Abstract

Objective: There has been no report on useful immunohistological markers for epithelioid sarcoma (ES) so far. The purpose of this study is to evaluate the positivity and specificity of CA125 as a marker for the correct diagnosis of ES.

Methods: This study was performed in 11 patients with ES (nine men and two women; distal type: 10 cases; proximal type: one case), 78 patients with other soft tissue tumors and nine with benign granulomas. The other soft tissue tumors consisted of six synovial sarcomas, six clear cell sarcomas, eight leiomyosarcomas, six rhabdomyosarcomas, five malignant peripheral nerve sheath tumors, ten malignant fibrous histiocytomas, 17 desmoid tumors, 14 liposarcomas, six squamous cell carcinomas (cutaneous SCC of the distal extremities), two rheumatoid nodules and seven foreign body granulomas. Immunohistochemical analysis for CA125 was performed for these 89 soft tissue tumors and nine granulomas using a labeled streptavidin biotin method. Immunohistochemical analysis of epithelial membrane antigen, cytokeratin, carcinoembrionic antigen, vimentin and CD34 was performed only for the 11 ES patients.

Results: CA125 was strongly expressed in 10 out of the 11 ES patients. EMA, cytokeratin and vimentin were also positive in all the cases. CEA was positive in two of the 11 patients. Immunohistochemical study in six ES patients showed expression of CD 34. The other 78 soft tissue tumors and nine granulomas did not express CA125.

Conclusion: This study clearly revealed the specificity and positivity of CA125 in ES. These data indicate that CA125 may be a useful tumor marker for diagnosing ES.

Received October 2, 2003; accepted January 9, 2004

INTRODUCTION

Epithelioid sarcoma (ES) is a rare mesenchymal soft tissue tumor with an epithelioid pattern. It often occurs in the extremities of young people. The tumors are presented as nodules or skin ulcers (1). Therefore, they are often misdiagnosed as benign granulomatous lesions. They may often be confused with other malignancies as well, especially squamous cell carcinoma and synovial sarcoma (1). This confusion often leads to a delay in initial treatment or inappropriate treatment. The histological origin of ES remains unknown. Immunohistochemical studies have been performed by previous investigators in order to characterize this tumor (26). However, unlike most other musculoskeletal tumors, ES has no known specific tumor marker for diagnosis. CA125 was discovered to be a serum antigen used as a marker for epithelial ovarian carcinoma (7). Its level rises in certain gynecological conditions and a few gastrointestinal carcinomas and endometriosis (8,9). However, there are few reports linking the expression of CA125 with musculoskeletal tumors. We had previously reported two cases of ES patients with elevated serum CA125 level in whom CA125 expression was also confirmed in the tumor cells (10). In this paper, we examined CA125 expression in ES and other soft tissue tumors and benign granulomas in order to evaluate the usefulness of CA125 as a marker in the diagnosis of ES. We suggest that CA125 expression is a novel method of differentiating ES from other soft tissue tumors and benign granulomas.

