Immunologic approaches to cancer prevention—current status, challenges, and future perspectives

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Immunologic Approaches to Cancer Prevention – Current Status, Challenges and Future Perspectives Malgorzata E. Wojtowicz, Barbara K. Dunn, Asad Umar

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S0093-7754(15)00243-2 http://dx.doi.org/10.1053/j.seminoncol.2015.11.001 YSONC51897

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Cite this article as: Malgorzata E. Wojtowicz, Barbara K. Dunn, Asad Umar, Immunologic Approaches to Cancer Prevention – Current Status, Challenges and Future Perspectives, Semin Oncol, http://dx.doi.org/10.1053/j.seminoncol.2015.11.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Immunologic Approaches to Cancer Prevention – Current Status, Challenges and Future Perspectives Malgorzata E Wojtowicz 1,* Barbara K Dunn 1 Asad Umar 1 1

Division of Cancer Prevention, National Cancer Institute, National Institutes of Health

*Corresponding author: [email protected]

Key Words: Tumor immunity, cancer immunoprevention, tumor antigens, cancer vaccines

Abstract The potential of the immune system to recognize and reject tumors has been investigated for more than a century. However, only recently impressive breakthroughs in cancer immunotherapy have been seen with the use of checkpoint inhibitors. The experience with various immune-based strategies in the treatment of late cancer highlighted the importance of negative impact advanced disease has on immunity. Consequently, use of immune modulation for cancer prevention rather than therapy has gained considerable attention, with many promising results seen already in pre-clinical and early clinical studies. Although not without challenges, these results provide much excitement and optimism that successful cancer immunoprevention could be within our reach. In this review we will discuss the current state of predominantly primary and secondary cancer immunoprevention, relevant research, potential barriers and future directions.

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I.

Introduction

Cancer immunoprevention refers to the modulation of the host immune system to prevent the initiation and progression to cancer. The knowledge that tumor growth can be regulated by the immune system dates back over 100 years (1) and has been further strengthened by the observations of "spontaneous regressions" seen in certain solid tumors (2, 3). Only recently, however, has a better understanding of the mechanisms and components involved in cancer immunology allowed for rapid development of the cancer immunotherapy field in general and immunoprevention in particular (4). Various immune-based strategies offer substantial promise, and in specific cases the clinical benefit has been meaningful enough to gain FDA approval for some of the newest members of the therapeutic armamentarium for several cancer types (e.g. T-cell checkpoint blockade) (5-10). Despite this exciting progress, however, the benefits of immunotherapy in most patients with advanced malignancy have been limited by the large tumor burden, tumor-induced tolerance mechanisms, and the local immune inhibitory factors within the tumor itself and its microenvironment. Therefore, there is a growing interest in applying cancer immunomodulatory strategies in the state of minimal residual disease and cancer prevention settings. Potential advantages to immune-based approaches over drug-based chemoprevention include: a) high specificity and adaptability of immune responses (adaptive immunity is specific to a given antigen and can adjust to changes within the antigenic repertoire); b) favorable toxicity profile (immune strategies - cancer vaccines in particular - appear non-toxic in the majority of cases (11, 12)); c) ability to generate immunological memory, providing long-term (potentially life-long) protection (not achievable with drugs); and d) ease of 2

administration (e.g. several vaccinations with occasional boosts vs. daily dosing for many years with chemopreventive agents). Despite this rapid-pace development of immunebased therapies for treatment of cancer, moving these approaches to cancer prevention (especially for primary cancer prevention where the goal is preventing onset of disease in high-risk individuals who are otherwise healthy) has proven to be much more difficult and has been considerably slower. In this review we will discuss the current state of predominantly primary and secondary cancer immunoprevention, relevant research, challenges and future perspectives.

II. Mechanistic Bases of Immune Modulation for Cancer Prevention A. Innate Versus Adaptive Immunity.

The concept of cancer immunomodulation is based on the physiologic immune responses observed during the natural course of disease.

In general, the immune

responses have been divided into two broad categories: innate ("native") and adaptive (“acquired”) immunity (13, 14) (Figure 1). The innate immune responses provide immediate, short-term protection against a variety of non-specific pathogens ("danger signals"). In contrast, adaptive immunity develops over a longer period of time, is antigen-specific, and provides long-term protection. Activation of innate and subsequent adaptive immunity is essential to effective immunological eradication of exogenous and endogenous pathogens as well as undesirable endogenous elements (e.g., cancer cells).

Although the essential components of both types of immunity differ, many of their activities overlap with the common goal of eliminating "danger". The components of the innate immune system include: 1) epithelial barriers, 2) phagocytic leukocytes, 3) 3

dendritic cells (DC), 4) natural killer (NK) cells, and 5) circulating plasma proteins (15). Components of the adaptive immune system fall into two functional categories: a) humoral immunity, mediated by the antibodies produced by B lymphocytes; and b) cellular immunity, mediated by T lymphocytes (16). Dendritic cells (DC) are a major component of both innate and adaptive immune responses.

Functionally, the innate immune system (also referred to as "non-specific") is the first, immediate line of defense, the body’s “gut reaction” (17), defending the host in a non-specific/non-targeted manner. Inflammation is one of the components of innate immunity. During innate immune reactions DCs scan for potential "danger signals" using toll-like receptors (TLRs), which are the membrane-bound pattern recognition receptors (PRRs) that detect foreign pathogen components that exhibit certain pathogen-associated molecular patterns (PAMPs) (18). Once the TLR recognizes and processes a given "danger signal", it activates the nuclear factor kappa B (NF-ƙB) and mitogen-activated protein kinase (MAPK) signal transduction pathways, which are the major players in the initiation and regulation of innate immune responses (19-21). Modulation of TLR immune responses is currently being pursued as a potential therapeutic target for various inflammatory diseases and cancer. In contrast, the adaptive (“acquired”) immune response (also referred to as "specific") is much more complex than the innate response as it continuously adapts to the new “specific danger signals”, i.e. antigens from infectious agents or tumor cells. This system requires presentation of the antigen in a very specific manner in order to induce activation of the key components of this arm of immunity: lymphocytes, both T- and B-

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cells (14, 22, 23). Most adaptive immune responses are triggered when naïve T cells, via their antigen-binding receptors and either CD4 or CD8 co-receptors, recognize antigens that are being adequately presented (in the proper context and presence of co-stimulation) by the professional antigen-presenting cells (APCs), among which DCs are the most potent. This generates signals that ultimately result in the activation, proliferation and differentiation of T-cell progeny into highly specific effector T cells. The CD8+ T cells are pre-destined to become cytotoxic T-lymphocytes (CTLs) and their main function is to kill “target cells” (cells expressing given antigen(s)). The naïve CD4+ T cells, however, can differentiate upon activation into either Th1 or Th2 cells, which differ in their function depending on the cytokines they produce. Th1 cells (type 1 helper cells, sometimes called inflammatory CD4+ T cells) are predominantly involved in stimulating macrophages and together with CD8+ CTLs are the key components of cellular immunity. The Th2 CD4+ T cells (type 2 helper cells) are mainly involved in stimulating Blymphocytes to produce antibodies, creating the key forces of humoral immunity. B-cell activation requires both binding of the antigen by the B-cell surface immunoglobulin (Bcell receptor) and interaction with antigen-specific T helper cells resulting in B-cell proliferation and differentiation. During differentiation of activated B cells, various changes take place, including somatic hypermutation in the hypervariable regions of the B-cell receptors and isotype switching, leading to generation of high-affinity, highly specific humoral immune responses.(14, 22, 23). In addition to its ability to generate distinct immune responses, adaptive immunity has a key advantage in inducing a robust, specific immunological B- and T-cell memory that allows quick responses to subsequent challenges by the same antigen (whether pathogen or cancer cell/antigen, etc). However,

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the fact that development of a fully effective adaptive immune response can take days, weeks or even months is a major limitation. The lagging adaptive response leaves a gap in time during which the innate immune system becomes critically important as part of the host's defense. Therefore, the two arms are complementing each other and are crucial for optimal immune protection. Consequently, they both are being targeted in the efforts to develop effective cancer immunotherapies and cancer immunoprevention strategies.

