Cancer/testis (CT) antigens are expressed in a range of malignant neoplasms, but with the exception of testis, not in normal tissues. 1,2 CT antigens elicit cellular 3 as well as humoral 4 -6 immune responses. Ten genes or gene families have been identified that code for CT antigens. 7 The first members of this class of tumor antigens, MAGE, BAGE and GAGE, were identified as targets for CD8 T cells in a patient with melanoma. 8 -10 Other CT antigens have been recognized by the cloning technique called SEREX, involving serological screening of cDNA expression libraries of tumors with patient sera as the source of antibody 11 and by representational-difference analysis. 12,13 NY-ESO-1 is a classic CT antigen discovered during a SEREX analysis of an esophageal cancer. 14 NY-ESO-1 is a 22 kD hydrophobic protein 14 coded for by a gene in the Xq28 region. 15 In normal tissues, expression of NY-ESO-1 mRNA as detected by RT-PCR is predominantly restricted to the testis, whereas in cancer NY-ESO-1 is found in a variable proportion of a wide range of different malignancies, including melanoma, cancers of the breast, ovary and lung. 14 The strong immunogenicity of NY-ESO-1 is another distinguishing feature of the antigen; 40 -50% of patients with advanced NY-ESO-1 mRNA positive tumors have humoral 6,16,17 or CD8 T cell 18 response to NY-ESO-1.Current knowledge about CT antigen expression in normal and malignant tissues is mainly based on RT-PCR analysis. Mouse monoclonal antibodies (MAbs) to MAGE gene products have been generated, 19 -23 and these have been used to analyze the pattern of MAGE expression in tissues. 24,25 Mouse MAbs recognizing NY-ESO-1 antigen have been generated against NY-ESO-1 recombinant protein. 6 In the present study, these NY-ESO-1 MAbs have been used to establish the expression pattern of NY-ESO-1 in normal and malignant tissues.
MATERIAL AND METHODS
AntibodiesTwo NY-ESO-1 monoclonal antibodies were described previously. 6 Clone E978 (IgG1) was generated against a 23 kD NY-ESO-1 recombinant protein and clone ES121 (IgG1) was generated against a shorter, 14 kD NY-ESO-1 recombinant protein.Initial analysis with testis and selected tumor tissues indicated a similar immunohistochemical staining pattern with these two MAbs. Because ES121 showed both the best and most consistent results in immunohistochemical staining, it was chosen as the reagent for our study.
TissuesFrozen and formalin-fixed paraffin-embedded tissues were provided by the Departments of Pathology of Memorial Sloan-Kettering Cancer Center and New York Hospital Cornell University Medical School. Frozen and 10% neutral buffered formalin-fixed paraffin-embedded tissues were used. The frozen specimens were embedded in O.C.T. (Tissue Tek, Torrance, CA) and snap-frozen in pre-cooled (Ϫ70°C) isopentane. Five m cuts were performed from frozen or paraffin blocks and applied to histology slides for immunohistochemistry (Superfrost Plus, Fisher Scientific, Pittsburgh, PA). Frozen sections were fixed in cold acetone for 10 min and air-dried befor...
