ANATOMIC PATHOLOGY Original Article
Immunohistochemical Characterization of Atypical Fibroxanthoma and Dermatofibrosarcoma Protuberans CHAN K. MA, M.D.,1 RICHARD J. ZARBO, M.D.,2 AND ALLEN M. GOWN, M.D.2
Atypical fibroxanthoma (AFX), first reported by Helwig1 in 1961, usually occurs in sun-damaged skin of the head and neck of elderly persons and appears as solitary cutaneous nodules or ulcers. Less commonly it occurs on the extremities and trunks of young persons.2 When examined histologically, it appears to be composed of spindle and pleomorphic polyhedral cells with many giant cells and mitotic figures. Despite the anaplastic appearance, AFX is regarded as a low-grade malignant tumor and generally follows a benign clinical course, although it may recur and rarely metastasizes.3,4 Dermatofibrosarcoma protuberans (DFSP) usually occurs in young or middleaged persons as large cutaneous nodules, most frequently on the trunk and proximal extremities.5 Histologic examination shows that this lesion is composed of relatively
for muscle-specific actin. None of the cases were reactive with melanoma antibodies HMB-45 and HMB-50; NKI/C3 strongly stained 26 of 27 tumors. Compared to HMB-45 and HMB-50, NKI/C3 cross-reacted with nonmelanocytic neoplasms. Two AFXs stained for alpha-1-antitrypsin and alpha-1-antichymotrypsin. This study confirms (1) the immunophenotypic similarity of AFX and DFSP, (2) the presence of myofibroblastic differentiation in both tumors, as reflected by HHF-35 staining, and (3) that AFX and DFSP are easily distinguished from spindle cell squamous carcinoma and desmoplastic melanoma by the absence of cytokeratin, HMB-45, and HMB-50 staining. (Key words: Atypical fibroxanthomas; Dermatofibrosarcoma protuberans; Immunohistochemical stains; Squamous cell carcinoma; Melanoma) Am J Clin Pathol 1992; 97:478-483
uniform spindle cells with a conspicuous storiform pattern. Dermatofibrosarcoma is also regarded as a low-grade malignant tumor: recurrence may be a problem, but metastasis is rare.6 The exact histogenesis is still uncertain, although most people classify both tumors as fibrohistiocytic neoplasms.7"9 These two lesions need to be distinguished from other forms of spindle cell tumors of the skin, including spindle cell squamous cell carcinoma, spindle or desmoplastic melanomas, and sometimes leiomyosarcomas. 10 " Although they have characteristic clinicopathologic features, their differentiation from other forms of spindle cell tumor of the skin can be difficult if not impossible, particularly if the biopsy specimen is small. Immunoperoxidase stains may be helpful to address this problem. To identify the characteristic immunophenotypes of these two lesions, we examined 12 cases of AFX and 15 cases of DFSP with a panel of antibodies. From the ' Department of Pathology, Henry Ford Hospital, Detroit, 2 Michigan, and the University of Washington, Seattle. Washington. MATERIALS AND METHODS Received April 10, 1991; manuscript accepted for publication May Based on accepted histopathologic criteria, 12 cases of 23, 1991. Presented in part at the American Society of Pathologists and College AFX and 15 cases of DFSP were selected from the files of American Pathologists Meeting, Washington, D.C., October 1989. Addressreprintrequeststo Dr. Ma: Henry Ford Hospital, Department of the Henry Ford Hospital after a careful review of the of Pathology K-6, 2799 West Grand Boulevard, Detroit, Michigan 48202. original biopsy material, excised specimens, and speci478
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Atypical fibroxanthoma (AFX) and dermatofibrosarcoma protuberans (DFSP) have generated undue interest regarding their histogenesis, biological behavior, and differentiation from other forms of spindle cell tumors of the skin, including spindle cell squamous carcinomas and desmoplastic melanomas. To identify characteristic immunophenotypes, 12 AFXs and 15 DFSPs were examined with a panel of antibodies against cytokeratin; vimentin; desmin; proteolytic enzymes (alpha-1-antitrypsin and alpha-1antichymotrypsin); melanoma-associated antigens defined by HMB-45, HMB-50, and NKI/C3; muscle-specific actin (HHF35); and S-100 protein. The staining patterns of these two tumors were nearly identical. All cases tested negative for cytokeratin, desmin, and S-100 protein and strongly positive for vimentin. Six (50%) AFXs and 12 (80%) DFSPs tested focally positive
MA, ZARBO, AND GOWN Immunohistochemical Characterization of AFX and DFSP
479
TABLE 1. ANTIBODY PANEL Antibody
Anti-cytokeratin cocktail KA4/UCDPR10.11 CAM 5.2 34 BE 12 AE1 AE3 HMB-45 HMB-50 NKI/C3 V9 Antiserum Clone 33 HHF-35 Antiserum Antiserum
Anti-melanoma Anti-melanoma Anti-melanoma Anti-vimentin Anti-S-100 Anti-desmin Anti-actin Anti-AAT Anti-AACT
Source
Specificity
Clone CK CK CK CK
14,15,16,19/8,18 8,18,19 1,5,10,14 10,14,15,16,19
CK 1,2,3,4,5,6,7,8 10KD MAA 10/98/110 kD MAA 25-110 kD MAA Vimentin Alpha/Beta subunit Desmin Muscle specific actins Alpha-1-antitrypsin (AAT) Alpha-1-antichymotrypsin (AACT)
Triton Biosciences Becton-Dickenson Enzo Biochemical ICN Immunobiologicals Boehringer-Mannheim Dr. A.M. Gown Dr. A.M. Gown San Bio Dako Corp. Dako Corp. San Bio Dr. A.M. Gown Dako Corp. Dako Corp.
