has high sensitivity (92.3%–100%) for detecting ZFTA fusion‐positive ependymomas [44, 51, 52]. However, it is not a specific marker, and demonstrates expression in many other tumor types, where it does not indicate an underlying ZFTA fusion [53]. Nuclear p65/RELA staining has variable sensitivity (68%–100%) and high specificity (92%‐100%) for its main fusion gene partner, RELA [51]. Combinatorial IHC approaches (L1CAM/p65) result in greatly improved sensitivity (88%–100%) and specificity (88%–92%) [44, 47, 51, 52] as a surrogate for ZFTA fusion‐positive tumors. Referral to an RL III center for surrogate IHC is also “highly recommended” to progress toward molecular subtyping of ST ependymoma.

The definitive diagnosis of ST ependymoma subtype relies on molecular genetic analyses to detect pathognomonic fusion genes involving ZFTA (C11orf95) (i.e., ST ependymoma, ZFTA fusion‐positive) or YAP1 (i.e., ST ependymoma, YAP1 fusion‐positive) [4, 5, 39, 48, 49]. Referral to an RL IV center for FISH or RT‐PCR “can be considered,” noting that local treatment decisions may not require this level of information. The diagnosis is also supported by the exclusion of morphologically similar tumors including BCOR‐altered tumors and MN1‐altered astroblastoma (Table 2). Therefore, definite diagnoses cannot be made by surrogate markers alone based on WHO CNS5 criteria, rendering an ADAPTR descriptive diagnosis of ST ependymoma, NOS, as the most appropriate diagnosis in RL III or lower settings.

TABLE 2.

Entities with ependymoma‐like morphology.

Tumor entity	Distinguishing morphological features from ependymoma	IHC profile	Molecular findings
Astroblastoma, MN1‐altered [54]	Astroblastic pseudorosette	D2‐40++ GFAP+ to +++ OLIG2+ to +++ EMA+ to +++	MN1 fusion (common partner—BEND2)
Ependymoma‐like neuroepithelial tumor with PLAGL1 fusions [55, 56]	Variable mixed SE‐ependymoma morphology	GFAP+++ OLIG2− SOX10− EMA+/− L1CAM−	PLAGL1 fusion (common partners—FOX01 [3′], EWSR1 [5′])
Neuroepithelial tumor with PATZ1 fusion [57, 58]	Major patterns include ependymoma‐like, glial tumor with multinucleated cells, spindle cell pattern	GATA2+/− GFAP + to +++ OLIG2+ to +++ CD34+ to +++ Synaptophysin/chromogranin+/−	PATZ1 fusion (common partners—EWSR1, MN1)
Gliomas with EP300::BCOR or CREBBP::BCORL1 fusions [59, 60, 61]	Major patterns include ependymoma‐like, oligodendroglioma‐like, microcystic appearances	BCOR+/− GFAP+/− OLIG2+/−	EP300::BCOR or CREBBP::BCORL1 fusions

Abbreviations: GFAP, glial fibrillary acidic protein; L1CAM, L1 cell adhesion molecule; SE, subependymoma.

3.2.2. Posterior fossa ependymoma (PF ependymoma)

Ependymal tumors of the posterior fossa (Figure 2) are currently classified into three groups: PFA ependymoma (Table S2), PFB ependymoma (Table S3), and subependymoma (SE) (see section below) [62]. The diagnostic flow charts for PFA and PFB ependymomas are combined in Figure 2. The RL I and RL II description is common to all subtypes and is “highly recommended,” with ADAPTR histological grade and specific location mentioned.

PFA and PFB subgroups are predominantly defined by gene expression and whole‐genome DNA methylation profiles. They differ in age of onset and, more importantly, in prognosis. PFA ependymomas predominantly occur in infants and young children, and are characterized in the current WHO Classification [3] by loss of H3 K27me3 expression in tumor cells and overexpression of EZHIP, a protein implicated in inhibition of the Polycomb Repressive Complex 2 (PRC2) [63]. These molecular changes drive epigenetic dysregulation, thus contributing to the aggressive nature of PFA ependymomas [47].

Referral to RL III centers for surrogate IHC is therefore “highly recommended,” as a WHO CNS5 diagnosis of PFA ependymoma can be achieved at this RL. For a diagnosis of PFA, loss of nuclear expression of H3 K27me3 in the current WHO Classification [3] is a highly specific and sensitive marker for PFA ependymoma (admixed non‐neoplastic cells, including endothelial and inflammatory cells, retain nuclear H3 K27me3 and serve as positive internal controls) [63]. It should be noted that in cIMPACT‐NOW update 11 [37], the terminology has now been clarified from “global reduction” [64, 65] to “loss.”

