Breast Disease

Human tumors are immunogenic and tumor-associated proteins that generate immunity in cancer patients have been defined. Many of these proteins are involved in the malignant transformation and play a role in either initiating or maintaining the malignant phenotype. Furthermore, due to technical advances in basic immunology over the last decade we have a better understanding of the immune effector cell phenotypes that are potentially involved in tumor eradication and have developed methods to quantitate and characterize these immune effectors. Breast cancer is an intriguing model tumor to target with active immunization. Dozens of breast cancer antigens have been defined [1]. Although many patients with breast cancer can be rendered free of disease with standard therapy such as surgery, radiation, and chemotherapy, some patients will have their disease recur. However, relapse may not occur for many months to years after definitive treatment giving an extended period of micrometastatic disease that may be amenable to immune eradication or modulation. Peptide based vaccines are one of the most commonly studied vaccine strategies targeting breast cancer.

Rapid progress in defining the molecular underpinnings of the antitumor immune response has laid the foundation for tumor immunotherapy, leading to multiple early clinical studies testing vaccines for the treatment of breast cancer. Together, these small trials have provided early evidence for the induction of clinically relevant vaccine-induced tumor-specific immunity in some patients. However, they have not convincingly demonstrated a significant impact on disease progression or overall survival in women with advanced breast cancer. These disappointing results are likely due to the negative impact of standard cancer treatments on vaccine-activated antitumor immunity, the limited potency of current tumor vaccine formulations against large burdens of established tumor, and the presence of pre-existing tumor-specific immune tolerance. It is increasingly clear that standard and novel breast cancer treatments can influence the antitumor immune response. Also, signaling pathways that regulate immune responses have emerged as novel targets for immune modulation. The use of preclinical models to elucidate the pharmacodynamic interactions of standard breast cancer treatment modalities and novel, targeted immunotherapeutics with breast cancer vaccines will facilitate the development of combinatorial immunotherapeutic strategies. Combined modality immunotherapies should maximize the potency of the antitumor immune response, thereby improving the outcome of breast cancer therapy.

Her-2/neu (HER2) is a protooncogene that is known to be amplified in a proportion of many different types of carcinomas. Overexpression of HER2 is generally associated with poor prognosis and resistance to chemo and radiotherapy. There exist many approaches that may be potentially utilized for therapy of HER2 overexpressing tumors. Understanding the biochemical and physiological function of HER2 in oncogenesis as well as its role in facilitating immune escape of tumor cells is critical for the improvement of existing strategies as well as developing new therapeutic candidates targeting HER2.

Development of the mammary gland is controlled by hormones and many other growth factors. Hyperplasia and neoplasia are thus a likely consequence of alterations in their number and type, or the number and function of their receptors.

p185neu, a member of the epidermal growth factor family, is coded by the ErbB-2 oncogene. It is expressed in the normal human breast, but overexpressed and associated with a poor prognosis in 15–30% of breast tumours. Employment of ErbB-2 sequences as vaccinal antigens to induce an immune rejection of such tumours is being investigated in several transgenic animal models. One of the most aggressive models of mammary p185neu-dependent carcinogenesis is that of BALB-neuT mice, which are transgenic for the rat transforming Her-2/neu oncogene (a homologue of the human ErbB-2 oncogene). The progression of their early neoplastic lesions can be prevented with both cellular and DNA vaccines coadjuvated by antiangiogenic and immunostimulatory molecules. This suggests that induction of a specific immune reaction against a tumor target antigen may provide a way of preventing the onset of tumours in subjects with a high genetic risk of developing cancer.

The heterogeneous nature of breast cancer and the correlation of myeloid cell infiltration with accelerated tumor progression were recognized early in breast cancer immunology research using murine model systems induced by the mouse mammary tumor virus, chemical carcinogens or hormones. Distinct cell lines established from a single mammary tumor attest to the challenges of controlling tumors with such complexity. Here, we test the feasibility of controlling breast cancer by active vaccination targeting a shared tumor-associated antigen, human ErB-2 (Her-2). Her-2 DNA vaccines were constructed and Her-2 transgenic mice were established. DNA vaccination overcomes Her-2 tolerance to induce anti-tumor immunity which is amplified by the removal of regulatory T cells, but is accompanied by a significant risk of autoimmunity. Her-2 vaccines combined with appropriate immune modulation to trigger in vivo priming to other tumor-associated antigens will be the key to improved breast cancer control.

