hMena (ENAH), an actin regulatory protein involved in the control of cell motility and adhesion, is modulated during human breast carcinogenesis. In fact, whereas undetectable in normal mammary epithelium, hMena becomes overexpressed in high-risk benign lesions and primary and metastatic tumors. In vivo, hMena overexpression correlates with the HER-2 +

Screening of a cDNA expression library from a primary breast tumor with the autologous patient serum led to the isolation of 6 cDNA clones corresponding to 3 different genes, including a novel gene that maps to chromosome 1 and encodes the human homologue of mouse Mena (hMena, cDNA clone RMNY-BR-55), a protein of the Ena/VASP family involved in the regulation of cell motility and adhesion. A cancer-restricted antibody response against hMena was demonstrated, since 18/93 cancer patient sera, the majority (10/ 52) from breast cancer, showed anti-hMena-specific IgG, while no antibodies were present in healthy donors. When hMena protein expression was analyzed by Western blot and immunohistochemistry, the antigen was overexpressed in the majority of breast cancer cell lines and in 75% of primary breast tumor lesions evaluated. Furthermore, when HLA-A2-restricted peptides from the hMena sequence were used to stimulate CD8 ؉ T cells, an hMena-specific response was found in 9 out of 12 HLA-A2 ؉ breast cancer patients. In 4 patients, this cell-mediated immune response was concomitant with antibody response to hMena. Furthermore, an hMena-specific T-cell line was established from an HLA-A2 Key words: tumor immunity; major histocompatibility complex; cytotoxic T lymphocyteThe identification of the repertoire of molecules recognized by the immune system of cancer patients at different stages of the disease is of major biologic and clinical relevance, 1 as the immune system continuously shapes the immunogenic phenotype of the developing tumor 2 by a complex process recently referred to as cancer immunoediting. 3 Genetic changes continuously occurring during tumor development and progression lead to a number of mutant and/or aberrantly expressed proteins, which can potentially function as tumor-associated antigens and elicit antitumor immune responses. 4 However, the dynamics and the consequences of these events have not yet been fully elucidated. The serologic analysis of cDNA expression libraries (SEREX) of human tumors has identified a broad spectrum of tumor proteins capable of eliciting a humoral immune response in tumor patients. 5 The majority of these SEREX-defined antigens do not show any detectable mutations and/or structural modifications. Although some tumor antigens show restricted expression in normal tissues, i.e., cancer/testis (CT) and melanoma differentiation antigens, to date, the results indicate that the overexpression of normal proteins in the tumor may be of major significance in eliciting a tumor-specific humoral immunity. 6 The isolation of tumor antigens recognized by high-titer IgG implies CD4 ϩ and CD8 ϩ T-cell recognition, as extensively demonstrated for the C/T antigen NY-ESO-1 that induces a concomitant humoral and cellular immune response in a high proportion of NY-ESO-1 ϩ cancer-bearing patients. [7][8][9][10] To identify and characterize new antigens in breast cancer, we applied the SEREX approach analyzing a primary breast tumor and the autologous serum of a long surviving patient. A novel...