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The Impact of Osteonectin for Differential Diagnosis of Bone Tumors
Path. Res. Pract. 186, 651-657 (1990)
The Impact of Osteonectin for Differential Diagnosis of Bone Tumors! An Immunohistochemical Approach A. Bosse, E. Vollmer, W. Boeker and A. Roessner Gerhard-Domagk-Institut fUr Pathologie, Munster, FRG
P. Wuisman and D. Jones Orthopadische Klinik, Westfalische Wilhelms-Universitiit Munster, FRG
L. W. Fisher NIH, Institute of Dental Research, BethesdalMd USA
SUMMARY 75 osteosarcoma at various grades of histologic differentiation, including chondroblastic and small cell variants, and 5 fibrosarcomas of bone, 5 Ewing's sarcomas, 5 malignant fibrous histiocytomas of bone, 8 chondrosarcomas, and 2 dedifferentiated chondrosarcomas, were investigated immunohistochemically for evidence of osteonectin. According to the results of our study, osteonectin is present in all osteosarcomas, with special topographic preponderance in the osteoblastic and chondroblastic variants. Evidence of osteonection was also found in all other bone tumors we had analysed so far. In chondrosarcomas, positive reactions appeared only in the vicinity of trabeculae and in dedifferentiated areas. Thus, osteonectin cannot be regarded as a bone-specific protein. Although a high affinity for the osseous matrix is one of its undoubted features, it is therefore unsuitable for differential diagnostic purposes.
Introduction Osteosarcomas are classified mainly according to the ground substance of their mesenchymal tissue. Consequently, the definition of bone-specific proteins plays an important role in the classification and diagnostics of bone tumors. Since osteoblasts are known to loose their capacity for osteoid formation under certain pathologic conditions, osteosarcomas will often show a strong resemblance to other mesenchymal bone tumors such as fibrosarcoma, I This work was supported by grant number Bo 990/1-1 from the Deutsche Forschungsgemeinschaft.
© 1990 by Gustav Fischer Verlag, Stuttgart
malignant fibrous histiocytoma, chondrosarcoma or Ewing's sarcoma. This variegated appearance often hampers histologic diagnosis, especially when it has to be based on rather small biopsy material 15 , 25. Immunohistochemistry has failed, so far, to provide helpful data for differential diagnosis, since the results were not exactly bone-specific; moreover, it was found that various types of collagen were synthesized under pathologic conditions 12 , 13. The attempts to produce specific antibodies against osteosarcoma have not been very successful either, since these would also react with other soft tissue tumors 2 . In view of the differential separation of osteosarcoma from other mesenchymal soft tissue tumors, much hope 0344-0338/90/0186-0651$3.50/0
652 . Bosse, A. et al. 40
7-
Table 1. Immunohistochemical results with ostonectin antibody in different types of osteosarcoma and other mesenchymal bone tumors
30
Tumor diagnosis
Number of cases total
Reaction intensity with anti-osteonectin
Osteosarcomas osteoblastic telangiectatic fibroblastic anaplastic chondroblastic small-cell
75 35 10
++ +++ +++ ++ ++ ++ ++ ++ + ++ (+)* +
20
10
o
o, ~., ',. 0,'.0<.'," ~..
.,,",,-
J...~J l".mT.?"",,
Siolo HS-Glyko- cones 1/11 Protein 1/11 protein
'00_
colla~enous proteins
Fig. 1. Non-collagenous bone proteins. Distribution in the developing bone tissue.
Fibrosarcoma MFH Ewing's Sarcoma Chondrosarcoma dediff. Chondrosarcoma
8 9
12 1 5 5 5
8
2
+++ +++ +++ +++ +++
* Positive immunoreactivity occuring only in areas of osteoid trabeculae; + = low, ++ = moderate; +++ = strong.
Fig. 2. Osteoblastic osteoblastoma with strong immune reaction in osteoblasts near trabeculae (anti-osteonectin AB, X 550).
Fig. 3. Chondroblastic osteosarcoma with strong marginal ~ immune expression and negative labeling in cartilaginous centers (anti-osteonectin AB, x 120).