The immune system is a very complex network of intricately connected, overlapping sub-systems that function together to maintain the host's immune homeostasis. It needs to recognize a full range of antigens (whether pathogens or abnormal/cancer cells), react only to those that pose "danger", and respond in a very specific manner to eliminate this “danger” without causing harm to the host. After elimination of a specific “threat” is completed, it is equally important for the immune system to have a mechanism in place for “turning off” all its stimulatory machinery in order to avoid overstimulation and inadvertent reaction against “normal self” which can cause autoimmunity. These “off switches” or immunological "breaks" are cumulatively referred to as "checkpoints" and are attractive targets of the newest cancer immunotherapeutic strategies (referred to as checkpoint blockade) in a variety of tumor types.

The key molecules within these checkpoints currently targeted for cancer

treatment are: CTLA-4, PD-1, and PD-L1 (6, 7, 24-27).

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B. Cancer Immunosurveillance and Immunoediting

The concept of immunological surveillance was initially introduced by Burnet and Thomas in the 1950’s (28). Only recently, however, has the development of new molecular techniques enabled further exploration of this hypothesis, especially in relation to the carcinogenesis process (29). This hypothesis proposes that both the innate and adaptive immune systems conduct constant immunosurveillance throughout the body to identify and eliminate aberrant cells and to build durable specific responses against them (30, 31). It also postulates how developing malignancies are still able to escape these host-protecting processes through various immune-evading mechanisms (referred to as cancer immunoediting), ultimately inducing self-protection and further growth. This cancer immunoediting is a dynamic process involving the full continuum of cancer progression and is generally divided into three phases: elimination, equilibrium and escape (Figure 2). Briefly, after malignant cells expressing their highly immunogenic antigens (viewed as clearly "abnormal") are destroyed (elimination phase), some of the remaining cancer cells expressing fewer or less immunogenic epitopes ("unclear signal") can persist although their further growth could still be controlled by immunologic mechanisms (equilibrium phase). With time, under constant immune selection pressure placed on genetically unstable tumor cells using various mechanisms (antigen loss, defects in antigen processing or presentation, etc.), tumor cell variants can develop that are no longer recognized and destroyed by immune mechanisms, allowing their further growth (escape phase) (Figure 2). Growing tumor further enhances its self-protection via multiple immune-evading mechanisms (production of immunosuppressive cytokines within the tumor microenvironment, recruitment of regulatory T-cells/T regs, myeloid7

derived suppressor cells/MDSCs, inadequate T-cell activation, compromised T-cell memory function, etc.), leading eventually to development of clinically detectable disease and its progression.

III. Immunoprevention of Cancer

In general, cancer prevention strategies include: risk reduction through elimination of environmental factors (e.g. tobacco, asbestos); life style changes (e.g. exercise, diet); and intervention in high risk populations either with the use of surgery (e.g. preventive colectomy, mastectomy, oophorectomy), chemopreventive agents (e.g. aspirin/COX (cyclooxygenase)-2 inhibitors, tamoxifen, raloxifene), and/or immunoprevention (e.g. vaccines to prevent infection with cancer causing viruses, etc.). There are various levels of prevention ranging from primary to secondary and tertiary (32). Although definitions for phases of prevention vary, in general, primary prevention involves interventions to prevent onset/initiation of disease, i.e. invasive disease or cancer, including treatment of high risk pre-malignant disease to prevent progression to cancer. Secondary prevention aims at prevention of second primary cancers in individuals who already have experienced a first primary and are therefore at increased risk of a second primary (e.g. contralateral breast cancer). Early detection of non-invasive cancer and intervention to treat and prevent progression to invasive disease is sometimes included in this category. Tertiary prevention is the use of treatment to improve the outcome of existing illness (invasive cancer), e.g. prevention of recurrence after curative cancer therapy (32). This review will focus mainly on primary and secondary prevention of cancer using immunological agents as the preventive interventions.

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Although there are several cancer immunotherapeutic strategies already approved for clinical use and many others are at various stages of development, thus far the most suitable for use in the primary prevention setting appear to be cancer vaccines. This is mainly due to their very good safety profiles and specificity, which is of major importance, as they are intended for use in high risk individuals who are otherwise healthy (11, 12).

A. Cancers associated with infectious agents

The most successful application of primary cancer immunoprevention to date has been vaccination against infectious agents that cause cancer as complicating sequelae of chronic infection. This is best established in cases of hepatitis B (HBV)-associated hepatocellular carcinoma (HCC) and human papillomavirus (HPV)-associated cancers. Vaccination against HBV, implemented as part of the nationwide vaccination program in Taiwan, led to a gradual decrease in the average annual incidence of HCC in children, with a corresponding decrease in the rates of HCC-associated mortality (33). For HPVassociated cervical and other gynecological cancers two international trials (FUTURE I/II) have evaluated the efficacy of the quadrivalent HPV vaccine (Gardasil: serotypes 6,11,16,18).

These trials have shown 96% efficacy in preventing grade 1 cervical

intraepithelial neoplasia (CIN) caused by viruses corresponding to the serotypes included in the vaccine and 100% protection for vulvar and vaginal intraepithelial neoplasia (VINs) (34). Similarly, the HPV-16/18 ASO4-adjuvant vaccine (Cervarix) was successful in preventing cervical cancer, CIN and cervical adenocarcinoma in situ (35). Vaccination against HPV also decreases the incidence of penile intraepithelial neoplasia (34) and anal

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intraepithelial neoplasia (36, 37). Currently, Cervarix, Gardasil and Gardasil 9 (covering serotypes 6, 11, 16, 18, 31, 33, 45, 52, 58) are FDA approved. It is estimated that vaccination against the seven “high-risk” HPV types found in the nonavalent vaccine can potentially prevent over 90% of cervical cancers and a similarly high percentage of other HPV-associated cancers of the vulva, vagina, anus, penis, and some head and neck cancers (38). HPV-associated head and neck cancer includes mainly cancers of the oropharynx, which are most frequently associated with HPV-16, although HPV-18, 31, and 33 are also causative but less common. The potential for prevention of these cancers is especially important since HPV-associated head and neck cancers are on the rise and their annual incidence is expected to surpass the annual number of cervical cancers by 2020 (39).

