Recent advances in medical therapy for metastatic urothelial cancer

Cytotoxic chemotherapy has been the mainstay of medical therapy for metastatic urothelial cancer. Currently, the gemcitabine/cisplatin regimen is widely used worldwide as the standard first-line medical treatment. Very recently, in 2017, pembrolizumab, a highly selective, humanized monoclonal IgG4κ isotype antibody against programmed death 1, was approved as a second-line treatment to be used after platina-based chemotherapy for metastatic urothelial cancer in Japan. Based on its promising anti-tumor efficacy and manageable safety profile as demonstrated in the phase III KEYNOTE-045 trial, pembrolizumab therapy is expected to be rapidly introduced for treating metastatic urothelial cancer in clinical practice. The paradigm of medical treatment for patients with metastatic UC is dramatically changing through the introduction of this and other immune-checkpoint inhibitors. In this article, we provide a brief overview of these immune-checkpoint inhibitors and a comprehensive summary of the use of cytotoxic chemotherapy for metastatic urothelial cancer, including ongoing clinical trials.


Introduction
Cytotoxic chemotherapy has been the mainstay of medical therapy for patients with metastatic urothelial cancer (UC) for a long time. Since 1985, when Sternberg et al. reported the excellent results of the cisplatin-based multi-agent chemotherapy regimen known as MVAC (methotrexate, vinblastine, adriamycin, cisplatin), no medical treatment has been more effective [1]. Only one clinical trial comparing a gemcitabine-plus-cisplatin regimen (GC) with MVAC demonstrated that the GC regimen had a treatment efficacy similar to that of MVAC while causing less toxicity than MVAC [2,3]. Currently, therefore, the GC regimen is widely used worldwide as the standard first-line medical treatment.
On December 25, 2017, pembrolizumab (Keytruda ® , Merck), a highly selective, humanized monoclonal IgG4κ isotype antibody against programmed death 1 (PD-1) that selectively inhibits the interaction between PD-1 (which is expressed on activated T cells) and PD-1 ligand 1 (PD-L1) and 2 (PD-L2) [which are expressed on antigen-presenting cells (APC) and cancer cells] was approved as a secondline treatment for use after platina-based chemotherapy for patients with metastatic UC in Japan.
We are currently on the verge of the second breakthrough in the medical treatment of metastatic UC since the discovery of MVAC therapy. The paradigm of medical treatment for metastatic UC is dramatically changing through the introduction of this and other immune-checkpoint inhibitors. The United States Food and Drug Administration (US-FDA) has approved five immune-checkpoint inhibitors including pembrolizumab, atezolizumab (Tecentriq ® , Roche), nivolumab (Opdivo ® , Ono/Bristol-Myers\Squibb), avelumab (Bavencio ® , Merck, Pfizer, Eli Lilly), and durvalumab (Imfinzi ® , Medimmune/AstraZeneca). Various clinical trials currently underway are attempting to increase the efficacy of each of these checkpoint inhibitors by combining them with other immunogenic agents and with cytotoxic chemotherapy. In this article, we provide a brief overview of these immune-checkpoint inhibitors and a summary of comprehensive medical treatment using cytotoxic chemotherapy for metastatic UC, including ongoing clinical trials. The doses and schedules of the regimens currently administered in Japan are shown in Table 1.

Immune-checkpoint inhibitors
PD-L1 and PD-L2, which are known to be expressed on the surfaces of APC and cancer cells, engage PD-1, which is expressed on CD8-positive cytotoxic T lymphocytes (CTLs), as negative immune regulators [4,5]. When the complex of PD-1 and PD-L1/PD-L2 is formed, immune tolerance is achieved. Novel immune therapy using immune-checkpoint inhibitors can destroy this immune tolerance (Fig. 1). When immune tolerance is broken by immune-checkpoint inhibitors, the CD8-positive CTLs can recognize the neoantigens from cancer cells that are presented on major histocompatibility class I (MHC-I) or class II (MHC-II) molecules. Thus, Cancer cell CTL they are activated and proliferate, leading to an antigen-specific immune response that kills neoantigen-bearing cancer cells [4,5]. This is the functional mechanism of this novel immune therapy (Fig. 1). In addition, the main results of the clinical trials using current immune-checkpoint inhibitors for the patients with metastatic UC are shown in Table 2.

