The Prostate 75:337^347 (2015)
Beyond Immune Checkpoint Blockade: New Approaches toTargeting Host^Tumor Interactions in Prostate Cancer: Report From the 2014 Coffey^Holden Prostate Cancer Academy Meeting Andrea K. Miyahira,1 Haydn T. Kissick,2 Jennifer L. Bishop,3 David Y. Takeda,4 Christopher E. Barbieri,5 Jonathan W. Simons,1 Kenneth J. Pienta,6,7,8 and Howard R. Soule1* 1
Prostate Cancer Foundation,Santa Monica,California 2 Emory University, Atlanta,Georgia 3 Vancouver Prostate Centre,Vancouver,British Columbia,Canada 4 Dana-Farber Cancer Institute,Boston, Massachusetts 5 Weill Cornell Medical College,NewYork,NewYork 6 Departmentof Urology,The James Buchanan Brady Urological Institute,Baltimore, Maryland 7 DepartmentofOncology,The Johns Hopkins,SchoolofMedicine,Baltimore, Maryland 8 Departmentof Pharmacologyand Molecular Sciences,The Johns Hopkins SchoolofMedicine,Baltimore, Maryland
INTRODUCTION. The 2014 Coffey–Holden Prostate Cancer Academy Meeting, held in La Jolla, CA from June 26 to 29, 2014, was themed: “Beyond Immune Checkpoint Blockade: New Approaches to Targeting Host–Tumor Interactions in Prostate Cancer.” METHODS. Sponsored by the Prostate Cancer Foundation (PCF), this annual, invitation-only meeting is structured as an action-tank, and brought together 72 investigators with diverse academic backgrounds to discuss the most relevant topics in the fields of prostate cancer immunotherapy and the tumor microenvironment. RESULTS. The questions addressed at the meeting included: mechanisms underlying the successes and failures of prostate cancer immunotherapies, how to trigger an effective immune response against prostate cancer, the tumor microenvironment and its role in therapy resistance and tumor metastasis, clinically relevant prostate cancer mouse models, how host– tumor interactions affect current therapies and tumor phenotypes, application of principles of precision medicine to prostate cancer immunotherapy, optimizing immunotherapy clinical trial design, and complex multi-system interactions that affect prostate cancer and immune responses including the effects of obesity and the potential role of the host microbiome. DISCUSSION. This article highlights the most significant recent progress and unmet needs that were discussed at the meeting toward the goal of speeding the development of optimal immunotherapies for the treatment of prostate cancer. Prostate 75:337–347, 2015. # 2014 Wiley Periodicals, Inc.
KEY WORDS: prostate cancer; immunotherapy; tumor microenvironment; therapeutics; immunosuppression
Disclosure statement: No authors declare any potential conflicts of interest.
Correspondence to: Howard R. Soule, Prostate Cancer Foundation, 1250 4th Street, Santa Monica, CA 90401. E-mail:
[email protected]
ß 2014 Wiley Periodicals, Inc.
Received 12 September 2014; Accepted 17 September 2014 DOI 10.1002/pros.22920 Published online 30 October 2014 in Wiley Online Library (wileyonlinelibrary.com).
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The Prouts Neck Prostate Cancer Meeting, held annually from 1985 to 2007 and sponsored by the NIH, was designed to be a topic-focused action-tank composed of a group of approximately 75 investigators that resulted in many significant advances in the prostate cancer field [1]. Continuing this legacy, the Prostate Cancer Foundation (PCF) reactivated this meeting in 2013 [2]. In 2014, as the Meeting is no longer held in Prouts Neck, Maine, PCF renamed the event the Coffey–Holden Prostate Cancer Academy Meeting in honor of Drs. Donald Coffey and Stuart Holden, two individuals who have made an immeasurable impact on the understanding and treatment of prostate cancer. The 2014 meeting was themed “Beyond Immune Checkpoint Blockade: New Approaches to Targeting Host–Tumor Interactions in Prostate Cancer,” and was held in La Jolla, CA, from June 26 to 29, 2014. The meeting was attended by 72 investigators, including 34 PCF-funded Young Investigators. The agenda consisted of an opening session on “big questions” that were discussed in length by attendees, sessions of 10 min talks followed by 20 min of discussion, and a discussion panel on the prospects and challenges faced in forwarding immunotherapy for prostate cancer patients. In the last 4 years, immunotherapy has shown striking efficacy in the treatment of solid tumors including melanoma, renal cell cancer, non-small cell lung carcinoma, and most recently in bladder cancer. However, while sipuleucel-T (Provenge) was the first FDA-approved therapeutic cancer vaccine, responses to immunotherapies in prostate cancer patients have never neared the dramatic, and in some cases curative outcomes observed in a subset of melanoma and other cancers, prompting immunotherapy to be the focus of this year’s meeting. In this article, we discuss highlights from the 2014 Coffey–Holden Prostate Cancer Academy Meeting, including the knowns and unknowns surrounding prostate cancer immunobiology, the successes and failures of prostate cancer immunotherapy clinical trials, the biggest unmet needs in prostate cancer immunotherapy, and strategies to bring immunotherapy to the forefront of treatment options for prostate cancer patients.