Fig. 5
Osteonectin in Osteosarcoma . 655
has focused therefore on the immunohistochemical demonstration of non-collagenous osseous structure proteins which are obviously characterised by high bone specificity. The protagonists of this group are osteocalcin7, 11, sialoproteins8, and osteonectin18,19 (Fig. 1). Osteonectin is a phosphorylated glycoprotein with a molecular weight of 38000 Dalton, isolated first from bovine bone 18 . In bone cell culture24 as well as in reactive bone lesions 9, osteonectin could be demonstrated immunohistochemically in osteoblasts. Further evidence was found in non-osteoblastic cells that should therefore be classified as osteo-progenitor cells 16 . The functional role and significance of osteonectin in the bone is still as undefined as that of the other non-collagenous osseous proteins. Obviously, they must have a potential to steer the mineralisation of tissue 21 . Osteonectin, for instance, shows a high affinity for hydroxylapatite and for type I collagen4. On account of their high bone specificity, the immunohistochemical demonstration of non-collagenous osseous structure proteins might help to differentiate osteosarcoma from other bone tumors. Some positive experience has been published 1o,17, but other groups have demonstrated osteonectin also in fibroblasts 22 and even in chorionic villi23 . In order to define the value and validity of osteonectin in this context, we surveyed its immunoexpression in a large material comprising osteosarcomas and other bone tumors. Material and Methods The study comprised 75 osteosarcomas, including chondroblastic and small cell variants, 5 fibrosarcomas, 5 malignant fibrous histiocytomas, 5 Ewing's sarcomas, 8 chondrosarcomas and two dedifferentiated chondrosarcomas. Antibodies were raised in rabbits against bovine and human osteonectin as described by Termine et al. ls . All immunohistochemical studies were performed on formol-fixed material (4% buffered formol) embedded in paraffin. A large part of the
... Fig. 4. Immunocytochemical results of osteonectin with different types of osteosarcomas. - a: Telangiectatic osteosarcoma with numerous positive marked cells that are often connected to osteoid islets. - b: Small cell osteosarcoma with some positive cells. - c: Fibroblastic osteosarcoma with intense marking in scattered disseminated tumor cells. - d: anaplastic osteosarcoma lacking osteoid production, with a strong marking in the cytoplasm of huge pleomorphic atypical osteoblasts (all antiosteonectin AB, x 550). ... Fig. 5. Immunohistochemical proof of osteonectin in mesenchymal bone tumors. - a: Fibrosarcoma with a strong positive immune reaction pattern in the cytoplasm of tumor cells. - b: Malignant fibrous histiocytoma with fainter marking of scattered disseminated tumor cells. - c: Ewing's sarcoma with positive marking of many tumor cells in the small cytoplasmic rim. d: Dedifferentiated chondrosarcoma with a small number of positive cells in dedifferentiated areas (all anti-osteonectin AB, x 550).
samples had been previously decalcified with 10% EDTA solution. The. tests with polyclonal antibodies were carried out on 2 ~m sections. Demonstration of osteonectin was performed using the APAAP method (alkaline phosphatase - antialkaline phosphatase) with naphthol salt and hexazodated neofuchsin 3• Deparaffinated sections were incubated for 45 minutes at room temperature with the primary antibody after optimal antibody dilution (1 :400) had been tested in preceding experiments. Trypsinase pretreatment had no influence on the immunocytochemical reaction which yielded identical results with human and bovine anti-osteonectin. Immunohistochemical demonstration of osteonectin in an aggressive osteoblastoma served as positive control specimen.
Results In all osteosarcoma specimens osteonectin was expressed. The intensity of immune reactions and the number of labeled tumor cells varied considerably (Table 1). Classical osteoblastic osteosarcomas showed a strong immunoexpression in atypical osteoblasts, especially in the vicinity of neoplastic osteoid trabeculae. No topographic foci of labeling could be identified in osteoblastic tumors, the general pattern of distribution being rather uniformly disseminated (Fig. 2). In chondroblastic osteosarcoma osteonectin immunolabeling was confined to marginal chondroblastic areas and to a smaller amount of fibroblasts (Fig. 3). Osteonectin was also found in the rare form of small cell osteoblastic osteosarcoma, with occasional perinuclear labeling of tumor cells in random distribution. The distribution pattern in telangiectatic osteosarcomas corresponded largely to that in osteoblastic osteosarcomas. Fibroblastic and anaplastic variants disclosed intense immunolabeling in cells that could not be classified as osteoblasts by purely histologic criteria, without any clear correlation to osteoblastic or bone differentiation (Fig. 4). In all other bone tumors osteonectin expression was observed. Fibrosarcoma showed strong intracytoplasmic reaction in a diffuse patchy pattern. In malignant fibrous histiocytomas, intensive osteonectin labeling was seen occasionally in some cells. In Ewing's sarcomas there again was a marked patchy immune reaction. In chondrosarcomas, however, osteonectin could be demonstrated only in isolated cells in close proximity to trabeculae, and occasionally in dedifferentiated chondrosarcoma. The chondroid tumor formation proper was plainly negative without exception (Fig. 5). Discussion The organic bone matrix is made up of type I collagen (90%) and non-collagenous osseous structure proteins (10%). Of these, osteonectin is one of the major components, produced by osteoblasts (among others) and obviously characterised by remarkable bone specificity18,19. Osteonectin was successfully demonstrated, so far, in numerous benign and malignant bone tumors as well as in reactive bone lesions, especially and preferentially in the topographic areas of enchondral ossification9,16,20.
