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Immunotherapies in Genitourinary Oncology: Where Are We Now? Where Are We Going?

Simple Summary Genitourinary malignancies include cancers along the urinary tract and the male reproductive tract, encompassing the adrenal glands, kidneys, bladder, prostate, and testicles. Immunotherapy, which treats cancer by using the immune system to attack malignant cells, has historically been successful in treating some types of genitourinary cancers, especially of the bladder and kidney. In the past decade, a more precise method of immunotherapy, known as immune checkpoint inhibition, has gained popularity as it enhances the immune system’s ability to recognize and destroy tumor cells. Several immune checkpoint inhibitors have achieved success in patients with advanced genitourinary cancers. This review provides a brief overview of traditional immunotherapies, focuses on how immune checkpoint inhibitors have achieved success in patients with advanced cancers, and investigates the role for immunotherapy in genitourinary malignancies in the future. Abstract For decades, limited options existed to treat metastatic genitourinary cancers, including treatment options that could be classified as immunotherapy. Historically, immunotherapy centered on systemic cytokines for the treatment of metastatic kidney cancer, which had several adverse effects, as well as the Bacillus Calmette–Guérin vaccine for non-metastatic bladder cancer. Within the past decade, advances in immunotherapy have led to several approvals from the United States Food and Drug Administration, particularly in the field of immune checkpoint inhibition. Immune checkpoint inhibitors (ICIs) are now being used extensively to treat multiple solid tumors, including kidney and bladder cancers, and they are also being tested in many other cancers. Despite encouraging data from phase 2/3 clinical trials, less is known about biomarkers that may predict better response to ICIs. The effect of ICIs in genitourinary cancers is heterogeneous, with some tumor types having little clinical data available, or ICIs having limited activity in other tumors. In this review, we briefly discuss approved immunotherapy agents prior to the time of ICIs. Then, given the emergence of this class of agents, we summarize the several important ICIs and the clinical trials that led to their approval. Finally, we mention ongoing and future clinical trials.


Introduction
Genitourinary (GU) malignancies affect many people worldwide, with millions of new cases diagnosed annually. According to the World Health Organization's Global Cancer Observatory, in the year 2020, there were over 1,400,000 new diagnoses of prostate cancer, over 550,000 new cases of bladder cancer (in this article, interchangeable with urothelial carcinoma, which accounts for 90% of bladder cancer cases), and over 400,000 new cases of kidney cancer (in this article, interchangeable with renal cell carcinoma, which accounts for 90% of kidney cancer cases) [1]. Testicular cancer, a less common cancer, was diagnosed in over 70,000 people that same year [1]. Adrenocortical carcinoma is an even rarer tumor, with an estimated incidence of 1.02 per one million people [2].
In recent decades, our understanding of cancer and its pathophysiology has evolved. It has been well established that cancer cells have developed multiple mechanisms to avoid the immune system. This includes downregulation of antigen-presenting complexes via the Major Histocompatibility Complex (MHC) class 1 pathway in tumor cells [3], as well as suppressed host immune activation via regulatory T cells [4]. Multiple cytokines produced by tumors in large quantities, such as transforming growth factor beta (TGF-β), can also dampen the host immune response by preventing organic T-cell and NK-cell response [5]. By interfering with pathways in the tumor microenvironment, immune checkpoint inhibitors (ICIs) allow the immune system to "ramp up" and effectively target malignant cells. This article delves into the role of immunotherapy in the treatment of GU malignancies: specifically urothelial, renal, prostate, and other rarer tumors. We will review established molecular targets for GU immunotherapies as well as explore up-and-coming targets for disease treatment. We summarize selected clinical trials that are complete, those that are still in progress, and those that are about to begin.

Immune Checkpoint Inhibitors
The concept of immune checkpoint inhibition began with the discovery of genes encoding cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) [6] and programmed cell death protein 1 (PD-1) [7] at the end of the 20th century. Both products are expressed on T cells; CTLA-4 binds to B7 ligand and PD-1 binds to programmed death ligand-1 (PD-L1). The downstream effect of these interactions suppresses the immune system through many mechanisms including diminishing T-cell immune response, decreasing the production of inflammatory cytokines, and increasing activity of regulatory T cells [8]. Cancer cells can take advantage of this signaling, allowing tumors to develop while avoiding attack from the immune system. The goal of immune checkpoint inhibition is to prevent the suppression of the immune system, resulting in its stimulation to destroy tumor cells.
A handful of ICIs have been approved for use in various malignancies by the Food and Drug Administration since 2011; these include one CTLA-4 inhibitor (ipilimumab), three PD-1 inhibitors (nivolumab, pembrolizumab, and cemiplimab), and three PD-L1 inhibitors (atezolizumab, avelumab, and durvalumab) [9]. Six of these agents have been successfully applied to various GU cancers as well, with all having received either full or accelerated approval in recent years (Table 1). Several ICIs targeting other pathways are currently in the developmental stages [10].

