Immune-related strategies driving immunotherapy in breast cancer treatment: a real clinical opportunity.

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Review

Immune-related strategies driving immunotherapy in breast cancer treatment: a real clinical opportunity Expert Rev. Anticancer Ther. Early online, 1–14 (2015)

Andrea Ravelli1, James M Reuben2, Francesco Lanza1, Simone Anfossi2, Maria Rosa Cappelletti3, Laura Zanotti3, Angela Gobbi3, Manuela Milani3, Daniele Spada3, Paolo Pedrazzoli4, Massimo Martino5, Alberto Bottini3, Daniele Generali*3 and on behalf of the Solid Tumor Working Party of the European Blood and Marrow Transplantation Society (EBMT) 1 U.O. Ematologia e CTMO, AZ. Istituti Ospitalieri di Cremona, Viale Concordia 1, 26100 Cremona, Italy 2 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 3 U.O. Multidisciplinare di Patologia Mammaria, U.S. Terapia Molecolare e Farmacogenomica, AZ. Istituti Ospitalieri di Cremona, Viale Concordia 1, 26100 Cremona, Italy 4 U.O. Ematologia con Trapianto di Midollo Osseo e Terapia Intensiva, Dipartimento di Oncologia, AZ. Ospedaliera Bianchi-Melacrino-Morelli, 89100 Reggio Calabria, Italy 5 S.C Oncologia, Dipartimento di Onco-Ematologia, Policlinico IRCCS San Matteo, Pavia, Italy *Author for correspondence: Tel.: +39 0372 408 042 Fax: +39 0372 401 72 [email protected]

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Because its original use as a treatment for hematologic disease, more recently immunotherapy has emerged as a novel effective therapeutic strategy for solid malignancies, such as melanoma and prostate carcinoma. For breast carcinoma, an immunologic therapeutic approach has not been well evaluated, even though there is evidence to suggest it would be a successful novel strategy, especially taking into account the high mortality rate of the most aggressive variants of this heterogeneous disease. Here, we briefly describe the most recently awarded immune-based therapies with a consolidated or potential implication for the treatment of solid malignancies. We focus on immune checkpoints and on the clinical potential of their abrogation, with a further overview of novel vaccine-based approaches and the most relevant immunotherapeutic techniques. We aim to provide an exhaustive review of the most promising immune-therapeutic agents that may have implications for breast cancer treatment. . breast cancer . breast cancer therapy . breast cancer vaccine ipilimumab . pidilizumab . tremelimumab . vaccination

KEYWORDS: adoptive T-cell therapy checkpoint blockade

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Breast carcinoma (BC) is the most prevalent cause of cancer-related death in women worldwide [1]. Although the less aggressive variants of this disease can today be treated through a series of well-consolidated therapy strategies with a generally good expectation of survival, the most severe disease forms – such as TripleNegative BC (TNBC) and Inflammatory BC (IBC) – currently do not benefit from wellassessed treatments, mainly due to their intrinsic lack of defined molecular targets and their high aggressiveness. Relatively recently, the immune system has acquired growing relevance in the context of cancer, and its overall role has been largely debated. It is now generally accepted that immune cells can exert both anti-tumor and tumor-promoting activities, but their primary function is to eradicate transforming cells during early carcinogenesis [2]. It has been demonstrated that the balance between the two effects can be changed at the molecular level to induce an anti-cancer immunity, therefore introducing new opportunities to counteract malignancies. Novel immune-based therapies can exploit several 10.1586/14737140.2015.1042864

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immune

mechanisms involved in the immune response to cancer; overall, the rationale that drives them is to boost or even to restore physiological machinery whose function represents a natural defense against malignancies. Immune strategies are, however, subordinated to many factors: first to the presence of anti-tumor immune cells, which are the first requirement for the effective establishment of an anti-cancer response, and second, of no less importance, to the immunogenicity of the tumor to be treated. Because it generally meets all these requirements, BC disease could be the perfect candidate for immunotherapy. Tumor-infiltrating lymphocytes & immunogenicity of BC

Immune cells are often found to infiltrate the tumor microenvironment in almost all BC patients [3]. Tumor-infiltrating lymphocytes (TILs) can be found directly inside the neoplasm or they can be located inside the stroma, and their clinical relevance depends mainly on their nature. Indeed, different populations of infiltrating lymphocytes are

2015 Informa UK Ltd

ISSN 1473-7140

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Review

Ravelli, Reuben, Lanza et al.

associated with diverse clinical outcomes: CD8+ T-cytotoxic cells, CD4+ Th1 cells, natural killer (NK) cells, immunogenic dendritic cells (iDCs), and type-1 macrophages are able to combat tumor growths, therefore representing positive predictive factors when detected. On the contrary, FOXP3 CD25+ CD17+ T-regulatory cells (Tregs), CD4+ Th2 cells, myeloidderived suppressor cells (MDSCs), tolerogenic DCs (tDCs), and type-2 macrophages are related to worst outcomes when observed to infiltrate solid tumors in consistent amounts [4]. In the context of BC, the presence of infiltrating-cytotoxic CD8+ T-cells and macrophages is considered an independent positive prognostic factor and an indicator of longer overall survival (OS) [5]; as a confirmation, the same kind of lymphocytic infiltration is also predictive of better responses to chemotherapy [6]. Experimentally, two randomized clinical trials recently highlighted that stromal lymphocytic infiltrates generally constitute a strong positive prognostic factor for BC patients undergoing adjuvant therapy, with particular regard to the triple-negative subset [7]. Moreover, in a multivariate analysis comprehensive of 1058 BC cases, the presence of CD8+ TILs was clearly related to favorable outcomes when anthracycline/ taxane-based neoadjuvant therapies were applied [8]. Instead, the presence of FOXP3 Treg cells is considered a negative prognostic factor, usually observed to be increased in strict association with the most severe BC subtypes [9]. Furthermore, metastatic BC patients are usually characterized by augmented levels of MDSCs, a cell population which is commonly observed to represent a negative prognostic factor [10]. Physiologically, both FOXP3 Treg cells and MDSCs are strongly implicated in the suppression of cytotoxic activities, and their presence is normally indispensable for maintaining host homeostasis because they efficiently keep under control cytotoxic lymphocytes and avoid the insurgence of deleterious auto-immune reactions. However, in the BC context, the amount of these immune-suppressive cells is usually found to be increased, and this abnormally high presence reflects the abrogation of antitumor cytotoxic activities; as discussed below, this phenomenon explains the cancer immune-evasion process. Immune evasion