Nagle,30 Chan31 Leader32 Gown33 Cooper34 Cooper34 Gown35 Gown (personal communication) Vennegoor36 Osborn37 Debus38 Tsukada' 6 "
— — —
MAA = melanoma-associated antigen.
mens from any recurrences. Formalin-fixed, paraffinembedded tissue sections from each case were studied by the avidin-biotin complex immunoperoxidase method using a panel of antibodies (Table 1). The sections were predigested with 0.4% pepsin (Sigma P-7000, Sigma Chemical Co. St. Louis, MO) in 0.1% N HC1 at 37°C for 30 minutes to unmask the binding sites before immunoperoxidase reactions for all primary antibodies, except S-100 protein, alpha-1-antichymotrypsin (AACT), and alpha-1-antitrypsin (AAT). An overnight incubation at 4 °C with primary antibodies was performed on all the cases. Biotinylated secondary antibodies and avidin-biotinylated horseradish peroxidase complex were obtained from Vector Laboratories (Burlingame, CA). The sections were developed with chromogen 3-amino, 9-ethyl-carbazole. The primary antibody was substituted with buffer and appropriate nonimmune immunoglobulins for negative case control. Hepatoblastoma was used for positive control for AACT and AAT; melanoma for HMB-45, HMB-50, and
NKI/C3; and a multitissue block for the remaining antibodies. RESULTS As expected, the AFX occurred mainly in sun-damaged skin of the heads of elderly persons or in the extremities of young adults (Table 2). Histologic examination revealed that these tumors were composed of various proportions of pleomorphic spindle cells and polygonal mononuclear cells, often with mononuclear or multinucleated giant cells. Mitosis was a conspicuous feature. The tumors involved mainly the dermis, with frequent extension into the subcutaneous tissue. The covering epidermis often demonstrated focal ulceration (Figs. \A and B). Dermatofibrosarcoma occurred mainly in the trunks and proximal extremities (Table 3). They usually involved the dermis and subcutaneous tissue. The morphologic findings were similar from case to case and included bland,
TABLE 2. RESULTS OF IMMUNOPEROXIDASE STAINS OF AFX Case
Site
Age/Sex
CK
VIM
NKI/C3
HMB-45
HMB-50
1 2 3 4 5 6 7 8 9 10 11 12
Nose Right thigh Cheek Left thigh Right scalp Forehead Left temple Left ala nasi Right toenail bed Right upper back Left thigh Scalp
34/F 45/M 89/M 24/F 73/M 91/F 63/F 66/M 47/F 59/F 33/F 72/M
0 0 0 0 0 0 0 0 0 0 0 0
4+ 4+ 4+ 4+ 4+ 3+ 4+ 4+ 4+ 4+ 4+ 4+
4+ 4+ 4+ 4+ 4+ 0 4+ 4+ 3+ 4+ 4+ 4+
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
' Scattered dendritic cells between tumor cells.
HHF-35 0
2+ 0 2+ 1+ 2+ 0 0 0 0 3+ 1+
DES
AAT
AACT
S-100
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 2+ 0 0 1+ 0 0
0 0 1+ 0 0 0 0
0* 0* 0 0 0 0 0* 0 0 0 0 0
2+ 0 0 0 0
AFX = atypical fibroxanthoma; VIM = vimentin; DES = desmin; AAT = alpha-1-antitrypsin; AACT = alpha-1-antichymotrypsin.
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CK = cytokcratin numbered according to the nomenclature of Moll and associates.*
Reference
480
ANATOMIC PATHOLOGY Original Article
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FlGS. 1A and B (upper). Atypical fibroxanthoma, composed of pleomorphic spindle and polygonal cells (hematoxylin and eosin stain; A, X5; B, X33). FlGS. 2A and B (lower). Dermatofibrosarcoma protuberans, infiltrating the subcutaneous tissue, was composed of uniform spindle cells with a conspicuous storiform pattern (hematoxylin and eosin stain; A. X5; B. X33).