Bladder/Urothelial Cancer
The use of immunotherapy in bladder cancer has been well studied. It began with approval of the Bacillus Calmette-Guérin (BCG) vaccine in 1990, which produced a robust immune response against bladder tumors. However, this malignancy remains difficult to treat, especially after it has metastasized. The advancement of ICIs has revolutionized treatment for bladder cancer. Several ICIs have been successfully used in different stages of bladder cancer, resulting in approval by the FDA for various stages and conditions ( Table 2). Abbreviations: mOS, median overall survival; mPFS, median progression-free survival; ORR, objective response rate; BCG, Bacillus Calmette-Guérin. * Complete response rate was the primary endpoint: 40.6%. ** Median disease-free survival was the primary endpoint: 20.8 months vs. 10.8 months in the intention-to-treat group.
3.1. Non-Muscle-Invasive Bladder Cancer (NMIBC) 3.1.1. BCG BCG was developed early in the 20th century as a live-attenuated vaccine that used Mycobacterium bovis against Mycobacterium tuberculosis to prevent clinical tuberculosis. BCG successfully inhibited tumor growth in the 1950s in mice that were infected with the vaccine [28]. Morales et al. [29] successfully used vesical and intradermal BCG in humans with superficial bladder cancer in the 1970s, which led to a successful prospective clinical trial [30]. The FDA approved intravesical BCG for patients with superficial bladder tumors in 1990, making it one of the most successful immunotherapy agents of the 20th century. It is thought that BCG causes innate and adaptive immune activation through cytokine and chemokine secretion, which leads to the recruitment of various immune cells to the bladder tumor site, resulting in death of bladder cancer cells [31]. BCG remains standard first-line treatment for high-risk NMIBC.

ICIs for NMIBC
Despite the success of BCG, up to 40% of patients with NMIBC fail this treatment [32]. These patients have limited options, including radical cystectomy, chemotherapy, or clinical trials with novel methods such as chemoablation and oncolytic viruses [32]. ICIs demonstrated promise in these patients as well.
The phase 2 trial KEYNOTE-057 (NCT02625961) enrolled patients with NIMBC who failed BCG to receive pembrolizumab [11]. After a median follow-up of 36.4 months, analysis of 96 patients showed a complete response rate (CRR) in 39 patients (40.6%) and median duration of response of 16.2 months (95% CI, 6.7-36.2), with no patients having progressed to muscle-invasive or metastatic disease. Based on this trial, pembrolizumab was approved in January 2020 for high-risk NMIBC with carcinoma in situ, with or without papillary tumors unresponsive to BCG therapy. KEYNOTE-676 (NCT03711032) is a phase 3 trial designed to expand on the findings from KEYNOTE-057 [33]. It is currently recruiting, and the primary outcome measures CRR and event-free survival.

Neoadjuvant Therapy for MIBC
MIBC is treated with radical cystectomy [36]. Neoadjuvant cisplatin-based chemotherapy regimens have traditionally improved survival outcomes [37]. Immunotherapy is being explored as an alternative for cisplatin-ineligible patients in the neoadjuvant setting, though no agents are currently FDA approved for this indication. Initial findings from the phase 2 PURE-01 trial (NCT02736266) showed that 21 of 50 patients (42%) who received 3 courses of pembrolizumab before radical cystectomy resulted in pT0 [38]. On extended follow-up, event-free survival rate at 24 months was 71.7% (95% CI, 62.7-82.0) and recurrence-free survival rate was 39.3% (95% CI, 19.2-80.5), suggesting that pembrolizumab may make a favorable neoadjuvant choice compared to standard chemotherapy [39]. In the phase 2 trial ABACUS (NCT02662309), two cycles of atezolizumab were given before cystectomy, and among 88 patients with evaluable data, 27 had a pathologic complete response (31%; 95% CI, 21-41), which met initial primary endpoint goals [40]. These results support further investigation and validation of the role of ICIs in this setting.

Adjuvant Therapy for MIBC
Unlike standard neoadjuvant therapy for MIBC, less is known regarding standard adjuvant chemotherapy due to poor clinical outcomes [41]. Phase 3 trials evaluating the use of adjuvant therapy with different ICIs are currently ongoing for MIBC patients.
In the IMvigor010 (NCT02450331) trial, 809 patients with MIBC who underwent radical cystectomy or nephroureterectomy were randomized to either adjuvant atezolizumab or observation [42]. There was no significant difference in median disease-free survival in the atezolizumab versus observation group (19.4 months vs. 16.6 months; HR 0.89; 95% CI, 0.74-1.08; p = 0.24), while serious adverse effects occurred for nearly double the patients who received atezolizumab compared to the observation group (31% vs. 18%). As these results were somewhat disappointing, they discourage the use of atezolizumab in this setting.
Other important studies are ongoing, such as the phase 3 trial AMBASSADOR (NCT03244384) which is evaluating pembrolizumab versus observation.

Bladder-Sparing Treatments Involving ICIs in MIBC
The 2021 ASCO Meeting provided new phase 2 clinical trials involving ICIs focusing on bladder-sparing techniques. In HCRN GU16-257 (NCT03558087), 76 patients with MIBC underwent treatment with gemcitabine, cisplatin, and nivolumab followed by clinical restaging; 31 of 64 patients had complete remission from the regimen, and 30 patients who opted not to undergo cystectomy received additional nivolumab and were alive at the time of data cutoff [43]. In another phase 2 trial (NCT02621151), 54 patients received one dose of pembrolizumab, followed by maximal transurethral resection of the bladder tumor (TURBT), followed by pembrolizumab again along with whole bladder radiation and gemcitabine. Forty-two patients (85%) completed the study and the estimated one-year bladder-intact disease-free survival rate was 77% [44]. In IMMUNOPRESERVE-SOGUG (NCT03702179), 32 patients received transurethral resection followed by durvalumab and tremelimumab, then normo-fractionated external beam radiation therapy. Twenty-six patients (81%) achieved complete remission and 12 month disease-free survival rate was 73% (95% CI, 59-91) [45]. These studies show promising preliminary data for patients with worsening MIBC and for those who are not candidates for radical cystectomy.