656 . Bosse, A. et al.
On account of the close relationship of osteonectin and bone matrix, and the proven synthesis by osteoblastic cells supported by immune electron microscopical investigations 1, 16, this protein is supposed to be truly bone specific. Certain previous studies based on a limited number of cases seemed to confirm this concept, since no evidence of osteonectin could be obtained from various mesenchymal tumors 10,17. Additional proof was seen in the fact that no proteins could be demonstrated so far, which would be manifested exclusively in osteoblasts. There had been no reliable mode for differentiating bone tumors, especially osteosarcomas, from other mesenchymal bone tumors by immunohistochemical methods, and even collagens were not found truly bone specific2, 12, 14. Since the diagnosis of bone tumors is often based on a rather small biopsy sample 15 ,25, accurate demonstration of bone specific proteins would be a powerful tool in differential diagnosis when osteosarcomas, frequently very heterogenous in appearance, have to be separated from other bone tumors. It had been shown, however, already in 1984 that osteonectin is also produced in vitro by fibroblasts from other tissues 22 . Subsequently, it could be demonstrated by a higly sensitive radioimmunoassay in non-osseous tissues 6 and even in chorionic villi, among others 23 . Prompted by these pointers to a disputed osseous specificity, we started the comparative investigation of 75 osteosarcomas against numerous other mesenchymal bone tumors with regard to their patterns of osteonectin expression. Our results have shown that all the osteosarcomas expressed osteonectin, independent of the grade of differention and even very rare variants such as small cell osteosarcoma and chondroblastic osteosarcoma. On the whole, immunohistochemistry reveals a very heterogenous picture. In particular, osteoblasts in the vicinity of trabeculae showed positive labeling, but the tumor cells of the fibroblastic, anaplastic, and even small cell variants also yielded distinct immune reactions in cells that were definitely non-osteoblastic by histomorphologic criteria. The chondroblastic variant of osteosarcoma showed stronger labeling in the marginal parts, with decreasing intensity towards the central cartilagineous areas (d. Fig. 3, 4). In fibrosarcomas, Ewing's sarcomas and malignant fibrous histiocytomas studied for comparison there was also a marked osteonectin expression, especially in fibrosarcomas where immune expression had a strong focal accentuation resembling that in fibroblastic osteosarcoma. Previous studies of chondrosarcoma 17 could be confirmed to a large extent; some positive atypical chondrocytes were found only in the immediate vicinity of trabeculae; some isolated evidence of osteonectin was also present in dedifferentiated chondrosarcomas (Fig. 5). With the exception of chondrosarcomas with their largely negative immune expression, our results are not in agreement with those of other work groups9, 10, 17. Since all were performed with identical antibodies from the research laboratories of NIH in Bethesda, the divergent results may be attribuable to methodological differences, such as different antibody dilutions, periods of incubation,
and temperatures. Moreover, the application of PAP technique, Avitin-Biotin complex method, or APAAP method as employed in our assay, the immunohistochemical approach is principally a different one. We have to infer that osteonectin, like collagen type I, cannot be considered as truly bone specific, being manifestly present in quite a number of other mesenchymal tumors. There is no doubt that osteonectin is produced predominantly by osteoblasts and has its outstanding localisation in osseous lesions where it may be taken as a marker of osteogenesis5, 14, 18. According to our results, however, it cannot be appraised as an exclusively osseous structure protein, since it is evident in several other types of