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  Table of Contents    
REVIEW ARTICLE  
Year : 2022  |  Volume : 65  |  Issue : 5  |  Page : 68-72
Overview of recent advances in the classification of ependymomas in WHO CNS5 classification: Simplified approach to their integrated diagnosis


1 Department of Pathology and Laboratory Medicine, All India Institute of Medical Sciences, Raipur, Chhattisgarh, India
2 Department of Pathology, Neuropathology Division, All India Institute of Medical Sciences, New Delhi, India

Click here for correspondence address and email

Date of Submission14-Dec-2021
Date of Decision28-Jan-2022
Date of Acceptance30-Jan-2022
Date of Web Publication11-May-2022
 

   Abstract 


Ependymomas can arise along the entire neuraxis; however, they possess site-specific unique molecular alterations and a methylome pattern which is directly related with the prognostic outcomes. Since 2016, when the updated fourth edition of World Health Organization (WHO) classification of tumors of the central nervous system was published, it has been emphasized to classify ependymomas by anatomic site and molecular signatures associated genetic alterations so that classification of the disease reflects its underlying biology. In continuation, the fifth edition of the WHO classification of CNS tumors introduces major changes, including site-specific molecular profiles as the basis of classifying ependymomas. Furthermore, an integrated tier system of reporting is recommended for better clinical correlation and predicting outcomes. WHO grading can still be included in a specific tier, along with molecular markers.

Keywords: C11orf95-RELA fusion, ependymoma, integrated tier reporting, WHO CNS5, YAP1-MAMLD1 fusion

How to cite this article:
Gupta RK, Sharma A, Sharma MC. Overview of recent advances in the classification of ependymomas in WHO CNS5 classification: Simplified approach to their integrated diagnosis. Indian J Pathol Microbiol 2022;65, Suppl S1:68-72

How to cite this URL:
Gupta RK, Sharma A, Sharma MC. Overview of recent advances in the classification of ependymomas in WHO CNS5 classification: Simplified approach to their integrated diagnosis. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 May 24];65, Suppl S1:68-72. Available from: https://www.ijpmonline.org/text.asp?2022/65/5/68/345052





   Introduction Top


Ependymomas are neuroepithelial malignancies occurring in both children and adults which can arise along the entire neuraxis.[1] These tumors are thought to arise from radial glial stem cells.[2] Before the updated fourth edition of World Health Organization (WHO) classification of tumors of the central nervous system (CNS) that appeared in 2016, ependymomas have traditionally been classified as grades I, II, and III based on their grade of anaplasia.[3] However, it was recognized that histological grading is insufficient with poor reproducibility and clinical utility in predicting outcomes. Later, Pajtler et al.[4] in their study identified different molecular signatures in site-specific ependymomas and proposed nine distinct molecularly defined groups. These molecular sub-groups show a close association with specific age groups. The 2016 updated fourth edition of WHO classification of tumors of the CNS described RELA fusion-positive ependymoma as a separate distinct entity and omitted cellular ependymoma variants from the classification.[5] The 2021 WHO classification of tumors of the CNS, fifth edition (WHO CNS5), has introduced major changes including molecular profiles as the basis of diagnosis in CNS tumor classification. This edition focuses mainly on a layered reporting system/integrated diagnosis based on a holistic approach including histological findings, WHO grading, immunohistochemistry (IHC), and molecular profiling.[6] Ependymomas are now classified according to a combination of histopathology features, anatomic sites, and molecular markers, thus dividing them into different molecular groups across the supratentorial (ST), posterior fossa (PF), and spinal compartments.[6] The molecular classification of ependymomas is based on novel diagnostic technologies such as DNA methylome profiling and DNA sequencing.

Evolution of changes in the classification of ependymoma from WHO classification of CNS tumors 2016 to WHO CNS5

The WHO 2016 classification of CNS tumors divided ependymomas into two main sub-groups, classic and anaplastic ependymoma, which correspond to traditional histological WHO grades II and III, respectively. A genetically defined supratentorial RELA fusion-positive ependymoma was described separately. Sub-ependymoma and myxopapillary ependymomas were described separately and graded as WHO grade I. In WHO CNS5, integrated reporting including histology, IHC, and site-specific molecular profiles is emphasized. On the basis of the results of large cohort methylome profiling-based studies, WHO CNS5 has classified ependymomas into site-specific molecular predictive groups which bear significant prognostic relevance.[4] WHO grading can be included; however, instead of Roman numerals, Arabic numerals are used for maintaining uniformity. Histological variants such as papillary, clear cell, and tanycytic are now considered as patterns. The anaplastic ependymoma term is obsolete. Myxopapillary ependymomas are now considered as WHO grade 2 tumors. Different molecular techniques such as real-time polymerase chain reaction (RTPCR), fluorescent-in-situ hybridization (FISH), DNA sequencing, RNA sequencing, and methylation arrays can be used to identify site-specific genetic alteration. For the tumors in which molecular work-up is not done or non-diagnostic, suffix NOS (not otherwise specified) is used, and for cases in which the results are not fitting to predictive molecular groups, suffix NEC (not elsewhere classified) is used.

