Novel therapies in urothelial carcinoma: a biomarker-driven approach

Article type: Review

Title: Novel therapies in urothelial carcinoma: a biomarker-driven approach

Authors: J. E. Rosenberg G. Iyer

Affiliations: Memorial Sloan Kettering Cancer Center, New York, NY, USA

Corresponding Author: Dr. Jonathan E. Rosenberg Memorial Sloan Kettering Cancer Center 1275 York Avenue New York NY 10065 USA Tel.: 001 646 422 4461 Email: [email protected]

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Review article word count: max. 4000

© The Author(s) 2018. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected].

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Abstract Urothelial malignancies, including carcinomas of the bladder, ureters, and renal pelvis comprised approximately 8% of new cancer cases in the United States in 2016. In the metastatic setting, 15% of patients exhibit long-term survival following cisplatin-based chemotherapy and in patients with recurrent disease, response rates to second-line chemotherapy are generally 15-20% with a 3-month progression-free survival. However, recent advances in immunotherapy represent an opportunity to significantly improve patient outcomes. Moreover, the advent of next-generation sequencing (NGS) has resulted in both an improved understanding of the fundamental genetic changes that characterize urothelial carcinoma (UC) and identification of several candidate biomarkers of response to various therapies. Incorporation of prospective genotyping into clinical trials will allow for the identification and enrichment of patients most likely to respond to specific targeted therapies and chemotherapy. Combining different therapeutic classes to enhance outcomes is also an area of active research in UC.

Keywords: Urothelial cancer, urinary bladder, metastasis, predictive biomarkers, gene sequencing

Key message: New developments in biomarker-driven approaches, based on an understanding of the genetic alterations underlying urothelial malignancies, are guiding the development of novel therapies and therapeutic strategies for this disease.

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Introduction In 2017, approximately 150,000 people will be diagnosed with UC in the United States and around 32,000 are likely to die from their disease [ACS 2017]. Urothelial malignancies include carcinomas of the bladder, ureters, and renal pelvis and are more common in men than women (3:1) and in Caucasians than African Americans (2:1) [Hurwitz et al. 2016]. The most common of the urothelial malignancies, accounting for an estimated 54% of cases and 52% of deaths, is urinary bladder cancer [ACS 2017]. Tobacco smoking and occupational exposures (e.g., arylamines) are the leading risk factors for developing urothelial bladder carcinoma in the United States and contribute to an increased risk of recurrence [Wilcox et al. 2016; Hurwitz et al. 2016]. Disease stage is closely related to prognosis. Patients with well-differentiated, non-invasive primary papillary lesions (Ta) without carcinoma in situ have a 95% survival rate, whereas those with higher-grade lesions that have invaded the sub-epithelial lamina propria (T1) have a 10-year survival rate of 50%; muscle-invasive urothelial carcinoma (T2) has a 5-year disease-specific survival rate of 40%–65% [Hurwitz et al. 2016]. However, a 5-year survival rate of only 0%–30% can be expected if lymph nodes are involved (Stage IV) [Hurwitz et al. 2016].

Patients with organ-confined disease are managed with surgical intervention, chemotherapy, radiotherapy, or a combination of these treatment modalities. Transurethral resection of the bladder tumor (TURBT) with or without intravesical therapy is generally recommended in patients with superficial bladder cancer [Bellmunt et al. 2014]. Patients with high-grade, recurrent nonmuscle invasive bladder cancer can be managed with radical cystectomy (RC) prior to the development of muscle invasion [Bellmunt et al. 2014; Hurwitz et al. 2016]. Patients with muscleinvasive disease are optimally managed with neoadjuvant cisplatin-based chemotherapy in eligible patients followed by RC and a bilateral pelvic lymph node dissection, or trimodality therapy consisting of a maximal TURBT followed by chemo-radiotherapy.

The chemotherapeutic management of patients with muscle-invasive and metastatic urothelial carcinoma (UC) has changed little in over 20 years. Platinum-based combinations remain the standard-of-care in perioperative (as neoadjuvant and adjuvant therapy) and first-line metastatic settings [Bellmunt et al. 2014]. Cisplatin-containing combination chemotherapy with either gemcitabine or methotrexate/vinblastine/doxorubicin is recommended for those patients with advanced surgically unresectable or metastatic disease who are able to tolerate cisplatin [Bellmunt et al. 2014]. Despite refinements in treatment schedules and toxicity management, median survival achieved with these treatments is 12-15 months, either in clinical trials [Loehrer et al. 1992; von der

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Masse et al. 2000; von der Masse et al. 2005] or in real-world settings [Robinson et al. 2016]. To address these limitations, intensification strategies including new triplet or quadruplet strategies, or dose-dense therapies, were developed with subsequent improvements in objective response rates but not median overall survival [Galsky et al. 2007; Milowsky et al. 2009; Bellmunt et al. 2012; Necchi et al. 2014].