Pembrolizumab
The essential clinical trial of pembrolizumab for metastatic UC is the KEYNOTE-045 study [6]. The KEYNOTE-045 study is an open-label, international, phase III clinical trial that randomly assigned 542 patients with advanced UC that had recurred or progressed after platinum-based chemotherapy to receive either pembrolizumab at a dose of 200 mg or the investigator's choice of chemotherapy with paclitaxel, docetaxel, or vinflunine every 3 weeks [6]. In this study, pembrolizumab was associated with significantly longer overall survival (OS) and a lower rate of adverse events (AEs) compared to chemotherapy as the second-line therapy for platinum-refractory advanced UC [6].  [6]. There was no significant difference in progression-free survival (PFS) period either among all treated patients or among high PD-L1 expression patients [6]. As for AEs, pembrolizumab-treated patients experienced significantly fewer events of any grade and significantly fewer events of grades 3, 4, and 5 (60.9 and 15.0%, respectively) compared to chemotherapy-treated patients (90.2 and 49.4%, respectively) [6]. Pembrolizumab has received accelerated approval from the US-FDA as a first-line therapy for patients with cisplatin-containing chemotherapy-ineligible metastatic UC. The KEYNOTE-052 study is an ongoing single-arm, openlabel trial for patients with locally advanced or metastatic UC who are ineligible for cisplatin-containing chemotherapy [7]. This study contains 370 patients. At a median followup of 7.8 months, the objective response rate (ORR) was approximately 29% and the median duration of response had not yet been reached. The most common side effects in the two completed trials included fatigue, musculoskeletal pain, decreased appetite, nausea, and diarrhea [6,7].
Currently, a multi-center international randomized clinical trial including data from Japan is comparing 1 3 pembrolizumab monotherapy and standard chemotherapy with or without pembrolizumab as first-line therapy for patients with previously untreated advanced UC.

Other checkpoint inhibitors
To date, as described above, five checkpoint inhibitors including pembrolizumab have been shown to be efficacious and have accordingly received approval from the US-FDA for metastatic UC. Here, we provide a brief overview of these other agents, although they have not yet been approved in Japan.

Nivolumab
Nivolumab is a fully human IgG4 anti-PD-1 monoclonal antibody similar to pembrolizumab. After its promising antitumor efficacy against metastatic renal cell cancer (RCC) and its manageable safety profile were demonstrated in the phase III Checkmate025 trial, nivolumab was launched in 2016 and has been rapidly introduced in clinical practice for metastatic RCC in Japan [8].
Nivolumab is currently being tested in two clinical trials for advanced UC in Japan. The first study is a phase II single-arm study for patients with advanced UC who were previously treated with at least one platinum-based chemotherapy regimen. The other is a phase III international, multi-institutional clinical trial comparing the combination of nivolumab and ipilimumab (Yervoy ® , Bristol-Myers Squibb), a checkpoint inhibitor of another type that blocks the complex of CTL-associated protein 4 (CTLA-4) and its ligand, with the combination of nivolumab and standard chemotherapy, and standard chemotherapy as first-line therapy for previously untreated patients.
Atezolizumab is currently being tested in a phase III international, multi-institutional clinical trial comparing atezolizumab monotherapy, a combination of atezolizumab and standard chemotherapy, and standard chemotherapy as first-line therapy for patients with previously untreated advanced UC.
Durvalumab is currently being tested in a phase III international, multi-institutional clinical trial to compare durvalumab monotherapy, a combination of durvalumab and tremelimumab (AstraZeneca), a fully human monoclonal antibody against anti-CTLA-4, and standard chemotherapy as first-line therapy for patients with previously untreated advanced UC.

Cytotoxic chemotherapy
Although these immune-checkpoint inhibitors have led to breakthroughs in medical therapy for patients with metastatic UC, cytotoxic chemotherapy is still the standard firstline therapy. Among the chemotherapeutic regimens, the GC regimen is the best known and most frequently used regimen.

Gemcitabine and carboplatin (GCa) regimen
The renal toxicity of platinum-based combinations presents a common problem for patients with metastatic UC. Gemcitabine and carboplatin regimens are used as an option for first-line therapy in cisplatin-ineligible patients with metastatic UC. In a phase II clinical trial for patients with previously untreated advanced UC (n = 60) with gemcitabine at a dose of 1000 mg/m 2 (days 1 and 8) and carboplatin at a dose of area under the concentration curve of 5 (AUC5, day 1), every 21 days for a total of six cycles, intent-to-treat analysis demonstrated an ORR of 38.4% (95% CI 26-51.8%) including 11.7% with CR. The median PFS and OS periods were 7.6 months (95% CI 4.5-10.7 months) and 16.3 months (95% CI 12-20.6 months), respectively [13]. The median OS appeared comparable to that reported for the MVAC and GC combination regimens. Severe (grade 3-4) treatment-related AEs included anemia (18%), thrombocytopenia (23%), and neutropenia (52%) including febrile neutropenia (11%), whereas non-hematologic toxicity was rare [13].

