Intracytoplasmic dot‐like inclusions as cytopathologically useful findings of ependymoma: Case report of adolescent supratentorial anaplastic ependymoma with clear cell morphology

Abstract Pediatric supratentorial ependymomas often have a clear cell morphology and reveal a RELA fusion. When a clear cell neoplasm is intraoperatively diagnosed, intracytoplasmic dot‐like inclusions by cytology are a useful cytopathological feature of ependymoma.


| INTRODUCTION
Ependymomas are major primary glial neoplasms that mainly affect children and young adults, accounting for 2%-9% of all neuroepithelial tumors. 1,2 Most ependymomas arise in the infratentorial area, including the fourth ventricle and spinal cord. However, although rare, supratentorial parenchymal ependymomas (ST-EPNs) can also occur, mainly affecting pediatric patients. 1,2 Furthermore, pediatric ST-EPNs often have clear cell morphology, a C11orf95-RELA fusion, and a biologically aggressive course in the clear cell variant. 1-3 Perivascular pseudorosettes are a major pathological hallmark of ependymomas. Additionally, intracytoplasmic dot-like inclusions are well-known in ependymoma cell histopathology. 2 However, these inclusions are not known as an important cytopathological finding for recognizing neoplastic cells as ependymoma cells. Therefore, in this report, we describe the detailed cytopathological and histopathological features of pediatric supratentorial anaplastic ependymoma with clear cell morphology and L1 cell adhesion molecule (L1CAM) immunoreactivity, suggesting RELA fusion-positive ependymoma. We believe that intracytoplasmic dot-like inclusions in neoplastic cells, in addition to perivascular pseudorosettes, might be a useful finding for ependymomas. Furthermore, we should recognize the presence of RELA fusion-positive ST-EPNs as a poor prognostic variant of ependymomas.

| CASE REPORT
An 8-year-old girl received symptomatic treatment for headache and vomiting in a local pediatric clinic. As her symptoms did not improve, she underwent brain computed tomography (CT) and magnetic resonance imaging (MRI) at a local hospital 6 days after onset. Brain CT and MRI revealed a | 381 HOMMA et Al.

F I G U R E 1
Cytopathological and histopathological features of the present case. A, High degree of cellular clusters without a necrotic background (original magnification: ×200; hematoxylin and eosin [H&E] stain). B, Neoplastic cells with relatively rich eosinophilic cytoplasm, rough fibrillary processes, and round to oval nuclei. On occasion, intranuclear pseudo-inclusions (white arrow) and/or intracytoplasmic eosinophilic dot-like inclusions (black arrow) are seen (original magnification: ×600; H&E stain). C, Small nest of neoplastic cells with eosinophilic dotlike inclusions in their cytoplasm (inset, black arrow) (original magnification: ×600; H&E stain). D, By Papanicolaou staining, intracytoplasmic dot-like inclusions of neoplastic cells are seen with well-demarcated shiny oval morphology (inset, black arrow) (original magnification: ×600; Papanicolaou stain). E, Frozen section: Cellular neoplasm with a rich capillary network is observed. Neoplastic cells form perivascular pseudorosette structures (original magnification: ×200; H&E stain). mass with a cyst at the right temporal lobe. Therefore, subtotal tumor resection was performed 10 days after onset, and the resected brain tumor was diagnosed as anaplastic ependymoma. After the operation, postoperative radiation therapy (59.4 Gy/33 Fr, 1 month) was performed at our hospital, following which the patient was observed at the outpatient ward of the hospital. However, 8 years after the operation, the right temporal tumor recurred and was resected again. As postoperative neuroradiological analysis revealed complete resection of the tumor, she received no postoperative therapy but was followed up at the outpatient ward of the hospital. We observed no tumor recurrence 1 month after the second operation.

