Path. Res. Pract. 164,385-4°1 (1979)
Department of Pathology, University of Muenster, Muenster, West Germany (Chairman: Prof. E. Grundmann, M. D.)
Malignant Fibrous Histiocytoma of Bone and Osteosarcoma. A Comparative Light and Electron Microscopic Study A. ROESSNER, H. P. HOBIK, and E. GRUNDMANN':'
Summary Malignant fibrous histiocytomas are well-described tumors of the soft tissues. Recent investigations have shown that malignant histiocytoma may also occur as a primary bone tumor. However, difficulties may arise to distinguish malignant histiocytoma of bone from other malignant bone tumors, such as osteosarcoma. In the present study, the ultrastructure of five cases of malignant fibrous histiocytoma of bone is compared with that of osteosarcoma. The results show that malignant fibrous histiocytoma is composed mainly of histiocytic cells and fibroblastic cells. In addition, xanthomatous cells, undifferentiated cells, and giant cells may be observed. By contrast, the predominant cell type in osteosarcoma is the neoplastic osteoblast, characterized by abundant rough endoplasmic reticulum. Signs of matrix calcification in the intercellular matrix between the collagen fibrils are regularly observed in osteosarcoma, but not in malignant histiocytoma. From these results it is concluded that the ultrastructure of malignant fibrous histiocytoma arising in bone is morphologically identical with the soft tissue counterpart of this tumor. The components of the tumor are derived from neoplastic histiocytes, This cytogenesis differs from that of osteosarcoma, which is derived from neoplastic osteoblasts. Therefore, from the ultrastructural point of view, malignant fibrous histiocytoma of bone should be accepted as a distinct histologic entity among bone tumors.
Introduction Fibrohistiocytic tumors showing bimodal differentiation into histiocytic and fibroblastic cells, such as fibrous histiocytoma and malignant fibrous histiocytoma, are well defined soft tissue tumors (Kempson et al., 1971; Ekfors et al., 1978). The ultrastructural characteristics of cell types involved in malignant fibrous histiocytoma of soft tissue have been described in several electron microscopic studies (Merkow et al., 197 I; Fu et al., ':. Dedicated to Prof. Dr. E. Vehlinger in honour of his anniversary, 8th August 1979
386 . A. Roessner, H. P. Hobik, and E. Grundmann
1975; Taxy et al., 1977). Recent investigations have suggested the possibility that malignant fibrous histiocytoma may occur as a primary tumor of bone (Spanier et al., 1975; Dahlin et al., 1977a). As far as we know, the use of electron microscopy for analyzing the cell types involved in malignant fibrous histiocytoma of bone has been described in only one case report of this disease (Johnson et al., 1978). It is sometimes difficult to distinguish malignant fibrous histiocytoma from other malignant bone tumors, such as osteosarcoma and fibrosarcoma (Dahlin et al., 1977a, b). Osteosarcoma, especially in its anaplastic phase, may show changes not unlike those in malignant fibrous histiocytoma (Uribe-Botero et al., 1977). Therefore Dahlin et al. (1977a) proposed to limit diagnosis of malignant fibrous histiocytoma of bone strictly to those cases where the entire tumor shows the characteristic pattern of fibrocytic and histiocytic cell differentiation. Another problem in diagnosis arises from the fact that malignant fibrous histiocytoma of bone may produce an osteoid-like ground substance (Dahlin et al., 1977a, b). Since all malignant bone tumors capable of osteoid production are usually classified as osteosarcoma, there are no absolute criteria for differentiating malignant fibrous histiocytoma of bone from osteosarcoma at the level of light microscopy. Thus in our present study of 5 cases, the ultrastructure of malignant fibrous histiocytoma of bone is compared to that of osteosarcoma. We sought to distinguish and describe the different cell types occurring in either of these lesions, and to determine to what extent the cytologic features of these cell types might point towards a difference in cell origins. Special interest was focussed on the value of electron microscopic investigation in the differential diagnosis of malignant fibrous histiocytoma of bone.
Material andMethods Case material Light microscopy
Tissues were fixed in neutral formalin, embedded in paraffin, and stained with hematoxylin and eosin, van Gieson, Ladewig, or Azan for light microscopic investigation. Electron mtcroscop y
Material for electron microscopic investigation was obtained in most cases at the time of frozen section diagnosis. Tissue specimes from various parts of the tumor were minced into 1 mm pieces immediately after removal, fixed in 2.25% glutaraldehyde (0.05 m phosphate buffer, pH 7.4) for 2 hrs at 4° C, then rinsed in buffer for 24 hrs. Postfixation was carried out in 1.33010 osmic acid with 0.05 m phosphate buffer, pH 7.4,
Malignant Fibrous Histiocytoma of Bone and Osteosarcoma . 3 87 The clinical data are summarized in the following table: Malignant fibrous histiocytoma of bone case no. 2 3 4 5
age
sex
localization
18 55 27 18 42
m m f m m
distal femur right posterior 5th rib proximal humerus ilium head of fibula
12 13 15 12 13
f f m m
distal femur distal femur proximal fibula proximal fibula proximal femur
Osteosarcoma
2 3 4
5
for 2 hrs. The tissues were dehydrated in graded ethanols, and embedded in Epon 812. Sections were cut with a Porter-Blum ultramicrotome, mounted on 3 mm copper grids, and poststained for 15 min with 5% uranyl acetate and 3 min with lead citrate. Specimens were examined with a Siemens Elmiskop I a electron microscope at 80 kV using the double condenser. Semithin sections from Epon-embedded tissue were stained with toluidine blue for light microscopy.
