Introduction
Secretory meningioma is an unusual histological subtype of meningiomas, characterized by focal epithelial and secretory transformation of meningothelial cells. It is a benign, slowly growing lesion, referred to as grade I tumour, according to the 2007 World Health Organization (WHO) classification of tumours of the central nervous system [24]. The incidence of this specific variant, reported in various studies, ranged from 1.1% to 4.4% of all meningiomas [7,12, 22,25,34,38]. Only one study documented a high incidence, accounting for 9.3% [1].
Secretory meningiomas are characterized by the presence of eosinophilic inclusions [20], which were originally described as hyaline bodies by Cushing and Eisenhart in 1938 (2nd edition 1962) [8] and later defined as “pseudopsammoma bodies” by Kepes in 1961 [14]. These inclusions are strongly stained with periodic acid-Schiff (PAS) and were immunopositive for some epithelial antigens, including cytokeratins (CKs), epithelial membrane antigens (EMA) and carcinoembryonic antigen (CEA) [2,5,7,25]. Moreover, pseudopsammoma bodies exhibit positivity for various secretory components, as immunoglobulins [4], -1-antitripsin (AT) and -1-antichymotrypsin (ACT) [7]. Ultrastructural features of secretory properties of the neoplastic cells were primarily demonstrated by Kepes [13] and confirmed by other electron microscopic findings [3,9,15-17,22]. Subsequently, the tumour was defined as “secretory meningioma” in 1986 by Alguacil-Garcia et al. [1]. From a clinical point of view, secretory meningiomas appear predominantly in women, with the most frequent location at the sphenoid ridge and frontal region. In comparison with other ordinary meningiomas, they are often associated with significant peritumoral oedema [5,12,25,26,36,38,40]. It has been speculated that some products of pseudopsammoma bodies might be involved in the development of cerebral oedema [23,26,36].
The aim of this report was to evaluate the clinico-pathological and ultrastructural features in the series of secretory meningiomas, including the immunohistochemical pattern of the lectin binding sites in tumour cells and pseudopsammoma bodies.
Material and methods
The study was performed on the surgical archival material from eight patients with postoperative diagnosis of secretory meningioma. Clinico-pathological characteristics of the patients are summarized in Table I.
All tumour specimens were fixed in 10% buffered formalin and routinely embedded in paraffin blocks. Sections were stained with haematoxylin and eosin (H&E) and periodic acid-Schiff (PAS).
Immunohistochemistry was performed on paraffin-embedded specimens; labelling was carried out by the avidin-biotin complex (ABC) method, with 3,3’-diaminobenzidine (DAB) as a chromogen. The following primary antibodies were used: anti-epithelial membrane antigen (EMA), anti-cytokeratin AE1/AE3 (CK), anti-CD34, anti-vimentin (vim) and anti-carcinoembryonic antigen (CEA); all reagents were obtained from Dako. Immunohistochemical staining of the lectin glycoconjugates was performed with 4 biotinylated lectins, as summarized in Table II: Peanut agglutinin (PNA), Soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), Concanavalin A (Con A) (obtained from Vector Laboratories, Burlingame, CA, USA). Briefly, the deparaffinized sections were incubated with lectins in concentration of 10 µg/ ml for 1 h, and then with the avidin-biotin peroxidase complex (ABC Kit) and 3’3’diaminobenzidine (DAB, Dako) reagents and counterstained with haematoxylin; details of the used method are presented in our previous report [33].
For electron microscopy, the small fragments of paraffin embedded specimens were selected, then deparaffinized, rinsed in 0.1 phosphate buffer, postfixed in solutions of 2% glutaraldehyde and 1% osmium tetroxide in the same buffer, dehydrated in graded alcohols and embedded in Epon 812. Ultrathin sections stained with uranyl acetate and lead citrate were examined with a JEM-1010 electron microscope (JEOL, Japan).
Results
Histopathological findings
All meningiomas were characterized by the presence of eosinophilic and PAS-positive pseudopsammoma bodies (Fig. 1A, B) that occurred on the background of various basic histopathological growth patterns, assessed as meningothelial, transitional, fibroblastic or angiomatous type (Table I). Pseudopsammoma bodies exhibited a wide spectrum of morphology, regarding their size and shape. The number and distribution of inclusions greatly varied within the same tumour. The bodies were loosely scattered or collected in groups and exhibited diverse forms, ranging from minute homogenous and brightly eosinophilic spheres to huge heterogeneous, weakly eosinophilic deposits, surrounded by clear “halo” (Fig. 1A).