Frontline Maintenance Therapy for Locally Advanced or Metastatic Bladder Cancer in Patients Who Previously Received Cisplatin Chemotherapy
After patients receive first-line chemotherapy, some patients will receive maintenance therapy to prevent disease progression or recurrence. This strategy has been applied to several malignancies [46]. Changing the initial induction chemotherapy agent to another agent with a different mechanism of action, commonly known as "switch maintenance," may avoid extra toxicities while providing synergistic disease benefit. The rationale for immune checkpoint inhibition as maintenance therapy after chemotherapy has been applied to different tumors including bladder cancer.
In a smaller phase 2 study, pembrolizumab achieved less success when used as switch maintenance after platinum-based chemotherapy in metastatic bladder cancer patients (NCT02500121) [47]. Of 108 patients enrolled in the trial, 55 received pembrolizumab and 53 received placebo. ORR was 23% with pembrolizumab versus 10% with placebo. The mPFS was significantly longer for pembrolizumab maintenance compared to placebo (5.4 months vs. 3.0 months; HR 0.65, p = 0.04), but the mOS was not significantly different (22 months vs. 18.7 months; HR 0.91; 95% CI, 0.52-1.59). In this trial, there was no significant benefit for PD-L1-positive patients (based on combined positive score ≥ 10) for either treatment (p = 0.8 for PFS and p = 0.9 for OS).
A single-arm phase 2 trial (NCT01524991) enrolled 36 patients with mUC and no prior chemotherapy to two cycles of gemcitabine and cisplatin followed by four cycles of gemcitabine, cisplatin, and ipilimumab [48]. This small study was not successful in meeting its primary endpoint of the lower bound of 90% CI for one-year OS >60%, with one-year OS of 0.61 (lower bound 90% CI of 0.51). The mOS was 13.9 months (95% CI, 10.5-23.4), and mPFS was 7.9 months (95% CI, 6.4-9.9).

Frontline Use in Combination with Chemotherapy
The combination of ICIs with chemotherapy, which has been successfully implemented in other solid tumors such as non-small-cell lung cancer [49], has been tested in mUC.
The phase 2 trial INDUCOMAIN (NCT03390595) evaluated 85 cisplatin-ineligible patients with untreated metastatic urothelial carcinoma; 42 patients received two cycles of induction therapy of avelumab followed by six cycles of avelumab with carboplatingemcitabine followed by two weeks of maintenance therapy with avelumab, compared to 43 patients who received six cycles of carboplatin-gemcitabine [52]. Preliminary results presented at the European Society of Medical Oncology (ESMO) Virtual Congress 2020 showed that the avelumab cohort performed worse than the standard chemotherapy cohort based on ORR (57% vs. 53%, p = 0.73), mPFS (6.9 months vs. 7.4 months, p = 0.712), and mOS (10.5 months vs. 13.2 months, p = 0.264).
Results to date have been disappointing, but more phase 3 trials of frontline ICI and chemotherapy combination regimens for mUC are underway. NILE (NCT03682068) is recruiting 1434 patients to three arms: a combination of tremelimumab, durvalumab, and standard of care (SoC) chemotherapy, durvalumab and SoC chemotherapy, or SoC chemotherapy alone. CheckMate 901 (NCT03036098) is recruiting 1290 patients that will also be assigned to different treatment arms: nivolumab and ipilimumab, nivolumab and SoC chemotherapy, or SoC chemotherapy alone.
Overall, ICIs combined with chemotherapy have not been successful in the setting of mUC. Pembrolizumab, nivolumab, and atezolizumab individually combined with chemotherapy have not produced statistically significant benefits in mOS and mPFS compared to chemotherapy alone when used as first-line therapy.

Frontline Use with Combination of Two ICIs
Combination immunotherapy in the frontline setting is another option for mUC and has been evaluated in multiple trials. The phase 3 trial DANUBE (NCT02516241) included 1032 previously untreated, locally advanced and mUC patients that were divided into three arms: durvalumab plus tremelimumab (n = 342), durvalumab monotherapy (n = 346), and chemotherapy (n = 344), and patients were followed for a median 41.2 months [53]. Neither of the primary endpoints (mOS in both the high PD-L1 cohort and overall intention-to-treat group) was met in the study, as chemotherapy appeared to be more favorable than immunotherapy. In the population with high PD-L1 expression (as defined by the authors of the study), mOS was not statistically different for the durvalumab group versus chemotherapy group (14.4 months vs. 12.1 months; HR 0.89; 95% CI, 0.71-1.11; p = 0.30). For the overall intention-to-treat group, mOS was not statistically significant between the durvalumab and tremelimumab combination versus the chemotherapy group (15.1 months vs. 12.1 months; HR 0.85; 95% CI, 0.72-1.02; p = 0.08). In the intentionto-treat population, mPFS was the longest for the chemotherapy group at 6.7 months (95% CI, 5.7-7.3) compared to the durvalumab group (2.3 months; 95% CI, 1.9-3.5) and the durvalumab plus tremelimumab group (3.7 months; 95% CI, 3.4-4.8).
Overall, the combination of durvalumab and tremelimumab provided disappointing results. Another immunotherapy combination, nivolumab and ipilimumab, has been successfully used in the frontline setting for advanced RCC [54], advanced melanoma [55], and advanced non-small-cell lung cancer [56]. This regimen is currently being evaluated in mUC in the phase 3 trial CheckMate 901 (NCT03036098), as mentioned above in the prior section.