Results Light microscopy
In all our cases of malignant fibrous histiocytoma of bone, tumor cells appeared densely packed, showing cytologic signs of malignancy: nuclei with irregular shapes and a wide variation in size. Nuclei were often hyperchromatic with prominent nucleoli. Mitotic figures were occasionally observed. Some tumor areas were composed of spindle cells arranged in the typical cartwheel or storiform pattern (Fig. I a). With Ladewig staining, the collagen fibers gave a picture similar to the osteoid-like ground substance in osteosarcoma. Some ossseous fragments were occasionally observed between tumor cells. In other areas, the tumor consisted largely of epitheloid cells and some multinucleated giant cells. Collagen fibers in these areas were less well developed and showed no similarity with the osteoid-like ground sub-
3 88 . A. Roessner, H. P. Hobik, and E. Grundmann
a
b
Fig. I. a. Typical area of malignant fibrous histiocytoma of bone showing the "storiform" pattern of tumor cells. Clusters of xanthomatous cells are observed between fibroblast-like cells. b. Typical area of osteosarcoma. Abundant neoplastic osteoid formation among irregularly shaped tumor cells. X 140.
Mal ignant Fibrous Hist iocytoma of Bon e and O steosarcoma . 3 89
a
b
Fig . 2 . a. Anaplastic area of malignant fibrous histiocytoma with sin gle giant cells . b. Anaplastic area of osteosarcoma lacking osteoid formation between tumor cell s. In these anaplastic areas, the two tumors can no t be d istin guish ed by light microscopic investigation. X 140. 26 Path. Res. Pract. Vol 164
390 . A. Roessner, H. P. Hobik, an d E. Grundrn ann
Fig. 3. Low power electron micrograph of a typical field of malignant fibrous histiocytoma of bone, showing mainly histiocytic cells. Note the lamellated inclusion bodies (L). X 3,4°0. Inset: H igher magnification of lamellated inclusion body. X 14,800.
Malignant Fibrous H istiocytoma of Bone and Osteosarcoma . 39 I
Fig. 4. Irregularl y shaped nucleus Mitochondria (M). X 6,5°0.
10
a histiocytic cell (N ). Lamellated inclusions (L).
stance in osteosarcoma (Fig. I b). Xanthomatous cells were a common feature in all tumors stud ied. They were usuall y scattered among the other tumor cells; one case showed them in large clusters. All cases of osteo sarcoma that were prepared for electron microscopy could readily be diagnosed by light microscopy. Most of them were of the osteoblastic type. It should be emphasized that, in some areas, the tumors showed an anaplastic appearance of the kind that is also observed in the anaplastic phase of malign ant fibrous histiocytoma (Fig. 2 b). It was sometimes not possible to distinguish the anaplastic areas of malignant fibrous histiocytoma from those of osteosarcoma. Cells in osteosarcoma show ed a marked pleomorphism, with hyperchromatic nuclei of irregular structure. Some irregularly shaped giant cells were occasionally observed. Some areas showing the typ ical light microscopic pattern of osteoid , were demonstr ated in the intercellular ground substance (Fig. 2 a). Fibrosarcomatous components were found in some areas of the tumors.
39 2 . A. Roessner, H . P. Hobik, and E. Grundmann
Fig. 5. Fibroblast-like cell in malignant fibrous histiocytoma. Elongated cytoplasm with well developed rough endoplasmic reticulum (ER). Ovally shaped cell nucleus (N). Note the occurrence of collagenous fibers in the surrounding matrix (C). Signs of matrix calcification are not observ ed. Mitochondria (M). Y 7,500 .
Mali gnant Fibro us H istiocytoma of Bone and Osteosarcoma . 393
Fig. 6. Xanthomatous cell with numerous lipid droplets filling the cytoplasm (Li). Well developed mitochondria (M) in the cyto plasm. Irregularly shaped nucleus (N) with a larg e nucleolus. Lamellated inclusion bodies (L). X 7,200.
Electron microscopy
In malignant fibrous histiocytoma of bone, several different cell types could be characterized: The most common type showed typical , irregularly shaped nuclei but well-developed cytoplasm. In the latter we found many particles of phagoc ytized mat erial (Fig. 3). These phagoc yt ized particles frequentl y exhib ited a fine, lamellat ed structure (Fig. 3, inset). We observed man y ribosomes and usually a well-developed Golgi app aratus in the cytoplasm. The amounts of both rough and smooth endopl asmic reticulum was variable. Intercellular connections were unusual; intercellular spaces
394 . A. Roessner. H . P. Hobik, and E. Grundmann
appeared electron lucent. There were only a few collagenous fibrils, or none at all, in the immediate vicinity of these cells. In general, this ultrastructural pattern is typical for histiocytic cells. In addition to these histiocytic cells with well developed cytoplasm and cell organelles, we observed another type of cells with only a small cytoplasmic rim, and a highl y irregular nucleus (Fig. 4). Although these cells possessed a rather poorly differentiated ultrastructure, some lamellated inclusion bodies resembling those in histiocytic cells could be found. Large areas of the tumor showed fibroblast-like structures (Fig. 5). Here the cells contained oval nuclei with small in vagi nations and condensed, marginal chromatin. The cytoplasm was spindle-shaped and contained well-developed, mostly dilated cisterns of rough endoplasmic reticulum. Mitochondria were rather scarce. No intracytoplasmic filaments with dense bodies were observed in our material. We observed xanthomatous cells in addition to typical histiocytic cells and fibroblastic cells. Their cytoplasm contained numerous lipid droplets, the Golgi apparatus was well developed, and nucleoli were prominent (Fig. 6). We observed characteristic collagenous bundles in an electron lucent matrix in close proximity to fibroblastic cells. In other areas of ground substance, however, collagen fibers were arranged not in bundles, but in criss-cross fashion showing a certain similarity to the electron microscopic appearance of osteoid. No signs of matrix calcification were discerned in the malignant fibrous histiocytomas of bone studied here. In osteosarcoma, the predominant cell type possessed irregularly shaped nuclei with condensed marginal chromatin and a well developed cytoplasm. One of the most striking features was the abundance of rough endoplasmic reticulum in dilated structures filled with electron lucent contents (Fig. 7). Mitochondria were frequent but had onl y a few cristae mitochondriales. The cytoplasm of certain tumor cells contained typical glycogen rosettes, and lipid droplets were observed occasionally. Some nuclei showed clusters of small spheroids approximately 400 A in diameter. It appears significant that no signs of phagocytosis were observed in this type of tumor cell. Free ribosomes and Golgi structures were frequent. The ultrastructural particularities of these cells suggest a similarity with the osteoblast, which is also characterized by an abundant, well developed rough endoplasmic reticulum. In addition to these cells with their characteristic electron microscopic features, we observed lastl y a more anaplastic kind of tumor cells without an y clearly developed organelles. The intercellular matrix in our osteosarcoma specimens showed several different patterns: In those areas that showed abundnat osteoid production under light microscopy, wide intercellular spaces were filled with a homo-
Malignant Fibrous H istiocytoma of Bone and Osteosar coma
39 5
Fig. 7. Low power electr on micrograph of osteosarcoma. The tumor cells are characterized by abundant rough endopl asmic reticu lum (Er). Collagenous bundle s are observed in the intercellular space (Co). Note the minera lization of tumor matrix (C). X 3,600.
