Immunohistochemical and Molecular Analysis of Human Melanomas for Expression of the Human Cancer-Testis Antigens NY-ESO-1 and LAGE-1 Hilary A. Vaughan, 1 Suzanne Svobodova, 2 Duncan MacGregor, 2,3 Sue Sturrock, 3 Achim A. Jungbluth, 4 Judy Browning, 1 Ian D. Davis, 1 Philip Parente, 1 Yao-Tseng Chen, 4 Elisabeth Stockert, 4 Fiona St. Clair, 1 Lloyd J. Old, 4 and Jonathan Cebon 1 1 Ludwig Institute for Cancer Research, Melbourne, Australia; 2 Division of Laboratory Medicine, Austin Health, Heidelberg, Victoria, Australia; 3 Department of Pathology, University of Melbourne, Austin Health, Heidelberg, Victoria, Australia; and 4 Ludwig Institute for Cancer Research, New York, New York ABSTRACT Purpose: NY-ESO-1 and LAGE-1 are homologous cancer- testis antigens, which are expressed in many different can- cers. It is essential to type tumors accurately to assess patient suitability for clinical trials which target these. This study evaluates typing strategies used to distinguish these two homologous but distinct antigens and to characterize and quantitate expression of each in clinical samples. Experimental Design: We typed 120 malignant melano- mas for the expression of NY-ESO-1 and LAGE-1 with immunohistochemistry, reverse transcription-PCR (RT- PCR), and quantitative real-time (qRT-PCR), which was also used to explore the relationship between NY-ESO-1 and LAGE expression. Results: The two monoclonal antibodies ES121 and E978 had very similar immunohistochemistry reactivities. Both were specific for NY-ESO-1 because neither bound to homologous LAGE-1 peptides despite 84% overall amino acid homology. Of 120 melanomas tested by immunohisto- chemistry, NY-ESO-1 was expressed in >50% of cells in 23 melanomas (19%), between 11 and 50% cells in 15 (12.5%), <11% cells in 16 (13.5%), and negative in 66 (55%). Al- though specific for both antigens, the PCR methods did not provide this information about microheterogeneity. Poly- morphisms in the LAGE-1 gene resulted in false negative LAGE-1 typing by qRT-PCR by inhibiting binding of oli- gonucleotide primers, thereby showing the exquisite speci- ficity of qRT-PCR as a typing method. Conclusions: For NY-ESO-1 typing, immunohisto- chemistry compared favorably with the RT-PCR, with the added advantage of being able to characterize heterogeneity of antigen expression. Because neither mAb bound LAGE and because there was no coordinate expression LAGE and NY-ESO-1, separate typing for each is required. INTRODUCTION To optimize vaccination approaches for cancer, it is im- portant to understand those factors that contribute to the success or failure of a vaccine. This includes understanding antigen distribution and the strengths and weaknesses of the tools avail- able for measuring these antigens. NY-ESO-1 is a cancer-testis antigen expressed in 20 to 70% of common cancers, including malignant melanoma (34%; ref. 1), bladder (32 to 80%; refs. 2, 3), lung (21%; ref. 3), and rarer cancers such as synovial cell sarcoma (80%; ref. 4). Initial immunohistochemical studies with the monoclonal antibody (mAb) ES121 have shown that tumors may express NY-ESO-1 heterogeneously. Expression can range from relatively few cells in the tumor to homogeneous expression in all cancer cells, when typed with this antibody (5). More recently, a highly homologous antigen called LAGE-1 has been previously described (6, 7), and because of homologies at both the mRNA and protein level, there is the potential for cross detection with molecular methods and anti- bodies against NY-ESO-1. NY-ESO-1 was identified by a SEREX (serological anal- ysis of tumor antigens by recombinant expression cloning) screen of an esophageal carcinoma (1). NY-ESO-1 shows a pattern of expression typical of cancer-testis antigens, which are present in various histologic types of tumors but not in any normal adult tissues, except spermatogonia (5). LAGE-1 was defined by representational difference analysis and has signifi- cant DNA and amino acid homology with NY-ESO-1. Both genes map to chromosome Xq28 and the fully spliced mRNAs share 84% homology (6). Their biological function is unknown. There are two known splice variants of the LAGE-1 antigen, “LAGE long” and “LAGE short” (6), and both the NY-ESO-1 and LAGE-1 genes are able to use an alternative open reading frame (8, 9). Both humoral and cellular responses to NY-ESO-1 have been described in patients with NY-ESO-1–positive tu- mors (10, 11), and a number of HLA class I-restricted epitopes have been identified (11, 12). More recently, a number of MHC class II epitopes have been described (13–20). NY-ESO-1 is therefore an attractive target for immunotherapeutic protocols and is the basis of several cancer vaccine clinical trials reported (19, 20, 21, 22) or currently in progress. In addition, HLA-A2– Received 4/25/04; revised 8/13/04; accepted 8/16/04. Grant support: This project was wholly supported by the Ludwig Institute for Cancer Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Jonathan Cebon, Ludwig Oncology Unit, Austin Health, Studley Road, Heidelberg VIC 3084, Australia. Fax: 61-3-9457- 6698; E-mail: jonathan.Cebon@ludwig.edu.au. ©2004 American Association for Cancer Research. 8396 Vol. 10, 8396 – 8404, December 15, 2004 Clinical Cancer Research Downloaded from http://aacrjournals.org/clincancerres/article-pdf/10/24/8396/1924130/zdf02404008396.pdf by guest on 14 November 2023