During carcinogenesis, the less immunogenic tumor cell variants are environmentally selected through a multi-step process termed ‘immunoediting’. This phenomenon is divided into an initial elimination phase, during which the innate arm is able to detect and destroy early-transforming cells, and continues to an equilibrium phase, a step which shows the contemporary elimination and survival of malignant cells, overall resulting in a temporary arrest of tumor growth. Immune escape, which is the last event, is characterized by the uncontrolled proliferation of those tumor clones which have acquired the ability to override immune-mediated recognition, thanks to surface antigens down-regulation and to a consequently reduced immunogenicity [11]. In addition to the increased ‘invisibility’, breast tumor cells appear to acquire the ability to actively suppress cytotoxic cells through the exploitation of the so-called inhibitory doi: 10.1586/14737140.2015.1042864

immune-checkpoints, a physiological machinery which works as a brake for cytotoxic cells and which normally keeps the immune system under control (that is, CTLA-4 and PD/ programmed cell death receptor ligand (PDL1)/PDL2, details described below). As a result of the typical over-exploitation of immune checkpoints operated by malignant cells, cytotoxic CD8+ T-cells, NK cells, and NKT cells are found to be reduced in amount and, if present, to be mostly ineffective [12]. Besides active mechanisms, BC cells can also indirectly escape from eradication through the selective promotion of immunosuppressive cell clones, such as FOXP3 Tregs and MDSCs. The proliferation of Treg cells is stimulated by cytokines, such as IL-10 and TGF-b, which are secreted by tumor cells and by Treg cells themselves. These molecules, in concert with VEGF, exert a strong immunosuppressive action on effector T-lymphocytes and are also able to further induce the expression of FOXP3 transcription factor on cytotoxic lymphocytes and epithelial cells [13,14]. Similarly, MDSCs are stimulated to proliferate and then exert a strong immunosuppressive activity through the production of reactive-oxygen species and cytokines release. MDSCs can also inhibit cytotoxic activity by overexpressing indoleamine 2.3-dioxygenase (IDO), a catabolic enzyme which acts on tryptophan as a substrate; overexpression of IDO results in tryptophan depletion and consequently in a lack of nutrients for effector T-lymphocytes [15]. The proliferation and survival of breast tumor cells is moreover enhanced in the context of chronic inflammation, because proinflammatory cytokines, such as TNF-a, IL-6 and IL-10 are massively released by both tumor cells and by some innate immune cells, overall mediating a proliferative effect on malignant cells through a mechanism involving NF-kb [16]. Persistent inflammation also boosts local invasion, induces metastasis development, and enhances angiogenesis, these phenomena being mainly caused by TGF-b and IL-17 in concert with other proinflammatory cytokines [17]. Chronic inflammation is also characterized by large amounts of PgE2, a highly immunosuppressive prostaglandin originated by COX-2 activity, an enzyme found to be highly expressed on breast tumor cells with a particular clinical relevance for the triple-negative subset [18]. Nevertheless, increased COX-2 expression levels are predictive of increased probabilities of relapse and are associated with a subgroup of high-risk ductal carcinoma in-situ patients [19]. In addition to its classic pro-inflammatory effect, PgE2 selectively promotes Treg cells proliferation through augmented expression of FOXP3 transcription factor on CD4+ T-cells located in the tumor microenvironment [20]. As a last parameter concurring in the establishment of proper conditions for tumor escape, dentritic cells (DCs) play a major role because they have been found to be switched toward an immunosuppressive activity in the BC contexture; DCs maturation and survival is inhibited by TGF-b, VEGF, and other inhibitory cytokines which are massively secreted by tumor cells. As a result, tumor-infiltrating DCs often exhibit an immature phenotype and circulating DCs have been observed to belong to the tolerogenic arm and to show a reduced Expert Rev. Anticancer Ther.

Immunotherapy and breast cancer

functionality. Moreover, circulating and infiltrating DCs are often found in a preapopoptotic state, and for BC this phenotype is directly associated with metastasis to the sentinel lymph-node and to the absence of CD8+ T infiltrates [21].


Immunomodulatory effects of standard chemotherapeutics

The immune signature of a patient can predict the outcome of response to chemotherapy. Because of this, the mechanisms of action of a large number of currently used chemotherapeutic agents have been re-evaluated [22]. Anthracyclines traditionally exert anti-tumor activity through DNA intercalation and inhibition of topoisomerase-II, therefore are categorized as antitumor antibiotics. However, since the first observation of their immunomodulatory effect, anthracyclines have been found to stimulate tumor cells to become more susceptible to cytotoxic attack, thereby requiring the presence of CD8+ T-cells as a necessary parameter for the antitumor activity [23]. During latestage apoptosis and after cell death, anthracyclines also induce tumor cells to expose calreticulin on their surface, to release chromatin-binding protein high-mobility group B1 (HMGB1) and ATP, which stimulate the activation of DCs through interaction via CD91 and TLR4 and give rise to immunogenic death [22,24]. Cyclophosphamide, a standard alkylating agent, when administered at metronomic doses mediates a strong immunestimulating effect through the inhibition of FOXP3 Treg cells. Specifically, at daily weak doses, cyclophosphamide mediates a down-regulation of the transcription factor FOXP3, thus, decreasing the number and the functionality of Treg cells. Treg cells inhibition then results in effector T-cells proliferation and in restoration of NK cells functionality, an innate immune-cell subpopulation whose cytotoxic activity is strongly limited by FOXP3 Treg cells [13]. Hence, metronomic doses of cyclophosphamide (usually 50 mg/day/month) have demonstrated a strong effectiveness, also in combination with vaccine therapies for treatment of metastatic diseases [25]. Nonetheless, estrogens can exert powerful immunemodulating effects also, but given the complexity of the variables involved into estrogen-mediated immunomodulation, the phenomenon is still under evaluation. In general, estrogens exert diverse effects in terms of immune differentiation and functionality: almost 20 years ago, it was demonstrated that estrogen administration is able to induce reversible thymic atrophy and to suppress CD4+ and CD8+ populations in a non-specific fashion in mice [26]. More recently, it has been documented that estrogenic stimulation can exert a potent apoptotic effect on T-cells, which are found to express estrogens receptors in a consistent amount [27,28]. The same expression profile has been detected on DCs and on B cells: estrogenic stimulation on DCs brings to the selective proliferation of the tolerogenic arm of these cells, and is accompanied by the contemporary up-regulation of immunosuppressive cytokines (IL-10 and TGF-b) and by an abnormal expression of PDL1/L2 [29]. B cells, then, have been observed to develop autoreactivity informahealthcare.com

