Immunotherapy Combinations and Sequences in Urothelial Cancer: Facts and Hopes Alejo Rodriguez-Vida1, Jose Luis Perez-Gracia2, and Joaquim Bellmunt1,3

Home , Atezolizumab, Enfortumab vedotin, Sacituzumab govitecan

Published OnlineFirst July 10, 2018; DOI: 10.1158/1078-0432.CCR-17-3108

Review Clinical Cancer Research Immunotherapy Combinations and Sequences in Urothelial Cancer: Facts and Hopes Alejo Rodriguez-Vida1, Jose Luis Perez-Gracia2, and Joaquim Bellmunt1,3

Abstract

Immune checkpoint inhibitors (ICI) have emerged as a maintained remissions. Active research is ongoing in sev- novel therapeutic strategy that achieves significant clinical eral fields, aiming to increase the number of patients that benefit in several tumor types, including urothelial cancer. benefit from ICI, and this research is largely based on the Overall, these agents have shown objective response rates development of biomarkers for personalized immunother- of around 20% to 23%, which indicates that a significant apy and novel combinations of ICI with other agents. proportion of patients do not benefit from immunother- This article will review ongoing efforts to develop combi- apy when given as monotherapy. Moreover, despite an nations of ICI with other therapeutic strategies in patients initial response to therapy and an improvement in the with urothelial cancer, including chemotherapy, targeted median duration of response compared with chemother- agents, other immunotherapy strategies, and radiotherapy. apy, still only half of the patients develop long-term Clin Cancer Res; 24(24); 6115–24. 2018 AACR.

Introduction urothelial cancer who are ineligible for cisplatin (8, 9), thus leading to ongoing randomized phase III trials in this setting. Immune checkpoint inhibitors (ICI) have become a major step Overall, these studies have shown objective response rates forward in the treatment of urothelial cancer. Indeed, significant (ORR) of around 20%–23%, which indicates that a significant clinical activity has been demonstrated since the first studies were proportion of patient does not benefit from immunotherapy reported in this tumor type, which also highlighted for the first when given as monotherapy. Moreover, despite an initial time the potential relevance of PD-L1 expression in tumor-infil- response to therapy and an improvement in the median duration trating immune cells (1). Subsequent phase II studies have of response compared with chemotherapy, still the majority of demonstrated significant activity and durable responses for ate- patients ultimately experience disease progression. Therefore, zolizumab, pembrolizumab, nivolumab, avelumab, and durva- additional strategies for improving the outcome of patients with lumab in patients with advanced urothelial cancer previously advanced urothelial cancer are urgently needed. In addition to treated with chemotherapy (2–5), and consequently all these personalized immunotherapy based on predictive biomarkers, agents have been approved for the treatment of this patient immunotherapy-based combinations (10) have emerged as one population by the FDA. Furthermore, pembrolizumab has shown of the most promising hopes to improve the outcomes of these increased overall survival (OS) in comparison with second-line patients, and great efforts are being undertaken in this regard. This chemotherapy in a randomized phase III trial in patients with review will focus on immunotherapy-based combinations under advanced urothelial cancer (6). However, with a similar phase III development for patients with advanced urothelial cancer. trial design, atezolizumab was not associated with significantly longer OS than chemotherapy, in patients with platinum-refractory metastatic urothelial cancer overexpressing PD-L1 (IC2/3), Combinations of ICIs and Chemotherapy although in an exploratory analysis, OS in the intention-to-treat Cisplatinum-based combination chemotherapy remains the population was numerically improved in the atezolizumab arm standard first-line treatment for patients with advanced urothelial (7). Finally, when exploring earlier stages of the disease in phase II cancer who are fit to tolerate cisplatin (11). Vinflunine is approved trials, both atezolizumab and pembrolizumab have shown prom- by the European Medicines Agency (EMEA) as second-line treat- ising activity in the first-line setting in patients with metastatic ment based on a limited survival benefit (12), and may have a role as maintenance treatment in the first-line setting (13). Therefore, it is not surprising that combinations of immunotherapy and 1Medical Oncology Department, Hospital del Mar, IMIM (Hospital del Mar chemotherapy have been included as experimental arms in several 2 Research Institute), Barcelona, Spain. Medical Oncology Department, Clinica of the ongoing phase III trials that are exploring the efficacy of ICI 3 Universidad de Navarra, Pamplona, Spain. Dana-Farber Cancer Institute, Brig- as first-line treatment of advanced urothelial cancer. The rationale ham and Women's Cancer Center, Harvard Medical School, Boston, Massachusetts. for such combinations is 2-fold. First, chemotherapy has shown synergistic activity when combined with ICI, which is based on the fi Note: A. Rodriguez-Vida and J.L. Perez-Gracia share rst authorship. ability of chemotherapy to induce immunogenic cell death, to Corresponding Author: Joaquim Bellmunt, Hospital Del Mar, Paseo Maritimo, attenuate immunosuppression in the tumor microenvironment Barcelona 08003, Spain. Phone: 349-3316-0750; Fax: 349-3316-0410; E-mail: [through the modulation of regulatory T cells, myeloid-derived [email protected] suppressor cells (MDSC), immunosuppressive cytokines, and doi: 10.1158/1078-0432.CCR-17-3108 immunosuppressive enzymes], and to enhance the performance 2018 American Association for Cancer Research. of antigen-presenting cells (10). Second, chemotherapy has well-