Frontline Use for Locally Advanced or Metastatic Cisplatin-Ineligible Patients
Different ICIs have been tested in the front line setting for cisplatin-ineligible patients in small, proof-of-concept studies. In cohort 1 of the phase 2 trial IMvigor210 (NCT02951767), 119 previously untreated cisplatin-ineligible patients with mUC received atezolizumab and had a mPFS of 2.7 months (95% CI, 2.1-4.2), mOS of 15.9 months (95% CI, 10.4-NE), and 23% ORR (95% CI, 16-31) at a median follow-up of 17.2 months [14]. Atezolizumab was granted accelerated approval in April 2017 based on these results. Updated follow-up time to a median of 29 months was presented at the 2018 ASCO Meeting, and results of mOS (16.3 months) and ORR (24%) were consistent with prior data [15].
In the phase 2 trial KEYNOTE-052 (NCT02335424), 89 of 370 patients (24%; 95% CI, 20-29) with no prior systemic chemotherapy received pembrolizumab and achieved an objective response [16]. Pembrolizumab was granted accelerated approval in May 2017 for use in these patients. With a minimum follow-up of two years, pembrolizumab continued to be effective in terms of ORR (28.6%; 95% CI, 24.1-33.5) and mOS (11.3 months; 95% CI, 9.7-13.1) [17]. The phase 3 trial KEYNOTE-361 (NCT02853305) included 1010 patients with advanced UC who received no prior systemic therapy were randomized to receive pembrolizumab with chemotherapy (n = 351), pembrolizumab only (n = 307), or chemotherapy only (n = 352). The preliminary data presented at the ESMO Virtual Congress 2020 showed that PFS and OS did not reach statistical significance between the pembrolizumab and chemotherapy group versus chemotherapy only [57]. In spite of KEYNOTE-361 and IMvigor310 (as mentioned in a prior section), at a recent Oncologic Drugs Advisory Committee meeting, that both pembrolizumab and atezolizumab retain accelerated approval as first-line therapy for advanced UC [58]. In August 2021, the FDA granted pembrolizumab full approval, given the lack of approved therapies in this frontline setting [59].
Results presented at the 2020 ASCO Meeting showed 45 patients with a median of 9 cycles of EV with pembrolizumab had an ORR of 73.3% (95% CI, 58.1-85.4), 12 month OS rate of 81.6% (95% CI, 62.0-91.8), and a mPFS of 12.3 months (95% CI, 7.98-NR) [18]. These results led the FDA to grant this combination as breakthrough designation therapy in February 2020.

Second-Line Therapy for Locally Advanced or Metastatic Bladder Cancer Patients after Cisplatin Chemotherapy
Before immune checkpoint inhibition, prognosis was grim for patients who relapsed after first-line platinum therapy due to a lack of available options [60], until ICIs provided renewed hope and improved patient outcomes in the second-line setting [61]. Several trials have been conducted with different ICIs as both monotherapy and as combination therapy.
The phase 2 trial CheckMate 275 (NCT02387996) evaluated nivolumab in patients with locally advanced or mUC with disease progression during, or following, platinumcontaining chemotherapy, or with disease progression within 12 months of neoadjuvant or adjuvant treatment with a platinum-containing chemotherapy [20]. Among 265 patients who received nivolumab, 52 (19.6%; 95% CI, 15.0-24.9) had a confirmed objective response. This led to accelerated approval for nivolumab for this indication in February 2017. On extended follow-up of at least 33.7 months, nivolumab continued to maintain its effectiveness [21].
In the phase 3 trial KEYNOTE-045 (NCT02256436), 542 patients with locally advanced or mUC with disease progression during, or following, platinum-containing chemotherapy, or within 12 months of receiving neoadjuvant or adjuvant treatment with platinumcontaining chemotherapy, were randomized to receive pembrolizumab (n = 270) or further chemotherapy (n = 272) [22]. The mOS (10.3 months vs. 7.4 months; HR 0.73; 95% CI, 0.59-0.91; p = 0.002) and ORR (21.1% vs. 11.4%, p = 0.001) both favored the group that received pembrolizumab. However, there was no significant difference in PFS between the two groups (HR 0.98; 95% CI, 0.81-1.19; p = 0.42). Based on the mOS and ORR, pembrolizumab received approval in this treatment setting in May 2017. With an extended median follow-up to 27.7 months, data continued to be consistent for OS and PFS, with maintained OS benefit and no statistical difference between PFS. Yet, at the 24 month cutoff, the PFS value was higher for the pembrolizumab group compared to the chemotherapy group (12.4% vs. 3.0%) [23]. Of note, pembrolizumab has been the only ICI to show a survival advantage compared with cytotoxic chemotherapy in this setting.
ICI combination has been investigated in the refractory disease setting. For example, the phase 1/2 trial CheckMate 032 (NCT01928394), which tested the safety and efficacy of nivolumab with and without ipilimumab, contained a cohort of 274 locally advanced or mUC patients that progressed on platinum chemotherapy were then treated with nivolumab monotherapy at a dose of 3 mg/kg (n = 78), nivolumab 3 mg/kg with ipilimumab 1 mg/kg (n = 104), or nivolumab 1 mg/kg with ipilimumab 3 mg/kg (n = 92) [65]. In the most recent data presented at the ESMO Virtual Congress 2020, the nivolumab 1 mg/kg with ipilimumab 3 mg/kg demonstrated superior clinical outcomes based on ORR, mPFS, and mOS, and patients with PD-L1 expression of ≥1% showing even better outcomes compared to patients with PD-L1 expression <1% [66].
In summary, within a one-year time span, atezolizumab, avelumab, nivolumab, pembrolizumab, and durvalumab were all approved by the FDA for second-line therapy, largely based on promising data from the phase 2 trials for patients in this stage. However, as the phase 3 data now available have been less encouraging than was initially expected for some of these agents, specifically for atezolizumab (IMvigor211) and durvalumab (DANUBE), there has been voluntary removal from drug manufacturers for this designation. Pembrolizumab remains the ICI of choice based on level 1 evidence.