Review

in response to estrogenic stimulation, a deleterious effect which, at its last stage, may give rise to systemic inflammation, a classical tumor-promoting condition [30,31]. Moreover, as it is well known that estrogens regulate Treg numbers in mice and promote the proliferation of human Tregs, it could be hypothesized that blocking estrogen receptor-alpha signaling would abrogate Tregs and be associated with response to hormonal therapy and increased survival in BC. It has been demonstrated that aromatase inhibitors have an indirect antitumor mechanism of action through reducing Tregs in breast tumors and may be of use in estrogen receptor-a-negative tumors in combination with immunotherapy approaches [32]. Among other classic chemotherapeutics, taxanes have been observed to work in concert with anti HER2 vaccines: paclitaxel demonstrated an overall augmented effectiveness of antiHER2 vaccines in combination with GM-CSF in transgenic mice, whereas docetaxel has been found to boost vaccine therapy only if administered before [33]. In a cohort of BC patients, taxane-based therapies have been associated with the proliferation and the improved functionality of T-cells and NK cells [34]. Also 5-fluorouracil seems to elicit an immunologic activity in addition to its standard anti-metabolite effect, being able to stimulate antigen uptake and cross-presentation on DCs [35]. Monoclonal antibodies (mAbs), one of the latest and smartest therapy options as of today, have also been seen to be strictly linked to the immune system too. Trastuzumab and pertuzumab – two mAbs targeting human epidermal growth factor receptor 2 (HER2/neu) in clinical use for HER2 positive BC – are typically able to stop tumor growth and to shrink the neoplasm through HER2-receptor blockade [36]. However, the pharmacodynamics of these drugs has been re-evaluated, revealing a strictly immunologic mechanism of action beside the classical receptor blockade: the Fc portion can indeed bind tumor cells and activate the innate immune system, therefore acting as a proper opsonin. The phenomenon, which is referred to as antibody-dependent cellular cytotoxicity, is now considered a major mechanism of action of trastuzumab and pertuzumab and seems to strongly contribute to explaining the improved anti-tumor activity observed when the two drugs are administered together [37]. Immune checkpoints

As briefly mentioned, the immune system regulates through several contemporary and subsequent interactions commonly referred to as ‘immune checkpoints’, which physiologically limit the extension and the severity of the immune response, especially regarding the adaptive arm. Immune checkpoints work in concert to maintain host homeostasis and to avoid autoimmune reactions, and overall can be classified into co-stimulatory and co-inhibitory regulatory loops. Major costimulatory checkpoints are represented by the binding of Tolllike receptor on T-cell surface with MHC-associated peptides, or either by the interaction between the T-cell CD28 surface molecule and CD80 (B7.1) or CD86 (B7.2) receptors on antigen-presenting cells (APCs) [38]. Comprehensively, these doi: 10.1586/14737140.2015.1042864

Review


MHC-Ag

TCR

APC

T-cell

CTLA-4


PD-1

IL-10 ROS Arginase

PD-L1

PD-L1 Tumor cell

PD-L1

MDSC

CD80 or CD86 gives rise to an inhibitory signaling pathway inside T-cells, which involves the engagement of two phosphatases (SHP2 and PP2A) and leads to the inhibition of protein tyrosine kinases activity. As a result, T-lymphocytes do not proliferate, undergo apoptosis and become anergic, a condition characterized by a total loss of cell functionality [41]. While the specific mechanism of action requires further investigation – although some data suggest a down-regulation of the TCR – it is clear that the steric blockade of CD80 and CD86 operated by CTLA-4 gives rise to a strong immunosuppression because it impedes the remaining lymphocytes from being activated, in addition to the effect of the outcompetition of CTLA-4 over CD28 [42,43].

