Combining prostate cancer radiotherapy with therapies targeting the androgen receptor axis

Background Prostate cancer ( pc a) is the most common non-dermatologic cancer and the 3rd leading cause of male cancer mortality in Canada. In patients with high-risk localized or recurrent pc a, management typically includes the combination of long-term androgen deprivation therapy ( adt ) and radiotherapy ( rt ). New androgen-receptor-axis targeted therapies ( arat s), which await validation, offer an option to intensify therapy. In this narrative review, we report the relevant history that has supported combining adt with rt . The literature in PubMed was searched for studies involving pc a and novel arat s (abiraterone acetate, enzalutamide, apalutamide, darolutamide) published between 1995 and 2019. Literature discussing clinical trials in which those modalities were combined was extracted and synthesized into a combined molecular and clinical discussion. Potential treatment intensification mechanisms and rationales are explored. Early results from three phase i / ii trials demonstrated that concurrent abiraterone acetate, adt , and rt is safe, improves the extent of chemical castration, and is associated with limited treatment failures. A single in vitro study implies synergy for radiosensitization beyond that facilitated by conventional adt . Studies investigating the combination of other arat s with rt are under way, including multiple phase iii trials, but short-term results are not yet available.


INTRODUCTION
Prostate cancer (pca) is the most common non-dermatologic malignancy affecting men in both Europe and North America 1 . Given the broad range of prognoses for patients with localized pca, stratification into low-, moderate-, and high-risk groups is usually performed ( Table i). In high-risk pca, a common treatment approach is to combine radiotherapy (rt) with androgen deprivation therapy (adt). Extensive evidence from randomized controlled trials and meta-analyses has demonstrated that adt with rt-generally external-beam rt (ebrt)-provides benefit for the full spectrum of pertinent oncologic outcomes in localized pca: overall survival (os), metastasis-free survival, biochemical progression-free survival, and local failure [2][3][4][5][6][7][8][9][10] .
Even though multiple relevant endpoints have been observed to be improved with dose-escalated ebrt (de-ebrt), patients in the highest-risk pca groups are still being considered for treatment intensification. Without such intensification, treatment with de-ebrt and long-term adt (lt-adt) will have failed for almost 50% of those patients by 6 years 11 . To refine the ability to design trials that further intensify therapy, a better understanding is required of adt's mechanisms for sensitizing pca to rt. In the present review, we walk through the relevant history of that therapeutic combination, we explore the potential mechanisms for its synergy, and we consider strategies for improved therapeutic combinations.

Optimal Duration of ADT in Combination with EBRT
In four randomized trials, it was observed that at least 4-6 months of adt were required for clinical benefit (Table iii). In those heterogeneous trials involving intermediate-and high-risk patients, biochemical control rates with st-adt and with ebrt alone were compared. The Trans-Tasman Radiation Oncology Group 96.01 trial demonstrated that 3 months and 6 months of adt were both superior to no adt 18 . Quebec L-101 and L-200 compared 3-5 or 10 months of adt, reporting improved biochemical control with both regimes 15 . However, only the Radiation Therapy Oncology Group 9910 trial performed inter-comparisons of st-adt durations. Extending st-adt to 9 months from 4 months was not found to further improve any biochemical or survival outcomes 19 .
The extension of adt to its present standard of 24-36 months in high-risk pca was largely based on the European Organisation for Research and Treatment of Cancer 22961 trial and the Radiation Therapy Oncology Group 9202 trial 16,17,22 . Those trials compared st-adt and lt-adt with conventional-dose ebrt in patients with predominantly high-risk pca. Both studies showed improved diseasespecific survival and os with lt-adt (28-36 months) as opposed to st-adt (4-6 months).
Two trials evaluated whether that length of adt was still necessary for patients with high-risk pca in the era of de-ebrt. The dart 01/05 trial found that extending adt to 28 months from 4 months improved biochemical control, metastasis-free survival, and os 20 . The pcs iv trial was designed to show-and in fact showed-that intermediateterm adt (18 months) was not inferior to lt-adt (36 months) with respect to os 21 . Given the gains in quality of life with a shorter course of adt, those results have prompted consideration of intermediate-term adt as a new standard of care for selected patients with high-risk pca treated with de-ebrt.