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confirmed activity in patients with advanced urothelial cancer, damage response mutations. On exploratory analysis, there was and the subset of patients that benefit from either chemotherapy a significant expansion of circulating CD4 T cells with the addition or ICI may not overlap. Moreover, chemotherapy retains high of ipilimumab and this correlated with an improved survival. The activity following first-line treatment with ICI (14), and the combination was safe with grade 3–4 adverse events (AE) occur- phenomenon of hyperprogression to immunotherapy has also ring in 81% of patients, the majority being hematologic. Despite been described among patients with urothelial cancer, which these promising results, the study did not achieve its primary could be potentially avoided with the combination (15, 16). endpoint of a lower bound of the 90% confidence interval for 1- Consequently, the strategy of delivering chemotherapy and ICI year OS greater than 60% and consequently further studies are to all patients might be able to capture all the clinical benefit that is needed to define optimal combinations and how to identify attainable with both treatment modalities. Hence, chemotherapy, patients most likely to benefit (18). with a quicker mechanism of action, could potentially counteract Several other trials combining chemotherapy and ICIs are still the deleterious delayed appearance of the response seen with ongoing. Table 1 lists the most relevant ongoing ICI–chemother- immunotherapy. Nevertheless, the potential disadvantages of apy combination studies. Three trials include an experimental combining ICI with chemotherapy might be increased toxicity arm that combines ICI and chemotherapy: IMVIGOR-130, which and potential unknown negative interactions of both therapeutic randomizes 1,200 patients to gemcitabine and platinum versus alternatives. Given the different toxicity profile of chemotherapy gemcitabine, platinum, and atezolizumab versus single-agent agents, the viability of combining new immunotherapy agents atezolizumab; Keynote 361, which is randomizing 990 patients and chemotherapy will depend greatly on the absence of to gemcitabine and platinum versus gemcitabine, platinum, and enhanced toxicity. Consequently, sequential administration of pembrolizumab versus single-agent pembrolizumab; and Check- both treatment modalities might also be a valid alternative to mate 901, which is randomizing 897 patients to gemcitabine and further explore, potentially limiting the risk of increased tox- platinum versus gemcitabine, platinum, and nivolumab versus icity. Despite the limitations of a retrospective data collection, nivolumab and ipilimumab (Table 1). Szabados and colleagues reported a 68% ORR to chemotherapy Another strategy is to compare treatment exclusively based on in patients with urothelial cancer previously treated with ICI, ICI with platinum-based chemotherapy. Such a strategy is also thus indicating a lack of cross resistance between both alter- being explored by the latter three studies, which include an arm in natives, and even suggesting increased efficacy of chemotherapy which patients are treated exclusively with ICI, either as single following ICI (14). Activity of single-agent ICI in urothelial agent (atezolizumab or pembrolizumab) or in combination cancer also seems to be maintained regardless of whether it is (nivolumab plus ipilimumab or durvalumab and tremelimumab, administered in the first-line setting or following one or several which will be reviewed in the following section; Table 1). These lines of chemotherapy (17). ICI-based arms should capture the benefit that is observed in Concomitant administration of chemotherapy and ICI is being patients that will respond to single-agent immunotherapy, spar- evaluated by several studies. The results of a small single-arm ing the adverse effects of chemotherapy, but might also miss any phase II study combining ipilimumab and cisplatin–gemcitabine potential clinical activity of chemotherapy that may not overlap in treatment-na€ve metastatic urothelial cancer patients were with ICI. recently published (18). Thirty-six patients were included and Finally, other ongoing studies use a sequential/maintenance treated with 2 cycles of cisplatin–gemcitabine followed by four strategy, administering in first place platinum-based chemo- cycles of cisplatin–gemcitabine plus ipilimumab. The ORR was therapy and randomizing patients that present clinical benefit 69% and the 1-year OS rate was 61% (lower bound of 90% after completing chemotherapy to receive single-agent avelu- confidence interval: 51%). Importantly, the response rate was mab (Javelin 100, 668 patients) or pembrolizumab significantly higher in patients with deleterious somatic DNA (NCT02500121, 200 patients) versus best supportive care.

Table 1. Ongoing phase II/III studies testing ICIs and chemotherapy combinations as ﬁrst-line treatment in advanced urothelial cancer Trial N Standard arm Experimental arm Objective Phase III trial 1,200 Gemcitabine–platinum Gemcitabine–platinum þ atezolizumab OS, PFS, safety IMVIGOR 130 Atezolizumab (NCT02807636) Phase III trial 990 Gemcitabine–platinum Pembrolizumab OS, PFS Keynote 361 Gemcitabine–platinum þ pembrolizumab (NCT02853305) Phase III trial 897 Gemcitabine–platinum Nivolumab þ ipilimumab OS, PFS Checkmate 901 Gemcitabine–platinum þ nivolumab (NCT03036098) Phase III trial 200 Chemotherapy followed by BSC in Chemotherapy followed by pembrolizumab 6-month PFS (NCT02500121) patients presenting response or in patients presenting response or stable disease stable disease Phase III trial 668 Chemotherapy followed by BSC in Chemotherapy followed by avelumab in OS Javelin 100 (NCT02603432) patients presenting response or patients presenting response or stable disease stable disease Phase II trial 80 Carboplatin-gemcitabine 2 cycles of induction avelumab prior to ORR, PFS, OS, safety (NCT03390595) 6 cycles of carboplatin-gemcitabine þ avelumab, followed by avelumab maintenance Abbreviations: BSC, best supportive care; PFS, progression-free survival.

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Importantly, the administration of chemotherapy prior to 17.7) 11.4) 16.2)

immunotherapy could lead to the upregulation of PD-L1 by – – – NA NA NA NA chemotherapy with a potential enhancement of the subse- 10.2 (4.5-NR)

quent immunotherapy. While this sequential strategy may Median OS 7.3 (5.6 9.7 (7.3 ﬁ 16.1 (10.5

bene t patients with an earlier administration of second-line (months, 95% CI) treatment and avoid the toxicities of concomitant therapy, it has the drawback that only patients presenting clinical benefit with chemotherapy will receive ICI as part of their first-line treatment, thus excluding patients that do not respond. 3.9) As previously commented, the sequential administration of ICI 5.9) 10.7) 8.2) – – – followed by chemotherapy has been associated with an increased – 4.3 NA NA NA NA efficacy of chemotherapy (14). Moreover, priming the chemo- (1.6 Median PFS 2.8 (1.5 2.6 (1.4 therapy response giving immunotherapy first could enhance the 7.1 (5.0 (months, 95% CI) overall response of the combination and prevent the detrimental effect of chemotherapy on immune cells. To test this concept

(sequential/combination/maintenance ICI chemotherapy), a cancer planned phase II study in ﬁrst-line "unﬁt" patients with advanced 35.5)

ﬁ 53.2) 66.3) urothelial cancer will evaluate the safety and ef cacy of sequenc- – – – 59.4) 48.4) 35.4) – – – ing 2 cycles of induction avelumab prior to combining carbopla- treatment-related adverse event. ﬁ rmed 35% 34% 5.4% 26.1% 38.5% – 24.4% (15.3 tin gemcitabine plus avelumab for 6 cycles, followed by avelu- (10.2 (20.2 Con mab maintenance, compared with carboplatin–gemcitabine ORR (95% CI) 53% (38.8 41% (29.3 alone (NCT03390595, 80 patients). 26.0% (17.9 The combination of pembrolizumab and gemcitabine with or without cisplatin is also being explored in a neoadjuvant study in 81 patients with T2-4a bladder urothelial cancer with pathologic

complete response at cystectomy as the primary objective þ

(NCT02365766). The neoadjuvant setting is particularly valuable þ for research on pharmacodynamic biomarkers, correlating them þ þ 5.0 mg/kg) 3 mg/kg) with pathologic response, and induction treatment with ICI may – enhance the activity of the immune response against the tumor, as compared with an adjuvant strategy (19, 20).

