Acta

Acta Neuropathol (Berl) (1987) 73:124-130

Heuropatholosca

9 Springer-Verlag 1987

Contribution of histiocytic cells to sarcomatous development of the gliosarcoma* An immunohistochemical study N. Kochi and H. Bodka

Neurological Institute, Universityof Vienna, Schwarzspanierstral3e17, A-1090 Wien, Austria

Summary. The expression of glial fibrillary acidic protein, fibronectin (FN), factor VIII-related antigen (FVIII/RAG), and of three monohistiocytic markers, lysozyme, ~-l-antitrypsin and ~-l-antichymotrypsin was examined in five gliosarcomas (GS) by peroxidase-antiperoxidase immunostaining of formalinfixed and paraffin-embedded specimens, and compared with vascular changes in 16 glioblastomas (GB). In contrast to GB, endothelial proliferations of GS were sheathed by sarcomatous tissue (perivascular sarcoma), which was contiguous with fibrosarcomatous areas. Cells with conspicuous intracytoplasmic FN content (FN § cells) were seen in the vascular stroma of GB and dominated in the sarcomatous parts of GS. Most FN § cells of GS were of varying size and shape and clearly neoplastic. Monohistiocytic markers were demonstrable in small infiltrating mononuclear cells as well as in many sarcomatous cells including FN § cells. FVIII/RAG was restricted to lumen-lining endothelium and was not found in sarcomatous cells. These results suggest that a major part of sarcoma in GS is less likely to develop from proliferated endothelial cells than from histiocytic cells in the perivascular spaces of GB. By FN mediation, histiocytic cells might also guide and promote sarcomatous proliferations of other mesenchymal cells, leading to fibrosarcomatous development. Prominent monstrous giant cells of one GS seemed to be degenerating glioma cells. Key words: Gliosarcoma -

Glioblastoma -

Fibro-

The designation of gliosarcoma (GS) was first used by Stroebe in 1895 [41]. It was implied later that Stroebe had misinterpreted the vascular hyperplasia of glioma as neoplastic; his concept was ignored until 1955, when Feigin and Gross presented true mixed tumors composed of both gliomatous and sarcomatous tissues [8]. Since then, Feigin's basic concept of possible sareomatous development from proliferating vessels has found general acceptance [9, 26, 35, 40]. However, Feigin's view that sarcoma cells of GS are derived from endothelial cells of glioblastoma (GB) vessels was challenged by Rubinstein, who regarded cells of some GS to originate from adventitial fibroblasts, and presented some cases with a reverse development, namely glioma arising in intracranial sarcoma (sarcoglioma) [22, 34, 35]. Endothelial proliferation is far more prominent in GB than in other tumors according to the microscopic angiogenesis grading system score of Brem et al. [3]. That correlates well with incidence: GS is more frequent than mixed tumors of all categories of other organs in man [9]. Recent immunohistochemical studies [24, 38, 39] gave contradictory results with regard to the expression of specific markers of endothelial cells in sarcoma cells of GS. Recently, when we studied the development of stroma in non-Hodgkin's malignant lymphomas of the CNS by means of immunocytochemistry, prominent appearance of histiocytic cells was noted [20]. That prompted us to evaluate the role of histiocytic cells in GS. This study demonstrates that histiocytic cells contribute not only to stromal construction in GB, but also to sarcomatous development in GS.

nectin - Histiocyte - Immunocytochemistry Materials and methods

* In part presented at the Xth InternationalCongressof Neuropathology, September 7-12, 1986, in Stockholm Offprint requests to: H. Budka (address see above)

GS was diagnosedin histologicalspecimensof ten operationsfor brain tumor in eightpatientsduringa 7-yearperiod, constituting 1.4% of 720 operated gliomasof all types and sites and 2.5% of 400 glioblastomas. In three of these GS patients, blocks were not available for immunohistochemistry.Bioptical tissue of 16 GB was selectedfor comparison of GS with GB.