To study immunology in breast tumors, we have utilized a mammary gland adenocarcinoma model in which mice develop spontaneous tumors of the mammary gland which are initiated at puberty and express a human tumor antigen, MUC1. MUC1 (CD227) is over-expressed in 90% of human breast cancers and its glycosylation status and pattern of expression in cancer cells is altered. Humoral and cellular responses to MUC1 have been reported in breast cancer patients and therefore, MUC1 is being evaluated as a target for immune intervention. This mouse model of spontaneous breast cancer allows the evaluation of anti-MUC1 immune responses at all stages of the disease. In this report, we review the model as it pertains to a) the development of the tumor, b) MUC1 expression, and the native immune responses against MUC1 as tumors progress, and c) the immune suppressive microenvironment within the developing tumor. Finally, we report our latest findings describing the therapeutic efficacy of adoptively transferred MUC1-specific cytotoxic T lymphocytes (MUC1-CTL) in these mice and discuss ways to increase their effectiveness by agonistic monoclonal antibody against CD137 T cell costimulatory molecule.

Continual attempts to stimulate the immune system against malignancies have led to the development of various strategies based on active immunotherapy treatments. Dendritic cells are the most potent antigen presenting cells with the capacity to stimulate naïve T cells and induce primary and secondary immune responses. Due to the pivotal role that DC play in eliciting and maintaining functional anti-tumor T cell responses, DC have been exploited as vaccines in an attempt to actively immunize patients. Initial solid tumor clinical trials involving DC-based immunization have shown progress in terms of eliciting T-cell reactivity and mediating tumor regression. These early promising data have led to multiple research endeavors to also employ DC immunotherapy for the treatment of poorly immunogenic malignancies such as breast cancer. Various strategies to load DC with tumor associated antigens in murine models of breast cancer as well as the state of human clinical trials are reviewed.

Continued progress in breast cancer immunotherapy, in particular breast cancer vaccines, depends on the identification of target molecules aberrantly expressed on breast cancer cells. Many different approaches to antigen discovery, including the recent developments in genomics and proteomics, have favored identification of protein tumor antigens. While some of these molecules provide important peptide epitopes recognized by T cells and antibodies, they represent only a small minority of potential targets. Considering that the majority of the cell proteins and therefore tumor cell proteins are glycosylated, tumor glycopeptides represent more important tumor-specific targets. Protein glycosylation is known to be dysregulated in cancer cells, leading to the accumulation of tumor-specific glycoproteins actively involved in tumor progression and metastasis. In addition to understanding the glycobiology of tumor cells and identifying tumor-specific glycoprotein antigens, better understanding is required of how the innate and the adaptive immune systems handle processing, presentation and recognition of glycoprotein antigens. We discuss here some of the new therapeutic strategies for exploiting abnormal glycosylation pathways in tumors and using defined carbohydrate and/or glycoprotein tumor antigens in active specific immunotherapy of breast cancer.

Two novel oral DNA-based vaccines provide immune protection against breast cancer in mouse model systems. These vaccines are delivered by attenuated Salmonella typhimurium to secondary lymphoid organs and are directed against novel targets such as transcription factor Fos-related antigen 1 (Fra-1) and endoglin (CD105). Both vaccines elicit suppression of angiogenesis in the breast tumor vasculature and break peripheral tolerance by eliciting potent cell-mediated protective immunity against these tumor self-antigens resulting in effective suppression of breast tumor growth and metastasis.