Gross pathologic features

Ependymomas are circumscribed tumors usually occurring within the ventricular system or in the periventricular region. Occasionally, they may occur within the cerebral hemisphere. Grossly, these are tan-colored, spongy and soft, and sometimes gritty in consistency because of calcification.

Microscopy

Classic ependymomas characteristically show pseudo-rosettes and occasionally true ependymal rosettes. The pseudo-rosettes are formed by the radial arrangement of tumor cells around blood vessels, which create anucleate zones of fine fibrillary processes. The ependymal rosettes are composed of bland tumor cells arranged around a central lumen or space. The tumor cells are monomorphic and characterized by round to oval nuclei with speckled nuclear chromatin and intra-tumoral variation in the cell density. Additionally, ST ependymomas (ST-EPs) may show a characteristic delicate capillary network with clear cell changes. Some PF ependymomas (PF-EPs) show a biphasic pattern, containing nodules of a high cell density with brisk mitotic activity.

Myxopapillary ependymomas are often encapsulated and lobulated with or without cytic components and gelatinous materials. They are composed of cuboidal to elongated tumor cells arranged radially around hyalinized fibro-vascular cores to form a papillary pattern. The myxoid ground substance can be highlighted by alcian–blue stain.

Sub-ependymomas are commonly present as small firm nodules bulging into the ventricle. Microscopically, these are characterized by clusters of small isomorphic uniform nuclei resembling sub-ependymal glial cells which are embedded in a dense fibrillary matrix of glial processes with frequent small cystic components.

Immunohistochemistry

GFAP is consistently positive in pseudo-rosettes but variably expressed in other components. S100 and vimentin are typically positive, and epithelial membrane antigens (EMAs) show reactivity along the luminal surface of the rosettes or dot-like perinuclear (golgi zone) or ring-like cytoplasmic positivity. NHERF1/EBP50, an adaptor protein localized at the apical plasma membrane of human epithelia, shows a similar staining pattern to EMA, which is a more sensitive marker for low-grade ependymomas. L1CAM is a surrogate IHC marker for C11orf95 rearrangement which can be expressed in ST-EP with ZFTA fusion. IHC for H3K27Me3 can be used to differentiate PF group A and group B ependymomas.

Molecular profile

Markedly variable molecular alterations are frequently reported in ependymomas which include cytogenetic, genetic, epigenetic, and transcriptomic changes. These changes are site-specific based on which ependymomas are broadly classified into nine groups by Pajtler et al.[4] This classification and further study of methylome patterns in the ependymomas led to the foundation of WHO CNS5 classification of ependymal tumors.

The WHO CNS5 classification of ependymal tumors

  1. Supratentorial ependymoma


    1. ST-EP, ZFTA fusion-positive
    2. ST-EP, YAP1 fusion-positive
    3. ST-EP grade 2/3


  2. Posterior fossa ependymoma


    1. PF-EP, group PFA
    2. PF-EP, group PFB
    3. PF-EP grade 2/3


  3. Spinal ependymoma


    1. Spinal ependymoma, MYCN-amplified
    2. Myxopapillary ependymoma grade 2
    3. Spinal ependymoma grade 2/3


  4. Sub-ependymoma grade 1


Supratentorial ependymoma (ST-EP)

WHO CNS5 classify ST-EP into two molecularly defined types: (1) ependymoma with ZFTA fusion and (2) ependymoma with YAP1 fusion. ZFTA is new designation for C11orf95, which is a better representative of the tumor type than RELA because it may be fused with partners other than RELA.

(1) Ependymoma with ZFTA fusion [Figure 1]
Figure 1: ST ZFTA fusion-positive ependymoma showing (a) thin-walled vascular channels and perinuclear clearing of the cytoplasm (HE x100), (b) Membranous L1CAM immunopositivity, (c) DNA sequencing showing RELA fusion, and (d) FISH study showing one normal and other fusion (separation of signals as break-apart probes is used)

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This accounts for approximately 70% of all childhood ST-EP. They do not have a specific morphology but often show a distinct vascular pattern of branching capillaries and clear-cell change.[5] These are immunoreactive for GFAP, EMA, and L1CAM (a surrogate IHC marker for RELA). L1CAM can be expressed by other brain tumors; hence, the C11orf95-RELA fusion gene should be confirmed by other techniques such as PCR, FISH, and so forth. C11orf95-RELA fusion is the result of chromothripsis, which leads to constitutive activation of the NF-kβ pathway.[7],[8] Rarely, C11orf95 can be fused with other genes; hence, the term RELA fusion-positive ependymoma is discouraged and not used in WHO CNS5.[6] These tumors have a poorer prognosis in comparison to ependymoma with YAP1 fusion.