There are many more known infectious agents (mostly viruses) recognized to cause cancer (summarized in Table 1), but to date there are no approved vaccines that can effectively prevent these infections. There are many challenges involved in development of such vaccines including, among others, high heterogeneity and mutability of these pathogens (i.e. hepatitis C virus), complex mechanisms of oncogenesis induced by them, and scarcity of available preclinical in vitro and in vivo models, to name the few. Despite these difficulties, active efforts in this field are beginning to provide new promising vaccine constructs that are currently at various stages of pre-clinical and clinical development.

Preventive (or prophylactic) vaccines are effective due to their ability to prevent primary infection with a causative agent when given prior to exposure. Another, more

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challenging, strategy to prevent cancer would be vaccination to treat already existing infection which, when eradicated, would potentially eliminate the relevant cancer risk (therapeutic vaccines). Such immune-based approaches, however, are still within the realm of research investigation. In the case of some cancer-causing infectious agents there are successful, FDA-approved drug-based and potentially curative therapies; for chronic hepatitis C, these include direct-acting antiviral agents (DAAs). However, these drugs still harbor considerable drawbacks such as their high cost, side effect profiles, viral resistance and risk for re-infection (40, 41). Therefore, immune-based therapies (i.e. vaccines) with their general properties of specificity, safety and ability to induce longlasting immune memory are still highly desirable and are the subject of active research. There are ongoing efforts to develop therapeutic vaccines for HBV (42), HCV (43, 44), Epstein-Barr Virus (EBV) – which is associated with epithelial cell malignancies (nasopharyngeal cancer and gastric cancer) as well as lymphoid malignancies (nonHodgkin’s lymphoma/NHL, Hodgkin’s lymphoma/HL, etc.) (45) and others. Among those that are more advanced in clinical development are several HPV therapeutic vaccines. In a landmark clinical trial, Kenter and colleagues vaccinated 20 women with HPV-16 positive high-grade vulvar intraepithelial neoplasia (VIN-3) with synthetic longpeptides from the HPV-16 viral oncoproteins E6 and E7 (46). At 3 months after the last vaccination 12 of 20 patients (60%) had clinical responses associated with symptomatic improvement (5 women had complete regression of lesions and 4 women cleared the HPV virus). At 12 months clinical responses were seen in 79%, with complete responses noted in 47% of patients. These complete responses were maintained at 24 months of follow-up and were correlated with induction of HPV-16-specific T-cell immunity. This

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strategy was safe with predominantly mild local reactions seen. Although not 100% effective, these data suggest an ability to treat HPV-positive early, pre-invasive malignant disease (secondary prevention) with the potential for viral clearance via vaccine-induced T-cell responses. Early promising results have also been seen with other HPV-directed vaccination strategies (47) evaluated not only in HPV-positive gynecologic pre-invasive cancers (48, 49) but also in head and neck malignancies (50). However, progress is proving to be slow, given complexities involving proper monitoring of generated immune responses, a need for better delineation of mechanisms involved in disease and viral clearance, and progressive recognition of the negative impact of the tumor microenvironment (even in early, pre-invasive lesions). All of these issues need to be addressed in order to achieve better clinical efficacy (51).

B. Cancers not associated with infections

Unlike cancers that are caused by infectious agents (viruses in the majority of cases), the development of non-viral-associated cancers involves gradual oncogenic progression emerging in large part from genetic alterations. These genetic changes accumulate over many years, leading to transformation of normal tissue through premalignant stages (in many cases) to subsequent development of pre-invasive and invasive disease ultimately culminating in development of clinically evident cancer (52). This gradual process provides a “window of opportunity” for primary and secondary immunoprevention in “high-risk” individuals. This approach has been gaining attention in recent years after unsuccessful efforts to target late cancers; as a result it is only in the

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early stages of development with a limited number of early-phase clinical trials initiated in patients.

Currently, only limited data support potential vaccine targets and platforms for preventive vaccinations for tumor-associated antigens, i.e. antigens that characterize noninfection-associated cancers. In addition, well-defined high-risk populations (based mainly on genetic risk) are sparse with limited numbers of subjects. This paucity of appropriate candidates for immunoprevention would create barriers to the conduct of “proof-of-principle” studies, even if suitable vaccines were available. Furthermore, our ability to identify alternative at-risk populations, i.e. those that are not genetically based and make up the majority of at-risk individuals, continues to challenge us and still has not been achieved for many types of cancers.

However, continued pursuit of cancer immunoprevention strategies is encouraged by pre-clinical experiments showing that vaccination of healthy mice at risk for cancer has resulted in either effective reduction or meaningful delay of carcinogenesis in a variety of model systems (reviewed in (53). These mouse models consist mainly of transgenic mice carrying oncogenes or knockout mice with altered tumor suppressor genes. The immune-based modification of critical cancer-related genes, and hence their protein products, in these genetically engineered mouse models implies that immunoprevention could be applicable to many tumor types not associated with infection.

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B.1. Potential for immunoprevention of non-infection associated cancers

B.1.a. Evidence supporting immune responsiveness in humans

A growing body of evidence points to immune (innate and adaptive) responses that are already operational in patients with pre-invasive lesions at high risk of progression to invasive cancer (reviewed in (54)). Tissue analyses of these pre-cancers demonstrate infiltration with lymphocytes capable of antigen recognition. The peripheral blood of individuals with such lesions shows immune responses directed against specific lesion/tumor-associated proteins. Finally, vaccines developed to target these pre-invasive antigens are delivering promising results in pre-clinical testing with early translational clinical investigations already initiated. Together these observations support the potential for successful development of immunopreventive strategies for non-infection associated tumors at early stages of development. B.1.b. Changes in antigenic repertoire during carcinogenesis – creation of immunologic targets

The selection of antigens for immunoprevention presents one of the most difficult challenges for cancers that are not associated with infections. The most promising antigens in immunotherapy of these cancers, thus far, are “self-antigens” or normal cellular proteins that have become immunogenic (“abnormal-self”) during malignant transformation. These antigens generally derive from the normal cell equivalent of a specific tumor tissue type. During carcinogenesis some crucial modification in the 14

antigen has taken place to make it characteristic of the malignant version of the cell type in question; it has essentially become a tumor-associated “neoantigen” (4, 11, 55). This critical modification should enable recognition of the neoantigen by the body’s immune system, thus providing a break through the barrier of tolerance to self-antigens; the modified antigen is different enough to allow this break in tolerance.

In addition, this

modification should offer an antigenic target that differs enough from the antigen expressed by the equivalent normal cell such that the antigen in normal tissue is not recognized and targeted; tolerance to normal tissue cells must be maintained in order to preempt autoimmune responses. Furthermore, the modified antigen should be highly immunogenic if it is to elicit an active immune response. Antigens characterized by these key attributes that are expressed on cancer cells are referred to as “tumor-associated antigens” (TAAs).