tissue. Obviously, immunohistochemical demonstration of osteonectin depends on its concentration in the respective tissue, which is liable to considerable variation 6, 23. Thus, osteonectin can be rather interpreted as a multilocular structure protein occurring in high concentration especially in osteoblasts. In this tissue, the varying patterns of distribution show clear topographic cumuli, especially in osteoblastic osteosarcoma in the vicinity of trabeculae and in the chondroblastic variant, where they suggest a special functional status of osteoblasts reflecting increased activity. Possibly the correlation of reaction intensity with the grade of osteosarcoma differentiation will offer a new approach to the differential, more subtle, and therapy-oriented classification of osteosarcomas. Some preliminary experience with another structure protein, bone morphogenetic protein (BMP) has been published26 • Osteonectin, however, is not specific enough to serve as differential marker against other bone mesenchymal tumors. Acknowledgements The authors want to thank Misses B. Duhm and P. Schafer for excellent technical assistance, and Mrs. E. Griffith and H. Lossmann for the preparation of the manuscript. References 1 Bonnucci E, Bianco P, Hayashi Y, Termine JD (1986) Ultrastructural immunohistochemical localization of non-collagenous proteins in bone, cartilage, and developing enamel. In: Ali Sy (Ed.) Cell Mediated Calcification and Matrix Vesicles. Elsevier, Amsterdam-New York, pp. 33-38 2 Bruland 0, Fudstad 0, Fundernd S, Pilil A (1986) New monoclonal antibodies specific for human sarcomas. Int J Cancer
38: 27-31
3 Cordell JL, Falini B, Erber WN, Ghosh AK, Abdulaziz Z, MacDonald S, Pulford KAF, Stein H, Mason DY (1984) Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem 32:
219-229
4 Fisher LW, Termine JD (1985) Noncollagenous proteins influencing the local mechanisms of calcification. Clin Orthop Rei Res 200: 362-385 5 Fisher LW, Hawkins GR, Tuross N, Termine JD (1987) Purification and partial characterization of small proteoglycans I and II, bone sialoproteins I and II, and osteonectin from the mineral compartment of developing human bone. J Bioi Chern
262: 9702-9708
Osteonectin in Osteosarcoma . 657 6 Gehron Robey P, Termine JD (1985) Human bone cells in vitro. Calcif Tissue Int 37: 453-460 7 Grundberg CM, Hauschka PV, Lian JB, Gallop PM (1984) Osteocalcin: Isolation, characterization, and detection. Meth Enzymol107: 526-544 8 Herring GM, Kent PW (1961) Acidic mucosubstances of cortical bone. Biochem J 81: 5P + 9 Jundt G, Berghauser KH, Termine JD, Schulz A (1987) Osteonectin differentiation marker of bone cells. Cell Tiss Res 248:409-415 10 Jundt G, Schulz A, Berghauser KH, Fisher LW, Gehron Robey P, TermineJD (1989) Immunocytochemical identification of osteogenic bone tumors by osteonectin antibodies. Virchows Archiv A 414: 345-353 11 Price P (1983) Osteocalcin. In: Peck WA (Ed.) Bone and Mineral Research Annual 1. Excerpta Medica, Princeton, pp. 157-190 12 Remberger K, Gay S (1977) Immunohistochemical 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 13 Roessner A, Voss B, RauterbergJ, Immenkamp M, Grundmann E (1983) Biologic characterization of human bone tumors. II. Distribution of different collagen types in osteosarcoma. J Cancer Res Clin Oncol106: 234-239 14 Romberg RW, Werners PG, Lollar P, Riggs BL, Mornukbi G (1985) Isolation and characterization of native adult osteonectin. J Bioi Chern 260: 2728-2736 15 Rosen G, Caparros B, Huvos AG, Kosloff C, Nirenberg A, Cacavio A, Marcove RC, Lane JM, Metha B, Urban C (1982) Preoperative chemotherapy for osteogenic sarcoma: Selection of postoperative adjuvant chemotherapy based on the response of the primary tumor to preoperative chemotherapy. Cancer 49: 1221-1230 16 Schulz A, Jundt G, Berghauser KH, Gehron Robey P, Termine JD (1988) Immunohistochemical study of osteonectin in various types of osteosarcoma. Am J Pathol 132: 233-238
