NY-ESO-1/LAGE-1 coexpression with MAGE-A cancer/testis antigens: A tissue microarray study Martin Bolli 1 * , Elke Schultz-Thater 1 , Paul Zajac 1 , Ulrich Guller 1 , Chantal Feder 1 , Francesca Sanguedolce 2 , Vincenza Carafa 2 , Luigi Terracciano 2 , Tvrtko Hudolin 3 , Giulio C. Spagnoli 1 and Luigi Tornillo 2 1 Institut Chirurgische Forschung und Spitalmanagement, Department Forschung, University of Basel, Switzerland 2 Institut fu¨r Pathologie, University of Basel, Switzerland 3 Department of Urology, KBC, Zagreb, Croatia The characterization of the expression pattern of different families of cancer/testis (C/T) antigens in different tumors, at the protein level, might be of relevance in the development of multiantigen vaccine preparations for active specific immunotherapy. We have used tissue microarray (TMA) technology to explore in large num- bers of tumor specimens the expression of NY-ESO-1/LAGE-1 C/T antigens and its correlation with MAGE-A expression by using D8.38 and 57B monoclonal antibodies (MAb). The epitopes recog- nized by these reagents in C/T antigens were identified by molecu- lar mapping by using a bacterial expression system. Out of 2,052 samples, 119 (5.8%) scored positive upon staining with D8.38 NY-ESO-1/LAGE-1-specific MAb. Expression in >10% of cases was detectable in melanoma and basalioma (31.6 and 18.2%, respec- tively), large cell carcinomas and adenocarcinomas of the lung (17.8 and 10.5%, respectively), stomach adenocarcinomas of the intestinal type (13.2%), pT2-4 bladder TCC (18.2%), nonsemi- nomatous carcinomas of the testis (10.4%) and liposarcomas (15.4%). Simultaneous expression of NY-ESO-1/LAGE-1 and MAGE-A C/T antigens was then addressed in a TMA where 101/845 and 73/845 samples (12 and 8.6%, respectively) showed evidence of MAGE-A or NY-ESO-1/LAGE-1 specific staining, respectively. In 35/845 specimens (4.1%) concomitant expression of MAGE-A and NY-ESO-1/LAGE-1 was observed ( p = 0.0002). Discrepancies in the expression of NY-ESO-1/LAGE-1 and MAGE-A were conspicuously detectable in squamous cell carci- nomas of the skin (MAGE-A positive but NY-ESO-1/LAGE-1 negative) and in liposarcomas (NY-ESO-1/LAGE-1 positive, but MAGE-A negative). Taken together, these data suggest novel areas of application of C/T antigens targeted active specific immunotherapy possibly based on multiantigen vaccine prepara- tions. ' 2005 Wiley-Liss, Inc. Key words: cancer/testis antigens; NY-ESO-1/Lage-1; MAGE-A; epitope mapping Cancer/testis (C/T) antigens are a group of proteins character- ized by a peculiar expression profile, mostly limited to male ger- minal cells and cancer cells of diverse histological origin. 1 Among C/T antigens, NY-ESO-1 appears to play an important role since it is probably the most immunogenic member of the group and it is also capable to induce humoral responses in addi- tion to specific cytotoxic T lymphocytes (CTL) and CD4þ T cells. 2,3 Remarkably, most recently a highly homologous deter- minant, LAGE-1, has been reported to be able to induce specific regulatory CD25þ/CD4þ in melanoma tumor infiltrating lympho- cytes (TIL). 4 Immunohistochemical detection of NY-ESO-1 has been made possible by the generation of monoclonal antibodies (MAb) capa- ble of recognizing the target protein in paraffin embedded tissues, as reported by us and others. 5,6 However, the fine specificity of these reagents at the molecular level was not clarified, the number of tissues tested was relatively limited and the issue of the coex- pression together with other C/T antigen families was not addressed. Here, we mapped epitopes recognized by reagents used to identify C/T antigens in clinical samples and explored NY-ESO-1/LAGE-1 expression by taking advantage of tissue microarrays (TMA), a technique allowing the fast assessment of the expression of determinants recognized by specific sero- logical reagents on large numbers of tissues. Furthermore, we comparatively evaluated the expression of NY-ESO-1/LAGE-1 as associated with MAGE-A C/T antigen expression. Material and methods Monoclonal antibodies Monoclonal antibodies (MAb) 57B and D8.38 were generated by using as immunogens recombinant MAGE-A3 and NY-ESO-1 proteins, respectively. 5,7 Epitope mapping The FliTrx random peptide library (Invitrogen, Basel, Switzer- land) is composed of 1.77  10 8 primary clones of E. coli with a dodecamer peptide sequence inserted within the Thioredoxin (TrxA) active site loop. The genes encoding TrxA-peptide fusion proteins are cloned into the nonessential domain of the major bac- terial flagellar protein (FliC) gene under the control of a PL pro- moter from bacteriophage g. When induced, the peptide sequence is expressed on the surface of the E. coli flagella with the N- and C-terminal ends constrained by a disulfide bond. This library was screened against the 2 different MAbs under investigation. Two milliliters of the FliTrx library were added to IMC me- dium containing 100 mg/ml ampicillin and grown by shaking at 258C for 15 hr. The peptide library was then induced by adding approximately 1  10 10 cells to IMC medium containing amp- icillin and 100 mg/ml tryptophan and shaken at 258C for further 6 hr. The target MAbs were diluted to 50 mg/ml in sterile water and adsorbed onto 60 mm tissue culture plates (Becton Dickinson, Franklin Lakes, NJ) by gentle agitation at 50 rpm for 1 hr. Plates were then rinsed with sterile water followed by gentle agitation for 1 hr with a blocking solution containing 100 mg/ml ampicillin, 1% nonfat dry milk, 150 mM NaCl and 1% a-methyl mannoside. After decanting the blocking solution, 10 ml of the induced E. coli culture in medium containing 1% nonfat drymilk, 150 mM NaCl and 1% a-methyl mannoside were added to culture plates and gently agitated at 50 rpm for 1 min followed by 1 hr of incu- bation with no agitation. The medium was then decanted and the plate gently washed 5  with IMC medium containing ampicillin and 1% a-methyl mannoside. Bacteria bound to the plates were eluted into 10 ml fresh culture IMC medium containing ampicillin by placing the plate on a vortex to shear the flagella and release the cells to the medium. E. coli were then cultured overnight and the complete procedure was repeated for 5 rounds of panning. Grant sponsor: Swiss Bridge foundation; Grant sponsor: Swiss National Science Foundation; Grant number: 3200B0-104060. *Correspondence to: Institut Chirurgische Forschung und Spitalma- nagement, ZLF, 20 Hebelstrasse, CH-4031, Basel, Switzerland. Fax: þ41-61-2655-3990. Received 22 July 2004; Accepted after revision 3 December 2004 DOI 10.1002/ijc.20953 Published online 4 March 2005 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 115, 960–966 (2005) ' 2005 Wiley-Liss, Inc. Publication of the International Union Against Cancer