WHAT ARE THE BARRIERS TO ANTI-TUMOR IMMUNE RESPONSES IN PROSTATE CANCER? A number of factors may cause the failure of the human immune system to recognize and eliminate prostate cancer, and these factors likely change during the long co-evolutionary period of the tumor and The Prostate
immune response. Anti-tumor T cells have been identified in prostate cancer patients, indicating that prostate cancer is immunogenic and prostate cancer-associated antigens are naturally recognized [3]. Why T cells fail to clear the tumor even in the setting of immunotherapy could be due to multiple mechanisms. Immunoediting may occur, in which the tumor mutates or loses expression of the antigens recognized by anti-tumor T cells. Prostate cancer progresses very slowly and as individuals age, multiple immunologic changes may cause waning efficiency in anti-tumor immune responses. Thymic involution and the subsequent reduction in output of na€ıve T cells, shifts in hematopoietic lineage outputs from myeloid and lymphoid to myeloid cells, and skewing of T cell subsets toward more terminally differentiated T cells with reduced replicative capacity were identified as mechanisms for reducing the capabilities of the immune response. The prostate appears to be a more immune privileged site than organs such as the skin, in which developing tumors are highly immunogenic, indicating differences in access or immune regulation by the tissue microenvironment. Androgens for instance, suppress anti-viral type immune responses that are also critical in antitumor immune responses [4,5]. Finally, the tumor microenvironment is directly immunosuppressive through multiple mechanisms mediated by tumor cells, infiltrating immune cells, and other tumor stromal factors. Overall, mechanisms of immune evasion and immune suppression in prostate cancer are likely to be unique compared with melanoma and other tumors and require alternative methods for immunotherapy to be effective. Results from ongoing and planned immunotherapy clinical trials are eagerly awaited, as they will illuminate which anti-tumor immunologic mechanisms are active versus shut down or absent in prostate cancer patients. As combination immunotherapies appear clinically superior to single therapy regimens in melanoma clinical trials, deciphering which mechanisms in prostate cancer patients need to be targeted or co-targeted to unleash the full power of the immune system will be critical in developing clinical immunotherapy strategies for prostate cancer patients. PROSTATE CANCER VACCINE STRATEGIES Provenge (Sipuleucel-T), a cellular vaccine in which patient dendritic cells are incubated ex vivo with a fusion protein consisting of prostate cancer-associated antigen PAP and GM-CSF before reinfusion into the patient in order to generate anti-PAP immune responses, was the first FDA-approved therapeutic vaccine for any cancer, with a 4.1 month improvement over placebo in median overall survival [6]. Other
Improving Prostate Cancer Immunotherapy vaccination strategies against various prostate cancer antigens are being tested in ongoing clinical trials. Most advanced in development is ProstVac-VF (Tricom), in which patients are injected with a modified vaccinia virus encoding PSA as the tumorassociated target antigen and LFA-3, ICAM-1 and CD80 as adjuvants [7]. A fowlpox vector encoding the same genes is then used for the booster vaccine to avoid neutralization by antibodies developed against the vaccinia vector backbone [7]. The Phase II trial of ProstVac-VF resulted in an improved survival of 8.5 months with an estimated hazard ratio of 0.56 [7]. The current Phase III trial of ProstVac-VF will randomize 1,200 patients into three groups receiving ProstVac-VF, ProstVac-VF þ GM-CSF, or an empty vector control. Accrual of patients for this trial has begun and initial results are anticipated in 2015. Discovering optimal antigen targets continues to be explored in order to improve the efficacy of therapeutic vaccines. Previously tested prostate cancer vaccines have focused on targeting antigens whose expression is restricted to prostate tissue such as PSA or PAP. However, if the target protein is not essential to the fitness of the tumor cell, tumor variants may undergo immune-editing to escape the host immune response. The androgen receptor (AR) is a key oncogenic driver of prostate cancer and is a central target of prostate cancer therapy, suggesting that AR can also be used as a therapeutic vaccine target. Anti-AR T cell and antibody responses are observed in prostate cancer patients [3] which strengthens this argument. A Phase I clinical trial is being planned to examine the safety and efficacy of a vaccine targeting AR (pTVG-AR) [8] in patients with metastatic prostate cancer undergoing androgen deprivation therapy (ADT), with an anticipated activation date in 2014. A second class of therapeutic vaccines related to mechanisms of AR-targeted treatment resistance is being considered. The expression of constitutively active AR-variants that lack the C-terminal ligandbinding domain (LBD) appear to be a treatment resistance mechanism in patients failing enzalutamide and abiraterone therapy [9,10]. Similar resistance mechanisms might be caused by AR vaccines that target the LBD, such as pTVG-AR. However, as long as full-length AR continues to be expressed, tumor cells should still be immunologically targeted. The Nterminus of AR has both sequence variation and lacks a defined structure, making the selection of an Nterminal AR peptide for an immunogenic vaccination target a complicated design consideration. An alternative precision medicine approach is to sequence patient tumors and design personalized vaccines against predicted tumor-specific proteincoding mutations. The obvious benefit of this ap-