Advances in the understanding and identification of genetic alterations underlying the spectrum of urothelial malignancies hold out hope for improved outcomes through the development of targeted therapies. Additionally, the recent approval of five immunotherapy agents in the United States has led to a transformation in the management of patients with advanced UC, although only a minority of patients displays responses to this class of agents and predictive biomarkers of sensitivity to immunotherapy have yet to be validated [Rosenberg et al 2016; Sharma et al, 2017]. Despite recent treatment advances, there remains an unmet need for the improved management of patients with UC. This paper provides an overview of the current state-of-the art of therapy for urothelial malignancies, provides insight into the paucity of successful treatments, and offers some perspectives on future directions for additional research, with a focus on the prospective benefits of using biomarker-driven approaches to patient management.

Immunotherapy for metastatic urothelial malignancies: Current state-of-the-art Immunotherapy has fundamentally altered the treatment paradigm for metastatic UC. Until recently, patients with metastatic disease had few treatment options after failure of platinum-based chemotherapy strategies. Single agent chemotherapies in the second-line setting generally display response rates of less than 30%, and more frequently 10-15%. An analysis of studies investigating doublet chemotherapy regimens as salvage therapy after initial platinum-based treatment showed no extended survival benefits compared with single agents [Raggi et al. 2016] and such regimens are frequently limited by toxicity [Oing et al. 2016]. In 2009, vinflunine, a fluorinated Vinca alkaloid, was approved by the European Medicines Agency (EMA) as monotherapy for the treatment of adult patients with advanced or metastatic urothelial carcinoma after failure of a prior platinumcontaining regimen [Javlor SmPC 2009] and is a recommended therapy in treatment guidelines [Bellmunt et al 2014]. This approval is based upon a phase III study of vinflunine plus best supportive care versus best supportive care alone for patients with metastatic UC who had progressed on platinum-based chemotherapy. A 2-month median survival advantage was observed for vinflunine plus best supportive care (6.9 months vs 4.3 months for best supportive care alone, p=0.040) in the

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eligible patient population. Significant improvements in overall response rate and progression-free survival were also noted with the addition of vinflunine.

In 2016, atezolizumab, a programmed death ligand 1 (PD-L1)-blocking antibody, received accelerated United States Food and Drug Administration (FDA) approval for locally advanced or metastatic UC in patients with disease progression during or following platinum-containing chemotherapy or within 12 months of peri-operative platinum-containing chemotherapy [Tecentriq PI 2016]. This approval was based on a phase 2 study in patients with metastatic UC who had progressed on prior platinum-based chemotherapy (Rosenberg et al, Lancet 2016). In this trial, the overall response rate was 15%, significantly higher than a historical response rate of 10% with chemotherapy. Responses were also organized based upon PD-L1 expression (using the Ventana SP142 assay) on immune infiltrating cells (IC) and were found to be highest in patients with ≥5% infiltration (IC2/3, 26%); notably, responses were also observed in IC0 (8%) and IC1 (10%) patients. At 11.7 months median follow-up, 84% of responders continued to show objective responses, underscoring a durability not usually seen with standard chemotherapy. In April 2017, accelerated approval was also granted for atezolizumab as a first-line treatment for patients with locally advanced or metastatic UC who are ineligible for cisplatin-based chemotherapy or who have disease progression at least 12 months after receiving perioperative chemotherapy [Tecentriq Approval History 2017]. In a phase 2 study of atezolizumab in this patient population, the objective response rate was 23%, including a 9% complete response rate, with a median overall survival of 15.9 months, all significantly higher than observed with carboplatin-based chemotherapy. In both studies, tumor mutation load was significantly higher in responders.

