2.1.2. IMvigor 210 Trial Cohort 1

On 17 April 2017, the FDA granted accelerated approval for atezolizumab in patients who are cisplatin-ineligible. This cohort consisted of 119 patients with a median age of 73 years old. The most common reason for cisplatin ineligibility was impaired kidney function. At a median follow-up time of 14.2 months the ORR was 23.5% (95% CI, 16.2–32.2%) in all treated patients [23]. Based on PD-L1 status, the ORRs were 28% (95% CI, 14–47%) and 21% (95% CI, 10–35%) for PD-L1 expression of ≥5% and <5% groups, respectively. The DOR was not reached in either subgroup. Responses were ongoing for 82% and 29% of responding patients at 5 months and 1 year, respectively. At the median follow-up of 29 months, the median ORR, OS, and DOR were 24%, 16.2 months, and not reached (95% CI: 30.4—N) (Table 1).

#### 2.1.3. IMvigor211 Trial

The phase III IMvigor211 trial compared atezolizumab with physician's choice of chemotherapy in patients with metastatic bladder cancer who had progressed after platinum-containing chemotherapy [24]. Again, patients were stratified based on PD-L1 expression. Unfortunately, patients with the greatest relative PD-L1 expression did not significantly survive longer when treated with atezolizumab (11.1 months) relative to chemotherapy (10.6 (8.4–12.2) months) [24]. There was also no significant difference in ORR. Thus, other patient cohorts were not evaluated. The most recent results are listed in Table 1.

#### 2.1.4. IMvigor130 Trial

This randomized trial enrolled 1213 patients with locally advanced or metastatic bladder cancer who were newly diagnosed or had received neoadjuvant or adjuvant chemotherapy more than 12 months prior to commencement of atezolizumab treatment [25]. The goal was to determine the therapeutic effectiveness of atezolizumab alone or in combination with chemotherapy versus chemotherapy alone. In addition, patients were stratified by PD-L1 status as previously described. Chemotherapy was gemcitabine with cisplatin and carboplatin for cisplatin-eligible and cisplatin-ineligible patients, respectively. Although cisplatin-ineligible patients were only originally recruited, the trial was amended to include cisplatin-eligible patients. Cisplatin-ineligible and eligible patients were randomized into three treatment arms: group A—atezolizumab plus open-label chemotherapy, group B—open-label atezolizumab monotherapy, or group C—masked placebo plus open-label chemotherapy. The two primary efficacy endpoints were OS and progression-free survival (PFS).

The most up-to-date results of the trial as reported by Galsky et al., did not statistically show that atezolizumab improved OS in all intention-to-treat patients [25]. The proportions (53–58%) of cisplatin-ineligible patients were similar among the three groups. At the median follow-up at 11.8 (6.1–17.2) months, the median OS among groups A and C were 16.0 (13.9–18.9) and 13.1 (11.7–15.1) months, respectively. This result did not cross the prespecified interim efficacy boundary for statistical significance. Group A did meet the co-primary PFS endpoint. Patients in group A had a statistically significant increased PFS of 8.2 (95% CI, 6.5–8.3) months. In contrast, patients in group C had a PFS of 6.3 (95% CI, 6.2–7.0) months. Unfortunately, the ORR for groups A and C were similar (47% (95% CI, 43–52%) and 44% (95% CI, 39–49%), respectively).

When evaluating atezolizumab alone versus chemotherapy alone, the results were not favorable. In all patients, the OS values for atezolizumab and chemotherapy alone were 15.7 (13.1–17.8) and 13.1 (11.7–15.1) months, respectively. Although significance for OS between groups B and C were not tested, the survival curves appear to show marginal benefit with atezolizumab alone. The median ORR for group B was 23% (95% CI, 19–28%), which was much lower than the previously described ORR values for groups A and C. The PFS was not reported.

For PD-L1 subgroups, atezolizumab alone and atezolizumab plus chemotherapy improved OS relative to chemotherapy alone in patients with PD-L1 expression IC2/3. The OS for the patients treated with atezolizumab and chemotherapy was 23.6 months versus 15.9 months for chemotherapy alone. The OS for the patients treated with atezolizumab alone was not estimable (17.7—not estimable) versus 17.8 (10.0—not estimable) months for chemotherapy alone. There were no survival advantages for both groups A and B relative to C in patients with PD-L1 expression scores of IC0 or IC1. Although OS was improved ORR was not. In patients with PD-L1 IC2/3 treated with atezolizumab or chemotherapy alone were 34% (95% CI, 28–50%) and 37% (95% CI, 33–55%), respectively.

IMvigor130 group B and IMvigor210 cohort 1 were the key trials that demonstrated a decreased response among patients with a PD-L1 status of <5% (i.e., IC0/1) when treated with atezolizumab alone, versus patients with the same PD-L1 status but who received cisplatin- or carboplatin-containing chemotherapy alone. Based on these results the FDA and the European Medicines Agency revised the indication for atezolizumab. Atezolizumab is now restricted for use in patients with locally advanced or metastatic bladder cancer who (i) are not eligible for cisplatin-containing chemotherapy, and whose tumors express PD-L1 ≥5% (based on the Ventana assay) and, (ii) are not eligible for any platinum-containing therapy regardless of PD-L1 status [37,38]. Notably, because atezolizumab did not reach its endpoints in the IMvigor211 trial, it is not indicated for cisplatin-eligible patients in the first-line regardless of PD-L1 tumor status.