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proach is that these antigens are unique targets occurring only in the tumor, theoretically avoiding both autoimmune targeting of any other tissue and mechanisms of central tolerance. The low somatic mutation rate in prostate cancer [11] implies that the antigen repertoire may be limited. However, it is of greater importance to determine the immunogenicity of mutations. Analysis of TCGA data from several solid tumors identified a correlation between patient survival and mutations with predicted immunogenicity but not with total somatic missense mutation rates [12]. A clinical trial underway at MD Anderson Cancer Center will test the efficacy of ipilimumab in generating T cell responses against tumor neoantigens in metastatic CRPC patients receiving standard hormone therapy. Patient-specific neoantigens will be identified by sequencing metastatic tumor tissue and patient T cells will be tested for reactivity to predicted immunogenic epitopes. The hypothesis of this approach is that mutant antigens that generated a strong T cell response could be used for that patient in subsequent personalized vaccine treatments. The issue of tumor heterogeneity may prevent vaccines that target a single antigen from being curative in an individual patient. Immunization against several antigens at once may avoid the development of tumor escape variants that have lost antigen expression and may also target preexisting tumor heterogeneity. Observations of the coexistence of adenocarcinomas and trans-differentiated clones with neuroendocrine features in individual patients [13] suggest the need to identify antigen expression on an individual tumor cell level to select optimal antigens for co-targeting vaccine strategies. WHYHAVECHECKPOINTINHIBITORS TODATE FAILEDINPROSTATECANCER? Checkpoint inhibitors have shown impressive results against a range of different cancers. The even greater success of combinations such as ipilimumab (anti-CTLA-4) plus nivolumab (anti-PD-1) to treat melanoma indicates that immunotherapy has powerful anti-cancer activity when optimally applied to a relevant tumor type [14,15]. However, the first randomized Phase III clinical trial of ipilimumab for CRPC, “Study of Immunotherapy to Treat Advanced Prostate Cancer” (CA184-043), did not meet the primary endpoint of overall survival [16]. As discussed in more detail later in this article, a subset analysis of this trial revealed an overall survival benefit in those patients without visceral metastases. Additionally, observations of tumor regressions in lung cancer patients treated on trials with epigenetic modulators followed by cross-over to nivolumab [17] indicated The Prostate
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that tumors escape immune surveillance and become resistant to checkpoint immunotherapy by epigenetically turning off the expression of antigens naturally targeted by T cells. These observations have prompted the planning of clinical trials combining epigenetic modulators with checkpoint inhibitors. Thus it is likely that checkpoint inhibitor therapy may have promise in prostate cancer if applied in the proper context. A full understanding of the mechanisms of checkpoint inhibitor function and failure will lead to improvements in clinical trial design including choosing drug combinations, optimizing timing, and selecting the appropriate patient population based on biological parameters. To understand the mechanisms of action for ipilimumab, the TCR repertoire was analyzed from prostate cancer and melanoma patients over the course of treatment with ipilimumab [18]. Ipilimumab lowered the threshold for T cell activation resulting in expansion of low-avidity T cells and enlargement of the available T cell repertoire, as opposed to preferentially expanding high-avidity T cells or causing depletion of regulatory T cells. Overall survival was reduced in patients with demonstrated loss of pre-existing high-frequency anti-viral T cell clones, indicating that targeting mechanisms which cause instability in T cell maintenance may improve responses to ipilimumab. Similar studies need to be performed for other checkpoint inhibitors including nivolumab to indicate which therapies will best apply to prostate cancer patients. It will be important to determine if reactivation of preexisting anti-tumor T cell clones versus de novo generation of anti-tumor T cells has more significant clinical effects in prostate cancer. This line of investigation may inform optimal therapeutic combinations with the strongest synergy.
spontaneously arising prostate tumors) that had received OVA-specific OT-I CD8 T cells were given a vaccine targeting OVA combined with anti-CTLA4 þ anti-OX40 mAbs which resulted in synergistic antitumor activity. With the addition of the vaccine to antiCTLA-4 þ anti-OX40, instead of TH2 cytokine induction, the expression of TH1 cytokines and chemokines was induced. As anti-tumor immune responses rely on TH1 immunity, agents that induce TH1 immunity or suppress TH2 immunity should be considered in immunotherapeutic combinations, which these studies support. A novel combination immunotherapy that is being developed are chimeric antigen receptor (CAR) T cells targeting the prostate antigen PSCA delivered with an siRNA against STAT3 coupled to CpG (CpG-STAT3 siRNA) to selectively silence STAT3 in TLR9þ cells. STAT3 is a cytokine-activated transcription factor that acts as an oncogene in tumor cells and as an immunosuppressive factor in immune cells [20]. TLR9 is expressed by prostate cancer and myeloid cells, thus STAT3 inhibition can block tumor cell growth while inhibiting immune suppression in antigen-presenting myeloid cells, manipulating the tumor microenvironment to allow maximal CAR T cell cytotoxic activity. Based on pre-clinical findings, Phase I clinical trials for this combination are proposed for 2016. Uncovering mechanisms of immune suppression by tumors and determining the immunological activities of immunotherapies will allow design of greater combination immunotherapeutic strategies. Many other novel strategies combining two immunotherapies or immunotherapy with targeted therapy, chemotherapy, or radiation therapy are in various phases of development and clinical trials.