MA, ZARBO, AND GOWN Immunohistochemkal Characterization of AFX and DFSP
481
TABLE 3. RESULTS OF IMMUNOPEROXIDASE STAINS OF DFSP Case
Site
Age/Sex
CK
VIM
NKI/C3
HMB-45
HMB-50
HHF-35
DES
AAT
AACT
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Suprasternal Right lower back Left buttock Left thigh Nose Right thigh Back Anterior chest Chest Right thigh Left shoulder Left shoulder Back Left (lank Buttock
61/M 43/F 48/M 38/F 34/F 13/M 20/M 68/M 36/F 82/M 33/M 27/F 32/M 49/F 35/M
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4+ 2+ 2+ 3+ 4+ 3+ 4+ 3+ 3+ 2+ 3+ 4+ 4+ 3+ 4+
4+ 4+ 4+ 4+ 3+ 3+ 4+ 4+ 3+ 4+ 4+ 4+ 4+ 3+ 3+
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2+ 1+ 1+ 1+
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1+ 2+ 2+ 0 2+ 0 0 2+
0* 0 0 0 0 0 0 0 0 0 0 0 0 0 0
DFSF = dermatofibrosarcoma protuberans: VIM = vimentin: DES = desmin: AAT = alpha-1antitrypsin: AACT = alpha-l-antichymotrypsin.
uniform interlacing fascicles of spindle cells with a conspicuous storiform pattern and some mitotic figures (Figs. 2A and B). Tables 2 and 3 list the results of immunostaining.
Quantitation was graded by the percentage of tumor cells stained using the following scale: 1 + (focal), 2+ (less than 25%), 3+ (26-75%), 4+ (more than 75%). All the cases were consistently and diffusely positive for vimentin and
FIG. 3 (left). Atypical fibroxanthoma; intense immunostaining with NKI/C-3 antibody is demonstrated (X50). FIG. 4 (right). Dermatofibrosarcoma protuberans stained with HHF-35 antibody, showing many positively stained cells (X50).
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* Scattered dendritic cells between tumor cells.
3+ 2+ 3+
S-100
482
ANATOMIC PATHOLOGY Original Article
negative for cytokeratin, S-100 protein, desmin, and melanoma-associated antigens (HMB-45 and HMB-50). However, NKI/C-3 was strongly positive in 26 of 27 cases (Fig. 3). Two cases of AFX were focally positive for AAT and two other cases were focally positive for AACT, whereas all 15 cases of DFSP were negative for AAT and AACT. Five AFXs and 12 DFSPs were focally positive for HHF-35; three of these had many positively stained cells (Fig. 4). Three AFXs and one DFSPs displayed scattered, positively stained dendritic cells by S-100 protein. DISCUSSION
HMB-45 HMB-50
-
Carcinoma
HMB-45 HMB-50
+ —•
Melanoma
As a result of our investigation, we made the following conclusions: (1) that AFX and DFSP have similar immunophenotypic characteristics; (2) most of both tumors studied demonstrated focal myofibroblasts differentiation identified by muscle-specific actin; (3) alpha-1-antitrypsin and AACT are not reliable markers for the identification of these two tumors; and (4) AFX and DFSP are easily distinguished from spindle cell squamous cell carcinoma and spindle or desmoplastic melanomas by lack of S-100 protein, cytokeratin, HMB-45, and HMB-50 staining.
REFERENCES Desmin HHF-35
HHF-35
+
-
—•
+/—
*
•
Leiomyosarc
AFX & DFSP
FIG. 5 Spindle cell neoplasm of the skin: algorithm.
1. Helwig EB. Atypical fibroxanthoma. Tumor seminar: Proceedings of 18th annual tumor seminar of San Antonio Society of Pathologists, 1961. Tex J Med 1963;59:664-667. 2. Fretin DF, Helwig EB. Atypicalfibroxanthomaof the skin: A clinicopathologic study of 140 cases. Cancer 1973;31:1541-1552. 3. Helwig EB, May D. Atypical fibroxanthoma of the skin with metastasis. Cancer 1986;57:368-376.
A.J.C.P. • April 1992
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Our study indicates that cytokeratin (CK), vimentin, desmin, HMB-45, HMB-50, S-100 protein, and HHF-35 are useful markers for distinguishing AFX and DFSP from other forms of spindle cell tumors of the skin. In daily practice, only cytokeratin, vimentin, and S-100 protein staining are required as routine procedures. Based on results of these stains, additional antibodies can be used to confirm the diagnosis (Fig. 5). However, the morphologic and immunostaining results should be integrated for proper interpretation. For example, melanoma and leiomyosarcoma have been reported to express cytokeratin12"14 and leiomyosarcomas can demonstrate immunoreactivity for S-100 protein.15 Six cases of AFX and 12 cases of DFSP were focally positive for muscle-specific actin; three of these had many positively stained cells. In addition to muscle cells, HHF-35 also reacts with pericytes, myoepithelial cells, and myofibroblasts.1617 In our cases, the positively stained cells probably are myofibroblasts. In addition to being identified in actively proliferatingfibroblastsin granulation tissue and benign fibroblastic proliferation-plantar fibromatosis, myofibroblasts also have been identified in malignant fibrous histiocytoma and other fibrohistiocytic tumors.18 Alpha-1-antitrypsin and AACT are recognized markers of reactive and
neoplastic histiocytes.19 However, both antibodies are nonspecific and have been reported to produce positive results in a variety of sarcomas, carcinomas, and melanomas.20'21 Among the recent studies of AFX, immunostaining for AAT and AACT were positive in most of the cases.21"24 Not many studies were performed on DFSP and results were inconsistent. Only three of six cases studied by du Boulay25 were positive for AAT and AACT. All 13 cases studied by Fletcher and co-workers6 were negative for AACT. A recent study by Eckert and colleagues26 revealed positive staining for AAT only in isolated cells in 8 of their 12 cases of AFX and 1 of 5 cases of DFSP. Only two of our AFX cases were focally positive for either AAT or AACT. All DFSP cases were negative for both AAT and AACT. These conflicting results may reflect differences in technique or antibodies used. On the other hand, this may support the contention that AFX and DFSP, particularly DFSP, histogenetically may be a heterogenous group of lesions.25'26 NKI/C-3 was strongly reactive in 26 of 27 cases. This antibody is an immunoglobulin G, murine monoclonal antibody raised against a membrane fraction of the human melanoma line MEWO. It was reported by several groups to react with almost all melanomas, but with very few nonmelanocytic tumors.27'28 Our study, however, clearly indicates that NKI/C-3 crossreacts with these nonmelanocytic spindle cell neoplasms. To exclude a diagnosis of spindle cell melanoma, the melanoma-specific antibodies HMB-45 and HMB-50 should be used to confirm specificity to S-100 protein stains. The identification of scattered S-100 protein-positive dendrictic cells in four of our cases (three AFX and one DFSP) have been observed by other authors and is believed to be reactive in nature.22,24
MA, ZARBO, AND GOWN Immunohistochemical Characterization of AFX and DFSP
22.