ADT Plus RT: Intracellular Mechanisms for Interaction
Numerous preclinical studies, in vitro and in vivo, have evaluated the interactions between adt and rt. Based on patient specimens, adt has been observed to induce apoptosis in epithelial cells and to inhibit proliferation 23 . In vitro studies of LNCaP cells [an androgen receptor (ar)-positive hormone-sensitive human pca cell line] showed that more apoptosis was produced during treatment with adt and rt than with either monotherapy 24 . Combination treatment with goserelin and rt using cultures of both LNCaP and PC3 (an ar-negative hormone-insensitive human pca cell line) inhibited cell proliferation by inhibition of the epidermal growth factor receptor 25 . However, neither study demonstrated the statistically significant reduction in survival required to demonstrate radiosensitivity.
Zietman et al. 26 were the first to show that adt lowers the tcd50 (the dose of rt necessary to control 50% of cultured tumour). They hypothesized two nonexclusive molecular mechanisms for that radiosensitization: suppression of tumour neovascularization improves blood flow through the more competent vasculature, and apoptosis-induced cytoreduction facilitates vascular access to hypoxic tissues. Tumour hypoxia has since been associated with impaired outcomes in pca 27 . Mechanistically, hypoxia stimulates angiogenic factors (for example, vascular endothelial growth factor), impairing tumour oxygenation secondary to the formation of incompetent vasculature 28 . As adt suppresses hypoxia-induced ar activity, the subsequent inhibition of hypoxia-inducible factor 1α transcription reduces the expression of vascular endothelial growth factor and limits neovascularization of hormone-sensitive pca cells, providing in vitro support for the latter hypothesis 29,30 .
It is important to appreciate that rt initially upregulates ar expression, with preclinical studies showing that rt induces the expression of ar-regulated proteins 31 . Work by Goodwin et al. 32 outlines how the addiction of castration-resistant pca to ar influences dna repair-and  thus, radioresistance. After rt, dna double-strand breaks activate the ar to enhance expression of numerous dna repair genes (PRKDC, KU70, PARP1) and dna repair protein rad51. More than 32 dna repair genes contain ar binding sites in their enhancer sequences 33 . Induction of those proteins can induce a positive feedback loop that causes radioresistance 32 . Initially, rt recruits dna protein kinase catalytic subunit (pkcs) to a double-strand break. Subsequent activation of dna pkcs also increases transcription of the ar. The ar then induces the expression and activity of additional dna pkcs through ar-mediated dna repair. Ultimately, the dna pkcs and ar upregulate each other in a process that expedites repair of the rt-caused doublestrand breaks. Importantly, intervention can interrupt the process. After castration, the decreased expression of KU70 in prostate tissue implies increased radiosensitivity of tumour 32 .