Combinations of ICIs and Other pembrolizumab 2 mg/kg dosages ipilimumab 1 mg/kg 10 mg/kg epacadostat 100 mg twice daily þ utomilumab (20 mg or 100 mg) ipilimumab 3 mg/kg Pembrolizumab 200 mg Utomilumab (0.45 PF-04518600 (0.1 – Sacituzumab govitecan Enfortumab vedotin at different Immunotherapy Agents Treatment arms ICI targeting the PD-1/PD-L1 pathway have shown a modest antitumor activity when administered as monotherapy in patient a b b d a with metastatic urothelial cancer both in the second-line and c N 78 Nivolumab 3 mg/kg 26 Nivolumab 1 mg/kg 81 41 37 23 104 Nivolumab 3 mg/kg cisplatinum-unﬁt ﬁrst-line settings (6–9). In addition to the 40 inhibitory PD-1/PD-L1 pathway, there are several other immune checkpoints, both stimulating and inhibitory, which are currently being investigated as potential therapeutic targets in combination with PD-1/PD-L1 inhibitors. These immune checkpoints induce tumor immune tolerance through additive and nonredundant mechanisms and therefore, a dual inhibition of two of these pathways could produce synergistic antitumor activity. Several

combinations of different ICIs are currently being investigated in Objective urothelial cancer. Table 2 summarizes the available preliminary Secondary: ORR, PFS, OS 67 Secondary: ORR Secondary: PFS, OS PFS, OS Secondary: ORR, PFS, OS results from clinical trials testing ICI combinations in advanced Secondary: ORR Primary: safety Primary: ORR, safety Secondary: TRAEs, DOR, Primary: safety Primary: safety Primary: ORR urothelial cancer. Table 3 lists the most relevant ongoing ICI Primary: safety combination studies.

Combinations with CTLA-4 inhibitors CTLA-4 and PD-1 inhibit antitumor immunity at different stages of the cancer immunity cycle, with CTLA-4 being mainly involved at the priming and activation phase, while PD-1 acts mostly at the effector phase of cancer cells killing (21). Targeting Trial both immune checkpoints has already shown excellent results in Preliminary results from phase I/II studies testing ICI combinations and other immunotherapy agents in advanced solid tumors including urothelial several solid tumors. Nivolumab plus ipilimumab combination therapy was associated with high antitumor activity in treatment- (NCT02315066) (31) (NCT02178722) (27) (NCT01631552) (33, 34) (NCT02091999) (36, 37) (NCT02179918) (30) (NCT01928394) (24, 52) All solid tumors. Patients previously treated with prior ICIs. Advanced urothelial cancer cohort. € Patients previously treated with prior chemotherapy and/or ICIs. Phase I trial Phase I/II trial Keynote 037 Table 2. a b c d Abbreviations: DOR, duration of response; NA, not available; NR, not reached; ORR, objective response rate; PFS, progression-free survival; TRAE, Phase I/II trial Phase Ib trial Keynote 0036 Phase I trial na ve patients with metastatic renal cell carcinoma and advanced Phase I/II trial Checkmate 032

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Table 3. Ongoing phase I/II/III studies testing ICI combinations and other immunotherapy agents in advanced solid tumors including advanced urothelial cancer Trial N Objective Treatment arms Phase III ﬁrst-line trial DANUBE 1,005 OS, PFS, ORR Gemcitabine–platinum (NCT02516241) Durvalumab Durvalumab þ tremelimumab Phase III ﬁrst-line trial 650 OS, PFS, ORR, safety Pembrolizumab þ epacadostat Keynote 672 (NCT03361865) Pembrolizumab þ placebo Phase III second-line trial 648 OS, PFS, ORR, safety Pembrolizumab þ epacadostat Keynote 698 (NCT03374488) Pembrolizumab þ placebo Phase I study (NCT03192943) 18 Safety, ORR BMS-986205 (IDO1 inhibitor) þ nivolumab JAVELIN Medley phase Ib/II study 560 Safety, ORR Arm A: avelumab þ utomilumab (4-1BB agonist) (NCT02554812) Arm B: avelumab þ PF-04518600 (OX40 agonist) Arm C: avelumab þ PD-0360324 (CSF-1 inhibitor) Arm D: avelumab þ utomilumab þ PF-04518600 Phase I/II study (NCT03217747) 188 Safety, ORR Arm A: avelumab þ utomilumab (4-1BB agonist) Arm B: avelumab þ PF-04518600 (OX40 agonist) Arm C: avelumab þ utomilumab þ PF-04518600 Arm D: avelumab þ utomilumab þ radiation therapy Arm E: avelumab þ PF-04518600 þ radiation therapy Arm F: avelumab þ utomilumab þ PF-04518600 þ radiation therapy Arm G: avelumab þ cisplatin þ radiation therapy Phase I/II study (NCT02452424) 80 Safety, ORR Pexidartinib (CSF1R inhibitor) þ pembrolizumab Phase Ib study (NCT02323191) 310 Safety, ORR Emactuzumab (CSF1R inhibitor) þ atezolizumab Phase 1b study EV-103 (NCT03288545) 85 Safety, ORR, PFS, OS Enfortumab vedotin þ pembrolizumab or atezolizumab Phase II study EV-201 (NCT03219333) 200 ORR, DOR, PFS, OS Enfortumab vedotin Phase I/IIa study (NCT02913313) 170 Safety, ORR BMS-986207 (anti-TIGIT) þ nivolumab Phase I study (NCT02794571) 300 Safety, ORR, DOR, PFS MTIG7192A (anti-TIGIT) þ atezolizumab Phase I/II study (NCT01968109) 1,000 Safety, ORR, DOR, PFS Relatlimab (anti-LAG3) alone or þ nivolumab Phase I study (NCT02966548) 45 Safety, ORR, DOR Relatlimab alone or þ nivolumab Phase I/II study (NCT03459222) 230 Safety, ORR, DOR, PFS Relatlimab þ nivolumab þ BMS-986205 (IDO1 inhibitor) or relatlimab þ nivolumab þ ipilimumab Phase I study (NCT03250832) 260 Safety, ORR, DOR, PFS TSR-033 (anti-LAG3) alone or þ PD-1/PD-L1 blockade Phase I study (NCT03005782) 301 Safety, ORR, DOR, PFS REGN3767 (anti-LAG3) alone or þ REGN2810 (anti-PD-1) Phase I/II study (NCT02460224) 515 Safety, ORR, DOR, PFS LAG525 (anti-LAG3) alone or þ PDR001 (anti-PD-1) Abbreviations: DOR, duration of response; PFS, progression-free survival.