A variety of mechanisms have been exploited by cancer cells to produce such “altered self” antigens. Among these alterations are: overexpression of otherwise normal antigens (i.e. Her2 in a subset of breast cancers); mutations in oncogenes (i.e. Ras) or tumor suppressor genes (i.e. p53) that confer a proliferative advantage; post-translational modifications (i.e. MUC1); and re-emergence of expression of proteins that are normally expressed early in development (or only under special circumstances) but normally decline with age (TAAs characterized by this last mechanism have been referred to as “retired proteins”) (56). Because they have “come out of retirement” and are expressed at immunologic levels in a tissue-specific manner only in cancer cells, the risk of inducing an autoimmune reaction towards normal cells via immune-based preventive intervention is very small. 15

Ideally, immunoprevention strategies should be aiming at specific antigen(s) to direct generated immune responses to cells designated for destruction while sparing healthy tissues (to limit toxicity). Identification of such antigens of not yet clinically evident cancer that would induce a diversified enough response to address tumor heterogeneity (for optimal eradication) but be specific enough to spare healthy tissue (for an acceptable safety profile) has proven very challenging.

Studying pre-invasive lesions, their antigenic repertoire, immunological milieu and immune responses at various stages of progression to invasive cancer is one way to advance this field and is proving very informative. Most advanced in this area are studies in breast and colon cancers.

B.2. Breast cancer immunoprevention

B.2.a. Immune responses at various stages of disease - the good, the bad and the “unknown” of immune infiltrates

Analysis of the spectrum of tissues ranging from normal breast to benign proliferative disease, to ductal carcinoma in situ (DCIS), to invasive breast cancer (IBC) demonstrates that infiltration with immune cells (CD3+/Th1 cells, FOXP3+/Th2 cells, CD68+/macrophages/innate immunity) is low in normal breast tissue but starts to rise in benign proliferative lesions with continuous proportional increases in DCIS and more so in IBC (54, 57, 58) (Figures 2, 3). The types of immune cell populations infiltrating preinvasive disease or IBC, relevant degree of infiltration, and the context of molecular tissue characteristics appear to have prognostic significance (and could potentially serve

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as immune biomarker).

Increased infiltration of invasive breast tumors with CD8+

cytotoxic T cells has been reported to be associated with improved breast cancer specific survival (59), whereas increased infiltration with FOXP3+ immunosuppressive T cells correlated with inferior survival (60). However, this relationship appears to be influenced by the molecular context of breast tumors. High levels of FOXP3+ regulatory tumor infiltrating lymphocytes (T regs) were associated with poor survival in estrogen receptor positive (ER+) invasive breast cancers (that also lacked CD8+ T-cell infiltrates) but were a favorable prognostic factor in the human epidermal growth factor receptor-2 (HER2)– positive/ER-negative breast tumors, particularly in those with co-existent CD8+ T-cell infiltrates (61).

In general, however, among specific subsets of tumor infiltrating

lymphocytes (TILs), high numbers of CD8+ cytotoxic T cells, CD4+ follicular helper T cells and CD20+ B cells are predictors of better patient survival across all breast cancer subtypes. In contrast, high numbers of FOXP3+ T regs are associated with higher tumor grade, ER-negativity, shorter relapse-free survival (RFS) and worse overall survival (OS) (reviewed in (62)). The presence of TILs also appears to predict response to systemic therapy in various clinical settings (neo-adjuvant, adjuvant therapy) where high numbers of TILs and a greater reduction in FOXP3+ T regs correlate with better response to treatment (60). Also, high levels of CD8+ TILs and lower FOXP3+ T reg numbers detected after completion of neo-adjuvant therapy were associated with improved recurrence free and overall survival (61). Similarly, in pre-invasive breast cancer (DCIS) prognosis was influenced by the type of infiltrating immune cells; increased FOXP3+ infiltrates in DCIS lesions were associated with shorter relapse-free survival (63). More recent reports suggest that elevated levels of infiltrating CD4+ cells secreting IL-17 (T

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helper/Th17 cells) in invasive breast cancer are associated with increased levels of regulatory T cells in these lesions. The presence of both cell types correlates with aggressive disease (64). However, Th17 cells play a complex and controversial role in tumor immunity and have been found to exhibit fluctuating characteristics within the context of cancer. They appear to exert different tumor effects - either promoting or suppressing tumor growth - depending on many factors (65). Whether Th17 cells adopt a pro- or anti- tumorigenic role appears to depend largely on the stimulation encountered by these cells. A better understanding of the signals that have an impact on the function and immunological fate of Th17 cells, with a better understanding of the mechanisms driving their anti-tumor effect, is important to the development of both effective immunebased cancer therapies and cancer immunopreventive strategies.

All these data suggest that tumor-mediated immune responses are generated early in the process of cancer development.

If we could modify these responses to induce

desirable Th1 type immunity against early pre-invasive lesions, including those that are pre-malignant, while decreasing (or eliminating) undesirable Th2 responses, we may achieve success in preventing recurrence of pre-malignant lesions and/or preventing transformation from pre-malignant to malignant disease.

As various preventive

vaccination strategies are already being tested pre-clinically and some even clinically, we need to keep in mind the complex and still largely unknown nature of immune regulation of cancer, starting with the earliest stages of cancer development, to induce truly protective and safe immune responses, as they are intended to be used in healthy although high risk individuals.

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B.2.b. Selecting antigen(s) for immunoprevention B.2.b.1. Her2/neu Human epidermal growth factor receptor 2 (Her2) is a transmembrane tyrosine kinase receptor and a member of the ErbB protein family. The Her2 gene product is overexpressed in 18-20% of invasive breast cancers (66) and 13-56% of DCIS lesions (67-69). Her2 is clearly a disease-driving oncogene. Her2-positive status in invasive disease is associated with more aggressive disease and poorer survival. Her2 positivity in DCIS is usually associated with ER/PR negativity, and this subtype of DCIS (ER/PR-, Her2+) has been shown to have a higher risk of disease recurrence. Therapeutic targeting of Her2 has been very effective in the management of all stages of Her2 amplified invasive disease, with two Her2 targeting monoclonal antibodies (trastuzumab and pertuzumab) and the toxin–carrying antibody trastuzumab emtansine approved for use in clinical practice. Other immune-based strategies targeting Her2 in the therapeutic setting have been tested pre-clinically and clinically with variable results. One promising Her2targeting strategy, not only for therapy but potentially also for breast cancer prevention, involves vaccines. Below, we discuss a few examples that are most advanced in clinical development and most promising for use in breast cancer prevention.