23.
24.
25.
26. 27.
28.
29.
30.
31. 32.
33. 34.
35. 36.
37. 38.
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melanomas. Its lack of specificity as a tumor marker. Am J Surg Pathol 1987;11:133-139. Kuwano H, Hashimoto H, Enjoji M. Atypical fibroxanthoma distinguishable from spindle cell carcinoma in sarcoma-like skin lesions. A clinicopatholgoic and immunohistochemical study of 21 cases. Cancer 1985;55:172-180. Leong ASY, Milios J. Atypical fibroxanthoma of the skin: A clinicopathological and immunohistochemical study and a discussion of its histogenesis. Histopathology 1987;11:463-475. Ricci A Jr, Cartun RW, Zakowski MF. Atypical fibroxanthoma. A study of 14 cases emphasizing the presence of Langerhans' histiocytes with implications for differential diagnosis by antibody panels. Am J Surg Pathol 1988; 12:591-598. du Boulay CEH. Demonstration of alpha-1-antitrypsin and alpha1-antichymotrypsin in fibrous histiocytomas using the immunoperoxidase technique. Am J Surg Pathol 1982;6:559-564. Eckert F, Burg G, Braun-Falco O. Immunostaining in atypical fibroxanthoma of the skin. Path Res Pract 1989; 184:27-34. Henzen-Logmans SC, Meijer JLM, Ruiter DJ, et al. Diagnostic application of panels of antibodies in mucosal melanomas of the Head and neck. Cancer 1988;61:702-711. Cochran AJ, Wen D, Morton DL. Occult tumor cellls in the lymph nodes of patients with pathological state I malignant melanoma. An immunohistological study. Am J Surg Pathol 1988; 12:612618. Moll R, Franke WW, Schiller DL, et al. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 1982; 31:11-24. Nagle RB, Bocker W, Davis JR, et al. Characterization of breast carcinomas by two monoclonal antibodies distinguishing myoepithelial from luminal epithelial cells. J Histochem Cytochem 1986;34:869-881. Chan R, Edwards BF, Hu R, et al. Characterization of two monoclonal antibodies in an immunohistochemical study of keratin 8 and 18 expression. Am J Clin Pathol 1988;89:472-480. Leader M, Patel J, Makin C, Henry K. An analysis of the sensitivity and specificity of the cytokeratin marker CAM 5.2 for epithelial tumours: Results of a study of 203 sarcomas, 50 carcinomas and 28 malignant melanomas. Histopathology 1986;10:1315-1324. Gown AM, Vogel AM. Monoclonal antibodies to human intermediate filament proteins: II. Distribution of filament proteins in normal human tissues. Am J Pathol 1984,114:309-321. Cooper D, Schermer A, Sun TT. Classification of human epithelia and their neoplasms using monoclonal antibodies to keratins: strategies, applications, and limitations. Lab Invest 1985;52:243256. Gown AM, Vogel AM, Hoak D, et al. Monoclonal antibodies specific for melanocyte tumors distinguish subpopulations of melanocytes. Am J Pathol 1986; 123:195-203. Vennegoor C, Calafat J, Hageman P, et al. Biochemical characterization and cellular localization of a formalin-resistant melanomaassociated antigen reacting with monoclonal antibody NK.I/C-3. Int J Cancer 1985;35:287-295. Osborn M, Debus E, Weber K. Monoclonal antibodies specific for vimentin. Eur J Cell Biol 1984;34:137-143. Debus E, Weber K, Osborn M. Monoclonal antibodies to desmin, the muscle-specific intermediate filament protein. EMBO J 1983;2:2305-2312.