SUBJECTS AND METHODS

Eleven specimens from ES patients from 13 to 51 years of age (average age: 31.4 years) were used in this study. All tumors but one originated in the extremities (lower leg: three cases; thigh: two cases; foot: two cases; elbow, palm, index finger and abdomen: one case each) (Table 1). All the eleven cases showed typical histological features of ES (distal type: 10 cases; proximal type: one case) (11). The tumors were characterized by a nodular growth and were arranged as compact sheets of round to oval cells with central necrosis (Fig. 1a and b). Aggregates of rhabdoid cells were observed in certain parts (Fig. 1c). To determine the specificity of CA125 for ES as opposed to other soft tissue tumors, 78 cases of soft tissue and nine cases of benign granuloma were investigated. These soft tissue tumors and granulomas consisted of six synovial sarcomas (biphasic type: four; monophasic type: two), six clear cell sarcomas, eight leiomyosarcomas, six rhabdomyosarcomas (embryonal type: two; alveolar type: two; pleomorphic type: two), five malignant peripheral nerve sheath tumors (MPNSTs), ten malignant fibrous histiocytomas (MFHs) (striform pleomorphic type: nine; mixoid type; one), 17 desmoid tumors, 14 liposarcomas (myxoid type: five; pleomorphic type: nine), six squamous cell carcinomas (cutaneous SCC of the distal extremities), two rheumatoid nodules and seven foreign body granulomas. The patients with epithelioid sarcoma had no associated malignancies of gynecological tumors, tumors of pleura or peritoneum or epithelial malignant tumors. The specimens were fixed in 10% buffered formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin. They were then examined immunohistochemically by labeled streptavidin biotin method (LSAB method) (12) with appropriate use of positive and negative controls throughout. Ovarian cancer was used as a positive control for CA125. Staining of more than 10% of the tumor cells was interpreted as positive for CA125. Nichirei Histofine® SAB-PO (M) kit (Nichirei Corporation, Tsukiji, Tokyo, Japan) was used for this purpose. In brief, deparaffinization of the sections was followed by blocking endogenous peroxidase activity with 3% hydrogen peroxide in absolute methanol. After pretreatment, ES sections were incubated overnight with 10% normal rabbit serum, followed by primary antibodies (Table 2) to vimentin (Dako Corporation, Carpinteria, CA, USA) (1:1000), cytokeratin (Dako Corporation, Carpinteria, CA, USA) (1:300), EMA (Dako Corporation, Carpinteria, CA, USA) (1:800), CEA (Mochida Pharmaceutical, Tokyo, Japan) (1:400), CD34 (Nichirei Corporation, Tokyo, Japan) (1:50), CA125 (Fujirebio Diagnostics, Malvern, PA, USA) (diluted), biotinylated rabbit anti-mouse antibody for 30 min and peroxidase labeled streptavidin complex for 30 min. Between the steps, the sections were thoroughly washed with phosphate buffered saline (pH 7.6). Antibody localization was visualized by a peroxidase reaction with 0.6% hydrogen peroxide and 0.03% diaminobenzidine. Only CA125 expression was evaluated in other soft tissue tumors by the same method.

RESULTS

Serum CA 125 level was measured serially in three out of the 11 cases (Cases 6, 7 and 10). High values of CA125 were reported in this study and the change in the values correlated well with the progression of the disease (Fig. 2a and b) (10). Case 10 had a high value of 755 U/ml (normal ≤35 U/ml) at recurrence after the initial treatment, which decreased to 25 U/ml after amputation of the affected leg (Fig. 2c). We found a strong positive staining of tumor cells for CA125 antigen in 10 of the 11 cases (90.9%) (Fig. 3a and b). In 11 cases (100%), the tumor cells stained positively for EMA, cytokeratin and vimentin. Further, only two of the 11 specimens (18.2%), stained positively for CEA, and six of the eight cases (75.0%) stained positively for CD34 (Table 3). In all the cases of non-ES mesenchymal soft tissue tumors including synovial sarcomas, clear cell sarcomas, leiomyosarcomas, rhabdomyosarcomas, MPNSTs, MFHs, desmoplastic fibromas, myxoid liposarcomas and pleomorphic liposarcomas, tissue samples were negative for anti-CA125 antibody. CA125 was not expressed in cutaneous squamous cell cancers, rheumatoid nodules and foreign body granulomas (Table 4).

DISCUSSION

ES is a rare histological subtype of sarcoma primarily affecting young adults and may sometimes be difficult to diagnose histologically. ES usually occurs in the soft tissues, particularly involving the hands and forearms, followed in frequency by the knees and lower legs. Most of the tumors present as firm and palpable masses in the subcutis or dermis. The tumor, which frequently appears as a painless nodular or ulcerated lesion, is slow growing and rarely more than 1 cm in diameter after weeks of development (1,13).