NOVEL IMMUNOTHERAPYCOMBINATIONS
EPIGENETIC REPROGRAMMING AND THERAPEUTIC TARGETING
The tumor microenvironment can be highly immunosuppressive and influence immune cells to support instead of inhibit tumor growth and limit the success of immunotherapies. Significant synergy may be achieved by combining agents that activate immune cells with those that block mechanisms of immune suppression. Combining the immune checkpoint inhibitor anti-CTLA-4 with an agonist antibody targeting the T cell activation molecule OX40 blocked tumor growth in mice, but efficacy was limited to early time points after tumor implantation [19]. This was found to be due to the induction of TH2 cytokine expression in the tumor microenvironment by the anti-CTLA-4 þ antiOX40 regimen, and the addition of anti-IL-4 profoundly boosted the anti-tumor effect of the regimen [19]. In another immunotherapy combination scheme, tumorbearing TRAMP-OVA mice (which express OVA on The Prostate
Recent clinical trials found that lung cancer patients who were treated with the demethylation agent Azacitidine exhibited tumor regression after cross-over to nivolumab [17]. While anecdotal, these observations have generated the hypothesis that the poor efficacy of checkpoint inhibitors in prostate cancer patients [21] may be due to epigenetic mechanisms that limit the anti-tumor immune response. Epigenetic “priming” may promote anti-tumor immunity by modulating both tumor cells and immune cells. DNA methylation can silence the expression of prostate tumor antigens, and antigen re-expression following epigenetic therapy may allow targeting of the tumor cells by pre-existing or de novo generated anti-tumor T cells [22]. Azacitidine upregulates the expression of anti-tumor immune response genes
Improving Prostate Cancer Immunotherapy including class I MHC for antigen presentation by tumor cells and IFN-g in immune cells [23]. However, expression of PD-1 and PD-L1 are also upregulated by epigenetic therapy [23], indicating that the addition of nivolumab may be necessary to activate immune responses following epigenetic therapy, generating the synergistic effects observed for this treatment sequence. This leads to questions regarding how other immunotherapies might synergize with epigenetic therapy as well as the differences in subsequent immune responses promoted by demethylating agents such as Azacitidine versus HDAC inhibitors. Clinical trials specifically designed to test the efficacy of ipilimumab and/or nivolumab with or without pre-treatment with epigenetic modifiers are being planned to further test these questions. The efficacy of combining epigenetic modifiers with vaccination, CAR T cells, and other immunotherapies will be of interest. Profiling tumor antigens that get reexpressed following epigenetic therapy might be a critical consideration for designing optimal vaccination strategies. RADIOTHERAPYAS A MODULATOR OF THE IMMUNE SYSTEM Tumor regression following local radiotherapy (RT) has been demonstrated in murine models to have an immune-dependent component, requiring CD8 T cells and type I interferons [24,25]. Cell death following RT is immunogenic, releasing tumor antigens and dangerassociated molecular patterns (DAMPs) such as HMGB1, and exposing calreticulin “eat me” signals on the surface of dying cells [26]. This results in activation of dendritic cells in the draining lymph node and generation of anti-tumor T cells that traffic back to the irradiated tumor site to mediate killing [26]. In rare cases, tumor regression is observed at distant sites, termed the abscopal effect (ab scopus ¼ away from the target), indicating that RT can induce systemic tumor immunosurveillance. Studies have begun testing the hypothesis that therapeutic synergy can be achieved by combining RT with immunotherapy. The addition of anti-CTLA-4 or anti-PD-L1 to RT significantly enhances regression of locally irradiated and abscopal tumors in mice [27,28]. Abscopal tumor regression following RT plus ipilimumab has been reported in rare melanoma patients [29]. Optimizing treatment to achieve abscopal effects more consistently and profoundly will allow RT to be more effective in the treatment of metastatic patients. The abscopal effect has been more apparent in smaller tumors, thus selection of patients and efficacy following cytoreduction needs to be studied. Differences in the immunogenic antigens expressed by local and
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various distant tumors may limit abscopal effects and need to be understood. RT can be immunosuppressive, for instance by inducing PD-1 and PD-L1 expression in the tumor microenvironment [27], so understanding the immune components involved and optimizing clinical trial design including RT dose, fractionation, and timing with respect to immunotherapeutics and other regimens will be critical. Finally, it is unclear if the mechanism of tumor cell death (via RT-induced DNA damage) is a unique component in the initiation of anti-tumor immune surveillance and the abscopal phenomenon, or if other cytotoxic modalities, such as high-frequency ultrasound ablation, microwave, cryoablation, or even androgen-targeting therapies can induce similar anti-tumor immune activities. WHAT IS THE EFFECT OF IMMUNOTHERAPY IN PATIENTS WITH VERSUS WITHOUT VISCERAL METASTASES? Successful immunotherapy is thought to be most beneficial in patients with low tumor burden allowing the immune system to clear minimal residual disease. Prostate cancer patients with visceral metastases (liver, lung or brain) have poorer prognosis than patients with metastatic lesions in bone or lymph nodes only [30] and may be less likely to benefit from all therapies, including immunotherapy. In the recent CA184-043 Phase III clinical trial, metastatic castrateresistant prostate cancer (mCRPC) patients who progressed after docetaxel treatment and had at least one symptomatic bone lesion, received a single dose of bone-directed radiotherapy and were then randomized to receive ipilimumab or placebo [16]. Results from this trial just missed statistical significance for overall survival with ipilimumab although there was a significant benefit for time to progression. Retrospective subgroup analysis revealed that patients with visceral metastases fared significantly worse for overall survival when treated with ipilimumab and radiation compared with placebo (HR: 1.2 for patients with visceral metastases vs. 0.73 for patients without visceral metastases) [16]. This suggests that if patients with visceral metastases had been excluded, the trial may have been positive and further implies that ipilimumab may promote instead of suppress prostate tumor growth in certain organs [16]. These results have led to subsequent trials testing ipilimumab vs. placebo that specifically exclude men with visceral metastases. Visceral metastases frequently occur in melanoma, yet patients still have a survival advantage with checkpoint immunotherapy, begging the question of what is different about the visceral tumor microenvironment in mCRPC [14,31]. The prostate tumor phenotype is known to be different at different metastatic The Prostate
