Expression of intermediate filaments in malignant fibrous histiocytomas

Expression of intermediate filaments in malignant fibrous histiocytomas

Expression of Intermediate Filaments in Malignant Fibrous Histiocytomas TAKANORI HIROSE, MD, EIJI KUDO, MD, TADASHI HASEGAWA, MD, JUN-ICHI ABE, MD AND...

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Expression of Intermediate Filaments in Malignant Fibrous Histiocytomas TAKANORI HIROSE, MD, EIJI KUDO, MD, TADASHI HASEGAWA, MD, JUN-ICHI ABE, MD AND KAZUO HIZAWA, MD The expression of intermediate filaments (IFS) in 34 malignant fibrous histiocytomas (MFHs) was studied immunohistochemitally and ultrastructurally. Using the avidin-biotin-peroxidase method, positive reactions were detected as follows: for desmin in 12 tumors, for neurofilament in two tumors, for cytokeratin in one tumor, and for vimentin in 30 tumors. Desmin immunoreactivity was found in tumors of all four histologic subtypes and cytokeratin immunoreactivity was found in one tumor of the myxoid type. Because of the cross-reactivity of antineurofilament antibody with reactive histiocytes, the immunoreactivity for neurofilament seemed to be non-specific. Ultrastructurally, five of 13 tumors studied contained some tumor cells showing myoftbroblastic or smooth muscle cell differentiation. A few tumor cells in one cytokeratin-positive tumor had tonofilaments in their cytoplasm. Desmin expression in some MFHs seemed to be due to myofibroblastic or smooth muscle cell differentiation of some tumor cells. Cytokeratin expression seemed to indicate epithelial differentiation in some MFHs. This varied expression of IFS in MFHs may reflect the heterogeneous nature of MFHs, and suggests that MFHs represent the final stages of dedifferentiation of several different types of sarcomas or, alternatively, represent forms of poorly differentiated sarcoma with the potential of developing into more differentiated sarcomas of heterogeneous origin. HUM PATHOL20:871-877. 0 1989 by W.B. Saunders Company.

Recently, immunohistochemical staining of the cytoskeleton, especially of intermediate filaments (IFS), has been applied in studies of the pathology of soft tissue tumors.1-3 However, expression of IFS in malignant fibrous histiocytomas (MFHs) has not been f u 11y investigated. MFHs, thought to derive from primitive mesenchymal or fibroblastic cells,4-6 are expected to express only vimentin,3 but Lawson et al,’ in an immunohistochemical study of ten MFHs found that six were desmin-positive, six were neurofilament-positive, and one was cytokeratin-positive. Moreover, Weiss et al8 reported a post-irradiation MFH expressing cytokeratin and suggested that sarcomas, including MFHs, may sometimes express cytokeratin protein. Also, we have previously reported a unique MFH showing both desmin and cytokeratin immunoreactivity.g To obtain information concerning these unusal IF expressions, we examined 34 MFHs immunohistochemically and ultrastructurally.

MATERIALS AND METHODS Thirty-four MFH cases from the file of the Department of Pathology at the University of Tokushima School of Medicine (Japan) were studied. The diagnosis was made microscopically based on the well-recognized criteria of MFH described by Enzinger and Weiss.‘O Twenty-two of these tumors were from men and 12 were from women. The patients were 28 to 83 years old (mean, 61.5 years). Eighteen tumors were located in the lower extremities, 11 in the trunk, four in the upper extremities, and one in the head. Histologically, 26 of the tumors were classified as storiform-pleomorphic, live as myxoid, two as giant cell, and one as inflammatory. Surgically resected specimens from 33 cases and autopsy materials from one case were fixed in 10% formalin and embedded in paraffin. Immunohistochemical stainings of paraffin sections were performed using the avidin-biotin-peroxidase complex (ABC) method of Hsu et al” with an ABC kit (Vector Laboratories, Burlingame, CA). The primary antibodies used are shown in Table 1. In addition to immunostaining for IFS and alpha smooth muscle actin (ASMA), desminpositive sections were stained for skeletal myosin and myoglobin. The expressions of desmin and ASMA were also examined in the preparations of ten myofibroblastic lesions; namely, five fibromatoses, four lesions of nodular fasciitis, and one lesion of proliferative fasciitis. For electron microscopic studies, tissues obtained at surgery from 13 tumors were fixed in 3% glutaraldehyde and processed in the usual manner. Thin sections stained with uranyl acetate and lead citrate were examined with an electron microscope. In particular, subcellular structures correlated with IF differentiation were investigated.

