**2. Cancer and the immune response**

132 Cancer Prevention – From Mechanisms to Translational Benefits

are designed to stimulate an immune response against pathogens from established tumors. The creation of a vaccine against an established tumor, either invasive or pre-invasive, is referred to as 'secondary cancer prevention'. The goal of secondary cancer prevention is to use active specific immunotherapy to eradicate cancer cells without causing harm to healthy tissues. Successful secondary cancer prevention can have of a number of goals including inhibiting the evolution of pre-invasive to invasive disease, impeding the progression of

Breast cancer remains the most common non-skin cancer diagnosis and the second leading cause of cancer related death in women [3]. Major improvements in the surgical and adjuvant treatment of breast cancer during recent decades have resulted in improved disease-free and overall survival for breast cancer patients. Morbidity from surgery, chemotherapy and radiation is substantial and even with optimal current treatments

A general challenge to constructing an effective cancer vaccine is that all tumor cells contain self-antigens that vary from normal tissue by mutation or expression level and therefore cancer cells are able to evade immune surveillance. It is essential to find tissue and tumor specific molecules that are capable of stimulating an immune response. Vaccination efforts are often focused on high risk cancers where the clinical impact can be the greatest. Immunotherapy, which involves actively manipulating the immune system to target tumors, promises the potential for a safe and effective adjuvant treatment for patients with

Elucidation of the molecular basis of carcinogenesis has identified that breast cancer and probably all solid tumors exist as discrete molecular subtypes rather than a single disease. Breast cancer is a heterogeneous disease and several microarray profiling studies have identified distinct subtypes of breast tumors that are associated with different clinical outcomes [4-7]. The implications of classifying tumors based on gene profiling are both therapeutic and predictive. Gene expression profiling facilitates both the prediction of patient

Breast tumors are typically classified into five distinct genetic subtypes based on immunphenotype and the expression of the following receptors; estrogen (ER), progesterone (PR), human epidermal growth factor receptor-2/neu (HER-2), cytokeratin 5/6 (CK5/6) and epidermal growth factor (EGFR). *Luminal A* cancers are ER positive and/or PR positive, HER-2 negative and Grades 1 or 2. *Luminal B* cancers are (a) ER positive and/or PR positive and HER-2 positive or (b) ER positive and/or PR positive and HER-2 negative and high grade. *HER-2 type* cancers stain negative for ER and PR and positive for HER-2. *Basallike* tumors have no staining for ER, PR and HER-2, but do stain positive for CK 5/6 and/or EGFR. Tumors that have no staining for all 5 markers are referred to as *Unclassified* [4, 8, 9]. These molecular phenotypes of breast carcinoma can be delineated with routine immunohistochemical markers. Substantial differences in the survival of patients with different subtypes have been reported. Luminal A tumors have a significantly better 5- and

outcome and the selection of patients that will benefit from specific adjuvant therapies.

disease and the formation of metastases, and increasing patient survival.

**1.3 Breast cancer background and potential for vaccine therapy** 

approximately 40,000 women a year succumb to breast cancer [3].

high risk breast cancer.

**1.4 Molecular phenotypes of breast cancer** 

The immune system is a complex and overlapping cellular network that protects against foreign pathogens and closely regulates self-tolerance. The innate system represents the first line of defense to tissue injury and foreign pathogens. It is comprised of natural barriers, cytokines, neutrophils, macrophages, dendritic cells (DCs), and natural killer (NK) cells [18, 19]. The innate response also includes the activation of the complement pathway. The early, antigen-nonspecific response of the innate immune system is necessary for the activation of the adaptive immune system which is comprised of B- and T- lymphocytes that express antigen-specific receptors and are ultimately responsible for producing and maintaining immunologic memory [20].

#### **2.1 Cancer response to the immune system: exploitation and evasion and editing**

In order for cancer cells to survive they must be able to either evade the immune system or to exploit it in a way that causes immune cells to actually enhance tumor growth. The immune response to neoplastic development is often described as paralleling the body's response to inflammation. It can be simplistically divided into an 'acute' and 'chronic' reaction. Epithelial cancer progression and eradication, similar to an inflammatory reaction, are regulated by both the innate and adaptive immune systems [21]. The specific immune cells involved paradoxically enhance and eliminate carcinogenesis. Accumulated data from animal and human studies has shown that the acute immune response to tumor growth is an anti-neoplastic process, comprised of CD8+ T cells, TH1 cells and NK cells [22].