The U.S. Military Cancer Institute Clinical Trials Group conducted two complementary phase 1-2 exploratory clinical trials evaluating human leukocyte antigen (HLA) A2/A3-restricted Her2 peptide, referred to as E75, with granulocyte-macrophage colony-stimulating factor (GM-CSF) used as an adjuvant. The lymph node- positive (dose escalation study) and lymph node-negative (dose optimization study) breast cancer patients whose tumors expressed any degree of Her2 were vaccinated after completion of 19

standard adjuvant therapy (70). Of 195 enrolled patients, 187 were assessable with 108 (57.8%) in the vaccinated group and 79 (42.2%) in the control group. The vaccination was safe with dose-dependent immune responses noted. Five-year disease free survival (DFS) was 89.7% in the vaccinated group vs. 80.2% in the control group (p=0.08). However, as a result of trial design, 65% of patients received less than the optimal vaccine doses. In optimally dosed patients, the five-year DFS was noted to be 94.6% yielding a p=0.05 when compared to the control group. In sub-optimally dosed patients the five-year DFS was 87.1%. Voluntary booster vaccinations were eventually allowed, were well tolerated and were able to re-stimulate E75 immunity in patients who had lost responsiveness. Only one disease recurrence occurred among 21 patients who were “optimally boosted”, i.e. women who prospectively received the boosters 6 months after completing the primary vaccination series. Of interest, this vaccine induced intra- and inter-antigen epitope spreading (71, 72). This immune phenomenon is believed to be important in generating immune responses that potentially translate into improved clinical benefits. Additional observations included decreased levels of T regs and TGF-β in the peripheral blood and increased memory CD4+ and CD8+ T cells (73, 74). Interestingly, while these trials enrolled patients with breast cancers expressing all levels of Her2, more robust and longer lasting immune responses were seen in patients with Her2-low disease (evidenced by immunohistochemistry (IHC) staining at 1+ or 2+ or fluorescent in situ hybridization (FISH) <2.0) than patients with Her2 overexpressing tumors (75). This is in contrast to what is observed with standard Her2 targeting strategies where benefits are seen almost exclusively in patients with Her2 overexpressing disease. At 24 months, DFS in E75 vaccinated patients with Her2-low

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disease was 94% vs. 79% for controls (p=0.04), while in patients with Her2 overexpressing tumors DFS in vaccinated patients was 90.3% vs 83.3% for controls (p=0.44). This E75 short peptide vaccine (now referred to as NeuVax or Nelipepimut-S ) was evaluated in the phase III study (PRESENT) in patients with early-stage nodepositive breast cancer with low–to-intermediate Her2 expression (IHC 1+ or 2+ by IHC/FISH) to test its ability to prevent disease recurrence after completion of standard therapy. Enrollment to this trial was just recently completed and data analyses are ongoing. Based on its very good safety profile and promising early results, the group is now planning to test NeuVax in pre-invasive disease (DCIS) in an attempt to move this vaccine earlier in the disease process for use in breast cancer prevention.

There are many vaccine platforms targeting Her2 that have been evaluated clinically in invasive breast cancer. They include (short- and long-) peptide-based vaccines, protein-based, plasmid-based, DNA-based, dendritic cell (DC)-based vaccines, vaccines that deliver Her2 in a viral or bacterial platform, etc. Among these, the strategy that has already been tested in pre-invasive disease (DCIS) is a DC-based vaccine. This vaccine construct included DCs that were activated in vitro with interferon (IFN)-γ and bacterial lipopolysaccharide to become highly polarized DC1-type DCs that secrete high levels of IL-12p70 and were pulsed with Her2 HLA class I and II peptides (76). Twenty-seven patients with Her2 overexpressing DCIS were vaccinated intranodally once a week for 4 weeks prior to surgical resection (76-78). This vaccination induced high numbers of IFNγ secreting T-cells specific for Her2. At surgery, 5 of 27 (18.5%) patients had no residual disease, 11 of 22 (50%) patients with remaining disease experienced a decrease in the size of the residual lesion and loss of Her2 expression. Of interest, the anti-Her 2 immune 21

responses were still observed up to 52 months post-vaccination. Although very promising, this experience suggests that targeting a single antigen may lead to selection of clones that do not overexpress the specific immunologic target allowing their persistence and thus, limit the overall efficacy of such vaccines.

B.2.b.2. Multivalent Preventive Vaccines

One of the ways to address lesion/tumor heterogeneity is to use multivalent vaccines that target several antigens in one vaccine construct/mix. Exploring large genomic data sets available for both pre-invasive and invasive lesions allows identification of potential candidate antigens for targeting in prevention of cancer. In breast cancer specifically, Her2/neu, insulin-like growth factor-binding protein 2 (IGFBP-2) and insulin-like growth factor receptor-1 (IGF1R) have been identified as mechanistically important proteins in breast oncogenesis and shown to be overexpressed in both pre-invasive and invasive breast tumors (79-81). Generally, overexpressed proteins are believed to be immunogenic by exposing hidden epitopes and by increasing the number of peptides available for immune recognition (57, 82). The Tumor Vaccine Group at the University of Washington has extensive experience, particularly in breast cancer vaccines. They are developing a multivalent vaccine for breast cancer prevention using Her2, IGFBP-2 (80) and IGF1R (81) as antigenic targets. Since induction of Th1 immunity provides the best anti-tumor response (by destroying current disease and generating immunologic memory), the group designs MHC class II vaccines that can stimulate Th1 CD4+ T cell responses that can both increase recruitment of CD8+ T cells to the tumor and create a Th1 immune environment (83). They have demonstrated that “self”- tumor antigenic proteins contain

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both Th1- and Th2-inducing epitopes. Screening of these proteins for immunosuppressive epitopes and exclusion of these sequences from the vaccine construct appears to be important for achieving the desired induction of Th1 immunity (81, 84, 85). They have reported that inclusion of any Th2 epitopes in a vaccine prevents development of effective anti-tumor immune responses (84). Accordingly, using their epitope prediction algorithms (86), the group designed a tri-antigen (Her2, IGFBP-2, IGF1R) vaccine which was tested in the Tg-MMTV-neu transgenic mouse mammary tumor model. In mid-life these mice develop atypical ductal hyperplasia (ADH), DCIS and invasive breast cancers that overexpress Her-2, IGFBP-2 and IGF1R, and are ER-low (87). This vaccine was able to prevent development of DCIS as well as progression of DCIS to invasive breast cancer. In vaccinated 18 week-old mice, this multi-antigen vaccine appeared to be safe and more effective in tumor prevention than vaccination with any single antigen or control. Among vaccinated mice 65% had no palpable lesions, and tumors that did develop grew slower and had increased CD8+ T-cell infiltration. Also, survival of vaccinated mice was significantly longer compared to controls, with the longest survival seen in multi-antigen vaccinated mice relative to any individual antigen or control. This protection was CD4+ T cell mediated and depletion of CD4+ cells led to loss of the protective tumor inhibitory effect (87).

These preclinical data demonstrated that the multi-antigen (Her2/IGFBP-

2/IGF1R) vaccine using Th1–specific MHC class II epitopes can be effective in preventing development of pre-invasive disease as well as invasive breast cancer. This tri-antigen vaccine will soon enter clinical testing in patients to evaluate its safety and immunogenicity.

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B.2.b.3. Novel Antigens for breast cancer prevention

B.2.b.3.a. Mammaglobin-A

Mammaglobin-A (MAM-A) is a secretory protein that is overexpressed in 80% of human breast cancers. Its near-universal expression in breast cancer as well as its exquisite tissue specificity makes it an attractive target for breast cancer immunopreventive and/or immunotherapeutic efforts. The evidence of pre-existing immunity to MAM-A in breast cancer patients supports its immunogenicity and suitability as a target for immune-based modulation. A vaccine research group at Washington University, St Louis developed a DNA-based vaccine designed to express the human breast cancer-associated MAM-A antigen. After generating strong pre-clinical data, the group tested this vaccine in an open label phase I study evaluating its safety and immunogenicity. Results demonstrate good vaccine safety with preliminary evidence to suggest that the mammaglobin-A vaccine is able to induce mammaglobin-A specific IFN-γ secreting CD8+T cells in treated patients. These vaccinated patients also demonstrated improved progression free survival (88, 89).

