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The fine structure of ependymomas
Practice Points
Stavros J Baloyannis*1 & Ioannis S Baloyannis2 Electron microscopy may be considered a very useful method in establishing a definitive diagnosis of
brain tumors. In ependymomas we might conclude that the most common ultrastructural findings of high diagnostic
value are: ūū The numerous microvilli and cilia sometimes incorporated in the cell body or extended freely into the
intracellular space; ūū The centriole or blepharoplast located in the base of the cilia; ūū The large number of the fragmented microtubules in the perikaryon and the cellular processes (any
small cellular projection into the neutrophil or intracellular space); ūū The junctional apparatuses between the neoplastic cells, such as zonula adherens, zonula occludens
and puncta adherentia; ūū The basement membrane-like structure in many papillary ependymomas and ependymomas of the
filum terminale. The elongated cells in loose intracellular space, commonly seen in myxopapillary ependymomas of the
filum terminale. The precise and definitive diagnosis of brain tumors is of great value for determining the proper
treatment for the patient, the application of the recently developed immunotherapeutic strategies will allow this.
SUMMARY Electron microscopy is a useful diagnostic technique in order to confirm or establish a definitive diagnosis in brain tumors that may have an atypical histological pattern, which requires a concrete diagnosis. In ependymomas, electron microscopy reveals morphological characters that have a pathognomonic diagnostic value, therefore allowing a definitive diagnosis. The main fine structural criteria of ependymomas consist of the numerous microvilli and cilia, which are incorporated in the cell body or extended freely in the intracellular space; the centriole or blepharoplast, which is located in the basis of the cilia; the large number of the fragmented microtubules in the perikaryon and the Aristotelian University Department of Neurology, Thessaloniki & Research Institute for Alzheimer’s disease, Iraklion Langada, Greece Aristotelian University, Department of Neurosurgery, Thessaloniki, Greece *Author for correspondence: Tel.: +30 231027043; Fax: +30 2310270434; [email protected] 1 2
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REVIEW Baloyannis & Baloyannis cellular processes (any small cellular projection into the neutrophil or intracellular space); the junctional apparatus between the cells, such as zonula adherens, zonula occludens and puncta adherentia; the basement membrane-like structure, seen in papillary ependymomas and ependymomas of the filum terminale; and the elongated cells in the loose intracellular space, commonly seen in myxopapillary ependymomas. Ependymomas, a general approach The CNS is a common place of cancerogenesis. Among the various tumors that may arise, gliomas are the most frequent primary brain neoplasms in humans. These are roughly divided, from a histological point of view, into astrocytomas, oligodendrogliomas and ependymomas [1]. Ependymomas account for 2–9% of all intracranial tumors. Although the majority of ependymomas are sporadic, a limited number can be seen as part of neurofibromatosis type 2 (or multiple inherited schwannomas, meningiomas and ependymomas [MISME] syndrome) [2]. Ependymomas may manifest at any age, from 1 month to 81 years [3]; however, they are mostly seen in children and are documented as the third most common primary brain tumor of childhood [4]. Thus, approximately 30% of pediatric ependymomas are diagnosed in children younger than 3 years of age, and in 60–70% of cases the tumor is located infratentorially, with a mean age presentation of approximately 6 years with no gender predilection [3]. Supratentorial ependymomas in children are commonly extraventricular [3,5]. Of those ependymomas that occur intraventricularly 58% originate in the fourth ventricle, whereas
Figure 1. Ependymoma cell from an ependymoma of the fourth ventricle. The cell has a large homogeneous nucleus with fine distribution of chromatin. The perikaryon is rich in organelles, 9000× magnification.
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the remaining 42% are located in the lateral and third ventricles [6]. In adults, 33% of brain ependymomas are infratentorial and 66% are located supratentorially [7]. In supratentorial ependymomas the mean age of the clinical manifestation of the tumors ranges between 18 and 24 years [5]. It is very rare to see supratentorial cortical ependymomas without any connection to the ventricular system. Nevertheless, in adults, 60% of ependymomas develop in the spinal cord, where they most frequently consist of intramedullary tumors of a peak incidence in the fourth decade of life, with males being more frequently affected than females. The majority of the ependymomas of the spinal cord are located at the conus medullaris, the filum terminale, the cauda equine or the cervical region of the spinal cord. Generally, intradural extramedullary ependymomas are highly unusual [8–10]. From a clinical point of view, the most common symptom of spinal ependymomas is lumbar, sacral or radicular pain, which is often worse at night or whilst in a recumbent position. This is also frequently associated with sensory changes, motor deficits, bladder and intestinal dysfunction, and impotence. The average duration of symptoms, preceding diagnosis, ranges from 1 to 9 years depending the location and the histological pattern of the tumor [11]. Ependymomas have also been described in the ovaries, soft tissues and mediastinum, although these are rare [12]. From the histological point of view ependymomas typically derive from the ependymal cells that line the ventricles of the brain, aqueduct and central canal of the spinal cord. The ventricles are surrounded by a lining of ependymal cells and subependymal glia [13], which may give rise to ependymomas, subependymomas and subependymal giant cell astrocytomas. However, ectopic ependymal cells are occasionally found within the brain itself, isolated from the ventricular system and trapped within the developing cerebral hemispheres during embryogenesis. These cells, may give rise to supratentorial cortical, lobar or extraventricular ependymomas, which, in the majority of the cases, do not retain any continuity with the ventricular system [3,14].
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The fine structure of ependymomas Intraventricular ependymomas, as well as any other intraventricular mass, usually induce increased intracranial pressure and hydrocephalus. Headache, vomiting, vertigo, cerebellar ataxia and gait disturbances are the most frequent clinical phenomena in ependymomas of the fourth ventricle, which are sometimes also associated with hemiparesis. In infants, a rapidly increasing head circumference is frequently noticed as one of the initial phenomena of the neoplasm and associated with sleepiness, vomiting, instability, vertigo and increased irritability. In supratentorial ependymomas, a substantial number of patients tend to frequently suffer from various focal neurologic deficits and seizures [13]. Children with intracranial ependymomas located in the fourth ventricle have a relatively less favorable prognosis than adults, despite recent advances in prompt diagnosis and treatment by microneurosurgical resection, radiosurgery, radiotherapy, chemotherapy and immunological methods. However, it is reasonable that the overall prognosis varies widely depending on the location, volume, histological characteristics and extent of tumor removal by surgery, which is normally the principal factor of tumor recurrence and patient survival. Infratentorial or anaplastic ependymomas may demonstrate a tendency (10–15%) to metastasize along the neuroaxis and through the cerebrospinal fluid [15]; however, there is no correlation between the grade and location of the tumor in the available data. Therefore, it is expected that mortality rates are higher in patients with metastasis than in patients with well-circumscribed primary lesions [16]. From a morphological point of view, ependymomas are soft, grayish or red pliable tumors that may contain cysts or small calcifications [13,17]. Ependymomas of the fourth ventricle mostly originated from the floor or the roof of the ventricle and then fill the ventricular lumen, they may occasionally extend through the foramen of Luschka into the cerebellopontine angle and possibly further into the adjacent parenchyma of the cerebellum, the pons and mesencephalon. Microscopically, ependymomas are cellular tumors characterized by the frequent formation of perivascular pseudorosettes and true ependymal rosettes. Perivascular pseudorosettes are composed of neoplastic cells radially surrounding the lumen of a capillary vessel, whereas true ependymal rosettes are composed of columnar cells arranged around a central lumen. Mitosis
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Figure 2. Ependymoma cell from an ependymoma of the fourth ventricle. The cellular process (any small cellular projection into the neutrophil or intracellular space) includes a large number of polymorphic mitochondria intermixed with twisted microtubules, 19,800× magnification.
