Virchows Archiv A Pathol Anat (1992) 421:229-238
VirchowsArchiv A PathologicalAnatomy and Histopathology 9 Springer-Verlag 1992
Osteosarcoma with features mimicking malignant fibrous histiocytoma Haruhiko Yoshida 1, Tokichi Yumoto 1, and Takeshi Minamizaki 2 Departments of 1 Pathology and 20rthopaedic Su9
Tottori University School of Medicine, Tottori, Japan
Received December 13, 1991 / Received after revision March 31, 1992 / Accepted April 1, 1992
Summary. Three osteosarcomas (OS) with features resembling malignant fibrous histiocytoma ( M F H ) were selected and investigated to identify any clinico-pathological similarities. In ail cases there was no significant difference from conventional OS on the radiography and laboratory data. The appearance of M F H - l i k e features within the whole t u m o u r tissue varied f r o m 7% to 55%. It was composed of spindle-shaped cells arranged in short irregular fascicles and a storiform pattern admixed with osteoclast-like giant cells, but devoid of neoplastic osteoid. Such spindle-shaped cells had a poorly developed rough endoplasmic reticulum and expressed a strong alkaline phosphatase activity as well as vimentin. A series of allografts to athymic mice using the M F H like tissues also showed histologically a proliferation of plump spindle-shaped cells with a storiform pattern lacking osteoclast-like giant cells, and intensely positive for alkaline phosphatase. These findings indicate that the M F H - l i k e features are identified as modulated OS. The constituting cells are most likely to be poorly developed with possible phenotypic alteration in the m a t u r a t i o n stage of osteoblastic cell lineage, but different from conventional M F H of bone as regards their distinct histochemical pattern. Key words: Osteosarcoma - Malignant fibrous histiocyt o m a - Transplantation
ly, additional histological subtypes and patterns of OS were variously proposed, all of which were hardly distinguishable f r o m other benign and malignant bone tumours (Mirra 1980; Yunis and Barnes 1986). On rare occasions OS shows an excessive proliferation of spindle-shaped cells with a pronounced pleomorphism in a storiform pattern without definite osteoid tissue. This pattern is similar to malignant fibrous histiocytoma ( M F H ) of bone, but the neoplastic osteoid and bone are identified only in other areas of the saine tumour. Mirra (1980) designated this M F H - l i k e OS to distinguish it from conventional M F H of bone. The percentage of patients with such M F H - l i k e OS in an entire series of OS varied f r o m 5% to 30% in a number of investigations (Mirra 1980; H u v o s 1986; Yunis and Barnes 1986). Ballance et al. (1988) emphasized the M F H subtype of OS as a distinct entity f r o m M F H of bone, and high-lighted the radiographic similarities to conventional, highly aggressive OS. The inclusion of an M F H - l i k e subtype in the classification of OS still remains debated. In this paper, we report three cases of OS resembling M F H of bone and describe the clinico-pathological features of MFH-like OS as c o m p a r e d with M F H of bone. We discuss whether the histogenetic concept should be listed in the classification of OS.
Materials and methods Introduction Osteosarcoma (OS) can exhibit a wide spectrum of histopathological appearances and on the basis of this a classification has been ruade into osteoblastic, chondroblastic and fibroblastic subtypes according to the m a j o r histological c o m p o n e n t (Dahlin and Unni 1977). RecentCorrespondence to: H. Yoshida, Department of Pathology, Tottori University School of Medicine, 86 Nishi-machi, Yonago, Tottori 683, Japan
Cases examined in this report were selected from 16 consecutive cases of OS (aged 9-75 years, mean 19.3 years) registered in the First Department of Pathology, Tottori University School of Medicine, Japan from 1975 to 1989. Every case was diagnosed by biopsy and histology of surgically resected material. From the•e, 3 cases of OS with MFH-like features were identifie& All clinical and radiographic records are li•ted in Table 1. For light microscopy, the surgically re•ected tumours were fixed with 10% buffered formalin, decalcified in a formic acid-9 buffer and embedded in paraffin. Further, large slab sections of maximally demonstrated areas were prepared for mapping of various histological patterns, including the MFH-like feature. Tissue blocks obtained from surgical sper were fixed with 4% para8 for 5 h at 4~ C, replaced and kept in gum-
230 Table 1. Clinical findings of osteosarcoma (OS) with malignant fibrous histiocytoma (MFH)-like features Case
Age/sex
Clinical sign
Location
Radiographic findings
Treatment
Prognosis
1
14/F
Right knee pain Serum ALPase 76 units/1 (37/107)
R-tibia proximal
Mixed sclerotic-lytic Sort tissue mass with calcifcation
Amputation above knee, neoadjuvant chemotherapy a
Alive, postop. 24 months dead lung metastases
2
11/F
Left knee pain Serum ALPase 250 units/1 (37/107)
L-femur distal
Mixed sclerotic-lytic Codman's triangle
Wide resection, limb salvation procedure (Kotz's prosthesis) neoadjuvant chemotherapy"
Alive, postop. 38 months disease-free
3
75/F
Left knee pain Serum ALPase 117 units/t (22-116)
L-femur distal
Lytic with focal sclerotic lesion
Amputation above the knee, postop. chemotherapy"
Alive, postop. 44 months disease-free
ALPase, Alkaline phosphatase Rosen T-12 protocol