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4. Starink TM, Hausman R, Van Delden L, Neering H. Atypical fibroxanthoma of the skin: Presentation of 5 cases and review of the literature. Br J Dermatol 1977;97:167-177. 5. Taylor HB, Helwig EB. Dermatofibrosarcoma protuberans. A study of 115 cases. Cancer 1962;15:717-725. 6. Fletcher CDM, Evans BJ, MacArtney JC, et al. Dermatofibrosarcoma protuberans: A clinicopathological and immunohistochemical study with a review of the literature. Histopathology 1985;9:921938. 7. Kindblom L, Jacobsen GK, Jacobsen M. Immunohistochemical investigations of tumors of supposed fibroblastic-histiocytic origin. Hum Pathol 1982;13:834-840. 8. O'Dowd J, Laidler P. Progression of dermatofibrosarcoma protuberans to malignant fibrous histiocytoma: Report of a case with implications for tumor histogenesis. Hum Pathol 1988; 19:368370. 9. Enzinger FM, Weiss SW: Soft Tissue Tumors. St Louis: CV Mosby, 1983. 10. Evans HL, Smith JL. Spindle cell squamous carcinomas and sarcoma-like tumors of the skin. A comparative study of 38 cases. Cancer 1980;45:2687-2697. 11. Eusebi V, Ceccarelli C, Piscioli F, Cristofolini M, Azzopardi JG. Spindle cell tumours of the skin of debatable origin. An immunocytochemical study. J Pathol 1984; 144:189-199. 12. Zarbo RJ, Gown AM, Nagle RB, Visscher DW, Crissman JD. Anomalous cytokeratin expression in malignant melanoma: One and two-dimensional Western Blot analysis and immunohistochemical survey of 100 melanomas. Mod Pathol 1990; 4:494501. 13. Gown AM, Boyd HC, Chang Y, Ferguson M, Reichler B, Tippens D. Smooth muscle cells can express cytokeratins of "simple" epithelium. Immunocytochemical and biochemical studies in vitro and in vivo. Am J Pathol 1988; 132:223-232. 14. Miettinen M. Immunoreactivity for cytokeratin and epithelial membrane antigen in leiomyosarcoma. Arch Pathol Lab Med 1988;112:637-640. 15. Swanson PE, Stanley MW, Scheithauer BW, Wick MR. Primary cutaneous leiomyosarcoma. A histological and immunohistochemical study of 9 cases, with ultrastructural correlation. J Cutan Pathol 1988;15:129-141. 16. Tsukada T, Tippens D, Gordon D, Ross R, Gown AM. HHF35, a muscle-actin-specific monoclonal antibody. I. Immunocytochemical and biochemical characterization. Am J Pathol 1987;126:51-60. 17. Tsukada T, McNutt MA, Ross R, Gown AM. HHF35, a muscle actin-specific monoclonal antibody. II. Reactivity in normal, reactive and neoplastic human tissue. Am J Pathol 1987; 127:389402. 18. Churg AM, Kahn LB. Myofibroblasts and related cells in malignant fiborus and fibrohistiocytic tumors. Hum Pathol 1977;8:205-218. 19. du Boulay CEH. Immunohistochemistry of soft tissue tumours: A review J Pathol 1985;145:77-94. 20. Silvis NG, Swanson PE, Manivel JC, Kaye VN, Wick MR. Spindlecell and pleomorphic neoplasms of the skin: A clinicopathologic and immunohistochemical study of 30 cases, with emphasis on "atypical fibroxanthomas". Am J Dermatopathol 1988; 10:9-19. 21. Leader M, Patel J, Collins M, Henry K. Anti-alpha 1 Antichymotrypsin staining of 194 sarcomas, 38 carcinomas and 17 malignant
483
Immunohistochemical Characterization of Atypical Fibroxanthoma and Dermatofibrosarcoma Protuberans CHAN K. MA, M.D.,1 RICHARD J. ZARBO, M.D.,2 AND ALLEN M. GOWN, M.D.2
Atypical fibroxanthoma (AFX), first reported by Helwig1 in 1961, usually occurs in sun-damaged skin of the head and neck of elderly persons and appears as solitary cutaneous nodules or ulcers. Less commonly it occurs on the extremities and trunks of young persons.2 When examined histologically, it appears to be composed of spindle and pleomorphic polyhedral cells with many giant cells and mitotic figures. Despite the anaplastic appearance, AFX is regarded as a low-grade malignant tumor and generally follows a benign clinical course, although it may recur and rarely metastasizes.3,4 Dermatofibrosarcoma protuberans (DFSP) usually occurs in young or middleaged persons as large cutaneous nodules, most frequently on the trunk and proximal extremities.5 Histologic examination shows that this lesion is composed of relatively