| Cytopathological findings
A cytopathological specimen was obtained during the second operation via tumor tissue squash-and-imprint and stained using the standard Papanicolaou procedure and hematoxylin and eosin (HE) staining ( Figure 1A-D). Cellular clusters without necrotic background were detected ( Figure 1A). Neoplastic cells had relatively rich eosinophilic cytoplasm with rough fibrillary processes and round to oval nuclei ( Figure 1B). Some neoplastic cells had intranuclear inclusions ( Figure 1B) and/or intracytoplasmic dot-like inclusions ( Figure 1B-C). The intracytoplasmic dot-like inclusions appeared as eosinophilic dot-like inclusions in HE staining ( Figure 1B-C) and with well-demarcated shiny oval morphology in Papanicolaou staining ( Figure 1D).

| Histopathological findings
In frozen section diagnosis, the tumor specimen had a high degree of cellular neoplasm with perivascular pseudorosettes formed by neoplastic cells elongating their processes into intermingled vessels ( Figure 1E, F). Those neoplastic cells often had intracytoplasmic dot-like eosinophilic inclusions and intranuclear pseudo-inclusions ( Figure 1F). Therefore, for the frozen section specimen, we diagnosed this as being consistent with recurrent anaplastic ependymoma. In permanent section diagnosis, the tumor specimen had a high degree of cellular neoplasm with microvascular proliferation ( Figure 1G). Neoplastic cells had irregular-shaped swollen round nuclei with perinuclear halos and showed clear cell morphology ( Figure 1H). Intracytoplasmic dot-like eosinophilic inclusions and intranuclear pseudo-inclusions were sometimes detectable in neoplastic cells ( Figure 1H). The neoplasm was immunopositive for glial fibrillary acidic protein (GFAP). Furthermore, neoplastic cells frequently had epithelial membrane antigen (EMA, Figure 1I) and/or podoplanin (D2-40) immunoreactive intracytoplasmic dotlike inclusions. According to previous reports describing intracytoplasmic dot-like eosinophilic inclusions of ependymomas, 4-7 we considered EMA-and/or podoplanin-positive dot-like inclusions as intracytoplasmic dot-like eosinophilic inclusions in the hematoxylin-eosin-stained sections. Nuclear expression of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) protein was observed, and no mutant isocitrate dehydrogenase 1 (IDH1)-R132H expression was observed. Neoplastic cells showed diffuse L1CAM expression ( Figure 1J). MIB-1 labeling index of the neoplasm was 7% at the hot spots. Among those pathological findings, the final diagnosis of the recurrent brain tumor was supratentorial anaplastic ependymoma with clear cell morphology, expressing L1CAM. RNA sequencing from the tumor tissue revealed a fusion of C11orf95 exon 3 and RELA exon 2, the most commonly identified fusion pattern. Therefore, integrated diagnosis of this pediatric supratentorial neoplasm was determined as "ependymoma, RELA fusion-positive" according to the revised 2016 World Health Organization (WHO) classification of central nervous system (CNS) tumors. 8