is very rare. Thus, ependymomas are considered WHO grade II lesions [18]. Several histological subtypes of ependymal cell tumors have been described, such as ependymomas (WHO grade II tumors with cellular papillary, epithelial, clear cell and mixed variants), anaplastic ependymoma (WHO grade III tumor) and myxopapillary ependymoma (WHO grade I tumors frequently located along the filum terminale with an occasional extension into the conus medullaris) [1,18].
Figure 3. Ependymoma cell from an ependymoma of the fourth ventricle. Near the nucleus several fragmented cisternae of the Golgi apparatus are interrelated with dilated cisternae of the smooth endoplasmic reticulum, 28,800× magnification.
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Figure 4. Ependymoma cells from the fourth ventricle. In a short cellular process a lysosome containing laminated osmiophilic material is shown, 14,600× magnification.
The 2007 WHO classification recognizes three grades of ependymal tumors: grade I, which includes subependymomas (which occur near a ventricle or the central canal of the spinal cord) and myxopapillary ependymomas; grade II corresponding to ependymoma; and grade III corresponding to anaplastic ependymoma. In addition, the same classification recognizes four variants of ependymomas: cellular, papillary, clear cell and tanycytic, usually belonging to grades I or II [18]. The myxopapillary subtype of ependymomas is a benign variant of the tumor, with a peak incidence of clinical manifestation between the
Figure 5. Ependymoma excised from the fourth ventricle. A congregation of microvilli is shown, which are crowded inside the soma of the cells in a parallel arrangement. Some microvilli protrude into narrow lumen-like spaces that are created between the adjacent cells, 9000× magnification.
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third and fifth decades of life. Myxopapillary ependymomas mostly develop in the thoracolumbar region, and are recognized as the most common subtype of ependymomas in the filum terminale [19], accounting for 13% of all spinal ependymomas and 90% of the tumors in the conus medullaris [20]. Myxopapillary ependymoma have also been described in extraspinal locations, such as subcutaneously and in the brain, although these are rare [21–24]. Myxopapillary ependymomas are also considered to represent grade I tumors, characterized, as a rule, by slow growth [18]. From a morphological point of view, macroscopically the tumor is often intradural, encapsulated and usually anatomically isolated from direct access to routes of possible dissemination, although local invasion and distant metastases have also been described [23]. Microscopically, the main histological characteristics of diagnostic value are the papillae surrounding blood vessels, which are formed by the arrangement of cuboidal or columnar cells, and the mucinous degeneration within the vascular connective tissue, which is presumably formed by an inundation of plasma proteins. Low cellular variants and hyaline changes of myxopapillary ependymomas have also been described [25]. Tanycytic ependymoma is a very rare subtype of ependymomas that was first described by Friede and Pollak in 1978 [26]. Tanycytic ependymoma is a WHO grade II tumor in which the typical ependymal rosettes and perivascular pseudorosettes are mostly replaced by fibrillar cells in a pseudo-palisading arrangement. Microvascular proliferation is also not an uncommon phenomenon in tanycytic ependymoma tumors. Anaplastic ependymomas are histologically characterized by increased cellularity and marked mitotic activity, and are occasionally invasive and metastasize through the cerebrospinal fluid pathway [27]. In general, in ependymomas the neoplastic cells usually retain the main morphological features of normal ependymal cells. Therefore, they have cilia and microvilli, and a deeply stained blepharoplast that aids in the histological and ultrastructural identification of the tumor. However, some ependymomas undergo rapid and extensive differentiation either to anaplastic types or to mixed tumors, due to participation of other glial elements in their general histological profile.
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The fine structure of ependymomas
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Figure 6. Cilia and microvilli. (A) Cilium of an ependymoma cell from the fourth ventricle. In the base of the cilium a centriole (blepharoplast) is shown, 28,500× magnification. (B) Microvilli and cilia in cross section in the intracellular space of an ependymoma of the fourth ventricle, 28,500× magnification.
The fine structure The electron microscopy study of ependymomas is an important diagnostic procedure for defining the fine structure of the neoplastic tissue for their differential diagnosis from mixed tumors and other rare neoplastic processes. To describe the fine structure we studied 45 cases of ependymomas that had been excised from various areas of the CNS. Twenty of them derived from the fourth ventricle, 15 from the lateral ventricles and 10 from the filum terminale. In 35 cases the histological diagnosis was based on the general microscopical pattern of the neoplastic tissue and the finding of blepharoplast, which was a valuable pathognomonic criterion of the histological origin of the tumor. In 10 cases the differential diagnosis from astrocytomas was determined by light microscopy and a definite diagnosis was determined using electron microscopy. All the specimens were fixed in Sotelo’s fixing solution, embedded in araldite, contrasted with an aqueous solution of saturated uranyl acetate and lead citrate, and were studied either in a Siemens Elmiscope I electron microscope (Siemens, Munich, Germany) or in a Zeiss 9aS electron microscope (Carl Zeiss Group, Oberkochen, Germany) . Most of the ependymal cells had large homogeneous nuclei with fine distribution of the chromatin (Figure 1). Nuclear envelops were even or they rarely had cytoplasmic invaginations. The perikaryon was very rich in organelles. It included a large number of mitochondria, which were round or elongated (Figure 1) and frequently demonstrating an atypical pattern of
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mitochondrial cristae (Figure 2). In some areas of the perikaryon some aggregations of elongated mitochondria were seen including osmiophilic material in a agranular laminated pattern. The endoplasmic reticulum demonstrated numerous frequently dilated cisternae (Figure 3). Near the nucleus, several fragmented cisternae of the Golgi apparatus connected with cisternae of the smooth endoplasmic reticulum were frequently seen in ependymomas of the fourth ventricle (Figure 3). In some of the ependymoma cells of the fourth ventricle lysosomes were seen that contained laminated osmiophilic material (Figure 4). A common finding in ependymoma cells of the fourth ventricle was the congregation of microvilli. They were
Figure 7. Cilia of an epedymoma cell from the lateral ventricles. The cilia retain their typical elongated straight pattern, protrude into the extracellular space and are intermixed with numerous microvilli, 14,600× magnification.