EZHIP, a well‐characterized inhibitor of PRC2 activity analogous to H3 K27M, demonstrates high diagnostic utility in PFA ependymoma [66, 67], with high sensitivity and specificity reported (99% and 98%, respectively) [68]. Demonstration of strong global nuclear positivity for EZHIP is currently listed as a desirable diagnostic criterion for PFA ependymoma in cIMPACT‐NOW Update 11 [37]. For PFA ependymoma, at RL IV, demonstration of 1q gain and/or 6q loss by FISH is indicative of a poorer prognosis and “can be considered” [37, 69].

In contrast, PFB ependymomas typically retain H3 K27me3 expression, are seen in older children, adolescents and adults, and exhibit a more indolent clinical course [62]. DNA methylation profiling is mandatory for a definitive WHO CNS5 diagnosis of PFB ependymoma [3]. Hence, cases that do not have methylation profiling, exhibit retention of nuclear H3 K27me3 (with a proposed cut‐off of >80% immunopositive cells) and/or are negative for EZHIP at RL III would be given an ADAPTR diagnosis of “PF ependymoma, NOS.” RL IV testing does not play a role in the diagnosis of PFB ependymoma. Given that access to radiotherapy can be limited in LICs/LMICs [15, 16, 17, 18], referral to an RL V centre with methylation profiling “can be considered” in reaching a WHO CNS5 diagnosis of PFB ependymoma if it will impact upon treatment decisions.

More recently, ependymomas of the posterior fossa with novel genetic signatures have been described, all of which would require at least RL IV to RL V testing. These include PF ependymomas that correspond to the methylation class of posterior fossa SE with TERT promoter mutation and/or chromosome 6 loss [70]; PF ependymomas with ZFTA fusion that correspond to the methylation class of ST ependymoma, ZFTA‐fusion positive [71]; and those with somatic heterozygous coding point mutations in the activin receptor type I (ACVR1) gene and retained H3 K27me3 expression [72].

SEs in the posterior fossa are seen in adults and are similar to those in the supratentorial and spinal compartments. They are described in detail below.

3.2.3. Spinal ependymoma (SP ependymoma)

The AOSNP‐ADAPTR recommendations for SP ependymomas and SP ependymoma, MYCN‐amplified (Figure 3; Table S4) largely follow those delineated in the ST ependymoma section. SP ependymomas share many of the features of ependymomas at other locations, including their circumscription and cytomorphology. Radiological surrogates may prove particularly helpful in this anatomical compartment, as aggressive radiological appearances are a feature of SP ependymoma‐MYCN, including extramedullary extension and leptomeningeal dissemination [73, 74, 75, 76].

The classic morphological features of an ependymoma are common to all RLs at RL I, where an ADAPTR descriptive diagnosis (suggestive of SP ependymoma, NOS, ADAPTR histological grade II/III) can therefore be made. The tanycytic morphological pattern should be kept in mind, because of its overrepresentation in SP ependymoma [3, 77]. A WHO CNS5 diagnosis of “SP ependymoma, CNS WHO grades 2 or 3” can be rendered at RL II, as basic IHC combined with histological findings meet the WHO CNS5 essential diagnostic criteria for SP ependymoma [3]. SOX10 can aid in distinguishing tanycytic ependymoma (SOX10 immunonegative) from schwannoma and pilocytic astrocytoma (SOX10 immunopositive) [3, 34]. OLIG2 can assist in distinguishing SP ependymoma (OLIG2 negative) from other gliomas (OLIG2 positive) [3, 34]. Although a WHO CNS5 diagnosis can be made at RL II, it is important to consider SP ependymoma, MYCN‐amplified in the setting of high‐grade histology and/or aggressive radiology [74, 75, 76]. Additionally, although SP ependymoma, MYCN‐amplified typically demonstrates high‐grade histological features, rare cases with CNS WHO grade 2 histological appearances have been reported [75].

