Prostate cancer patients on androgen deprivation therapy develop persistent changes in adaptive immune responses

Abstract

Prostate cancer is a significant cause of morbidity and mortality among men worldwide. The cornerstone treatment for metastatic prostate cancer is androgen deprivation, which has known effects on prostate tissue apoptosis and thymic regrowth. These findings, together with interest in developing immune-based treatments for prostate cancer, lead us to question whether androgen deprivation causes changes in the adaptive immune responses of prostate cancer patients, and whether the timing of changes has implications for the sequencing of immunotherapies in combination with androgen deprivation. Peripheral blood mononuclear cells were obtained from patients before beginning androgen deprivation therapy (ADT) and at several time points thereafter. These cells were analyzed for the frequency of specific lymphocyte populations and their response to stimulation. The development of prostate antigen-specific immune responses was assessed using SEREX (serological identification of antigens by recombinant expression). Patients developed expansion of the naive T-cell compartment persisting over the course of androgen deprivation, together with an increase in effector-cell response to stimulation, and the generation of prostate tissue-associated IgG antibody responses, implying a potential benefit to the use of ADT in combination with prostate cancer-directed immunotherapies. The optimal timing and sequence of androgen deprivation with immune-based therapies awaits future experimental evaluation.

Introduction

Prostate cancer is a significant cause of morbidity and mortality in the United States and remains the second leading cause of cancer-related death in men [1]. The last decade has observed enormous progress in the treatment of metastatic prostate cancer. Docetaxel was approved by the FDA as the only contemporary chemotherapeutic agent demonstrated to prolong survival in patients with androgen-independent, metastatic prostate cancer [2], [3]. Despite this advance, many patients and physicians believe that the small survival benefit provided by chemotherapy may not warrant its use in all individuals because of its potential negative impact on the quality of life [4]. Consequently, there is great interest in the development of treatments with fewer side effects. Immunologic therapies, and vaccines (also known as “active immunotherapies”) in particular, are one such treatment option being investigated [5], [6]. Recent clinical trials have demonstrated an increase in overall survival of patients treated with prostate cancer vaccines [7], [8], better than that observed with chemotherapeutic agents, and FDA approval for at least one prostate cancer vaccine is anticipated in the near future.

Androgen deprivation therapy (ADT) is the cornerstone of treatment for patients with metastatic prostate cancer, and the concurrent use of ADT is the standard of care for all other treatments for metastatic prostate cancer. ADT is achieved clinically through orchiectomy or with administration of chemical castrants, such as gonadotropin-releasing hormone (GnRH) agonists, and/or antiandrogens. GnRH agonists function through continuous stimulation of the GnRH receptor, which ultimately induces desensitization and blockade of the pituitary–gonadal axis achieving castrate levels of testosterone [9]. The nonsteroidal antiandrogens compete directly with testosterone and dihydrotestosterone for the ligand-binding domain of the androgen receptor. In addition to competitive inhibition of the androgen receptor, the steroidal antiandrogens have antigonadotrophic effects that suppress testosterone synthesis [10]. Although the individual therapies function through different mechanisms to inactivate the androgen receptor, the ultimate outcome is atrophy and death of androgen-dependent cells of the prostate [11], [12].

In the case of orchiectomy, prostatic glandular cells undergo apoptosis characterized by DNA fragmentation, cell surface blebbing, and the formation of apoptotic bodies [13]. The apoptotic bodies are recognized by scavenger receptors, phagocytized and digested by macrophages [14]. However, an expansion of dendritic cells have been reported at the prostate following orchiectomy as well [15], implicating a possible dendritic-cell role in the phagocytosis and presentation of prostate antigens. Furthermore, prostate cancer patients undergoing androgen deprivation experience a T-cell infiltration of the prostate by 1 month post-treatment, and the infiltrates appear to be of an oligoclonal nature. These findings imply that an adaptive response to the prostate may arise after androgen deprivation [15]. In the case of androgen deprivation achieved by chemical castration, tumor-cell vacuolization and glandular atrophy are observed. However, the levels of apoptosis appear unchanged, implying that the predominant form of cell death may be achieved through an alternative mechanism [11].

Numerous observations have also demonstrated an effect of androgens on lymphocyte numbers and function. Briefly, thymic involution can be reversed in mouse [16] and rat [17] models after orchiectomy. This regrowth is characterized by an increase in the weight and cellularity of the thymus and can be reversed when androgens are re-administered to the castrate subject [18]. The observed thymic regrowth has been attributed to an expansion of the thymic epithelia expressing increased levels of CCL25, which promotes immigration of early thymic progenitors to the thymus [19]. A robust expansion of the common lymphoid progenitor cells can also be observed in castrated mice given hematopoietic stem-cell transplants compared to sham-treated animals [20], with data suggesting immunosuppressive effects of testosterone. In human beings, administration of GnRH agonists in prostate cancer patients has been reported to induce expansion of signal-joint thymic recent emigrant cells (sj TRECs) [21]. This T-cell expansion can occur late in life when sj TRECs are reported to be at their lowest levels [22], and the risk for prostate cancer increases. These observations suggest that androgens have an immunosuppressive effect, and ADT might function to reverse this suppression late in life.

The observations that ADT can affect T lymphocytes and elicit prostate tissue-associated inflammation suggest that ADT might be combined with active immunotherapies. It was found that in a mouse model of prostate cancer, tumor-bearing animals receiving an adoptive transfer of prostate tumor-specific T-cells, followed by castration and vaccination, had greater T-cell expansion and development of an effector phenotype in the adoptively transferred cells compared with tumor-bearing mice similarly treated but with sham castration [23]. Roden et al. demonstrated splenocytes from castrated mice receiving an ovalbumin-specific vaccine proliferated more robustly in response to ovalbumin stimulation compared to splenocytes of similarly vaccinated controls from noncastrated mice [24]. Koh et al. reported that mice vaccinated with a dendritic-cell vaccine, and then surgically castrated, had a greater number of antigen-specific IFN-γ-secreting cells compared to vaccinated mice receiving sham surgery [25]. Taken together, these results from multiple investigator groups suggest that it might be clinically beneficial to combine active immunotherapies with androgen deprivation [26]. However, timing the administration of these therapies to gain maximum benefit needs to be experimentally determined, especially if the effects of androgen deprivation on the adaptive arm of the immune system are persistent over time.

To investigate the effects of ADT on the adaptive immune system and whether they are persistent, our analysis focused on the frequency of circulating T-cell subsets collected from prostate cancer patients at various time points up to 24 months after beginning androgen deprivation. We then characterized the ability of T-cell subsets to proliferate and express cytokines after receptor or mitogen activation. Finally, given the observations that ADT elicits T-cell infiltration of prostate tissue [15], we asked if antigen-specific responses to proteins expressed in the prostate develop after ADT, which proteins were recognized, and whether these responses are persistent over the course of therapy. For these studies, we employed the SEREX (serological identification of antigens by recombinant expression) methodology [27]. We report an alteration to the T-cell repertoire develops following ADT, with an expansion of naive T-cells and recent thymic emigrants (RTEs). This T-cell expansion is detectable at least by 1 month after beginning ADT, and the expansion was detectable up to 2 years later in specific individuals. Similarly, IgG responses were elicited to prostate tissue antigens as early as 1 month after beginning ADT, as well as after many months of treatment. Together, our findings suggest that changes in the adaptive immune system following androgen deprivation may occur early after beginning treatment, and may be persistent for a long time. These observations may suggest that active immunotherapies might be used in sequence with androgen deprivation and/or might be affected by the concurrent use of androgen deprivation.