(2) Ependymoma with YAP1 fusion

Tumors in this sub-group occur mainly in toddlers and frequently in female patients. On magnetic resonance imaging, they are large and commonly intra-ventricular, showing a prominent cystic part with multi-nodular solid components, signs of hemorrhage, iso-intense on T1 and T2 weighted images, and delineate heterogeneous contrast enhancement.[9] On microscopy, they are richly cellular, comprising cells with angulated nuclei forming pseudo-rosettes with or without true rosettes, a varying number of mitoses, calcification, and frequent vascular proliferation and commonly correspond to WHO grade 3. They may also show clusters of eosinophilic cells, eosinophilic granular bodies (EGBs), and peripheral gliosis. The YAP1-MAMLD1 fusions can be detected by RTPCR, sequencing, or FISH analysis. On FISH, two patterns can be observed, a classical re-arrangement with one fused signal and one break-apart signal and unbalanced rearrangements with one fused signal and loss of one YAP1 signal, indicating deletion. YAP1 is the main downstream effector of the Hippo signaling pathway, which is de-regulated in several malignancies.[10] Moreover, YAP1 can also act via activation of alternative Wnt signaling or canonical Wnt/β-catenin pathways.[11]

Posterior fossa ependymoma (PF-EP)

There are two demographically, clinically, and genetically distinct sub-groups of ependymomas in the PF, which have been designated as group A (PFA/pediatric type) and group B (PFB/adult type).[12] PFA are usually laterally located in PF, occur predominantly in infants, and show a balanced genome, with a poor clinical outcome. In contrast, PFB usually occur in older children and adults and associated with a better prognosis.[3] Mack et al.[13] identified a higher extent of CpG island methylation in PFA compared with PFB ependymomas and suggested that PFA and PFB ependymomas can be referred to as CpG island methylator phenotype (CIMP)-positive and CIMP-negative ependymomas, respectively. Moreover, both H3K27 trimethylation (H3K27Me3) and H3K27Me3 target genes can also be characteristically found in PFA-CIMP+ but not in the PFB-CIMP- ependymomas; hence, IHC for H3K27Me3 is used for their differentiation [Figure 2].[3] In analyzing the signal pathways differentiating PFA and PFB, Wu et al.[3] found Laminin alpha-2 (LAMA-2) and neural epidermal growth factor-2 (NELL-2) biomarkers distinctly up-regulated in PFA and PFB, respectively. PFA ependymomas show frequent chromosome 1q gain which is correlated with a poor prognosis but do not delineate other copy number aberrations.[14] However, PFB ependymomas frequently show copy number changes such as chromosomal instabilities such as loss of chromosome 6, which is seen in most cases.[14]
Figure 2: PF-EPs; PFA (a) histopathology image showing classical ependymal rosettes (HE x100) and (b) IHC showing loss of H3K27me3 and PFB (c) showing more pleomorphic cells with coarse chromatin (HE x100) with (d) intact H3K27me3

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Spinal ependymoma

Ependymomas are the most common intra-medullary spinal cord tumors in adults. These tumors most commonly occur in the cervical or cervicothoracic spine. The WHO CNS5 classification system defines a distinct group 'spinal ependymoma; MYCN amplified' as these have a dismal prognosis in comparison to other spinal ependymomas [Figure 3]. Spinal ependymomas also show association with neurofibromatosis type 2, and sporadic cases also show an increased frequency of NF2 gene mutation.[15] In comparison to intra-cranial ependymomas, spinal ependymomas show over-expression of homeobox (HOX) family genes and insulin-like growth factor 1 (IGF1).[16] Korshunov et al.[17] reported increased expression of homeobox B5 (HOXB5), phospholipase A2 group 5 (PLA2G5), and CDKN2A in spinal ependymoma.
Figure 3: Photomicrographs of a grade 3 spinal (D3-4 region) ependymoma with N myc amplification in a 32-year-old man; (a) histopathology image showing ependymoma with significant mitotic activity (arrow) (HE x100), (b) endothelial proliferation (HE x100), (c) high MIB1 labeling index of approximately 40%, and (d) N Myc amplification on the FISH study (arrow) (red signals)