Given that high‐grade histology may not always be present in SP ependymoma, MYCN‐amplified, MYCN IHC is “highly recommended” at RL III. MYCN IHC has excellent concordance with molecular testing for MYCN amplification and is therefore listed as a surrogate IHC marker of MYCN amplification at RL III [74, 75, 78]. All studies used the D4B2Y clone, Cell Signaling Technology (Danvers, Massachusetts, USA). Interestingly, Ghasemi and colleagues noted differential patterns of MYCN immunopositivity, with stronger expression observed in histologically high‐grade areas of tumor [75]. Cases positive for MYCN IHC can be designated an AOSNP‐ADAPTR descriptive diagnosis (suggestive of SP ependymoma, MYCN‐amplified) in the presence of an exophytic mass on MRI [74, 75, 76, 78]. An ADAPTR histological grade has not been assigned to align with WHO CNS5 grading of this entity [3].

It should be noted that diffuse midline glioma, H3 K27‐altered can also harbor MYCN amplification, and therefore IHC for H3 K27M and K27me3 should also be undertaken at RL III [79]. Notably, variable retention of H3 K27me3 nuclear staining has been demonstrated in one study of SP ependymoma, MYCN‐amplified [74]. Another important recommendation at RL II includes if the case is OLIG2 negative and shows H3 K27me3 loss, radiological correlation is “highly recommended” to exclude a drop metastasis from PFA ependymoma.

At RL IV, FISH testing for MYCN amplification would be sufficient to fulfil the WHO essential and desirable diagnostic criteria for SP ependymoma, MYCN‐amplified and SP ependymoma, respectively [75, 76, 78, 80]. Therefore, referral to an RL IV center “can be considered” in suspicious cases where feasible, and where knowledge of the MYCN status is likely to influence treatment decisions. More recently, stratification of SP ependymoma by NF2 status has been reported to have prognostic significance [81], and may play a role in RL IV testing in the future.

3.2.4. Myxopapillary ependymoma

Myxopapillary ependymoma (MPEs) (Figure 4; Table S5) have a distinct morphological appearance, typified by cuboidal to columnar glial cells arranged around hyalinized fibrovascular cores, with perivascular aggregation of myxoid material [3, 82]. MPE also has a characteristic anatomical distribution, with the vast majority of cases found in the region of the conus and filum terminale [82, 83]. The WHO CNS5 designates MPE as CNS WHO grade 2 [3]. Exceptional examples of “anaplastic” MPE have been documented in the literature, displaying increased cellularity, reduced myxoid material, and high‐grade histological features [84].

A WHO CNS5 diagnosis of MPE can be made on the basis of typical histology and GFAP immunopositivity [3]. In practice, some senior neuropathologists diagnose these tumors on H&E staining alone when the morphology is typical. Therefore, at RL I, the classic features of MPE should be seen, in which case an ADAPTR descriptive diagnosis of “suggestive of MPE, NOS” can be made. At RL II, a WHO CNS5 diagnosis can be achieved with positive GFAP staining and is therefore “highly recommended.” RL III level testing and higher becomes relevant where there is morphological ambiguity, and therefore, “can be considered” in this setting. HOXB13 is proposed as a surrogate IHC marker (RL III) to distinguish between MPE and SP ependymoma in instances of morphological overlap [85, 86]. In a recent single institutional study of 143 spinal tumors, it has shown 100% sensitivity and specificity for the diagnosis of MPE [85, 86]. HOXB13 is also expressed in cauda equina neuroendocrine tumor, an entity readily discernible from MPE by its neuroendocrine features and immunoprofile [85, 86]. Basic molecular testing at RL IV does not have much utility in MPE. Methylation profiling at RL V “can be considered” and may provide useful information in cases of unresolved lesions, as MPE has a discrete methylation profile [85, 86, 87]. Cases with ependymoma appearances that align with the MPE methylation profile likely represent MPE with non‐classic morphology [85, 86, 87].

3.2.5. Subependymoma

Subependymoma (SE, Figure 5; Table S6) has a well‐known, near pathognomonic morphological appearance, with WHO CNS5 essential diagnostic criteria enabling a diagnosis on morphology as seen on H&E staining (i.e., RL I) [3]. Basic IHC (RL II) can be performed to confirm their subependymal glial origin (diffuse GFAP immunopositivity and focal EMA dot‐like immunoreactivity). However, given the distinct morphological appearance, if the pathologist is confident in making a diagnosis of SE on histology, RL I fulfills the essential diagnostic criteria for WHO CNS5.

Tumors with SE‐type morphology may warrant consideration of RL II testing, although this is designated “can be considered” by the ADAPTR ependymoma Working Group. RL III testing with surrogate IHC markers was therefore considered unnecessary for a diagnosis of SE.