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B.2.b.3.b. α-Lactalbumin

α-Lactalbumin is a protein normally expressed only in lactating breast tissue but with a much greater expression level in breast cancer cells. It is one of the examples of “retired proteins” (55, 56). Jaini et al. (90) reported preclinical efficacy of vaccination with αlactalbumin in a transgenic mouse model of breast cancer. This vaccination strategy generated impressive long-term protection against development of breast tumors. None of the vaccinated mice developed breast cancer, while the control group showed 100% tumor incidence. Also, vaccination with α-lactalbumin was very successful in prophylactically inhibiting the growth of implanted 4T1 breast cancer cells. This elicited immune effect was characterized by influx of TILs into the tumor (predominantly CD4+ T cells as well as CD8+ T cells) with production of large amounts of IFN-γ; all of these are desired immune effects in successful vaccination. The CD8+ T cells were also capable of directly killing 4T1 cells in vitro (90). The authors have proposed a potential application of α-lactalbumin vaccination in prevention of breast cancer in women of post child-bearing years when lactation is readily avoidable.

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B.3. Colorectal cancer immunoprevention

B.3.a. Evolution of adaptive immunity during disease development

Unlike breast cancer, colon carcinogenesis and the development of pre-malignant and pre-invasive lesions (colon adenomas) and invasive colon cancer often occur in an inflammatory microenvironment where bacteria present in the gut provide constant immune stimulation. Therefore, proper self-regulation is required to control these bacteria-induced inflammatory responses, explaining the high prevalence of regulatory T cells and Th2 cells in the normal colonic epithelium (Figure 3). Inflammatory bowel disease (especially ulcerative colitis), which is associated with development of colon cancer, may, in part, reflect a failure of this regulatory immune system to control inflammation induced by gut bacteria. Evaluation of immune infiltrates in colonic polyps and adenomas (including low-grade and high-grade dysplasia) has demonstrated increasing infiltration with inflammatory cells with increasing degree of cell dysplasia and adenoma size (91).

The importance of adaptive immunity in invasive colon cancer is well established. In particular, the density of cytotoxic CD8+ T cells and memory CD45RO+ lymphocyte infiltration in early stage colon cancers was shown to be an independent predictor of complete remission and improved overall survival; the greater the immune cell density, the better the clinical outcome (92). In addition, an immune-based scoring system, where higher scores reflect greater density of CD8+ and CD45RO+ T cells, was reported to

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have superior prognostic significance in colon cancer stages I to IV than the standard staging system (American Joint Committee on Cancer/International Union Against Cancer-TNM -AJCC/UICC-TNM) (93, 94). However, clinical efficacy of tested cancer vaccination strategies used for therapy of advanced disease have proved disappointing despite their ability to induce strong immune responses. This is mainly due to many immunosuppressive mechanisms deployed by advanced cancer, as previously discussed, which present the major obstacle to effective use of cancer vaccines in such settings (95). These experiences have encouraged re-direction of interest in vaccines use to prevention rather than therapy of colon cancer.

B.3.b. Antigen selection for colon cancer immunoprevention

B.3.b.1. MUC1 as a prototypic antigen

One of the strategies to select antigen(s) for immune modulation is to select them based on differential levels of expression between normal and malignant tissue. In addition, post-translational modifications of expressed antigens may generate TAAs suitable for immune targeting. An example of a TAA that represents both mechanisms is Mucin 1 (MUC1) (96). MUC 1 is a large transmembrane protein that is expressed at low levels in glycosylated form on normal epithelia but is overexpressed and aberrantly glycosylated on many human adenocarcinomas including breast, colon, pancreas, lung, ovary and prostate. MUC-1 is a compelling tumor antigen; it ranked second on the NCI cancer antigens prioritization list (96). The aberrantly glycosylated (tumor) form of MUC1 is also overexpressed on pre-malignant lesions, such as adenomatous polyps, and its expression increases with higher degrees of dysplasia. MUC1 overexpression has also 27

been demonstrated in inflammatory bowel disease (IBD), which is associated with inflammation, tumor promotion, and progression to colitis-associated colon cancer (CACC). The aberrantly glycolysated MUC1 acts as a neoantigen and is thus, recognized by the human immune system. It can be targeted for immunotherapy/immunoprevention. Vaccination with MUC1 peptide has been shown to flatten existing polyps and reduce the number of large polyps in transgenic mice expressing human MUC1 (97). Chronic inflammation as well as adenocarcinomas have been shown to induce endogenous antiMUC1 antibodies, which have been associated with survival benefits in cancer patients (98). In pre-clinical experiments Beatty et. al. demonstrated that active immunization against MUC1 can prevent development of colon cancer in a MUC1-expressing transgenic mouse model of inflammatory bowel disease that progresses to malignancy (99). This active immunization against MUC1 did not result in increased inflammation, either by symptoms or histology, and caused no significant autoimmunity. Based on these results, this group initiated a pilot clinical study investigating their vaccination strategy for the prevention of colon cancer (100). They used the same synthetic 100-amino acid MUC1 peptide that had been previously tested with various adjuvants in advanced colon cancer where it showed a good safety profile and the ability to induce MUC1-directed immune responses. Individuals with a history of advanced colon adenomas, who are at high risk of subsequent colorectal cancer, were vaccinated with the 100-mer synthetic MUC1 peptide along with the adjuvant Poly-ICLC (Hiltonol, a TLR-3 agonist). The results showed good immunogenicity in 17 of 39 (43.6%) vaccinated individuals, with high expression levels of elicited anti-MUC1 IgG antibody and long-lasting immune memory. Lack of response in 22 of 39 individuals correlated with high levels of

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circulating immunosuppressive MDSCs that pre-existed the vaccination. This immunization was safe with no adverse reactions seen. Aside from positive immunological results, this study illustrated the importance of an immunosuppressive environment that already exists in some pre-malignant settings. This would necessitate moving prevention strategies to either much earlier stages of oncogenesis, pre-selecting candidates for immunoprevention based on whether or not they have already developed immunosuppression, or using a combination of anti-cancer vaccination with a strategy effectively reversing the immunosuppressive environment (discussed later).