Microscopically, aggregates of chronic inflammatory cells are present along the peripheral margin of the tumor nodules in most cases. Therefore, it is often mistaken to be a granulomatous lesion such as a foreign body granuloma, necrotizing infectious granuloma, necrobiosis lipoidica, granuloma annulare or rheumatoid nodule (14,15). ES may also be mistaken for a wide array of epithelioid-appearing malignant soft tissue neoplasms such as an epithelioid malignant peripheral nerve sheath tumor (MPNST) or a melanoma. In some cases, ES is often thought to be difficult to distinguish microscopically from synovial sarcoma and ulcerating squamous cell carcinoma (15). This difficulty in establishing the diagnosis can lead to a long delay before surgical excision of the lesion.

Immunohistochemically, most ES samples stain for low and high-molecular weight cytokeratins, EMA, and vimentin (26). Moreover, CEA was also positive in a small number of cases (3,13,16). Up to 60–70% of ES cases stain for CD34 (6,17). The results of the present study are consistent with those of the previous reports (26,13). Despite the reports of various histopathological characteristics and several immunohistochemical studies on ES, there is still no useful marker for ES that can precisely differentiate it from other mesenchymal tumors.

CA125 is the antigenic determinant identified by a murine monoclonal antibody raised specifically against non-mucinous epithelial ovarian carcinoma (18). CA125 is a differentiation antigen associated with coelomic epithelium and its normal and neoplastic derivatives (19). However, in this report, it is also shown that normal adult and fetal ovarian surface epithelium failed to express CA125 despite the presence of CA125 in normal mesothelial cells and tumors derived from surface epithelium, suggesting the presence of a distinct differentiation pathway for ovarian surface epithelium (19).

Zotter et al. (20) performed immunohistochemical analysis of CA125 in cancers and found it positive in all of 10 cases of ovarian cancer, four out of 10 cases in breast cancer, nine out of 10 cases in squamous cell carcinoma, three out of 10 cases in stomach cancer, eight out of 10 cases in pancreatic cancer, four out of 10 cases in colon cancer and five out of 10 cases in thyroid cancer (20). It is noteworthy that this study also reported that some sarcomas gave positive reactions to anti-CA125 antibody. The immunoreactivity of a tumor with CA125 does not necessarily indicate its epithelial or ovarian origin (20). However, there have been very few papers reporting an increase in serum CA125 in non-epithelial malignant tumors or other stromal tumors. Following are the reports on mesenchymal soft tissue tumors with elevation of serum CA125: leiomyoma and leiomyosarcoma (21), rhabdomyosarcoma (22,23) and desmoplastic small round cell tumor (DRCT) (24,25). Among these reports, immunohistochemical staining of CA125 was confirmed only for a rhabdomyosarcoma (22) and a DRCT (25).

Until now, there has been no report on CA125 as an immunohistological marker for ES. In the present study, CA125 expression was not observed in 78 cases of soft tissue tumors and nine granulomas except ES. Ten out of 11 ES cases expressed CA125, which suggests high positivity and specificity of CA125 in epithelioid tumors. In three out of these 10 cases, serum levels of CA125 were measured and confirmed to be elevated. In one of the three patients, the serum level of CA125 was detected to be 840 U/ml, which is far greater than 35 U/ml of CA125 found in the early stage of the disease (10). These results indicate that the serum level of CA125 is closely related to the progress of ES and that the tumor cells produce CA125. Serum CA125 values can be easily measured. CA125 immunohistochemical staining in ES is easily performed using the LSAB method in paraffin embedded blocks. We believe that CA125 can be a very useful marker in the differential diagnosis of ES from other mesenchymal soft tissue tumors.

Acknowledgments

The authors thank Drs Osamu Dohi, Masami Hosaka, Toshihisa Osanai and Hiroshi Orui for their kind suggestions and Mr Katsuyoshi Shoji for his technical help.