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Figure 8. Cellular processes (any small cellular projection into the neutrophil or intracellular space) of ependymoma of the lateral ventricle. (A) Inside the perikaryon and in a considerable number of cellular processes numerous, fragmented or twisted microtubules are shown, which are intermixed with dilated cisternae of the smooth endoplasmic reticulum, 9300× magnification. (B) Twisted, fragmented microtubules in the cellular processes of ependymoma cells of the lateral ventricle, 14,600× magnification.
crowded inside the soma of the cells in a parallel arrangement or were occasionally protruding into the narrow lumen-like spaces between the adjacent cells, which were filled with numerous short microvilli (Figure 5) or cilia arising from the surrounding cells. Frequent congregations of microvilli in the extracellular space were composed of well-distinguished spheroid masses.
In close proximity to the microvilli were the centriole and the cilia. The centriole is located, as a rule, in the base of the cilia in the outer zone of the perikaryon (Figure 6). The cilia retained their typical elongated straight pattern and protruded into the extracellular space (Figure 7). Congregation of the cytoplasmic processes is a common phenomenon seen mostly in ependymomas of
Figure 9. Intercellular junctions. (A) Ependymoma cells of the fourth ventricle that developed zonula adherens and puncta adherentia between adjacent cells, 5000× magnification. (B) Junctional apparatuses, such as puncta adherentia, zonula adherens and zonulas occludants, between cell bodies and cellular processes of the adjacent cells of an ependymoma of the fourth ventricle, 5000× magnification.
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The fine structure of ependymomas the fourth ventricle. Elongated processes are arranged in a concentric orientation around an ovoid cellular profile. Inside the perikaryon, as well as in a considerable number of cellular processes (any small cellular projection into the neutrophil or intracellular space), we cobserved numerous fragmented or twisted microtubules intermixed with dilated cisternae of the smooth endoplasmic reticulum (Figure 8). Frequently the cells of the ependymomas were arranged in solid compact masses and many junctional apparatuses were seen between the cell bodies and the cellular processes of the adjacent cells. The majority of those structures appeared to be zonula adherens, puncta adherenta and zonula occludens (Figure 9). In proximity of some of the zonulae adherentes condensations of microfibrils and small round vesicles were occasionally seen. In the papillary type of ependymomas and the ependymomas of the filum terminale a typical basement membrane-like structure was seen (Figure 10). This consisted of three layers a filamentous outer one, an intermediate light layer, poor in filaments, and a dense inner one, which was directly connected with the plasma membrane of the cell (Figure 10). In some of the ependymomas of the filum terminale several large vesicles were seen inside the cellular processes. Some of the vesicles had an osmiophilic center that was encased by a light outer zone surrounded by a single membrane (Figure 10). In the myxopapillary ependymomas of the filum terminale many elongated cells with long, thin processes in a parallel arrangement were frequently seen in the loose intracellular space (Figure 11). Among the ciliated cells of the ependymomas, several large cells with irregular nuclear contours and abundant perikaryon, containing large number of multivesicular bodies, dilated cisternae of the smooth endoplasmic reticulum and numerous fragmented microtubules, were also seen (Figure 12). In some ependymomas of the lateral ventricles, neoplastic cells with long cellular processes containing bands of fibrils were occasionally intermixed with the ciliated ependymoma cells typically seen (Figure 13). Clear round cells, which might have been oligodendroglioma-like cells, were sometimes seen in ventricular ependymomas, demonstrating that microvilli, cilia and intercellular junctions advocate in favor of their ependymal origin (Figure 14).
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Figure 10. Typical basement membrane-like structure in a papillary-type ependymoma of the lateral ventricle. 9300× magnification.
Electron microscopy may be considered as a useful method in establishing a definitive diagnosis of brain tumors. In ependymomas we might conclude that the most common ultrastructural findings with a high diagnostic value are: the numerous microvilli and cilia sometimes incorporated into the cell body or extended freely into intracellular space; the centriole or blepharoplast located in the basis of the cilia; the large number of the fragmented microtubules in the perikaryon and the cellular processes; the junctional apparatuses between neoplastic cells, such as zonula adherens, zonula occludens and puncta adherentia; the basement membrane-like structure in many papillary ependymomas and
Figure 11. Elongated cells of a myxopapillary ependymoma of the filum terminale. These are characterized by numerous thin, long processes in a parallel arrangement, 4500× magnification.
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Figure 12. Large cells with irregular nuclear contours and abundant perikaryon. The cells contain a large number of multivesicular bodies, dilated cisternae of the smooth endoplasmic reticulum and numerous fragmented microtubules in a papillary ependymoma of the lateral ventricles, 5000× magnification.
ependymomas of the filum terminale; and the elongated cells in the loose intracellular space commonly seen in myxopapillary ependymomas of the filum terminale. The diagnostic value of most of these findings has also been previously demonstrated [28–31]. In addition, the abnormal mitochondria, fragmentation of the Golgi apparatus, dilatation of the cisternae of the smooth endoplasmic reticulum, multivesicular bodies prescence, twisted or fragmented microtubules, large osmiophilic
Figure 13. Neoplastic cells with long cellular processes. They contain bands of fibrils in an ependymoma of the lateral ventricles, 5000× magnification.
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vesicles and large numbers of small cellular processes may also be considered as common findings in ependymomas, although without a high diagnostic value as they may also be seen elsewhere, since during the neoplastic process the organelles of the perikaryon may undergo several morphological changes. A rather unusual ultrastructural finding was the enwrapping of the cellular processes, which composed laminated figures of a concentric arrangement. That pattern has been already described in oligodendrogliomas [32,33] and we believe this might be rarely observed in ependymomas. The ependymomas of the filum terminale usually retain some morphological features of the choroid plexuses, such as the basement membrane and the large number of cilia on their surface [34]. These cells rarely express neurosecretory activity, and therefore accumulate numerous large osmiophilic dense-coated vesicles, similar to those found in the pituitary gland and the human infundibulum [35]. Similar large neurosecretory cells have been also described in oligodendrogliomas of the posterior fossa [36]. Neurosecretory elements in the ependymomas of the filum terminale have also been described previously by Miller and Torack [37], who correlated the cellular elements of the caudal section of the spinal cord with Dahlgren cells, which are also of ependymal origin [38]. Generally, the secretory capacity of glial cells, ologodendrocytes or ependymal cells is a crucial problem that demands further investigation. In myxopapillary ependymomas, Lim et al. hypothesized that the ‘proteinaceous’ material and the hyaline vascular alterations may be a consequence of the increased vascular permeability that leads to diffusion of plasma proteins into the extravascular spaces [39]. The basement membrane seen in electron microscopy may be due to location of the tumor in juxtaposition to the collagen, which exists normally in the conus medullaris and the filum terminale. Although myxopapillary ependymomas are most often benign [40], malignant transformation may seldom occur [27], demanding a differential diagnoses from other more aggressive spinal cord tumors and metastases [41]. The contact of ependymonal cells with zonula adhaerens, zonula occludens and pucta adherentia is a frequent finding in well-differentiated ependymomas of the fourth ventricle and the filum terminale, as well as in papillary ependymomas of the lateral ventricles, whereas it is rarely seen in
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The fine structure of ependymomas poorly differentiated and anaplastic ependymomas. The zonula adhaerens is a predominant finding in cells near microvilli or cilia, which protrude into the intercellular space, although the zonula occludens is seen in the majority of the contacting cells in close proximity to zonula adhaerens. In addition, in cases of myxopapillary ependymoma of the filum terminale, some authors described septate junctions, a common type of junction of invertebrate epithelium. The junctions are characterized by a ladder-like appearance and demonstrate electron-dense septa arranged in a periodicity of 40–50 nm [42]. Hemiseptate-like junctions, zonula adherens and gap junctions were also described in the vicinity of the septate junctional structures [42]. However, the most common form of contact was the simple apposition of cells with a cleft approximately 200 Å wide, between their cell membranes [42]. The luminal surface of ependymal cells is characterized by the presence of cilia with a typical 9 + 2 arrangement of microtubules, a feature that is also seen in a substantial number of ependymoma cells. However, abnormal cilia, such as giant, compound, swollen, bizarre cilia, cilia with dynein arm defect, cilia with random orientation of microtubular doublets and abnormal axial microtubules were frequently seen in malignant ependymomas of the fourth ventricle and the vermis of the cerebellum [43,44]. However, disorders in cilia orientation and aberrant cilia may occasionally be seen in common ependymal cells. It is important to emphasize that, regarding their fine structure, ependymomas are mostly morphologically similar at any location of the axis of the CNS, although they might have distinct chromosomal imbalances [45,46], participate in different oncogenic pathways and display varying clinical behaviors depending on their location [47]. Shuangshoti et al. found that there was no significant relation between histopathology, Ki-67 proliferation index, p53 immunolabeling, tumor ploidy and biological behavior of ependymomas [7]. Frequently, several secondary lysosomes are seen in the soma of a substantial number of ependymomas. In addition, glial filaments, glycogen granules, intracytoplasmic lumina ciliated [48], might be seen in the perikaryon of ependymoma cells, resulting from invagination of the extracellular space within the cytoplasm. Rarely, rectangular or square crystalloid bodies, which are frequently seen in oligodendrogliomas [33], are intermixed with dense bodies and abnormal
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Figure 14. Clear round cell of ependymoma of the lateral ventricles. The perikaryon includes many abnormal mitochondria and microvilli profiles, 9300× magnification.