125

N. Kochi and H. Budka: Histiocytic cells contribute to gliosarcoma Table 1. Clinicopathological data of five patients with gliosarcoma (GS) Case

Age

Sex

Location

Course and histological diagnoses GS GB + LMI developing to GS (2 operations, interval 4 months) GS AC III developing to GB + LMI and further to GS + LMI (total 5 operations, 3 x GS, intervals 2, 3, 3 and 2 months, respectively) GS

1

65

M

It F

2 3 4

44 65 41

M M M

rt FT rt T rt F

5

61

M

rt T

M: Male; lt: left; rt: right; F: frontal; T: temporal; AC III: anaplastic astrocytoma; GB: glioblastoma; LMI: leptomeningeal invasion

Tumor specimens were fixed in buffered formalin, embedded and stored in paraffin. Immunostained sections were compared with sections stained by hematoxylin-eosin, Gomori's, Van Gieson's, toluidine blue and phosphostungic acid-hematoxylin stains. Sixteen GB specimens and seven GS specimens were immunostained by the peroxidase-antiperoxidase (PAP) technique for fibronectin (FN) [19], factor VIII-related antigen (FVIII/RAG) [20] and glial fibrillary acidic protein (GFAP) [14] as previously described. All GS specimens and one GB were immunostained in addition for lysozyme (LZ), e-l-antitrypsin (A1AT) and c~-l-antichymotrypsin (AIACT) [20]. Specificity controls included substitution of the primary antiserum by nonimmune rabbit serum, and substitution of further steps by buffer. These control sections showed no immunoreactivity. Results

Clinicopathological findings of five GS cases are summarized in Table 1. The three primary cases were described as well-circumscribed tumors similar to metastatic brain tumors. There was no adhesion to the dura. Sharp demarcation of tumor was confirmed by histology, exhibiting a fibrous capsule surrounded by gliotic brain tissue, qn two cases, GS developed after previous operations with histological diagnoses of malignant glioma. After diagnoses of GB, cases 2 and 4 underwent radio- and chemotherapies. Material from the last operation o f case 4 revealed diffuse leptomeningeal spread of GS. All GS had distinct fibrosarcomatous parts, characterized by spindle-shaped large cells with irregular elongated nuclei in parallel rows, surrounded by abundant reticulin and collagen fibers. Cartilagineous and osseous metaplasia was found in case 3. With the exception of case 3, all specimens showed a few to moderately many giant cells; case 5 was characterized by abundance o f monstrous giant cells. Immunostaining for G F A P and F N (Fig. l a) demonstrated a mosaic-like arrangement o f gliomatous and sarcomatous parts o f most GS. Only case 4 showed dense intermingling growth of spindleshaped G F A P + and F N + cells, which could be differentiated as gliomatous or sarcomatous only by immunostaining (Fig. 2c, d).

Perivascular sarcoma Moderate to strong staining for F N was found in intratumorous reticulin fibers, vascular walls and basement membranes. Stromal parts o f GB and sarcomatous tissue of GS were diffusely stained for F N (Fig. 1 a). In contrast, F V I I I / R A G was restricted to lumen-lining endothelial cells (Fig. 1 b). The three primary GS cases showed sarcomatous changes around all vessels, most prominently in case 3, where all vessels were covered by thick, lattice-like sarcomatous tissue (Fig. 1 a,b). In the center of GS, endothelial proliferations of linear, serpentine and glomeruloid patterns were surrounded by sheaths of sarcomatous cells (Fig. 2a, b; e, f) which were contiguous with typical fibrosarcomatous areas (Fig. 2e, f). These features o f perivascular sarcoma and fibrosarcoma were distinct from frequent vascular proliferations in GB, which were usually restricted to F V I I I / R A G § endothelial cells.

Histiocytic cell proliferation in sarcomatous tissue of GS GS cells with conspicuous intracytoplasmic F N content (FN + cells) were scattered from the vicinity of vascular walls to the outer rims of perivascular sarcomatous tissue (Fig. I a). They were of varying size and shape, included mitotic figures, and corresponded to the cellular anaplasia of sarcoma cells (Fig. I f). A few monstrous cells contained occasional F N + cytoplasmic inclusions (Fig. i f). Many sarcoma cells, including F N + cells, were stained for A1AT (Fig. 1 c) and A1ACT (Fig. 1 d), including mitotic stages (Fig. 1 c). LZ was restricted to scattered small cells (Fig. l e). F N + cells and cells stained for monohistiocytic markers were more frequent in perivascular sarcomatous parts than in compact fibrosarcomatous parts. The cartilagineous and osseous metaplasia in case 3 was negative for all markers. Monohistiocytic markers were absent from endothelial cells, pericytes, smooth muscle cells,

