growth hormone or cortisol. Carcinoid tumors secrete serotonin. Some tumors of the pancreas secrete insulin. Serial measurements can also monitor treatment for these tumors.
profile many breast cancer tumors has revealed that certain tumors demonstrate a high level of immunoregulatory gene activation (Rody et al., 2009; Ascierto et al., 2012). It has been demonstrated that patients with tumors, which elicit a stronger T helper 1 (Th1) cytotoxic T-lymphocyte (CTL) response, have a better prognosis than do those whose tumors skew toward a T helper 2 (Th2) response or trigger a larger influx of tumor-associated macrophages via colony-stimulating factor 1 (CSF-1) secretion (DeNardo et al., 2011).
Studies have suggested that T cells found in high density within the tumor parenchyma are also correlated with a survival benefit. The type of adaptive immune response implicated in improved cancer outcomes is a type 1 response. That is, adaptive immunity associated with T cells that secrete pro-inflammatory cytokines, such as IFN-γ, which can not only support a proliferative antigen specific T cell response but also enhance "cross priming" by activating antigen presenting cells local to the tumor site.
Also, cells with a greater influx of tumor-infiltrating lymphocytes tend to respond better to neoadjuvant chemotherapy compared with less immunogenic tumors (Denkert et al., 2010).
These observations suggest that intrinsic properties exist within certain breast tumors that provoke a beneficial CTL response, which synergizes with chemotherapy. Other tumors can manipulate inflammatory pathways to promote metastatic tumor spread. The goal would be to manipulate the cytokine milieu within the tumor microenvironment to trigger a beneficial immune response during neoadjuvant therapy, thereby enhancing pathological complete response rates and reducing metastatic tumor spread.
Immunogenicity in cancer patients and restricted tissue expression are characteristics used to define antigenic targets for cancer vaccines. Gene discovery based on CD8+ and CD4+ T cell epitope cloning (van der Bruggen et al., 1991; Wang et al., 1999), and serum antibody expression cloning (SEREX) (Sahin et al., 1995), have led to the identification of tissue-restricted tumor antigens that are recognized by the immune systems of cancer patients and have added to the list of target antigens applicable to breast cancer. One of the first target molecules to be examined in the context of a breast cancer vaccine is carcino-embrionic antigen (CEA), a differentiation antigen of the gut, expressed exclusively in normal colonic epithelium and approximately 50% of breast cancers (Hodge, 1996). Objective responses in patients with metastatic disease, including breast cancer, following immunization with dendritic cells pulsed with an human leukocyte antigen (HLA)-A2 restricted peptide of CEA has been observed. This was quickly followed by the discovery of a new differentiation antigen of the breast, NY-BR-1, identified by SEREX analysis and was
found to be expressed exclusively in normal testis and breast, as well as in 80% of breast cancers (Jäger et al., 1998). The NY-BR-1 can induce cellular immune responses and high titered serum IgG antibodies has been demonstrated in breast cancer
Target antigens must first be presented as processed peptides bound to MHC class I and class II molecules. These MHC-peptide complexes on the surface of antigen presenting cells (APCs) are then recognized by antigen-specific T lymphocytes, and together with additional co-stimulation, leads to the proliferation of antigen-specific CD8+ and CD4+ T cells capable of lytic and immunostimulatory functions. Many antigen-specific cancer vaccines have been prepared as MHC class I binding peptides and administered intradermally, along with adjuvant and cytokines, in order to enhance uptake by APCs and augment the immune response. Encouraging results have been recorded with cancer/testis antigens (NY-ESO1, MAGE-3) and mutated and amplified antigens (HER-2/neu).
Strategies in the delivery and presentation of target antigens include continuous antigen administration by lymph node perfusion (Scalan et al., 2001), direct targeting of antigen presenting cells (APCs) with recombinant Listeria monocytogenes that has been engineered to express tumor-associated antigens, and dendritic cell (DC) vaccines (Mule, 2000; Palucka and Banchreau, 2013). Dendritic cells are highly proficient APCs, expressing elevated levels of MHC class I and class II molecules, as well as important co-stimulatory molecules, and they also produce a variety of immunostimulatory cytokines (Mule, 2000). Dendritic cells can be generated in vitro from precursors present in peripheral blood and subsequently used to present tumor antigens in-vivo, when pulsed with antigenic peptide or transfected with DNA constructs encoding appropriate antigens. Dentritic cells can be stimulated to activate a cytotoxic response towards an antigen. They can be harvested from a patient and then either pulsed with an antigen or transfected with a viral vector. Upon transfusion back into the patient these activated cells present tumour antigen to effector lymphocytes (CD4+
T cells, CD8+ T cells, and B cells). This initiates a cytotoxic response to occur against cells expressing tumour antigens (against which the adaptive response has now been primed).
The cancer vaccine Sipuleucel-T is one example of this approach (Overes et al., 2009).
