The Journal of Dermatology Vol. 18: 340-344, 1991
Recurrent Malignant Fibrous Histiocytoma with Expression of Cytokeratin Isamu Murakami, Satoshi Itami, Sakuhei Fujiwara, Hiroto Terashi, Sotaro Kurata, Tomohito Honda*, Hiroshi Shinkai and Susumu Takayasu Abstract A case of locally recurrent malignant fibrous histiocytoma was documented in a 70-year-old man. He first noticed a subcutaneous nodule forty years previously. The tumor was surgically removed four times during the last four years with local recurrence on every occasion. In the recurrent tumors, the tumor cells almost completely replaced the whole dermis and invaded skeletal muscles. They were composed of pleomorphic spindle cells arranged in a storiform pattern and bizarre histiocytic cells, which were present principally in the deeper portions of the tumor. Both types of tumor cells showed marked nuclear atypicality. In the primary tumor, surrounding a large necrotic area, spindle-shaped cells were arranged in a storiform pattern. These tumor cells exhibited only mild nuclear atypia. The recurrent tumor was strongly positive for vimentin and alpha-I-antichymotrypsin. Most tumor cells were also weakly positive for KLl, a monoclonal antibody for keratin. A Western-blot analysis revealed the presence of two bands (62 and 69 Kd) reacting with KLI in the fractions which were obtained from the tumor according to the method for keratin extraction.
Key words: malignant fibrous histiocytoma; intermediate-size filaments; eytokeratin; alpha-lantichymotrypsin Introduction Malignant fibrous histiocytoma (MFH), also referred to as malignant fibrous xanthoma (1), has been defined as a unique soft tissue sarcoma (2). MFH is the most common type of soft tissue sarcoma in late adult life and has variable histological appearances (2-4). The cellular origin of MFH remains controversial; various authors have proposed different sources, such as histiocytes, fibroblasts, or primitive mesenchymal cells (5-7). Furthermore, recent studies have suggested an epitheReceived March 22, 1991; accepted for publication April 30, 1991. Presented in part at the 41st Annual Meeting of Western Japanese Dermatological Society, Kurume, October 7, 1989. Department of Dermatology, Medical College of Oita, Oita 879-55,Japan. *Division of Plastic Surgery, Osaka Seamen's Insurance Hospital, Minato-ku, Osaka,Japan. Reprint requests to: I. Murakami, M.D., Department of Dermatology, Medical College of Oita, 1-1 Idaigaoka, Hasama-cho, Oita 879-55,japan.
Hal differentiation of the tumor cells based upon positive immunoreactivity to cytokeratin and the presence of the desmosome-like or tonofilament-like structures (8-10). This paper describes a case of a storiform-pleomorphic type of MFH with local recurrence and a positive reaction of the tumor cells to cytokeratin indicated by immunohistochemical staining. Moreover, we confirmed that the keratin fraction from the tumor mass reacted with an anti-keratin monoclonal antibody (KL1) (11), by means of Western blotting (12).
Case Report A 70-year-old man with a persistent tumor in the nucha was admitted to our hospital on October 18, 1988. He first noticed a small subcutaneous nodule about forty years previously. The gradually enlarging nodule reached walnut size and was resected in December of 1983 for the first time. Thereafter, the tumor was excised three more times before October of 1986, because of local recurrence. In October of 1987, the tumor became ulcerated in the center, accompanied by lacerating pain. At adminission, a
Malignant Fibrous Histiocytoma and Cytokeratin
341
deep ulcer 45 x 25 mm in diameter with an irregular margin was present on the nucha (Fig. 1). The skin surrounding the ulcer was slightly elevated, and the hard tumor was fixed to the underlying tissues. Neither cervical nor supraclavicular lymph nodes were palpable. No distant tumor metastasis was detected by chest X-ray film, and Garium and bone scintigram. The tumor was widely excised on December 1, 1988. The patient died on January 15, 1989, because of cerebellar bleeding which occurred 2 days after operation. Autopsy was not performed.