mitochondria in the soma of ependymoma cells. Pinocytotic vesicles have also been seen near the junctional apparatuses, which may suggest a possibility of cellular transport of fluid [48]. In tanycytic ependymomas, the neoplastic cells originate from the tanycytes, which are bipolar cells located plentiful in the vicinity of the lateral wall of the third ventricle, in the lateral ventricles, the fourth ventricle and the spinal cord [49], and may demonstrate a dumbbell arrangement [50,51]. Multifocal ependymomas are rare subvariants, belonging almost exclusively to the myxopapillary subtype [52]. Among the ependymoma cells, some astrocytes with long fibrillary cellular processes may also participate in the pattern of the tumor, representing mostly reactive cells [53]. At an experimental level, ependymomas may be developed by using chemical agents or certain viruses. However, xenografts in mouse models of human ependymomas may be considered the most useful experimental model [54]. Conclusion In conclusion, electron microscopy may reveal all the morphological characteristics of brain tumors [55] and can be used to determine the origin of the neoplastic cells [56,57], therefore, enabling a definitive diagnosis, which is essential for patient treatment [58,59], particularly allowing the application of recently developed immunotherapeutic strategies [60–63]. Future perspective Electron microscopy remains a very valuable method for providing a definitive diagnosis
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REVIEW Baloyannis & Baloyannis of neoplasms of the CNS. This is particularly relevant for immunohistochemical identification and classification of the various types and subtypes of brain tumors in the future enabling the expansion of the immunotherapeutic strategies. In the field of experimental cancerogenesis electon microscopy may also be instrumental in revealing, for example, the morphological alterations of organelles, protein trafficking, protein interactions, the role of mitochondria and Golgi apparatus, membrane receptors and cell–cell interactions during the neoplastic process, resulting in further
understanding of the biological behavior of brain tumors. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
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brain tumors, an electron microscopic study. J. Neurosurg.19, 731–753 (1962). 30 Brightman MW, Palay SL. The fine
structure of ependymal cells in the brain of the rat. J. Cell Biol. 19, 415–439 (1963). 31 Rawlinson DE, Herman MM, Rubinstein
LJ, The fine structure of myxopapillary ependymoma of the filum terminale. Acta Neuropath. (Berl.) 25, 1–13 (1973).
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lamination of glial processes in oligodendrogliomas. J. Cell Biol. 15, 313–334 (1962). 33 Baloyannis SJ. The fine structure of the
isomorphic oligodendroglioma. Anticancer Res. 1, 243–248 (1981). 34 Baloyannis SJ. The fine structure of the
choroid plexus of the rat. Ann. Med. Sch. Aristotle Univ. 12, 278–286 (1979). 35 Bergland RM, Torack RM. An electron
microscopic study of the lumen infundibulum. Z. Zellforsch. 99, 1–12 (1969). 36 Baloyannis SJ, Diacoyannis A.
Oligodendroglioma of neurosecretory function Ann. Med. Sch. Aristotle Univ. 11, 1283–1295 (1977). 37 Miller CA, Torack RM. Secretory
ependymoma of the filum terminale. Acta Neuropath. (Berl.) 15, 240–250 (1970). 38 Frieberg G, Bern HA. The hypophysis and
the caudal neurosecretory system of the fishes. Biol. Rev. 43, 175–199 (1968). 39 Lim SC, Jang SJ. Myxopapillary ependymoma
of the fourth ventricle. Clin. Neurol. Neurosurg. 108, 211–214 (2006). 40 Kaner T, Sasani M, Oktenoglu T, Solmaz B,
Sarloglu AC, Ozer AF. Clinical analysis of 21 cases of spinal cord ependymoma: positive clinical results of gross total resection. J. Korean Neurosurg. Soc. 47, 102–106 (2010). 41 Volpp PB, Han K, Kagan AR, Tome M.
Outcomes in treatment for intradural spinal cord ependymomas. Int. J. Radiat. Oncol. Biol. Phys. 69, 1199–1204 (2007). 42 Ho KL. Intercellular septate-like junction of
neoplastic cells in myxopapillary ependymoma of the filum terminale, Acta Neuropath. 79, 432–437 (1990). 43 Ho KL. Abnormal cilia in a fourth ventricular
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44 Kubota T, Ishise J, Yamashima T, Yamamoto
S. Abnormal cilia in a malignant ependymoma. Acta Neuropath. 71, 100–105 (1986). 45 Schneider D, Monoranu CM, Huang B et al.
Pediatric supratentorial ependymomas show more frequent deletions on chromosome 9 than infratentorial ependymomas: a microsatellite analysis. Cancer Genet. Cytogenet. 191, 90–96 (2009). 46 Puget S, Grill J, Valent A et al. Candidate
genes on chromosome 9q33–34 involved in the progression of childhood ependymomas. J. Clin. Oncol. 27, 1884–1892 (2009). 47 Andreiuolo F, Puget S, Peyre M et al. Neuronal
differentiation distinguishes supratentorial and infratentorial childhood ependymomas. Neuro Oncology 12, 1126–1134 (2010). 48 Ho K L, Caccamo DV, Garcia JH.