In spite of the demonstrated coexistence of tumor-associated rejection antigens expressed by breast tumors along with T cells that are capable of recognize them, breast cancers arises in immunocompetent hosts, outmaneuver immune recognition and ultimately progress to widely disseminated disease. In recent years, several explanations have been proposed to account for the inability of the immune system to recognize and reject antigenic breast tumors. Among them, the immune tolerance mechanisms have gained particular attention. Here, we discuss the increasing evidence pointing to tolerance towards tumor antigens as an important explanation for the failure of the immune system to reject breast tumors. A better understanding of the cellular and molecular mechanisms involved in breast tumor-induced antigen-specific T-cell tolerance may lead to approaches to effectively harness the immune system against this malignancy.

Metastatic disease is the principle cause of death for most patients with breast cancer. Conventional therapies including radiation therapy and chemotherapy are largely uneffective against metastatic disease. It is now generally appreciated that the immune system can destroy tumor cells, and numerous novel immunotherapies are currently under development. Many of these immunotherapies are dependent on activation of the host's immune system so the success of a cancer vaccine will depend on the immune status of the patient. Tolerance to tumor antigens, tumor-induced immune suppression, and the presence of immunomodulatory genes that block the development of tumor-specific immunity can potentially interfere with the therapeutic efficacy of immune-based therapies. Studies from the authors' laboratory demonstrate that although mice with bulky primary mammary tumors are immunosuppressed for T cell and antibody-mediated immunity, surgical removal of the primary tumor reverses the suppression, even when disseminated metastatic disease is present. The post-surgical reversal is associated with a large decrease in myeloid suppressor cells. In addition to tumor-induced suppression, two genes, the Stat6 and CD1 genes, are also associated with inhibiting tumor-specific immunity, since mice deficient for these genes have dramatically enhanced resistance to metastatic mammary carcinoma. Therefore, optimal delivery of immunotherapy should be coordinated with methodology that decreases immune suppression and eliminates or blocks inhibitory factors.

Breast cancers, like other malignancies, commonly express a repertoire of both chemokines and chemokine receptors. While some are more often expressed in certain histological types, a few general concepts are emerging that provide clues to the pathobiological role of these ligand receptor pairs. The receptor CXCR4 is often expressed in solid tumors and evidence is growing that this receptor plays a role in the growth and lymph node metastasis of breast and other cancers responding to ligand expressed at metastatic sites. Likewise, CCR7 is expressed in breast and other cancers and, in some cases, is associated with more aggressive disease. Like chemokine receptors, some ligands also modulate tumor behavior. CCL5 expression is associated with more aggressive breast cancers. CXC chemokines containing the ELR motif are expressed endogenously by some cancers, act as autocrine growth factors and support tumor angiogenesis. ELR-negative CXC chemokines inhibit tumor growth and metastasis when expressed at high levels by attracting immune effector cells and inhibiting angiogenesis. The roles of other chemokine receptors and ligands are under active investigation.

It has been reported that in epithelial cancer invasion, most matrix metalloproteinases (MMPs) are made by stromal cells of the host, and not the neoplasm itself. Findings from several laboratories indicate that immune cell-derived MMPs may be advantageous to developing tumors by promoting angiogenesis, neoplastic cell proliferation, and progression to malignancy. We have found a dramatic up-regulation of MMP-9 secretion by splenic and tumor-infiltrating T lymphocytes from D1-DMBA-3 mammary tumor-bearing mice compared to T cells from normal animals. Furthermore, tumor-derived vascular endothelial growth factor induced the up-regulation of MMP-9 in T cells, corroborating the suggestion that tumor cells may "conscript" inflammatory cells to make contributions to the tumor phenotype. Some investigators propose that the resulting degradation of the extracellular matrix by lymphocyte-derived proteases might be used by tumor cells to establish a blood supply and to metastasize. The outcome of MMP activity in the tumor microenvironment may be dependent on a variety of factors including tumor phenotype; the presence of other proteases and cytokines; and the time, level, and site of MMP production. In light of recent findings that MMPs are also capable of generating anti-angiogenic molecules, further investigation will bear out whether inflammatory cell-derived MMPs are friend or foe to developing tumors.