IL-10

Programmed death-1 & PDL1/PDL2

T-reg cell

PD1 – or CD279, programmed death 1 – is a tyrosine kinase receptor, which is IL-10 mainly expressed on DCs, B-lymphoIL-35 cytes, activated macrophages, NK cells, TGF-beta and activated T-lymphocytes. Its ligands Figure 1. Representation of immune checkpoints and major regulatory loops PDL1 (or B7-H1) and PDL2 (or within the tumor microenvironment. B7-H2), in addition to their presence on APC: Antigen-presenting cell; MDSC: Myeloid-derived suppressor cell; hematopoietic cells, are widely expressed ROS: Reactive-oxygen species; TCR; T-cell receptor. on vascular endothelial cells and on the majority of peripheral tissues, whereas interactions represent some essential steps for the engagement PDL2 is mostly expressed on hematopoietic cells [44]. Similarly of a T-cell adaptive response (FIGURE 1). Negative checkpoints, on to what was observed with CTLA-4, PDL1 also binds the contrary, are based on inhibitory interactions that regulate CD80 (B7.1) with much greater affinity than CD28, giving immune activity in a negative fashion and that are equally fun- rise to an out-competition [45]. The binding between PD1 and damental to limit lymphocytes cytotoxicity. Two major recep- its ligands represents an inhibitory loop: when a binding tors whose binding with their respective ligands gives rise to an occurs, the intracellular domain – which consists of an inhibiinhibitory-regulatory loop have been identified as cytotoxic tory motif united to a switch one – undergoes a phosphorylaT-lymphocyte antigen 4 (CTLA-4) and programmed-death-1 tion that leads to the inhibition of PI3K [46]. Although the (PD1). Their structure is typical of immunoglobulin-like trans- intracellular enzymatic pathways are slightly different, PD1 and membrane proteins and the binding between them and their CTLA-4 share the same immunosuppressive effect because they ligands results in T (and B)-cell exhaustion and loss of func- both induce an anergic, ineffective T-cell response [47]. tionality (anergy). On behalf of their biological function, CTLA-4 and PD1 are therefore known also with the title of Clinical potential of immune checkpoints in BC ‘immunological brakes’ [39]. The preclinical discovery and characterization of immune checkpoints makes it possible to reverse the immunosuppression typiCytotoxic T-lymphocyte antigen 4 cal of the microenvironment of the majority of solid CTLA-4 is a member of the immunoglobulin-superfamily trans- neoplasms [39]. Immune-checkpoint abrogating agents and their membrane receptors. It is expressed on the majority of immune targets (FIGURE 2). The CTLA-4 and PD1/PDL1/L2 inhibitory cells, especially on both CD4+ and CD8+ T-lymphocytes. Spe- pathways represent some of the most important mechanisms cifically, CTLA-4 translocates on T-cell surface when the T-cell exploited by malignant cells to take advantage over the immune receptor (TCR) is stimulated by a strong or durable stimulus, system. Many solid neoplasms have indeed been observed to and then it competitively binds to CD80 (B7.1) and express both PD1 and its ligand PDL1 at high levels, often with CD86 (B7.2) located on professional APCs with more affinity the contemporary presence of anergic infiltrating CD8+ T-lymand avidity than CD28 [40]. The binding between CTLA-4 and phocytes [48,49]. Furthermore, both PD1 and CTLA-4 act as FOXP3

CTLA-4

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Expert Rev. Anticancer Ther.



Review

positive proliferation modulators for Ipilimumab FOXP3 Treg cells, whence they are highly expressed [50]. All these observations perTremelimumab mitted the development of abrogating agents that specifically target immune CTLA -4 checkpoints. The result of the inhibition T-cell of an immune-inhibitory pathway is theo-4 LA CT retically an immune-stimulating effect, and this hypothesis has been already confirmed in preclinical models [51]. As of T-reg cell today, a CTLA-4-abrogating agent (ipilimumab) is in clinical use for the treatment PD-1 MEDI4736 of advanced melanoma, a highly immunoMPDL3280A genic malignancy. The drug demonstrates PD-L1 a high efficacy and is commonly administered in combination with other immuneNivolumab PD-L1 based or standard therapeutic regimens [52]. MK-3475 However, the tier that brought to the PD-L1 (Pembrolizumab) approval of ipilimumab has been characPidilizumab terized by a considerable amount of diffiMDSC culties. In preclinical models, the initial silencing of CTLA-4 resulted in strong Tumor cell lymphoproliferative events both in vitro and in vivo involving CD4+ and CD8+ T-lymphocyte expansion, with special regard to CD4+ T-cells. As a consequence, CTLA-4 knockout mice died after 3–4 Figure 2. Immune-checkpoint abrogating agents and their tatgets. weeks due to the development of autoimMDSC: Myeloid-derived suppressor cell. mune syndromes [53,54]. However, an antitumor effect was also observed, suggesting that a better understanding of the mechanisms related to Since the observation that a subgroup of melanoma patients was CTLA-4 abrogation would eventually retrieve some kind of suc- more likely to benefit from this therapeutic agent, this has furcess, especially in terms of reduction of toxicity rates. Presently, ther highlighted the need for clinically useful biomarkers that ipilimumab shows documented antitumor activity with an opti- could help in determining how best to incorporate these new mal dose of 3 mg/kg and exhibits a relatively low toxicity, partic- agents into treatment algorithms for patients with specific diseases. A series of potential markers and clinical prediction panels ularly if compared with classic chemotherapeutics [52]. The clinical potential of anti PD/PDL agents arises from the have been individuated: first, a high presence of TILs is strongly fact that a great number of solid tumors express PD-L1, predictive of good response to anti CTLA-4 therapy, especially including breast, colorectal, lung, renal-cell, and pancreatic car- if accompanied by high IDO and FOXP3 expression levels. cinomas [55–57]. In human hepatocellular carcinoma, overexpres- Also, an incrementation of absolute lymphocyte count has been sion of PDL1 is associated with worst prognoses, reflecting the found to increase the probabilities of response to treatment, as influence of the highly immunosuppressive microenviron- well as the up-regulation of HLA-DR/CD45RO on T-cells surment [58]. For BC, up-regulation of PDL1 is clearly predictive face [61,62]. These observations have been retrieved from a group of worst clinical outcomes, and has been found to be typical of of melanoma patients, but may putatively be extended to BC. the more aggressive tumor subtypes, especially of TN basal-like Moreover, a standardization of the cellular immune assays across carcinomas [59]. The overexpression of PDL1 is often correlated trials would be very important for predicting the effects of therawith the contemporary presence of high PD1-expressing TILs peutic cancer treatment. Currently, assays mostly target detecwithin the tumor microenvironment, and analysis of PD1/ tion of T-cell function, such as proliferation and cytokine PDL1 expression could predict patient’s prognosis [60]. Accord- release; however, T-cell phenotype analysis in peripheral blood ingly to the immunoediting process, PDL1 overexpression has and/or tumor sites may also be considered in the future. been putatively related to a long-lasting, chronic inflammatory response, which indirectly selects tumor cell clones that better Immune checkpoint blockade Cytotoxic T-lymphocyte antigen 4 up-regulate inhibitory checkpoints. However, the research of measurable markers to assess the Ipilimumab (Yervoy) is a recently approved CTLA-4-abropotential clinical efficacy of ipilimumab is currently ongoing. gating agent, a fully humanized IgG1 monoclonal antibody informahealthcare.com

