The major problem in the immunohistochemical study of mesotheliomas is most antibodies used recognise epitopes present with greater frequency in adenocarcinoma than in malignant mesothelioma. The diagnosis of mesothelioma therefore is supported by a series of negative results (1).
Adenocarcinoma often produce epithelial mucins. These can be neutral or acidic and can be demonstrated using mucicarmine or PAS following diasase treatment (to remove glycogen). Mesotheliomas may contain glycogen. Mesotheliomas on the other hand may produce acidic mucins of the connective tissue type, namely hyaluronic acid. This can be demonstrated using alcian blue (pH 2.5) with and without hyaluronidase pretreatment .
Wick etal (2) found that only 63% of adenocarcinomas with pleural involvement had epithelial mucin whilst only 41% of the mesotheliomas had hyaluronidase sensitive mucins.
Robb (3) described one case of mesothelioma that had a marked amount of hyaluronic acid and mucicarmine positive cytoplasmic vacuoles. These vacuoles failed to stain with mucicarmine following hyaluronidase pretreatment. Robb (3) and Triol & Chandler (4) suggest that cases containing heavy deposits of hyaluronic acid are most likely to give false positive staining reactions with mucicarmine.
Several epithelial cell markers have been used to aid in the differential diagnosis of adenocarcinoma and mesothelioma. CEA, B72.3 and Leu-M1 have sufficiently high specificity and sensitivity for adenocarcinoma so that, when used in a panel, they are highly effective in distinguishing most cases of malignant mesothelioma from adenocarcinoma (1). The use of a combination of antibodies significantly decreases the chance of a false negative result (1).
Wick etal (3) found Leu-M1 in all 52 adenocarcinoma of the lung, CEA was present in 96%, B72.3 labelled 84% and Lewis blood group antigen was present in 67%. Brown etal (5) found CEA to be the best single marker for adenocarcinoma, being present in 97% of adenocarcinoma. Since a few cases of mesothelioma were also positive, they advocated the use of a panel of CEA, B72.3, and Leu-M1 which further reduced the risk of false negative results.
Robb (3) found that aberrent CEA or Leu-M1 staining may occur in mesotheliomas rich in hyaluronic acid. He recommends the use of hyaluronidase predigestion to remove this unexpected staining. Stirling & Henderson (6) found that false positive CEA staining was focal and weak, and found to be diffuse within the cytoplasm or linear along the plasma membrane.
Some antibodies to membrane glycoproteins, such as HMFG-2 and epithelial membrane antigen (EMA), frequently stain both mesothelioma and adenocarcinoma. However the pattern of reactivity is usually quite different. In mesothelioma staining is almost always membranous, while in adenocarcinoma both components generally stain equally well. These antibodies may demonstrate a thick, brush border-like membrane pattern over much of the free cell surface, in contrast to the absence of such a pattern in adenocarcinoma. This staining probably correlates with the abundant, long microvilli present on the surface of mesothelial and mesothelioma cells, a long recognised useful feature in the evaluation of these cells by electron microscopy (1).
Sheibani etal (7) found 87% of adenocarcinoma positive for Ber-EP4 whilst only one of 115 mesotheliomas were positive. Staining in mesotheliomas is generally limited to focal staining (1). Lewis antigen was found to be diffusely positive in all 18 lung carcinoma tested but stained only rare cells in 23% of mesothelioma (1).
All three major morphologic subtypes of mesothelioma are immunoreactive with anti cytokeratin antibodies. This is particularly useful in distinguishing sarcomatoid mesothelioma from soft tissue sarcoma involving the pleura or from primary malignant fibrous tumours of the pleura (10).
Cytologically, distinguishing carcinoma cells from reactive mesothelial cells is important. Fibronectin is a glycoprotein found in connective tissues. Lee etal (8) found that positive fibronectin staining was highly specific for mesothelial cells, with all 28 carcinoma negative. However fibronectin will not distinguish between neoplastic and reactive mesothelials. Lee etal (8) found that reactive mesothelials did not express EMA.
Attanoos etal (9) examined the expression of three putative mesothelioma-binding antibodies, thrombomodulin, OV-632, and HBME-1 in 42 mesotheliomas and 32 adenocarcinomas. They found thrombomodin in 22 mesotheliomas where as it was only present in 2 adenocarcinomas. OV-632 was present in 27 mesotheliomas and 20 adenocarcinomas. HBME-1 was found in 26 mesotheliomas but was also seen in 23 adenocarcinomas.
Thrombomodulin is a 75kD transmembrane glycoprotein expressed by mesothelial cells, endothelial cells and placental syncytiotrophoblast. HBME-1 appears to react with some undetermined epitope in mesothelial microvilli.
Attanoos etal (9) recommend that thrombomodulin be used in a panel for the evaluation of mesothelioma, whilst OV-632 and HBME-1 are considered unsuitable.