#### *2.2. Pembrolizimab (Keytruda; Merck; Kenilworth, NJ, USA)*

#### 2.2.1. KEYNOTE-045 Trial

On 18 May 2017, the FDA granted accelerated approval to pembrolizumab, a PD-1-targeting mAb, for use in patients with bladder cancer who have either received platinum-containing chemotherapy or who are cisplatin-ineligible [39]. The phase III KEYNOTE-045 clinical trial enrolled patients with advanced or metastatic bladder cancer previously treated with any platinum-containing chemotherapy [40]. Patients (542) were randomly assigned to receive pembrolizumab or the investigator's choice of chemotherapy containing paclitaxel, docetaxel, or vinflunine. PD-L1 expression status was assessed using the Agilent PD-L1 IHC22C3 pharmDx assay. PD-L1 expression scores were defined as a 'tumor proportion score' (TPS), which is defined as the combined positive score determined from the percentage of PD-L1-expressing tumor cells and ICs relative to the total number of tumor cells. TPS values of <1%, 1–49%, ≥50% corresponding to PD-L1 expression of no expression, 'positive' expression, and 'high' expression [41].

In all patients treated with pembrolizumab the ORR was 21.1% (95% CI, 16.4–26.4%). In contrast patients treated with chemotherapy the ORR was 11.4% (95% CI, 7.9–15.8%). Pembrolizumab treatment also extended the median OS to 10.3 months (95% CI, 8.0–11.8). In comparison, the OS for patients in the chemotherapy-alone group was 7.4 (95% CI, 6.1–8.3) months. When focusing on patients whose tumor biopsies had a TPS ≥ 10%, the median OS was 8.0 (95% CI, 5.0–12.3) months compared to 5.2 (95% CI, 4.0–7.4) months for chemotherapy alone. The ORRs for the PD-L1-positive and chemotherapy alone groups were 21.6% (95% CI, 12.9–32.7%) and 6.7% (95% CI, 2.5–13.9%). Hence, there was a significant therapeutic response advantage for patients whose tumors with PD-L1 TPS ≥ 10%.

Necchi et al. and Fradet et al., recently reported the most up-to-date follow-ups of the KEYNOTE-045 trial [26,27]. The median ORR, OS, and DOR values were 21.1%, 10.1, and 29.7 months for patients treated with pembrolizumab (Table 1). In comparison, the updated median ORR, DOR, and OS values for patients in the chemotherapy group were 11%, 7.3, and 4.4 months [26]. In contrast to the earlier reported findings, patients with PD-L1 tumor TPS ≥ 10 had a lower ORR (20.3%) compared to all patients. When the effectiveness of both pembrolizumab and chemotherapy were evaluated in patients who did respond to treatment, the OS increased to 39.6 versus 17.7 months, respectively. This indicated that in patients who had been heavily pretreated with prior platinum-containing chemotherapy, have a clear increased benefit by being treated with pembrolizumab versus chemotherapy, and if responding to pembrolizumab, can survive out to >3-years. However, benefit from pembrolizumab appeared to be independent of PD-L1 expression status [40].

### 2.2.2. KEYNOTE-052 Trial

This phase II trial was a single-arm study designed to evaluate the efficacy of pembrolizumab in patients (370) with advanced bladder cancer who were ineligible for cisplatin-containing chemotherapy [42]. The up-to-date ORR in all treated patients was 28.6% (95% CI, 24.1–33.5%) (Table 1). Responses remained ongoing in 84% of patients. Patients in the PD-L1-high expression subgroup responded better to pembrolizumab compared to the PD-L1-low subgroup. Specifically, the ORR was 47% (95% CI, 38–57%) and 21% (95%, 16–26%) in patients with PD-L1 expression TPS ≥ 10 and <10, respectively. The median DOR for both subgroups was not reached. Among all responding patients, 52% and 7% continued responding at 6 months and at 1 year. Thus, these results strengthen the use of pembrolizumab in the first-line setting for cisplatin-ineligible patients with locally advanced and unresectable or metastatic bladder cancer.

#### 2.2.3. KEYNOTE-361 Trial

This current phase III trial (NCT02853305) is a randomized evaluation to verify the first-line effectiveness of pembrolizumab from the KEYNOTE-052 trial. Patients were stratified by their tumors either having ≥10 or <10 PD-L1 score. Unfortunately, pembrolizumab did not meet its two primary endpoints of OS or PFS [43]. As of 9 June 2020, patients receiving pembrolizumab and whose tumors were PD-L1-low had decreased survival compared to patients receiving cisplatin- or carboplatin-containing chemotherapy. We have not found any reports providing additional information on other patient cohorts.