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in clinical use for metastatic melanoma treatment [52]. Outside melanoma, however, data on its clinical efficacy are much less abundant and, for BC specific cases, presently only preclinical data are available: promising results were obtained in a study of ipilimumab monotherapy on EMT6 BC cell lines, in which tumor regression was observed in 40% of mice. In the same study, the efficacy of a combinatorial treatment between ipilimumab and ixabepilone has been evaluated, showing an impactful 100% of mice undergoing complete tumor regression [63] Moreover, ipilimumab activity on murine BC models was tested in association with fractionated radiotherapy, revealing a strong synergistic effect and an impressive increase in terms of tumor regression and metastases inhibition, probably because radiations dictate immunogenic death of cancer cells and also eliminate immunosuppressive cells [64,65]. Tremelimumab – a human IgG2 monoclonal antibody – is a novel CTLA-4 antagonist whose pharmacodynamics and toxicity are actually under evaluation. When in a Phase III trial, tremelimumab monotherapy was compared with standard chemotherapy combination regimens, the abrogating agent alone demonstrated an increase in terms of response duration (35.8 vs 13.7 months) with no advantage to OS in any case [66]. Tremelimumab activity has also been tested in BC patients, being the first CTLA-4 abrogating agent to be investigated for this disease. Its activity was evaluated in combination with exemestane for metastatic BC disease, demonstrating stable responses in 42% of cases with mild adverse reactions (diarrhea or constipation, pruritus, and fatigue) without any benefit to OS. An overall increase of serum T-cells (CD4+ and CD8+) was also observed and associated with a reduced amount of FOXP3+ Treg cells [67]. Programmed death-1 blockade

Blockade of receptor (PD1) or its ligands (PDL1 or PDL2) does not lead to analogous effects, due to the complexity of the inhibitory loop. Abrogation of the receptor indeed leaves PDL1 free to bind B7.1 and, similarly, PDL1 abrogation allows the receptor to bind PDL2 [68]. PD1 blockade is the mechanism of action of nivolumab (alias BMS-936558), an IgG4 fully human monoclonal antibody. Its antitumor activity has been evaluated in two remarkable clinical trials. In a Phase I trial, nivolumab activity was studied on advanced melanoma, with the observation of stable and lasting responses. Those promising results were also related to less severe adverse reactions, such as dermatitis, asthenia, anemic syndromes, and nausea, the same as seen in preclinical models. In a Phase I/II trial, objective responses were documented for NSCLC as 14 of 76 (18%), renal-cell carcinoma as nine of 33 (27%) and melanoma as 26 of 94 (28%) and clearly associated with PDL1 positivity of tumor cells, whereas PDL1 absence was related to no clinical objective response [69]. For BC, no preclinical or clinical data are available at the moment. However, a Phase I/II clinical trial is actually recruiting participants to assess the safety and efficacy of nivolumab as a single agent or doi: 10.1586/14737140.2015.1042864

associated with ipilimumab in advanced solid tumors, including BC (TABLE 1). Another recently investigated anti PD1 agent is MK-3475, an IgG4 humanized monoclonal antibody, also called pembrolizumab, formerly lambrolizumab. Its activity has been evaluated at different dosages on NSCLC, rectal cancer, melanoma, and sarcoma, but has not been correlated to a maximum tolerated dose. Immune-related adverse reactions occurred at high percentages, so the safety of pembrolizumab monotherapy is still under investigation [70]. At the moment, there are two ongoing clinical trials that aim to evaluate the activity of this compound on trastuzumab-resistant and triple-negative BC diseases (TABLE 1). Pidilizumab (CT-011) is a recently developed humanized IgG-1k recombinant monoclonal antibody specifically targeting PD1. First, investigated for diffuse large B-cell lymphoma treatment with promising results in a Phase II trial, pidilizumab activity has been also evaluated in metastatic melanoma, colorectal cancer, and many other solid malignancies [71]. Currently many clinical trials are ongoing; for BC, pidilizumab activity is being evaluated in association with a p53 vaccine as combinatorial therapy (TABLE 1). PDL1 blockade

The effects of PDL1 blockade are less predictable due to the duplex affinity of PDL1 both for PD1 and for B7.1. mAbs specifically targeting PDL1 have been found to elicit antitumor activity in melanoma and renal-cell, NSCL, and ovarian carcinomas, eliciting objective responses in 17, 12, 10, and 6% of patients, respectively [72]. For BC, there are no preclinical or clinical data available in literature at the moment. MEDI4736 is a human IgG1 monoclonal antibody which abrogates PDL1 interaction both with PD1 and B7.1 through direct antagonism with PDL1. In preclinical models, MEDI4736 combined with chemotherapy caused a pathologic complete response in more than one-half of mice and augmented the OS values when administered alone. Solid tumors included melanoma, colorectal cancer, renal cancer, and NSCLC [73]. As of today, a Phase I/II clinical trial is ongoing with the aim of testing the efficacy and tolerability of the compound on advanced solid tumors, including TNBC (TABLE 1). MPDL3280A is another anti-PDL1 monoclonal antibody whose activity has been evaluated in a Phase I trial for metastatic melanoma only. Response ratios were encouraging and adverse reactions occurred with a reduced incidence [71]. MPDL3280A is actually under evaluation in an ongoing Phase I clinical trial for the treatment of locally advanced or metastatic solid malignancies, including BC (TABLE 1). Vaccines

Traditionally, a vaccine is a harmful portion of a pathogen agent which, after inoculation, stimulates the establishment of a specific adaptive immunity without eliciting any damage to the host. Today, cancer has become the new frontier for vaccinebased therapies. While an anticancer vaccine would be unlikely Expert Rev. Anticancer Ther.