Fig. 1 a, b. Peroxidase-antiperoxidase (PAP) stain for fibronectin (FN) (a) and factor VIII-related antigen (FVIII/RAG) (b) in gliosarcoma (GS) case 3. Mosaic-like arrangement of gliomatous islands surrounded by strongly FN-positive sarcomatous network (a). The lumen of all vessels stained for FVIII/RAG (b) is covered with thick sarcomatous tissue. Slight hemalum counterstain, x 75. c - e PAP stain for c~-l-antitrypsin (A1AT) (c), c~-l-antichymotrypsin (A1ACT) (d) and lysozyme (LZ) (e) in GS case 3. Many sareomatous cells show strong granular expression of A I A T and A1ACT including mitotic figures (arrow). LZ is distributed only in few infiltrating small mononuclear cells, x 300. f P A P stain for F N in GS case 5. Cells with conspicuous intracytoplasmic F N content (FN + cells) are scattered in sarcomatous tissue of GS. Shape and size of F N + ceils vary. Some monstrous giant cells contain F N + material, x 300

Fig. 2a, b. PAP stain for F N (a) and F V I I I / R A G (b) in serial sections of GS case 5. There are vigorous endothelial proliferations of glomeruloid vascular structures sheathed by sarcomatous F N + cells, x 190. e, d PAP stain for F N (c) and G F A P (d) in GS case 4. Adjacent sections show intermingling growth o f F N + and G F A P + cells of similar shape. • 300. e,f Case 4. Perivascular F N + sarcoma is contiguous with fibrosarcoma (lower right) (e); immunoreactivity for FVIII/RAG is restricted to lumen-lining endothelia in an adjacent section (f). x 100

128 fibroblasts or leptomeningeal cells, but were seen in polymorphonuclear leukocytes. Giant cells in GS

Giant cells were usually found in gliomatous areas. Some monstrous giant cells in case 5 were stained for GFAP, but many lacked expression of GFAP or other markers. As compared with monstrous cells of GB cases, differences in staining behavior were not apparent. Many small infiltrating mononuclear cells, which stained for monohistiocytic markers, were seen in gliomatous islands, frequently surrounding monstrous cells. Discussion

Mixed tumors have been classified into collision, composite and dependent types [10], but these types are difficult to distinguish by histology. Ahnost all intracranial mixed tumors were reported as dependent tumors; most are gliosarcomas [8, 9, 26, 35]. All GS cases of this study showed contiguity from perivascular sarcoma to fibrosarcoma. FVIII/RAG + capillary proliferations, comparable to those of GB, had FN + sarcomatous sheaths. It thus appears that in GS, sarcoma develops perivascularly and further progresses to fibrosarcoma. Sarcomatous cells of GS were considered to originate from endothelial cells [9]. This assumption was recently tested by immunohistochemical studies [24, 38, 39]. FVIII/RAG is considered as a specific marker of endothelial cells [24]. McComb et al. [24] reported expression of FVIII/RAG only by lumenlining endothelial cells of GS but not by adventitial or sarcomatous cells; they concluded that sarcomatous cells in GS derive from pericytes, perivascular fibroblasts or endothelial cells, which do not express FVIII/RAG. Schiffer et al. [38] described expression of FVIII/RAG, which became weaker in cells distant from the lumen when endothelial cells composed multiple layers; sarcomatous cells of GS were considered to originate from endothelial cells, which lost antigenicity for FVIII/RAG. That interpretation was supported by Slowik et al. [39]. This study showed FVIII/RAG restricted to lumen-lining endothelial cells whenever endothelial cells could be distinguished from adventitial and sarcomatous cells. However, immunoreactivity for FVIII/RAG at some distance from lumen-lining endothelia [38,39] is difficult to accredit to specific structures at the light microscopical level. FVIII/RAG is also distributed in plasma and basement membranes [16], which thus may contribute to some immunoreactivity for FVIII/RAG in tissue. Ulex europaeus

