**1. Introduction**

130 Cancer Prevention – From Mechanisms to Translational Benefits

Zhang, Y. W.; Morita, I.; Ikeda, M.; Ma, K. W. & Murota, S. (2001). Connexin43 suppresses

p27. *Oncogene*, vol.20, No.31, (July 2001), pp. 4138–4149, ISSN 0950-9232. Ziech, D.; Franco, R.; Pappa, A.; Malamou-Mitsi, V.; Georgakila, S.; Georgakilas, A. G. &

1872-7786.

proliferation of osteosarcoma U2OS cells through post-transcriptional regulation of

Panayiotidis, M.I. (2010). The role of epigenetics in environmental and occupational carcinogenesis. *Chem Biol Interact.*, Vol. 188, No. 2, (July 2010), pp. 340-349, ISSN

#### **1.1 Role of vaccines in disease prevention and cancer prevention**

Vaccine therapy is traditionally designed to be given prophylactically in order to prevent infectious diseases. Vaccines formulated against pathogens known to cause disease have been successfully created and implementation policies have been effective in the prevention and eradication of many life-threatening diseases. A well-know example of the success that is possible with a well constructed vaccine and an efficient vaccination strategy is the poliomyelitis vaccine. Poliomyelitis was first documented in the late 19th century and it quickly reached the level of causing annual global endemics. The vaccine was introduced in the United States in 1955 and was associated with an immediate reduction in the disease and eventual the elimination of wild-type polioviruses in the United States by 1972. Subsequent global expansion of polio vaccination has resulted in a drastic reduction of documented cases of the disease as well as eradication of wild type 2 poliovirus [1].

A natural next step after the development of prophylactic vaccines to prevent infectious disease was the emergence of vaccines used against specific infectious agents that are known to cause cancer. An estimated 12% of human cancers are attributable to viral infections [2]. Viral infections that are known to cause cancer include human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpes virus (KSHV). Safe and effective vaccines have been developed against two oncovirus, HPV to prevent HPV-associated cervical carcinoma and HBV to prevent HBV-associated hepatocellular carcinoma.

#### **1.2 Primary versus secondary prevention**

A small number of cancers are directly associated with exposure to an oncovirus. Vaccinating against these viruses and preventing infection and subsequent cancer formation is referred to as 'primary cancer prevention'. This term also encompasses the theoretic potential of vaccinating against non-infectious cancers prior to tumorigenesis. Most cancers do not have a direct link to one specific pathogen. Therefore, experimental cancer vaccines

Targeting Molecular Pathways

type breast cancers.

immunologic memory [20].

**2. Cancer and the immune response** 

for Prevention of High Risk Breast Cancer: A Model for Cancer Prevention 133

10- year survival compared to Luminal B, HER-2, Basal-like and unclassified tumors [4]. In addition, certain ductal carcinoma in situ (DCIS) lesions over express HER-2, which results in a more rapid progression to invasive disease [11] and higher risk of recurrence [10, 13]. Anti-estrogen therapy is used for primary and secondary prevention of luminal tumors, but there are currently no similar options for prevention of the high risk tumors (Luminal B, Basal, HER-2). Trastuzumab is a human epidermal HER-2-targeted monoclonal antibody that has been shown to decrease recurrence and improve survival when used in the adjuvant setting combined with chemotherapy to treat patients with invasive disease that over-express the HER-2 protein [14]. The effect of trastuzumab has been postulated to be mediated by antibody-dependent cytotoxicity (ADCC) [15]. Unfortunately, this regimen is often not curative [14, 16] and patients can become resistant to therapy and ultimately fail [17] . A protein in the HER family would be an ideal target for a breast cancer vaccine. The HER family of tyrosine receptor kinases of which HER-1, HER-2, HER-3 and HER-4 are members make intriguing targets as these molecules are implicated in HER-2 and Basal-type breast cancers and also play a significant role in the development of some of the Luminal-

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

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

an anti-neoplastic process, comprised of CD8+ T cells, TH1 cells and NK cells [22].

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

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

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

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

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

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 high risk breast cancer.

#### **1.4 Molecular phenotypes of breast cancer**

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

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 10- year survival compared to Luminal B, HER-2, Basal-like and unclassified tumors [4]. In addition, certain ductal carcinoma in situ (DCIS) lesions over express HER-2, which results in a more rapid progression to invasive disease [11] and higher risk of recurrence [10, 13].

Anti-estrogen therapy is used for primary and secondary prevention of luminal tumors, but there are currently no similar options for prevention of the high risk tumors (Luminal B, Basal, HER-2). Trastuzumab is a human epidermal HER-2-targeted monoclonal antibody that has been shown to decrease recurrence and improve survival when used in the adjuvant setting combined with chemotherapy to treat patients with invasive disease that over-express the HER-2 protein [14]. The effect of trastuzumab has been postulated to be mediated by antibody-dependent cytotoxicity (ADCC) [15]. Unfortunately, this regimen is often not curative [14, 16] and patients can become resistant to therapy and ultimately fail [17] . A protein in the HER family would be an ideal target for a breast cancer vaccine. The HER family of tyrosine receptor kinases of which HER-1, HER-2, HER-3 and HER-4 are members make intriguing targets as these molecules are implicated in HER-2 and Basal-type breast cancers and also play a significant role in the development of some of the Luminaltype breast cancers.