39 6 . A. Roessner, H. P. H obik, and E. Grundmann
Fig. 8. Higher power electron micrograph of osteosarcoma. Small calcified regions (C) in the tumor matrix with abundant collagenous bundles (Co). Well developed rough endoplasmic reticulum in the cytoplasm of the adjacent tumor cell (Er). X 29,000.
genous ground substance. The latter was densely interspersed with fibrils whose characteristic 640 A periodicity marked them as collagen. These fibrils were arranged in collagenous bundles in some areas; in other areas their orientation was random and crisscrossed. The matrix near the collagen fibers revealed calcification at varying stages. Crystals of hydroxyapatite (Fig. 8) were a significant finding.
Malignant Fibrous Histiocytoma of Bone and Osteosarcoma . 397
Discussion We examined the ultrastructure of specimens taken from 5 cases of malignant fibrous histiocytoma of bone, and compared it with the ultrastructure of osteosarcoma. In general, our results revealed an ultrastructure in malignant fibrous histiocytoma of bone very similar to that of its counterpart in soft tissue. In a recent paper, Johnson et a1. (1978) arrived at the same conclusion after investigating one case of malignant fibrous histiocytoma in the acromion by electron microscopy. Two main cell types are observed at the ultrastructural level in malignant fibrous histiocytoma of bone: histiocytic cells and fibroblastic cells. Tissue culture studies of malignant fibrous histiocytoma and fibrous xanthoma have shown that the neoplastic histiocyte can act as a facultative fibroblast; the same capacity has been demonstrated in the normal histiocyte (Ozello et a1., 1963). Moreover, previous electron microscopic studies (Merkow et a1., 197 I) had shown that fibroblastic and histiocytic tumor cells may be considered as two manifestations or stages of the same cell type. The inference is that the fibroblastic component of a malignant fibrous histiocytoma results from differentiation of neoplastic histiocytes in the direction of facultative fibroblasts. The giant cells which are regularly found in the malignant fibrous histiocytoma both of soft tissue and bone show certain signs of phagocytosis which suggest a histiocytic origin. Furthermore, in those xanthomatous cells whose cytoplasm is not completely filled by lipid droplets, certain organelles are observed which are typical for histiocytic cells. Consequently, we may assume that histiocytic cells can transform into xanthomatous cells. In accordance with other electron microscopic investigations of malignant fibrous histiocytoma of soft tissue (Fu et a1., 1975; Merkow et a1., 1971; Taxy et a1., 1977), we interpret our results as follows: The most commonly observed cells in these tumors, i.e. histiocytic cells, fibroblastic cells, giant cells, and xanthomatous cells, are probably all derived from the histiocytic cell by differentiation. Thus, the neoplastic histiocyte is regarded as initial or basic cell of malignant fibrous histiocytoma both in bone and soft tissue (Johnson et a1., 1978). Langerhans bodies, which are described in histiocytosis X (Basset et a1., 1966), were not found in histiocytic cells in the malignant fibrous histiocytoma cases in our study. This is in accordance with the results of Fu et a1. (1975). In this context it must be emphasized that Huhn et a1. (1978) did not find Langerhans bodies in malignant histiocytosis either. As regards the differential diagnosis of malignant fibrous histiocytoma of bone versus osteosarcoma, there may be some difficulties at the light
39 8 . A. Roessner, H. P. Hobik, and E. Grundmann
microscopic level: Osteoid is produced by tumors of this type even in soft tissue, and the malignant fibrous histiocytoma of bone in particular may contain an osteoid-like ground substance (Dahlin et al., 1977a, b). In our cases of malignant fibrous histiocytoma of bone, osteoid-like ground substance was also observed by light microscopy. On the other hand, osteosarcoma may sometimes show anaplastic foci very much like those found in malignant fibrous histiocytoma. If this observation were substantiated by further investigations, the inference would be that malignant fibrous histiocytoma of bone should not be seen as a histologic entity among bone tumors, but rather as a dedifferentiation phenomenon from osteosarcoma (Uribe-Botero et al., 1977)' In opposition to the above hypothesis, our electron microscopic studies yielded further evidence for the character of malignant fibrous histiocytoma of bone as a true histologic entity, which will now be discussed. When areas showing osteoid-like patterns in light microscopy were reexamined under the electron microscope, they were found to contain collagen fibers in an amorphous electron lucent ground substance. A similar picture is found in osteoid-rich regions of osteosarcoma; this fact was confirmed in our study, and reported in previous investigations (Ghadially et al., 1970; Williams et al., 1976). However, this electron microscopic picture of collagen fibrils in a homogenous ground substance is in no way specific for osteoid, but may be found in many other connective tissues. In recent immunofluorescent studies of both normal bone and osteosarcoma, it was demonstrated that osteoid and bone trabeculae in normal bone react exclusively to collagen type I. Osteoid in osteosarcoma shows the same reaction (Remberger et al., 1977). Thus, in order to identify a possible osteoid nature of the matrix in malignant fibrous histiocytoma of bone, immunofluorescent studies for distinguishing collagen types are indicated, since routine electron microscopic preparation is unable to differentiate collagen types in connective tissue. Our comparative electron microscope studies however, revealed one important and distinctive difference between malignant fibrous histiocytoma of bone and osteosarcoma: the absence of matrix calcification, manifested in matrix vesicles and hydroxyapatite crystals, in malignant fibrous histiocytoma. Those signs of early calcification are regularly observed during mineralization of primary bone, and also in osteosarcoma (Bonucci, 1970; Williams et al., 1976). It is this absence in malignant fibrous histiocytoma which may be used as an argument against the osteoblastic nature of neoplastic cells in this tumor. In our electron microscopic observations of osteosarcoma the neoplastic osteoblast was the predominant type of cell. It was described in several