| DISCUSSION
Recently, genetic alterations of ST-EPNs have been reported. 3 ST-EPNs with a C11orf95-RELA fusion, in particular, are defined as distinct variants of ependymoma, named "ependymoma, RELA fusion-positive," in the revised 2016 WHO classification of CNS tumors. 8 Histopathologically, RELA fusion-positive ependymomas usually have anaplasia, including high cellularity, nuclear atypia, high mitotic activity, microvascular proliferation, and/or tumor necrosis. 3 Furthermore, they reveal features of clear cell ependymoma (CC-EPN), classic ependymoma, or papillary ependymoma. 3 Immunohistochemically, L1CAM expression is known as a surrogate marker of RELA fusion in ST-EPNs. 3,8 This ependymoma variant is clinically recognized as showing aggressive behavior and a poor prognosis. 8 In fact, the RELA fusion-positive ST-EPN affecting this 16-year-old girl recurred 8 years after the first operation even though postoperative radiation therapy had been administered. In addition, its histopathology was anaplastic ependymoma with clear cell morphology, resembling CC-EPN. Furthermore, the neoplasm revealed L1CAM immunoreactivity. These clinicopathological and immunohistochemical features are compatible with a diagnosis of "ependymoma, RELA fusion-positive." 3,8 The ependymoma, in this case, was morphologically characterized by clear cell features. CC-EPN is a rare histological variant of ependymoma, accounting for 6% of ependymomas. 1 Most CC-EPNs affect the supratentorial region of | 383 HOMMA et Al. young patients. 1 Histopathologically, clear cell morphology with perivascular pseudorosettes and/or ependymal rosettes is the characteristic feature of CC-EPNs. 1 Genetically, supratentorial CC-EPNs often have RELA fusion, 3 suggesting the association between supratentorial CC-EPNs and RELA fusion-positive ependymoma. 3,8,9 Therefore, when diagnosing supratentorial clear cell neoplasms, we have to consider the following differential diagnoses: CC-EPN,  The major points of the differential diagnoses of CC-EPN, OD, CN, and ccRCC are summarized in Table 1. 1,[10][11][12][13][14][15][16][17][18][19][20] First, clinically, CC-EPNs affect young patients whereas the other tumors tend to occur in middle-aged patients. 1,11,12 The foramen of Monro is the area most frequently affected by CNs, which differs from CC-EPNs, OD, or metastatic ccRCC. Cytopathologically, neoplastic cells of CC-EPNs show a cohesive tendency and often form cell clusters suggesting perivascular aggregation. 2 Furthermore, by detailed cytopathological observation of CC-EPNs, we found that some neoplastic cells had intracytoplasmic dot-like inclusions, as has been seen by histopathology. 2 Histopathologically, intracytoplasmic dot-like eosinophilic inclusions showing EMA immunoreactivity are reported as helpful diagnostic findings of ependymoma. 6 These intracytoplasmic dot-like eosinophilic inclusions of ependymoma were confirmed by electron microscopy as microscopic intracytoplasmic lumina. 6 Furthermore, these intracytoplasmic dot-like eosinophilic inclusions could be found in various degrees among various ependymoma subtypes, including ordinary (65%), papillary (33%), clear cell (80%), tanycytic (50%), myxopapillary (20%), and anaplastic ependymomas (66%). 6 However, to our knowledge, cytopathological descriptions of intracytoplasmic eosinophilic inclusions of ependymoma are rare. 21 Otani et al reported two cases of ependymoma with intracytoplasmic inclusions: One was an ordinary WHO grade Ⅱ ependymoma with perivascular pseudorosettes, and the other was ependymoma characterized by signet-ring-like neoplastic cells, and they suggested that the presence of intracytoplasmic inclusions in cytological specimens of brain tumors could be useful for the diagnosis of ependymomas. 21 Although there are very limited cases, we also suggest that the presence of intracytoplasmic dot-like inclusions in cytology might be a useful cytological finding suggesting ependymal neoplasm even when no distinct perivascular pseudorosettes and/or ependymal rosettes are found in a brain tumor. Further case accumulation of cytological specimens of ependymomas and detailed cytopathological analyses of these intracytoplasmic dot-like eosinophilic inclusions of ependymomas are needed to consider them as cytologically useful findings of ependymoma.
Immunohistochemically, CC-EPN shows GFAP immunoreactivity 2 and EMA-immunopositive intracytoplasmic dot-like inclusions. 3 Furthermore, as seen in our case, supratentorial CC-EPN tends to express L1CAM, suggesting the presence of C11orf95-RELA fusion. 3 These immunohistochemical features are also useful for excluding OD, CN, and ccRCC.
In conclusion, there are several histologic types of brain tumors showing clear cell morphology. Among them, some show biologically aggressive behavior and require suitable postoperative chemoradiation therapies. One of those tumors is ST-EPN with RELA fusion. It is difficult to diagnose a RELA fusion-positive ependymoma by intraoperative-frozen pathological diagnosis. However, using the patient's age, tumor location, and several cytopathological findings including the intracytoplasmic dot-like inclusions of neoplastic cells, we could suggest a diagnosis of ependymoma with a possible C11orf95-RELA fusion: the poor prognostic variant of ependymoma.