The human homolog of the rat neu oncogene, HER2 (also termed c-erbB2) has been demonstrated in amplified form in human breast tumors with poor prognosis. Although amplification of the gene correlates with expression of a 185-kDa transmembrane glycoprotein, no extensive information is available regarding the extent of tissue and tumor specificity of this gene product. We have addressed this issue by immunohistochemically evaluating the expression of p185 HER2 in normal tissue and various tumors using monoclonal antibodies (MAbs) to distinct epitopes of its extracellular domain. No detectable levels of p185 HER2 were found in fetal tissues analyzed, with the exception of renal tubules in 2 out of 3 specimens tested and in intestinal epithelium. In adult tissues, detectable levels of this glycoprotein were found in a restricted number of cell types, the expression being heterogeneous among individuals and cell histotypes. Among the neoplasms assayed p185 HER2 was expressed in 46% of primary breast cancers, in 28% of ovarian tumors and in 30% of colon rectum malignancies. No male breast adenocarcinomas were p185-positive. A large number of other tumors tested revealed only a low incidence of expression of the p185. In metastatic breast tumors p185 HER2 was demonstrated homogeneously among multiple autologous lesions and almost invariably (80%) the expression of p185 in the primary lesion correlated with that of the deriving metastases. Our findings indicate that the expression of the p185 HER2 represents a tumor marker of clinical relevance in breast cancer. Whether this holds true for other malignancies remains to be explored.

Introduction Estrogen receptor-alpha (ER-α) and progesterone receptor (PgR) are consolidated predictors of response to hormonal therapy (HT). In contrast, little information regarding the role of estrogen receptor-beta (ER-β) in various breast cancer risk groups treated with different therapeutic regimens is available. In particular, there are no data concerning ER-β distribution within the novel molecular breast cancer subtypes luminal A (LA) and luminal B (LB), HER2 (HS), and triplenegative (TN).

Purpose: hMena (ENAH), a cytoskeleton regulatory protein involved in the regulation of cell motility and adhesion, is overexpressed in breast cancer. The aim of this study was to define at what stage of breast carcinogenesis hMena is overexpressed and to correlate hMena overexpression with established prognostic factors in breast cancer, focusing on human epidermal growth factor receptor-2 (HER-2). Experimental Design: hMena expression was assessed immunohistochemically in a prospective cohort of cases (n = 360) encompassing a highly representative spectrum of benign breast diseases associated with different risk of transformation, in situ, invasive, and metastatic tumors. Correlations with conventional pathologic and prognostic variables, such as proliferation index, hormonal receptor status, and HER-2 overexpression, were also evaluated. In vitro experiments were done to study the effect of neuregulin-1and Herceptin treatments on hMena expression. Results: hMena protein is undetectable in normal breast and is weakly expressed in a small percentage of low-risk benign diseases (9%), but displays a progressive and significant increase of positivity in benign lesions at higher risk of transformation (slightly increased risk 43%; moderate increased risk 67%), in in situ (72%), invasive (93%), and metastatic breast cancer (91%).A significant direct correlation with tumor size (P = 0.04), proliferation index (P < 0.0001), and HER-2 overexpression (P < 0.0001) and an inverse relationship with estrogen (P = 0.036) and progesterone receptors (P = 0.001) are found in invasive carcinomas. In vitro experiments show that neuregulin-1up-regulates, whereas Herceptin down-regulates, hMena expression. Conclusions: Our data provide new insights into the relevance of actin-binding proteins in human breast carcinogenesis and indicate hMena overexpression as a surrogate indicator in breast disease management. Management of breast cancer, and other malignancies, islikely to benefit from the identification of early markers of transformation. In this context, major efforts are ongoing in the areas of genomic and proteomic profiling aimed at identifying genetic or biochemical markers related to proliferative lesions heralding the development of breast cancer. In view of the increasing evidence that the host immune response contributes to the editing of the tumor phenotype (1), we have recently isolated, by serologic analysis of cDNA expression libraries (SEREX), hMena (ENAH) protein, the human orthologue of murine Mena, which is overexpressed in over 70% of primary breast cancers (2). Mena belongs to the Ena/VASP protein family, which, by controlling the geometry of the actin filament network (3, 4), represents key regulator molecules of cell movement and shape in a large variety of cell types and organisms (5). It has been suggested that Ena/VASP proteins are members of the adherens junction structures and are required for the actin dynamics necessary to seal membranes into epithelial sheets (6), a process frequently deregula...

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...

Analysis of surgical biopsies with monoclonal antibodies (mAbs) to framework determinants of major histocompatibility complex class I antigens has shown that malignant transformation is frequently associated with a marked loss of these cell surface molecules. The present study sought to determine whether more selective losses of major histocompatibility complex class I expression occur. Multiple specimens from 13 different types of primary and metastatic tumors were tested utilizing mAb BB7.2, which recognizes a polymorphic HLA-A2 epitope. In each case, expression of HLA-A,B,C molecules was determined by testing with mAb W6/32 directed to a framework HLA class I determinant. We have found that