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sites, with adenocarcinomas invading bone while visceral sites may be primarily occupied by tumor cells which have trans-differentiated into a highly aggressive, sometimes AR-independent tumor with neuroendocrine features [13]. Studies comparing the phenotype and functions of tumor cells, stromal cells, and immune cells, and their interactions in different metastatic tissues are of critical importance. For instance, immunotherapy resistance of visceral metastases could be due to differential tumor-antigen expression, an immunologically uncontrollable rate of tumor growth, characteristics of the tumor microenvironment that cause induction of pro-tumor instead of anti-tumor immune responses, or other immunosuppressive mechanisms. A major consideration is whether prostate cancer patients with visceral metastases should be broadly excluded from immunotherapy clinical trials or if other clinical trial designs should be considered. FACTORS THAT PROMOTE THE ESTABLISHMENT OF METASTATIC TUMOR NICHES In order to optimize treatment of metastatic patients, it is important to determine how metastases emerge after years of dormancy, the effects of any crosstalk between tumors in different tissues, and how treatment of the primary tumor affects metastatic sites. In murine breast cancer xenograft models, effects of primary tumors on secondary tumors have been studied by implanting a “primary” tumor cell line and subsequently either systemically injecting or contralaterally implanting a cell line with a different aggressive index [32]. Aggressive primary tumors established a systemic pro-tumorigenic environment, promoting the growth of tumors from subsequently administered indolent cells and influencing the histopathology of the second tumor [32–34]. The distant effects mediated by the primary tumor proceeded via release of molecules including osteopontin that either circulated to the bone marrow or were absorbed by platelets and delivered to the bone marrow [32,33]. These factors induced bone marrow cells to mobilize and be recruited to dormant micrometastatic tumors where they contributed to tumor outgrowth by production of growth and angiogenic factors [32–34]. Both prostate cancer and hormone-responsive breast cancer have a tropism for bone, and it is tempting to speculate that prostate cancer uses similar mechanisms to promote metastases. Accordingly, in prostate cancer mouse models, platelets were found to transport bone-modifying molecules including TGF-b, RANK/RANKL, and MMPs via a-granules from tumors to bone marrow to induce mobilization of bone marrow cells The Prostate
that are then recruited to tumor sites [35]. Thus, platelets can act as delivery vehicles, increasing the concentration of tumor-secreted molecules that are delivered to a specific site. How to target platelets for therapy or their utility as tumor biomarkers will be important to determine. Of note, many studies have been performed using immunodeficient murine xenograft models. New murine models need to be established to examine these mechanisms in fully immunocompetent systems. REACTIVE STROMAL CELLS AND THE INFLAMMATORY MICROENVIRONMENT The tumor microenvironment is composed of tumor cells along with a stroma consisting of heterogeneous cell types including immune cells, endothelial cells, and fibroblasts, which modulate tumor growth and disease progression. The majority of prostate biopsies both from benign and prostate cancer specimens exhibit inflammatory cell infiltration possibly related to tissue injury, infection, urine reflux, hormonal changes, or diet [36]. However, the role of prostatic inflammation in the initiation and progression of prostate cancer remains unclear. Epidemiologic studies support both a positive and negative association between inflammation in prostate biopsies and the development of cancer. One potential source of discrepancies between studies is the difficulty in classifying the nature of the inflammation in prostate biopsies. Prostatic inflammation is heterogeneous, multifocal, and distributed throughout different regions in the prostate. Standardizing methodology to identify and quantify tumor-infiltrating immune cell types and determine their functional activity is critical. As therapies begin to incorporate more immune-based strategies, it will become increasingly relevant to understand the role of prostatic inflammation in prostate cancer, effects on sensitivity or resistance to immunotherapies, and how to incorporate this information into clinical trials. An understanding of the sources of prostatic inflammation may also identify pathogenic causes that could be potentially treated and decrease the risk of prostate cancer. The reactive stromal response is a wound healing mechanism observed in prostate tumors that is characterized by activated cancer-associated fibroblasts and myofibroblasts. Efforts to isolate and characterize stromal components have identified cell populations that can proliferate in response to signaling molecules secreted by the prostate cancer cells and reciprocally stimulate androgen signaling in cancer cells. Studies to understand the co-evolution of tumors and reactive stroma have indicated the role of reactive microvasculature as a source of CD34þ/vimentinþ fibroblasts [37].