17 Schulz A, Romanowski R, Jundt G, Berghauser KH (1987) Immune electron microscopy of cell and matrix antigens in bone cells and bone tumors. Calcif Tiss Int 41 (Suppl. 2): 18 + 18 Termine JD (1987) Non-collagenous proteins of enamel and bone. Calcif Tiss Int 41 (Suppl. 2): 810 + 19 Termine JD, Kleinman HK, Whitson SW, Conn KM, McGarvey ML, Martin GR (1981) Osteonectin, a bone-specific protein linking mineral to collagen. Cell 26: 99-105 20 Tung PS, Domenicucci C, Wasi S, Sodek J (1985) Specific immunohistochemical localization of osteonectin and collagen types I and III in fetal and adult porcine dental tissues. J Histochem Cytochem 33: 531-540 21 Urist R, Delange RJ, Finerman GAM (1983) Bone cell differentiation and growth factors. Science 220: 680-686 22 Wasi S, Otsuka K, Yao KL, Tung PS, Aubin JE, Sodek J, Termine JD (1984) An oste.onectin-like protein in porcine peridontal ligament and its synthesis by peridontal ligament fibroblasts. Can J Biochem Cell Bioi 62: 470-478 23 Wewer UM, Abrechtsen P, Fisher LW, Young MF, Termine JD (1988) Osteonectin/SPARC/BM-40 in human decidua and carcinoma, tissues characterized by de novo formation of basement membrane. Am J Pathol132: 345-355 24 Whitson SW, Harrison W, Dunlap MK, Bower DE jr, Fisher LW, Gehron Robey P, Termine JD (1984) Fetal bovine bone cells synthesize bone-specific matrix proteins. J Cell BioI 99: 607-614 25 Winkler K, Beron G, Kotz R, Salzer-Kuntschik M, Beck J, Beck W, Brandeis W, Ebell W, Ettmann R, Gobel U, Havers W, Henze G, Hinderfeld C, Hocher P, Jubke A, Jiirgens H, Kabisch H, Preusser P, Prindull G, Ramach W, Ritter J,SekeraJ, TreunerJ, Wiist G, Landbeck G (1984) Neoadjuvant chemotherapy for osteogenic sarcoma: Results of a cooperative German-Austrian study. J Clin Oncol2: 614-624 26 Yoshikawa H, Takaoka K, Masuhara K, Ono K, Sakamoto Y (1988) Prognostic significance of bone morphogenetic activity in osteosarcoma tissue. Cancer 61: 569-573
Received November 24, 1989 . Accepted in revised form February 1, 1990
Key words: Osteosarcoma - Osteonectin - Differential diagnosis - Osteogenesis Dr. med. Alexander Bosse, Gerhard-Domagk-Institut fiir Pathologie, Domagkstr. 17, D-4400 Miinster, FRG
The Impact of Osteonectin for Differential Diagnosis of Bone Tumors! An Immunohistochemical Approach A. Bosse, E. Vollmer, W. Boeker and A. Roessner Gerhard-Domagk-Institut fUr Pathologie, Munster, FRG
P. Wuisman and D. Jones Orthopadische Klinik, Westfalische Wilhelms-Universitiit Munster, FRG
L. W. Fisher NIH, Institute of Dental Research, BethesdalMd USA
SUMMARY 75 osteosarcoma at various grades of histologic differentiation, including chondroblastic and small cell variants, and 5 fibrosarcomas of bone, 5 Ewing's sarcomas, 5 malignant fibrous histiocytomas of bone, 8 chondrosarcomas, and 2 dedifferentiated chondrosarcomas, were investigated immunohistochemically for evidence of osteonectin. According to the results of our study, osteonectin is present in all osteosarcomas, with special topographic preponderance in the osteoblastic and chondroblastic variants. Evidence of osteonection was also found in all other bone tumors we had analysed so far. In chondrosarcomas, positive reactions appeared only in the vicinity of trabeculae and in dedifferentiated areas. Thus, osteonectin cannot be regarded as a bone-specific protein. Although a high affinity for the osseous matrix is one of its undoubted features, it is therefore unsuitable for differential diagnostic purposes.
Introduction Osteosarcomas are classified mainly according to the ground substance of their mesenchymal tissue. Consequently, the definition of bone-specific proteins plays an important role in the classification and diagnostics of bone tumors. Since osteoblasts are known to loose their capacity for osteoid formation under certain pathologic conditions, osteosarcomas will often show a strong resemblance to other mesenchymal bone tumors such as fibrosarcoma, I This work was supported by grant number Bo 990/1-1 from the Deutsche Forschungsgemeinschaft.