Malignant Fibrous Histiocytoma of Bone and Osteosarcoma . 399
studies (Williams et al., 1976; Ghadially et al., 1970). Its main characteristic is the abundance of rough endoplasmic reticulum in the cytoplasm. On the electron microscopic level, however, not all of the tumor cells will show this typical pattern. In the anaplastic parts we may find certain cells with an ultrastructurally undifferentiated appearance. It appears possible that these were the cells that U ribe- Botero et aI. (1977) observed light microscopically in anaplastic areas of osteosarcoma. Viewed under the electron microscope, however, anaplastic tumor cells in osteosarcoma failed to show any signs of phagocytosis or any of the organelles that are typical for the histiocytic cell, and were clearly recognized in malignant fibrous histiocytoma. Therefore, evidence of neoplastic histiocytic cells with signs of phagocytosis can be interpreted as a characteristic feature of malignant fibrous histiocytoma in differential diagnosis of a questinable tumor. The tumor cells in osteosarcoma sometimes showed a certain similarity to those of chondrosarcoma and fibrosarcoma. The abundance of rough endoplasmic reticulum which characterizes osteoblasts, is not restricted to these, but also observed in cells of chondrosarcoma (Schajowicz et al., 1974; Steiner et al., 1973) and fibrosarcoma (Crocker et al., 1969; jakobiec et al., 1974). Based on these electron microscopic results, Williams et al, (1976) proposed that all these neoplastic cells could be arising from one common progenitor cell. We mentioned above that malignant fibrous histiocytoma of bone contains certain areas of neoplastic fibroblast-like cells which, in this tumor, probably have developed via differentiation from histiocytic cells. The apparent similarity of neoplastic osteoblasts, fibroblasts and chondroblasts in the electron microscopic picture may provide an explanation for the confusing ultrastructural likeness of neoplastic cells in some regions of both malignant fibrous histiocytoma and osteosarcoma. But if all regions of the tumor are subjected to a careful electron microscopic scrutiny, neoplastic histiocytic cells will be observed only in malignant fibrous histiocytoma, but not in osteosarcoma. The results of our study lead to the conclusion that the ultrastructure of malignant fibrous histiocytoma in bone, is generally identical to that of the soft tissue counterpart of this tumor. The components of the tumor are derived from neoplastic histiocytic cells. This cytogenetic pattern permits a clear distinction from osteosarcoma whose cells are derived from neoplastic osteoblasts. With regard to its ultrastructural characteristics, malignant fibrous histiocytoma of bone should be interpreted and accepted as a distinct histologic entity of bone tumors. On the electron microscopic level the presence of neoplastic histiocytes with signs of phagocytosis can be counted as a diagnostic criterion for differentiating malignant fibrous histiocytoma of bone from osteosarcoma.
40 0 . A. Roessner, H. P. Hobik, and E. Grundmann
References Basset, F., et Nezelof, c.: Presence en microscopie electronique de structures filamenteuses originales dans les lesion pulmonaires et osseuses de l'histiocytose "X". Bull. Soc. med, Hop. Paris II7, 413-423 (1966) Bonucci, E.: Fine structure and histochemistry of "calcifying globules" in epiphyseal cartilage. Z. Zellforsch. 103, 192-217 (1970) Crocker, D. ]., and Murad, T. M.: Ultrastructure of fibrosarcoma in male breast. Cancer 23, 89 1-899 (19 69) Dahlin, D. c., Unni, K. K., and Matsuno, T.: Malignant (fibrous) histiocytoma of bone - fact or fancy? Cancer 39, 15°8-1516 (1977a) Dahlin, D. c., and Unni, K. K.: Osteosarcoma of bone and its important recognizable varieties. Amer.]. Surg. Path. 1,61-72 (1977b) Ekfors, T. 0., and Rantakokko, V.: An analysis of 38 malignant fibrous histiocytomas in the extrerneties. Acta path. microbiol. scand. Sect. A 86, 25-35 (1978) Erlandson, R. A., and Huvos, A. G.: Chondrosarcoma. A light and electron microscopic study. Cancer 34, 1642-1652 (1974) Fu, Y. S., Gabbiani, G., Kaye, G. I., and Lattes, R.: Malignant soft tissue tumors of probable histiocytic origin (malignant fibrous histiocytomas): General considerations and electron microscopic and tissue culture studies. Cancer 35, 176-198 (1975) Ghadially, F. N., and Mehta, P. N.: Ultrastructure of osteogenic sarcoma. Cancer 25, 1457- 1467 (197 0 ) Huhn, D., Meister, P., Thiel, E., Bartl, R., and Theml, H.: Maligne Histiocytose, Dtsch. med. Wschr. 103, 55-61 (1978) Jakobiec, F. C., and Tannenbaum, M.: The ultrastructure of orbital fibrosarcoma. Amer. ]. Ophthalmol. 77, 899-9 17 (1974) Johnson, W. W., Coburn, T. P., Pratt, C. B., Smith, ]. W., Maheshkumar, A. P., and Dahlin, D. c.: Ultrastructure of malignant histiocytoma arising in the acromion. Human Path. 9, 199-209 (1978) Kempson, R. L., and Kyriakos, M.: Fibroxanthosarcoma of the soft tissues. A type of malignant fibrous histiocytoma. Cancer 29, 961-976 (1972) Merkow, L. P., Frich, ]. c., Slifkin, M., Kyreages, C. G., and Pardo, M.: Ultrastructure of a fibroxanthosarcoma (malignant fibroxanthoma). Cancer 28, 372-383 (1971) Ozello, L., Stout, A. P., and Murray, M. R.: Cultural characteristics of malignant histiocytomas and fibrous xanthomas. Cancer 16, 331-334 (1963) Rernberger, K., and Gay, S.: Immunhistochemical demonstration of different collagen types in the normal epiphyseal plate and in benign and malignant tumors of bone and cartilage. Z. Krebsforsch. 