B.3.b.2. Frame shift mutations as immunologic targets Immune-based prevention for colon cancer is also being investigated in the setting of the familial disease, Lynch Syndrome (LS). Inherited mutation in one of the mismatch repair (MMR) genes, MLH1, MSH2, MSH6 and PMS2, accounts for 80-90% of LSassociated colorectal cancers (101). The specific effect imparted by the faulty DNA repair enzymes encoded by mutated MMR genes involves ubiquitous mutations at microsatellite sequences (simple repetitive sequences) in the DNA of somatic tissue (102). The insertion/deletion mutations in coding microsatellites in genes affected by the defective MMR process are ultimately translated into novel C-terminal peptides containing frameshift mutations and hence frameshift peptides that constitute neoantigens (103). The potential of these neopeptides to serve as TAAs for vaccine development is supported by their high immunogenicity. Colorectal cancers that are characterized by MSI are densely infiltrated with lymphocytes. Furthermore, LS individuals with MSI colorectal cancer generate potent T cell and humoral immune responses targeted to frameshift peptides derived from coding microsatellite-containing genes (103, 104). The 29

fact that MSI colorectal cancers have a better prognosis, with a lower incidence of distal organ metastases, than MSI-stable cancer suggests a protective role for the observed immune responses. In sum, the spontaneous tumor infiltration with TILs that target frameshift neoantigens in MSI colorectal cancers provides the basis for current work in developing vaccines that will elicit comparable frameshift directed immune responses in LS patients.

B.4. Pancreatic cancer: immune modulation attempts to alter disease process

Pancreatic ductal adenocarcinoma (PDA), a particularly lethal cancer with only 5% of patients living to 5 years from diagnosis, poses unique challenges to prevention in general and immunoprevention in particular. In contrast to “immunogenic” cancers such as renal cell carcinoma or melanoma, PDA is generally viewed as a “non-immunogenic” cancer that usually lacks tumor-infiltrating effector lymphocytes within the tumor microenvironment (105). PDA is less responsive to immunologic therapy such as single agent immune checkpoint inhibitors (antagonists of CTLA-4, PD-1/PD-L1). These agents have, however, demonstrated positive results in other tumor types such as non-small cell lung cancer that were initially also believed to be “non–immunogenic”(6, 7, 106, 107). In addition, because few tumor antigens have been identified in PDA, the entire tumor cell has frequently been used in vaccine constructs in immunotherapy trials for this disease. The group at Johns Hopkins University has a long history of investigating immunotherapy and now immunoprevention strategies in PDA. They developed GVAX, an allogeneic GM-CSF-secreting whole-cell pancreatic tumor vaccine. The immunologic benefit of this vaccine is evident in its ability to induce intratumoral lymphoid aggregates.

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These immune structures reflect ongoing adaptive immune responses and appear within 2 weeks of GVAX treatment in 85% of resectable PDA patients vaccinated prior to and after surgical resection.

This response indicates that the tumor microenvironment

undergoes considerable change in response to this immunization strategy (108). Furthermore, this GM-CSF-secreting whole-cell vaccine has been shown to elicit immune responses targeted to mesothelin, an antigen expressed essentially in all PDAs (105, 108, 109). In a phase 2 trial of adjuvant GVAX for resectable PDA, mesothelinspecific CD8+ T-cell responses were shown to correlate with improved disease-free survival when compared to historical controls (108, 110). Low-dose cyclophosphamide has been combined with GVAX to inhibit T regs and alleviate their immunosuppressive effects, resulting in improved activity on initial analyses (105, 108, 111). Primary immunoprevention of PDA is currently being studied in mouse models genetically engineered to express mutant Kras and p53 in the pancreas, which drives PDA development. An attenuated intracellular Listeria monocytogenes vaccine that expresses the entire mutant KrasG12D gene product (LM-Kras) was shown to induce CD4+ and CD8+ T-cell immune responses against multiple mutated antigenic epitopes (108).

B.5. Human Telomerase Reverse Transcriptase (hTERT) as a potential universal target for cancer prevention

Telomeres are repetitive non-coding DNA sequences located at the ends of chromosomes. Most human somatic cells progressively lose telomeric DNA with each cell division leading to replicative senescence and limited potential for proliferation (112). Telomerase is an enzyme that maintains telomeric DNA that would otherwise be

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progressively lost during repetitive cell divisions. Most normal human cells do not express telomerase activity and therefore gradually lose their telomeric DNA over successive cell divisions. However, most human malignancies (>85% regardless of cancer type) do exhibit strong telomerase function (113) with high expression seen also in putative cancer stem cells (114). Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase and a rate-limiting component of the telomerase complex. Its expression is tightly correlated with telomerase activity. hTERT is not only highly expressed in the majority of human cancers (115, 116) but has also been shown to be one of the defined genetic factors allowing the conversion of normal human epithelial cells and fibroblasts into tumor cells (115, 117). Inhibition of telomerase activity in hTERT-positive tumors resulted in progressive shortening of telomere length and death by apoptosis (115, 118). Also, hTERT has been reported to be immunogenic both in vitro and in vivo with naturally occurring hTERT-specific immunity detected in cancer patients with various malignancies (115, 119).

All these important attributes make

hTERT an attractive and possibly “universal” antigen for immune targeting in treatment and/or prevention of many human cancers. Extensive pre-clinical data together with several early clinical studies evaluating various vaccination strategies (peptide-based, DC-based, etc.) to target hTERT in advanced cancers have shown consistently good safety and immunogenicity of this approach (115, 120). Although clinical benefits were scarce, as expected in advanced malignancy, several long-term survivors participating in these trials have been noted, with all of them demonstrating durable vaccine-specific Tcell responses. These responses appear to be enhanced and sustained by repeated booster vaccinations with no additional toxicity observed. However, some normal tissues such as

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bone marrow stem cells, colonic crypt epithelial cells, basal keratinocytes and gonadal cells (113, 120, 121) do express hTERT, posing potential safety concerns. Thus far, these toxicity concerns are not supported by available clinical trial data where mainly flulike symptoms and local reactions at the injection site have been seen despite good immunogenicity of the tested hTERT vaccine formulations. Furthermore, the study evaluating an hTERT vaccine in non-small cell lung cancer (122) included assessment of progenitor cell activity in bone marrow samples where no decreased colony-forming ability was detected. Also, spontaneous anti-hTERT immune responses in cancer patients are not shown to be accompanied by autoimmunity. Pre-clinical toxicity testing has also demonstrated that hTERT-specific cytotoxic T lymphocytes (CTLs) do not lyse telomerase–positive CD34+ hematopoietic progenitor cells (115). Although experience with hTERT vaccines is still relatively limited as compared to other approaches, to date safety and early efficacy data are encouraging. Currently, the second-generation vaccines are addressing strategies to enhance cellular immunity against hTERT. Close monitoring is ongoing in order to assess these more robust immune responses for improved efficacy, while still maintaining acceptable side effect profiles (123).

IV. Challenges and Future Perspectives

Our increasing understanding of the role played by the immune system in carcinogenesis has enabled the design of improved immunological interventions for the therapy and prevention of cancer. However, cancer prevention, rather than therapy, with its lower level of pre-existing immunosuppression appears to be the most promising setting for the use of immuno-modulatory strategies. Nevertheless, in order to optimize

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such preventive applications, we need to better understand the mechanisms of early carcinogenesis and their immune regulation, which are virtually unknown in the majority of tumor types. Current investigations of pre-malignant and pre-invasive lesions, their antigenic repertoire, immunological milieu and immune modulation, identification of regulators of their progression and regression should provide a stepping stone to better appreciation of the earlier stages of human oncogenesis and its interplay with immunity.