Improving Prostate Cancer Immunotherapy Understanding how interactions between tumor and stroma contribute to disease progression, castrateresistance, and effects on anti-tumor immune responses will be important in developing novel therapies aimed at the supportive stroma. Although making up a small percentage of cells in the tumor microenvironment, mesenchymal stem cells (MSCs) have generated significant interest for their role in modulating virtually all components of the immune system. MSCs function as key negative regulators of the immune response and likely contribute to the immunosuppressive properties of the tumor microenvironment. Ongoing studies suggest that tumor-associated MSCs are fundamentally different than those from normal prostate tissue including their lineage differentiation potential and gene expression profile [38]. They may also be a source of protumorigenic cancer-associated fibroblasts. Moving forward it will be critical to determine the role of MSCs in tumor progression and whether they hold prognostic or predictive value. WHAT ARE THE ROLES OF HORMONES AND ADT IN MODULATING IMMUNE RESPONSES? Androgens are known to have a negative immunomodulatory effect. Men exhibit poorer viral vaccine responses compared with women and decreased vaccine responses correlate with higher levels of testosterone [5]. In prostate cancer patients, androgen deprivation therapy (ADT) induces prostatic inflammation, characterized by T cell infiltration [39]. When tumor vaccination is preceded by ADT, the numbers of anti-tumor T cells generated is increased, indicating that androgens promote tolerance or reduce responsiveness of T cells [40]. ADT enables thymic reemergence and enhanced thymic generation of na€ıve T cells [41], indicating that the increased numbers of anti-tumor T cells observed following ADT may be related to having a larger pool of T cells to initiate antitumor responses. During the course of ADT, prostate cancer cells are thought to die by apoptosis, but whether ADT induces immunogenic cell death is unclear. The role of ADT-induced damage to prostate tissues and vessels in inducing prostatic inflammation and the timing and nature of immune infiltration into prostates following ADT should be closely examined to determine how immunotherapy can best be applied in combination with androgen axis-targeting therapies. ADT is being tested in combination with immunotherapies in multiple prostate cancer clinical trials. Considerations for clinical trial design include sequencing versus concomitant treatment, adjuvant versus neoadjuvant settings, and patient selection. Sequencing and timing of therapy is important, as
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ADT may release tumor-associated antigens and both prostatic inflammation and thymic reemergence following ADT may be transient phenomena. A clinical trial of ADT combined with Provenge is underway, in which the question of sequencing will be addressed by comparing the initiation of ADT before versus after Provenge. Finally, understanding the androgen-driven immune effects versus the cytotoxic and tissue damaging effects of ADT on immunity will clarify the most appropriate patients and settings in which to apply immunotherapy. THE ROLE OF OBESITY IN INFLAMMATION AND PROSTATE CANCER Epidemiologic studies have linked adult obesity with more aggressive prostate cancer and reduced survival [42]. A number of obesity-related factors may drive prostate cancer including altered metabolism and the induction of a state of chronic inflammation by adipose tissue which can manifest systemically. Obesity and chronic prostatic inflammation positively correlate in patients, prompting questions on whether and how obesity modulates tumor–immune interactions and responses to immunotherapies. Factors produced by adipose cells may influence infiltration of the tumor by different immune cell types and promote an immunosuppressive microenvironment. Obesity also modulates hormone levels—increasing estrogen and lowering androgens, thereby influencing immune responses by hormone-dependent mechanisms. Specific populations of obese men have been found to be at higher risk for lethal prostate cancer, including African American men and patients harboring the TMPRSS2: ERG genomic fusion [43,44]. TMPRSS2:ERG-positive tumors were found to express higher levels of inflammation and adiposity-related genes, indicating that promotion of inflammation underlies the association between obesity and TMPRSS2:ERG in increasing prostate cancer risk [43]. How ERG and other genetic factors modify inflammatory genes in obese but not healthy weight patients is an important question. Teasing apart the relationships between host and/or tumor genetic factors and obesity-related effects on inflammation and metabolism in driving prostate cancer progression is complex and will require welldesigned model systems. Studies examining the role of diet in murine xenograft models indicate that caloric and carbohydrate restriction increase overall survival [45], however, the effects of diet alterations on prostate cancer outcomes need to also be explored in immune-competent models. Diet and exercise-based interventions are being tested in clinical trials. Pharmacologic inhibitors of mechanisms linking obesity to prostate cancer may be promising. The Prostate