melanoma in phase III trials (22, 23). A similar approach is platinum-based chemotherapy in the same setting (Table 3). currently being tested in patients with advanced urothelial cancers The main endpoints of both studies are OS and progression- in several clinical trials. Checkmate 032 is a randomized phase I/II free survival (PFS). study evaluating the safety and efficacy of nivolumab monotherapy (3 mg/kg) or nivolumab plus ipilimumab (at two different Combinations with IDO1 inhibitors dosages schedules) for four cycles followed by nivolumab mono- IDO1 is an IFNg-induced intracellular enzyme that induces therapy in advanced or metastatic solid tumors (NCT01928394). tryptophan degradation and production of kynurenine leading to The study has included a urothelial cancer cohort of 208 patients a shift toward an immunosuppressive microenvironment (25). with locally advanced or metastatic disease previously treated Upregulation of IDO1 has been described as a potential immune with a platinum-based combination and preliminary results were evasion mechanism in solid tumors. Given its different and recently presented (24). Combination treatment was associated complementary role to the PD-1/PD-L1 pathway, it has been with significant antitumor activity, including an ORR of around hypothesized that targeting both pathways could lead to 26%–38.5% and a complete response (CR) rate of 2.9%–3.8% enhanced immune-mediated antitumor activity (26). Epacado- (Table 2). Moreover, most responses occurred early and were stat, a potent and selective oral inhibitor of IDO1, is currently maintained (70%–80% of patients showing ongoing response at being investigated in combination with PD-1/PD-L1 inhibitors in the time of analysis). The combination was well tolerated, with several clinical trials. The Keynote 037 is an ongoing phase most treatment-related AEs being of low grade and manageable I/II study evaluating the safety and efficacy of epacadostat in (24). combination with pembrolizumab across multiple tumors In view of these promising results, this combination is (NCT02178722). Preliminary results from the urothelial cancer currently being investigated in a randomized phase III trial, expansion cohort were recently presented (27). The study includ- the Checkmate 901 study (NCT03036098), which will com- ed 40 patients with locally advanced or metastatic urothelial pare as mentioned earlier, standard first-line platinum-based cancer progressing to first-line platinum-based chemotherapy chemotherapy alone or with nivolumab versus nivolumab 1 (Table 2). Treatment combination was associated with an ORR mg/kg plus ipilimumab 3 mg/kg for four cycles followed by of 35%, including a CR rate of 8%. A higher ORR was observed in nivolumab monotherapy in patients with treatment-na€ve PD-L1–positive patients (using a combined positive score 1%), metastatic or unresectable urothelial cancer (Table 1). Simi- although responses were also noted in PD-L1–negative patients larly, the DANUBE study (NCT02516241) is a randomized (64% vs. 13%, respectively). Most responses occurred early and phase III trial comparing durvalumab monotherapy or in were maintained, with a median duration of response (DOR) of combination with tremelimumab versus standard first-line 30.6 weeks. Treatment combination was well tolerated with a low

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frequency of grade 3–4 AEs (22.5%) and safety profile similar to that of pembrolizumab monotherapy. Given these promising results, this combination is currently being investigated in two randomized phase III trials both in the first- and second-line settings (Table 3). The Keynote 672 study is a randomized, double-blind trial of pembrolizumab in fi

Objective combination with epacadostat or placebo as rst-line therapy in cisplatin-ineligible patients with advanced urothelial cancer (NCT03361865). Similarly, the Keynote 698 study is assessing the same strategy as second-line treatment for recurrent or pro-

survival rate gressive advanced urothelial cancer in patients who have failed a Safety ORR Safety, PFS, DCR Safety 12-month PFS (versus historical control) Clinical CR rate Two-year bladder-intact disease-free Feasibility Two-year rate FFS ﬁrst-line platinum-containing chemotherapy (NCT03374488). Both studies have coprimary endpoint of OS and PFS and several secondary endpoints including ORR, safety, and quality of life. Other IDO1 inhibitors are also being investigated in combination with PD-1/PD-L1 inhibitors (Table 3). While a recent press release has reported that the Keynote 252 phase III trial testing pembrolizumab plus epacadostat versus single-agent pembrolizumab in patients with unresectable or metastatic melanoma failed to improve PFS leading to the premature closing of the trial (28), results of studies testing the same combination in different dis- eases are awaited. Population

Combinations with 4-1BB agonists 4-1BB is a costimulatory immune checkpoint receptor found þ þ on CD8 and CD4 T cells as well as in natural killer cells. The expression of 4-1BB is induced upon T-cell receptor (TCR) acti- monotherapy chemotherapy chemotherapy vation and it promotes T-cell activation and enhances cytotoxic Metastatic UC Stage IV genitourinary malignancies Muscle-invasive bladder UC T2-4 N0-2 bladder UC Localized/locally advanced bladder UC ineligible for Advanced, platinum-refractory UC Stage IV UC that has progressed on anti-PD-1/PD-L1 Localized muscle-invasive bladder UC T-cell responses, natural killer cell expansion, and antibody- dependent cell-mediated cytotoxicity. Utomilumab is a novel, fully human IgG2 agonist mAb that binds with high afﬁnity to 4-1BB, stimulating and increasing T-cell proliferation and cyto- kine production and enhancing antitumor immune function. In view of this different mechanism of action to the PD-1/ PD-L1 pathway, it has been hypothesized that targeting both immune checkpoints could produce an additive or synergistic concurrent

