TOWARD A MORE COMPLETE RECOGNITION OF IMMUNOREACTIVE ANTIGENS IN SQUAMOUS CELL LUNG CARCINOMA Isabel DIESINGER 1 , Christine BAUER 1 , Nicole BRASS 1 , Hans-Joachim SCHAEFERS 2 , Nicole COMTESSE 1 , Gerhard SYBRECHT 3 and Eckart MEESE 1 * 1 Department of Human Genetics, Medical School, University of Saarland, Homburg/Saar, Germany 2 Department of Thorax, Heart and Vascular Surgery, Medical School, University of Saarland, Homburg/Saar, Germany 3 Department of Pneumology, Medical School, University of Saarland, Homburg/Saar, Germany There is very limited knowledge about the antibody re- sponse against tumor-expressed antigens in lung cancer. To arrive at a more complete picture of lung cancer antigens, we generated 2 cDNA libraries from squamous cell lung carci- noma and isolated 15 immunogenic antigens using autolo- gous sera. Among the antigens most frequently identified were the lymphoid blast crisis oncogene (LBC), an unknown hypothetical protein and the p53-binding protein (TP53 BP), which have already been associated with tumor develop- ment. Of the immunogenic antigens, 6 map to chromosomes that are frequently altered in squamous cell lung carcinoma. SEREX database analysis showed that 7 of the identified immunogenic antigens have been associated with the hu- moral immune response in other human tumors. Screening with heterologous sera of patients with lung carcinoma iden- tified 4 antigens, including human protein kinase C and TP53 BP, which have also been found by autologous screening. Only 1 of the 15 identified antigens reacted with any of the 36 control sera, which were taken from individuals without known disease. Sera from adenocarcinoma and large cell carcinoma of the lung were not reactive for the antigens. In summary, using 2 newly established cDNA libraries, we iso- lated 15 novel antigens, which were subsequently evaluated for the frequency of their corresponding antibodies in autol- ogous, normal and heterologous sera; their chromosomal localization; and their correlation with survival after surgery. © 2002 Wiley-Liss, Inc. Key words: lung cancer; SEREX; autologous sera; heterologous sera There is mounting evidence that many tumors express antigens that elicit an immune response in cancer patients. Target antigens of tumor-related autoantibodies can be oncoproteins, tumor-sup- pressor proteins or proliferation-associated antigens. 1 The most prominent example of an autoimmune response against a tumor- associated protein is the antibody response against the tumor- suppressor TP53. Examples of TP53-directed antibodies in cancer patients include antibodies in 50% of bladder cancer patients, in 12% of B-cell lymphoma patients, in 23% of ovarian cancer patients, in 32% of colon cancer patients and in 42% of breast cancer patients. 2–6 In addition, there is an increasing number of other tumor or proliferation-associated gene products that cause an antibody response, including, e.g., c-myb with an antibody re- sponse in 43% of breast cancer patients and cyclin B1 in 15% of hepatocellular carcinoma patients. 7,8 There are limited data on the autoantibody response in human lung cancer patients, specifically patients suffering from squamous cell lung carcinoma, which constitutes 40 –50% of lung cancer patients. Lung cancer is the most frequent lethal tumor in Western societies, with a 5-year survival rate of approximately 13%. 9 The most frequent autoantibodies in lung cancer patients are directed against TP53, with an antibody response detected in 14.3% of lung cancer patients and 21% of small cell lung cancer patients, and against L-myc and c-myc, in 10% and 13.2% of lung cancer patients, respectively. 10 –13 Previously, we reported an antibody response against the eukaryotic translation initiation factor 4G (eIF4G) in 15% of squamous cell lung carcinoma sera. 14 Still, a large number of antigens remain to be identified in this tumor type. To further investigate the autoantibody repertoire in lung cancer, we generated 2 cDNA expression libraries derived from squamous cell lung carcinomas. The libraries were screened by serologic analysis of recombinant cDNA expression libraries (SEREX), which allows detection of antibodies directed against tumor-ex- pressed antigens. SEREX has successfully detected novel antigens in many human cancers, as shown for gastric cancer by Obata et al., 15 for renal cell carcinoma by Scanlan et al., 16 for small cell lung cancer by Gure et al. 17 and for several other human tumors by Tureci et al. 18 Eichmuller et al. 19 identified 15 novel antigens in cutaneous T-cell lymphoma. Our study was aimed at increasing the knowledge of tumor-expressed immunogenic antigens in squa- mous cell lung carcinoma. The novel antigens may serve as prog- nostic parameters in this tumor, which is generally associated with very poor prognosis. MATERIAL AND METHODS Tumor tissue and sera Tumor tissue and sera were obtained from the Institute of Pneumology (Homburg, Germany) and the Institute of Thorax, Heart and Vascular Surgery (Homburg, Germany). Tissue samples were frozen in liquid nitrogen immediately after surgery and stored at –70°C. Two cDNA expression libraries were generated from 2 squamous cell lung carcinoma specimens, termed PEC6 and L102, which were staged as T2N2M0 and T1N1GII, respectively. His- topathology showed moderate differentiation of tumors L102 and PEC6. RNA isolation Frozen tissue was homogenized, proteins were extracted by phenol-chloroform and RNA was precipitated twice with isopro- panol and finally resuspended in diethylpyrocarbonate-treated H 2 O. Integrity and concentration of RNA were evaluated by form- aldehyde gel electrophoresis. cDNA expression library Total RNA was applied to oligo(dT) cellulose push columns, and poly(A) mRNA was eluted according to the Poly(A) Quick Kit (Stratagene, La Jolla, CA). cDNA synthesis was performed using the ZAP Express cDNA synthesis kit (Stratagene) as described previously. 20 To generate the libraries, we inserted reverse-tran- scribed poly(A) mRNA into the ZAP Express expression vector in sense orientation with respect to the lacZ promoter. Grant sponsor: Deutsche Krebshilfe; Grant number: 10-1526-ME3. *Correspondence to: Department of Human Genetics, Medical School, University of Saarland, Building 60, 66421 Homburg/Saar, Germany. Fax: +6841-162-6186. E-mail: hgemee@med-rz.uni-sb.de Received 12 October 2001; Revised 24 June 2002; Accepted 2 August 2002 DOI 10.1002/ijc.10714 Published online 7 October 2002 in Wiley InterScience (www. interscience.wiley.com). Int. J. Cancer: 102, 372–378 (2002) © 2002 Wiley-Liss, Inc. Publication of the International Union Against Cancer