Adoptive immunotherapy relies on in-vitro immunization, whereby tumor infiltrating lymphocytes are harvested from surgical specimens and propagated in-vitro in the presence of interleukin-2 (IL-2) and appropriate antigen. The resultant CTL clones are then reintroduced into the autologous patient (Kawakami et al., 1995; Takayama et. al; 2000;
Yang 2003; Egawa 2004). This is a form of passive immunization. There are multiple ways of producing and obtaining tumour targeted T-cells. T-cells specific to a tumor antigen can either be removed from a tumor sample (tumour-infilterating lymphocytes) or T-cells can be removed from the blood and genetically engineered to be tumor specific. Subsequent activation and expansion of these cells is performed outside the body (ex-vivo) and then they are transfused into the recipient. Although research has made major advances in this form of therapy, there is no known approved adoptive T-cell therapy as yet (June 2007;
Retifo et al., 2012; Wright 2012).
An attempt using autologous tumor-infiltrating lymphocytes was found effective in treatment for patients with metastatic melanoma, (Wojtowicz-Praga, 1996). This was achieved by taking T cells that are found with the tumor of the patient, which are trained to attack the cancerous cells. These T cells are referred to as tumour-infiltrating lymphocytes (TIL) are then encouraged to multiply in-vitro using high concentrations of IL-2, anti-CD3 and allo-reactive feeder cells. These T cells are then transferred back into the patient along with exogenous administration of IL-2 to further boost their anti-cancer activity (Gardner et. al; 2012). Genetically engineered T cells are created by infecting patient's cells with a virus that contain a copy of a T cell receptor (TCR) gene that is specialized to recognize tumour antigens. The virus is not able to reproduce within the cell however integrates into the human genome. This is beneficial as new TCR gene remains stable in the T-cell. A patient's own T cells are exposed to these viruses and then expanded non-specifically or stimulated using the genetically engineered TCR. The cells are then transferred back into the patient and ready to have an immune response against the tumour. Morgan et al., (2006) demonstrated that the adoptive cell transfer of lymphocytes transduced with retrovirus encoding TCRs that recognize a cancer antigen are able to mediate anti-tumour responses in patients with metastatic melanomas and this can be adapted in breast cancer.
The tumor specific T-cells used for treatment will be specific for a particular antigen present within the tumor, or for the stroma or vasculature, which the tumor may be dependent on. Examples of T-cell targets are tissue differentiation antigens, mutant protein antigens, oncogenic viral antigens, cancer-testis antigens and vascular or stromal specific antigens. Tissue differentiation antigens are those that are specific to a certain type of tissue. T-cells specific to these antigens will target normal cells that contain these antigens as well as cancer cells (e.g. carcino-embryonic antigen; CEA). Mutant protein antigens are
likely to be much more specific to cancer cells because normal cells shouldn't contain these proteins. Normal cells will display the normal protein antigen on their MHC molecules, whereas cancer cells will display the mutant version. T-cells can differentiate between these two, selectively targeting the cancer cell. Some viral proteins are implicated in forming cancer (oncogenesis), and therefore T-cells that are specific to viral antigens can be used to attack infected cells (which will include cancer cells).
Other immunization strategies include the use of DNA vaccines, either in the form of viruses (adenovirus, vaccinia virus) or naked DNA, to deliver genes encoding tumor antigens (Restifo, 1996). Such vectors contain the coding sequence for a particular target antigen and may also contain sequences encoding targeting motifs for MHC class I and class II pathways, immunostimulatory cytokines, and co-stimulatory molecules. One major concern with using viral vectors is the presence of neutralizing antiviral antibodies in the recipient, resulting from a prior immunization (e.g. smallpox vaccine), which would negate vaccination.
Breast cancer is one of the major cancer types for which new immune-based cancer treatments are being developed and is a good model for vaccination (Disis and Park, 2009).
First, the tumor is immunogenic and many tumor antigens have been identified that are expressed in breast cancer (Ludvig et al., 2005; Chaudhuri et al., 2009; Mishra and Verma, 2010). Secondly, breast cancer is amenable to standard therapies such as surgery and chemotherapy. For this reason the patients can be treated to a very minimal residual disease state where the amount of tumor burden is potentially readily eradicated by antigen competent T cells. Finally, breast cancer is slow growing and often the doubling rate can be expressed in terms of years rather than weeks (Disis and Park, 2009). The slow growth of the disease allows for the ability to expand T cells over time with repeated booster immunizations potentially achieving therapeutically effective levels before the disease begins to become bulky or metastasize.
When diagnosed early, breast cancer treatment generally involves surgery, which, depending on the stage and molecular characteristics of the cancer, may be followed by chemotherapy, radiation therapy, or targeted therapy (including hormone therapy, such as tamoxifen or the aromatase inhibitors; letrozole, anastrozole, or exemestane).
