Fibulin-1 (Fbln-1) is an immunogenic breast cancerrelated glycoprotein identified by serological analysis of cDNA expression library (SEREX) strategy. Here, we show that dendritic cells from two breast cancer patients elicited a CD4 þ -mediated T-cell response to Fbln-1 presentation. In both patients, an antibody response to Fbln-1 was also found. By contrast, a Fbln-1-seronegative patient and a weakly seropositive patient demonstrated no such T-cell response. Analysis of human breast cancers for Fbln-1 RNA and protein expression revealed the presence of Fbln-1C and -1D variants. Fbln-1 was detected in the cytoplasm and at the cell surface of different human breast carcinoma cell lines. Immunohistochemical analysis of 528 archival primary breast carcinomas showed the expression of Fbln-1 in 35% of the cases. When the immunohistochemical findings were compared against pathobiological information associated with each specimen, an inverse relationship between Fbln-1 and cathepsin D expression was observed (P ¼ 0.04). Furthermore, even though long-term survival was similar between Fbln-1-positive and -negative cases, the survival of Fbln-1-positive cases improved when a lymphoid infiltrate was present at the tumour site. Taken together, our findings of an Fbln-1-specific immunity and the improved survival associated with Fbln-1 expression in the presence of lymphoid infiltration point to a role of Fbln-1 in tumour immunosurveillance.

Our data demonstrate different leukocytic types in the infiltrate of breast tumors with different prognostic significance.

Screening of a breast cancer cDNA library from SKBR3 human breast cancer cells by SEREX (serological analysis of cDNA expression library) using a preselected serum from a breast cancer patient revealed 13 genes, two of which, INT-MI-1 and INT-MI-2, encode novel gene products, while the remaining 11 genes and their products are identical with or highly homologous to known GenBank entries. Immunoscreening of the 13 clones using 20 allogeneic sera from breast cancer patients and 20 samples from age- and gender-matched healthy donors showed that lactate dehydrogenase-A (LDH-A), lactate dehydrogenase-B (LDH-B), fibulin-1, and thyroid hormone-binding protein (THBP) were recognized principally by the breast cancer patient sera, indicating the immunogenicity of these molecules in vivo. The other antigens were similarly recognized by normal and patients sera, and thus not tumor-restricted immunologically. RT-PCR analysis revealed strong expression of fibulin-1 in tumor cell lines and surgical specimen whereas in the same experimental conditions, normal tissues scored negative. Also THBP expression was found in various tumors whereas in normal tissues, its expression is restricted to the testis and, at lower levels, in ovary, liver, and spleen. In contrast, LDH-A and LDH-B were ubiquitously expressed in normal and tumor tissues, with LDH-B levels considerably lower and heterogeneous in normal samples compared to those expressed in tumor cell lines. The differential expression of fibulin-1 between the normal tissues and breast carcinoma cell lines (5/6) and surgical specimens (5/6) suggests the possible involvement of the overexpression of this extracellular matrix-associated glycoprotein in the pathogenesis of this neoplasm.

Fibulin-1 (Fbln-1) is an extracellular matrix (ECM) and plasma glycoprotein. Considering the growing evidence indicating that Fbln-1 plays a role in cancer we sought to develop monospecific antibodies to better facilitate further studies of the function of Fbln-1 in breast cancer. Using a plasmid expression vector encoding full-length human Fbln-1D as an immunogen and CpG oligodeoxyribonucleotides as adjuvant a monoclonal antibody (MAb) against Fbln-1 was produced. This MAb, designated MEM-2 was of IgM isotype and reacted with bacterially expressed Fbln-1. Furthermore, MEM-2 reacted with Fbln-1 expressed in the ECM released by cultured human breast carcinoma SKBR-3 cells in ELISA, and also with Fbln-1 present in SKBR-3 cell extract in immunoprecipitation and Western blotting. MEM-2 also reacted with Fbln-1 in human breast carcinoma specimens. These findings illustrate the utility of genetic immunization as a means of generating monoclonal antibodies to tumor-related ECM proteins. MEM-2 represents a useful new tool for the study of Fbln-1 in breast cancer.

Plasmid DNA vectors encoding the full-length (VR1012/HER-2-FL) or only the extracellular and transmembrane domains (VR1012/HER-2-ECD-TM) of human (h) HER-2/neu proto-oncogene were used to vaccinate HER-2/neu transgenic mice (N202) engineered to overexpress the rat (r) neu proto-oncogene product (r-p185neu). Both the full-length and the deleted vaccines were significantly (P = 0.0001 and P = 0.06, respectively) more active than the empty vector (VR1012/EV) in preventing and delaying HER-2/neu-driven mammary carcinogenesis. A low-level intratumoral infiltrate of dendritic cells, macrophages, CD8 T cells and polymorphonuclear granulocytes in association with low-level cytokine production was observed, which was not detected in tumors from control mice. Morphologic analyses showed that vaccination with VR1012/HER-2-FL or ECD-TM also efficiently hampered the development of terminal ductal lobular units (TDLU). Analyses of sera from vaccinated mice revealed high titers of antihuman HER-2/neu antibodies, which correlated with the delayed time of tumor onset (P = 0.002). These antibodies did not cross-react with r-p185neu. Nontransgenic mice treated with the vaccines produced autoreactive antibodies targeting mouse (m)-p185neu and showed impaired function of the lactating mammary gland and accelerated involution of the gland after weaning. Together, these data indicate that xenogeneic DNA immunization breaks tolerance against the endogenous m-p185neu, impairing the development of mammary TDLU in which m-p185neu expression is concentrated. The reduction in the number of TDLU decreases the number of glandular structures available for r-p185neu-dependent mammary carcinogenesis, resulting in a significant inhibition of mammary carcinoma development.