90, 95-106 (1977) Schajowicz, F., Cabrini, R. C; and Simes, R. ].: Ultrastructure of chondrosarcoma. Clin. Orthop. 100,378-386 (1974) Spanier, S. S., Enneking, W. F., and Enriquez, P.: Primary malignant fibrous histiocytoma of bone. Cancer 36, 2084-2098 (1975) Steiner, G. c., Mirra, ]. M., and Bullough, P. G.: Mesenchymal chondrosarcoma - A study of the ultrastructure. Cancer 32,926-939 (1973) Taxy, J. B., and Battifora, H.: Malignant fibrous histiocytoma. An electron microscopic study. Cancer 4°,254-267 (1977) Uribe-Botero, G., Russell, W.O., Surow, W. W., and Martin, R. G.: Primary osteosarcoma of bone. A clinicopathologic investigation of 243 cases with necropsy studies in 54. Amer.]. Clin. Path. 67, 427-435 (1977)
Malignant Fibrous Histiocytoma of Bone and Osteosarcoma . 401 Williams, A. H., Schwinn, C. P., and Parker, J. W.: The ultrastructure of osteosarcoma. A review of twenty cases. Cancer 37,1293-1301 (1976) Received July 31,1978' Accepted in revised form November 17, 1978
Key words: Histiocytoma of bone - Osteosarcoma - Electron microscopy - Comparative study Dr. med. A. Roessner, Pathologisches Institut der Universirat, Westring 17, D-4400 Munster i. W.
Department of Pathology, University of Muenster, Muenster, West Germany (Chairman: Prof. E. Grundmann, M. D.)
Malignant Fibrous Histiocytoma of Bone and Osteosarcoma. A Comparative Light and Electron Microscopic Study A. ROESSNER, H. P. HOBIK, and E. GRUNDMANN':'
Summary Malignant fibrous histiocytomas are well-described tumors of the soft tissues. Recent investigations have shown that malignant histiocytoma may also occur as a primary bone tumor. However, difficulties may arise to distinguish malignant histiocytoma of bone from other malignant bone tumors, such as osteosarcoma. In the present study, the ultrastructure of five cases of malignant fibrous histiocytoma of bone is compared with that of osteosarcoma. The results show that malignant fibrous histiocytoma is composed mainly of histiocytic cells and fibroblastic cells. In addition, xanthomatous cells, undifferentiated cells, and giant cells may be observed. By contrast, the predominant cell type in osteosarcoma is the neoplastic osteoblast, characterized by abundant rough endoplasmic reticulum. Signs of matrix calcification in the intercellular matrix between the collagen fibrils are regularly observed in osteosarcoma, but not in malignant histiocytoma. From these results it is concluded that the ultrastructure of malignant fibrous histiocytoma arising in bone is morphologically identical with the soft tissue counterpart of this tumor. The components of the tumor are derived from neoplastic histiocytes, This cytogenesis differs from that of osteosarcoma, which is derived from neoplastic osteoblasts. Therefore, from the ultrastructural point of view, malignant fibrous histiocytoma of bone should be accepted as a distinct histologic entity among bone tumors.
Introduction Fibrohistiocytic tumors showing bimodal differentiation into histiocytic and fibroblastic cells, such as fibrous histiocytoma and malignant fibrous histiocytoma, are well defined soft tissue tumors (Kempson et al., 1971; Ekfors et al., 1978). The ultrastructural characteristics of cell types involved in malignant fibrous histiocytoma of soft tissue have been described in several electron microscopic studies (Merkow et al., 197 I; Fu et al., ':. Dedicated to Prof. Dr. E. Vehlinger in honour of his anniversary, 8th August 1979
386 . A. Roessner, H. P. Hobik, and E. Grundmann
1975; Taxy et al., 1977). Recent investigations have suggested the possibility that malignant fibrous histiocytoma may occur as a primary tumor of bone (Spanier et al., 1975; Dahlin et al., 1977a). As far as we know, the use of electron microscopy for analyzing the cell types involved in malignant fibrous histiocytoma of bone has been described in only one case report of this disease (Johnson et al., 1978). It is sometimes difficult to distinguish malignant fibrous histiocytoma from other malignant bone tumors, such as osteosarcoma and fibrosarcoma (Dahlin et al., 1977a, b). Osteosarcoma, especially in its anaplastic phase, may show changes not unlike those in malignant fibrous histiocytoma (Uribe-Botero et al., 1977). Therefore Dahlin et al. (1977a) proposed to limit diagnosis of malignant fibrous histiocytoma of bone strictly to those cases where the entire tumor shows the characteristic pattern of fibrocytic and histiocytic cell differentiation. Another problem in diagnosis arises from the fact that malignant fibrous histiocytoma of bone may produce an osteoid-like ground substance (Dahlin et al., 1977a, b). Since all malignant bone tumors capable of osteoid production are usually classified as osteosarcoma, there are no absolute criteria for differentiating malignant fibrous histiocytoma of bone from osteosarcoma at the level of light microscopy. Thus in our present study of 5 cases, the ultrastructure of malignant fibrous histiocytoma of bone is compared to that of osteosarcoma. We sought to distinguish and describe the different cell types occurring in either of these lesions, and to determine to what extent the cytologic features of these cell types might point towards a difference in cell origins. Special interest was focussed on the value of electron microscopic investigation in the differential diagnosis of malignant fibrous histiocytoma of bone.