Materials and Methods Tissue materials: The primary, third, and fourth recurrent tumors, fixed in formalin and embedded in paraffin, were available for the hematoxylin and eosin and immunohistochemical PAP stainings (13). The fourth recurrent tumor we removed surgically was cut into pieces; most of them were frozen at -80°C until used for keratin extraction. Commercial antibodies usedfor immunohistochemistry: The following antibodies were used: anti-alpha-lantichymotrypsin polyclonal rabbit antibodies from DAKO Corp., Santa Barbara, CA, USA, and antikeratin (KL1) (11) and anti-vimentin (V9) (14) monoclonal antibodies from Immunotech, Marseille, France and DAKO, and PAP KITTM System fromDAKO. Extraction ofkeratins: After thawing, the deep part of the tumor mass was processed for keratin extraction by a slight modification of the method deSCribed by Winter et al. (15). Briefly, the minced tissue mixed with finely broken glass pieces was ground with a mortar in an ice bath under liquid nitrogen gas in 105M KCI, 10mM NaCl, 2mM dithiotbreitol (DTT), 0.5mM phenylmethylsulfonylfluoride, 0.5% Triton X-lOO, and 10mM Tris-HCl, pH 7.5, followed by homogenization with seven strokes using a glass homogenizer. The homoJenate was stirred for 12 hrs at room temperature and centrifuged at 14,000 rpm for I'hr at 4°C with a Hitachi RPR 20-2-1059rotor. The pellet was washed three times with the same buffer, and was solubilized by stirring for 14 hrs at 3°C in 8M urea, 10mM DTT, 50mM Tris-HCI, pH 9.0. After removing the glass pieces by centrifugation, the supernatant was centrifuged at 25,000rpm for 1 hr at 4°C with a Hitachi RP42-45I rotor. The clear
Fig. 1. Clinical picture of the fourth recurrent tumor. A hard tumor with ulceration measuring 45 x 25 mm on the nucha. supernatant was dialyzed extensively against 2mM Tris-HCI, pH 7.6, lOmM 2-mercaptoethanol, and deionized water. The lyophilized powder of the material in a dialysis bag was dissolved in the sample buffer (20mM Tris-HCI, pH 6.8, 4% SDS, 5mM DTT, 20% sucrose, 0.0015%bromphenol blue) for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by heating for 2 min at 90°C. For the control, the epidermis-rich tissue overlying the tumor mass, including a small amount of the dermis and the upper portion of tumor mass as well, and the sole callus from a healthy person were similarly processed for keratin extraction. SDS-PAGE and Wes-tern blotting: The test samples and molecular marker proteins were applied onto 8.7% acrylamide slab gel according to the method of Laemmli (16). The separated polypeptides were transferred to the membrane (Immobilou'Y, Millipore Corp., Bedford, MA) by the method described by Towbin et al. (12). The membrane was cut into each slot A slot of marker proteins was directly stained with amido black. For immunoblot, each slot was reacted with KLl, V9, or with normal mouse IgGl as the first antibody. The DAKO products (DAKO PAP KITTM System) were used as the second and third antibodies. Finally, bands were visualized with diaminobenzidine and H 20 2 •
Results
Histopathologic findings: In the fourth recurrent tumor (Figs. 2a-c), tumor cells almost completely replaced the skin and invaded the superficial pans of the muscle. In the upper
342
Murakami et al Table 1. Immunohistochemical profile of primary and recurrent tumors Tumor Primary Recurrent third fourth
Vimentin Keratin
ACT
Control
ND ND
+
+ +
+
ACT: alpha-1-antichymotrypsin, ND: Not determined, +/-: positive/negative staining.