Continued epithelial cancer development leads to dysregulation between the two subsets of the immune system and excessive activation results in an immune response that is similar to the body's response to chronic inflammation. Chronic activation of innate immune cells is associated with an ongoing infiltration cells that facilitate the survival of neoplastic cell survival by stimulating angiogenesis, inflammation and proliferation [19-23]. The chronic activation of the innate immune system also directly contributes to cancer development by

Targeting Molecular Pathways

cancers in both early and late stages.

longer survival in patients with invasive breast cancers.

systemic.

for Prevention of High Risk Breast Cancer: A Model for Cancer Prevention 135

administered prior to disease exposure and effective immunity is created before infection. In contrast, cancer vaccines are intended to stimulate active immunity only after tumors cells are already present and established. Oftentimes, vaccines are not given until the cancer has spread systemically. Also, unlike bacteria or other microbes for which vaccines are used, all tumor cells also express antigens that are very similar to established self-antigens. The ideal vaccine target would be an antigen that is present only on cancer cells and not on normal cells. Since this is uncommon, one of the largest challenges in vaccine development is breaking immune tolerance without inducing autoimmune reaction that would be harmful to healthy tissues. Several vaccine approaches have been established for a multitude of

**3. Immune response in relation to invasive and in situ breast cancer** 

Breast cancer, both the *in situ* and invasive forms, is an ideal target for vaccine therapy since this disease creates a significant public health burden. There is potential for vaccines to inhibit the progression of in situ disease into invasive cancer. The ultimate goal would be to prevent the formation of breast cancer altogether. Central to success of using immunotherapy to treat breast cancer is that breast tumors have already been established to be relatively immunogenic and the growing tumors are subject to immunosurveillance. Tumor antigens that are over-expressed or mutated in breast cancer cells initiate the development of a tumor-specific adaptive immune response [23, 37, 38]. T-cells that recognize these antigens have been isolated from breast cancer patients [39, 40]. As further evidence that the cell-mediated immune reaction has an important role in breast cancer development and clinical outcome, lymphocyte infiltration has been shown to be associated with improved survival in breast cancer patients [41]. Recent data by Mahmoud *et al*  confirmed that the presence of an efficient T-cell-mediated immune response is associated with breast cancer outcomes [42]. This study, a retrospective review of immunohistochemical staining from nearly 2000 patients with invasive breast cancer who received standard surgical and adjuvant treatment revealed that a higher number of CD8+ T lymphocytes infiltrating the tumor of adjacent stroma was independently associated with

In addition to being immunogenic, other aspects of breast cancer make it a good model for the development of a high-impact cancer vaccine, especially for patients with early stage disease. First, solid tumor cancer vaccines have had limited success when used to treat advanced or metastatic disease [43]. Breast cancer is most frequently treated with surgery and radiation therapy, which greatly decreases the disease burden, even in advanced cases. This tumor debulking provides a greater potential for disease eradication by competent immune cells. Second, the typical slow growing nature of most breast cancers allows for the expansion of immune cells over time with repeated vaccine boosters. Therefore, effective levels of active and immune competent cells can be achieved before the disease becomes

Although most cancer vaccines have been developed to treat metastatic and systemic disease, there are a number of theoretical benefits to instead delivering vaccine therapy to patients with limited, microscopic cancer burden (as neoadjuvant therapy) in the absence of bulky disease. For instance, immune-competent patients would be able to produce a significant response comprised of antigen-competent T-cells that can rapidly expand when

suppressing the anti-tumor adaptive immune response (CD8+ T cells, TH1 cells, NK cells) and allowing tumor cells to escape from surveillance. One type of innate immune cells, myeloid suppressor cells, are known to accumulate in the peripheral blood of patients with cancer [24, 25]. Myeloid suppressor cells directly inhibit T lymphocytes and therefore inhibit the anti-tumor environment produced by innate immune cells [25, 26]. These cells also promote tumor growth by assisting in angiogenesis [27].

In addition to suppressing the anti-tumor effects of the adaptive immune system, chronic activation of the cells of the innate immune (B cells, TH2 cells) response actually promotes tumor development [21]. Multiple population based studies have definitively linked chronic inflammatory conditions with the development of certain cancers. For example *Helibacter pylori* infection and gastric cancer, inflammatory bowel disease and colon cancer, and hepatitis and hepatocellular carcinoma [28-30]. Subsequent studies that revealed an inverse relationship between long-term usage of anti-inflammatory medications and a decreased cancer risk support the support the direct association between chronic inflammation and cancer development [31]. Through a variety of cellular mediators, chemokines and cytokines, innate immune cells and TH2 cells are able to create a pro-tumor microenvironment that favors cell proliferation, genomic instability and malignant conversion [32].

In addition to exploiting the immune system to stimulate tumor growth, cancer cells must also be able to evade the immune response. Prolonged activation of the innate immune cells results in subsequent suppression of the anti-tumor adaptive immune response, therefore allowing tumors cells to avoid specific immune surveillance. Neoplastic lesions attract regulatory T cells that suppress cytotoxic T cells [33]. Furthermore, cancer cells avoid immune surveillance by over expression or mutation of self-peptides. These non-foreign antigens are only weakly immunogenic and thus evade the host immune response or do not induce an immune specific response in the same way that a completely foreign antigen would.