Following the approval of atezolizumab, the results of an open-label, multi-center phase III study were reported (IMvigor211) in which patients with progressive metastatic urothelial carcinoma following platinum chemotherapy were randomized to receive either atezolizumab or treating investigator’s choice of paclitaxel, docetaxel, or vinflunine [Powles et al. 2018]. The primary endpoint of overall survival within a predefined patient population with IC2/3 PD-L1 expression was not significantly different between atezolizumab and chemotherapy (median OS 11.1 months vs 10.6 months, respectively, HR 0.87, p=0.41). The objective response rate was also similar in both groups, but the rate of ongoing responses was higher with atezolizumab therapy (62% vs 20% with chemotherapy). Potential reasons for the lack of improvement in OS include the observation that survival with vinflunine was higher than expected within the statistical model for this study and the fact that IC2/3 patients displayed improved survival and response independent of treatment

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modality. The association of tumor mutation burden with survival was evaluated in an exploratory analysis of the intent to treat patient population. High tumor mutation burden (defined as being higher than the median total mutation burden within assessable patients) was associated with a non-significant improvement in overall survival with atezolizumab therapy (11.3 months vs 8.3 months with chemotherapy, HR 0.68). Comparison of survival between treatment arms within high total mutation burden patients with IC2/3 PD-L1 expression showed a longer median survival with atezolizumab (17.8 months) versus chemotherapy (10.6 months).

Nivolumab, a PD-1 immune checkpoint inhibitor antibody, received accelerated approval from the FDA in February 2017 in patients with locally advanced or metastatic UC whose disease has progressed on platinum chemotherapy [Opdivo PI]. The phase 2 CHECKMATE 275 study explored nivolumab monotherapy in patients with metastatic, pre-treated UC and showed a response rate of 19.6% across all patient subgroups. When segregated by PD-L1 expression status on tumor cells, the response rate was 28.4% in patients with ≥5% expression versus 16.1% in patients with <1% PD-L1 expression. The median overall survival was 8.74 months (11.3 months in patients with ≥1% PD-L1 expression versus 5.95 months in patients with <1% expression). Nivolumab has also been studied in two dose combinations with ipilimumab (an anti-CTLA-4 antibody) in patients with locally advanced or metastatic UC previously treated with platinum-based chemotherapy; interim results showed that high response rates were achieved with combination therapy, but with higher toxicity [Sharma 2016a] rates, which is similar to observations in treatment-naïve melanoma patients [Larkin et al. 2015; Hodi et al. 2016]. Further study is needed to evaluate the clinical utility of this combination.

Durvalumab was investigated in 191 patients with advanced or metastatic UC following progression on prior chemotherapy or who were ineligible or refused chemotherapy in a phase I/II multi-center trial. In the latest evaluation, patients with Stage IV disease (98 PD-L1–positive, 79 PD-L1–negative [1 patient with unknown PD-L1 status]) were treated with intravenous durvalumab 10 mg/kg every 2 weeks for up to 12 months, and 99.5% had received one or more prior therapies. After a median follow-up of 5.8 months, the overall response rate (ORR) was 17.8% (27.6% in the PD-L1–positive subgroup versus 5.1% in the PD-L1–negative subgroup). The initial 20 patients on study were enrolled regardless of PD-L1 expression, while subsequent patients were required to have ≥5% PD-L1 expression (using the Ventana SP263 assay) on tumor cells for enrolment. PD-L1 expression was defined as positive if either ≥25% of tumor cells or ≥25% of immune cells expressed PD-L1. Using this definition, the ORR in PD-L1 positive patients was 46.4% versus 0% in PD-L1 negative patients.

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Avelumab, a fully humanized monoclonal PD-L1 antibody, is in phase 1b investigation in patients with solid tumors (JAVELIN trial program); an analysis of patients with metastatic UC showed promising and durable clinical activity in heavily pre-treated patients [Apolo et al. 2017]. In this study, the unconfirmed ORR was 17.4%, and 25.4% in PD-L1–positive patients (defined using a ≥5% cutoff for tumor cell staining, Dako PD-L1 IHC kit) versus 13.2% in PD-L1–negative patients. Avelumab is also being assessed in a study (JAVELIN Bladder 100) as maintenance treatment for patients with locally advanced/metastatic UC whose disease did not progress after first-line platinum-based treatment [Powles et al. 2016a], with primary outcome results expected in the latter half of 2019.

Pembrolizumab is a humanized monoclonal antibody directed against PD-1. In a phase 3 study of pembrolizumab as second-line therapy in patients with advanced UC that recurred or progressed after platinum-based chemotherapy versus investigator’s choice of docetaxel, paclitaxel, or vinflunine (KEYNOTE 045), pembrolizumab was associated with significantly longer overall survival (10.3 months vs 7.4 months, Hazard Ratio 0.70, p<0.001) [Bellmunt et al. 2017; Bajorin et al. 2017]. Notably, the median duration of response was longer (not reached) in the pembrolizumab treated patients versus 4.4 months with chemotherapy. PD-L1 expression using a 10% or greater combined positive score in tumor and infiltrating immune cells (22C3 pharmDx assay, Dako) was not associated with a statistically significant difference in response rate. This led to full FDA approval for patients previously treated with platinum-based chemotherapy. The phase 2 KEYNOTE 052 study investigated pembrolizumab in cisplatin-ineligible patients; the overall response rate was 24%, including 5% complete responses [Balar et al. 2017]. At an 8-month median follow-up, 74% of patients continued to respond and the median response duration was not reached. The results of this study supported an accelerated FDA approval in cisplatin-ineligible metastatic urothelial carcinoma patients.