Fig. 2. Histopathologic pictures of the fourth recurrent tumor. (a) Dense infiltration of fibroblastic tumor cells arranged in a storiform pattern and intermingled with lymphocytes in the upper to middle dermis. H.E. Original magnification xlOO. (b) Pleomorphic spindle cells with nuclear atypicality. H.E. Original magnification x400. (c) Histiocytic cells with large, atypical nuclei and bizarre giant cells (arrowhead) at the deep portion of the tumor. H.E. Original magnification x400.
and middle dennis, the tumor cells were arranged in a pleomorphic and storifonn pattern and invaded between hyalinized collagen bundles. Their nuclei, varying in form and size, were pale and contained one or two prominent nucleoli. Most of these cells were spindleshaped. The deeper portion of the tumor was mainly composed of histiocytic cells possessing large, pale nuclei with prominent nucleoli and abundant eosinophilic cytoplasm. Bizarre giant cells were scattered among these histiocytic cells. Moderate numbers of lymphocytes were present throughout the tumor. The primary tumor, surgically removed in 1983, contained a large area of necrosis in the center. Surrounding this necrotic mass, spindle-shaped cells without obvious atypia were arranged in a fascicular or storifonn pattern. A large amount of hemosiderin was present between the cells or phagocytized by macrophages. The third recurrent tumor extirpated in 1986 showed similar histologic findings to the fourth one shown in Figures 2a-c, except for smaller numbers of tumor cells and more prominent hyalinization of collagen bundles. Immunohistochemical findings: The results of immunohistochemical study on the primary and recurrent tumors are summarized in Table 1. Cells of the fourth recurrent tumor gave positive reactions to vimentin with V9, keratin with KL1, and alpha-1-antichymotrypsin (ACT) (Figs. 3a-c); the primary tumor was negative for both keratin and ACT. Intensity of keratin and/or ACT immunostainings varied among these tumor cells. Western blotting: As shown in Figure 4, in the keratin fraction obtained from the fourth recurrent tumor, two bands with estimated
343
Malignant Fibrous Histiocytoma and Cytokeratin
Fig. 3. Immunohistochemical PAP stainings of the fourth recurrent tumor. More than eighty percent of tumor cells were positive for vimentin with V9 (a), and alpha-l-antichymotrypsin with variable staining intensity (b). Tumor cells were weaklybut definitely positive for keratin with KLI (c).Arrowheads in a, b and c indicate the representative immunopositive cells. Original magnification x200. molecular weights of 69 and 62 Kd (slot 1, bands 1 and 2) were stained with KLI. Band 1 was more strongly blotted than band 2. In the epidermis-rich fraction and the sole callus, these two bands were also observed, but the immunoreactivity of band 2 was stronger than that of band 1 (slots 2 and 3).
Discussion The recurrent tumors in the present case can be diagnosed as a storifonn-pleomorphic type of MFH from the following histopathologic findings: invasion into skeletal muscles, plump spindle cells arranged in a storifonn pattern, and pleomorphic histiocytic cells intermingled With bizarre giant cells. For the primary tumor, 1t is difficult to make a definite diagnosis of MFH, because of the rare atypicality of nuclei of spindle tumor cells. Results of the immunohistochemical study On the fourth recurrent tumor, such as positive staining for vimentin and ACT, are consistent \vith previous reports (17, 18). It is interesting that more than eighty percent of the tumor cells in this recurrent tumor expressed cyto-
1
2
3
4
Fig. 4. Western blot analyses of the keratin fraction from the fourth recurrent tumor by PAP stainings. Slots 1 and 4: keratin fraction from the tumor; 2: keratin fraction of epidennis-rich tissue overlying the tumor; 3: sole callus. Slots 1-3 were immunoblotted with KLl, while slot 4 with normal mouse IgG}. Arrows indicate positions of marker proteins (downward): IgG (150 Kd), bovine serum albumin (68 Kd), ovoalbumin (43 Kd).