Intracytoplasmic lumina in ependymomas: an ultrastructural study. Ultrastr. Path. 18, 371–380 (1994). 49 Sato KT, Kubota M, Ishida M, Handa Y.
Spinal tanycytic ependymoma with hematomyelia – case report. Neurol. Med. Chir. (Tokyo) 45, 168–171 (2005). 50 Ishihama H, Nakamura M, Funao H et al. A
rare case of spinal dumbbell tanycytic ependymoma. Spine 36, E612–E614 (2011). 51 Salomão JF, Vianna de Andrade C, Bellas AR,
Protzenko Cervante T. The nature of double concomitant myxopapillary ependymoma: report of a case. Childs Nerv. Syst. doi:10.1007/ s00381-013-2251-2250 (2013) (Epub ahead of print). 52 Andoh H, Kawaguchi Y, Seki S et al. Multi-
focal myxopapillary ependymoma in the lumbar and sacral regions requiring craniospinal radiation therapy: a case report. Asian Spine J. 5, 68–72 (2011). 53 Hirano A, Matsui T, Zimmerman HM. [The
fine structure of ependymona]. No Shinkei Geka 3, 557–563 (1975).
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ultrastructural studies of two cases. Ultrastruct. Pathol. 15, 159–166 (1991). 55 Vaquero J, Zurita M. Ependymoma models
animal models of brain tumors. Neuromethods 77, 153–161 (2013). 56 Hirano A. Some contributions of electron
microscopy to the diagnosis of brain tumors. Acta Neuropath. 43, 119–128 (1978). 57 Kawano N, Yada K, Aihara M, Yagishita S.
Oligodendroglioma-like cells (clear cells) in ependymoma. Acta Neuropath. 62, 141–144 (1983). 58 Kawano N, Yada K, Yagishita S. Clear cell
ependymoma. Virchows Arch. A Pathol. Anat. Histopathol.415, 467–472 (1989). 59 Mrak RE. The Big Eye in the 21st century:
the role of electron microscopy in modern diagnostic neuropathology. J. Neuropathol. Exp. Neurol. 61, 1027–1039 (2002). 60 Ho L, Kee CC, Heon KC, Hoon YS et al.
Long-term outcomes of surgical resection with or without adjuvant radiation therapy for treatment of spinal ependymoma: a retrospective multicenter study by the Korea Spinal Oncology. Research Group. Neuro Oncol. 15, 921–929 (2013). 61 Donson AM, Birks DK, Barton VN et al.
Immune gene and cell enrichment is associated with a good prognosis in ependymoma. J. Immunol. 183, 7428–7440 (2009). 62 Pollack IF, Jakacki RI, Butterfield LH, Okada
H. Ependymomas: development of immunotherapeutic strategies. Expert Rev. Neurotherap. 13 (10), 1089–1098 (2013). 63 Yeung JT, Hamilton RL, Okada H, Jakacki R
I, Pollack IF. Increased expression of tumor-associated antigens in pediatric and adult ependymomas: implication for vaccine therapy. J. Neurooncol. 111(2), 103–111 (2013).
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The fine structure of ependymomas
Practice Points
Stavros J Baloyannis*1 & Ioannis S Baloyannis2 Electron microscopy may be considered a very useful method in establishing a definitive diagnosis of
brain tumors. In ependymomas we might conclude that the most common ultrastructural findings of high diagnostic
value are: ūū The numerous microvilli and cilia sometimes incorporated in the cell body or extended freely into the
intracellular space; ūū The centriole or blepharoplast located in the base of the cilia; ūū The large number of the fragmented microtubules in the perikaryon and the cellular processes (any
small cellular projection into the neutrophil or intracellular space); ūū The junctional apparatuses between the neoplastic cells, such as zonula adherens, zonula occludens
and puncta adherentia; ūū The basement membrane-like structure in many papillary ependymomas and ependymomas of the
filum terminale. The elongated cells in loose intracellular space, commonly seen in myxopapillary ependymomas of the
filum terminale. The precise and definitive diagnosis of brain tumors is of great value for determining the proper
treatment for the patient, the application of the recently developed immunotherapeutic strategies will allow this.
SUMMARY Electron microscopy is a useful diagnostic technique in order to confirm or establish a definitive diagnosis in brain tumors that may have an atypical histological pattern, which requires a concrete diagnosis. In ependymomas, electron microscopy reveals morphological characters that have a pathognomonic diagnostic value, therefore allowing a definitive diagnosis. The main fine structural criteria of ependymomas consist of the numerous microvilli and cilia, which are incorporated in the cell body or extended freely in the intracellular space; the centriole or blepharoplast, which is located in the basis of the cilia; the large number of the fragmented microtubules in the perikaryon and the Aristotelian University Department of Neurology, Thessaloniki & Research Institute for Alzheimer’s disease, Iraklion Langada, Greece Aristotelian University, Department of Neurosurgery, Thessaloniki, Greece *Author for correspondence: Tel.: +30 231027043; Fax: +30 2310270434; [email protected] 1 2
10.2217/CNS.13.64 © 2014 Future Medicine Ltd
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REVIEW Baloyannis & Baloyannis cellular processes (any small cellular projection into the neutrophil or intracellular space); the junctional apparatus between the cells, such as zonula adherens, zonula occludens and puncta adherentia; the basement membrane-like structure, seen in papillary ependymomas and ependymomas of the filum terminale; and the elongated cells in the loose intracellular space, commonly seen in myxopapillary ependymomas. Ependymomas, a general approach The CNS is a common place of cancerogenesis. Among the various tumors that may arise, gliomas are the most frequent primary brain neoplasms in humans. These are roughly divided, from a histological point of view, into astrocytomas, oligodendrogliomas and ependymomas [1]. Ependymomas account for 2–9% of all intracranial tumors. Although the majority of ependymomas are sporadic, a limited number can be seen as part of neurofibromatosis type 2 (or multiple inherited schwannomas, meningiomas and ependymomas [MISME] syndrome) [2]. Ependymomas may manifest at any age, from 1 month to 81 years [3]; however, they are mostly seen in children and are documented as the third most common primary brain tumor of childhood [4]. Thus, approximately 30% of pediatric ependymomas are diagnosed in children younger than 3 years of age, and in 60–70% of cases the tumor is located infratentorially, with a mean age presentation of approximately 6 years with no gender predilection [3]. Supratentorial ependymomas in children are commonly extraventricular [3,5]. Of those ependymomas that occur intraventricularly 58% originate in the fourth ventricle, whereas
Figure 1. Ependymoma cell from an ependymoma of the fourth ventricle. The cell has a large homogeneous nucleus with fine distribution of chromatin. The perikaryon is rich in organelles, 9000× magnification.
