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Mesothelin is a differentiation antigen that was first described as the antigenic target of the monoclonal antibody K1. Using this antibody, it was demonstrated that mesothelin is strongly expressed in normal mesothelial cells, mesotheliomas, nonmucinous ovarian carcinomas, and some other malignancies. Immunostaining with the K1 antibody was suggested to be useful in the diagnosis of mesothelioma in the early 1990s. This, however, could not be further explored until recently because of the lack of commercially available anti-mesothelin antibodies. In a recent investigation by this author, all epithelioid mesotheliomas and about 40% of the lung adenocarcinomas reacted with the 5B2 anti-mesothelin antibody, which has only recently become commercially available. It was concluded that immunostaining with this antibody has limited value in discriminating between these conditions. The aim of the current study was to further investigate the potential application of the 5B2 antibody in tumor diagnosis. Mesothelin expression was evaluated in formalin-fixed, paraffin-embedded samples of normal tissues and in 471 tumors of various origins. The carcinomas that most frequently exhibited strong mesothelin reactivity were nonmucinous carcinomas of the ovary (14 of 14 serous, 3 of 3 endometrioid, 6 of 8 clear cell, and 4 of 4 transitional cell carcinoma), and adenocarcinomas of the pancreas (12 of 14), the ampulla of Vater (3 of 3), endometrium (7 of 11), lung (14 of 34), and liver (7 of 19 cholangiocarcinomas). The carcinomas that did not express mesothelin included renal cell carcinomas, hepatomas, carcinomas of the thyroid, adrenal cortical carcinomas, prostatic adenocarcinomas, and carcinoid tumors. All germ cell tumors, with the exception of teratomas, were consistently negative for mesothelin. Because of the strong mesothelin expression in nonmucinous carcinomas of the ovary, but not in a variety of tumors with which these lesions may be confused (eg, clear cell carcinoma of the ovary versus endodermal sinus tumor or renal cell carcinoma, clear cell type; transitional cell carcinoma of the ovary versus TCC of the urinary tract), immunostaining for this marker could be useful in establishing the differential diagnosis. The strong mesothelin expression in the large majority of pancreatic ductal adenocarcinomas (12 of 14), but not in normal pancreas, confirms that this marker may have some diagnostic utility in discriminating between neoplastic and nonneoplastic pancreatic ductal epithelium. The mesothelin expression in about one-third of the cholangiocarcinomas, but not in hepatomas, suggests that this marker may have some utility in distinguishing between these two malignancies when they are poorly differentiated. In the group of small round blue cell tumors, only desmoplastic small round cell tumors exhibited mesothelin positivity (7 of 12). Of the soft tissue tumors, only the epithelial component of biphasic synovial sarcomas (9 of 9) expressed mesothelin. These findings indicate that, in some instances, mesothelin immunostaining can assist in the diagnosis of these tumors. Finally, the strong mesothelin reactivity seen in the adenomatoid tumors (3 of 3) provides further support for a mesothelial derivation for this lesion.