© 1990 by Gustav Fischer Verlag, Stuttgart
malignant fibrous histiocytoma, chondrosarcoma or Ewing's sarcoma. This variegated appearance often hampers histologic diagnosis, especially when it has to be based on rather small biopsy material 15 , 25. Immunohistochemistry has failed, so far, to provide helpful data for differential diagnosis, since the results were not exactly bone-specific; moreover, it was found that various types of collagen were synthesized under pathologic conditions 12 , 13. The attempts to produce specific antibodies against osteosarcoma have not been very successful either, since these would also react with other soft tissue tumors 2 . In view of the differential separation of osteosarcoma from other mesenchymal soft tissue tumors, much hope 0344-0338/90/0186-0651$3.50/0
652 . Bosse, A. et al. 40
7-
Table 1. Immunohistochemical results with ostonectin antibody in different types of osteosarcoma and other mesenchymal bone tumors
30
Tumor diagnosis
Number of cases total
Reaction intensity with anti-osteonectin
Osteosarcomas osteoblastic telangiectatic fibroblastic anaplastic chondroblastic small-cell
75 35 10
++ +++ +++ ++ ++ ++ ++ ++ + ++ (+)* +
20
10
o
o, ~., ',. 0,'.0<.'," ~..
.,,",,-
J...~J l".mT.?"",,
Siolo HS-Glyko- cones 1/11 Protein 1/11 protein
'00_
colla~enous proteins
Fig. 1. Non-collagenous bone proteins. Distribution in the developing bone tissue.
Fibrosarcoma MFH Ewing's Sarcoma Chondrosarcoma dediff. Chondrosarcoma
8 9
12 1 5 5 5
8
2
+++ +++ +++ +++ +++
* Positive immunoreactivity occuring only in areas of osteoid trabeculae; + = low, ++ = moderate; +++ = strong.
Fig. 2. Osteoblastic osteoblastoma with strong immune reaction in osteoblasts near trabeculae (anti-osteonectin AB, X 550).
Fig. 3. Chondroblastic osteosarcoma with strong marginal ~ immune expression and negative labeling in cartilaginous centers (anti-osteonectin AB, x 120).
Fig. 5
Osteonectin in Osteosarcoma . 655
has focused therefore on the immunohistochemical demonstration of non-collagenous osseous structure proteins which are obviously characterised by high bone specificity. The protagonists of this group are osteocalcin7, 11, sialoproteins8, and osteonectin18,19 (Fig. 1). Osteonectin is a phosphorylated glycoprotein with a molecular weight of 38000 Dalton, isolated first from bovine bone 18 . In bone cell culture24 as well as in reactive bone lesions 9, osteonectin could be demonstrated immunohistochemically in osteoblasts. Further evidence was found in non-osteoblastic cells that should therefore be classified as osteo-progenitor cells 16 . The functional role and significance of osteonectin in the bone is still as undefined as that of the other non-collagenous osseous proteins. Obviously, they must have a potential to steer the mineralisation of tissue 21 . Osteonectin, for instance, shows a high affinity for hydroxylapatite and for type I collagen4. On account of their high bone specificity, the immunohistochemical demonstration of non-collagenous osseous structure proteins might help to differentiate osteosarcoma from other bone tumors. Some positive experience has been published 1o,17, but other groups have demonstrated osteonectin also in fibroblasts 22 and even in chorionic villi23 . In order to define the value and validity of osteonectin in this context, we surveyed its immunoexpression in a large material comprising osteosarcomas and other bone tumors. Material and Methods The study comprised 75 osteosarcomas, including chondroblastic and small cell variants, 5 fibrosarcomas, 5 malignant fibrous histiocytomas, 5 Ewing's sarcomas, 8 chondrosarcomas and two dedifferentiated chondrosarcomas. Antibodies were raised in rabbits against bovine and human osteonectin as described by Termine et al. ls . All immunohistochemical studies were performed on formol-fixed material (4% buffered formol) embedded in paraffin. A large part of the
... Fig. 4. Immunocytochemical results of osteonectin with different types of osteosarcomas. - a: Telangiectatic osteosarcoma with numerous positive marked cells that are often connected to osteoid islets. - b: Small cell osteosarcoma with some positive cells. - c: Fibroblastic osteosarcoma with intense marking in scattered disseminated tumor cells. - d: anaplastic osteosarcoma lacking osteoid production, with a strong marking in the cytoplasm of huge pleomorphic atypical osteoblasts (all antiosteonectin AB, x 550). ... Fig. 5. Immunohistochemical proof of osteonectin in mesenchymal bone tumors. - a: Fibrosarcoma with a strong positive immune reaction pattern in the cytoplasm of tumor cells. - b: Malignant fibrous histiocytoma with fainter marking of scattered disseminated tumor cells. - c: Ewing's sarcoma with positive marking of many tumor cells in the small cytoplasmic rim. d: Dedifferentiated chondrosarcoma with a small number of positive cells in dedifferentiated areas (all anti-osteonectin AB, x 550).