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the remaining 42% are located in the lateral and third ventricles [6]. In adults, 33% of brain ependymomas are infratentorial and 66% are located supratentorially [7]. In supratentorial ependymomas the mean age of the clinical manifestation of the tumors ranges between 18 and 24 years [5]. It is very rare to see supratentorial cortical ependymomas without any connection to the ventricular system. Nevertheless, in adults, 60% of ependymomas develop in the spinal cord, where they most frequently consist of intramedullary tumors of a peak incidence in the fourth decade of life, with males being more frequently affected than females. The majority of the ependymomas of the spinal cord are located at the conus medullaris, the filum terminale, the cauda equine or the cervical region of the spinal cord. Generally, intradural extramedullary ependymomas are highly unusual [8–10]. From a clinical point of view, the most common symptom of spinal ependymomas is lumbar, sacral or radicular pain, which is often worse at night or whilst in a recumbent position. This is also frequently associated with sensory changes, motor deficits, bladder and intestinal dysfunction, and impotence. The average duration of symptoms, preceding diagnosis, ranges from 1 to 9 years depending the location and the histological pattern of the tumor [11]. Ependymomas have also been described in the ovaries, soft tissues and mediastinum, although these are rare [12]. From the histological point of view ependymomas typically derive from the ependymal cells that line the ventricles of the brain, aqueduct and central canal of the spinal cord. The ventricles are surrounded by a lining of ependymal cells and subependymal glia [13], which may give rise to ependymomas, subependymomas and subependymal giant cell astrocytomas. However, ectopic ependymal cells are occasionally found within the brain itself, isolated from the ventricular system and trapped within the developing cerebral hemispheres during embryogenesis. These cells, may give rise to supratentorial cortical, lobar or extraventricular ependymomas, which, in the majority of the cases, do not retain any continuity with the ventricular system [3,14].
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The fine structure of ependymomas Intraventricular ependymomas, as well as any other intraventricular mass, usually induce increased intracranial pressure and hydrocephalus. Headache, vomiting, vertigo, cerebellar ataxia and gait disturbances are the most frequent clinical phenomena in ependymomas of the fourth ventricle, which are sometimes also associated with hemiparesis. In infants, a rapidly increasing head circumference is frequently noticed as one of the initial phenomena of the neoplasm and associated with sleepiness, vomiting, instability, vertigo and increased irritability. In supratentorial ependymomas, a substantial number of patients tend to frequently suffer from various focal neurologic deficits and seizures [13]. Children with intracranial ependymomas located in the fourth ventricle have a relatively less favorable prognosis than adults, despite recent advances in prompt diagnosis and treatment by microneurosurgical resection, radiosurgery, radiotherapy, chemotherapy and immunological methods. However, it is reasonable that the overall prognosis varies widely depending on the location, volume, histological characteristics and extent of tumor removal by surgery, which is normally the principal factor of tumor recurrence and patient survival. Infratentorial or anaplastic ependymomas may demonstrate a tendency (10–15%) to metastasize along the neuroaxis and through the cerebrospinal fluid [15]; however, there is no correlation between the grade and location of the tumor in the available data. Therefore, it is expected that mortality rates are higher in patients with metastasis than in patients with well-circumscribed primary lesions [16]. From a morphological point of view, ependymomas are soft, grayish or red pliable tumors that may contain cysts or small calcifications [13,17]. Ependymomas of the fourth ventricle mostly originated from the floor or the roof of the ventricle and then fill the ventricular lumen, they may occasionally extend through the foramen of Luschka into the cerebellopontine angle and possibly further into the adjacent parenchyma of the cerebellum, the pons and mesencephalon. Microscopically, ependymomas are cellular tumors characterized by the frequent formation of perivascular pseudorosettes and true ependymal rosettes. Perivascular pseudorosettes are composed of neoplastic cells radially surrounding the lumen of a capillary vessel, whereas true ependymal rosettes are composed of columnar cells arranged around a central lumen. Mitosis
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Figure 2. Ependymoma cell from an ependymoma of the fourth ventricle. The cellular process (any small cellular projection into the neutrophil or intracellular space) includes a large number of polymorphic mitochondria intermixed with twisted microtubules, 19,800× magnification.
is very rare. Thus, ependymomas are considered WHO grade II lesions [18]. Several histological subtypes of ependymal cell tumors have been described, such as ependymomas (WHO grade II tumors with cellular papillary, epithelial, clear cell and mixed variants), anaplastic ependymoma (WHO grade III tumor) and myxopapillary ependymoma (WHO grade I tumors frequently located along the filum terminale with an occasional extension into the conus medullaris) [1,18].
Figure 3. Ependymoma cell from an ependymoma of the fourth ventricle. Near the nucleus several fragmented cisternae of the Golgi apparatus are interrelated with dilated cisternae of the smooth endoplasmic reticulum, 28,800× magnification.
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Figure 4. Ependymoma cells from the fourth ventricle. In a short cellular process a lysosome containing laminated osmiophilic material is shown, 14,600× magnification.
The 2007 WHO classification recognizes three grades of ependymal tumors: grade I, which includes subependymomas (which occur near a ventricle or the central canal of the spinal cord) and myxopapillary ependymomas; grade II corresponding to ependymoma; and grade III corresponding to anaplastic ependymoma. In addition, the same classification recognizes four variants of ependymomas: cellular, papillary, clear cell and tanycytic, usually belonging to grades I or II [18]. The myxopapillary subtype of ependymomas is a benign variant of the tumor, with a peak incidence of clinical manifestation between the
Figure 5. Ependymoma excised from the fourth ventricle. A congregation of microvilli is shown, which are crowded inside the soma of the cells in a parallel arrangement. Some microvilli protrude into narrow lumen-like spaces that are created between the adjacent cells, 9000× magnification.
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third and fifth decades of life. Myxopapillary ependymomas mostly develop in the thoracolumbar region, and are recognized as the most common subtype of ependymomas in the filum terminale [19], accounting for 13% of all spinal ependymomas and 90% of the tumors in the conus medullaris [20]. Myxopapillary ependymoma have also been described in extraspinal locations, such as subcutaneously and in the brain, although these are rare [21–24]. Myxopapillary ependymomas are also considered to represent grade I tumors, characterized, as a rule, by slow growth [18]. From a morphological point of view, macroscopically the tumor is often intradural, encapsulated and usually anatomically isolated from direct access to routes of possible dissemination, although local invasion and distant metastases have also been described [23]. Microscopically, the main histological characteristics of diagnostic value are the papillae surrounding blood vessels, which are formed by the arrangement of cuboidal or columnar cells, and the mucinous degeneration within the vascular connective tissue, which is presumably formed by an inundation of plasma proteins. Low cellular variants and hyaline changes of myxopapillary ependymomas have also been described [25]. Tanycytic ependymoma is a very rare subtype of ependymomas that was first described by Friede and Pollak in 1978 [26]. Tanycytic ependymoma is a WHO grade II tumor in which the typical ependymal rosettes and perivascular pseudorosettes are mostly replaced by fibrillar cells in a pseudo-palisading arrangement. Microvascular proliferation is also not an uncommon phenomenon in tanycytic ependymoma tumors. Anaplastic ependymomas are histologically characterized by increased cellularity and marked mitotic activity, and are occasionally invasive and metastasize through the cerebrospinal fluid pathway [27]. In general, in ependymomas the neoplastic cells usually retain the main morphological features of normal ependymal cells. Therefore, they have cilia and microvilli, and a deeply stained blepharoplast that aids in the histological and ultrastructural identification of the tumor. However, some ependymomas undergo rapid and extensive differentiation either to anaplastic types or to mixed tumors, due to participation of other glial elements in their general histological profile.