MVAC regimen
The classic MVAC regimen, which consists of methotrexate, vinblastine, doxorubicin, and cisplatin, was proposed in 1985 by Sternberg et al., and has been used since then to treat metastatic UC [1]. Treatment consists of 4-week (28 days) cycles of 30 mg/m 2 of methotrexate (day 1), followed by 3 mg/m 2 of vinblastine, 30 mg/m 2 of doxorubicin, and 70 mg/m 2 of cisplatin (day 2), and concluded with repeat vinblastine and methotrexate on days 15 and 22 (Table 1). In this first report, excellent treatment results of tumor regression were noted including 71% ORR and 50% CR [1,14]. Until its replacement by GC regimen, the MVAC regimen was the standard first-line chemotherapy for a long time; nowadays, it is used as an optional first-line therapy as well as a second-line therapy after the GC regimen.

High-dose MVAC (HD-MVAC) regimen
To increase the treatment results, a dose-dense schedule of the MVAC regimen has also been proposed. A randomized trial was conducted to evaluate the anti-tumor activity of and survival associated with high-dose-intensity chemotherapy with methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) plus granulocyte colony-stimulating factor (HD-MVAC) versus MVAC in patients with advanced UC [15]. A total of 263 patients with metastatic or advanced UC who had no prior chemotherapy were randomized to HD-MVAC (2-week cycles) or MVAC (4-week cycles) [15]. Using an intent-to-treat analysis, at a median follow-up of 38 months, on the HD-MVAC arm, there were 62% (95% CI 54-70%) including 21% CRs [15]. On the MVAC arm, there were 50% (95% CI 42-59%) including 9% CRs [15]. The median PFS periods were 9.1 and 8.2 months on the HD-MVAC and MVAC arms, respectively [15]. Although the PFS period was significantly better in the HD-MVAC group (P = 0.037, HR 0.75, 95% CI 58-98), there was no statistically significant difference in OS [15]. Nowadays, HD-MVAC is frequently used as neo-adjuvant chemotherapy for patients with locally advanced UC before radical cystectomy or nephroureterectomy.

Taxane-including regimen
The taxane chemotherapy agents include docetaxel and paclitaxel. Taxane-including regimens have been frequently used as second-line chemotherapy for patients with metastatic UC who progressed after first-line chemotherapy. However, most studies investigating second-line regimens for advanced UC have targeted patients who received MVAC as first-line chemotherapy. Therefore, there is not yet any established regimen to be followed after the failure of GC therapy, which has been widely accepted as a first-line therapy for advanced UC due to its equivalent efficacy and lower toxicity compared to MVAC, which was formerly regarded as the standard first-line chemotherapy prior to the introduction of GC, as described above. Considering the mechanism mediating the acquisition of the resistant phenotype to chemotherapeutic agents, second-line regimens are not likely to include agents integrated into the first-line chemotherapy. Nowadays, most institutions in Japan administer paclitaxel-based chemotherapy (as single agents or combinations) to patients with advanced UC refractory to first-line GC therapy [16]. In a large retrospective study, combination chemotherapy was possibly significantly associated with improved OS compared with single-agent therapy (HR 0.60, P = 0.001) [16]. When pembrolizumab therapy is used as a second-line therapy, taxane-including regimens may be used as third-line therapies.

Ramucirumab and docetaxel
Ramucirumab (Cyramza ® , Eli Lilly) is a human IgG1 monoclonal antibody against vascular endothelial growth factor receptor (VEGFR)-2. A ramucirumab plus docetaxel regimen demonstrated superior PFS period over chemotherapy in patients with platinum-refractory metastatic UC [19]. This evidence validates the inhibition of VEGFR-2 signaling as a potential new therapeutic treatment option for patients with UC. In a randomized double-blind, phase III trial in patients with metastatic UC who had progressed during or after platinum-based chemotherapy, patients received intravenous docetaxel 75 mg/m 2 plus either intravenous ramucirumab 10 mg/kg (n = 263) or matching placebo (n = 267) on day 1 of each repeating 21-day cycle [19]. The PFS period was prolonged significantly in patients treated with ramucirumab plus docetaxel versus placebo plus docetaxel (median 4.07 versus 2.76 months, HR 0.757, P = 0.0118) [18]. ORR was achieved by 24.5% (95% CI 18.8-30.3%) and 14.0% (95% CI 9.4-18.6%) of patients treated with ramucirumab and placebo, respectively [19]. Regarding treatment-related AEs, the frequency of severe (grade 3-4) AEs was similar in patients treated with ramucirumab and placebo (60 versus 62%) [19]. Ramucirumab has not yet been approved in Japan for the treatment of metastatic UC. Eventually, however, as these positive results are likely to permit its approval, the combination of ramucirumab and docetaxel will become an important option for second-line as well as third-line therapy after pembrolizumab.