sucrose solution at 4~ C. The tissues then were quickly frozen using dry-ice with addition of hexane, sectioned to 4-6 gm thickness with a cryostat (Bright) at - 2 0 ~ and stained for the following enzymes: alkaline phosphatase (ALPase, by Bengt et al.), acid phosphatase (ANAE, by Leder), adenosine triphosphatase (ATPase, by Mfiller-Hermelink), 5'-nucleotidase (5'-N, by Muller-Hermelink) and beta-glucuronidase (beta-Gl, by Hayashi et al.). Immunohistochemical staining was carried out on paraffin sections of 6 gin thickness employing the avidin-biotin peroxidase complex technique after being reacted with antibodies against collagen I (Col I), aminopeptidase M(APM), and dipeptidyl peptidase IV (DDP IV). These antibodies were all primarily prepared in our laboratory (Aki 1989). Their specificities were determined and characterized by recognizing fibroblasts selectively. Anti-factor XIIIa (Hoechst-Behring, Marburg, FRG) for fibroblast and osteoblast marker, anti-alpha-l-antitrypsin (AAT), anti-alpha-l-antichymotrypsin (AACT), anti-lysozyme (Lys, Dakopatts, Copenhagen, Denmark) for monocyte-macrophage marker, and anti-vimentin (Dakopatts) for mesenchymal marker. Antibodies against Col I were used at a dilution of i : 100. The optimal dilution of other primary antibodies used in this study was as follows: 1:50 for the anti-factor XIIIa; 1:2 for the antiAPM and DDP IV; original kit of anti-AAT, AACT and Lys; and 1 : 10 for anti-vimentin. The sections were incubated with primary antibodies for 30 min at room temperature, followed by biotinylated anti-mouse/anti-rabbit immunoglobulin for 20 min and then avidin-biotin peroxidase complex (Dakopatts) for 20 min. Finally, the sections were immersed with 0.3 % (v/v) hydrogen peroxide and 0.6% (w/v) 3.37-diaminobenzidine solution and counterstained with methyl green. Osteoblastic tumour cells in conventional OS served as controls for 3 cases with MFH-like features to be compared as regards the intensity and extent of staining reactions. The fresh samples were fixed with 3% glutaraldehyde solution (buffered pli 7.4) for l h at 4~ C and were post-fixed with 2% phosphate-buffered osmium tetroxide for 1 h. After dehydration with an alcohol series, they were embedded in epoxy resin. Ultrarhin sections were stained with lead citrate and uranyl acetate and were observed with a H-800 electron microscope (Hitachi, Japan). For transplantation study, inbred nu/nu mice of Balb/c genetic background were used at the age of 8 weeks. The tissue fragments with MFH-like features in case 1 were transplanted subcutaneously onto the flank. The transplanted mice were bred and kept under special pathogen-free conditions. A new generation was obtained by transplantation of the xenografted tumour tissue for 5 months.
The tissue obtained from the nude mice was examined in the saine way as the original tumour.
Results T h e clinical d a t a o f each case are s u m m a r i z e d in Table 1. T h e p a t i e n t s were all female, r a n g i n g in age f r o m 11 to 74-years. A l l p a t i e n t s c o m p l a i n e d o f p a i n a n d swelling with a s y m p t o m d u r a t i o n o f f r o m 2 weeks to 6 m o n t h s . The s e r u m A L P a s e level in each case was f r o m 76 to 250 units/1; case 2 s h o w e d a significant elevation a n d the o t h e r two cases (cases I a n d 3) were within n o r m a l limits. R a d i o g r a p h i c findings (Fig. 1 A - C ) s h o w e d m i x e d o s t e o l y t i c a n d , in p a r t , o s t e o s c l e r o t i c lesions o f the m e t a physis o f f e m u r (cases 2 a n d 3) a n d t i b i a (case 1). D e s t r u c t i o n o f the c o r t i c a l b o n e was o b v i o u s in all cases. G r o s s l y , the t u m o u r s were solid, white-greyish a n d g r i t t y to b o n e - h a r d w i t h v a r i a b l e a m o u n t s o f h a e m o r r h a g e a n d necrosis (Fig. 2 A - C ) . T h e m a r r o w c a v i t y o f the m e t a p h y s i s was a l m o s t c o m p l e t e l y o b l i t e r a t e d b y the a b o v e tissue affecting the e p i p h y s e a l p l a t e (cases 1 a n d 2) o r d i a p h y s i s (case 3). T h e t u m o u r e x t e n d e d directly t h r o u g h the a d j a c e n t c o r t i c a l b o n e i n t o the s u r r o u n d i n g soft tissues in ail cases. C a r t i l a g i n o u s t u m o u r tissue w i t h g r o u n d - g l a s s a p p e a r a n c e was d e t e c t e d in the soft tissue c o m p o n e n t a n d d e l i n e a t e d clearly f r o m the h a r d tissue c o m p o n e n t (case 2). H i s t o l o g i c a l l y , the M F H - l i k e f e a t u r e c o n s i s t e d o f f i b r o b l a s t - l i k e s p i n d l e - s h a p e d cells a n d r o u n d cells int e r m i n g l e d with v a r y i n g n u m b e r s o f o s t e o c l a s t - l i k e g i a n t cells, a n d f a s c i c u l a t e d g r o w t h p a t t e r n o f s p i n d l e - s h a p e d cells in a s t o r i f o r m p a t t e r n (Fig. 3). O s t e o c l a s t - l i k e g i a n t cells were s c a t t e r e d m o r e o r less a m o n g the t u m o u r cells (Fig. 4). O c c a s i o n a l l y , the t u m o u r cells m o d u l a t e d their m o r p h o l o g i c a l f e a t u r e s f r o m s p i n d l e - s h a p e d to r o u n d cells with a b u n d a n t c y t o p l a s m a n d b e c a m e i n c o m p l e t e in a s t o r i f o r m a r r a n g e m e n t (all cases), thus i m p a i r i n g the i m p r e s s i o n o f g i a n t cell t u m o u r o r g i a n t cell-rich M F H w i t h m a r k e d cellular p l e o m o r p h i s m (Fig. 5). H o w -
231
Fig. 1 A-C. Radiographic findings: A AP view of proximal tibia of 14-year-old female (case 1). Eccentric, loculated, sclerotic lesion is obvious with extension to knee joint; B 11-year-old female (case 2) with mixed sclerotic and lytic lesion of the metaphysis of right femur. Periosteal reaction is visible showing Codman's triangle; C 75-year-old female (case 3) with mixed sclerotic and lytic lesion of the metaphysis of left femur
232
Fig. 2A-C. Gross findings: A (case 1) centrally located, hard tumour extending directly into the surrounding soft tissue, followed by large, white mass; B (case 2), intraosseous solid, white-greyish tumour with central necrosis and haernorrhage; C (case 3) the marrow cavity of the metaphysis is completely obliterated by yellow-greyish tumour tissue
ever, phagocytic figures were not found. Atypical mitotic figures were frequently encountered. These were ill-defined from the osteoblastic area and lacked neoplastic osteoid and bone, but the lace-like osteoid tissue was frequently recognized among the tumour cells adjacent to the osteoblastic component (Fig. 6). An osteoblastic, bone-rich area was found largely in the tumour component located in the marrow cavity (all cases). In case 1, malignant neoplastic chondroid tissue was observed
in the sofl tissue component and well-delineated from areas of malignant cell proliferation containing bone and osteoid tissue and MFH-like feature (Fig. 7). According to the histological mapping of the whole tumour tissue, the incidence of MFH-like feature in the three cases was 55%, 40% and 7% (Fig. 8 ~ C ) , respectively. Those areas were hardly distinguishable from the osteoblastic area in two cases (cases 1 and 2) and were relatively delineated in case 3.