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IS THERE A ROLE FOR THE MICROBIOME IN PROSTATE CANCER? The human microbiome comprises the bacterial community that lives on and in our bodies, and is being increasingly recognized for its regulatory role in many aspects of human biology and disease, from metabolism and inflammation to neuropathy and cancer. The microbiome, particularly the community housed in the gastrointestinal tract, is a major modulator of host immunity. Homeostatic interactions are established with the immune system, resulting in generation or maintenance of immune cell types including TH17 cells and mucosal regulatory T cells [46]. The loss of homeostasis is referred to as dysbiosis, and can be caused by pathogen infections, failed epithelial barrier functions, or aberrant activation of microbial pattern recognition pathways including the TLR, NOD, and NLRP/inflammasome pathways, and results in a state of chronic inflammation [46]. Recent studies have indicated that the microbiome plays a role in promoting the efficacy of chemotherapy and immunotherapy through its effects on the host immune response [47,48]. A number of cancers are now linked with either direct (production of genotoxins and oncogenic virulence factors) or indirect (modulation of host immunity, inflammation, or metabolism) interactions with a dysbiotic microbiome [49]. While the normal prostate is not known to harbor its own resident microbial flora, prostate infections, urinary tract infections, microbial components entering the circulation, microbe-regulated metabolites, and hormone modulation may all be mechanisms by which the microbiome affects aspects of prostate cancer directly or through modulation of host immunity. The role of microbiome-regulated factors in prostate cancer development, progression, and therapeutic response has not been studied and may be a topic of great interest for future studies.
NOVEL MOUSE MODELS TO STUDY TUMOR^IMMUNE INTERACTIONS There is a general consensus that the field lacks mouse models that accurately recapitulate many features of human prostate cancer biology, and the use of xenograft models negates any insight into the role of the immune system. Immune-competent animal models often examine immune responses against implanted tumor cells, which express non-physiologic levels of foreign antigens, generating observations that may not apply in human patients. The establishment of improved, immune-competent murine models of prostate cancer that faithfully imitate features of human disease is critical. The Prostate
One important aspect of human prostate biology that is difficult to model in mice is the prostatic inflammation commonly observed in biopsies. It has been hypothesized that repeated episodic inflammation contributes to the development of prostate cancer. Modeling this process is challenging, as many agents are able to incite prostatic inflammation. Furthermore, inflammation is a chronic process that occurs over the lifetime of a man. A novel Hoxb13/rtTAþ/|TetO/ IL1 b þ/ mouse model has been developed in which Hoxb13 regulatory elements drive inducible expression of prostate specific expression of rTA [50], which controls expression of the pro-inflammatory cytokine IL-1b. This inducible system allows control over the timing and intensity of IL-1b expression to model the waxing and waning of chronic inflammation. Characterization of this model will be valuable in defining the role of inflammation in prostate cancer and generate hypotheses on the interplay between environmental factors and genetic background. Genetic screening in mammalian cells was made possible by the advent of RNAi technology. Recently, a novel high-throughput method was created to screen for critical inhibitory pathways in tumor-specific CD8 T cells in vivo [51]. CD8þ OT1 cells transduced with single shRNA molecules from a shRNA library were injected into B16-OVA tumor-bearing mice [51]. Subsequent deep sequencing of tumor infiltrating T cells for enriched clones identified genes that act to inhibit antitumor T cell responses [51]. Ppp2r2d phosphatase and a number of other genes were identified and represent potential new targets for immunotherapy [51]. These results suggest that models may not need to recapitulate every aspect of human disease to be useful. Accurately modeling specific aspects of the immune response such as T cell expansion or function can allow one to generate and test hypotheses relevant to human disease. Mouse models are important for generating preclinical toxicity and efficacy data and for modeling the sequencing, timing, and dose of immunotherapies and immunotherapy-drug combinations, to discover mechanisms and biomarkers of response, resistance, metastasis and recurrence, and for understanding the biology of tumor–immune–stromal interactions in the tumor microenvironment. IDENTIFYING AND STANDARDIZING PREDICTIVE BIOMARKERS FOR IMMUNOTHERAPYCLINICALTRIALS A significant challenge in clinical trials for cancer immunotherapies is a lack of biomarkers that predict responsiveness or resistance that can be used to monitor patients and as intermediate efficacy end-