Murakami et al
344
keratin. This is the third reported instance of keratin expression by MFH (8, 9). As reviewed by LJ. McGuire et al. (19), the reported incidence of co-expression of vimentin and cytokeratin in neoplastic tissues is rapidly expanding. The presence of 62- and 69-Kd components reactive with KLI in MFH was proven for the first time by a more sophisticated method, Western blot analysis. It is unlikely that this positive reaction to KLI by immunoblotting is due to contamination by epidermis or its appendages, since epidermal components were not observed in the deep part of the tumor, which was used for the keratin extraction, under light microscopy, and the relative immunoreactive intensity of these two 62- and 69-Kd bands in the tumor differed from that in the epidermis-rich fraction. Crossreaction of vimentin with KL1 seemed unlikely, because vimentin(s) copurified with the keratin fraction of the present tumor were blotted with V9 at different positions from 62- and 69-Kd (unpublished data, Murakami). Furthermore, epithelial differentiation in MFH was also suggested by a ultrastructural study (8, 10). Thus, coexpression of vimentin, cytokeratin, and ACT might imply an ability for multidirectional differentiation of neoplastic cells (19). Acknowledgments We wish to thank Drs. Munetomo Enjoji and Hiroshi Hashimoto, Second Department of Pathology, Faculty of Medicine, Kyushu University, japan, for their valuable suggestion on the histopathological diagnosis. We are also grateful to Mr. Shuji Imi, Research Laboratory Center, Medical College of Oita, for taking the photomicrographs.
References 1) O'Brien jE, Stout AP: Malignant fibrous xanthomas, Cancer; t.7: 1445-1455, 1964. 2) Weiss SW, Enzinger FM: Malignant fibrous histiocytoma: An analysis of 200 cases, Cancer, 41: 22502266,1978. 3) Hashimoto H: Malignant fibrous histiocytoma: A clinicopathologic study of 130 cases, Fukuoka Acta Med, 70: 585-613, 1979. (in Japanese) 4) Enzinger FH, Weiss SW: Soft Tissue Tumors, C.V. Mosby, St. Louis, 1983, pp 166-198.
5) Ozzello L, StoutAP, Murray MR: Cultural characteristics of malignant histiocytomas and fibrous xanthomas, Cancer, 16: 331-344,1963. 6) Roholl PjM, Kleynej, UnnikjAMV: Characterization oftumor cells in malignant fibrous histiocytomas and other soft-tissue tumors, in comparison with malignant histioeytes, Am] Pathol, 121: 269-274,1985. 7) Iwasaki H, Isayama T,johzaki H, Kikuchi M: Malignant fibrous histiocytoma: Evidence of perivascular mesenchymal cell origin; Immunocytochemical studies with monoclonal anti-MFH antibodies, Am] Pathol, 128: 528-537,1987. 8) Hirose T, Sano T, Abej, Hizawa K: Malignant fibrous histiocytoma with epithelial differentiation?, UltrastructPatkoi, 12: 529-536, 1988. 9) Weiss SW, Bratthauer GL, Morris PA: Postirradiation malignant fibrous histiocytoma expressing cytokeratin: Implications for the immunodiagnosis of sarcomas, Am] Surg Patkoi, 12: 554-558, 1988. 10) Merkow LP, Frich JC, Slifkin M, Kyreages CG, Pardo M: Ultrastructure of a fibroxanthosarcoma (Malignant fibroxanthoma), Cancer, 28: 372-383, 1971. 11) Viacj, Reano A, Brochier j, Staquet M:f, ThivoletJ: Reactivity pattern of a monoclonal antikeratin antibody (KLl),] Invest Dermatol, 81: 351-354, 1983. 12) Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc Nat! Acad sa USA, 76: 4350-4354, 1979. 13) Sternberger LA, Hardy PHjr, CuculislJ, Meyer HG: The unlabeled antibody enzyme method of immunohistochemistry: Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes,] Histochem Cytoche1n, 18: 315-333, 1970. 14) Osborn M, Debus E, Weber K: Monoclonal antibodies specific for vimentin, Eur ] Cell Bioi, 34: 137-143,1984. 15) Winter H, Schweizer J, Goerttler K: Keratins as markers of malignancy in mouse epidermal tumors, Carcinogenesis, 1: 391-398, 1980. 16) Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227: 680-685, 1970. 17) Miettinen M, Lehto V-P, Badley RA, Virtanen I: Expression of intermediate filaments in soft-tissue sarcomas, Int] Cancer, 30: 541-546, 1982. 18) Meister P, Nathrath W: Immunohistochemical markers of histiocytic tumors, Hum Pathol, 11: 300-301, 1980. 19) McGuire LJ, Ng JPW, Lee JCK: Coexpression of cytokeratin and vimentin, Appi Pathol, 7: 73-84, 1989.