The application of immune-based strategies to cancer prevention must be accompanied by a strong safety profile, given the healthy state of the target population. At this time clinical experience with various immunomodulation strategies points towards cancer vaccines as the most suitable approach. However, it is becoming progressively evident that even in the earlier stages of cancer development (e.g. pre-malignant, preinvasive lesions) immunosuppression is already operational. For this reason it is suspected that cancer vaccines alone may not be enough to produce satisfactory results. They will likely require a combination with other approaches to overcome these immunosuppressive forces. Possible combination partners could include either standard cancer chemopreventive drugs or other well-tested and available agents that exert immunomodulatory properties. Examples could include aspirin and COX-2 inhibitors (124-126), aromatase inhibitors, curcumin, retinoids or bisphosphonates (124).

In

addition, although not acceptable in currently approved dosing due to potential toxicity, checkpoint inhibitors are, to date, the most potent agents for overcoming cancer-induced immunosuppression. Therefore, combining cancer vaccines with checkpoint inhibitors at altered doses/schedules, to maintain their efficacy while minimizing/eliminating toxicity, appears to be a very attractive approach. 34

Additional challenges remain, in particular the development of improved tools for defining truly at risk populations, reliable predictive/prognostic biomarkers of immune responses/side effects and clinical benefits from immunopreventive strategies. Also, optimal timing of immune intervention to intercept cancer early enough to be effective and safe, optimal duration/schedule of intervention/vaccinations, and optimal design of clinical trials to evaluate these strategies in primary cancer prevention still are not defined and must be addressed. However, these approaches hold great promise, and as a result cancer immunoprevention appears to be within our reach.

Finally, immunopreventive measures must be integrated into the larger domain of cancer preventive interventions, specifically screening. This need is exemplified most starkly by the recent success of HPV prophylactic vaccines. For years Papanicolaou smears (PAP smears) have been administered, with considerable mortality benefit, to detect early stage cervical cancer and precancerous lesions. The addition of HPV testing to this old standard screen improved the positive predictive value of overall screening for cervical cancer. Now, with the implementation in appropriate at-risk groups of the three currently available HPV vaccines, new strategies that integrate the various screening modalities with the new chemopreventive interventions are critical (127). The same idea of integrating the two basic approaches to prevention, chemoprevention and screening, will be necessary for optimal cancer prevention at other disease sites once effective preventive interventions, including immunopreventives, are developed.

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adaptive immunity. However, with further mutations and changes in the tumor microenvironment, immunoediting further shapes the immune responses. This, in turn, results in an equilibrium of actions in which elimination of rogue clones are juxtaposed against changes in the immune environment. The interplay of these processes eventually lead to escape of some clones due primarily to tumor-associated immunosuppression. Figure 3. Evolution of adaptive immunity during progression of breast and colon carcinogenesis. Modified from Marquez et al. Cancer Prevention Research 2015, with permission from the corresponding author: 0 (no infiltrate), ↑ (low infiltrate), ↑↑ (intermediate infiltrate), ↑↑↑ (extensive infiltrate), ↑↑↑↑ (very extensive infiltrate).

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Table 1. Infectious Agents Associated with Cancer Infectious Agents:

Associated Cancers:

Viruses Epstein-Barr virus (EBV)

Burkitt lymphoma Hodgkin lymphoma Non-Hodgkin lymphoma Nasopharyngeal carcinoma NK/T-cell lymphoma

Hepatitis B virus (HBV)

Hepatocellular carcinoma

Hepatitis C virus (HCV)

Hepatocellular carcinoma Non-Hodgkin lymphoma

Human papillomavirus types 16, 18, and others (HPV)

Anal cancer Cervical cancer Oral cancer Oropharyngeal cancer (cancer of the base of the tongue, tonsils, soft palate, posterior pharyngeal wall) Penile cancer Vaginal cancer Vulvar cancer

Human immunodeficiency virus 1 (HIV 1)

A variety of immunosuppression-related cancers Anal cancer Cervical cancer Conjunctiva cancer (the eye) Hodgkin lymphoma Kaposi sarcoma Non-Hodgkin lymphoma

Human T-cell lymphotropic virus 1 (HTLV1)

Adult T-cell leukemia/lymphoma Lymphoma

Kaposi sarcoma herpesvirus/ human herpesvirus 8 (KSHV/HHV 8)

Kaposi sarcoma Primary effusion lymphoma

Merkel cell polyomavirus (MCV)

Merkel cell carcinoma (type of skin cancer)

Other infectious agents Helicobacter pylori (bacterium)

Gastric (stomach) cancer

Liver flukes (parasite)

Cholangiocarcinoma

Schistosomes (parasite)

Bladder cancer

45

Mast cell

Macrophage Natural killer cell Eosinophil Neutrophil Basophil

gd T cell

B cell

Dendritic cell

Antibodies

T cell

Natural killer T cell CD4+ T cell

Cytokines Complement proteins

CD8+ T cell

Figure 1: Major Cell Types in Innate versus Adaptive Immunity. Major players in Innate immune response include Dendritic cells, Mast cells, Macrophages, Natural killer cells, Eosinophils, Neutrophils, Basophils, cytokines and Complement proteins. Natural killer T cells and T cells are common to both innate and adptive immune response while B cells and T cells are major players for adaptive immune response alone forming both humoral and cellular immune responses.

Escape

Elimination

Equilibrium

Immune response

Cancer growth

Figure 2: Immune Response During Carcinogenesis. In the early stages most pre-cancerous lesions are intercepted by immune responses via a well defined mechanism called immunosurveillance. These responses involve both innate and anti-tumor adaptive immunity. However, with further mutations and changes in the tumor microenvironment, immunoediting further shapes the immune responses. This, in turn, results in an equilibrium of actions in which elimination of rogue clones are juxtaposed against changes in the immune environment. The interplay of these processes eventually lead to escape of some clones due primarily to tumor-associated immunosuppression.

46

Breast cancer Colorectal cancer

CD3/Th1 cells

0

↑

↑↑

↑↑↑

↑↑↑↑

FoxP3/Th2 cells

↑

↑

↑

↑↑

↑↑↑↑

CD68 cells

0

↑

↑

↑↑

↑↑↑↑

Th17 cells

0

0

0

0

↑↑

Normal

Hyperplasia

Atypical hyperplasia / dysplasia

Carcinoma in situ

Invasive carcinoma

↑

↑

↑↑

↑↑↑

↑↑↑↑

↑↑↑↑

↑↑↑↑

↑↑↑↑

↑↑↑

↑↑

CD68 cells

↑

↑

↑

↑↑

↑↑

Th17 cells

↑

↑↑↑

↑↑↑

↑↑

↑↑

CD3/Th1 cells

FoxP3/Th2 cells

Figure 3. Evolution of adaptive immunity during progression of breast and colon carcinogenesis. Modified from Marquez et al. Cancer Prevention Research 2015, with permission from the corresponding author: 0 (no infiltrate), ↑ (low infiltrate), ↑↑ (intermediate infiltrate), ↑↑↑ (extensive infiltrate), ↑↑↑↑ (very extensive infiltrate).

47