RESULTS lmmunohistochemistry The results of immunostainings of the 34 MFHs are shown in Table 1. Many cells from the 30 tumors were vimentinpositive. All the vimentin-negative tumors, except one that showed desmin positivity, were also negative for all other IFS. Desmin-positive tumor cells were present in 12 tumors. Three different anti-desmin antibodies (DP1, DP-2, and DM) reacted with some tumor cells in 12, seven, and eight tumors, respectively. Six tumors were positive for all three antibodies and three tumors were positive for two antibodies. In most tumors, small numbers of desmin-positive tumor cells were present focally, although a few tumors contained increased numbers of DP- l-positive cells. There were no remarkable differences in microscopic

From the First Department of Pathology, The University of Tokushima School of Medicine, Tokushima, Japan. Accepted for publication March 29, 1989. Key words: malignant fibrous histiocytoma, intermediate filament, immunohistochemistry, electron microscopy. Address correspondence and reprint requests to Takanori Hirose, MD, First Department of Pathology, The University of Tokushima School of Medicine, Kuramoto-cho 3, Tokushima 770, Japan. 0 1989 by W.B. Saunders Company. 0046-8 177/89/2009-0009$5.00/O



Volume 20. No. 9 (September1989)

TABLE 1. lmmunohistochemical Primary Antibodv


Features of 34 MFHs


No.of Positive Cases











12 7 8

6 3 4

3 2 1

2 1 2

1 1



Desmin DP-1 DP-2 DM

Polyclonal (our laboratoryg) Polyclonal (DAKO) Monoclonal (DAKO)

x 1000 x500 x50

Cytokeratin Keratin PKKl 34BEl2

Polyclonal (DAKO) Monoclonal (Labsystems) Monoclonal (Enzo)

x500 x100

1 1

Monoclonal(Biomakor) Monoclonal(Biomakor)

x100 x100

0 2

Monoclonal (Biomakor) Polyclonal (DAKO) Monoclonal (Biomakor) Monoclonal (Biomakor) Polyclonal (DAKO)

x100 x500 x 1000 x 1000 x 500

0 0 12 0 0

Neurofilament 68 kD 160 kD 200 kD GFAP ASMA SM Myoglobin


Abbreviations: SP. storiform-nleomorphic; protein; ASMA, alpha’smooth m&le act&;

x 1000

M, myxoid; G, giant SM, skeldtal myosiny


1 1 1


cell; 1, inflammatory;


2 9

kD, kilodalton;




glial fibrillary


Electron Microscopy

features between desmin-positive and desminnegative tumors (Fig 1, top left). Desmin immunoreactivity was not related to histologic subtypes: six desmin-positive tumors were storiform-pleomorphic, three were myxoid, two were giant cell, and one was inflammatory (Table 1). Solid areas of both the storiform-pleomorphic and myxoid types contained desmin-positive tumor cells with polygonal or spindle shapes (Fig 1, top right and bottom left). Short spindle-shaped tumor cells and ganglion-like stellate cells in tumors of the giant cell and inflammatory types, respectively, were positive for desmin (Fig 1, bottom right). Only one tumor of myxoid type showed cytokeratin immunoreactivity. All three anti-cytokeratin antibodies showed dot-like reactions in the perinuclear region of some polygonal tumor cells (Fig 2). Numerous cells that reacted with monoclonal antibodies for 160-kilodalton neurofilaments were found in two tumors. The reaction products were seen in small polygonal cells, but not in bizarre tumor cells (Fig 3). These 160-kilodalton neurofilamentpositive cells seemed to be reactive histiocytes, not tumor cells, because in seven other tumors this antibody also reacted with xanthoma cells and hemosiderin-laden macrophages, usually located around the tumors. No tumors showed GFAP immunoreactivity. ASMA immunoreactivity was seen in 12 tumors, of which seven were also desmin-positive. Reactive substances were present in many spindle-shaped tumor cells and also in the pericytes and smooth muscle cells of the vessels (Fig 4). Myoglobin and skeletal myosin were not detected by immunostaining in any of the 12 desmin-positive tumors examined. With regard to the ten myofibroblastic lesions, the three anti-desmin antibodies (DP-1, DP-2, and DM) reacted with five, one, and three lesions, respectively, and the ASMA antibody reacted with all ten lesions (Table 2).