Variant Histologic Subtypes of Urothelial Carcinoma and Response to ICIs
Most bladder cancer histology is pure urothelial carcinoma (PUC); however, there has been increased recognition of many other histological subtypes, collectively known as variant urothelial carcinomas (VUC) [67]. VUC may be more aggressive than PUC, and patients with VUC appear to have a worse prognosis [68,69]. Evidence for optimal treatments is lacking in VUC, as patients with VUC are generally excluded from clinical trials. In a retrospective multi-institutional study, 286 patients with PUC responded to ICIs at similar rates to 120 patients with VUC based on ORR (28% vs. 29%, p = 0.90), mOS (11.0 months vs. 10.1 months, p = 0.60), and mPFS (4.1 months vs. 5.2 months, p = 0.43), while patients with VUC with neuroendocrine features had the lowest PFS and OS [70]. More prospective clinical data are necessary to better evaluate ICIs in patients with VUC.

Kidney Cancer
The most common type of kidney cancer is renal cell carcinoma (RCC), which can be categorized as clear cell (cc) or non-clear cell (ncc) based on histology. There are now several ICI options available to treat RCC (Table 3). However, in the past, few therapies existed to treat metastatic RCC for many decades due to its lack of sensitivity to chemotherapy and radiation [71]. Systemic cytokine therapy was used to treat RCC prior to the development of targeted therapies and ICIs. Interleukin-2 (IL-2) and Interferon (IFN) alfa-2a were two cytokines that received FDA approval for metastatic RCC.
High-dose IL-2 was approved as a treatment for metastatic RCC by the FDA in 1992. In one of the earliest published studies of high-dose IL-2, among 149 patients with metastatic RCC that had failed prior treatment, there was an overall response of 20% [72]. A randomized phase 3 trial compared high-dose IL-2 with subcutaneous IL-2 combined with IFN alfa-2b; overall response rate was 23.2% versus 9.9% (p = 0.018) and durable complete response was 7.4% versus 0% in favor of high-dose IL-2. There was no significant difference in mPFS (p = 0.082) or OS (p = 0.211), although the data also favored high-dose IL-2 [73]. Overall, high-dose IL-2 therapy was associated with severe adverse effects such as capillary leak syndrome which historically limited its use to young and fit patients.
IFN alfa-2a received FDA approval in 2009 to treat metastatic RCC when combined with bevacizumab based on the results of the randomized phase 3 trial AVOREN [74,75]. This study enrolled 649 patients with previously untreated metastatic RCC to receive IFN alfa-2a with bevacizumab or IFN alfa-2a with placebo. Median duration of PFS for interferon with bevacizumab was significantly longer compared to the control group (10.2 months vs. 5.4 months; HR 0.63; 95% CI, 0.52-0.75; p = 0.0001), and ORR was 31% to 13% in favor of interferon with bevacizumab (p = 0.001) [74]. The mOS between the two groups showed no statistically significant benefit for interferon with bevacizumab [75]. p < 0.001) and ORR (29% vs. 52%, p < 0.001). For an extended follow-up minimum of four years, the mPFS and mOS remained advantageous for ipilimumab plus nivolumab over sunitinib for the intermediate-and poor-risk populations, but less effective than sunitinib for the favorable-risk population [77].

Combination of ICI with Tyrosine Kinase Inhibitor
Many ICIs have now been approved by the FDA as front-line therapy for metastatic ccRCC. This includes ICIs used in combination with a tyrosine kinase inhibitor (TKI) or an antibody that targets vascular endothelial growth factor (VEGF) or its associated receptor (VEGF-R). These therapies include pembrolizumab with axitinib, atezolizumab with bevacizumab, avelumab with axitinib, pembrolizumab with lenvatinib, and nivolumab with cabozantinib. Understanding angiogenesis has played a key role in the development of TKIs which target VEGF-R [87]. The combination of ICI and VEGF inhibitors together is based on the discovery that VEGF plays a role in immune suppression of the tumor microenvironment; hence, VEGF inhibition could provide a synergistic benefit with ICIs to reverse immune suppression and allow the immune system to more effectively destroy cancer cells [88][89][90]. Phase 3 clinical trials all compared these combinations with sunitinib monotherapy, which itself had been shown to be more effective than IFN as first-line therapy [91].
The phase 3 trial KEYNOTE-426 (NCT02853331) enrolled 861 advanced ccRCC patients to either pembrolizumab and axitinib (n = 432) or sunitinib (n = 429) [78]. The mPFS, 12 month overall survival, and ORR were all significantly better in the pembrolizumabaxitinib group compared to the sunitinib group, with benefit seen in all IMDC risk groups. The phase 3 trial JAVELIN Renal 101 (NCT02684006) enrolled 886 patients, randomized to either avelumab and axitinib (n = 442) or sunitinib (n = 444) [82]. In the trial, avelumab and axitinib were superior to sunitinib in terms of mPFS in the PD-L1+ subgroup and the overall intention to treat group. Avelumab plus axitinib received approval in May 2019 based on these results. Updated results with a minimum follow-up of 13 months continued to show patients had a significantly better mPFS when they received avelumab with axitinib (13.3 months vs. 8.0 months; HR 0.69; 95% CI, 0.57-0.83; p < 0.0001) [83]. This trial so far has not revealed significant differences in OS between groups (HR 0.796; 95% CI, 0.616-1.027; p = 0.0392), although OS data remain immature.
So far, the combinations of pembrolizumab plus axitinib, pembrolizumab plus lenvatinib, and nivolumab plus cabozantinib (in addition to ipilimumab plus nivolumab) have demonstrated statistically significant improvement in mOS and mPFS over sunitinib, while avelumab plus axitinib and atezolizumab plus bevacizumab have demonstrated statistically significant improvement in mPFS only. With several options recently approved in the frontline setting, there is now debate over the optimal regimen to use. No trials exist that directly compare combinations of ICI with VEGF/VEGF-R inhibitors, and the differences in demographics among patients enrolled in these trials prevent direct comparisons retrospectively. Patients enrolled in CLEAR who received lenvatinib and pembrolizumab had similar or improved quality of life over sunitinib [93]. Notably, the patients enrolled in CheckMate 9ER [94] and CheckMate 214 [95] who received ICI combination had superior quality of life over sunitinib. All regimens showed superior activity in tumors with sarcomatoid and rhabdoid features. Ultimately, the optimal treatment regimen is based on multiple factors including risk group, tumor burden, location of metastases, efficacy endpoints and patient and physician preference.