233
Fig. 3. Malignant fibrous histiocytoma (MFH)-like feature in case 2. Bundles of spindle-shaped cells with pleomorphism are arranged in a typical storiform pattern. H&E, x 100
Fig. 4. Osteoclast-like giant cells are scattered among spindle-shaped tumour cells. H&E, x 200 Fig. 5. In this area, osteoclast-like giant cells are prominent as in giant cell tumour (case 1). H&E, x 100
234
Fig. 6. MFH-like area is generally ill-defined from central osteoblastic area. H&E, • 100 Fig. 7. MFH-like area is well-delineated from the neoplastic chondroid tissue (case 1). H&E
Fig. 8. Schematic diagram of the tumours in each case; the percentage incidence of MFH-like area is shown and determined at a rate of 55% (A: case 1), 40% (B: case 2) and 7% (C: case 3)%, respectively
The results of immunohistochemical staining are shown in Table 2 with additional findings of M F H of soft tissue origin. Spindle-shaped tumour cells in a storiform pattern reacted significantly with anti-AACT antibody for monocyte-macrophage marker and anti-vimentin antibody for mesenchymal marker. In contrast, all
types of tumour cells showed negative reaction for the anti-Lys antibody. Both anti-Col I and anti-factor XIIIa antibodies reacted with a small number of cells which were negative for the anti-APM and anti-DDP IV antibodies. Table 3 shows the cumulative enzyme histochemical
235
Fig. 9. ALPase reaction of MFH-like area (cases 1 and 2). Most of the tumour cells are significantly positive except for osteoclast-like giant cells. ALPase, x 200 Fig. 10. ATPase reaction of the same areas as those shown in Fig. 8. Positive reaction is shown in osteoclast-like giant cells and a small number of histiocytic cells but not in most of the tumour cells. ATPase, x 200 Fig. 11. Transplanted tumour tissue showing a fasciculating growth pattern of fibroblast-like spindle-shaped cells often with a storiform pattern (case 1, 5th generation). H&E, x 100
236 Table 2. Comparative immunohistochemical findings between OS with MFH-likc feature and MFH of bone
Antibodies against Col I
XIIIa
OS with MFH-like lesion Case I + Case 2 + + + + + Case 3 + + + MFH of bone Case 1 Case 2 . Case3 ++ Case 4 Case 5 +
.
+ + . ++ -
APM
DDPIV
AACT
AAT
Lys
Vim
+ -
+ -
+ + +
+ +
-
+ + + + + + +
-
-
+ -
+ + -
+ + + + ++ + +
+ + + + ++ +
+ -+ +
+ + + ++ + +
.
Intensity of reaction: + § § strong; + § moderate; + , slight and focal; - , negative. In strong and moderate reactions, the reactivity extended diffusely on the overall specimens, whereas in slight reaction it was present in some focal areas. OS, Osteosarcoma; MFH, malignant fibrous histiocytoma of bone; Col I, collagen I; XlIIa, factor XIIIa; APM, aminopeptidase M; DDP IV, dipeptidyl peptidase IV; AACT, alpha-l-antichymotripsin; AAT, alpha-l-antitripsin; Lys, lysozyme; Viro, vimentin
Table
3. Comparative enzyme histochemical findings between OS with MFH-like feature and MFH of bone
Tumour cell components
OS with MFH-like lesion (n = 3) Osteoblastic cells Fibroblast-like spindle cells MFH of bone (n=4) Fibroblast-like spindle cells Histiocyte-like round cells
Enzymes ALPase
ACPase
ANAE
ATPase
5'-N
Beta-G1
+ + + + +
+
--
++
+++
+
--
+
-
+
-
+ ++
+ +++
+ ++
+ +
+ +
Intensity of reaction: + + + , strong; + - , moderate; + , slight and focal; - , negative. In the strong and moderate reactions, the activity extended diffusely on the overall specimens, whereas in slight reaction, it was present in some focal areas
ALPase, Alkaline phosphatase; ACPase; ANAE; ATPase, adenosine triphosphatase; 5'-N, 5'-nucleotidase; beta-G1, beta-glucoronidase
results. T h e s p i n d l e - s h a p e d cells a n d b i z a r r e g i a n t cells were s t r o n g l y positive for A L P a s e r e a c t i o n (Fig. 9). O t h e r e n z y m e activities were as f o l l o w s : s t r o n g l y positive for 5 ' - N a n d w e a k l y positive for A T P a s e o n the cell surface a n d w e a k l y positive for A C P a s e close to the nucleus. A small n u m b e r o f histiocytic cells w h i c h were i n t e r m i n g l e d a n d p o s i t i v e for A T P a s e (Fig. 10) a n d A N A E s e e m e d to be in the p r o c e s s o f reaction. U l t r a s t r u c t u r a l l y m o s t o f the t u m o u r cells in M F H like foci d i s p l a y e d a s p i n d l e - s h a p e d c o n f i g u r a t i o n with w e l l - d e v e l o p e d r o u g h e n d o p l a s m i c r e t i c u l u m d i l a t e d to a v a r y i n g degree in the e l o n g a t e d c y t o p l a s m as d e s c r i b e d in a p r e v i o u s r e p o r t ( M i n a m i z a k i et al. 1990). A m o r p h o u s m a t e r i a l s w i t h b u n d l e s o f c o l l a g e n fibres were r a r e l y seen in the e x t r a c e l l u l a r area. O s t e o c l a s t - l i k e g i a n t cells were f o u n d m o r e o r less w i t h a b u n d a n t i n t r a c y t o p l a s m i c vesicles. T h e y h a d i r r e g u l a r l y i n d e n t e d nuclei with p e r i n u c l e a r c h r o m a t i n c l u m p i n g . T h e l a t e n c y o f t r a n s p l a n t s v a r i e d f r o m 155 to 280 days. T b e M F H - l i k e a r e a in o s t e o s a r c o m a (case ~) was
successfully g r a f t e d a n d t r a n s p l a n t e d for rive generations. H i s t o l o g i c a l findings o f the tissue o b t a i n e d f r o m the n u d e mice were a l m o s t similar to t h o s e o f t h e original t u m o u r , s h o w i n g a p r o l i f e r a t i o n o f i n t e r l a c i n g fascicles o f f i b r o b l a s t - l i k e s p i n d l e - s h a p e d cells with a s t o r i f o r m p a t t e r n in a large p r o p o r t i o n o f the tissues (Fig. ~1). T h e lace-like o s t e o i d tissue was r e c o g n i z e d largely in the central p o r t i o n o f the t u m o u r mass. T h e r e was a c o m p l e t e l a c k o f o s t e o c l a s t - l i k e g i a n t cells. T h e constituent cells o f the t r a n s p l a n t e d t u m o u r were s t r o n g l y positive for A L P a s e reaction.