for muscle-specific actin. None of the cases were reactive with melanoma antibodies HMB-45 and HMB-50; NKI/C3 strongly stained 26 of 27 tumors. Compared to HMB-45 and HMB-50, NKI/C3 cross-reacted with nonmelanocytic neoplasms. Two AFXs stained for alpha-1-antitrypsin and alpha-1-antichymotrypsin. This study confirms (1) the immunophenotypic similarity of AFX and DFSP, (2) the presence of myofibroblastic differentiation in both tumors, as reflected by HHF-35 staining, and (3) that AFX and DFSP are easily distinguished from spindle cell squamous carcinoma and desmoplastic melanoma by the absence of cytokeratin, HMB-45, and HMB-50 staining. (Key words: Atypical fibroxanthomas; Dermatofibrosarcoma protuberans; Immunohistochemical stains; Squamous cell carcinoma; Melanoma) Am J Clin Pathol 1992; 97:478-483
uniform spindle cells with a conspicuous storiform pattern. Dermatofibrosarcoma is also regarded as a low-grade malignant tumor: recurrence may be a problem, but metastasis is rare.6 The exact histogenesis is still uncertain, although most people classify both tumors as fibrohistiocytic neoplasms.7"9 These two lesions need to be distinguished from other forms of spindle cell tumors of the skin, including spindle cell squamous cell carcinoma, spindle or desmoplastic melanomas, and sometimes leiomyosarcomas. 10 " Although they have characteristic clinicopathologic features, their differentiation from other forms of spindle cell tumor of the skin can be difficult if not impossible, particularly if the biopsy specimen is small. Immunoperoxidase stains may be helpful to address this problem. To identify the characteristic immunophenotypes of these two lesions, we examined 12 cases of AFX and 15 cases of DFSP with a panel of antibodies. From the ' Department of Pathology, Henry Ford Hospital, Detroit, 2 Michigan, and the University of Washington, Seattle. Washington. MATERIALS AND METHODS Received April 10, 1991; manuscript accepted for publication May Based on accepted histopathologic criteria, 12 cases of 23, 1991. Presented in part at the American Society of Pathologists and College AFX and 15 cases of DFSP were selected from the files of American Pathologists Meeting, Washington, D.C., October 1989. Addressreprintrequeststo Dr. Ma: Henry Ford Hospital, Department of the Henry Ford Hospital after a careful review of the of Pathology K-6, 2799 West Grand Boulevard, Detroit, Michigan 48202. original biopsy material, excised specimens, and speci478
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 8, 2016
Atypical fibroxanthoma (AFX) and dermatofibrosarcoma protuberans (DFSP) have generated undue interest regarding their histogenesis, biological behavior, and differentiation from other forms of spindle cell tumors of the skin, including spindle cell squamous carcinomas and desmoplastic melanomas. To identify characteristic immunophenotypes, 12 AFXs and 15 DFSPs were examined with a panel of antibodies against cytokeratin; vimentin; desmin; proteolytic enzymes (alpha-1-antitrypsin and alpha-1antichymotrypsin); melanoma-associated antigens defined by HMB-45, HMB-50, and NKI/C3; muscle-specific actin (HHF35); and S-100 protein. The staining patterns of these two tumors were nearly identical. All cases tested negative for cytokeratin, desmin, and S-100 protein and strongly positive for vimentin. Six (50%) AFXs and 12 (80%) DFSPs tested focally positive
MA, ZARBO, AND GOWN Immunohistochemical Characterization of AFX and DFSP
479
TABLE 1. ANTIBODY PANEL Antibody
Anti-cytokeratin cocktail KA4/UCDPR10.11 CAM 5.2 34 BE 12 AE1 AE3 HMB-45 HMB-50 NKI/C3 V9 Antiserum Clone 33 HHF-35 Antiserum Antiserum
Anti-melanoma Anti-melanoma Anti-melanoma Anti-vimentin Anti-S-100 Anti-desmin Anti-actin Anti-AAT Anti-AACT
Source
Specificity
Clone CK CK CK CK
14,15,16,19/8,18 8,18,19 1,5,10,14 10,14,15,16,19
CK 1,2,3,4,5,6,7,8 10KD MAA 10/98/110 kD MAA 25-110 kD MAA Vimentin Alpha/Beta subunit Desmin Muscle specific actins Alpha-1-antitrypsin (AAT) Alpha-1-antichymotrypsin (AACT)
Triton Biosciences Becton-Dickenson Enzo Biochemical ICN Immunobiologicals Boehringer-Mannheim Dr. A.M. Gown Dr. A.M. Gown San Bio Dako Corp. Dako Corp. San Bio Dr. A.M. Gown Dako Corp. Dako Corp.
Nagle,30 Chan31 Leader32 Gown33 Cooper34 Cooper34 Gown35 Gown (personal communication) Vennegoor36 Osborn37 Debus38 Tsukada' 6 "
— — —
MAA = melanoma-associated antigen.