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Myxopapillary ependymoma

These tumors uniquely arise in the region of the conus medullaris, cauda equina, and filum terminale but rarely in the intra-cranial location. They are identified by their characteristic histological features of elongated, fibrillary processes arranged radially around hyalinized vascular channels, microcyst formation, and abundant Alcian blue positive mucoid materials in the stroma. They are associated with chromosomal instability, particularly copy number alterations (gains) across multiple chromosomes; however, there are no clear clinical indicators proving better utility of these molecular alterations than morphological classification.[18],[19],[20] Some studies suggest that there are no differences in the clinical outcomes of classic and myxopapillary ependymomas in adults as local recurrence is very common and rarely can metastasize; hence, the WHO CNS5 classification system assigned the latter as grade 2 tumors.[2],[21]

Sub-ependymoma

These are rare relatively indolent ependymomas found ubiquitously in any compartment identified by their characteristic morphology. Histologically, they show a fibrillary matrix with clusters of ependymal nuclei haphazardly arranged within a solid fibrillary or microcystic background.[22] In sub-ependymomas, the relationship between the methylome pattern and morphology is imprecise, and incorporation of molecular profiles does not have any proven clinical advantage over morphological classification.[18] Pajtler et al.[4] in their study found balanced genomes in ST and PF groups of sub-ependymomas and recurrent chromosomal abnormality and 6q deletion in spinal sub-ependymomas.

Apart from the molecularly defined site-specific ependymomas, other cases can be labeled by anatomic location but not by a molecular alteration. This situation arises either when molecular analysis shows non-defined molecular alteration or when molecular analysis is unavailable. The NEC suffix is used when necessary diagnostic testing is performed, but the results do not conform to a standard WHO diagnosis. NEC is a descriptive diagnosis which uses a non-WHO diagnosis to categorize the tumor.

The NOS suffix is used when diagnostic information (histological or molecular) required to assign a specific WHO diagnosis is not available. It may result from either non-availability of the test in resource-poor laboratories or non-diagnostic/negative results because of technical problems.

Papillary, clear cell, and tanycytic morphological variants are now considered as patterns rather than sub-types of ependymoma in the histopathological description. There is a long-standing controversy about the reproducibility and clinical utility of grading ependymal tumors.[23] However, similar to other brain tumors as per WHO CNS5, a pathologist can choose to assign a WHO grade (2 or 3) to an ependymoma based on its histopathological features.

Prognostic factors

Children have poor outcomes in comparison to adults and 5-year overall survival increases with increasing age in ependymoma. This may be partly related to the site as most of the pediatric ependymomas occur in the PF compartment. Sites are also major determinants of outcomes which might be actually related to molecular alterations (as these are site-specific). Spinal ependymomas have much better prognosis in comparison to intra-cranial ependymomas. Among intra-cranial ependymomas, ST-EP-ZFTA and PFA groups particularly show poor outcomes. Extent of resection serves as an independent prognostic factor.

Final approach and criteria to diagnose ependymomas

Considering radiological, intra-operative, gross finding, and characteristic microscopic features, a histological diagnosis of ependymoma can be made with or without using basic IHC markers such as GFAP, S100, vimentin, EMA, and EBP50. Further, to classify these tumors into site-specific predictive molecular groups, a few distinct IHC markers such as L1CAM and H3K27Me3 can be used along with basic molecular work-up such as RTPCR and/or FISH. If the tumor cannot be placed into the particular predictive groups based on the results of the above investigations, then such cases may be further considered for advanced ancillary tests such as DNA sequencing, methylation arrays, and RNA sequencing [Figure 4].[24]
Figure 4: Flowchart describing the approach to classify ependymomas using basic ancillary techniques such as IHC, FISH, and RTPCR, subsequently followed by advanced molecular tests such as DNA sequencing and methylation arrays as per the requirement. Caption: IHC, immunohistochemistry; FISH, fluorescent in-situ hybridization; RTPCR, reverse transcription polymerase chain reaction; MW, molecular weight; SE, spinal ependymoma

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To conclude, unlike other glial tumors, ependymomas show site-specific molecular signatures with distinct clinical, demographic, and radiological features which are directly related to the predictive outcomes, tumor behavior, and prognosis. A layered integrated diagnosis should be offered for ependymomas. In cases of non-availability of molecular work-up facilities or inconclusive/non-defined results, appropriate suffixes such as NEC or NOS may be used. In an integrated diagnosis, the CNS WHO grade can be presented in a specific tier.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Mehar C Sharma
Professor, Department of Pathology, Neuropathology Division, All India Institute of Medical Sciences, New Delhi – 110 029
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijpm.ijpm_1212_21

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