Material andMethods Case material Light microscopy
Tissues were fixed in neutral formalin, embedded in paraffin, and stained with hematoxylin and eosin, van Gieson, Ladewig, or Azan for light microscopic investigation. Electron mtcroscop y
Material for electron microscopic investigation was obtained in most cases at the time of frozen section diagnosis. Tissue specimes from various parts of the tumor were minced into 1 mm pieces immediately after removal, fixed in 2.25% glutaraldehyde (0.05 m phosphate buffer, pH 7.4) for 2 hrs at 4° C, then rinsed in buffer for 24 hrs. Postfixation was carried out in 1.33010 osmic acid with 0.05 m phosphate buffer, pH 7.4,
Malignant Fibrous Histiocytoma of Bone and Osteosarcoma . 3 87 The clinical data are summarized in the following table: Malignant fibrous histiocytoma of bone case no. 2 3 4 5
age
sex
localization
18 55 27 18 42
m m f m m
distal femur right posterior 5th rib proximal humerus ilium head of fibula
12 13 15 12 13
f f m m
distal femur distal femur proximal fibula proximal fibula proximal femur
Osteosarcoma
2 3 4
5
for 2 hrs. The tissues were dehydrated in graded ethanols, and embedded in Epon 812. Sections were cut with a Porter-Blum ultramicrotome, mounted on 3 mm copper grids, and poststained for 15 min with 5% uranyl acetate and 3 min with lead citrate. Specimens were examined with a Siemens Elmiskop I a electron microscope at 80 kV using the double condenser. Semithin sections from Epon-embedded tissue were stained with toluidine blue for light microscopy.
Results Light microscopy
In all our cases of malignant fibrous histiocytoma of bone, tumor cells appeared densely packed, showing cytologic signs of malignancy: nuclei with irregular shapes and a wide variation in size. Nuclei were often hyperchromatic with prominent nucleoli. Mitotic figures were occasionally observed. Some tumor areas were composed of spindle cells arranged in the typical cartwheel or storiform pattern (Fig. I a). With Ladewig staining, the collagen fibers gave a picture similar to the osteoid-like ground substance in osteosarcoma. Some ossseous fragments were occasionally observed between tumor cells. In other areas, the tumor consisted largely of epitheloid cells and some multinucleated giant cells. Collagen fibers in these areas were less well developed and showed no similarity with the osteoid-like ground sub-
3 88 . A. Roessner, H. P. Hobik, and E. Grundmann
a
b
Fig. I. a. Typical area of malignant fibrous histiocytoma of bone showing the "storiform" pattern of tumor cells. Clusters of xanthomatous cells are observed between fibroblast-like cells. b. Typical area of osteosarcoma. Abundant neoplastic osteoid formation among irregularly shaped tumor cells. X 140.
Mal ignant Fibrous Hist iocytoma of Bon e and O steosarcoma . 3 89
a
b
Fig . 2 . a. Anaplastic area of malignant fibrous histiocytoma with sin gle giant cells . b. Anaplastic area of osteosarcoma lacking osteoid formation between tumor cell s. In these anaplastic areas, the two tumors can no t be d istin guish ed by light microscopic investigation. X 140. 26 Path. Res. Pract. Vol 164
390 . A. Roessner, H. P. Hobik, an d E. Grundrn ann
Fig. 3. Low power electron micrograph of a typical field of malignant fibrous histiocytoma of bone, showing mainly histiocytic cells. Note the lamellated inclusion bodies (L). X 3,4°0. Inset: H igher magnification of lamellated inclusion body. X 14,800.
Malignant Fibrous H istiocytoma of Bone and Osteosarcoma . 39 I
Fig. 4. Irregularl y shaped nucleus Mitochondria (M). X 6,5°0.
10
a histiocytic cell (N ). Lamellated inclusions (L).
stance in osteosarcoma (Fig. I b). Xanthomatous cells were a common feature in all tumors stud ied. They were usuall y scattered among the other tumor cells; one case showed them in large clusters. All cases of osteo sarcoma that were prepared for electron microscopy could readily be diagnosed by light microscopy. Most of them were of the osteoblastic type. It should be emphasized that, in some areas, the tumors showed an anaplastic appearance of the kind that is also observed in the anaplastic phase of malign ant fibrous histiocytoma (Fig. 2 b). It was sometimes not possible to distinguish the anaplastic areas of malignant fibrous histiocytoma from those of osteosarcoma. Cells in osteosarcoma show ed a marked pleomorphism, with hyperchromatic nuclei of irregular structure. Some irregularly shaped giant cells were occasionally observed. Some areas showing the typ ical light microscopic pattern of osteoid , were demonstr ated in the intercellular ground substance (Fig. 2 a). Fibrosarcomatous components were found in some areas of the tumors.
39 2 . A. Roessner, H . P. Hobik, and E. Grundmann
Fig. 5. Fibroblast-like cell in malignant fibrous histiocytoma. Elongated cytoplasm with well developed rough endoplasmic reticulum (ER). Ovally shaped cell nucleus (N). Note the occurrence of collagenous fibers in the surrounding matrix (C). Signs of matrix calcification are not observ ed. Mitochondria (M). Y 7,500 .
Mali gnant Fibro us H istiocytoma of Bone and Osteosarcoma . 393
Fig. 6. Xanthomatous cell with numerous lipid droplets filling the cytoplasm (Li). Well developed mitochondria (M) in the cyto plasm. Irregularly shaped nucleus (N) with a larg e nucleolus. Lamellated inclusion bodies (L). X 7,200.