Discussion
M F H - l i k e OS was first p r o p o s e d b y M i r r a (1980) as a different entity f r o m M F H o f b o n e , w h i c h was welld e s c r i b e d a n d w i d e l y a c c e p t e d ( H u v o s et al. 1985). This c o n c e p t was also s u p p o r t e d b y Ballance et al. (1988), w h o e m p h a s i z e d t h a t it was difficult to distinguish it
237 from conventional and giant cell tumour-like M F H s without radiographic correlation, and that this type of OS showed a predominantly fibro-histiocytic soft tissue component. Our 3 cases out of 16 OS showed histologically similar features to conventional M F H in varying proportions of 55%, 40% and 7%. These were largely in the soft tissue components of the whole tumour tissue and in addition to the central component of conventional OS, and were not clinically different 9 conventional OS. In the previous literature, the incidence of M F H subtype, including MFH-like foci, varied from 8% to 30% among all OS (Mirra 1980; Yunis et al. 1986). Huvos et al. (1986) recorded the occurrence at 5-10% of all OS of both bone and soft tissue origin in patients older than 60 years. We encountered 3 cases of OS with M F H like feature (18.8%) in 16 OS which were all examined by making slab sections of the whole tumour. Therefore, such an area may be noticed in OS much more frequently if sampling is extensive. OS is subsequently reported as a mixed tumour of one or more histological components that may cause difficulties in determining the subtype. Two of 3 OS with MFH-like foci mentioned here represented MFH-like feature in nearly half or more of the whole tumour and thus could be defined as a putative category of MFH-like OS. Histologically, such an area seems to be fibro-histiocytic with a marked pleomorphism and devoid of malignant tumour osteoid and bone. The histological distinction from fibroblastic OS in which the tumour osteoid and woven bone are observed in intimate relation with fibrosarcomatous proliferation without histiocytic reaction is obvious (Mirra 1980). However, Unni (1988) stated that MFH-like OS is within the category of fibroblastic OS. The essential difference between MFH-like and fibroblastic OS may be attributed to a histiocytic reaction, probably due to chemotactic factor produced and secreted by tumour cells. It was reported that the ALPase enzyme, sensitive to heat treatment, is a better tool to identify and diagnose OS with various histological subtypes (Jeffree and Pice 1965; Yoshida et al. 1982; Yumoto et al. 1988). This is actively demonstrated in normal osteoblasts and their neoplastic counterparts responsible for bone and osteoid formation and was proposed as an intrinsic property of osteoblastic cell lineage (Yoshida et al. 1982). Our cases showed ALPase activity but at varying degrees in spindle-shaped cells exhibiting a characteristic storiform pattern and constituting MFH-like feature. Thus, they were diagnosed correctly as a variant of OS with biopsy specimens. The well-developed rough endoplasmic reticulum seen on electron microscopy implies that the spindle-shaped cells manifest the character of osteoblastic cell lineage. The osteoblastic property was further appreciated from the evidence of positive Col I, factor XIIIa, APM and D D P IV for fibroblast and osteoblast markers. This hypothesis should be attributed to our transplantation study to nude mice. The xenografted tissue also showed an ALPase-positive proliferation of spindle-shaped cells arranged in a storiform pattern and lacking osteoclast-like giant cells and histiocytic cells
which seemed likely to be reactive but not neoplastic. It may also be proposed that the spindle-shaped cell is in an early stage of maturation from the evidence of negative staining reaction for osteocalcin (data not shown). The presence of MFH-like foci has been increasingly reported by accumulating numbers of malignant neoplasms of bone and soft tissue origin such as chondrosarcoma (Jaworski 1984; Wick et al. 1987; Breiwiss and Kaneko 1988), leiomyosarcoma (Hashimoto et al. 1986) and liposarcoma (Kim et al. 1989), and is now understood as a phenomenon of de-differentiation which undergoes anaplastic transformation by successive modulation with a progressive loss of the original histological characters and represents an MFH-like appearance during the final c o m m o n pathway (Brooks 1986; Roholl et al. 1988; Kosmell 1990). Wick et al. (1987) suggested this phenomenon to be the evolution of a second distinct neoplastic cell clone with more primitive attributes by the observation of dedifferentiated chondrosarcoma having some markers shared by chondrocytes. In the three cases presented here, de-differentiation is not suitable because of a gradual transition from osteoblastic to MFH-like areas. Possible appearance of genuine MFH-like OS, however, is expected as reported in other mesenchymal tumours. Such a particular OS should be limited to the case in which specific ALPase activity bas been lost. Further careful study will be necessary to establish the entity of M F H subtype of OS. In conclusion, MFH-like feature in OS can be defined as the proliferation of osteoblastic cell lineage in the poorly developmental stage, imitating the histological feature of M F H of bone, but differs from conventional Os and M F H . Thus, careful observation is required to distinguish it from conventional M F H of bone and OS in the soft tissue component.
Acknowledgements. This study was presented at the 79th Annual Meeting of the Japanese Society of Pathology held on 29 March 1990 at Fukuoka. The authors thank Miss Norieh Tanaka for her technical assistance in enzyme histochemistry and secretarial work. This study was supported in part from a grant of The Vehicle Racing Commemorative Foundation, Tokyo, Japan.