Recurrent Malignant Fibrous Histiocytoma with Expression of Cytokeratin Isamu Murakami, Satoshi Itami, Sakuhei Fujiwara, Hiroto Terashi, Sotaro Kurata, Tomohito Honda*, Hiroshi Shinkai and Susumu Takayasu Abstract A case of locally recurrent malignant fibrous histiocytoma was documented in a 70-year-old man. He first noticed a subcutaneous nodule forty years previously. The tumor was surgically removed four times during the last four years with local recurrence on every occasion. In the recurrent tumors, the tumor cells almost completely replaced the whole dermis and invaded skeletal muscles. They were composed of pleomorphic spindle cells arranged in a storiform pattern and bizarre histiocytic cells, which were present principally in the deeper portions of the tumor. Both types of tumor cells showed marked nuclear atypicality. In the primary tumor, surrounding a large necrotic area, spindle-shaped cells were arranged in a storiform pattern. These tumor cells exhibited only mild nuclear atypia. The recurrent tumor was strongly positive for vimentin and alpha-I-antichymotrypsin. Most tumor cells were also weakly positive for KLl, a monoclonal antibody for keratin. A Western-blot analysis revealed the presence of two bands (62 and 69 Kd) reacting with KLI in the fractions which were obtained from the tumor according to the method for keratin extraction.
Key words: malignant fibrous histiocytoma; intermediate-size filaments; eytokeratin; alpha-lantichymotrypsin Introduction Malignant fibrous histiocytoma (MFH), also referred to as malignant fibrous xanthoma (1), has been defined as a unique soft tissue sarcoma (2). MFH is the most common type of soft tissue sarcoma in late adult life and has variable histological appearances (2-4). The cellular origin of MFH remains controversial; various authors have proposed different sources, such as histiocytes, fibroblasts, or primitive mesenchymal cells (5-7). Furthermore, recent studies have suggested an epitheReceived March 22, 1991; accepted for publication April 30, 1991. Presented in part at the 41st Annual Meeting of Western Japanese Dermatological Society, Kurume, October 7, 1989. Department of Dermatology, Medical College of Oita, Oita 879-55,Japan. *Division of Plastic Surgery, Osaka Seamen's Insurance Hospital, Minato-ku, Osaka,Japan. Reprint requests to: I. Murakami, M.D., Department of Dermatology, Medical College of Oita, 1-1 Idaigaoka, Hasama-cho, Oita 879-55,japan.
Hal differentiation of the tumor cells based upon positive immunoreactivity to cytokeratin and the presence of the desmosome-like or tonofilament-like structures (8-10). This paper describes a case of a storiform-pleomorphic type of MFH with local recurrence and a positive reaction of the tumor cells to cytokeratin indicated by immunohistochemical staining. Moreover, we confirmed that the keratin fraction from the tumor mass reacted with an anti-keratin monoclonal antibody (KL1) (11), by means of Western blotting (12).
Case Report A 70-year-old man with a persistent tumor in the nucha was admitted to our hospital on October 18, 1988. He first noticed a small subcutaneous nodule about forty years previously. The gradually enlarging nodule reached walnut size and was resected in December of 1983 for the first time. Thereafter, the tumor was excised three more times before October of 1986, because of local recurrence. In October of 1987, the tumor became ulcerated in the center, accompanied by lacerating pain. At adminission, a
Malignant Fibrous Histiocytoma and Cytokeratin
341
deep ulcer 45 x 25 mm in diameter with an irregular margin was present on the nucha (Fig. 1). The skin surrounding the ulcer was slightly elevated, and the hard tumor was fixed to the underlying tissues. Neither cervical nor supraclavicular lymph nodes were palpable. No distant tumor metastasis was detected by chest X-ray film, and Garium and bone scintigram. The tumor was widely excised on December 1, 1988. The patient died on January 15, 1989, because of cerebellar bleeding which occurred 2 days after operation. Autopsy was not performed.