þ antitumor effect. Preclinical studies have indicated that costimulation signals through an agonist 4-1BB antibody increases the efﬁcacy of anti-PD-L1 or CTLA-4 therapy (29). The Keynote

Treatment 0036 phase Ib tested the safety and efﬁcacy of utomilumab in gemcitabine hypofractionated RT conventional or hypofractionated RT Stage II/III bladder UC ineligible or refusing combination with pembrolizumab in patients with advanced RT þ þ þ RT

þ solid tumors (NCT02179918). Twenty-three patients with RT þ

þ advanced solid tumors were included and treated with the

pembrolizumab combination with no dose-limiting toxicities observed (Table þ 2). Treatment-related AEs were mostly grades 1 to 2, with no hypofractionated RT treatment-related discontinuations. The ORR was 26.1% with a Pembrolizumab Pembrolizumab Nivolumab SBRT SBRT before or concurrent with pembrolizumab Atezolizumab Nivolumab followed by chemo-RT CR rate of 8.7% (30). This favorable safety proﬁle and preliminary clinical activity has led to design of further clinical trials assessing the role of combining utomilumab plus pembrolizumab in advanced urothelial cancer. Two phase I/II studies, the JAVELIN Medley study (NCT02554812) and NCT03217747

Study design study are evaluating the safety and preliminary antitumor activity of avelumab in combination with other cancer immunotherapies, such as utomilumab in patients with locally N advanced or metastatic solid tumors, including urothelial cancer (Table 3).

Combinations with OX40 agonists Ongoing phase I/II studies exploring combinations of radiotherapy and ICIs in urothelial cancer OX40 is a costimulatory immune checkpoint receptor, which is þ expressed on activated CD4 and regulatory T cells (Tregs). The NCT03287050Abbreviations: DCR, disease control rate; 20 FFS, failure-free survival; RT, radiotherapy; SBRT, stereotactic body radiation therapy; Phase UC, I urothelial cancer. Metastatic UC NCT03115801 112 Phase II NCT02880345NCT02826564 14 20 Pilot study Phase I Pembrolizumab NCT03171025NCT03419130 28NCT02621151 60 Phase II Phase 54 II Phase II Table 4. Trial Localized/locally advanced UC NCT02891161NCT03421652 42 34 Phase I/II Phase II Durvalumab costimulation of the TCRs in the presence of OX40 leads to an

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enhanced T-cell antitumor response. Targeting OX40 with ago- Pexidartinib, an oral small-molecule inhibitor of the CSF1R, is nistic mAbs can reduce the inhibitory effect of Tregs in the tumor currently being investigated in combination with pembrolizumab microenvironment. PF-04518600, a fully human IgG2 agonist in a phase I/IIa study in a panel of solid tumors, including mAb that binds to human OX40, is currently being investigated in urothelial cancer (NCT02452424). Another phase Ib study is several solid tumors including urothelial cancer. A phase I dose- assessing the safety and efficacy of emactuzumab, a humanized escalation study is testing the safety and preliminary anticancer mAb of IgG1 subclass directed against CSF1R, in combination activity of PF-04518600 in combination with utomilumab in with atezolizumab in patients with locally advanced or metastatic advanced solid tumors (NCT02315066; Table 2; ref. 31). Because solid tumors that are not amenable to standard treatment, includ- OX40 acts predominantly on CD4 T-cell function, while 4-1BB ing urothelial cancer (NCT02323191). Finally, the previously primarily impacts on CD8 T-cell and natural killer cell function, mentioned Medley study (NCT02554812) also includes a treat- dual stimulation of both OX40 and 4-1BB has been postulated as ment arm evaluating the safety and preliminary antitumor activity a promising treatment combination. Preclinical models have of avelumab in combination with PD-0360324, a mAb targeting confirmed a potential synergistic activity of this combination in the CSF-1. þ terms of induction of clonal expansion of CD8 and cytotoxic þ CD4 T cells. The study has recruited 42 patients, including 5 Antibody–drug conjugates previously treated patients with urothelial cancer, who were Antibody–drug conjugates (ADC) are comprised of active treated with increasing doses of PF-04518600 and utomilu- therapeutic agents linked to antibodies that target cell surface mab. The combination showed no unexpected toxicity beyond antigens and can selectively deliver toxic payload to cancer cells. what might be expected for either agent alone and no dose- Several ADCs are being investigated as potential antitumor agents limiting toxicity (DLT) was observed. Treatment-related AEs in solid cancers, two of which, sacituzumab govitecan and enfor- were mostly grade 1–2 and included fatigue (9.5%), nausea tumab vedotin, are being evaluated in patients with advanced (7.1%), and anemia (4.8%) as the most common toxicities. urothelial cancers. Among the 37 evaluable patients, the ORR was 5.4% with a Sacituzumab govitecan is a second-generation ADC comprising stable disease rate of 29.7%. Regarding the 4 evaluable urothe- a humanized anti-Trop-2 mAb (hRS7) conjugated with the active lial cancer patients, 2 experienced stable disease (50%) and 2 metabolite of irinotecan, SN-38.9. Trop-2 is overexpressed on progressive disease (50%) as best response. Biomarker analysis many epithelial tumors, particularly more aggressive types, showed that combination treatment resulted in greater included in urothelial cancer (33). A phase I/II study has tested increases in expression of activation and proliferation markers the safety and activity of sacituzumab govitecan in patients with on CD8 memory T cells, in particular, and memory T-cell previously treated advanced solid tumors and has included an subsets overall, than with PF-04518600 alone (31). expansion cohort of 41 patients with advanced urothelial cancer On the basis of these results, this study will continue to further (NCT01631552; refs. 33, 34). It was a single-arm, open-label evaluate this combination, particularly in a setting of resistance to study evaluating sacituzumab govitecan in patients with meta- ICIs. In addition, the two previously mentioned multiarm trials, static urothelial cancer who progressed after one or more prior the Medley study (NCT02554812) and NCT03217747 study, also systemic therapies. Sacituzumab govitecan was administered at include a treatment arm evaluating the safety and preliminary 10 mg/kg on days 1 and 8 of 21-day cycles, until progression or antitumor activity of avelumab in combination with PF- unacceptable toxicity (Table 2). The recruited patients were heavi- 04518600 with or without utomilumab or radiotherapy in ly pretreated, with a median of 3 prior therapies (range 1–6), patients with advanced solid tumors, including urothelial cancer including platinum combinations (93%) and ICI (34%). In spite (Table 3). of that, sacituzumab govitecan was associated with a significant antitumor activity with an ORR of 34%, 2 patients achieving a CR Combinations with CSF-1 inhibitors (4.8%), and 72% of patients showing some shrinkage of target Tumor-associated macrophages (TAM) support tumor lesions in CT scans. Median time to response was 1.9 months growth and have been identified as an independent poor (range 1.7–7.4) and median DOR was 12.6 months (95% CI, 7.5– prognostic factor in several cancer types (32). Similarly, MDSCs 12.9) with 4 long-term responders of more than 1 year. Of note, inhibit antitumor immunity andcancauseresistancetoPD-1/ among patients with liver metastasis, the ORR was 39%. A clinical PD-L1 blockade. Both TAMs and MDSCs are partially regulated benefit, defined as CR, partial response (PR), and stable disease by colony-stimulating factor 1 (CSF-1) and its receptor (SD) 6 months occurred in 49% of patients. For patients (CSF1R). CSF-1 plays also a key role in the regulation of the previously treated with three or more treatment lines, the ORR proliferation, differentiation, and survival of monocytes. Sev- was 30% and the clinical benefit rate 45%. Patients previously eral inhibitory agents targeting CSF-1 or CSF1R are currently treated with an ICI had an ORR of 29% and a clinical benefit rate being investigated, both in monotherapy and in combination of 43%. A significant efficacy was also seen in terms of survival, with other ICI (Table 3). By preventing CSF1R activation and including a median PFS of 7.1 months (95% CI, 5.0–10.7) and CSF1R-mediated signaling in these cells, these agents induce median OS of 16.1 months (95% CI, 10.5–17.7). Treatment was the inhibition of monocyte differentiation, block the activity of well tolerated, with most toxicities being grade 1–2 and most TAMs, and reduce their production of inflammatory mediators, common grade 3–4 being neutropenia (39%), anemia (10%), which reduces inflammation and angiogenesis. By blocking the fatigue (7%), and diarrhea (7%; ref. 34). In view of these prom- activity and proliferation of TAMs in the tumor microenviron- ising results, further evaluation of sacituzumab govitecan in ment, CSF-1 inhibitors reduce TAM-mediated immune sup- advanced urothelial cancer is warranted. pression, decrease Tregs and MDSCs, reactivate antitumor Enfortumab vedotin is a fully humanized mAb against nectin-4 immunity, and improve antitumor cell responses mediated by conjugated with a microtubule-disrupting agent monomethyl increasing infiltration by cytotoxic T cells (32). auristatin E (MMAE). Nectin-4, a transmembrane polypeptide