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VirchowsArchiv A PathologicalAnatomy and Histopathology 9 Springer-Verlag 1992
Osteosarcoma with features mimicking malignant fibrous histiocytoma Haruhiko Yoshida 1, Tokichi Yumoto 1, and Takeshi Minamizaki 2 Departments of 1 Pathology and 20rthopaedic Su9
Tottori University School of Medicine, Tottori, Japan
Received December 13, 1991 / Received after revision March 31, 1992 / Accepted April 1, 1992
Summary. Three osteosarcomas (OS) with features resembling malignant fibrous histiocytoma ( M F H ) were selected and investigated to identify any clinico-pathological similarities. In ail cases there was no significant difference from conventional OS on the radiography and laboratory data. The appearance of M F H - l i k e features within the whole t u m o u r tissue varied f r o m 7% to 55%. It was composed of spindle-shaped cells arranged in short irregular fascicles and a storiform pattern admixed with osteoclast-like giant cells, but devoid of neoplastic osteoid. Such spindle-shaped cells had a poorly developed rough endoplasmic reticulum and expressed a strong alkaline phosphatase activity as well as vimentin. A series of allografts to athymic mice using the M F H like tissues also showed histologically a proliferation of plump spindle-shaped cells with a storiform pattern lacking osteoclast-like giant cells, and intensely positive for alkaline phosphatase. These findings indicate that the M F H - l i k e features are identified as modulated OS. The constituting cells are most likely to be poorly developed with possible phenotypic alteration in the m a t u r a t i o n stage of osteoblastic cell lineage, but different from conventional M F H of bone as regards their distinct histochemical pattern. Key words: Osteosarcoma - Malignant fibrous histiocyt o m a - Transplantation
ly, additional histological subtypes and patterns of OS were variously proposed, all of which were hardly distinguishable f r o m other benign and malignant bone tumours (Mirra 1980; Yunis and Barnes 1986). On rare occasions OS shows an excessive proliferation of spindle-shaped cells with a pronounced pleomorphism in a storiform pattern without definite osteoid tissue. This pattern is similar to malignant fibrous histiocytoma ( M F H ) of bone, but the neoplastic osteoid and bone are identified only in other areas of the saine tumour. Mirra (1980) designated this M F H - l i k e OS to distinguish it from conventional M F H of bone. The percentage of patients with such M F H - l i k e OS in an entire series of OS varied f r o m 5% to 30% in a number of investigations (Mirra 1980; H u v o s 1986; Yunis and Barnes 1986). Ballance et al. (1988) emphasized the M F H subtype of OS as a distinct entity f r o m M F H of bone, and high-lighted the radiographic similarities to conventional, highly aggressive OS. The inclusion of an M F H - l i k e subtype in the classification of OS still remains debated. In this paper, we report three cases of OS resembling M F H of bone and describe the clinico-pathological features of MFH-like OS as c o m p a r e d with M F H of bone. We discuss whether the histogenetic concept should be listed in the classification of OS.
Materials and methods Introduction Osteosarcoma (OS) can exhibit a wide spectrum of histopathological appearances and on the basis of this a classification has been ruade into osteoblastic, chondroblastic and fibroblastic subtypes according to the m a j o r histological c o m p o n e n t (Dahlin and Unni 1977). RecentCorrespondence to: H. Yoshida, Department of Pathology, Tottori University School of Medicine, 86 Nishi-machi, Yonago, Tottori 683, Japan
Cases examined in this report were selected from 16 consecutive cases of OS (aged 9-75 years, mean 19.3 years) registered in the First Department of Pathology, Tottori University School of Medicine, Japan from 1975 to 1989. Every case was diagnosed by biopsy and histology of surgically resected material. From the•e, 3 cases of OS with MFH-like features were identifie& All clinical and radiographic records are li•ted in Table 1. For light microscopy, the surgically re•ected tumours were fixed with 10% buffered formalin, decalcified in a formic acid-9 buffer and embedded in paraffin. Further, large slab sections of maximally demonstrated areas were prepared for mapping of various histological patterns, including the MFH-like feature. Tissue blocks obtained from surgical sper were fixed with 4% para8 for 5 h at 4~ C, replaced and kept in gum-
230 Table 1. Clinical findings of osteosarcoma (OS) with malignant fibrous histiocytoma (MFH)-like features Case
Age/sex
Clinical sign
Location
Radiographic findings
Treatment
Prognosis
1
14/F
Right knee pain Serum ALPase 76 units/1 (37/107)
R-tibia proximal
Mixed sclerotic-lytic Sort tissue mass with calcifcation
Amputation above knee, neoadjuvant chemotherapy a
Alive, postop. 24 months dead lung metastases
2
11/F
Left knee pain Serum ALPase 250 units/1 (37/107)
L-femur distal
Mixed sclerotic-lytic Codman's triangle
Wide resection, limb salvation procedure (Kotz's prosthesis) neoadjuvant chemotherapy"
Alive, postop. 38 months disease-free
3
75/F
Left knee pain Serum ALPase 117 units/t (22-116)
L-femur distal
Lytic with focal sclerotic lesion
Amputation above the knee, postop. chemotherapy"
Alive, postop. 44 months disease-free
ALPase, Alkaline phosphatase Rosen T-12 protocol
sucrose solution at 4~ C. The tissues then were quickly frozen using dry-ice with addition of hexane, sectioned to 4-6 gm thickness with a cryostat (Bright) at - 2 0 ~ and stained for the following enzymes: alkaline phosphatase (ALPase, by Bengt et al.), acid phosphatase (ANAE, by Leder), adenosine triphosphatase (ATPase, by Mfiller-Hermelink), 5'-nucleotidase (5'-N, by Muller-Hermelink) and beta-glucuronidase (beta-Gl, by Hayashi et al.). Immunohistochemical staining was carried out on paraffin sections of 6 gin thickness employing the avidin-biotin peroxidase complex technique after being reacted with antibodies against collagen I (Col I), aminopeptidase M(APM), and dipeptidyl peptidase IV (DDP IV). These antibodies were all primarily prepared in our laboratory (Aki 1989). Their specificities were determined and characterized by recognizing fibroblasts selectively. Anti-factor XIIIa (Hoechst-Behring, Marburg, FRG) for fibroblast and osteoblast marker, anti-alpha-l-antitrypsin (AAT), anti-alpha-l-antichymotrypsin (AACT), anti-lysozyme (Lys, Dakopatts, Copenhagen, Denmark) for monocyte-macrophage marker, and anti-vimentin (Dakopatts) for mesenchymal marker. Antibodies against Col I were used at a dilution of i : 100. The optimal dilution of other primary antibodies used in this study was as follows: 1:50 for the anti-factor XIIIa; 1:2 for the antiAPM and DDP IV; original kit of anti-AAT, AACT and Lys; and 1 : 10 for anti-vimentin. The sections were incubated with primary antibodies for 30 min at room temperature, followed by biotinylated anti-mouse/anti-rabbit immunoglobulin for 20 min and then avidin-biotin peroxidase complex (Dakopatts) for 20 min. Finally, the sections were immersed with 0.3 % (v/v) hydrogen peroxide and 0.6% (w/v) 3.37-diaminobenzidine solution and counterstained with methyl green. Osteoblastic tumour cells in conventional OS served as controls for 3 cases with MFH-like features to be compared as regards the intensity and extent of staining reactions. The fresh samples were fixed with 3% glutaraldehyde solution (buffered pli 7.4) for l h at 4~ C and were post-fixed with 2% phosphate-buffered osmium tetroxide for 1 h. After dehydration with an alcohol series, they were embedded in epoxy resin. Ultrarhin sections were stained with lead citrate and uranyl acetate and were observed with a H-800 electron microscope (Hitachi, Japan). For transplantation study, inbred nu/nu mice of Balb/c genetic background were used at the age of 8 weeks. The tissue fragments with MFH-like features in case 1 were transplanted subcutaneously onto the flank. The transplanted mice were bred and kept under special pathogen-free conditions. A new generation was obtained by transplantation of the xenografted tumour tissue for 5 months.