Improving Prostate Cancer Immunotherapy points. Unlike conventional therapies, median overall survival benefits seen in response to cancer immunotherapeutics including Siplucel T, ProstVac, and ipilimumab in melanoma patients, do not correspond with improvements in median progression-free survival times or PSA declines [52]. In fact, PSA levels may continue to increase following treatment and radiographic scans may worsen, despite improvement in overall survival [52]. In order to identify biomarkers predictive of clinical responses to immunotherapies, a comprehensive assessment of immune components needs to be performed during clinical trials. Knowing what immune cells are present within the tumor dynamically, their functional activity including cytokine and chemokine signatures and their relevance to outcome is critical. Preliminary studies have indicated a cytokine signature (IL-10 and MCSF) that associates with increased survival in patients treated with ADT and ipilimumab. Addition of ADT to ipilimumab treatment increased both CD4þ T cell and CD8þ T cell infiltration into tumors and strong TH1 and T follicular effector responses. PD-1/PD-L1 may need to be expressed to elicit responses to PD-1/PD-L1-blockade [53]. As expression of PD-L1 is induced in the tumor and immune system in response to IFNg, the expression of IFNg may predict response to PD-1/PD-L1 blockade [53]. Systematic assessment to identify biomarkers of immunotherapy responses should also extend to include stromal cells within the tumor microenvironment and to other tumor and host biomarkers. Novel and more sophisticated techniques, such as molecular imaging, multi-color/photon immunohistochemistry or mass cytometry to map the spatial and temporal dynamics of assessed parameters will speed the identification of predictive biomarkers and the understanding of their role in prostate cancer immune biology. One of the most critical steps in identifying rational immunotherapy biomarkers is to develop standardized immune and tumor monitoring protocols performed before, during, and post-treatment on clinical trials. Currently, different trials use different methods to monitor immune responses, making it difficult to use results from other trials as validation for the value of promising biomarkers. In addition to an immune signature, common indicators already in use for prostate cancer, such as PSA levels, disease burden, the presence of visceral metastases, or responsiveness to ADT, should be studied for their utility in stratifying patients into groups that would predict response to immunotherapy. Standardizing protocols to assess biomarkers across immunotherapy clinical trials will speed the validation of predictive biomarkers, improve patient selection and the design of clinical trials, and
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accelerate the development of new immunotherapeutic drugs for prostate and other cancers. CONCLUSION In the past few years, cancer immunotherapy has made huge strides in the treatment of solid tumors, most notably in melanoma. Deep and durable tumor responses have been observed in subsets of melanoma and some other tumor types, and a number of patients from early clinical trials remain tumor-free today, indicating that immunotherapy may have the unique power to cure patients with aggressive, otherwise lethal disease. Extending these successes to prostate cancer patients is a critical goal in order to improve outcomes and achieve cures. At the 2014 Coffey– Holden Prostate Cancer Academy Meeting, the current state of the most critical topics and the next steps that need to be taken to harness the power of the immune system to cure prostate cancer were thoroughly and vigorously discussed. Some of the most over-arching unmet needs discussed include development of immunologic biomarkers that predict the efficacy of immunotherapies in clinical trials, generation of immune-competent mouse models that recapitulate human prostate cancer biology, and developing a thorough understanding of how the immune system interacts with the tumor, tumor stroma, altered systemic host factors, and other therapies. Progress in these areas and others discussed in this article will hopefully accelerate the development of effective immunotherapeutic regimens that will extend the lives of prostate cancer patients. The theme of the 2015 Coffey–Holden Prostate Cancer Academy will be “Multidisciplinary Intervention of Early, Lethal Metastatic Prostate Cancer.” REFERENCES 1. Keller ET, Rowley DR, Tomlins SA, Drake CG, Kantoff PW, Pienta KJ, Montie JE, Carter HB, Hruszkewicz AM, Gomez J, Mohla S, Getzenberg RH. Eleventh Prouts Neck Meeting on Prostate Cancer: Emerging strategies in prostate cancer therapy. Cancer Res 2007;67(20):9613–9615. 2. Pienta KJ, Walia G, Simons JW, Soule HR. Beyond the androgen receptor: New approaches to treating metastatic prostate cancer. Report of the 2013 Prouts Neck Prostate Cancer Meeting. Prostate 2013;74(3):314–320. 3. Olson BM, McNeel DG. CD8þ T cells specific for the androgen receptor are common in patients with prostate cancer and are able to lyse prostate tumor cells. Cancer Immunol Immunother 2011;60(6):781–792. 4. Muehlenbein MP, Cogswell FB, James MA, Koterski J, Ludwig GV. Testosterone correlates with Venezuelan equine encephalitis virus infection in macaques. Virol J 2006;3:19. 5. Furman D, Hejblum BP, Simon N, Jojic V, Dekker CL, Thiebaut R, Tibshirani RJ, Davis MM. Systems analysis of sex The Prostate
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