Immunohistochemistry
Immunohistochemical studies revealed a strong immunoreactivity for CK, limited to the part of the cytoplasm of tumour cell that bordered the pseudopsammoma bodies. This CK expression usually displayed the form of a strong ring-like pattern around pseudopsammoma bodies (Fig. 2A). Immunoreactivity for CEA, of varying intensity, was evidenced both in the pseudopsammoma bodies and in the surrounding cytoplasm of neoplastic cells (Fig. 2B). Pseudopsammoma bodies were also immunopositive for EMA (Fig. 2C) but they were negative for vimentin, what was in contrast to overall positivity of tumour cells (Fig. 2D).
Immunohistochemical staining with the lectins showed that the majority of pseudopsammoma bodies and surrounding neoplastic cell cytoplasm were strongly labelled with PNA (Fig. 3A) and SBA (Fig. 3B), although the individual bodies remained unstained or were only weakly stained with the both lectins. Immunolabelling with Con A showed irregular staining of pseudopsammoma bodies. It was intense mainly in small bodies and weak in larger ones, whereas several bodies and the cytoplasm of neoplastic cells were unlabelled (Fig. 3C). Immunostaining with DBA was rarely positive in pseudopsammoma bodies and was negative in the tumour cells cytoplasm, however, the slight reactivity for DBA was seen in the nuclei of neoplastic cells (Fig. 3D).
Electron microscopy
Electron microscopic examination revealed the presence of numerous neoplastic cells, exhibiting intracytoplasmic lumina filled with microvilli and aggregations of homogenous or heterogeneous material, corresponding to pseudopsammoma bodies (Fig. 4A, B). The cytoplasm of these cells was characterized by increased electron-density, rich endoplasmic reticulum and Golgi complex vesicles and conspicuous bundles of tonofilaments, often in the close connection with intracytoplasmic lumina and microvilli (Fig. 4A, B). Furthermore, distinct interdigitations of cell processes with desmosomes and numerous microvilli, projecting from the plasmalemma to the extracellular space, were seen (Fig. 4C, D). The size of the pseudopsammoma bodies and content of intracytoplasmic lumina greatly varied. Small lumina usually contained moderately electron dense, fine filamentous and rather homogenous substance, similar to those covering the surface of microvilli (Fig. 4B). Enlarged lumina were filled with polymorphic, filamentous or granular electron dense material, either homogenous or exhibiting a mixture of heterogeneous vesicles, membranaceous structures and dense bodies (Fig. 5A, B). Numerous lumina of different size, filled with polymorphic pseudopsammoma bodies, were often seen in the cytoplasm of the same cell. Some pseudopsammoma bodies seemed to be located extracellularly and lacked the obvious lumina. They were associated with numerous microvilli, extending into intercellular space (Fig. 5C). Pseudopsammoma bodies were frequently surrounded by numerous fascicles of tonofilaments (Fig. 5D).
Discussion
All secretory meningiomas in our series showed focal epithelial and secretory transformation with accumulation of pseudopsammoma bodies. The same features have been previously demonstrated in other reports [13,22,29,35,38]. Pseudopsammoma bodies are most frequently associated with the meningothelial pattern of the tumour, however they might occur also in other histological subtypes of benign meningiomas, including lipomatous or vacuolated component [18,20,21,39]. The secretory meningiomas of the present study, comprised tumours with the basic growth pattern of the meningothelial type (4 cases), angiomatous type (2 cases), transitional type (1 case) and fibroblastic type (1 case). The mechanism, by which the individual neoplastic cells of any subtype of meningioma might be transformed into the secretory cells, is unknown. The most characteristic cellular abnormalities, corresponding to the epithelial and secretory features, consist of the presence of intracytoplasmic lumina with accumulation of secretory products [6,7,11,19]. Epithelial and glandular-like differentiation of the cells has been also indicated with CK, EMA and CEA immunoreactivity in many studies [1,2,25,32,35]. Moreover, our present study demonstrated the abnormal glycosylation pattern of secretory meningioma cells.