The tissue obtained from the nude mice was examined in the saine way as the original tumour.
Results T h e clinical d a t a o f each case are s u m m a r i z e d in Table 1. T h e p a t i e n t s were all female, r a n g i n g in age f r o m 11 to 74-years. A l l p a t i e n t s c o m p l a i n e d o f p a i n a n d swelling with a s y m p t o m d u r a t i o n o f f r o m 2 weeks to 6 m o n t h s . The s e r u m A L P a s e level in each case was f r o m 76 to 250 units/1; case 2 s h o w e d a significant elevation a n d the o t h e r two cases (cases I a n d 3) were within n o r m a l limits. R a d i o g r a p h i c findings (Fig. 1 A - C ) s h o w e d m i x e d o s t e o l y t i c a n d , in p a r t , o s t e o s c l e r o t i c lesions o f the m e t a physis o f f e m u r (cases 2 a n d 3) a n d t i b i a (case 1). D e s t r u c t i o n o f the c o r t i c a l b o n e was o b v i o u s in all cases. G r o s s l y , the t u m o u r s were solid, white-greyish a n d g r i t t y to b o n e - h a r d w i t h v a r i a b l e a m o u n t s o f h a e m o r r h a g e a n d necrosis (Fig. 2 A - C ) . T h e m a r r o w c a v i t y o f the m e t a p h y s i s was a l m o s t c o m p l e t e l y o b l i t e r a t e d b y the a b o v e tissue affecting the e p i p h y s e a l p l a t e (cases 1 a n d 2) o r d i a p h y s i s (case 3). T h e t u m o u r e x t e n d e d directly t h r o u g h the a d j a c e n t c o r t i c a l b o n e i n t o the s u r r o u n d i n g soft tissues in ail cases. C a r t i l a g i n o u s t u m o u r tissue w i t h g r o u n d - g l a s s a p p e a r a n c e was d e t e c t e d in the soft tissue c o m p o n e n t a n d d e l i n e a t e d clearly f r o m the h a r d tissue c o m p o n e n t (case 2). H i s t o l o g i c a l l y , the M F H - l i k e f e a t u r e c o n s i s t e d o f f i b r o b l a s t - l i k e s p i n d l e - s h a p e d cells a n d r o u n d cells int e r m i n g l e d with v a r y i n g n u m b e r s o f o s t e o c l a s t - l i k e g i a n t cells, a n d f a s c i c u l a t e d g r o w t h p a t t e r n o f s p i n d l e - s h a p e d cells in a s t o r i f o r m p a t t e r n (Fig. 3). O s t e o c l a s t - l i k e g i a n t cells were s c a t t e r e d m o r e o r less a m o n g the t u m o u r cells (Fig. 4). O c c a s i o n a l l y , the t u m o u r cells m o d u l a t e d their m o r p h o l o g i c a l f e a t u r e s f r o m s p i n d l e - s h a p e d to r o u n d cells with a b u n d a n t c y t o p l a s m a n d b e c a m e i n c o m p l e t e in a s t o r i f o r m a r r a n g e m e n t (all cases), thus i m p a i r i n g the i m p r e s s i o n o f g i a n t cell t u m o u r o r g i a n t cell-rich M F H w i t h m a r k e d cellular p l e o m o r p h i s m (Fig. 5). H o w -
231
Fig. 1 A-C. Radiographic findings: A AP view of proximal tibia of 14-year-old female (case 1). Eccentric, loculated, sclerotic lesion is obvious with extension to knee joint; B 11-year-old female (case 2) with mixed sclerotic and lytic lesion of the metaphysis of right femur. Periosteal reaction is visible showing Codman's triangle; C 75-year-old female (case 3) with mixed sclerotic and lytic lesion of the metaphysis of left femur
232
Fig. 2A-C. Gross findings: A (case 1) centrally located, hard tumour extending directly into the surrounding soft tissue, followed by large, white mass; B (case 2), intraosseous solid, white-greyish tumour with central necrosis and haernorrhage; C (case 3) the marrow cavity of the metaphysis is completely obliterated by yellow-greyish tumour tissue
ever, phagocytic figures were not found. Atypical mitotic figures were frequently encountered. These were ill-defined from the osteoblastic area and lacked neoplastic osteoid and bone, but the lace-like osteoid tissue was frequently recognized among the tumour cells adjacent to the osteoblastic component (Fig. 6). An osteoblastic, bone-rich area was found largely in the tumour component located in the marrow cavity (all cases). In case 1, malignant neoplastic chondroid tissue was observed
in the sofl tissue component and well-delineated from areas of malignant cell proliferation containing bone and osteoid tissue and MFH-like feature (Fig. 7). According to the histological mapping of the whole tumour tissue, the incidence of MFH-like feature in the three cases was 55%, 40% and 7% (Fig. 8 ~ C ) , respectively. Those areas were hardly distinguishable from the osteoblastic area in two cases (cases 1 and 2) and were relatively delineated in case 3.