Cancers over-expressing HER2 (HER2 3+ or FISH positive) may be treated with targeted therapies such as trastuzumab (Herceptin) and, in the case of advanced cancer, lapatinib (Tykerb), pertuzumab (Perjeta), or TDM-1 (Kadcyla) {Wright, 2012}. Of these the newest treatment options are pertuzumab and TDM-1. Pertuzumab (Perjeta) which was approved by the FDA in 2012 for first-line treatment of HER2+ metastatic breast cancer in combination with trastuzumab (Herceptin) and the chemotherapy docetaxel (Taxotere).
Wright, (2012) also demonstrated in the laboratory that combining trastuzumab with an antibody directed against a different part of HER2 is more effective than trastuzumab alone against HER2+ breast cancer.
Trastuzumab is a monoclonal IgG1 humanized antibody specific for the epidermal growth factor receptor 2 protein (HER2). It received FDA-approval in 1998, and is clinically used for the treatment of breast cancer. The use of trastuzumab is restricted to patients whose tumours over-express HER-2, as assessed by immunohistochemistry (IHC) and either chromogenic or fluorescent in situ hybridization (FISH), as well as numerous PCR-based methodologies.
HER-2 is a member of the epidermal growth factor receptor (EGFR) family of transmembrane tyrosine kinase, and is normally involved in regulation of cell proliferation and differentiation (Jones et al., 1999). Amplification or overexpression of HER-2 is present in 25-30% of breast carcinomas and has been associated with aggressive tumour phenotype, poor prognosis, non-responsiveness to hormonal therapy and reduced sensitivity to conventional chemotherapeutic agents (Slamon et al., 1989).
Although treatment with trastuzumab has shown efficacy, particularly in combination with chemotherapy, only patients with the highest levels of HER-2 expression, representing approximately 20% of breast cancer patients, are eligible for trastuzumab and other HER-2-targeted therapies. Studies with trastuzumab therapy show that many patients progress despite treatment or relapse, requiring novel approaches to increase anti-HER-2 antibody efficacy and to induce more sustained immune responses against HER-2 (Slamon et al., 1989; Jones et al., 1999).
In patients who do not express HER2, or who express it at lower levels (HER2 1+ or 2+ or FISH negative), targeted therapeutic options remain limited, and new strategies specific for alternative breast cancer markers and pathways will be required to improve treatment
outcomes for breast cancer patients. There is need therefore, for intensive research towards proteomics of breast cancer antigens which will be of benefit to the management of breast cancer.
Overall, immunotherapy holds several key advantages over conventional chemotherapeutic treatments, as well as over new targeted therapies. Not only does immunotherapy generally confer fewer side effects, enabling it to be administered for longer periods of time and/or in combination with other agents without added toxicity, but patients may also be less likely to develop resistance to immunotherapeutic approaches because of the immune system‘s ability to target multiple cancer antigens and pathways simultaneously and to offer longer-term protection due to its capacity for memory (Friedmann, 2005).
Some immunotherapies that have shown promise in recent clinical trials include NeuVax (nelipepimut-S or E75) that is under investigation to prevent breast cancer recurrence among patients with low to intermediate levels of HER2 expression (HER2 1+ and 2+) following surgery (Wrights, 2012). A vaccine targeting the AE37 peptide is also undergoing testing in a phase II trial involving 600 women who have completed all primary standard treatment and are without clinical evidence of disease. Nelipepimut-S (formerly known as E75) is an immunogenic peptide from the HER2 protein that is highly expressed in breast cancer. The NeuVax™ (Galena, OR, USA) vaccine, nelipepimut-S plus granulocyte-macrophage colony-stimulating factor is designed for the prevention of clinical recurrences in high risk, disease-free breast cancer patients. Although cancer vaccines such as NeuVax represent promising approaches to cancer immunotherapy, much remains to be elucidated regarding their mechanisms of action: particularly given that multiple cancer vaccine trials have failed to demonstrate a correlation between immunologic data and clinical outcome (Schenebe et al., 2014). The GVAX, a therapeutic vaccine made from breast cancer cell lines irradiated and engineered to express the immune molecule GM-CSF, is being tested in a phase II trial in patients with stage IV breast cancer that does not overexpress HER-2 (Wrights, 2012).
Breast cancer therefore, is an immunogenic tumor and multiple immunotherapeutic strategies are being tested as new clinical modalities which may improve disease outcome.
It has been shown that eliciting an immune response similar to what is observed in tissue rejection may indicate a beneficial outcome in cancer patients (Palucka et al., 2013). The CD4+ Th1 cells are uniquely capable of creating such a rejection signal. In secreting high
levels of type 1 cytokines these cells not only support the expansion of antigen specific cytotoxic T cells but also have the capability of directly modulating the tumor microenvironment to generate epitope spreading which would lead to tissue destruction.
Clinical strategies focused on eliciting tumor antigen specific Th1 are showing promise in terms of benefiting clinical outcomes in patients with breast cancer.