Materials and Methods Tissue materials: The primary, third, and fourth recurrent tumors, fixed in formalin and embedded in paraffin, were available for the hematoxylin and eosin and immunohistochemical PAP stainings (13). The fourth recurrent tumor we removed surgically was cut into pieces; most of them were frozen at -80°C until used for keratin extraction. Commercial antibodies usedfor immunohistochemistry: The following antibodies were used: anti-alpha-lantichymotrypsin polyclonal rabbit antibodies from DAKO Corp., Santa Barbara, CA, USA, and antikeratin (KL1) (11) and anti-vimentin (V9) (14) monoclonal antibodies from Immunotech, Marseille, France and DAKO, and PAP KITTM System fromDAKO. Extraction ofkeratins: After thawing, the deep part of the tumor mass was processed for keratin extraction by a slight modification of the method deSCribed by Winter et al. (15). Briefly, the minced tissue mixed with finely broken glass pieces was ground with a mortar in an ice bath under liquid nitrogen gas in 105M KCI, 10mM NaCl, 2mM dithiotbreitol (DTT), 0.5mM phenylmethylsulfonylfluoride, 0.5% Triton X-lOO, and 10mM Tris-HCl, pH 7.5, followed by homogenization with seven strokes using a glass homogenizer. The homoJenate was stirred for 12 hrs at room temperature and centrifuged at 14,000 rpm for I'hr at 4°C with a Hitachi RPR 20-2-1059rotor. The pellet was washed three times with the same buffer, and was solubilized by stirring for 14 hrs at 3°C in 8M urea, 10mM DTT, 50mM Tris-HCI, pH 9.0. After removing the glass pieces by centrifugation, the supernatant was centrifuged at 25,000rpm for 1 hr at 4°C with a Hitachi RP42-45I rotor. The clear
Fig. 1. Clinical picture of the fourth recurrent tumor. A hard tumor with ulceration measuring 45 x 25 mm on the nucha. supernatant was dialyzed extensively against 2mM Tris-HCI, pH 7.6, lOmM 2-mercaptoethanol, and deionized water. The lyophilized powder of the material in a dialysis bag was dissolved in the sample buffer (20mM Tris-HCI, pH 6.8, 4% SDS, 5mM DTT, 20% sucrose, 0.0015%bromphenol blue) for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by heating for 2 min at 90°C. For the control, the epidermis-rich tissue overlying the tumor mass, including a small amount of the dermis and the upper portion of tumor mass as well, and the sole callus from a healthy person were similarly processed for keratin extraction. SDS-PAGE and Wes-tern blotting: The test samples and molecular marker proteins were applied onto 8.7% acrylamide slab gel according to the method of Laemmli (16). The separated polypeptides were transferred to the membrane (Immobilou'Y, Millipore Corp., Bedford, MA) by the method described by Towbin et al. (12). The membrane was cut into each slot A slot of marker proteins was directly stained with amido black. For immunoblot, each slot was reacted with KLl, V9, or with normal mouse IgGl as the first antibody. The DAKO products (DAKO PAP KITTM System) were used as the second and third antibodies. Finally, bands were visualized with diaminobenzidine and H 20 2 •
Results
Histopathologic findings: In the fourth recurrent tumor (Figs. 2a-c), tumor cells almost completely replaced the skin and invaded the superficial pans of the muscle. In the upper
342
Murakami et al Table 1. Immunohistochemical profile of primary and recurrent tumors Tumor Primary Recurrent third fourth
Vimentin Keratin
ACT
Control
ND ND
+
+ +
+
ACT: alpha-1-antichymotrypsin, ND: Not determined, +/-: positive/negative staining.