Most tumors were composed mainly of atypical, large fibroblastic cells and small, bland-like histiocytic cells.‘* Spindle-shaped and polygonal fibroblastic cells contained well-developed, rough endoplasmic reticulum, prominent Golgi complexes, numerous polysomes, some small lysosomes, occasional lipid droplets, and variable amounts of intermediate filaments (Fig 5). In five tumors, some fibroblastic tumor cells had a few bundles of myofilaments with dense bodies (Fig 5). The relationships between myofibroblastic differentiation and immunohistochemical reactivities for desmin and ASMA are shown in Table 3. The results show a close relationship between myofibroblastic differentiation and expression of ASMA, but not of desmin. Distinct rhabdomyoblastic differentiation was not observed, but a few smooth muscle cell-like tumor cells were seen in one tumor. These tumor cells had large amounts of myofilaments and pinocytotic vesicles and were covered by fragmented external lamina-like material (Fig 6). Occasional tumor cells in the myxoid area of the cytokeratinpositive tumor had tonofilaments in the cytoplasm.g No neuronal differentiation was seen. DISCUSSION The only report of systematic studies on the expression of IFS on MFHs is that of Lawson et al.’ Confirming their findings, in the present immunohistochemical study we observed varied IF expression in 34 MFHs, ie, expression of vimentin in 30 tumors, of desmin in 12 tumors, of neurofilament in two tumors, and of cytokeratin in one tumor. Immunoreactivity for the neurofilaments seemed to be a nonspecific cross-immunoreaction, because the monoclonal antibody used also reacted with reactive histiocytes. These varied expressions of IFS indicate 872


FIGURE 1. Desmin immunoreactivii in MFH. (Top left) A desmin-positive MFH of storiform-p&morphic tVpe showing typical features of MFH. (Hematoxylin-eosin stain; magnification x 200.) (Top right] Storiform-pleomorphic type. A few tumor cells of the same tumor shown in the top left panel demonstrattng posittvity for desmin. (Magnification x 400.) [Bottom left) Myxoid type. The solid area of the myxoid MFH contains some positive cells. (Magnification X 330.) [Bottom right) Inflammatory type. Ganglion-like tumor cells that are positive for desmin are seen among many neutrophils and xanthoma cells. (Magnification x 660.)



Volume 20, No. 9 [September 1989)



FIGURE 2. An MFH of myxoid type showing cytokeratin positivily. (Left] A myxoid area abutting a cellular area. (Hematoxylin-eosin stain; magnification x 200.) (Right) Cytokeratin immunoreactivity. Some tumor cells have dot-like reaction products in their cytoplasm. (Magnification x 500.)

FIGURE 3. One hundred sixly kilodalton nerofilament immunoreactivity. Bland-appearing histiocytic cells are positive for neurofilament, whereas bizarre tumor cells are not. (Magnification x 500.)