+

For reprints and all correspondence: Masahito Hatori, M.D., Department of Orthopedic Surgery, Tohoku University School of Medicine, 1–1 Seiryomachi, Aobaku, Sendai 980-8574, Japan. E-mail: mhato@mail.tains.tohoku.ac.jp

Figure 1 (a) Microphotograph showing nodular growth pattern with central necrosis (×4). (b) Microphotograph showing compact sheets of round to oval and polygonal cells with eosinophilic cytoplasm growing in an epithelial pattern (×40). (c) Microphotograph showing rhabdoid cells with eccentric vesicular nuclei with prominent nucleoli and eosinophilic inclusion body (arrow).

Figure 1 (a) Microphotograph showing nodular growth pattern with central necrosis (×4). (b) Microphotograph showing compact sheets of round to oval and polygonal cells with eosinophilic cytoplasm growing in an epithelial pattern (×40). (c) Microphotograph showing rhabdoid cells with eccentric vesicular nuclei with prominent nucleoli and eosinophilic inclusion body (arrow).

Figure 2 (a) Case 6, the figure is quoted from reference 10, (b) Case7, the figure is quoted from reference 10 and (c) Case 10. Clinical course and serum CA125 level.

Figure 2 (a) Case 6, the figure is quoted from reference 10, (b) Case7, the figure is quoted from reference 10 and (c) Case 10. Clinical course and serum CA125 level.

Figure 3 (a) (Case 8), (b) (Case 10); Microphotographs showing strong CA125 immunoreactivity in the tumor cell membrane and cytoplasm.

Figure 3 (a) (Case 8), (b) (Case 10); Microphotographs showing strong CA125 immunoreactivity in the tumor cell membrane and cytoplasm.

Table 1.

Epithelioid sarcomas: summary of eleven cases

(ES) Case no. Age Sex Location Subtype Ulcer Follow-up Outcome 
18 Palm Distal 9 y 6 mo DOD 
32 Lower leg Distal 2 y 5 mo DOD 
17 Foot sole Distal – 6 y 4 mo NED 
13 Elbow Distal – 10 y NED 
48 Lower leg Distal 2 y NED 
26 Lower leg Distal 9 y 6 mo DOD 
29 Thigh Proximal – 5 y DOD 
51 Abdominal wall Distal – 2 mo DOD 
43 Index finger Distal 7 y 5 mo NED 
10 25 Foot sole Distal – 1 y 2 mo NED 
11 43 Thigh Distal – 3 y 9 mo AWD 
(ES) Case no. Age Sex Location Subtype Ulcer Follow-up Outcome 
18 Palm Distal 9 y 6 mo DOD 
32 Lower leg Distal 2 y 5 mo DOD 
17 Foot sole Distal – 6 y 4 mo NED 
13 Elbow Distal – 10 y NED 
48 Lower leg Distal 2 y NED 
26 Lower leg Distal 9 y 6 mo DOD 
29 Thigh Proximal – 5 y DOD 
51 Abdominal wall Distal – 2 mo DOD 
43 Index finger Distal 7 y 5 mo NED 
10 25 Foot sole Distal – 1 y 2 mo NED 
11 43 Thigh Distal – 3 y 9 mo AWD 

Average age: 31.4 years, average follow-up period: 5 years 2 months.

M: Male, F: Female, DOD: death of disease, NED: no evidence of disease, AWD: alive with disease.

Table 2.