Fig. 2. Histopathologic pictures of the fourth recurrent tumor. (a) Dense infiltration of fibroblastic tumor cells arranged in a storiform pattern and intermingled with lymphocytes in the upper to middle dermis. H.E. Original magnification xlOO. (b) Pleomorphic spindle cells with nuclear atypicality. H.E. Original magnification x400. (c) Histiocytic cells with large, atypical nuclei and bizarre giant cells (arrowhead) at the deep portion of the tumor. H.E. Original magnification x400.
and middle dennis, the tumor cells were arranged in a pleomorphic and storifonn pattern and invaded between hyalinized collagen bundles. Their nuclei, varying in form and size, were pale and contained one or two prominent nucleoli. Most of these cells were spindleshaped. The deeper portion of the tumor was mainly composed of histiocytic cells possessing large, pale nuclei with prominent nucleoli and abundant eosinophilic cytoplasm. Bizarre giant cells were scattered among these histiocytic cells. Moderate numbers of lymphocytes were present throughout the tumor. The primary tumor, surgically removed in 1983, contained a large area of necrosis in the center. Surrounding this necrotic mass, spindle-shaped cells without obvious atypia were arranged in a fascicular or storifonn pattern. A large amount of hemosiderin was present between the cells or phagocytized by macrophages. The third recurrent tumor extirpated in 1986 showed similar histologic findings to the fourth one shown in Figures 2a-c, except for smaller numbers of tumor cells and more prominent hyalinization of collagen bundles. Immunohistochemical findings: The results of immunohistochemical study on the primary and recurrent tumors are summarized in Table 1. Cells of the fourth recurrent tumor gave positive reactions to vimentin with V9, keratin with KL1, and alpha-1-antichymotrypsin (ACT) (Figs. 3a-c); the primary tumor was negative for both keratin and ACT. Intensity of keratin and/or ACT immunostainings varied among these tumor cells. Western blotting: As shown in Figure 4, in the keratin fraction obtained from the fourth recurrent tumor, two bands with estimated
343
Malignant Fibrous Histiocytoma and Cytokeratin
Fig. 3. Immunohistochemical PAP stainings of the fourth recurrent tumor. More than eighty percent of tumor cells were positive for vimentin with V9 (a), and alpha-l-antichymotrypsin with variable staining intensity (b). Tumor cells were weaklybut definitely positive for keratin with KLI (c).Arrowheads in a, b and c indicate the representative immunopositive cells. Original magnification x200. molecular weights of 69 and 62 Kd (slot 1, bands 1 and 2) were stained with KLI. Band 1 was more strongly blotted than band 2. In the epidermis-rich fraction and the sole callus, these two bands were also observed, but the immunoreactivity of band 2 was stronger than that of band 1 (slots 2 and 3).
Discussion The recurrent tumors in the present case can be diagnosed as a storifonn-pleomorphic type of MFH from the following histopathologic findings: invasion into skeletal muscles, plump spindle cells arranged in a storifonn pattern, and pleomorphic histiocytic cells intermingled With bizarre giant cells. For the primary tumor, 1t is difficult to make a definite diagnosis of MFH, because of the rare atypicality of nuclei of spindle tumor cells. Results of the immunohistochemical study On the fourth recurrent tumor, such as positive staining for vimentin and ACT, are consistent \vith previous reports (17, 18). It is interesting that more than eighty percent of the tumor cells in this recurrent tumor expressed cyto-
1
2
3
4
Fig. 4. Western blot analyses of the keratin fraction from the fourth recurrent tumor by PAP stainings. Slots 1 and 4: keratin fraction from the tumor; 2: keratin fraction of epidennis-rich tissue overlying the tumor; 3: sole callus. Slots 1-3 were immunoblotted with KLl, while slot 4 with normal mouse IgG}. Arrows indicate positions of marker proteins (downward): IgG (150 Kd), bovine serum albumin (68 Kd), ovoalbumin (43 Kd).