N. Kochi and H. Budka: Histiocyticcells contribute to gliosarcoma agglutinin (UEA) lectin seems to be a more sensitive marker for neoplastic endothelial cells than FVIII/ RAG, as inferred from strong expression of UEA in angiogenic solid tumors, even by cells which are remote from the lumen [2]. The occasional demonstration of UEA-reactive sarcomatous cells in GS [39] suggests that endothelia indeed may contribute to the sarcomatous growth in GS, whereas extent and regularity of such endothelial contribution to GS have remained controversial. Here, abundance of monohistiocytic markers and F N in sarcomatous areas of GS, including immunoreactivity of anaplastic and mitotic cells, as well as absence of FVIII/RAG, suggest a prominent role of histiocytic rather than endothelial cells for the development of GS. This interpretation is compatible with an electron microscopic study which showed abundant lysosomes as the most characteristic feature of sarcomatous cells of GS [29]. Sources of histiocytic cells could be resident macrophages in the cerebrospinal fluid, described as fibroblastic pial cells [30], perivascular cells, epiplexus and supraependymal cells [27], and blood monocytes belonging to the mononuclear phagocytic system [27, 31, 32]. FN production, as demonstrated here in the sarcoma of GS, was described in macrophages [17, 42, 43] and in monocytes in culture [1]. Conspicuous cytoplasmic FN content of histiocytes was repeatedly reported [4, 5, 28]. FN + histiocytic cells were described as contributing to production of reticulin fibers in lymph nodes [33] and stroma of Hodgkin's lymphomas [25] and of malignant non-Hodgkin's lymphomas of the CNS [20]. Confusion with mast cells was excluded by absence of metachromasia in toluidine blue stain. The marked difference in numbers of histiocytic cells stained for A1AT and/or A1 ACT and those stained for LZ is in accordance with previous reports [6, 28]: positive staining of LZ is limited to a small portion of non-neoplastic reactive histiocytes [32]. In this study, monohistiocytic markers were clearly restricted to the sarcomatous portion of GS. It was recently emphasized by Kepes [18] that, under some circumstances, glioma cells may acquire monohistiocytic properties including immunoreactivity for monohistiocytic markers; however, in this series such immunoreactivity was absent in gliomatous parts. Histiocytic cell markers were less frequent in typical fibrosarcomatous areas than in perivascular sarcoma. This finding might be interpreted in two ways: first, malignant histiocytic cells could represent "facultative fibroblasts" [11], which lose antigenicity for monohistiocytic markers after differentiating to fibrosarcoma. Second, a major role of histiocytic cells could be to guide and promote sarcomatous proliferations of other mesenchymal cells. FN was found

N. Kochi and H. Budka: Histiocytic cells contribute to gliosarcoma to p r o m o t e cell migration in vitro [16], in wound healing [12] and formation of granulation tissue [15, 21]. Mesenchymal cells influenced by such F N mediated histiocytic p r o m o t i o n could be perivascular fibroblasts, leptomeningeal cells or, in accordance with findings by Schiffer et al. [38] and Slowik et al. [39], also some endothelial cells. Anyhow, the perivascular space around small vessels of G B is the likely cradle of sarcomatous development leading to GS. Most intracranial giant cell tumors are regarded now as giant cell glioblastoma [36] according to electron microscopy [13, 23] and G F A P immunohistochemistry [7]. However, Schiffer et al. [37] suggested that b o t h glioma and sarcoma cells could develop to monstrous cells. Although we found F N + cytoplasmic inclusions in some monstrous cells of case 5, we would interpret this as secondary phagocytic uptake rather than true F N production. Moreover, some monstrous cells were immunoreactive for G F A P . Thus monstrous cells are more likely to belong to the glioma than to the sarcomatous parts of GS. Prominent vascular proliferations in G B must be separated from GS, which is m u c h less frequent than GB. Incidence o f GS a m o n g all gliomas according to Feigin et al. [9] is 2% for fully developed GS, which is very similar to 1.4% o f our series, and 5% for all GS including "partially developed sarcomatous changes". Later, M o r a n t z et al. [26] described 5% GS in astrocytomas and 8% GS in GB; the latter figure is higher than the 2.5% o f GS in our GB series. Since stromal and vascular proliferative changes cover a wide spectrum in GB, it can be understood that diagnostic criteria for GS vary between observers. In conclusion, histiocytes seem to contribute prominently to sarcomatous development of GS; however, this does not necessarily mean that the sarcoma of GS should be regarded as a malignant fibrous histiocytoma ( M F H ) . M F H is the m o s t c o m m o n soft tissue sarcoma of late adult life and contains both fibroblast-like and histiocyte-like cells in varying proportions [44]. Future studies using specific monoclonal antibodies against different phenotypes of cells belonging to the mononuclear phagocytic system might elucidate the relation between M F H and GS.

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Received October 29, 1986/Accepted December 2, 1986