BMC Cancer 2004, 4:78 http://www.biomedcentral.com/1471-2407/4/78 develop skin papillomas with 100% penetrance [22]. Moreover, a Sos1 mutant, lacking four functionally important proline-rich (SH3 binding) regions was reported to be responsible for gingival fibromatosis [23]. AKAP450 is a member of the A-kinase anchor proteins family. It is located in the centrosome [24], and acts as a microtubule nucleation site [25] and as a scaffold for pro- teins involved in mitotic process [26]. Other selected antigens with known or predicted intracel- lular location are alpha-6-fucosyltransferase (clone T6-7) and zinc finger protein 258 (ZNF258, clone T11-6). Alpha-fucosyltransferase catalyzes the transfer of GDP- fucose to oligosaccharide chains linked to proteins, lipids and sugars [27] and resides in the luminal compartment of trans-Golgi vesicles [28]. The predicted protein product ZNF258 contains zinc-binding motif repeats [29]. If ZNF258, together with structural homology, also shares biological properties with zinc finger proteins, thus recog- nizing and interacting with DNA, it should have a nuclear localization. The presence on our antigen list of proteins with predicted intracellular residence is in agreement with findings from the other groups [30,5] and is related to possible tissue necrosis and cell lysis associated with tumor growth. The human myc oncogene is transcribed from four alter- native promoters giving rise to mRNAs for Myc1, Myc2, MYCHEX1 and 5'ORF [31]. Clone T5-18 is the result of the translation of an alternative frame to Myc1, Myc2 and does not correspond to any known protein product of myc oncogene transcription. It is not clear whether selec- tion of this clone is an artifact of the experiment, or the result of an aberrant genome rearrangement in the tumor cells used for library construction. Among isolated antigens, there are 4 clones (T5-9, T9-21, T9-27 and T11-7) having between 55–91% sequence identity with that of a reverse transcriptase homolog (Figure 4). Viral antigens corresponding to human endog- enous retrovirus were previously isolated from renal can- cers and melanomas by SEREX [9]. It is interesting to note that all these clones, isolated with sera from breast cancer patients, derive from libraries constructed with cDNA from every different origin utilized: i.e. cell lines (T5), solid tumor (T9), testis (T11). We have no explanation for the transcription of reverse transcriptase gene in normal testis tissue. Eight proteins in the tumor antigen panel are unknown, or hypothetical proteins with unknown functions (T5-2, T5-15, T5-19, T6-2, T6-6, T7-1, T11-5, T11-9). The four underlined gene products from the list in parenthesis were analyzed for mRNA expression in tumors and normal breast tissue. The mRNA expression levels were analyzed by PCR from SMART-cDNA template in 7–10 breast can- cer specimens, one metastasized lymph node, normal breast, testis and peripheral lymphocytes from healthy donors. Two of these 4 unknown antigens and T11-3 were found to be frequently overexpressed in breast cancer. In particular clone T7-1, which was classified as encoding for an unknown protein since it has 100% identity only with KIAA1288 from EST database, was found to be overex- pressed in breast carcinomas. This finding, together with the good reactivity of T7-1 protein with sera from tumor patients, identifies this antigen among the most promis- ing targets for diagnosis of the disease. In contrast to the other antigens, which are overexpressed in breast cancer, mRNAs of T5-13, T5-15 and T11-5 appear to be underexpressed in 50–90% of breast cancer specimens, in comparison with normal breast tissue. How the immune system succeeds in responding to such anti- gens is still not clear. However, this finding is common to several SEREX-defined antigens, such as LU-12 [32], REN- 9, REN-10 [33] and BR-41 [15], representing a group of TAAs deleted or downregulated in tumors. Lu-12, REN-9, Ren-10 map within cancer tumor suppressor gene locus at chromosome 3p21.3, a region often deleted in small cell lung cancer as well as in renal cancer. Downregulated anti- gen BR-41 was identified as SNT-1, a membrane-associ- ated adaptor protein interacting with Sos1 [15]. In the present work we show that Sos1 (T5-13) is also downreg- ulated in 50% of breast cancer samples. The downregu- lated antigens T11-5, T5-13 (Sos1), and T5-15 do not react with sera from patients B82-B96 analyzed for tumor mRNA expression. Furthermore, tumor biopsies from patients with good response for these antigens were not available for expression analysis. Thus, at present, it is not possible to determine whether T11-5, T5-13 (Sos1), and T5-15 are normally expressed, or downregulated, in patients showing an immune response for the corre- sponding antigen. Sequence comparison of T11-5 and T5-15 clones with the EST database revealed identity with the hypothetical pro- teins MGC4170 and KIAA1735. We have derived the aa sequence of the corresponding ORFs and predicted the whole sequence architecture by computer analysis using the SMART program http://smart.embl-heidel berg.de[34,35]). MGC4170 encodes for two NL domains, while KIAA1335 encodes for a 389 aa protein bearing a DIX domain at the carboxy-terminus. The presence of such structural domains indicates that both of these still unknown proteins (corresponding to clones T11-5 and T5-15), which we found downregulated in several tumor specimens, may be involved in the signal transduction machinery. Page 8 of 10 (page number not for citation purposes)