Considering the morphology of pseudopsammoma bodies, it ought to be noted that histochemical and ultrastructural patterns of pseudopsammoma bodies exhibit the striking resemblance to the inclusions, observed within intracellular lumina in some epithelial neoplasms, especially in mammary and gastro-intestinal carcinoma or mesothelioma [4,13,16]. In epithelial cancer cells, the intracytoplasmic lumina appears as spherical cavities with microvilli and accumulation of secretion products, and they are usually surrounded by a filamentous network [27,28]. Various types of intracytoplasmic lumina have been demonstrated in electron microscopic study of breast carcinoma: type A with many microvilli on the luminal surface and abundant secretion within the lumen; type B with fewer microvilli and little or abundant secretion, and a third type of large intracytoplasmic lumina with abundant secretion and compression of the nucleus [30,31].
The present ultrastructural investigations of secretory meningiomas documented that the pseudopsammoma bodies were predominately located within large intracytoplasmic lumina, lined by microvilli. The same ultrastructural features of pseudopsammoma bodies were also demonstrated in other studies [3,9,13,16,37]. Less frequently the pseudopsammoma bodies were observed within extracellular space, filling by numerous projecting microvilli [13,15]. Both intracellular and extracellular secretory material might exhibit various forms of homogenous deposits of moderately electron-dense fine filamentous and granular substances or polymorphic large inclusions with a mixture of microvesicles, lamellar structures and electron dense clumps. These findings indicated that ultrastructural features of the secretory type of meningioma differ from the fine structure of other types of meningothelial tumours [10].
We found also some cells with predominance of small intracytoplasmic cavities, accompanied by intraluminal protrusions of microvilli and scanty secretory material. These features probably represent an initial stage of secretory activity. Sometimes, the small lumina coexisted with larger and polymorphic pseudopsammoma bodies. These observations indicate the dynamic secretory process and support the suggestion that formation of the large polymorphic bodies represent the final stage of accumulation and degradation of secretory products [3,15-17,22].
The previous histochemical reports showed that the products of secretion are composed mainly of conjugated glycoproteins [9,13,17] and are reminiscent to the glycoproteinaceous coating seen on the surface of intestinal and respiratory epithelia [16]. Immunohistochemical characteristics of pseudopsammoma bodies with one lectin were given by Kuratsu et al. [17]. The authors used only Concanavalin A staining and postulated that secretory bodies in meningiomas belong to mucosubstances of class II and III.
In the present study we demonstrated the reaction of the pseudopsammoma bodies and the tumour cells with four lectins, including glucose and mannose binding Con A and three lectins specific to galactose/N-acetylgalactosamine (GalNAc) glycoconjugates with differential binding sites, i.e. PNA, SBA and DBA. Our results showed that pseudopsammoma bodies and surrounding cells presented a large range of glycan structures. Pseudopsammoma bodies were stained more intensely with PNA and SBA, moderately with Con A and weakly with DBA, whereas the cytoplasm of the surrounding neoplastic cells was labelled mostly with PNA and SBA. Very small secretory granules, probably corresponding to small intracytoplasmic lumina, were also labelled with the lectins and visualized especially with Con A. These findings and our previous study of the lectin binding in various subtypes of meningioma indicated that the pattern of lectin glycoconjugates in secretory meningioma differs from other variants of meningothelial tumours [33]. Moreover, the variations in lectin bindings were demonstrated between the secretory cells and other neoplastic cells within the same tumour. Enhanced expression of the lectin glycoconjugates in the meningioma secretory cells probably reflects the selective changes in tumour cell glycosylation, that may be important for their phenotypic heterogeneity. Expression of CEA, which is a highly N-glycosylated glycoprotein, is also the characteristic finding in secretory meningiomas [1,5,22,36,37]. We demonstrated a similar pattern of the immunoreactivity for CEA and the binding sites for PNA in the pseudopsammoma bodies and the cells surrounding them.
Concluding, the present study documented the heterogenous ultrastructural and lectin binding pattern of pseudopsammoma bodies and illustrated the status of altered glycosylation in the neoplastic cells of secretory meningiomas.
Disclosure
Authors report no conflict of interest.
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