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belonging to the nectin family of cell adhesion molecules, is superfamily. LAG3 is expressed on TILs, leading to T-cell dysfunc- highly expressed in cancer cells, especially in urothelial cancers tion and tumor-mediated immune suppression. Blocking LAG3 (35). Enfortumab vedotin selectively binds to nectin-4 expressed receptor may activate antigen-specific T lymphocytes and enhance on target cells, triggering the internalization of the ADC–nectin-4 cytotoxic T-cell–mediated tumor cell lysis. LAG3 and PD-1 recep- complex, which is transported to the lysosomal compartment tors are overexpressed and/or coexpressed on TILs and therefore a where MMAE is released. MMAE then disrupts the microtubule dual blockade of both checkpoints may synergistically restore T- network inducing cell-cycle arrest and apoptotic death (36). A cell activation and enhance antitumor immunity. Moreover, prior phase I dose escalation/dose-expansion study (NCT02091999) anti-PD-1 therapy may increase the expression of LAG3 and evaluated the safety and efficacy of enfortumab vedotin in patients promote T-cell exhaustion and resistance to anti-PD-1 therapy. with advanced urothelial cancer treated with 1 or more chemo- Targeting LAG3 in combination to PD-1/PD-L1 might therefore therapy regimens or who were cisplatin ineligible (ref. 37; Table 2). help prevent the occurrence of primary resistance or contribute to The trial included 81 patients and 97% had positive IHC for nectin- resensitize patients developing resistance to anti-PD-1 monother- 4. Patients were heavily pretreated, with 62% having received 2 apy (39). Several phase I/II studies are currently assessing the prior therapies, 95% have had prior platinum combinations, 46% safety and efficacy of LAG3 inhibitors in combination with PD1-/ prior ICI, and 43% prior taxanes. The study showed a promising PD-L1 inhibitors in solid tumors, including urothelial cancers antitumor activity, with an ORR of 41%, a CR rate of 4%, and (Table 3). disease control rate (PR, CR, and SD) of 72%. Median DOR was 24 weeks (range 0.14–40.3). The ORRs were 44%, 41%, and 47%, in Combinations with personalized vaccines patients previously treated with ICI, taxanes, and with liver metas- Effective antitumor immunity in humans has been associated tases, respectively. Treatment was generally well tolerated, with with the presence of T cells directed at cancer neoantigens, a class nausea, pruritus, and fatigue being the most commonly reported of autologous human leukocyte antigen (HLA)-bound peptides treatment-related AEs (37). Following these promising results, a that arise from tumor-specific somatic mutations (40). Genera- dose expansion cohort of ICI-pretreated advanced urothelial cancer tion of a personalized, multipeptide neoantigen vaccines for patients was initiated. A total of 67 patients were included, and a patients with high-risk melanoma has shown promising results similar ORR of 53% was described (36). These results warrant (40). With this novel approach, somatic mutations are identified further development of enfortumab vedotin of use in patients with by whole-exome sequencing in tumor tissue and germline DNA advanced urothelial cancer. Of note, two studies have recently been and their expression confirmed by tumor RNA sequencing. The initiated (Table 3): the EV-103 study, a phase 1b dose-escalation mutated peptides that are predicted to bind with high-affinity to study of enfortumab vedotin in combination with pembrolizumab the HLA molecules are selected as potential neoantigens. Subse- or atezolizumab in patients with advanced urothelial cancer pre- quently, pools of synthetic long immunizing peptides selected on treated with platinum-based chemotherapy or cisplatin ineligible the basis of HLA binding are given as vaccination to patients. In a (NCT03288545) and the EV-201 study, a single-arm phase II study preliminary report, 4 of 6 vaccinated patients had no recurrence at of enfortumab in advanced urothelial cancer previously treated 25 months and the recurring patients had a subsequent CR after with an ICI (NCT03219333). receiving treatment with an ICI (40). These findings have provid- ed a strong rationale for further development of this approach, Combinations with TIGIT inhibitors alone and in combination with checkpoint blockade or other T-cell immunoreceptor with immunoglobulin and immunor- immunotherapies. A similar trial is ongoing in renal cell carci- eceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT) noma, melanoma, and metastatic urothelial cancer. As highlight- is a coinhibitory molecule and immune checkpoint, expressed on ed by the authors, the advantages of a personal neoantigen vaccine various immune cells, particularly on tumor-infiltrating T lym- is to broaden the repertoire of tumor neoantigens inducing a T-cell phocytes (TIL). TIGIT plays a key role in the suppression of T-cell response in comparison with nonantigen-directed immunother- proliferation and activation and it is involved in tumor cell apy, such as ICI or tumor-infiltrating lymphocyte therapy (40). immune evasion, and the inhibition of antiviral immune Moreover, because neoantigens arise from tumor-specific muta- responses. Because TIGIT also acts as an inhibitory immune tions, they are not present in normal tissues and consequently checkpoint on NK cells, targeting it with an ICI provides an vaccines can selectively target the tumor, respecting healthy tissues opportunity to attack both the adaptive and innate arms of the and minimizing toxicities. Finally, personal neoantigen vaccines immune system (38). Although clinical development is still in have shown the ability to induce de novo T-cell clones that can preliminary stages, several anti-TIGIT inhibitors are under detect multiple individual-specific neoantigens thus targeting a investigation in combination with PD-1/PD-L1 inhibitors. diversity of malignant clones per patient, successfully addressing BMS-986207, a mAb targeting TIGIT, is currently being inves- tumor heterogeneity and minimizing the chance of tumor escape tigated in a phase I/IIa trial in combination with nivolumab by loss of antigen (40). in advanced solid tumors including urothelial cancer (NCT02913313; Table 3). Similarly, MTIG7192A, a fully Combinations of ICIs and Targeted human mAb that binds to TIGIT, is being evaluated both as monotherapy and in combination atezolizumab in a two-step Therapies phase I study in patients with advanced tumors including Immune escape mechanisms are activated by oncogenic events urothelial cancers (NCT02794571). in cancer cells through the activation of multiple immunoinhi- bitory receptors and tyrosine kinase receptors. Consequently, Combinations with LAG3 inhibitors immunoncology agents and targeted therapies are two treatment Lymphocyte activation gene-3 (LAG3) is an inhibitor immune modalities that have the potential to deliver synergy when used in checkpoint receptor and a member of the immunoglobulin combination (41). The administration of targeted therapies that