FIGURE 4. ASMA immunoreactivity. Many tumor cells show ASMA immunoreactiiity. (Magnification x 330.)


lmmunohistochemical Features of Ten Myofibroblastic Lesions Positive

Primary Antibody Desmin DP-1 DP-2 DM ASMA

Nodular Fasciitis

l/4 o/4 l/4 414


Proliferative Fasciitis

O/l Oil O/l l/l

three antibodies showed similar distribution. Ultrastructural study of this tumor demonstated a few tumor cells containing tonofilaments in the perinuclear cytoplasm. Details of this tumor have already been described elsewhere.g Thus, in addition to synovial sarcomas,i6 malignant rhabsarcomas,15 e ithelioid which are aldoid tumors,’ ! and leiomyosarcomas,i8 ready known to show cytokeratin immunoreactivity, MFHs may also occasionally express cytokeratin. 1,2,7,14,19-21 have demon_ Many investigators strated desmin immunoreactivities in some MFHs. Leader et a120 and Lawson et al’ interpreted the desmin-positive reaction of MFHs as being due to myofibroblastic differentiation of tumor cells. However, the only IF expressed by myofibroblasts in culture, granulation tissue, stroma of invasive carcinoma, and fibromatosis has been thought to be vimentin.22,23 Recently, desmin-positive cells have been found in desmoid tumors, however, and the existence of a desmin-positive subset of myofibroblasts has been suggested. 21 In the present immunohistochemical study, five of ten myofibroblastic lesions, including four desmoid tumors, were desmin-positive. As described by Miettinen21 myofibroblasts may be subdivided into two subsets by their IF expression: one subset expresses only vimentin and the other expresses desmin (or desmin and vimentin). Similar heterogeneous expression of desmin is well-known in smooth muscle cells. Some vascular smooth muscle cells are desmin-negative, whereas those of the digestive, respiratory, and urogenital tracts express desmin.24 The present ultrastructural study also suggested the heterogeneity of IF expression by myofibroblastic tumor cells; only three of five tumors in which myofibroblastic differentiation was demonstrated contained desmin-positive tumor cells (Table



415 l/5 215 515


5/10 l/10 3110 lO/lO

the flexibility of IF expression in tumor cells. The type of IF is not rigidly determined in a certain cell type and can change under various conditions, including normal development, repair, in vitro growth, and neoplastic transformation.13 However, it seems unusual that a tumor expected primarily to express vimentin should express other IFS. Three MFHs showing cytokeratin immunoreactivity have recently been reported: the tumor reported by Lawson et al’ was also positive for desmin, neurofilament, and vimentin; the tumor reported by Regezi et all4 was an angiomatoid MFH of the thigh; and the tumor reported by Weiss et al* was an MFH of the sacrum of a female complicating the course of radiation therapy for endometrial carcinoma. The immunoreactivity of this last tumor was examined with four antibodies (three monoclonal and one polyclonal), and the reactivity was abolished when the antiserum was preabsorbed with keratin protein, excluding the possibility of cross-reactivity of the antibodies with other IFS such as vimentin.8 In this study, the cytokeratin-positive tumor was also found to react with three different antibodies (two monoclonal and one polyclonal), and the reaction products with these

FIGURE 5. A myofibroblastic tumor cell containing a welldeveloped rough endoplasmic reticulum, prominent Golgi areas, and a few bundles of myofllaments.(Magnification x 12,470.)



Volume 20, No. 9 [September19891

TABLE 3. Relationship Between Myofibroblastic Differentiation and lmmunohistochemical Features of MFHs Desmin Myofibroblastic Differentiation + (n = 5) - (n = 8)

No. Positive 3 3

also difficult. Smooth muscle cells are characterized by the presence of thin actin filaments with focal densities, abundant pinocytotic vesicles, subplasmalemma1 density, and distinct external laminae.*” The presence of these organellae is also a characteristic of myofibroblasts. Myofibroblasts can be distinguished from smooth muscle cells only by the degree of development of these organellae and the abundance of elements of the Golgi complex and rough endoplasmic reticulum.26 It is hard to differentiate neoplastic smooth muscle cells from neoplastic myofibroblasts ultrastructurally, because the quantities of their organellae are readily altered in the neoplastic state. Therefore, some myofibroblastic cells in MFHs might be true smooth muscle cells. Because of recent reports of the heterogeneous nature of some MFHs, it is not surprising that tumor cells of MFHs show various IF expressions. Miettinen2r and De Jong et all9 observed the immunohistochemical properties of smooth muscle cells in MFHs. Jabi et al *? obtained ultrastructural evidence of lipogenic, neurogenic, and granular cell differentiation in tumors diagnosed histologically as MFHs. Moreover, Roholl et a12* reported that five xenografted sarcomas taken from seven MFHs showed leiomyogenic (three tumors) or schwannian (two tumors) differentiation. These reports suggest that some MFHs may have tumor cells expressing several types of mesenchymal cells other than primitive, fibroblastic, and histiocytic cells. This heterogeneous nature of MFHs suggests that some primitive tumor cells of MFHs have the capacity to differentiate into cells of other lineages of soft part, such as smooth muscle cell, lipoblast, Schwann cell, etc.21.28-s0 Another possibility is that MFHs represent the final