Murakami et al
344
keratin. This is the third reported instance of keratin expression by MFH (8, 9). As reviewed by LJ. McGuire et al. (19), the reported incidence of co-expression of vimentin and cytokeratin in neoplastic tissues is rapidly expanding. The presence of 62- and 69-Kd components reactive with KLI in MFH was proven for the first time by a more sophisticated method, Western blot analysis. It is unlikely that this positive reaction to KLI by immunoblotting is due to contamination by epidermis or its appendages, since epidermal components were not observed in the deep part of the tumor, which was used for the keratin extraction, under light microscopy, and the relative immunoreactive intensity of these two 62- and 69-Kd bands in the tumor differed from that in the epidermis-rich fraction. Crossreaction of vimentin with KL1 seemed unlikely, because vimentin(s) copurified with the keratin fraction of the present tumor were blotted with V9 at different positions from 62- and 69-Kd (unpublished data, Murakami). Furthermore, epithelial differentiation in MFH was also suggested by a ultrastructural study (8, 10). Thus, coexpression of vimentin, cytokeratin, and ACT might imply an ability for multidirectional differentiation of neoplastic cells (19). Acknowledgments We wish to thank Drs. Munetomo Enjoji and Hiroshi Hashimoto, Second Department of Pathology, Faculty of Medicine, Kyushu University, japan, for their valuable suggestion on the histopathological diagnosis. We are also grateful to Mr. Shuji Imi, Research Laboratory Center, Medical College of Oita, for taking the photomicrographs.
References 1) O'Brien jE, Stout AP: Malignant fibrous xanthomas, Cancer; t.7: 1445-1455, 1964. 2) Weiss SW, Enzinger FM: Malignant fibrous histiocytoma: An analysis of 200 cases, Cancer, 41: 22502266,1978. 3) Hashimoto H: Malignant fibrous histiocytoma: A clinicopathologic study of 130 cases, Fukuoka Acta Med, 70: 585-613, 1979. (in Japanese) 4) Enzinger FH, Weiss SW: Soft Tissue Tumors, C.V. Mosby, St. Louis, 1983, pp 166-198.
5) Ozzello L, StoutAP, Murray MR: Cultural characteristics of malignant histiocytomas and fibrous xanthomas, Cancer, 16: 331-344,1963. 6) Roholl PjM, Kleynej, UnnikjAMV: Characterization oftumor cells in malignant fibrous histiocytomas and other soft-tissue tumors, in comparison with malignant histioeytes, Am] Pathol, 121: 269-274,1985. 7) Iwasaki H, Isayama T,johzaki H, Kikuchi M: Malignant fibrous histiocytoma: Evidence of perivascular mesenchymal cell origin; Immunocytochemical studies with monoclonal anti-MFH antibodies, Am] Pathol, 128: 528-537,1987. 8) Hirose T, Sano T, Abej, Hizawa K: Malignant fibrous histiocytoma with epithelial differentiation?, UltrastructPatkoi, 12: 529-536, 1988. 9) Weiss SW, Bratthauer GL, Morris PA: Postirradiation malignant fibrous histiocytoma expressing cytokeratin: Implications for the immunodiagnosis of sarcomas, Am] Surg Patkoi, 12: 554-558, 1988. 10) Merkow LP, Frich JC, Slifkin M, Kyreages CG, Pardo M: Ultrastructure of a fibroxanthosarcoma (Malignant fibroxanthoma), Cancer, 28: 372-383, 1971. 11) Viacj, Reano A, Brochier j, Staquet M:f, ThivoletJ: Reactivity pattern of a monoclonal antikeratin antibody (KLl),] Invest Dermatol, 81: 351-354, 1983. 12) Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc Nat! Acad sa USA, 76: 4350-4354, 1979. 13) Sternberger LA, Hardy PHjr, CuculislJ, Meyer HG: The unlabeled antibody enzyme method of immunohistochemistry: Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes,] Histochem Cytoche1n, 18: 315-333, 1970. 14) Osborn M, Debus E, Weber K: Monoclonal antibodies specific for vimentin, Eur ] Cell Bioi, 34: 137-143,1984. 15) Winter H, Schweizer J, Goerttler K: Keratins as markers of malignancy in mouse epidermal tumors, Carcinogenesis, 1: 391-398, 1980. 16) Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227: 680-685, 1970. 17) Miettinen M, Lehto V-P, Badley RA, Virtanen I: Expression of intermediate filaments in soft-tissue sarcomas, Int] Cancer, 30: 541-546, 1982. 18) Meister P, Nathrath W: Immunohistochemical markers of histiocytic tumors, Hum Pathol, 11: 300-301, 1980. 19) McGuire LJ, Ng JPW, Lee JCK: Coexpression of cytokeratin and vimentin, Appi Pathol, 7: 73-84, 1989.