mens from any recurrences. Formalin-fixed, paraffinembedded tissue sections from each case were studied by the avidin-biotin complex immunoperoxidase method using a panel of antibodies (Table 1). The sections were predigested with 0.4% pepsin (Sigma P-7000, Sigma Chemical Co. St. Louis, MO) in 0.1% N HC1 at 37°C for 30 minutes to unmask the binding sites before immunoperoxidase reactions for all primary antibodies, except S-100 protein, alpha-1-antichymotrypsin (AACT), and alpha-1-antitrypsin (AAT). An overnight incubation at 4 °C with primary antibodies was performed on all the cases. Biotinylated secondary antibodies and avidin-biotinylated horseradish peroxidase complex were obtained from Vector Laboratories (Burlingame, CA). The sections were developed with chromogen 3-amino, 9-ethyl-carbazole. The primary antibody was substituted with buffer and appropriate nonimmune immunoglobulins for negative case control. Hepatoblastoma was used for positive control for AACT and AAT; melanoma for HMB-45, HMB-50, and
NKI/C3; and a multitissue block for the remaining antibodies. RESULTS As expected, the AFX occurred mainly in sun-damaged skin of the heads of elderly persons or in the extremities of young adults (Table 2). Histologic examination revealed that these tumors were composed of various proportions of pleomorphic spindle cells and polygonal mononuclear cells, often with mononuclear or multinucleated giant cells. Mitosis was a conspicuous feature. The tumors involved mainly the dermis, with frequent extension into the subcutaneous tissue. The covering epidermis often demonstrated focal ulceration (Figs. \A and B). Dermatofibrosarcoma occurred mainly in the trunks and proximal extremities (Table 3). They usually involved the dermis and subcutaneous tissue. The morphologic findings were similar from case to case and included bland,
TABLE 2. RESULTS OF IMMUNOPEROXIDASE STAINS OF AFX Case
Site
Age/Sex
CK
VIM
NKI/C3
HMB-45
HMB-50
1 2 3 4 5 6 7 8 9 10 11 12
Nose Right thigh Cheek Left thigh Right scalp Forehead Left temple Left ala nasi Right toenail bed Right upper back Left thigh Scalp
34/F 45/M 89/M 24/F 73/M 91/F 63/F 66/M 47/F 59/F 33/F 72/M
0 0 0 0 0 0 0 0 0 0 0 0
4+ 4+ 4+ 4+ 4+ 3+ 4+ 4+ 4+ 4+ 4+ 4+
4+ 4+ 4+ 4+ 4+ 0 4+ 4+ 3+ 4+ 4+ 4+
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
' Scattered dendritic cells between tumor cells.
HHF-35 0
2+ 0 2+ 1+ 2+ 0 0 0 0 3+ 1+
DES
AAT
AACT
S-100
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 2+ 0 0 1+ 0 0
0 0 1+ 0 0 0 0
0* 0* 0 0 0 0 0* 0 0 0 0 0
2+ 0 0 0 0
AFX = atypical fibroxanthoma; VIM = vimentin; DES = desmin; AAT = alpha-1-antitrypsin; AACT = alpha-1-antichymotrypsin.
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CK = cytokcratin numbered according to the nomenclature of Moll and associates.*
Reference
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ANATOMIC PATHOLOGY Original Article
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FlGS. 1A and B (upper). Atypical fibroxanthoma, composed of pleomorphic spindle and polygonal cells (hematoxylin and eosin stain; A, X5; B, X33). FlGS. 2A and B (lower). Dermatofibrosarcoma protuberans, infiltrating the subcutaneous tissue, was composed of uniform spindle cells with a conspicuous storiform pattern (hematoxylin and eosin stain; A. X5; B. X33).
MA, ZARBO, AND GOWN Immunohistochemkal Characterization of AFX and DFSP
481
TABLE 3. RESULTS OF IMMUNOPEROXIDASE STAINS OF DFSP Case
Site
Age/Sex
CK
VIM
NKI/C3
HMB-45
HMB-50
HHF-35
DES
AAT
AACT
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Suprasternal Right lower back Left buttock Left thigh Nose Right thigh Back Anterior chest Chest Right thigh Left shoulder Left shoulder Back Left (lank Buttock
61/M 43/F 48/M 38/F 34/F 13/M 20/M 68/M 36/F 82/M 33/M 27/F 32/M 49/F 35/M
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4+ 2+ 2+ 3+ 4+ 3+ 4+ 3+ 3+ 2+ 3+ 4+ 4+ 3+ 4+
4+ 4+ 4+ 4+ 3+ 3+ 4+ 4+ 3+ 4+ 4+ 4+ 4+ 3+ 3+
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2+ 1+ 1+ 1+
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1+ 2+ 2+ 0 2+ 0 0 2+
0* 0 0 0 0 0 0 0 0 0 0 0 0 0 0
DFSF = dermatofibrosarcoma protuberans: VIM = vimentin: DES = desmin: AAT = alpha-1antitrypsin: AACT = alpha-l-antichymotrypsin.
uniform interlacing fascicles of spindle cells with a conspicuous storiform pattern and some mitotic figures (Figs. 2A and B). Tables 2 and 3 list the results of immunostaining.
Quantitation was graded by the percentage of tumor cells stained using the following scale: 1 + (focal), 2+ (less than 25%), 3+ (26-75%), 4+ (more than 75%). All the cases were consistently and diffusely positive for vimentin and
FIG. 3 (left). Atypical fibroxanthoma; intense immunostaining with NKI/C-3 antibody is demonstrated (X50). FIG. 4 (right). Dermatofibrosarcoma protuberans stained with HHF-35 antibody, showing many positively stained cells (X50).
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* Scattered dendritic cells between tumor cells.