ICI Monotherapy
Although combination immunotherapy and ICI synergy with an anti-angiogenic agent have demonstrated success, adverse effects that lead to dose adjustments, treatment interruption, and diminished quality of life are common concerns. Hence, the concept of ICI monotherapy is being trialed as an alternative for patients who likely will not tolerate combination therapy. Pembrolizumab was recently tested as monotherapy in the open-label phase 2 trial KEYNOTE-427 (NCT02853344), in clear cell (cohort A) [96] and non-clear cell (cohort B) RCC [97]. In cohort A (n = 110), the ORR was 36.4% (95% CI, 27.4-46.1), mPFS was 7.1 months (95% CI, 5.6-11.0), and 24 month overall survival rate was 70.8% (mOS not reached). From a safety perspective, pembrolizumab was favorably tolerated. Nivolumab has been tested as first-line monotherapy in the phase 2 studies of HCRN GU16-260 (NCT03117309) [98] and OMNIVORE (NCT03203473) [99], which produced modest results and had adverse effects similar to prior studies involving nivolumab. Retrospective experience with ICI monotherapy has reported similar outcomes [100]. While these are proof-of concept studies, they raise the question of whether ICI monotherapy is an effective treatment strategy.
The combination of lenvatinib and pembrolizumab as second-line therapy for 104 metastatic ccRCC patients, who previously progressed during, or following, ICI therapy was studied in the phase 2 expansion cohort of Study 111/KEYNOTE-146 (NCT02501096) [101]. For 91 patients with an evaluable response, the ORR was 51% (95% CI, 39.9-62.2). The mPFS for 103 patients was 11.7 months (95% CI, 9.5-NE). The study suggests that lenvatinib with pembrolizumab may warrant further investigation as a second-line option for relapsed metastatic ccRCC treated with prior PD-1/PD-L1 inhibitors. Other phase 3 trials investigating the addition of ICI to TKI in the refractory setting including TiNivo2 [102], which combines tivozanib and nivolumab, and CONTACT-03 (NCT04338269) [103], which combines atezolizumab and cabozantinib, are currently ongoing or being planned.

Adjuvant Therapy after Nephrectomy
Few options exist for patients with high-risk RCC who previously underwent curativeintent nephrectomy [104]. Sunitinib is the only TKI approved in this setting based on the phase 3 trial S-TRAC (NCT00375674) which revealed a statistically significant prolonged disease-free survival over placebo despite the adverse effects of sunitinib [105]. However, this approval was controversial, as the phase 3 trial ASSURE (NCT00326898) showed that sunitinib did not have any survival benefit relative to placebo in the adjuvant setting [106]. Pembrolizumab is the first ICI to provide promising data as an adjuvant therapy, based on the phase 3 trial KEYNOTE-564 (NCT00375674) [107]. In this trial, 994 patients with ccRCC at high risk for recurrence after nephrectomy were randomized to adjuvant pembrolizumab (n = 496) or placebo (n = 498). Disease-free survival was significant improved in patients who received pembrolizumab (77.3% vs. 68.1%; HR for recurrence or death 0.68; 95% CI, 0.53-0.87; p = 0.002). Overall survival data remain immature. Based on these promising results, the FDA granted pembrolizumab in the adjuvant therapy setting priority review in August 2021.

Non-Clear Cell Renal Cell Carcinoma
When ccRCC and nccRCC tumors possess sarcomatoid or rhabdoid features, they are often more aggressive. Clinical implications include shorter time to the development of metastases and decreased overall survival time [108,109]. Because of their rarity and aggressive nature, no definitive treatments exist for these pathologic subtypes [110,111].
In single-institution studies, ICIs appear to have better outcomes for sarcomatoid RCC (sRCC) compared to targeted therapies like VEGF inhibitors [112,113]. In a meta-analysis of sRCC in JAVELIN Renal 101, IMmotion151, KEYNOTE-426, and the intermediate/poor patient group of CheckMate 214, the conclusion is support for ICI-driven therapy for these patients over sunitinib [114]. More prospective data are needed for patients with RCC with sarcomatoid and/or rhabdoid features treated with ICIs.
Similarly, for nccRCC, the best treatment options remain unclear due to underrepresentation in clinical trials. Most of the evidence-based data are limited to retrospective studies. Chahoud et al. [115] analyzed 40 patients (including 8 ccRCC patients with rhabdoid features) who received nivolumab at one institution. The mPFS was 4.9 months (95% CI, 3.53-10.27) and mOS was 21.7 months (95% CI, 7.83-NR). In one multi-institutional retrospective study, Koshkin et al. [116] analyzed 41 patients (including 5 patients with sarcomatoid features) with nccRCC who received at least one dose of nivolumab (median treatment duration 3.0 months). The mPFS was 3.5 months (95% CI, 1.9-5.0) and overall survival at 10 months was 68%. In another multi-institutional study, McKay et al. [117] included 43 patients (11 patients with rhabdoid and/or sarcomatoid features) who received PD-1/PD-L1 inhibitors. Overall, eight patients (19%) had an objective response, median time-to-treatment failure was 4.0 months (95% CI, 2.8-5.5), and mOS was 12.9 months (95% CI, 7.4-NR). All these studies underscore the urgent need for prospective studies in this heterogenous population.
KEYNOTE-427 (NCT02853344), as mentioned in a prior section, had a nccRCC cohort that received pembrolizumab monotherapy as first-line treatment. This is one of the first trials with nccRCC patients with published data. For 165 patients with previously untreated advanced nccRCC, the ORR was 26.7%, mPFS was 4.2 months (95% CI, 2.9-5.6), and mOS was 28.9 months (95% CI, 24.3-NR) [97]. These data suggest that immunotherapy may be effective as a first-line treatment for nccRCC.