No. Negative 2 5

No. Positive 5 1

No. Negative 0 7

3). Desmin expression in some MFHs, therefore, seems to be due to myoftbroblastic differentiation of some tumor cells. As another explanation for desmin-positive cells, true skeletal muscle or smooth muscle cell differentiation of tumor cells must be taken into account. Miettinen et al2 found that five of 30 MFHs were desmin-positive, and suggested that focal muscle cell differentiation had occurred in these tumors. De Jong et alI9 reclassified five desmin-positive pleomorphic tumors which were originally diagnosed as MFHs to be pleomorphic leiomyosarcomas. In the present study, rhabdomyoblastic differentiation of tumor cells could be ruled out because none of the 12 desmin-positive tumors reacted with anti-myoglobin or anti-skeletal myosin antibodies. Immunohistochemical or ultrastructural identification of true smooth muscle cell differentiation is usually difficult. Anti-ASMA antibody is considered to be useful in studies of smooth muscle differentiation in normal and pathologic conditions,25 but ASMA was present in many myofibroblastic cells of all ten myofibroblastic lesions examined. Ultrastructural differentiation between smooth muscle cells and myofibroblasts is

FIQURE 6. A tumor cell showing smooth muscle cell-like features.The cell has plentiful myofilaments,pinocytotlcvesicles, and intermediate fllaments and is coveredby fragmented external lamina-llke materials. (Magnification x 7,568.)



OF MFH (Hirose et al)

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stage of dedifferentiation of several different types of sarcomas.21~2g*30 The varied IF expression in MFHs demonstrated in this study seemed to be due to the heterogeneity of the constituent tumor cells. REFERENCES 1. Miettinen M, Lehto VP, Badley RA, et al: Expression of intermediate filaments in soft-tissue sarcomas. Int J Cancer 30:541546, 1982 2. Miettinen M, Lehto VP, Virtanen I: Antibodies to intermediate filament proteins in the diagnosis and classification of human tumors. Ultrastruct Pathol 7:83-107. 1984 3. Denk H, Krepler R, Artlieb’U, et al: Proteins of intermediate filaments: An immunohistochemical and biochemical approach to the classification of soft tissue tumors. Am J Path01 110:193-208, 1983 4. Fu YS, Gabbiani G, Kaye GI, et al: 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 5. Hoffman MA, Dickersin GR: Malignant fibrous histiocytoma: An ultrastructural study of eleven cases. HUM PATHOL 14:913-922, 1983 6. Wood GS, Beckstead JH, Turner RR, et al: Malignant fibrous histiocytoma tumor cells resemble fibroblasts. Am J Surg Pathol 10:323-335, 1986 7. Lawson CW, Fisher C, Gatter KC: An immunohistochemical study of differentiation in malignant fibrous histiocytoma. Histopathology 11:375-383, 1987 8. Weiss SW, Bratthauer CL, Morris PA: Postirradiation malignant fibrous histiocytoma expressing cytokeratin: Implications for the immunodiagnosis of sarcomas. Am J Surg Path01 12:554558, 1988 9. Hirose T, Sano T, Abe J, et al: Malignant fibrous histiocytoma with epithelial differentiation? Ultrastruct Pathol 12:529-536, 1988 10. Enzinger Mosby, 1983

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