3+ 2+ 3+
S-100
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ANATOMIC PATHOLOGY Original Article
negative for cytokeratin, S-100 protein, desmin, and melanoma-associated antigens (HMB-45 and HMB-50). However, NKI/C-3 was strongly positive in 26 of 27 cases (Fig. 3). Two cases of AFX were focally positive for AAT and two other cases were focally positive for AACT, whereas all 15 cases of DFSP were negative for AAT and AACT. Five AFXs and 12 DFSPs were focally positive for HHF-35; three of these had many positively stained cells (Fig. 4). Three AFXs and one DFSPs displayed scattered, positively stained dendritic cells by S-100 protein. DISCUSSION
HMB-45 HMB-50
-
Carcinoma
HMB-45 HMB-50
+ —•
Melanoma
As a result of our investigation, we made the following conclusions: (1) that AFX and DFSP have similar immunophenotypic characteristics; (2) most of both tumors studied demonstrated focal myofibroblasts differentiation identified by muscle-specific actin; (3) alpha-1-antitrypsin and AACT are not reliable markers for the identification of these two tumors; and (4) AFX and DFSP are easily distinguished from spindle cell squamous cell carcinoma and spindle or desmoplastic melanomas by lack of S-100 protein, cytokeratin, HMB-45, and HMB-50 staining.
REFERENCES Desmin HHF-35
HHF-35
+
-
—•
+/—
*
•
Leiomyosarc
AFX & DFSP
FIG. 5 Spindle cell neoplasm of the skin: algorithm.
1. Helwig EB. Atypical fibroxanthoma. Tumor seminar: Proceedings of 18th annual tumor seminar of San Antonio Society of Pathologists, 1961. Tex J Med 1963;59:664-667. 2. Fretin DF, Helwig EB. Atypicalfibroxanthomaof the skin: A clinicopathologic study of 140 cases. Cancer 1973;31:1541-1552. 3. Helwig EB, May D. Atypical fibroxanthoma of the skin with metastasis. Cancer 1986;57:368-376.
A.J.C.P. • April 1992
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Our study indicates that cytokeratin (CK), vimentin, desmin, HMB-45, HMB-50, S-100 protein, and HHF-35 are useful markers for distinguishing AFX and DFSP from other forms of spindle cell tumors of the skin. In daily practice, only cytokeratin, vimentin, and S-100 protein staining are required as routine procedures. Based on results of these stains, additional antibodies can be used to confirm the diagnosis (Fig. 5). However, the morphologic and immunostaining results should be integrated for proper interpretation. For example, melanoma and leiomyosarcoma have been reported to express cytokeratin12"14 and leiomyosarcomas can demonstrate immunoreactivity for S-100 protein.15 Six cases of AFX and 12 cases of DFSP were focally positive for muscle-specific actin; three of these had many positively stained cells. In addition to muscle cells, HHF-35 also reacts with pericytes, myoepithelial cells, and myofibroblasts.1617 In our cases, the positively stained cells probably are myofibroblasts. In addition to being identified in actively proliferatingfibroblastsin granulation tissue and benign fibroblastic proliferation-plantar fibromatosis, myofibroblasts also have been identified in malignant fibrous histiocytoma and other fibrohistiocytic tumors.18 Alpha-1-antitrypsin and AACT are recognized markers of reactive and
neoplastic histiocytes.19 However, both antibodies are nonspecific and have been reported to produce positive results in a variety of sarcomas, carcinomas, and melanomas.20'21 Among the recent studies of AFX, immunostaining for AAT and AACT were positive in most of the cases.21"24 Not many studies were performed on DFSP and results were inconsistent. Only three of six cases studied by du Boulay25 were positive for AAT and AACT. All 13 cases studied by Fletcher and co-workers6 were negative for AACT. A recent study by Eckert and colleagues26 revealed positive staining for AAT only in isolated cells in 8 of their 12 cases of AFX and 1 of 5 cases of DFSP. Only two of our AFX cases were focally positive for either AAT or AACT. All DFSP cases were negative for both AAT and AACT. These conflicting results may reflect differences in technique or antibodies used. On the other hand, this may support the contention that AFX and DFSP, particularly DFSP, histogenetically may be a heterogenous group of lesions.25'26 NKI/C-3 was strongly reactive in 26 of 27 cases. This antibody is an immunoglobulin G, murine monoclonal antibody raised against a membrane fraction of the human melanoma line MEWO. It was reported by several groups to react with almost all melanomas, but with very few nonmelanocytic tumors.27'28 Our study, however, clearly indicates that NKI/C-3 crossreacts with these nonmelanocytic spindle cell neoplasms. To exclude a diagnosis of spindle cell melanoma, the melanoma-specific antibodies HMB-45 and HMB-50 should be used to confirm specificity to S-100 protein stains. The identification of scattered S-100 protein-positive dendrictic cells in four of our cases (three AFX and one DFSP) have been observed by other authors and is believed to be reactive in nature.22,24
MA, ZARBO, AND GOWN Immunohistochemical Characterization of AFX and DFSP
22.
23.
24.
25.
26. 27.
28.
29.
30.
31. 32.
33. 34.
35. 36.
37. 38.
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