Based on KEYNOTE-052 and KEYNOTE-361 results, the FDA issued an alert to health care professionals and oncology clinical investigators due to the substantial uncertainty concerning efficacy as a monotherapy to treat bladder cancer patients whose tumor express low (TPS < 10%) amounts of PD-L1 [44]. Cisplatin-ineligible patients should receive pembrolizumab only if their tumors express PD-L1 TPS ≥ 10. However, if patients are not eligible for any platinum containing chemotherapy, pembrolizumab can be used regardless of PD-L1 tumor status.

#### *2.3. Nivolumab (Opdivo; Bristol-Myers Squibb; New York, NY, USA)*

#### Checkmate 275 Trial

Nivolumab is a PD-1 blocking mAb that was approved in 2017 based on its performance in the multicenter, single-arm phase II Checkmate 275 clinical trial [45,46]. The trial evaluated Nivolumab in 270 patients with metastatic or surgically unresectable locally advanced bladder cancer, or with progression or recurrence after at least one platinum-based regimen, or within 12 months of perioperative platinum treatment for muscle-invasive disease. [46] PD-L1 expression was assessed using the Dako PD-L1 IHC 28-8 pharmDx kit [47]. PD-L1-positive staining is defined as complete and/or partial plasma membrane staining of tumor cells at any intensity. A minimum number of 100 viable tumor cells should be present in the PD-L1 stained tumor slide. Notably, infiltrating ICs that may stain positive for PD-L1 are not included in the scoring for the determination of PD-L1 positivity.

At the median follow-up of 7 (3.0–8.8) months, nivolumab treatment resulted in an ORR of 19.6%, (95% CI, 15.0–24.9%). Regarding, PD-L1 status: PD-L1 expression scores of ≥5%, ≥1% and <5%, and <1% of tumor cells, ORR values were 24.8% (95% CI, 18.9–39.5%), 23.8% (95% CI, 16.5–32.3%) and 16.1% (95% CI, 10.5–23.1%) respectively. The median OS was 8.7 months (95% CI, 6.1 to not reached) in the overall population, 11.3 (8.7 to not reached) months in the patients with PD-L1 expression of ≥1% and 6.0 (95% CI, 4.3–8.1%) months in patients with tumors with low PD-L1 expression of <1%. DOR was not reached (7.4—NR) in all patients at the moment of the publication of these results.

Based on these results, the FDA granted accelerated approval to durvalumab for the treatment of patients with locally advanced or metastatic bladder cancer who have disease progression during or following platinum-containing chemotherapy or have disease progression within 12 months of neoadjuvant or adjuvant treatment with platinum-based chemotherapy [45].

The most recent follow-up reported in Galsky et al., reported on the effectiveness of nivolumab [29]. At 33.7 months minimum follow-up, the ORR was 20.7% (Table 1), with complete responses in 6.7% of patients. Importantly, when the efficacy of nivolumab was evaluated in patients whose PD-L1 status was either <1% (*n* = 146) or ≥1% (*n* = 124), the ORR values were 16.4% (95% CI, 10.8–23.5%) and 25.8% (95% CI, 18.4–34.4%), respectively. When PD-L1 expression was evaluated at <5% (*n* = 187) and ≥5% (*n* = 83) the ORR values were 16.0% (95% CI, 11.1–22.1%) and 31.3% (95% CI, 21.6–42.4%), respectively. Out of the responding population, 73.2% (41/56) and 58.9% (33/56) had responses lasting ≥6- and ≥12-months, respectively. At the time of the evaluation, 25% of patients had ongoing responses.

Importantly, TMB was an important factor for successful patient response. A 'high' TMB was defined as ≥170 mutations per tumor. Low and medium TMB was defined as <85 and 85–169 mutations per tumor, respectively. Cox proportional hazards regression models were used to assess the dependence of PFS and OS on TMB alone and in combination with PD-L1 scoring. The ORR was 13.0%, 19.6%, and 31.9% in patients with low, medium, and high TMBs. High TMB tumors showed a positive association with ORR (odds ratio (95% CI): 2.13 (1.26–3.60), *p* < 0.05) regardless of PD-L1 scoring. However, futher evaluation including sufficiently large numbers of patients will be needed to determine the nuances if there is a benefit in ORR with subgroups evaluating combinations of low, medium, high TMB ≥1% and <1% PD-L1. PFS and OS were longer in patients with high TMB values compared to low and medium TMB tumors. Interestingly, when TMB was analyzed in combination with PD-L1 status there were diverging trends with PFS and OS. For PFS, there was approximately a 1 month increase in patients with ≥1% PD-L1 with tumors that were both low and high TMB. However, patients with ≥1% PD-L1 had poorer OS compared to patients with <1% PD-L1 for low, medium, and high TMB.

This study at almost 3-years of minimum follow-up indicated that nivolumab is not only effective for treating metastatic bladder cancer, but high TMB was strongly associated with improved outcomes relative to all treated patients. Moreover, TMB and PD-L1 could be used in combination as biomarkers for predicting PFS and OS. As a result this study is the first to show for ICI treatment of bladder cancer, TMB may prove a superior biomarker than PD-L1. For example, patients can be grouped into low, medium, and high TMB levels as opposed to the 1% cutoff for PD-L1.
