BMC Cancer 2004, 4:78 http://www.biomedcentral.com/1471-2407/4/78 In spite of the fact that several promising antigens were identified from cDNA library constructed from testis mRNA, none of the antigens derived from testis or other libraries could be classified as specific testis/cancer (CT) antigen, because of their low expression in testis (T11-9, T7-1) or expression in other tissues as well. In this work we analyzed the frequency of the immune response to the 21 identified antigens by using a panel of sera from tumor patients and healthy donors. In general, we observed a low frequency of serum reactivity with the antigens, which was expected and is similar to that of the vast majority of SEREX-identified clones [36]. A signifi- cant number of sera from tumor patients, in comparison with healthy individuals, efficiently recognized five of the identified antigens (T6-2, T6-7, T7-1, T9-21, T9-27). Clones T9-21 and T9-27, isolated from breast carcinoma library, respectively show 70% homology (55% identity) and 62% homology (68% identity) to reverse tran- scriptase homolog (PO8547). T7-1 is a protein having an unknown function, which was found to be overexpressed in breast carcinoma. Taken together, these results lead us to believe that analy- sis of a complex panel of serologically-defined TAAs, with very large panels of sera from patients classified according to clinical parameters, i.e., age of patient, stage, extent and outcome of disease, etc. could lead to a much clearer understanding of the role, specificity and significance of the immune response versus disease in cancer patients. Conclusions We demonstrated that a lambda display-based approach permits the efficient identification of tumor antigens, potential immunological targets in breast cancer. The list of 21 antigens identified in this work contains eight pro- teins of still unknown function. Three of the genes (T7-1, T11-3, T11-9) were found to be overexpressed in tumors as compared to normal breast. Five of the selected anti- gens (T6-2, T6-7, T7-1, T9-21, T9-27) were recognized spe- cifically by breast cancer patient sera. List of abbreviations aa, amino acids; Ag, antigen; EST, expressed sequence tags; ds cDNA, double-stranded cDNA; SEREX, serological identification of antigens by recombinant expression cloning; PAGE, polyacrylamide gel electrophoresis; PEG, polyethylene glycol; pfu, plaque-forming units; Sos1, son of sevenless homolog 1; TAA, tumor-associated antigen. Competing interests EP, PV, AP, GM, EB, FF and OM are salaried by an organ- ization holding two patents relating to the content of the manuscript. Authors' contributions EP and PV contributed equally to the work. EP, PV and AP carried out cDNA library experiments, recombinant pro- tein production, immunoassays and database search. GM and EB performed selection experiments. SB, MLD and MC contributed to immunological analysis of tumor anti- gens. ADPC and AL performed clinical studies. EC coordi- nated all medical aspects of the work. OM planned and performed molecular biology experiments, and teamed with FF in design and coordination of the entire project. All authors read and approved the final manuscript. Additional material Additional File 1 Table 2. List of identified TAAs. The File is given in Microsoft Word for- mat. The table contains information about selected antigens. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2407-4-78-S1.doc] Acknowledgements We wish to thank Dr. Nicola Gargano and Dr. Manlio Di Cristina for very insightful critical discussion during this work, and Mr. Luca Bruno for excel- lent technical assistance. We also thank Ms. M. Deutsch for the linguistic revision of the text. References 1. Lake DF, Huynh WC, Hersh EM: Natural and induced human antibody response to cancer. Cancer investigation 2000, 18(5):480-489. 2. Sahin U, Tureci O, Schmitt H, Cochlovich B, Johannes T, Schmits R, Stenner F, Luo G, Schobert I, Pfreundschuh M: Human neoplasms elicit multiple specific immune responses in the autologous host. Proc Natl Acad Sci USA 1995, 92:11810-11813. 3. Sioud M, Hansen MH: Profiling the immune response in patients with breast cancer by phage-displayed cDNA libraries. Eur J Immunol 2001, 31:716-725. 4. Hansen MH, Ostenstad B, Sioud M: Identification of immuno- genic antigens using a phage-displayed cDNA library from an invasive ductal breast carcinoma tumor. Int J Oncol 2001, 19:1303-1309. 5. Somers VA, Brandwijk RJ, Joosten B, Moerkerk PT, Arends J-W, Men- heere P, Pieterse WO, Claessen A, Scheper RJ, Hoogenboom HR, Hufton SE: A panel of candidate tumor antigens in colorectal cancer revealed by the serological selection of a phage dis- played cDNA expression library. J Immunol 2002, 169(5):2772-2780. 6. Minenkova O, Pucci A, Pavoni E, De Tomassi A, Fortugno P, Gargano N, Cianfriglia M, Barca S, De Placido S, Martignetti A, Felici F, Cortese R, Monaci P: Identification of tumor-associated antigens by screening phage-displayed human cDNA libraries with sera from tumor patients. Int J Cancer 2003, 106(4):534-544. 7. Santi E, Capone S, Mennuni C, Lahm A, Tramontano A, Luzzago A, Nicosia A: Bacteriophage lambda display of complex cDNA libraries: a new approach to functional genomics. J Mol Biol 2000, 296:497-508. 8. Pfreundschuh M: Exploitation of the B cell repertoire for iden- tification of human tumor antigens. Cancer Chemother Pharmacol 2000, 46:S3-S7. 9. Chen Y-T: Cancer vaccine: identification of human tumor antigens by SEREX. Cancer J Sci Am 2000, 3:S208-217. 10. Tureci O, Sahin U, Zwick C, Koslowski M, Seitz G, Pfreundschuh M: Identification of a meiosis-specific protein as a member of Page 9 of 10 (page number not for citation purposes)