Vinflunine
Vinflunine is a microtubule inhibitor that is effective for patients with metastatic UC although this agent has not yet been approved in Japan. A randomized phase III study compared vinflunine (n = 253, PS = 0: 320 mg/m 2 , every 3 weeks; PS = 0 with previous pelvic radiation and PS = 1: 280 mg/m 2 subsequently escalated to 320 mg/m 2 ) in combination with best supportive care (BSC) and BSC (n = 117) alone in the treatment of patients with metastatic UC who had experienced progression after a first-line platinumcontaining regimen [20]. In the intent-to-treat population, the objective of a median 2-month survival advantage (6.9 months for vinflunine plus BSC versus 4.6 months for BSC alone) was achieved (HR, 0.88, 95% CI 0.69-1.12) but was not statistically significant (P = 0.287) [20]. However, multivariate Cox analysis adjusting for prognostic factors showed a statistically significant effect of vinflunine on OS (P = 0.036, HR 0.77, 95% CI 0.61-0.98) [20]. In the eligible population (n = 357), the median OS was also significantly longer for vinflunine plus BSC than it was for BSC alone (6.9 versus 4.3 months), with the difference being statistically significant (P = 0.040) [20].

Biomarkers
Among the biomarkers for metastatic urothelial cancer, Bajorin score, which incorporates Karnofsky performance status (KPS) less than 80% and presence of visceral (lung, liver, or bone) metastasis, is the best known and most frequently used stratification factor in the clinical trials [21]. In a phase II study of patients with metastatic UC (n = 203) who were undergoing MVAC chemotherapy, predictive prognostic factors were retrospectively analyzed by multivariate regression analysis [21]. Two factors were extracted as independent indicators of poor prognosis: KPS less than 80% and the presence of visceral metastasis [21]. Median survival times for patients who had zero, one, and two risk factors were 33, 13.4, and 9.3 months, respectively (P = 0.0001) [21].
Regarding the view from the genetic factors, expression level of various drug resistance and susceptible genes have been disclosed to associate with efficacy of cytotoxic chemotherapy [22][23][24]. Excision repair cross complementing 1 (ERCC1) is the nucleotide excision repair enzyme, which is involved in cisplatin-resistance [22]. Ribonucleotide reductase subunit M1 (RRM1) functions DNA repair after chemotherapy damage [22,23]. These chemotherapy resistance genes are considered to inhibit the efficacy of cytotoxic chemotherapy for the patients with metastatic UC [22,23].
On the other hand, the human equilibrative nucleoside transporter 1 (hENT1) functions major nucleoside transporter and facilitates efficient delivery of gemcitabine into cancer cells [24]. Hence, it might increase the efficacy of the combination of gemcitabine and platinum-based chemotherapy [24]. Various genes including these genes should be associated with the efficacy of medical therapy. Recent advances of precision medicine using the next generation sequencer may shed light to predict the response and prognosis. Furthermore, as described above, on current, various clinical trials, which is comparing immune-checkpoint inhibitors monotherapy and combination of immune-checkpoint inhibitors with standard chemotherapy as first-line therapy for patients with advanced UC. Expression level of these drug function and/or resistance associated genes might become one of the key factors whether we decide to undergo checkpoint inhibitors monotherapy or combination therapy with cytotoxic chemotherapy.
At the same time, there are several other biomarkers for immune-checkpoint inhibitor therapy, as we introduced very recently [25]. Based on the mechanism of efficacy, the number of neoantigens and expression of MHC molecules are strong candidate biomarkers [25]. Despite the various interference factors, which include antibody used, immunohistochemical procedure, cut-off point of stained sample, newly corrected specimen versus archival tumor sample, heterogeneity between primary and metastatic sites, and heterogeneity among metastatic sites, PD-1/PD-L1 expression can be considered a potential biomarker [25]. As more treatment options become available, more biomarkers need to be established.

Conclusion
In this review, we introduced the current US-FDA-approved immune-checkpoint inhibitors, including pembrolizumab, which has just become available for clinical practice in the treatment of metastatic UC in Japan. Based on its promising anti-tumor efficacy and manageable safety profile as demonstrated in the phase III KEYNOTE-045 trial, pembrolizumab therapy is expected to be rapidly introduced in clinical practice for metastatic UC in Japan. In addition, we summarized the cytotoxic chemotherapies as they still represent the mainstay of first-line therapy as well as useful options for second or later lines. The options proposed for current (2018) possible medical therapy for patients with metastatic UC in Japan are listed in Table 3. At this time, various combination therapies including various combinations of immune-checkpoint inhibitors with cytotoxic chemotherapy and combinations of double immune-checkpoint inhibitors are in clinical trials. We await the results of these trials with high hopes for new therapies. Taxane-including regimen such as GP (gemcitabine and paclitaxel)