Review

Table 1. Immune checkpoint blockade clinical trials. Drug

Aim of the study

Conditions

Status

ClinicalTrials. gov ID

Immune checkpoint blockade clinical trials


CTLA-4 blockade Ipilimumab

Evaluation of ipilimumab activity after allogeneic stem cell transplant

Persistent or progressive cancer, including stage II/IV BC

Completed, Phase I

[93]

CTLA-4 monoclonal antibody

Evaluation of Immunochemotherapy ± hypofractionated radiation treatment for complete response

TNBC, metastatic NSCLC, metastatic colon cancer

Pilot study, withdrawn

[94]

Ipilimumab

Evaluation of ipilimumab activity in eventual association with cryoablation

Early-stage, resectable BC

Pilot study, ongoing

[95]

Nivolumab, Nivolumab + Ipilimumab

Evaluation of safety and efficacy of nivolumab monotherapy and its association with ipilimumab

TNBC, gastric cancer, pancreatic cancer, SCLC, bladder cancer

Phase I/II

[96]

MK-3475 (pembrolizumab)

Evaluation of MK-3475 activity in Advanced, trastuzumab-resistant, HER2 positive BC

Metastatic BC

Phase I/II

[97]

MK-3475 (pembrolizumab)

Evaluation of safety, tolerability and anti-tumor activity of pembrolizumab

TNBC, advanced head and neck cancer, advanced urothelial cancer, advanced gastric cancer

Phase I

[98]

CT-011 (pidilizumab)

Evaluation of CT-011 activity in association with p53 genetic vaccine

Solid malignancies not responding to standard treatments, including BC

Phase I

[99]

Evaluation of safety, tolerability and pharmacokinetics of MPDL3280A monotherapy

Locally advanced or metastatic solid malignancies including BC, hematologic malignancies.

Phase I, recruiting patients

PD-1 blockade

PD-L1 blockade MPDL3280A

[100]

BC: Breast carcinoma; NSCLC: Non-small-cell lung carcinoma; SCLC: Small-cell lung carcinoma; TNBC: Triple-negative breast carcinoma.

to totally eradicate the risk of developing a certain type of neoplasm during life, it may more realistically reduce the risk of relapse after surgery or the probabilities of developing metastases, with obvious enormous benefits for patients. To set up an antitumor vaccine, identification of tumor antigens (TAs) is firstly required. TAs can be expressed by a unique tumor type and be restricted to a few individuals, or they can be founded on all kinds of malignancies. These last antigens can in turn be divided in tumor-specific TAs – also referred to as cancer/testis (CT) antigens – which are expressed on malignancies only, and tumor-associated antigens (TAAs) which are overexpressed on cancer cells and observed at lower levels on healthy tissues. A large variety of TAAs is actually published on a web database [74] and categorized on the bases of expression profile. An ideal vaccine should target CT-like tumor-specific antigens and should be directed against more than one of them: tumor cells can indeed delete or down-regulate genes encoding for TAs, and as a result they can escape immune recognition. Targeting multiple TAs can therefore improve success probabilities [75].


Among BC antigens, MAGE-A3, NY-ESO-1, and MUC-1 have been identified. MAGE-A3 and NY-ESO-1 are categorized as CT antigens and are observed among many solid malignancies. For BC, these antigens are characteristically expressed on estrogen-receptor (ER) negative and/or triplenegative subvariants [76]. Their peculiar expression profile among BC subtypes has been largely evaluated through immunohistochemistry, and often related to high-grade disease variants and poorer outcomes [77]. Moreover, triple-negative lesions have been found to express NY-ESO-1 at higher levels than what is observed in other disease forms, usually in association with massive CD8+ T-cell infiltration; these observations suggest a high immunogenicity for this particular BC subvariant, which is also the most dangerous [78]. Another antigen that could be targeted for vaccine-based therapies is MUC-1, a widely expressed mucine typical of the majority of epithelial cells. Its overexpression and aberrant glycosylation usually observed on breast tumor cells renders it a good candidate as an antigenic target [79]. In BC patients, aberrant glycosylated cells have been frequently observed and their

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antigenicity has been confirmed by high levels of MUC-1-directed circulating antibodies and by the finding and isolation of CD8+ TILs reactive against MUC-1 [80]. As now, MUC-1 is the target of Stimuvax (L-BLP25), a liposome-based vaccine which elicits an immunogenic action majorly through a peptide derived from MUC-1, BLP25. Its effectiveness, alone or in association with standard treatments, was evaluated through a series of Phase II/III clinical trials in NSCLC, resulting in a general OS increase especially for combinatorial therapy [81]. For BC, BLP25-mediated induction of immune response against MUC-1 has been evaluated in association with tamoxifen and letrozole in murine models, revealing some benefits for letrozole-based therapy lines [82]. While BC currently does not benefit from this kind of therapies, as of today, a cancer vaccine is clinically available for asymptomatic or weakly symptomatic metastatic castrationresistant prostate carcinoma: Sipuleucel-T (Provenge) consists of autologous APCs targeting a prostatic acid phosphatase, which is found to be overexpressed on the majority of prostate cancer cells [83]. The effects of vaccine-based strategies on BC are being currently evaluated in many clinical trials. In a pilot study, the immunogenicity of a synthetic vaccine comprising nine peptides (Poly-ICLC) is being tested (TABLE 2). Other trials mainly involve techniques which aim to boost the effectiveness of vaccine-based strategies, usually because the strongly immunosuppressive tumor microenvironment makes it difficult to establish an anti-tumor response. Hence, vaccine-based strategies can involve the administration of in vitro activated DCs, a practice referred to as DC vaccination: DCs are first cultured from peripheral human blood and then pulsed with natural or synthetic peptides to increase antigen-presentation power [84]. For BC, a Phase II clinical trial is testing autologous peptidepulsed DC vaccination in combination with other drugs, and two Phase I trials are respectively evaluating the vaccination through AdHER2 and oncofetal-antigen-pulsed DCs (TABLE 2 for details). Another method to improve the efficacy of a vaccination is based on the co-administration of immunostimulatory cytokines, such as GM-CSF, IL-2, IL-12, or IFN-a, which are able to promote the activation and the proliferation of such antitumor immune cells. Many experiments on murine models evidenced the effectiveness of a prophylactic strategy based on the co-administration of a vaccine and GM-CSF. Interestingly, encouraging results were obtained from the administration of GVAX, an autologous tumor cell vaccine genetically modified to secrete GM-CSF [85]. For BC, several Phase I/II clinical trials are evaluating the activity of GM-CSF-secreting vaccines, usually in association with standard chemotherapy or with anti HER2 mAbs (TABLE 2). Adoptive T-cell therapy