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inhibit immunosuppressive immune cells or disrupt oncogenic 38.5% (44). In view of these results, the future of acalabrutinib in signals in cancer cells may result in enhanced antitumor combination with pembrolizumab remains uncertain. responses. Several studies are currently investigating the role of combining ICI with multi TKIs and other targeted therapies in Biomarker-driven combinations patients with advanced urothelial cancer. The BISCAY trial, an open-label randomized multi-drug biomarker-directed phase Ib study, is evaluating different treatment combinations in patients with metastatic urothelial cancer who Combinations with cabozantinib have progressed on prior treatment (NCT02546661). The study Cabozantinib, a multi TKI targeting VEGFR2, MET, and AXL consists of a number of modules, each evaluating the safety, has shown clinical activity in pretreated patients with meta- tolerability, and efficacy of the PD-L1 inhibitor durvalumab in static urothelial cancer. Cabozantinib promotes the upregula- combination with a specific targeted therapy in patients whose tion of MHC class 1 on tumor cells, downregulates Tregs and tumors express specific genomic alterations relevant to the tar- MDSC numbers, and increases circulating CD8 T cells. These geted therapy under investigation. Patients with tumors showing immunomodulatory properties of cabozantinib may counter- mutations in an homologous recombination repair gene panel act tumor-induced immunosuppression providing a robust will be allocated in module B and will receive durvalumab in rationale for combination with ICIs. Some preliminary prom- combination with the PARP inhibitor olaparib. Patients with ising results were recently presented from a phase I expansion tumors showing mutations in genes involved in cell-cycle regu- study evaluating the safety and efficacy of cabozantinib plus lation will be allocated in module C and treated with durvalumab nivolumab alone or with ipilimumab in patients with meta- in combination with AZD1775, a WEE1 inhibitor. Patients with static urothelial cancer (NCT02496208). Seventy-five patients tumors showing genomic alterations that have potential to with genitourinary malignancies were recruited in the study, respond to mTOR inhibitors (RICTOR amplification, TSC1/2 including 23 patients with urothelial cancer (42). The phase I mutations) will be allocated in module E and treated with cohort had 7 dose levels while in the expansion cohort, patients durvalumab in combination with vistusertib, a selective mTORC1 were treated with cabozantinib 40 mg daily, nivolumab 1 mg/ and mTORC2 inhibitor. Finally, patients with no specific genomic kg, and ipilimumab 3 mg/kg. The efficacy data in the urothelial alterations may be allocated in module D and receive durvalumab cancer cohort was significant, with an ORR of 39%, a CR rate of monotherapy or in module F and receive durvalumab plus 8.7%, and a clinical benefit rate of 82.6%. These data were even AZD9150, a next-generation antisense oligonucleotide inhibitor better for the subgroup of patients not previously treated with of STAT3. Importantly, new additional modules and agents might an ICI (n ¼ 19), with an ORR of 42.1%, a CR rate of 10.5%, and be added as part of this multi-drug study as information on aclinicalbenefit rate of 84.2%. Regarding the different treat- potential attractive combinations become available. ment combinations, the ORRs were 50% and 28.6%, the CRs rate 8.3% and 14.3% and the clinical benefit rates 83.3% and Combinations with FGFR inhibitors 85.7% for the cabozantinib–nivolumab (n ¼ 12) and cabo- Dysregulation of the fibroblast growth factor receptor zantinib–nivolumab–ipilimumab (n ¼ 7) arms, respectively (FGFR) pathway via genetic modifications including muta- (42). The urothelial cancer cohort had a median PFS of 12.8 tions, amplifications, and gene fusions have been described months (95% CI 1.8–undefined) and a 12 months OS of 70.2% in several solid tumors including urothelial cancers. The pres- (95% CI 44.4–85.8%). Both treatment combinations were safe ence of these oncogenic drivers might confer sensitivity to and well tolerated with few grade 3–4 toxicities (moderate to FGFR inhibitors (45). Inhibition of FGFR-mediated signaling severe toxicities). can result in an antiproliferative activity and may have an antiangiogenic effect. FGFR3 pathway alterations have been Combinations with acalabrutinib associated with noninflamed bladder tumors and inversely þ Activation of the Bruton tyrosine kinase (BTK), a key B-cell and correlates with CD8 T cells (46). Moreover, luminal cluster macrophage kinase, has been linked to B-cell and macrophage- I urothelial cancers, which hypothetically poorly respond to mediated T-cell suppression and tumor growth in cancer (43). PD-L1 blockade (2), overexpress FGFR pathway alterations in Targeting the BTK might therefore restore T-cell–dependent anti- the TCGA classification (47). Consequently, combinations of tumor immune response and improve responsiveness to ICIs. ICI and FGFR inhibitors might have the potential to enhance Acalabrutinib, a novel second-generation BTK inhibitor, is being antitumor activity. Several clinical trials are currently testing investigated in combination with pembrolizumab in a phase II different FGFR inhibitors in combination with ICI in patients study, the Keynote 143 (NCT02351739) in patients with plati- with metastatic urothelial cancer. The BISCAY multi-drug num-refractory metastatic urothelial cancer (44). Seventy-five phase Ib trial, early mentioned, also includes a module (mod- patients have been randomized to receive pembrolizumab alone ule A) of durvalumab in combination with AZD4547, a potent or in combination with acalabrutinib. However, the results pre- and selective inhibitor of FGFR-1, 2, and 3, in patients presented so far have been somewhat disappointing. Acalabrutinib senting FGFR3 mutations or FGFR fusions (NCT02546661). plus pembrolizumab did not improve the efficacy over pembro- Two other clinical trials are assessing the safety and efficacy of lizumab alone, with an ORR of 25.7% with pembrolizumab and pembrolizumab in combination with B-701, a novel and 20% with acalabrutinib–pembrolizumab. The CR rate was similar specific mAb against FGFR3, in patients with metastatic between treatment arms, 9% and 10%, respectively. The 12- urothelial cancer. The first study is a single-arm phase I trial month DOR rate was longer with the treatment combination, (NCT02925533) evaluating the combination in a biomarker- 63.5% and 83.3%, respectively. However, median OS was longer unselected population with cisplatinum-refractory urothelial with pembrolizumab monotherapy, 11.4 months versus 6.3 cancer. As an exploratory analysis, FGFR3 status will be ret- months, respectively, as was the 12-month OS rate, 44.1% versus rospectively assessed by IHC and correlated with efficacy. The