Strategies for Improving Therapeutic Combinations of ADT and EBRT
Relative to older anti-androgens, which were efficacious despite achieving only partial antagonism of the ar 34,35 , modern ar antagonists (for example, apalutamide, enzalutamide) have significantly improved binding affinity, can penetrate the cell for intracytosolic binding, and inhibit nuclear translocation of the ar 36,37 . Preclinically, our group demonstrated that a modern ar antagonist induced radiosensitization beyond that seen with adt alone 38 . Enzalutamide alone potentiated the response to radiation in LNCaP cells and, in combination with adt, in C4-2 hormone-resistant human pca cells. Dose-enhancement factors were 1.75 and 1.30 respectively. Maximal radiosensitization was achieved when enzalutamide was given concurrently with-as opposed to before or after-rt, and increased expression of phosphoγ-H2AX was consistent with enhanced dna damage.  The other new agents in this class of modern arats are abiraterone and darolutamide. Darolutamide is also an ar antagonist, but maintains efficacy against the ar F876L, ar W741L, and ar T877A resistance mutations that limit the efficacy of apalutamide and enzalutamide 39 . Also, limited access by darolutamide to the cerebral circulation has produced a modest neurocognitive adverse effects profile 40 . Abiraterone differs significantly in its mechanism,. Despite the achievement of castrate levels of serum testosterone by adt in most patients, production of intraprostatic or adrenal androgens (dehydroepiandrosterone and androstenedione) are sufficient to maintain expression of androgen-responsive genes 41,42 . Abiraterone selectively and irreversibly reduces both of those androgen biosynthesis pathways by potent inhibition of cyp17a1, suppressing the predominant remaining pathway for androgen biosynthesis 43 . Theoretically, that suppression could better potentiate the synergistic benefits seen with less-complete suppression of the androgen axis 26 .
The most recent clinical trial investigating de-ebrt and lt-adt in a high-risk population in need of further intensification showed worrisome rates of relapse approaching 50% at 6 years after treatment 11 . Although treatment intensification with chemotherapy can improve survival, the benefit came at the cost of increased toxicity, including treatment-related deaths. Implementing next-generation arats offers a more tolerable route to treatment intensification in the localized setting, preserving docetaxel as an effective choice for metastatic pca [44][45][46] . Because the arats have demonstrated clinical efficacy in more advanced clinical settings [47][48][49][50][51] and because there is preclinical evidence that these agents are radiosensitizers 38 , their combination with rt is the next logical step for treatment intensification in patients with high-risk pca.

METHODS
Relevant articles resulting from a literature search of PubMed for 1995-2019 were reviewed. These search terms and phrases were used individually and in combination: "localized prostate cancer," "androgen deprivation therapy," "radiation therapy," "randomized trial," "review," "high-risk prostate cancer," "intensification," "enzalutamide," "abiraterone acetate," "apalutamide," "darolutamide," and "clinical trials." All published, presented, or registered trials addressing the concurrent use of novel arats with rt were extracted for further review. For extracted works that combined rt with a novel arat, populations, interventions, and outcomes were extracted and summarized.

Clinical Trials Combining Abiraterone Acetate with RT
Three trials of abiraterone combined with rt were found (Table iv). A single phase i study investigated the safety of combining abiraterone with salvage rt 54 and two phase ii trials evaluated efficacy based on the extent of castration as assessed by testosterone level 52,53 . The two phase ii trials varied in their populations and adt durations. At a median follow-up of 21 and 23 months, a single treatment failure  had occurred in the phase ii studies. Notably, in the one 2-arm study, which compared abiraterone monotherapy with combined therapy using adt, castration levels of testosterone were achieved in only 78% of men receiving monotherapy compared with 100% of those receiving combination therapy. Toxicity data showed a 64% cumulative incidence of grade 3 lymphopenia during de-ebrt 52 .

Clinical Trials Combining Enzalutamide with RT
Eight ongoing clinical trials assessing the combination of enzalutamide with rt were found ( Table v) Two studies are randomized controlled trials: enzarad (NCT02446444, n = 802, accrual complete) and NCT02203695 (target accrual n = 122). NCT02203696, a multicentre trial in patients who are receiving salvage rt and st-adt is randomizing participants to either enzalutamide or placebo and has a primary endpoint of biochemical control. The fully accrued phase iii enzarad trial is randomizing patients with high-risk pca to receive either enzalutamide or placebo for 24 months in addition to de-ebrt and lt-adt. Its primary outcome is os, and based on the timing of its accrual, it is expected to be the first phase iii trial to provide insight into whether an arat can safely and effectively intensify de-ebrt and adt.

Clinical Trials Combining Apalutamide with RT
Seven ongoing trials, all either phase ii or iii, are evaluating the combination of apalutamide with rt ( Table vi). Three of the seven trials are combining apalutamide with de-ebrt. Another two are the only trials identified in our search to be combining stereotactic body rt with a novel arat (NCT02772588, NCT03503344), and two trials are combining salvage radiotherapy with an arat (NCT03311555, NCT03141671). Notably, two phase iii randomized controlled trials are including patients with high-risk pca.
The fully accrued atlas trial has a primary outcome of metastasis-free survival, an established surrogate for os 56 . In atlas, the accepted standard of de-ebrt and lt-adt has been intensified, randomizing participants to either apalutamide or placebo bicalutamide. In contrast, the European Organisation for Research and Treatment of Cancer's upcoming phase iii randomized controlled trial limits adt to the neoadjuvant and concurrent period, with a primary outcome of disease-free survival, but will not consider biochemical failure to be disease progression. Patients with intermediate-and high-risk pca will receive adt and de-ebrt and will be randomized to receive either apalutamide or placebo while on adt.