Growing tumors are phenotypically sculpted by the immune system. One of the risks of cancer immunotherapy is that it can result in 'immunoediting', whereby the immune response sculpts cancer cells into a more aggressive phenotype [34, 35]. Surviving tumor cells acquire the ability to evade immune recognition through selective pressures that favor the survival and reproduction of cancer cells that lack the selected antigen. In considering an immunotherapy target it is important to select an antigen that is central to the survival of the tumor cell or that contributes to the aggressiveness of the disease. Therefore, when the cells adapt, only variants that do not express the antigen will survive. This creates a tumor that consists of cells that are unable to survive or that have a phenotype that is associated with a better clinical prognosis [36]. This process of immunoediting explains in part why targeting only single antigens has resulted in limited clinical success.

#### **2.2 General potential for vaccine therapy**

The intimate relationship between cancer and the immune system illustrates the potential for an effective immunotherapeutic agent, such as a cancer vaccine, to harness the immune system and then to manipulate the immune cells to create a strong anti-tumor environment. Traditional vaccines are designed to be prophylactic. The immunogens in the vaccines are

suppressing the anti-tumor adaptive immune response (CD8+ T cells, TH1 cells, NK cells) and allowing tumor cells to escape from surveillance. One type of innate immune cells, myeloid suppressor cells, are known to accumulate in the peripheral blood of patients with cancer [24, 25]. Myeloid suppressor cells directly inhibit T lymphocytes and therefore inhibit the anti-tumor environment produced by innate immune cells [25, 26]. These cells also

In addition to suppressing the anti-tumor effects of the adaptive immune system, chronic activation of the cells of the innate immune (B cells, TH2 cells) response actually promotes tumor development [21]. Multiple population based studies have definitively linked chronic inflammatory conditions with the development of certain cancers. For example *Helibacter pylori* infection and gastric cancer, inflammatory bowel disease and colon cancer, and hepatitis and hepatocellular carcinoma [28-30]. Subsequent studies that revealed an inverse relationship between long-term usage of anti-inflammatory medications and a decreased cancer risk support the support the direct association between chronic inflammation and cancer development [31]. Through a variety of cellular mediators, chemokines and cytokines, innate immune cells and TH2 cells are able to create a pro-tumor microenvironment that favors cell proliferation, genomic instability and malignant

In addition to exploiting the immune system to stimulate tumor growth, cancer cells must also be able to evade the immune response. Prolonged activation of the innate immune cells results in subsequent suppression of the anti-tumor adaptive immune response, therefore allowing tumors cells to avoid specific immune surveillance. Neoplastic lesions attract regulatory T cells that suppress cytotoxic T cells [33]. Furthermore, cancer cells avoid immune surveillance by over expression or mutation of self-peptides. These non-foreign antigens are only weakly immunogenic and thus evade the host immune response or do not induce an immune specific response in the same way that a completely foreign antigen

Growing tumors are phenotypically sculpted by the immune system. One of the risks of cancer immunotherapy is that it can result in 'immunoediting', whereby the immune response sculpts cancer cells into a more aggressive phenotype [34, 35]. Surviving tumor cells acquire the ability to evade immune recognition through selective pressures that favor the survival and reproduction of cancer cells that lack the selected antigen. In considering an immunotherapy target it is important to select an antigen that is central to the survival of the tumor cell or that contributes to the aggressiveness of the disease. Therefore, when the cells adapt, only variants that do not express the antigen will survive. This creates a tumor that consists of cells that are unable to survive or that have a phenotype that is associated with a better clinical prognosis [36]. This process of immunoediting explains in part why targeting

The intimate relationship between cancer and the immune system illustrates the potential for an effective immunotherapeutic agent, such as a cancer vaccine, to harness the immune system and then to manipulate the immune cells to create a strong anti-tumor environment. Traditional vaccines are designed to be prophylactic. The immunogens in the vaccines are

promote tumor growth by assisting in angiogenesis [27].

only single antigens has resulted in limited clinical success.

**2.2 General potential for vaccine therapy** 

conversion [32].

would.

administered prior to disease exposure and effective immunity is created before infection. In contrast, cancer vaccines are intended to stimulate active immunity only after tumors cells are already present and established. Oftentimes, vaccines are not given until the cancer has spread systemically. Also, unlike bacteria or other microbes for which vaccines are used, all tumor cells also express antigens that are very similar to established self-antigens. The ideal vaccine target would be an antigen that is present only on cancer cells and not on normal cells. Since this is uncommon, one of the largest challenges in vaccine development is breaking immune tolerance without inducing autoimmune reaction that would be harmful to healthy tissues. Several vaccine approaches have been established for a multitude of cancers in both early and late stages.