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The fine structure of ependymomas
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Figure 6. Cilia and microvilli. (A) Cilium of an ependymoma cell from the fourth ventricle. In the base of the cilium a centriole (blepharoplast) is shown, 28,500× magnification. (B) Microvilli and cilia in cross section in the intracellular space of an ependymoma of the fourth ventricle, 28,500× magnification.
The fine structure The electron microscopy study of ependymomas is an important diagnostic procedure for defining the fine structure of the neoplastic tissue for their differential diagnosis from mixed tumors and other rare neoplastic processes. To describe the fine structure we studied 45 cases of ependymomas that had been excised from various areas of the CNS. Twenty of them derived from the fourth ventricle, 15 from the lateral ventricles and 10 from the filum terminale. In 35 cases the histological diagnosis was based on the general microscopical pattern of the neoplastic tissue and the finding of blepharoplast, which was a valuable pathognomonic criterion of the histological origin of the tumor. In 10 cases the differential diagnosis from astrocytomas was determined by light microscopy and a definite diagnosis was determined using electron microscopy. All the specimens were fixed in Sotelo’s fixing solution, embedded in araldite, contrasted with an aqueous solution of saturated uranyl acetate and lead citrate, and were studied either in a Siemens Elmiscope I electron microscope (Siemens, Munich, Germany) or in a Zeiss 9aS electron microscope (Carl Zeiss Group, Oberkochen, Germany) . Most of the ependymal cells had large homogeneous nuclei with fine distribution of the chromatin (Figure 1). Nuclear envelops were even or they rarely had cytoplasmic invaginations. The perikaryon was very rich in organelles. It included a large number of mitochondria, which were round or elongated (Figure 1) and frequently demonstrating an atypical pattern of
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mitochondrial cristae (Figure 2). In some areas of the perikaryon some aggregations of elongated mitochondria were seen including osmiophilic material in a agranular laminated pattern. The endoplasmic reticulum demonstrated numerous frequently dilated cisternae (Figure 3). Near the nucleus, several fragmented cisternae of the Golgi apparatus connected with cisternae of the smooth endoplasmic reticulum were frequently seen in ependymomas of the fourth ventricle (Figure 3). In some of the ependymoma cells of the fourth ventricle lysosomes were seen that contained laminated osmiophilic material (Figure 4). A common finding in ependymoma cells of the fourth ventricle was the congregation of microvilli. They were
Figure 7. Cilia of an epedymoma cell from the lateral ventricles. The cilia retain their typical elongated straight pattern, protrude into the extracellular space and are intermixed with numerous microvilli, 14,600× magnification.
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Figure 8. Cellular processes (any small cellular projection into the neutrophil or intracellular space) of ependymoma of the lateral ventricle. (A) Inside the perikaryon and in a considerable number of cellular processes numerous, fragmented or twisted microtubules are shown, which are intermixed with dilated cisternae of the smooth endoplasmic reticulum, 9300× magnification. (B) Twisted, fragmented microtubules in the cellular processes of ependymoma cells of the lateral ventricle, 14,600× magnification.
crowded inside the soma of the cells in a parallel arrangement or were occasionally protruding into the narrow lumen-like spaces between the adjacent cells, which were filled with numerous short microvilli (Figure 5) or cilia arising from the surrounding cells. Frequent congregations of microvilli in the extracellular space were composed of well-distinguished spheroid masses.
In close proximity to the microvilli were the centriole and the cilia. The centriole is located, as a rule, in the base of the cilia in the outer zone of the perikaryon (Figure 6). The cilia retained their typical elongated straight pattern and protruded into the extracellular space (Figure 7). Congregation of the cytoplasmic processes is a common phenomenon seen mostly in ependymomas of
Figure 9. Intercellular junctions. (A) Ependymoma cells of the fourth ventricle that developed zonula adherens and puncta adherentia between adjacent cells, 5000× magnification. (B) Junctional apparatuses, such as puncta adherentia, zonula adherens and zonulas occludants, between cell bodies and cellular processes of the adjacent cells of an ependymoma of the fourth ventricle, 5000× magnification.
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The fine structure of ependymomas the fourth ventricle. Elongated processes are arranged in a concentric orientation around an ovoid cellular profile. Inside the perikaryon, as well as in a considerable number of cellular processes (any small cellular projection into the neutrophil or intracellular space), we cobserved numerous fragmented or twisted microtubules intermixed with dilated cisternae of the smooth endoplasmic reticulum (Figure 8). Frequently the cells of the ependymomas were arranged in solid compact masses and many junctional apparatuses were seen between the cell bodies and the cellular processes of the adjacent cells. The majority of those structures appeared to be zonula adherens, puncta adherenta and zonula occludens (Figure 9). In proximity of some of the zonulae adherentes condensations of microfibrils and small round vesicles were occasionally seen. In the papillary type of ependymomas and the ependymomas of the filum terminale a typical basement membrane-like structure was seen (Figure 10). This consisted of three layers a filamentous outer one, an intermediate light layer, poor in filaments, and a dense inner one, which was directly connected with the plasma membrane of the cell (Figure 10). In some of the ependymomas of the filum terminale several large vesicles were seen inside the cellular processes. Some of the vesicles had an osmiophilic center that was encased by a light outer zone surrounded by a single membrane (Figure 10). In the myxopapillary ependymomas of the filum terminale many elongated cells with long, thin processes in a parallel arrangement were frequently seen in the loose intracellular space (Figure 11). Among the ciliated cells of the ependymomas, several large cells with irregular nuclear contours and abundant perikaryon, containing large number of multivesicular bodies, dilated cisternae of the smooth endoplasmic reticulum and numerous fragmented microtubules, were also seen (Figure 12). In some ependymomas of the lateral ventricles, neoplastic cells with long cellular processes containing bands of fibrils were occasionally intermixed with the ciliated ependymoma cells typically seen (Figure 13). Clear round cells, which might have been oligodendroglioma-like cells, were sometimes seen in ventricular ependymomas, demonstrating that microvilli, cilia and intercellular junctions advocate in favor of their ependymal origin (Figure 14).
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Figure 10. Typical basement membrane-like structure in a papillary-type ependymoma of the lateral ventricle. 9300× magnification.
Electron microscopy may be considered as a useful method in establishing a definitive diagnosis of brain tumors. In ependymomas we might conclude that the most common ultrastructural findings with a high diagnostic value are: the numerous microvilli and cilia sometimes incorporated into the cell body or extended freely into intracellular space; the centriole or blepharoplast located in the basis of the cilia; the large number of the fragmented microtubules in the perikaryon and the cellular processes; the junctional apparatuses between neoplastic cells, such as zonula adherens, zonula occludens and puncta adherentia; the basement membrane-like structure in many papillary ependymomas and
Figure 11. Elongated cells of a myxopapillary ependymoma of the filum terminale. These are characterized by numerous thin, long processes in a parallel arrangement, 4500× magnification.