Clinical Trials Combining Darolutamide with RT
Our search methods did not identify any clinical trials combining darolutamide with rt directly. Outside our established search, abstracts made reference to the upcoming Darolutamide Augments Standard Therapy for Localized High-Risk Cancer of the Prostate, a randomized phase iii trial in patients with high-risk pca receiving rt. Participants will be randomized to receive concurrent darolutamide or placebo with rt and lt-adt [Canadian Cancer Trials Group (cctg). Darolutamide augments standard therapy for localized high-risk cancer of the prostate (dasl-hicap). Kingston, ON: cctg; 2019].

DISCUSSION
This review of the clinical and preclinical evidence highlights the influence of rt on ar-mediated protein expression and the ar's role in enhancing dna repair and radioresistance 31,32 . Such data outline how the combination of adt and rt can disrupt those interactions to facilitate the survival benefits seen in patients with pca 33 . Despite combination therapy, key trials still show that an unacceptable proportion of men with high-risk pca will not achieve longterm disease control 11,16,57 .
Preclinical work has provided a limited demonstration that an arat can provide further synergy beyond adt's known potentiation of rt-mediated dna damage. Combined with the known clinical efficacy of those agents, the rationale to combine them with rt to facilitate treatment intensification is strong. The review of the literature in the Results section demonstrate that a multitude of studies exploring this concept are under way. Although studies combining rt with abiraterone have been completed and have not been followed with phase iii trials, randomized phase iii trials are evaluating rt combined with apalutamide [atlas (NCT03488810)], darolutamide [Canadian Cancer Trials Group (cctg). Darolutamide augments standard therapy for localized high-risk cancer of the prostate (dasl-hicap). Kingston, ON: cctg; 2019], and enzalutamide [enzarad (NCT02446444)]. Of those trials, atlas and enzarad have both fully accrued, but even early results are still awaited. Another incidental observation is that upcoming assessments in oligometastatic pca might also produce clinical data about subgroups that received an arat in combination with rt (NCT03784755). A notable absence is any phase iii trial that is accruing patients exclusively in the salvage setting, although four randomized phase ii trials are exploring that setting (NCT02057939, NCT02203695, NCT03311555, and NCT03141671).
Reflecting on past preclinical data can direct the field's next steps to intelligently fill this rapidly crowding clinical trials space. In consideration of arat and rt scheduling, the timing of bicalutamide treatment relative to rt affects the radiosensitivity of hormone-sensitive cell lines 34 . Furthermore, our group's preclinical work demonstrated that concurrent enzalutamide-compared with neoadjuvant enzalutamide, adjuvant enzalutamide, or adt-provides the most potent radiosensitization 38 .
Such observations direct the field to a few key areas that should be considered for preclinical investigation before clinical trials: n Establishing the most effective radiosensitizers of the novel arats n Scheduling of the agents to optimize radiosensitization With those studies completed, the duration and timing of a novel arat could be optimized and then compared  with the various available agents. It would be reasonable to expect that maximally suppressing the androgen axis with the combination of abiraterone and a modern ar antagonist could further potentiate radiosensitization. Such preclinical studies could have a signal adequate to support a clinical trial. The ongoing clinical work and the opportunities for preclinical studies hold great promise to direct and establish novel strategies that will enhance outcomes for patients with high-risk pca.

CONCLUSIONS
Suppressing the function of the ar (historically with the use of adt) remains an essential component in treating advanced pca. Although adt works synergistically with rt to provide further benefit, whether the use of novel arats could further potentiate that interaction is unknown. Early preclinical experiments and phase i/ii studies have implied that such combinations might be efficacious. Multiple phase iii trials in patients with high-risk pca are ongoing and will more firmly address those hypotheses.