samples had been previously decalcified with 10% EDTA solution. The. tests with polyclonal antibodies were carried out on 2 ~m sections. Demonstration of osteonectin was performed using the APAAP method (alkaline phosphatase - antialkaline phosphatase) with naphthol salt and hexazodated neofuchsin 3• Deparaffinated sections were incubated for 45 minutes at room temperature with the primary antibody after optimal antibody dilution (1 :400) had been tested in preceding experiments. Trypsinase pretreatment had no influence on the immunocytochemical reaction which yielded identical results with human and bovine anti-osteonectin. Immunohistochemical demonstration of osteonectin in an aggressive osteoblastoma served as positive control specimen.
Results In all osteosarcoma specimens osteonectin was expressed. The intensity of immune reactions and the number of labeled tumor cells varied considerably (Table 1). Classical osteoblastic osteosarcomas showed a strong immunoexpression in atypical osteoblasts, especially in the vicinity of neoplastic osteoid trabeculae. No topographic foci of labeling could be identified in osteoblastic tumors, the general pattern of distribution being rather uniformly disseminated (Fig. 2). In chondroblastic osteosarcoma osteonectin immunolabeling was confined to marginal chondroblastic areas and to a smaller amount of fibroblasts (Fig. 3). Osteonectin was also found in the rare form of small cell osteoblastic osteosarcoma, with occasional perinuclear labeling of tumor cells in random distribution. The distribution pattern in telangiectatic osteosarcomas corresponded largely to that in osteoblastic osteosarcomas. Fibroblastic and anaplastic variants disclosed intense immunolabeling in cells that could not be classified as osteoblasts by purely histologic criteria, without any clear correlation to osteoblastic or bone differentiation (Fig. 4). In all other bone tumors osteonectin expression was observed. Fibrosarcoma showed strong intracytoplasmic reaction in a diffuse patchy pattern. In malignant fibrous histiocytomas, intensive osteonectin labeling was seen occasionally in some cells. In Ewing's sarcomas there again was a marked patchy immune reaction. In chondrosarcomas, however, osteonectin could be demonstrated only in isolated cells in close proximity to trabeculae, and occasionally in dedifferentiated chondrosarcoma. The chondroid tumor formation proper was plainly negative without exception (Fig. 5). Discussion The organic bone matrix is made up of type I collagen (90%) and non-collagenous osseous structure proteins (10%). Of these, osteonectin is one of the major components, produced by osteoblasts (among others) and obviously characterised by remarkable bone specificity18,19. Osteonectin was successfully demonstrated, so far, in numerous benign and malignant bone tumors as well as in reactive bone lesions, especially and preferentially in the topographic areas of enchondral ossification9,16,20.
656 . Bosse, A. et al.