Today the adoptive T-cell therapy (ATCT) is considered a powerful immune-based therapeutic option capable of inducing a durable immune response in cancer patients. Historically, active immune therapies such as IL-2 produced durable doi: 10.1586/14737140.2015.1042864

responses in only a small minority of patients while being associated with significant, nearly universal toxicity. This has limited their applicability to selected patients, usually with either melanoma or renal cell cancer [86]. Briefly, ATCT consists of the administration of cytotoxic T-cells after in vitro or ex vivo isolation and activation, thus permitting the establishment of an effector immunity, which recognizes and kills malignant cells. According to the different techniques developed up to now, the adoptive immunity can be obtained as nonspecific or specifically cancer-targeted. The allogeneic hematopoietic progenitor cell transplantation was the first ATCT technique adopted, but its safety was very low. The advent of leukocyteactivated killer and cytokine-induced killer cell-based adoptive therapies partially solved the problem in terms of safety, because these strategies demonstrated to elicit reduced graftversus-host reactions and to improve patient’s recurrence-free survival. Moreover, the independence from classical MHCrestriction allowed them to kill tumor cells in a more effective and prompt way. The best results with leukocyte-activated killer cells were obtained for hepatocellular carcinoma, especially in terms of time to first recurrence and recurrence-free survival [87], whereas cytokine-induced killer cells elicited an enhanced cytotoxic activity in in vitro tumor models [88]. However, tumor specificity is characteristic of more recent techniques, such as TIL therapy, TAA-specific lymphocytes, and engineered T-cells. TIL therapy exploits the already arranged specificity of TILs to obtain an activated cancer-directed lymphocyte population after ex vivo expansion, but, despite the high response rates obtained (49–72%), the main limit is the difficulty in obtaining enough TILs from cancer patients [89]. Another approach is the generation of T-cell populations specific for TAAs, which can be useful also to enhance the effectiveness of a vaccination. Lymphocytes are often CD8+ T-cytotoxic, but recently also CD4+ T-helper 1 awarded some successes, due to their immune-activator function through direct contact or cytokines release (mainly IL-2) [90]. Moreover, the most recent ATCT strategy relies on the engineering of TCRs, a technique that allows us to set-up lymphocyte populations specific for almost every kind of TAAs. Chimeric antigen receptor (CAR) T-cells targeting HER2 have been adopted in a clinical trial for TNBC disease, but the results obtained were unsatisfactory. Moreover, the recent discovery of mesothelin as a potential therapeutic target in TNBC disease provided new opportunities to improve the CAR-based technique which, however, still requires further ameliorations in terms of avidity of the receptor and in signal transduction efficiency [91,92]. Immunotherapy & BC: why just now?

With the exception of trastuzumab and pertuzumab, BC treatment currently does not involve immunotherapeutics among the available therapies, whereas other neoplasms such as melanoma are benefiting from immune-checkpoint blockers (the most recent advance in the field) for nearly 5 years as of today. Why does BC treatment still not involve immunotherapeutics, although there is a rational basis for it? Expert Rev. Anticancer Ther.


Review

Table 2. vaccine-based clinical trials. Vaccine

Aim of the study

Conditions

Status

ClinicalTrials. gov ID


Vaccine-based clinical trials Poly-ICLC

Evaluation of immunogenicity of a 9 peptidesconsisting BC vaccine

BC

Pilot study, recruiting patients

[101]

Allogeneic GM-CSF secreting BC vaccine

Evaluation of GM-CSF-secreting vaccine in association with doxorubicin and cyclophosphamide

BC

Phase I, completed

[102]


Evaluation of GM-CSF secreting vaccine in association with Trastuzumab and Cyclophosphamide

BC

Phase II

[103]


Evaluation of vaccine side effects in association with trastuzumab

BC

Phase II, ongoing, not recruiting patients

[104]


Comparison between two combinatory therapies to evaluate side effects: 1. Vaccine + cyclophosphamide 2. Vaccine + cyclophosphamide + trastuzumab

Metastatic BC

Phase II, Recruiting patients

[105]

Autologous AdHER2 DC vaccination

Evaluation of safety and activity of AdHER2 DC vaccination

BC Adenocarcinoma Metastatic HER2+ve solid tumors

Phase I

[106]

OFA-pulsed DC vaccination

Evaluation of oncofetal antigen- pulsed DC vaccination

Metastatic BC

Phase I

[107]

DC vaccination

Time to progression in BC patients after peptide-pulsed autologous DC vaccination in combination with adjuvant aromatase inhibitor, Thymosin 1-alpha and IL-2

BC

Phase II

[108]

BC: Breast carcinoma; GM-CSF: Granulocyte macrophage-colony stimulating factor; DC: Dendritic cell.

The preclinical evaluation and development of immunotherapeutics for BC has become a field of interest just recently. This delay is primarily due to the fact that BC immunogenicity has been proven just very recently, whereas formerly this disease was considered a typical nonimmunogenic neoplasm. Data obtained so far were often confused and controversial due to the high heterogeneity of BC subtypes reflected on different immune-profiles, and furthermore complicated by the fact that systemic treatments were likely to unbalance immune-signatures, an evidence that has been proven very recently too. Thus, immunotherapeutics for BC have been thought not to be effective for a long period of time. Second, first immune-checkpoint abrogating agents were likely to elicit high rates of toxicities, therefore they were preferably tested on those malignancies with welldefined immune signatures and consequently with putatively more predictable responses. After 5 years of clinical practice, and after novel links and evidences about BC immunogenicity came to light, the potential use of immune-checkpoints abrogating agents treatment has finally become theoretically possible also for BC. informahealthcare.com

Conclusions

Immune-based therapies have demonstrated to be effective in the treatment of solid malignancies as well as hematologic diseases. The key of the success of an immunotherapeutic agent is, above all, the immunogenicity of the tumor to be treated: in this setting, BC has been demonstrated to be highly immunogenic, despite the immunosuppressive conditions that seem to contradict this statement at first sight. The presence of TILs indicates that the target of a potential immunotherapy is present and is at the right place, and the anergic, harmful condition in which lymphocytes are frequently found provides the rationale that the exploitation of immune checkpoints can achieve some kind of success. Due to the immunogenicity of BC, vaccines are also a valid potential therapeutic chance, and methods to improve their effectiveness have the advantage to be personalized on the basis of the patient’s immune-profile. As a second important point, BCs seem to show specific immune signatures, and the different immune profiles are clearly linked to the molecular BC subvariants; thus, basal-like carcinomas can potentially benefit from immunotherapies better than less aggressive variants, as well as HER2 positive and doi: 10.1586/14737140.2015.1042864