Prostate Cancer
Prostate cancer tends to progress more slowly and is usually less lethal than other cancers, but after it metastasizes, the five-year survival rate drops significantly [118]. Metastatic castration-resistant prostate cancer (mCRPC), which is incurable, justifies the need for effective novel therapies. Unlike kidney and bladder cancer, few immunotherapies have been approved for prostate cancer due to disappointing results such as in phase 3 trials where participants received ipilimumab [119,120]. In KEYNOTE-199 (NCT02787005), which included 258 mCRPC patients who previously received docetaxel and endocrinetargeted therapy and were given pembrolizumab, the ORR was only 3% and 5% in two of the study cohorts with response evaluation criteria in solid tumors (RECIST)-measurable disease, but those patients who benefited had a robust response [121]. The limited role for ICIs in prostate cancer could be explained by a lower mutational burden and decreased homing targets for the immune system in the tumor microenvironment [122].
Sipuleucel-T is a vaccine-based therapy that utilizes host antigen-presenting cells (APCs) that are exogenously exposed to prostatic acid phosphatase (PAP) and granulocytemacrophage colony-stimulating factor (GM-CSF). PAP and GM-CSF are processed by the APCs, and the activated cells are infused into the recipient. T cells recognize the activated APCs and become primed for tumor attack. In the phase 3 IMPACT trial (NCT00065442), 512 mCRPC patients with little to no symptoms were treated with either sipuleucel-T (n = 341) or placebo (n = 171) [123]. For patients who received sipuleucel-T, there was a longer mOS (25.8 months vs. 21.7 months), and the adjusted hazard ratio for death was 0.78 (95% CI, 0.61-0.98; p = 0.03). However, the time to objective disease progression (14.6 weeks vs. 14.4 weeks; HR 0.95; 95% CI, 0.77-1.17; p = 0.63) or clinical disease progression (HR 0.92; 95% CI, 0.75-1.12; p = 0.40) did not differ significantly between the groups. The data helped lead to FDA approval of sipuleucel-T in patients with asymptomatic or minimally symptomatic mCRPC in April 2010.
Prostate cancer genetics has helped inform treatment. Liquid biopsies from blood samples evaluating circulating tumor DNA have been implemented successfully in a multiinstitutional case series of advanced prostate cancer, in which 15 of 460 patients (3.7%) had high levels of microsatellite instability (MSI-H) based on liquid biopsies [124]. In the study, nine patients with mCRPC received pembrolizumab, and three of five patients evaluated had a radiographic response to the treatment.
Pembrolizumab is the one ICI approved for a special group of cancer patients regardless of primary site based on genetic testing. In an initial phase 2 trial (NCT01876511) that enrolled 41 patients with various metastatic cancers (not including any GU cancers) with or without mismatch-repair deficiency, pembrolizumab showed a clinical benefit in patients with mismatch-repair gene deficiency [125]. The authors reported further success from an analysis of 86 patients, which included one prostate cancer patient that had a complete response [126]. Based on promising data from 149 patients in five trials, pembrolizumab was granted accelerated approval in May 2017 to treat MSI-H/dMMR (deficient mismatch repair) disease that had progressed on prior therapy, including unresectable or metastatic prostate cancer [127]. In June 2020, pembrolizumab received accelerated approval for adult and pediatric cancer patients with a high tumor mutational burden (TMB) (≥10 mutations/megabase) that had progressed despite prior treatment, based on results from the phase 2 trial KEYNOTE-158 (NCT02628067) [128]. The trial enrolled 1073 patients with advanced solid tumors which included 102 patients with a high tissue TMB and 688 patients in the non-high tissue TMB group. The ORR was 29% (95% CI, [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39] in the high tissue TMB cohort compared to 6% (95% CI, [5][6][7][8] in the non-high tissue TMB cohort. The field of immunotherapy in prostate cancer has expanded to include chimeric antigen receptor (CAR-T) cells and bispecific T-cell engagers (BiTe). In pre-clinical studies, engineered CAR-T cells were shown to effectively combat prostate cancer by targeting prostate specific membrane antigen (PSMA) [129]. More recently, there have been preclinical studies combining CAR-T cells with chemotherapy (docetaxel) that showed antitumor activity in a xenograft model [130]. In humans, there was a small phase 1 study where anti-PSMA CAR-T cells were infused into five patients with metastatic prostate cancer, and there was a partial reduction in PSA in two patients [131]. Pasotuxizumab, a bispecific T-cell engager developed to target prostate cancer cells via PSMA, was given to 47 patients with advanced prostate cancer either subcutaneously or through continuous IV (cIV) in a phase 1 study (NCT01723475) [132]. In the cIV cohort, there was a dose-dependent reduction in the level of PSA in 14 out of 16 patients, illustrating that BiTe may be a therapy option for CRPC, though further clinical studies are necessary.
For neuroendocrine mCRPC, there is a Category 3 recommendation from the National Comprehensive Cancer Network that the combination of atezolizumab, carboplatin, and etoposide can be used as a treatment strategy. This is an extrapolation from a study that showed that this treatment combination resulted in longer OS and PFS in patients with extensive-stage small-cell lung carcinoma [133]. In the phase 2 trial DART SWOG S1609 (NCT02834013), 32 patients with non-pancreatic neuroendocrine tumors were enrolled, including two patients with prostate cancer, who received ipilimumab and nivolumab [134]. Patients with high-grade tumors had a significantly better overall response rate compared to patients with low/intermediate grade tumors (p = 0.004).