RL IV testing for SE could be undertaken in situations of mixed ependymoma‐SE morphology in the posterior fossa tumors, or where the SE‐like tumor occurs in the brainstem, spine, or other midline locations. In a small series of SEs (n = 24), gliomas with SE‐type histology in the medulla and pons were found to harbor H3 p.K27M variants (n = 4) [88], and form a cluster distinct from both PF‐ependymoma and diffuse midline glioma, H3 K27‐altered on subsequent methylation analysis [89]. With a median follow‐up of 3.2 years, the clinical outcome of these cases did not align with a CNS WHO grade 4 prognosis [88]. Clinical and radiological correlation in such cases is therefore imperative. IHC for H3 K27M and/or H3 K27me3 may therefore provide useful additional information in the setting of a brainstem or midline location [88]. Molecular testing (RL IV) in the unique situation of mixed ependymoma‐subependymoma morphology in the posterior fossa can include sequencing for TERT promoter mutation and assessment of chromosome 6 loss, which have shown an association with this mixed morphological appearance and shorter PFS [70].

At RL V, DNA methylation profiling, including classification and chromosomal copy number variant calls derived from whole‐genome DNA methylation data, could be considered for unresolved lesions. SEs have distinct methylation profiles as per their locations (ST‐SE, PF‐SE, and SP‐SE), with chromosome 19 loss common to all locations [90]. In the setting of mixed ependymoma‐subependymoma tumors of the posterior fossa, DNA‐methylation profiling may be helpful in classification and prognostication [70]. The proportion of ependymoma‐type histology can be variable, and it is unclear at present as to whether the ependymoma component dictates the grade [70, 91, 92].

3.3. Diagnostic flow charts

Diagnostic flow charts were generated for ST ependymoma (Figure 1), PFA/PFB ependymomas (Figure 2), SP ependymoma (Figure 3), MPE (Figure 4), and SE (Figure 5). The diagnostic flow charts provide a reference for reporting pathologists based on RL and indicate when an ADAPTR descriptive diagnosis is suitable or a WHO CNS5 diagnosis is attainable at a particular RL.

The ADAPTR Ependymoma Working Group also considered entities with ependymoma‐like morphology (Table 2). Some of these are already included in WHO CNS5, including astroblastoma, MN1‐altered [3]. cIMPACT‐NOW update 10 [93] gives recommendations for minimum criteria for recognizing new (provisional) tumor types as such. Using those criteria, neuroepithelial tumor with PATZ1 fusion [57, 58] and ependymoma‐like neuroepithelial tumor with PLAGL1 fusions [55, 56] meet the criteria for provisional tumor types, while gliomas with EP300::BCOR or CREBBP::BCORL1 fusions did not at the time this update was published [59]. There is significant overlap in the degrees of GFAP, EMA, and OLIG2 staining between these entities, with many harboring ependymoma‐like appearances as part of a polymorphous morphological spectrum (Table 2). Patient outcome data for these new tumor types requires further follow‐up, although neuroepithelial tumor with PATZ1 fusion are said to have a better prognosis compared to glioblastoma, IDH‐wildtype [57, 58]. The accurate diagnoses of these entities will rely on RL V testing, although it is possible that an ADAPTR descriptive diagnosis, along with relevant surrogate IHC performed at an RL III center, could address these newer diagnoses in the future.

4. CONCLUSION

The AOSNP‐ADAPTR ependymoma recommendations provide a resource‐stratified, histology‐oriented diagnostic framework that enables neuropathologists to deliver WHO CNS5‐aligned diagnoses even in resource‐restrained regions. These guidelines complement existing ADAPTR frameworks for adult‐type diffuse gliomas and collectively aim to harmonize CNS tumor diagnostics in resource‐restrained regions. We note the emergence of artificial intelligence‐assisted digital pathology tools for the prediction of molecular subtyping in ependymoma and its potential benefit in LICs/LMICs [94]. We acknowledge that while ADAPTR descriptive diagnoses and histological grading will be of great assistance to pathologists in LICs/LMICs, working toward more equitable access to molecular testing, including advocacy for pathology spending in healthcare, is vital in the Asian‐Oceanian region and elsewhere.

AUTHOR CONTRIBUTIONS

LS, MEB, GC, KM, and JS contributed to the study concept, design, and manuscript writing. TK, VS, MA‐H, S‐HP, TT, H‐KN, CS, DNL, and PW revised the guidelines and edited the manuscript. All authors contributed to Asian Oceanian Society of Neuropathology guidelines and approved the final manuscript.

Supporting information

Data S1. Supporting Information.

DATA AVAILABILITY STATEMENT

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.