Review


inflammatory subtypes. On the basis of the recent approval of immunotherapeutic agents for melanoma and urogenital diseases, the achievements in understanding the mechanisms involved in regulating whether lymphocytes can promote or combat carcinogenesis in BC and exploit them to obtain therapeutic success are actually an additional gear that should be engaged. Identifying factors that predict the subpopulations of patients and breast tumor subtypes that respond to a specific immunotherapy will be critical to both understanding the mechanism of action of immunotherapy and the efficient development of novel immunotherapy combinations with standard treatment. Expert commentary

With the exception of trastuzumab and pertuzumab, BC treatment strategies actually do not involve any immune-based therapies; these monoclonal antibodies are effective only in 30% of HER2-expressing carcinomas and the high percentage of fatalities highlights the need for that novel approaches. Immune checkpoints are now known to be exploited by tumor cells to shape lymphocyte-mediated responses and to render vain immune response. Moreover, cancers actively recruit immune-suppressive cells, such as Tregs and MDSCs, rendering the comprehensive framework more prone to the development of a new growth. The high levels of immunosuppressive cells found in BC patients suggest that these phenomena are ongoing on BC, and implicates that immune-based therapies can elicit a powerful reversal of cancer immunosuppression. If we consider the significant amount of TILs, especially cytotoxic CD8+ T-cells and NKs which are found in almost all BC cases, we can speculate that the use of immune-based strategies can significantly improve clinical outcomes, as already seen in melanoma and prostate cancer. Therefore, immunoprofiles could become part of the diagnostic pathway of a patient and the selection of a personalized therapy could be better defined. Immune checkpoint-abrogating agents can be exploited, for example, to reduce standard chemotherapy doses and to alleviate related toxicities, as well as a vaccination which could strongly decrease the risk of relapse after surgery. A great general advantage of immune-based therapies is that they enhance an already existing mechanism of defense of a patient: if cancer cells were not able to actively avoid immune attack, the development of malignancies would be a more difficult process. Immune-therapies, basically, take advantage of the same tools that cancer cells use to avoid immune system attack. The great potential of these new therapeutic strategies has still to be exploited in BC, and the amount of deaths from BC is an occurrence that cannot be ignored.

doi: 10.1586/14737140.2015.1042864

Five-year view

In the near future, an explosion of activity in the cancer immunotherapy field will cover diverse therapeutic platform technologies and cancer. Novel immune checkpoint blockades and immune-stimulating agents will be developed and approved as monotherapies or as adjuncts to targeted therapies in metastatic disease. The development and licensing of the first T-cell-based immune interventions and bispecific engineered antibodies will, hopefully, be ready for clinical purposes, more likely first in leukemias and lymphomas where there are fewer immune-inhibiting mechanisms to overcome. Vaccines will be predominantly tested in minimal residual disease in various cancer types and as adjunct to other forms of therapy facilitating a safer and more effective treatment. The efforts of researchers will focus on the development of immune interventions that achieve objective and durable clinical responses in individual patients surpassing, for the first time, the activities in support of immunotherapies that only achieve a slowdown of disease. The recent US FDA approval of the use of ipilimumab in the treatment of melanoma makes it likely that this agent will be used for patients in the treatment of several advanced solid tumors. It is likely that the therapeutic efficacy of agents targeting other inhibitory receptors expressed on tumor-infiltrating lymphocytes, such as PD1 will receive further attention. The evolution of personalized therapy with the use of biomarkers to predict response to immunotherapy, allowing for tailoring of targeted and individualized therapy to maximize response, is attractive; however, there is currently a paucity of information on relevant markers, thus research at the molecular level is warranted. More detailed molecular analysis of tumors, including mutational analysis, is already beginning to be adopted in profiling solid cancer to have a benefit from immunotherapy. Further genetic analysis of pathways involving immune cell function may provide useful prognostic information and therapeutic targets. Acknowledgements

Andrea Ravelli and Laura Zanotti wish to thank ARCO Onlus-Cremona-Italy and AMOS Onlus-Pavia-Italy for their support. Financial & competing interests disclosure

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.



Review

Key issues .

Immune-based therapies have emerged in the past decades for the treatment of solid malignancies, including melanoma and prostate carcinoma.

.

Breast carcinoma (BC) is a putatively perfect candidate for immunotherapy, because its immunogenicity is supported by a large amount of literature and because no well-assessed treatments are currently available for most-aggressive variants, such as Triple-Negative BC and Inflammatory BC.

.

Immune system is heavily involved in combating BC, because its original role is to eradicate early-transforming cells. The immuneevasion phenomenon, however, permits malignant cells to proliferate through the progressive development of an immunosuppressive microenvironment, where cytotoxic lymphocytes are not effective at all.


.

Immune system has been observed to play a primary role also in the mechanism of action of standard chemotherapeutic agents, which previously were thought to mediate only an immunomodulatory effect. Moreover, the observation that antibody-dependent cell-mediated cytotoxicity is a main mechanism of action of trastuzumab highlights the importance of immune system in cancer.

.

The recent discovery of the mechanisms at the basis of immune-escape has rendered possible the reversal of immunosuppression and to boost cytotoxic lymphocytes activity instead; immune checkpoint blockade represents a way to enhance cytotoxic cells and to limit immunosuppression at the same time, moreover with a safety already confirmed by the clinical practice of ipilimumab for melanoma treatment.

.

The development of a BC vaccine is an ongoing research field which promises some kind of success. MAGE-A3, NY-ESO-1, and MUC-1 are three tumor-associated antigens specific for BC. Their evaluation as vaccines in clinical trials is systematically ongoing, as well as many methods to enhance their effectiveness.

independent predictor of response to neoadjuvant chemotherapy in breast cancer. J Clin Oncol 2010;28(1):105-13

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