Electron microscopy
In malignant fibrous histiocytoma of bone, several different cell types could be characterized: The most common type showed typical , irregularly shaped nuclei but well-developed cytoplasm. In the latter we found many particles of phagoc ytized mat erial (Fig. 3). These phagoc yt ized particles frequentl y exhib ited a fine, lamellat ed structure (Fig. 3, inset). We observed man y ribosomes and usually a well-developed Golgi app aratus in the cytoplasm. The amounts of both rough and smooth endopl asmic reticulum was variable. Intercellular connections were unusual; intercellular spaces
394 . A. Roessner. H . P. Hobik, and E. Grundmann
appeared electron lucent. There were only a few collagenous fibrils, or none at all, in the immediate vicinity of these cells. In general, this ultrastructural pattern is typical for histiocytic cells. In addition to these histiocytic cells with well developed cytoplasm and cell organelles, we observed another type of cells with only a small cytoplasmic rim, and a highl y irregular nucleus (Fig. 4). Although these cells possessed a rather poorly differentiated ultrastructure, some lamellated inclusion bodies resembling those in histiocytic cells could be found. Large areas of the tumor showed fibroblast-like structures (Fig. 5). Here the cells contained oval nuclei with small in vagi nations and condensed, marginal chromatin. The cytoplasm was spindle-shaped and contained well-developed, mostly dilated cisterns of rough endoplasmic reticulum. Mitochondria were rather scarce. No intracytoplasmic filaments with dense bodies were observed in our material. We observed xanthomatous cells in addition to typical histiocytic cells and fibroblastic cells. Their cytoplasm contained numerous lipid droplets, the Golgi apparatus was well developed, and nucleoli were prominent (Fig. 6). We observed characteristic collagenous bundles in an electron lucent matrix in close proximity to fibroblastic cells. In other areas of ground substance, however, collagen fibers were arranged not in bundles, but in criss-cross fashion showing a certain similarity to the electron microscopic appearance of osteoid. No signs of matrix calcification were discerned in the malignant fibrous histiocytomas of bone studied here. In osteosarcoma, the predominant cell type possessed irregularly shaped nuclei with condensed marginal chromatin and a well developed cytoplasm. One of the most striking features was the abundance of rough endoplasmic reticulum in dilated structures filled with electron lucent contents (Fig. 7). Mitochondria were frequent but had onl y a few cristae mitochondriales. The cytoplasm of certain tumor cells contained typical glycogen rosettes, and lipid droplets were observed occasionally. Some nuclei showed clusters of small spheroids approximately 400 A in diameter. It appears significant that no signs of phagocytosis were observed in this type of tumor cell. Free ribosomes and Golgi structures were frequent. The ultrastructural particularities of these cells suggest a similarity with the osteoblast, which is also characterized by an abundant, well developed rough endoplasmic reticulum. In addition to these cells with their characteristic electron microscopic features, we observed lastl y a more anaplastic kind of tumor cells without an y clearly developed organelles. The intercellular matrix in our osteosarcoma specimens showed several different patterns: In those areas that showed abundnat osteoid production under light microscopy, wide intercellular spaces were filled with a homo-
Malignant Fibrous H istiocytoma of Bone and Osteosar coma
39 5
Fig. 7. Low power electr on micrograph of osteosarcoma. The tumor cells are characterized by abundant rough endopl asmic reticu lum (Er). Collagenous bundle s are observed in the intercellular space (Co). Note the minera lization of tumor matrix (C). X 3,600.
39 6 . A. Roessner, H. P. H obik, and E. Grundmann
Fig. 8. Higher power electron micrograph of osteosarcoma. Small calcified regions (C) in the tumor matrix with abundant collagenous bundles (Co). Well developed rough endoplasmic reticulum in the cytoplasm of the adjacent tumor cell (Er). X 29,000.
genous ground substance. The latter was densely interspersed with fibrils whose characteristic 640 A periodicity marked them as collagen. These fibrils were arranged in collagenous bundles in some areas; in other areas their orientation was random and crisscrossed. The matrix near the collagen fibers revealed calcification at varying stages. Crystals of hydroxyapatite (Fig. 8) were a significant finding.
Malignant Fibrous Histiocytoma of Bone and Osteosarcoma . 397
Discussion We examined the ultrastructure of specimens taken from 5 cases of malignant fibrous histiocytoma of bone, and compared it with the ultrastructure of osteosarcoma. In general, our results revealed an ultrastructure in malignant fibrous histiocytoma of bone very similar to that of its counterpart in soft tissue. In a recent paper, Johnson et a1. (1978) arrived at the same conclusion after investigating one case of malignant fibrous histiocytoma in the acromion by electron microscopy. Two main cell types are observed at the ultrastructural level in malignant fibrous histiocytoma of bone: histiocytic cells and fibroblastic cells. Tissue culture studies of malignant fibrous histiocytoma and fibrous xanthoma have shown that the neoplastic histiocyte can act as a facultative fibroblast; the same capacity has been demonstrated in the normal histiocyte (Ozello et a1., 1963). Moreover, previous electron microscopic studies (Merkow et a1., 197 I) had shown that fibroblastic and histiocytic tumor cells may be considered as two manifestations or stages of the same cell type. The inference is that the fibroblastic component of a malignant fibrous histiocytoma results from differentiation of neoplastic histiocytes in the direction of facultative fibroblasts. The giant cells which are regularly found in the malignant fibrous histiocytoma both of soft tissue and bone show certain signs of phagocytosis which suggest a histiocytic origin. Furthermore, in those xanthomatous cells whose cytoplasm is not completely filled by lipid droplets, certain organelles are observed which are typical for histiocytic cells. Consequently, we may assume that histiocytic cells can transform into xanthomatous cells. In accordance with other electron microscopic investigations of malignant fibrous histiocytoma of soft tissue (Fu et a1., 1975; Merkow et a1., 1971; Taxy et a1., 1977), we interpret our results as follows: The most commonly observed cells in these tumors, i.e. histiocytic cells, fibroblastic cells, giant cells, and xanthomatous cells, are probably all derived from the histiocytic cell by differentiation. Thus, the neoplastic histiocyte is regarded as initial or basic cell of malignant fibrous histiocytoma both in bone and soft tissue (Johnson et a1., 1978). Langerhans bodies, which are described in histiocytosis X (Basset et a1., 1966), were not found in histiocytic cells in the malignant fibrous histiocytoma cases in our study. This is in accordance with the results of Fu et a1. (1975). In this context it must be emphasized that Huhn et a1. (1978) did not find Langerhans bodies in malignant histiocytosis either. As regards the differential diagnosis of malignant fibrous histiocytoma of bone versus osteosarcoma, there may be some difficulties at the light