233
Fig. 3. Malignant fibrous histiocytoma (MFH)-like feature in case 2. Bundles of spindle-shaped cells with pleomorphism are arranged in a typical storiform pattern. H&E, x 100
Fig. 4. Osteoclast-like giant cells are scattered among spindle-shaped tumour cells. H&E, x 200 Fig. 5. In this area, osteoclast-like giant cells are prominent as in giant cell tumour (case 1). H&E, x 100
234
Fig. 6. MFH-like area is generally ill-defined from central osteoblastic area. H&E, • 100 Fig. 7. MFH-like area is well-delineated from the neoplastic chondroid tissue (case 1). H&E
Fig. 8. Schematic diagram of the tumours in each case; the percentage incidence of MFH-like area is shown and determined at a rate of 55% (A: case 1), 40% (B: case 2) and 7% (C: case 3)%, respectively
The results of immunohistochemical staining are shown in Table 2 with additional findings of M F H of soft tissue origin. Spindle-shaped tumour cells in a storiform pattern reacted significantly with anti-AACT antibody for monocyte-macrophage marker and anti-vimentin antibody for mesenchymal marker. In contrast, all
types of tumour cells showed negative reaction for the anti-Lys antibody. Both anti-Col I and anti-factor XIIIa antibodies reacted with a small number of cells which were negative for the anti-APM and anti-DDP IV antibodies. Table 3 shows the cumulative enzyme histochemical
235
Fig. 9. ALPase reaction of MFH-like area (cases 1 and 2). Most of the tumour cells are significantly positive except for osteoclast-like giant cells. ALPase, x 200 Fig. 10. ATPase reaction of the same areas as those shown in Fig. 8. Positive reaction is shown in osteoclast-like giant cells and a small number of histiocytic cells but not in most of the tumour cells. ATPase, x 200 Fig. 11. Transplanted tumour tissue showing a fasciculating growth pattern of fibroblast-like spindle-shaped cells often with a storiform pattern (case 1, 5th generation). H&E, x 100
236 Table 2. Comparative immunohistochemical findings between OS with MFH-likc feature and MFH of bone
Antibodies against Col I
XIIIa
OS with MFH-like lesion Case I + Case 2 + + + + + Case 3 + + + MFH of bone Case 1 Case 2 . Case3 ++ Case 4 Case 5 +
.
+ + . ++ -
APM
DDPIV
AACT
AAT
Lys
Vim
+ -
+ -
+ + +
+ +
-
+ + + + + + +
-
-
+ -
+ + -
+ + + + ++ + +
+ + + + ++ +
+ -+ +
+ + + ++ + +
.
Intensity of reaction: + § § strong; + § moderate; + , slight and focal; - , negative. In strong and moderate reactions, the reactivity extended diffusely on the overall specimens, whereas in slight reaction it was present in some focal areas. OS, Osteosarcoma; MFH, malignant fibrous histiocytoma of bone; Col I, collagen I; XlIIa, factor XIIIa; APM, aminopeptidase M; DDP IV, dipeptidyl peptidase IV; AACT, alpha-l-antichymotripsin; AAT, alpha-l-antitripsin; Lys, lysozyme; Viro, vimentin
Table
3. Comparative enzyme histochemical findings between OS with MFH-like feature and MFH of bone
Tumour cell components
OS with MFH-like lesion (n = 3) Osteoblastic cells Fibroblast-like spindle cells MFH of bone (n=4) Fibroblast-like spindle cells Histiocyte-like round cells
Enzymes ALPase
ACPase
ANAE
ATPase
5'-N
Beta-G1
+ + + + +
+
--
++
+++
+
--
+
-
+
-
+ ++
+ +++
+ ++
+ +
+ +
Intensity of reaction: + + + , strong; + - , moderate; + , slight and focal; - , negative. In the strong and moderate reactions, the activity extended diffusely on the overall specimens, whereas in slight reaction, it was present in some focal areas
ALPase, Alkaline phosphatase; ACPase; ANAE; ATPase, adenosine triphosphatase; 5'-N, 5'-nucleotidase; beta-G1, beta-glucoronidase
results. T h e s p i n d l e - s h a p e d cells a n d b i z a r r e g i a n t cells were s t r o n g l y positive for A L P a s e r e a c t i o n (Fig. 9). O t h e r e n z y m e activities were as f o l l o w s : s t r o n g l y positive for 5 ' - N a n d w e a k l y positive for A T P a s e o n the cell surface a n d w e a k l y positive for A C P a s e close to the nucleus. A small n u m b e r o f histiocytic cells w h i c h were i n t e r m i n g l e d a n d p o s i t i v e for A T P a s e (Fig. 10) a n d A N A E s e e m e d to be in the p r o c e s s o f reaction. U l t r a s t r u c t u r a l l y m o s t o f the t u m o u r cells in M F H like foci d i s p l a y e d a s p i n d l e - s h a p e d c o n f i g u r a t i o n with w e l l - d e v e l o p e d r o u g h e n d o p l a s m i c r e t i c u l u m d i l a t e d to a v a r y i n g degree in the e l o n g a t e d c y t o p l a s m as d e s c r i b e d in a p r e v i o u s r e p o r t ( M i n a m i z a k i et al. 1990). A m o r p h o u s m a t e r i a l s w i t h b u n d l e s o f c o l l a g e n fibres were r a r e l y seen in the e x t r a c e l l u l a r area. O s t e o c l a s t - l i k e g i a n t cells were f o u n d m o r e o r less w i t h a b u n d a n t i n t r a c y t o p l a s m i c vesicles. T h e y h a d i r r e g u l a r l y i n d e n t e d nuclei with p e r i n u c l e a r c h r o m a t i n c l u m p i n g . T h e l a t e n c y o f t r a n s p l a n t s v a r i e d f r o m 155 to 280 days. T b e M F H - l i k e a r e a in o s t e o s a r c o m a (case ~) was
successfully g r a f t e d a n d t r a n s p l a n t e d for rive generations. H i s t o l o g i c a l findings o f the tissue o b t a i n e d f r o m the n u d e mice were a l m o s t similar to t h o s e o f t h e original t u m o u r , s h o w i n g a p r o l i f e r a t i o n o f i n t e r l a c i n g fascicles o f f i b r o b l a s t - l i k e s p i n d l e - s h a p e d cells with a s t o r i f o r m p a t t e r n in a large p r o p o r t i o n o f the tissues (Fig. ~1). T h e lace-like o s t e o i d tissue was r e c o g n i z e d largely in the central p o r t i o n o f the t u m o u r mass. T h e r e was a c o m p l e t e l a c k o f o s t e o c l a s t - l i k e g i a n t cells. T h e constituent cells o f the t r a n s p l a n t e d t u m o u r were s t r o n g l y positive for A L P a s e reaction.