On account of the close relationship of osteonectin and bone matrix, and the proven synthesis by osteoblastic cells supported by immune electron microscopical investigations 1, 16, this protein is supposed to be truly bone specific. Certain previous studies based on a limited number of cases seemed to confirm this concept, since no evidence of osteonectin could be obtained from various mesenchymal tumors 10,17. Additional proof was seen in the fact that no proteins could be demonstrated so far, which would be manifested exclusively in osteoblasts. There had been no reliable mode for differentiating bone tumors, especially osteosarcomas, from other mesenchymal bone tumors by immunohistochemical methods, and even collagens were not found truly bone specific2, 12, 14. Since the diagnosis of bone tumors is often based on a rather small biopsy sample 15 ,25, accurate demonstration of bone specific proteins would be a powerful tool in differential diagnosis when osteosarcomas, frequently very heterogenous in appearance, have to be separated from other bone tumors. It had been shown, however, already in 1984 that osteonectin is also produced in vitro by fibroblasts from other tissues 22 . Subsequently, it could be demonstrated by a higly sensitive radioimmunoassay in non-osseous tissues 6 and even in chorionic villi, among others 23 . Prompted by these pointers to a disputed osseous specificity, we started the comparative investigation of 75 osteosarcomas against numerous other mesenchymal bone tumors with regard to their patterns of osteonectin expression. Our results have shown that all the osteosarcomas expressed osteonectin, independent of the grade of differention and even very rare variants such as small cell osteosarcoma and chondroblastic osteosarcoma. On the whole, immunohistochemistry reveals a very heterogenous picture. In particular, osteoblasts in the vicinity of trabeculae showed positive labeling, but the tumor cells of the fibroblastic, anaplastic, and even small cell variants also yielded distinct immune reactions in cells that were definitely non-osteoblastic by histomorphologic criteria. The chondroblastic variant of osteosarcoma showed stronger labeling in the marginal parts, with decreasing intensity towards the central cartilagineous areas (d. Fig. 3, 4). In fibrosarcomas, Ewing's sarcomas and malignant fibrous histiocytomas studied for comparison there was also a marked osteonectin expression, especially in fibrosarcomas where immune expression had a strong focal accentuation resembling that in fibroblastic osteosarcoma. Previous studies of chondrosarcoma 17 could be confirmed to a large extent; some positive atypical chondrocytes were found only in the immediate vicinity of trabeculae; some isolated evidence of osteonectin was also present in dedifferentiated chondrosarcomas (Fig. 5). With the exception of chondrosarcomas with their largely negative immune expression, our results are not in agreement with those of other work groups9, 10, 17. Since all were performed with identical antibodies from the research laboratories of NIH in Bethesda, the divergent results may be attribuable to methodological differences, such as different antibody dilutions, periods of incubation,
and temperatures. Moreover, the application of PAP technique, Avitin-Biotin complex method, or APAAP method as employed in our assay, the immunohistochemical approach is principally a different one. We have to infer that osteonectin, like collagen type I, cannot be considered as truly bone specific, being manifestly present in quite a number of other mesenchymal tumors. There is no doubt that osteonectin is produced predominantly by osteoblasts and has its outstanding localisation in osseous lesions where it may be taken as a marker of osteogenesis5, 14, 18. According to our results, however, it cannot be appraised as an exclusively osseous structure protein, since it is evident in several other types of tissue. Obviously, immunohistochemical demonstration of osteonectin depends on its concentration in the respective tissue, which is liable to considerable variation 6, 23. Thus, osteonectin can be rather interpreted as a multilocular structure protein occurring in high concentration especially in osteoblasts. In this tissue, the varying patterns of distribution show clear topographic cumuli, especially in osteoblastic osteosarcoma in the vicinity of trabeculae and in the chondroblastic variant, where they suggest a special functional status of osteoblasts reflecting increased activity. Possibly the correlation of reaction intensity with the grade of osteosarcoma differentiation will offer a new approach to the differential, more subtle, and therapy-oriented classification of osteosarcomas. Some preliminary experience with another structure protein, bone morphogenetic protein (BMP) has been published26 • Osteonectin, however, is not specific enough to serve as differential marker against other bone mesenchymal tumors. Acknowledgements The authors want to thank Misses B. Duhm and P. Schafer for excellent technical assistance, and Mrs. E. Griffith and H. Lossmann for the preparation of the manuscript. References 1 Bonnucci E, Bianco P, Hayashi Y, Termine JD (1986) Ultrastructural immunohistochemical localization of non-collagenous proteins in bone, cartilage, and developing enamel. In: Ali Sy (Ed.) Cell Mediated Calcification and Matrix Vesicles. Elsevier, Amsterdam-New York, pp. 33-38 2 Bruland 0, Fudstad 0, Fundernd S, Pilil A (1986) New monoclonal antibodies specific for human sarcomas. Int J Cancer
38: 27-31
3 Cordell JL, Falini B, Erber WN, Ghosh AK, Abdulaziz Z, MacDonald S, Pulford KAF, Stein H, Mason DY (1984) Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem 32:
219-229
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Received November 24, 1989 . Accepted in revised form February 1, 1990
Key words: Osteosarcoma - Osteonectin - Differential diagnosis - Osteogenesis Dr. med. Alexander Bosse, Gerhard-Domagk-Institut fiir Pathologie, Domagkstr. 17, D-4400 Miinster, FRG