39 8 . A. Roessner, H. P. Hobik, and E. Grundmann
microscopic level: Osteoid is produced by tumors of this type even in soft tissue, and the malignant fibrous histiocytoma of bone in particular may contain an osteoid-like ground substance (Dahlin et al., 1977a, b). In our cases of malignant fibrous histiocytoma of bone, osteoid-like ground substance was also observed by light microscopy. On the other hand, osteosarcoma may sometimes show anaplastic foci very much like those found in malignant fibrous histiocytoma. If this observation were substantiated by further investigations, the inference would be that malignant fibrous histiocytoma of bone should not be seen as a histologic entity among bone tumors, but rather as a dedifferentiation phenomenon from osteosarcoma (Uribe-Botero et al., 1977)' In opposition to the above hypothesis, our electron microscopic studies yielded further evidence for the character of malignant fibrous histiocytoma of bone as a true histologic entity, which will now be discussed. When areas showing osteoid-like patterns in light microscopy were reexamined under the electron microscope, they were found to contain collagen fibers in an amorphous electron lucent ground substance. A similar picture is found in osteoid-rich regions of osteosarcoma; this fact was confirmed in our study, and reported in previous investigations (Ghadially et al., 1970; Williams et al., 1976). However, this electron microscopic picture of collagen fibrils in a homogenous ground substance is in no way specific for osteoid, but may be found in many other connective tissues. In recent immunofluorescent studies of both normal bone and osteosarcoma, it was demonstrated that osteoid and bone trabeculae in normal bone react exclusively to collagen type I. Osteoid in osteosarcoma shows the same reaction (Remberger et al., 1977). Thus, in order to identify a possible osteoid nature of the matrix in malignant fibrous histiocytoma of bone, immunofluorescent studies for distinguishing collagen types are indicated, since routine electron microscopic preparation is unable to differentiate collagen types in connective tissue. Our comparative electron microscope studies however, revealed one important and distinctive difference between malignant fibrous histiocytoma of bone and osteosarcoma: the absence of matrix calcification, manifested in matrix vesicles and hydroxyapatite crystals, in malignant fibrous histiocytoma. Those signs of early calcification are regularly observed during mineralization of primary bone, and also in osteosarcoma (Bonucci, 1970; Williams et al., 1976). It is this absence in malignant fibrous histiocytoma which may be used as an argument against the osteoblastic nature of neoplastic cells in this tumor. In our electron microscopic observations of osteosarcoma the neoplastic osteoblast was the predominant type of cell. It was described in several
Malignant Fibrous Histiocytoma of Bone and Osteosarcoma . 399
studies (Williams et al., 1976; Ghadially et al., 1970). Its main characteristic is the abundance of rough endoplasmic reticulum in the cytoplasm. On the electron microscopic level, however, not all of the tumor cells will show this typical pattern. In the anaplastic parts we may find certain cells with an ultrastructurally undifferentiated appearance. It appears possible that these were the cells that U ribe- Botero et aI. (1977) observed light microscopically in anaplastic areas of osteosarcoma. Viewed under the electron microscope, however, anaplastic tumor cells in osteosarcoma failed to show any signs of phagocytosis or any of the organelles that are typical for the histiocytic cell, and were clearly recognized in malignant fibrous histiocytoma. Therefore, evidence of neoplastic histiocytic cells with signs of phagocytosis can be interpreted as a characteristic feature of malignant fibrous histiocytoma in differential diagnosis of a questinable tumor. The tumor cells in osteosarcoma sometimes showed a certain similarity to those of chondrosarcoma and fibrosarcoma. The abundance of rough endoplasmic reticulum which characterizes osteoblasts, is not restricted to these, but also observed in cells of chondrosarcoma (Schajowicz et al., 1974; Steiner et al., 1973) and fibrosarcoma (Crocker et al., 1969; jakobiec et al., 1974). Based on these electron microscopic results, Williams et al, (1976) proposed that all these neoplastic cells could be arising from one common progenitor cell. We mentioned above that malignant fibrous histiocytoma of bone contains certain areas of neoplastic fibroblast-like cells which, in this tumor, probably have developed via differentiation from histiocytic cells. The apparent similarity of neoplastic osteoblasts, fibroblasts and chondroblasts in the electron microscopic picture may provide an explanation for the confusing ultrastructural likeness of neoplastic cells in some regions of both malignant fibrous histiocytoma and osteosarcoma. But if all regions of the tumor are subjected to a careful electron microscopic scrutiny, neoplastic histiocytic cells will be observed only in malignant fibrous histiocytoma, but not in osteosarcoma. The results of our study lead to the conclusion that the ultrastructure of malignant fibrous histiocytoma in bone, is generally identical to that of the soft tissue counterpart of this tumor. The components of the tumor are derived from neoplastic histiocytic cells. This cytogenetic pattern permits a clear distinction from osteosarcoma whose cells are derived from neoplastic osteoblasts. With regard to its ultrastructural characteristics, malignant fibrous histiocytoma of bone should be interpreted and accepted as a distinct histologic entity of bone tumors. On the electron microscopic level the presence of neoplastic histiocytes with signs of phagocytosis can be counted as a diagnostic criterion for differentiating malignant fibrous histiocytoma of bone from osteosarcoma.
40 0 . A. Roessner, H. P. Hobik, and E. Grundmann
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Malignant Fibrous Histiocytoma of Bone and Osteosarcoma . 401 Williams, A. H., Schwinn, C. P., and Parker, J. W.: The ultrastructure of osteosarcoma. A review of twenty cases. Cancer 37,1293-1301 (1976) Received July 31,1978' Accepted in revised form November 17, 1978
Key words: Histiocytoma of bone - Osteosarcoma - Electron microscopy - Comparative study Dr. med. A. Roessner, Pathologisches Institut der Universirat, Westring 17, D-4400 Munster i. W.