Primary antibodies used and pretreatment

Marker Clone Antibody type Source Dilution Pretreatment 
CA125  M11 Fujirebio Diagnostics, Malvern, PA Prediluted No 
EMA E29 Dako Corporation, Carpinteria, CA 1:800 No 
Cytokeratin AE1/AE3 Dako Corporation, Carpinteria, CA 1:300 Trypsynization 
CEA CEM010 Mochida Pharmaceutical, Tokyo 1:400 Trypsynization 
Vimentin V9  Dako Corporation, Carpinteria, CA 1:1000 Autoclave 
CD34 NU-4A1  Nichirei Corporation, Tokyo 1:50 No 
Marker Clone Antibody type Source Dilution Pretreatment 
CA125  M11 Fujirebio Diagnostics, Malvern, PA Prediluted No 
EMA E29 Dako Corporation, Carpinteria, CA 1:800 No 
Cytokeratin AE1/AE3 Dako Corporation, Carpinteria, CA 1:300 Trypsynization 
CEA CEM010 Mochida Pharmaceutical, Tokyo 1:400 Trypsynization 
Vimentin V9  Dako Corporation, Carpinteria, CA 1:1000 Autoclave 
CD34 NU-4A1  Nichirei Corporation, Tokyo 1:50 No 

Antibody type M: monoclonal antibody.

Table 3.

Immunohistochemical findings in eleven cases of epithelioid sarcoma

Case no. CA125 EMA Cytokeratin CEA Vimentin CD34 
– 
– 
– ND 
– 
– – 
– ND 
– – 
10 – 
11 – ND 
Positivity(%) 90.9 100 100 18.2 100 75 
Case no. CA125 EMA Cytokeratin CEA Vimentin CD34 
– 
– 
– ND 
– 
– – 
– ND 
– – 
10 – 
11 – ND 
Positivity(%) 90.9 100 100 18.2 100 75 

EMA, Epithelial membrane antigen; CEA, Carcinoembryonic antigen; ND, not done.

Table 4.

Immunohistochemistry for CA125

Tumor and granuloma subtype Case CA125 positive (%) 
Synovial sarcoma 0/6 (0%) 
 Monophasic type: 2   
 Biphasic type: 4   
Clear cell sarcoma  0/6 (0%) 
Leiomyosarcoma 0/8 (0%) 
Rhabdomyosarcoma 0/6 (0%) 
 Embryonal type: 2   
 Alveolar type: 2   
 Pleomorphic type: 2   
Malignant fibrous histiocytoma 10 0/10 (0 %) 
 Striform pleomorphic type: 9   
 Myxoid type: 1   
Malignant peripheral nerve sheath tumor 0/5 (0%) 
Desmoid tumor 17 0/17 (0%) 
Liposarcoma 14 0/14 (0%) 
 Myxoid type: 5   
 Pleomorphic type: 9   
Squamous cell carcinoma (cutaneous) 0/6 (0%) 
Rheumatoid nodule 0/2 (0%) 
Foreign body granuloma 0/7 (0%) 
Tumor and granuloma subtype Case CA125 positive (%) 
Synovial sarcoma 0/6 (0%) 
 Monophasic type: 2   
 Biphasic type: 4   
Clear cell sarcoma  0/6 (0%) 
Leiomyosarcoma 0/8 (0%) 
Rhabdomyosarcoma 0/6 (0%) 
 Embryonal type: 2   
 Alveolar type: 2   
 Pleomorphic type: 2   
Malignant fibrous histiocytoma 10 0/10 (0 %) 
 Striform pleomorphic type: 9   
 Myxoid type: 1   
Malignant peripheral nerve sheath tumor 0/5 (0%) 
Desmoid tumor 17 0/17 (0%) 
Liposarcoma 14 0/14 (0%) 
 Myxoid type: 5   
 Pleomorphic type: 9   
Squamous cell carcinoma (cutaneous) 0/6 (0%) 
Rheumatoid nodule 0/2 (0%) 
Foreign body granuloma 0/7 (0%) 

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Author notes

1Department of Orthopedic Surgery, 2Department of Pathology and 7Department of Dermatology, Tohoku University School of Medicine, Sendai, Japan, 3University of Tennessee-Campbell Clinic, Memphis, Tennessee, USA, 4Department of Orthopedic Surgery, Niigata University School of Medicine, Niigata, 5Department of Orthopedic Surgery, Niigata Prefectural Cancer Center, Niigata and 6Department of Orthopedic Surgery, Miyagi Prefectural Cancer Center, Natori, Miyagi, Japan