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second study is a randomized phase Ib/II trial Conclusions and Future Directions (NCT03123055) of B-701 alone or in combination with Treatment with ICI is now firmly established as a therapeutic pembrolizumabinpatientswithFGFR3alteredadvancedUCs. strategy for patients with advanced urothelial cancer previously treated with cisplatin-based chemotherapy or unfit to receive Combinations of ICIs and Radiotherapy cisplatin. Results of ongoing studies may move forward the use Radiotherapy exerts potent coadjuvant effects when admin- of ICI to earlier stages, including the first-line, adjuvant, neoad- istered concomitantly with immunotherapy with ICI, because it juvant, and even nonmuscle-invasive settings. Different combi- prompts immunogenic tumor cell death, inducing the release nations of ICI with other immunotherapy strategies, chemother- of tumor antigens and turning the tumor into an in situ vaccine, apy, targeted agents, and radiotherapy are intensively being triggering proinflammatory signals that activate tumor-specific studied aiming to maximize the benefits of these new therapeutic T cells, and enhance tumor infiltration by lymphocytes (48). class of drugs, and active research is ongoing to develop new Combinations of ICI and radiotherapy may allow the devel- predictive biomarkers to select the most active treatments for opment of abscopal responses, (i.e., outside of the irradiated patients, according to the molecular profile of their disease (51). field), as has been reported in the preclinical (49) and clinical settings (50). Several ongoing trials are evaluating ICI and fl radiotherapy in patients with urothelial cancer (Table 4). Some Disclosure of Potential Con icts of Interest of them target patients presenting localized disease, while A. Rodriguez-Vida reports receiving commercial research support from MSD, Pfizer, and Takeda; speakers bureau honoraria from Astellas, AstraZeneca, Bayer, others include patients with metastatic tumors, thus pursuing Bristol-Myers Squibb, Janssen, MSD, Pfizer, Roche, and Sanofi-Aventis; and is a abscopal activity. These studies evaluate concomitant and consultant/advisory board member for Astellas, Bayer, Bristol-Myers Squibb, sequential administration of radiotherapy and ICI, as well as Janssen, MSD, Pfizer, and Roche. J.L. Perez-Gracia reports receiving speakers different techniques of administration of radiotherapy, includ- bureau honoraria from and is a consultant/advisory board member for Bristol- ing conventional external-beam radiotherapy, hypofractio- Myers Squibb and Roche. J. Bellmunt is a consultant/advisory board member for fi nated radiation, or stereotactic body radiation therapy (SBRT) AstraZeneca, Bristol-Myers Squibb, Genentech, Merck, Pierre Fabre, and P zer. and will help to understand the role and the value of combin- Received May 2, 2018; revised June 15, 2018; accepted July 5, 2018; ing these therapeutic modalities. published first July 10, 2018.

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6124 Clin Cancer Res; 24(24) December 15, 2018 Clinical Cancer Research

Immunotherapy Combinations and Sequences in Urothelial Cancer: Facts and Hopes

Alejo Rodriguez-Vida, Jose Luis Perez-Gracia and Joaquim Bellmunt

Clin Cancer Res 2018;24:6115-6124. Published OnlineFirst July 10, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-17-3108

Cited articles This article cites 47 articles, 15 of which you can access for free at: http://clincancerres.aacrjournals.org/content/24/24/6115.full#ref-list-1

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