BMC Cancer 2004, 4:78 http://www.biomedcentral.com/1471-2407/4/78 Background A recent development in tumor immunology is based on the idea that the immune system can distinguish between normal and tumor tissues. Various studies suggest that both the cellular and humoral components of the immune system are able to recognize tumors (see review of Lake et al.) [1]. The presence of natural antibodies against cancer cells in peripheral blood of tumor patients probably plays a protective role against tumor develop- ment. The latest advances in molecular techniques further support the existence of natural antibodies against cancer antigens. The SEREX approach, based on the serological screening of cDNA expression libraries generated from tumor tissues of various origin, led to the molecular defi- nition of immunogenic tumor proteins (tumor-associated antigens, TAAs) based on their reactivity with autologous patient sera [2]. This type of screening of a cDNA expres- sion library is quite a laborious procedure requiring the preparation of a large number of membrane filters blotted with bacteriophage plaques, which are then screened with sera from cancer patients, usually available in limited quantity. In contrast to SEREX, phage display strategy is based on the selection and enrichment of antigens dis- played on the phage surface. A physical link between a dis- played fusion protein and the DNA encoding for it makes this phage target selectable through affinity purification. Phage display technology has been successfully applied to the screening of cDNA libraries from different tumors using the antibody repertoire of cancer patients [3-6]. In these experiments different phage display systems were used. Some of the authors used the C-terminus of a fila- mentous phage minor protein pVI for expression of cDNA libraries from breast cancer cell lines T47D and MCF-7 [3] and from colorectal cancer cell line HT-29 [5]. However, the filamentous phage display system imposes some bio- logical bias for the expression and display of fusion pro- teins, since a filamentous phage-based library displays only those recombinant proteins able to pass through the inner bacterial membrane during filamentous phage assembly. To overcome this potential problem the lytic bacteriophages T7 [4] and λ [6] were used. By using these latter systems, the phage capsid is assembled in the cyto- plasm of bacteria and mature phage particles are released by cell lysis. For example, Hansen and co-workers in their studies screened a commercially available (Novagen) human breast cancer cDNA library cloned in T7 vector [4], identifying positive clones. Usually cDNA libraries are generated as C-terminal fusions. When such a library is panned on a serum, the presence of a complex antibody repertoire gives to out-of- frame or antisense-derived cross-reactive short peptide sequences a good chance of being enriched. In our previ- ous work [6] we designed a new-concept lambda vector for the display of cDNA-encoded protein fragments as fusion to the N-terminus of bacteriophage gpD, allowing us to overcome this obstacle. In this vector, phage clones display a given protein fragment on the phage surface only when the insert's correct reading frame matches that of gpD. The size of the cloned DNA fragments in our libraries was adjusted to an average of 200–300 base pairs, which is of a size reasonably sufficient to potentially encode for a protein domain. The vast majority of out-of- frame sequences of the above-mentioned length most probably contains at least one in-frame stop codon. Thus, these inserts are not expressed as D fusion, are conse- quently not displayed on the phage surface and cannot be selected. In such cases, phage capsid contains only wt gpD encoded by lambda genome D gene. The N-terminal dis- play system greatly reduces the selection of artifactual peptides, in comparison with a C-terminal fusion library displayed on lambda ([7] and our unpublished data). By employing the SEREX approach numerous tumor anti- gens from different human neoplasms were identified [8,9]. Analysis of TAA expression in tumor samples and normal tissue led to the identification of a group, called cancer/testis antigens. Members belonging to this family are aberrantly expressed in human cancers and only in normal testis, but not in other normal tissue. For this rea- son, in addition to tumor samples and tumor cell lines, testicular cDNA libraries are also a convenient source of antigens which can be identified by screening with sera derived from tumor patients [10,11]. In the present work we report the construction of lambda- displayed cDNA libraries from breast cancer cell lines MCF-7, MDA-MB-468, from human breast carcinomas and from human testis, generated according to an improved protocol. These libraries were screened by using sera from breast cancer patients. The list of 21 identified antigens contains eight proteins with still unknown func- tions. Three of the genes (T7-1, T11-3 and T11-9) were found to be overexpressed in tumors as compared to nor- mal breast. Recognition by human sera of five of the selected antigens (T6-2, T6-7, T7-1, T9-21 and T9-27) was associated with cancer diagnosis. Methods Tissue and serum samples Specimens of breast carcinoma and autologous sera from breast cancer patients (B81-B96) were obtained from M. G. Vannini Hospital, Rome. A panel of human sera from breast cancer patients B1-B20, B36-B80 was provided by the Division of Medical Oncology, Federico II University of Naples. All the human biological samples were obtained through informed consent. Page 2 of 10 (page number not for citation purposes)