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Figure 12. Large cells with irregular nuclear contours and abundant perikaryon. The cells contain a large number of multivesicular bodies, dilated cisternae of the smooth endoplasmic reticulum and numerous fragmented microtubules in a papillary ependymoma of the lateral ventricles, 5000× magnification.
ependymomas of the filum terminale; and the elongated cells in the loose intracellular space commonly seen in myxopapillary ependymomas of the filum terminale. The diagnostic value of most of these findings has also been previously demonstrated [28–31]. In addition, the abnormal mitochondria, fragmentation of the Golgi apparatus, dilatation of the cisternae of the smooth endoplasmic reticulum, multivesicular bodies prescence, twisted or fragmented microtubules, large osmiophilic
Figure 13. Neoplastic cells with long cellular processes. They contain bands of fibrils in an ependymoma of the lateral ventricles, 5000× magnification.
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vesicles and large numbers of small cellular processes may also be considered as common findings in ependymomas, although without a high diagnostic value as they may also be seen elsewhere, since during the neoplastic process the organelles of the perikaryon may undergo several morphological changes. A rather unusual ultrastructural finding was the enwrapping of the cellular processes, which composed laminated figures of a concentric arrangement. That pattern has been already described in oligodendrogliomas [32,33] and we believe this might be rarely observed in ependymomas. The ependymomas of the filum terminale usually retain some morphological features of the choroid plexuses, such as the basement membrane and the large number of cilia on their surface [34]. These cells rarely express neurosecretory activity, and therefore accumulate numerous large osmiophilic dense-coated vesicles, similar to those found in the pituitary gland and the human infundibulum [35]. Similar large neurosecretory cells have been also described in oligodendrogliomas of the posterior fossa [36]. Neurosecretory elements in the ependymomas of the filum terminale have also been described previously by Miller and Torack [37], who correlated the cellular elements of the caudal section of the spinal cord with Dahlgren cells, which are also of ependymal origin [38]. Generally, the secretory capacity of glial cells, ologodendrocytes or ependymal cells is a crucial problem that demands further investigation. In myxopapillary ependymomas, Lim et al. hypothesized that the ‘proteinaceous’ material and the hyaline vascular alterations may be a consequence of the increased vascular permeability that leads to diffusion of plasma proteins into the extravascular spaces [39]. The basement membrane seen in electron microscopy may be due to location of the tumor in juxtaposition to the collagen, which exists normally in the conus medullaris and the filum terminale. Although myxopapillary ependymomas are most often benign [40], malignant transformation may seldom occur [27], demanding a differential diagnoses from other more aggressive spinal cord tumors and metastases [41]. The contact of ependymonal cells with zonula adhaerens, zonula occludens and pucta adherentia is a frequent finding in well-differentiated ependymomas of the fourth ventricle and the filum terminale, as well as in papillary ependymomas of the lateral ventricles, whereas it is rarely seen in
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The fine structure of ependymomas poorly differentiated and anaplastic ependymomas. The zonula adhaerens is a predominant finding in cells near microvilli or cilia, which protrude into the intercellular space, although the zonula occludens is seen in the majority of the contacting cells in close proximity to zonula adhaerens. In addition, in cases of myxopapillary ependymoma of the filum terminale, some authors described septate junctions, a common type of junction of invertebrate epithelium. The junctions are characterized by a ladder-like appearance and demonstrate electron-dense septa arranged in a periodicity of 40–50 nm [42]. Hemiseptate-like junctions, zonula adherens and gap junctions were also described in the vicinity of the septate junctional structures [42]. However, the most common form of contact was the simple apposition of cells with a cleft approximately 200 Å wide, between their cell membranes [42]. The luminal surface of ependymal cells is characterized by the presence of cilia with a typical 9 + 2 arrangement of microtubules, a feature that is also seen in a substantial number of ependymoma cells. However, abnormal cilia, such as giant, compound, swollen, bizarre cilia, cilia with dynein arm defect, cilia with random orientation of microtubular doublets and abnormal axial microtubules were frequently seen in malignant ependymomas of the fourth ventricle and the vermis of the cerebellum [43,44]. However, disorders in cilia orientation and aberrant cilia may occasionally be seen in common ependymal cells. It is important to emphasize that, regarding their fine structure, ependymomas are mostly morphologically similar at any location of the axis of the CNS, although they might have distinct chromosomal imbalances [45,46], participate in different oncogenic pathways and display varying clinical behaviors depending on their location [47]. Shuangshoti et al. found that there was no significant relation between histopathology, Ki-67 proliferation index, p53 immunolabeling, tumor ploidy and biological behavior of ependymomas [7]. Frequently, several secondary lysosomes are seen in the soma of a substantial number of ependymomas. In addition, glial filaments, glycogen granules, intracytoplasmic lumina ciliated [48], might be seen in the perikaryon of ependymoma cells, resulting from invagination of the extracellular space within the cytoplasm. Rarely, rectangular or square crystalloid bodies, which are frequently seen in oligodendrogliomas [33], are intermixed with dense bodies and abnormal
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Figure 14. Clear round cell of ependymoma of the lateral ventricles. The perikaryon includes many abnormal mitochondria and microvilli profiles, 9300× magnification.
mitochondria in the soma of ependymoma cells. Pinocytotic vesicles have also been seen near the junctional apparatuses, which may suggest a possibility of cellular transport of fluid [48]. In tanycytic ependymomas, the neoplastic cells originate from the tanycytes, which are bipolar cells located plentiful in the vicinity of the lateral wall of the third ventricle, in the lateral ventricles, the fourth ventricle and the spinal cord [49], and may demonstrate a dumbbell arrangement [50,51]. Multifocal ependymomas are rare subvariants, belonging almost exclusively to the myxopapillary subtype [52]. Among the ependymoma cells, some astrocytes with long fibrillary cellular processes may also participate in the pattern of the tumor, representing mostly reactive cells [53]. At an experimental level, ependymomas may be developed by using chemical agents or certain viruses. However, xenografts in mouse models of human ependymomas may be considered the most useful experimental model [54]. Conclusion In conclusion, electron microscopy may reveal all the morphological characteristics of brain tumors [55] and can be used to determine the origin of the neoplastic cells [56,57], therefore, enabling a definitive diagnosis, which is essential for patient treatment [58,59], particularly allowing the application of recently developed immunotherapeutic strategies [60–63]. Future perspective Electron microscopy remains a very valuable method for providing a definitive diagnosis
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REVIEW Baloyannis & Baloyannis of neoplasms of the CNS. This is particularly relevant for immunohistochemical identification and classification of the various types and subtypes of brain tumors in the future enabling the expansion of the immunotherapeutic strategies. In the field of experimental cancerogenesis electon microscopy may also be instrumental in revealing, for example, the morphological alterations of organelles, protein trafficking, protein interactions, the role of mitochondria and Golgi apparatus, membrane receptors and cell–cell interactions during the neoplastic process, resulting in further
understanding of the biological behavior of brain tumors. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
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