Discussion
M F H - l i k e OS was first p r o p o s e d b y M i r r a (1980) as a different entity f r o m M F H o f b o n e , w h i c h was welld e s c r i b e d a n d w i d e l y a c c e p t e d ( H u v o s et al. 1985). This c o n c e p t was also s u p p o r t e d b y Ballance et al. (1988), w h o e m p h a s i z e d t h a t it was difficult to distinguish it
237 from conventional and giant cell tumour-like M F H s without radiographic correlation, and that this type of OS showed a predominantly fibro-histiocytic soft tissue component. Our 3 cases out of 16 OS showed histologically similar features to conventional M F H in varying proportions of 55%, 40% and 7%. These were largely in the soft tissue components of the whole tumour tissue and in addition to the central component of conventional OS, and were not clinically different 9 conventional OS. In the previous literature, the incidence of M F H subtype, including MFH-like foci, varied from 8% to 30% among all OS (Mirra 1980; Yunis et al. 1986). Huvos et al. (1986) recorded the occurrence at 5-10% of all OS of both bone and soft tissue origin in patients older than 60 years. We encountered 3 cases of OS with M F H like feature (18.8%) in 16 OS which were all examined by making slab sections of the whole tumour. Therefore, such an area may be noticed in OS much more frequently if sampling is extensive. OS is subsequently reported as a mixed tumour of one or more histological components that may cause difficulties in determining the subtype. Two of 3 OS with MFH-like foci mentioned here represented MFH-like feature in nearly half or more of the whole tumour and thus could be defined as a putative category of MFH-like OS. Histologically, such an area seems to be fibro-histiocytic with a marked pleomorphism and devoid of malignant tumour osteoid and bone. The histological distinction from fibroblastic OS in which the tumour osteoid and woven bone are observed in intimate relation with fibrosarcomatous proliferation without histiocytic reaction is obvious (Mirra 1980). However, Unni (1988) stated that MFH-like OS is within the category of fibroblastic OS. The essential difference between MFH-like and fibroblastic OS may be attributed to a histiocytic reaction, probably due to chemotactic factor produced and secreted by tumour cells. It was reported that the ALPase enzyme, sensitive to heat treatment, is a better tool to identify and diagnose OS with various histological subtypes (Jeffree and Pice 1965; Yoshida et al. 1982; Yumoto et al. 1988). This is actively demonstrated in normal osteoblasts and their neoplastic counterparts responsible for bone and osteoid formation and was proposed as an intrinsic property of osteoblastic cell lineage (Yoshida et al. 1982). Our cases showed ALPase activity but at varying degrees in spindle-shaped cells exhibiting a characteristic storiform pattern and constituting MFH-like feature. Thus, they were diagnosed correctly as a variant of OS with biopsy specimens. The well-developed rough endoplasmic reticulum seen on electron microscopy implies that the spindle-shaped cells manifest the character of osteoblastic cell lineage. The osteoblastic property was further appreciated from the evidence of positive Col I, factor XIIIa, APM and D D P IV for fibroblast and osteoblast markers. This hypothesis should be attributed to our transplantation study to nude mice. The xenografted tissue also showed an ALPase-positive proliferation of spindle-shaped cells arranged in a storiform pattern and lacking osteoclast-like giant cells and histiocytic cells
which seemed likely to be reactive but not neoplastic. It may also be proposed that the spindle-shaped cell is in an early stage of maturation from the evidence of negative staining reaction for osteocalcin (data not shown). The presence of MFH-like foci has been increasingly reported by accumulating numbers of malignant neoplasms of bone and soft tissue origin such as chondrosarcoma (Jaworski 1984; Wick et al. 1987; Breiwiss and Kaneko 1988), leiomyosarcoma (Hashimoto et al. 1986) and liposarcoma (Kim et al. 1989), and is now understood as a phenomenon of de-differentiation which undergoes anaplastic transformation by successive modulation with a progressive loss of the original histological characters and represents an MFH-like appearance during the final c o m m o n pathway (Brooks 1986; Roholl et al. 1988; Kosmell 1990). Wick et al. (1987) suggested this phenomenon to be the evolution of a second distinct neoplastic cell clone with more primitive attributes by the observation of dedifferentiated chondrosarcoma having some markers shared by chondrocytes. In the three cases presented here, de-differentiation is not suitable because of a gradual transition from osteoblastic to MFH-like areas. Possible appearance of genuine MFH-like OS, however, is expected as reported in other mesenchymal tumours. Such a particular OS should be limited to the case in which specific ALPase activity bas been lost. Further careful study will be necessary to establish the entity of M F H subtype of OS. In conclusion, MFH-like feature in OS can be defined as the proliferation of osteoblastic cell lineage in the poorly developmental stage, imitating the histological feature of M F H of bone, but differs from conventional Os and M F H . Thus, careful observation is required to distinguish it from conventional M F H of bone and OS in the soft tissue component.
Acknowledgements. This study was presented at the 79th Annual Meeting of the Japanese Society of Pathology held on 29 March 1990 at Fukuoka. The authors thank Miss Norieh Tanaka for her technical assistance in enzyme histochemistry and secretarial work. This study was supported in part from a grant of The Vehicle Racing Commemorative Foundation, Tokyo, Japan.
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