Penile/Testicular/Adrenocortical Cancer
With rare GU tumors including penile, testicular, and adrenocortical cancers, there are indications for immunotherapy in advanced disease that has responded poorly to prior treatment in specific circumstances. Like prostate cancer with MSI-H/dMMR, pembrolizumab can be used for these cancers with MSI-H/dMMR [127]. In a case series from a phase 2 basket trial (NCT02721732), pembrolizumab was effective in achieving partial response in one patient with penile SCC and MSI-H, while two other patients with microsatellite stability progressed [135].
Overall, sufficient data on immunotherapy are lacking for these cancers. Results have been limited to phase 1/2 trials, and so far, ICIs have not shown to be very effective in treating these tumors. In a phase 2 study that enrolled 12 patients with relapsed metastatic germ-cell tumors (11 of testicular origin) refractory to first line and salvage chemotherapy who were treated with pembrolizumab, there was no significant partial response or complete response in any of the patients [136]. In the phase 2 trial APACHE (NCT03081923), 22 patients were randomized to receive either durvalumab with tremelimumab (n = 11) versus durvalumab alone (n = 11), and nearly all the patients had progressive disease regardless of TMB and PD-L1 expression [137]. More trials have enrolled patients with tumors testing other ICIs, including a phase 1 trial (NCT02496208) evaluating optimal dosing for cabozantinib, nivolumab, and ipilimumab that enrolled six patients with germ cell tumor and one patient with Sertoli cell tumor [138].
Likewise, adrenocortical carcinomas (ACC) are a heterogeneous group of characteristically late-presenting tumors for which treatment options remain limited. There are data from four phase 2 trials in ACC for the efficacy of pembrolizumab (NCT02721732 and NCT02673333) [139,140], nivolumab (NCT02720484) [141], and nivolumab with ipilimumab (NCT03333616) [142]. These phase 2 trials all demonstrated ICIs possess some activity against ACC. The largest ICI trial is the phase 1b trial JAVELIN Solid Tumor (NCT01772004) of avelumab which enrolled 50 patients, of which 25 patients remained on the adrenolytic mitotane [143]. With a median follow-up time of 16.5 months, the disease control rate was 48.0%, OS was 10.6 months (95% CI, 7.4-15.0), and mPFS (95% CI, 1.4-4.0) was 2.6 months. ORR was 16.7% among 12 patients with PD-L1+ tumors and 3.3% among 30 patients with PD-L1-tumors. Larger trials involving ICIs are necessary to further examine their efficacy in ACC.

Potential Predictive Biomarkers for ICIs
Despite the rapid advancements in immunotherapy, many patients who receive ICIs either continue to have their disease progress despite treatment, or their cancer may initially respond to therapy but then relapse. Because of this, there is an urgent need for predictive biomarkers for ICIs. Three promising biomarkers approved by the FDA for patient selection to respond to ICIs, as mentioned in this article, include PD-L1, MSI-H/dMMR, and TMB [144]. Although data support the notion that the presence of these biomarkers may render cancer more susceptible ICIs, conflicting data exist. There are no accepted guidelines, cutoffs, or standard assays for PD-L1 expression, TMB, or MSI-H/dMMR. which vary among different cancers and were studied differently in clinical trials [144][145][146]. Indeed, for ICI clinical trials involving mUC and RCC, cutoffs to determine PD-L1 status for immune cells and/or tumor cells, and the assays used to detect PD-L1 status, varied widely. Presently, these biomarkers are not reliable predictors for ICI efficacy in GU oncology. Several emerging biomarkers currently being studied include mutations in phosphatase, polymerase, and transcription factor proteins, as well as combining multiple biomarkers for improving predictive performance [144,145].

Future Immune Checkpoint Inhibition Trials
ICIs have made major strides in GU oncology in the past decade. New trials are active and enrolling, aiming to steadily increase the median overall survival and progression-free survival in patients with metastatic GU cancers (Table 4). A large proportion of upcoming immune checkpoint inhibition trials will evaluate for synergistic benefit among ICIs and other treatment strategies. These include EZH2 and arginase inhibitors, hypomethylating agents, novel decoy receptors, and new recombinant human interleukins. Phase 2/3 trials are underway to evaluate ICIs as front-line therapy in combination with tyrosine kinase inhibitors in the GU tumors, building upon the success of this combination in RCC.

Conclusions
In summary, the field of immunotherapy has rapidly expanded in the past decade, especially involving immune checkpoint inhibition. This has made for groundbreaking strides in the world of GU oncology. ICIs are now at the forefront of cancer therapy, especially in front-line metastatic RCC and in multiple settings of bladder/urothelial cancer. The use of ICIs in prostate cancer, testicular cancer, penile cancer, and adrenocortical cancer has not been as heavily explored, but in certain situations may provide clinical benefit. However, there are still a few setbacks with ICIs, as not all phase 3 trials have been shown to be superior to traditional therapy, and not all long-term responses have remained durable. This suggests that further research is still necessary to determine which patients are most likely to benefit from immunotherapy. Looking towards the future, several more clinical trials are underway in GU oncology evaluating the combination of ICIs with other targeted therapies. The goal is to enhance "precision therapy" to increase overall survival and progression-free survival. With the successes that have stemmed from the intersection of GU oncology and immunotherapy over the last decade, the future of these fields is bright.