The observation that patients without PD-1 or PD-L1 expression may still exhibit responses to immune checkpoint inhibitors underscores the fact that these biomarkers cannot be used in isolation to make treatment decisions. Moreover, several caveats to the application of routine PD-1/ PD-L1 staining exist, including the use of different assays to test for PD-1 and PD-L1 expression, variability in the definitions and cutoffs for positivity (the use of tumor-cell vs immune infiltrating cell surface expression), differences in familiarity with interpreting test results, intratumor heterogeneity of expression, and the impact of prior treatments upon expression levels over time. As discussed below in more detail, several other tumor-specific factors likely contribute to checkpoint blockade sensitivity in UC, including expression subtyping and mutation profile (for example, the presence of

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DNA damage response gene alterations or FGFR3 alterations). Additionally, host-specific factors, such as T-cell receptor clonality, tumor-infiltrating lymphocytes, and others, also modulates response to checkpoint blockade. A significant challenge will be to understand the relative impact of each of these biomarkers in predicting for checkpoint blockade sensitivity and resistance and implementing testing for these biomarkers in a rapid, prospective fashion prior to initiation of therapy.

Immunotherapies in development A number of other checkpoint inhibitors, interleukins (ILs), and novel immune molecules have been identified as potential immunotherapeutic agents in muscle-invasive and metastatic urothelial cancer (Figure 1).

Novel therapies and therapeutic strategies in urothelial malignancies Identification of prognostic biomarkers (to provide information on overall cancer outcome in patients and/or to facilitate cancer diagnosis) and predictive biomarkers (to inform treatment decisions) are integral to a personalized oncology approach in cancer. A number of strategies have been used to identify clinically useful biomarkers for UC. The Cancer Genome Atlas (TCGA) analyzed 412 muscle-invasive, high-grade bladder cancers using multiple platforms, including whole exome sequencing, RNA sequencing, DNA methylation profiling, and others. The total mutation burden was high, similar to that of melanoma and non-small cell lung cancers; moreover, the most mutations were clonal and were detected within the context of an APOBEC mutation signature. (Figure 2) [Robertson 2017]. RNA expression subtyping delineated 5 distinct molecular subtypes of bladder cancer, including luminal, luminal-papillary, luminal-infiltrated, basal-squamous, and neuronal. The neuronal subtype was associated with the worst survival of the 5 subtypes and was enriched for TP53 and RB1 alterations while the luminal infiltrated subtype has been correlated with response to clinical trials of immune checkpoint blockade in urothelial carcinoma. While several groups have identified similar molecular subtypes based upon expression profiling of urothelial carcinoma, the predictive and prognostic characteristics of these subtypes are as yet unclear and require prospective validation. A novel gene expression algorithm, Co-eXpression ExtrapolatioN (COXEN), is an informatics-based approach to predict sensitivity of independent cell line panels and patient responses to therapeutic agents [Smith et al. 2010; Smith et al. 2011]. The COXEN model combines in vitro and in vivo molecular profiling of cancer cell lines and drug responsiveness using the NCI-60 and BLA-40 cancer cell line panels (Figure 3). For the purposes of prospective validation, the COXEN algorithm is being incorporated into the SWOG S1314 study in

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which muscle-invasive bladder cancer patients are randomized to two different neoadjuvant chemotherapy regimens. The results of COXEN will be correlated with response to chemotherapy at cystectomy to gauge the capability of the algorithm to predict for chemo-sensitivity. Additional correlative analysis of pre-treatment specimens and post-chemotherapy residual tumors is planned, including whole transcriptome profiling to facilitate correlation of the published molecular subtypes with response and survival.

Next-generation sequencing has enabled a more detailed analysis of the underlying genetic changes characterizing UC and identification of potential new therapeutic targets. Although most tumors harbor potentially tractable genomic alterations that may predict for response to target-selective agents, substantial genomic heterogeneity has been identified in urothelial bladder cancer [Faltas et al 2016]. Additionally, subclonal mutational heterogeneity, which may contribute to chemotherapy resistance [Liu et al. 2017], poses an obstacle to the effective application of targeted agents.

Among the promising therapeutic targets, FGFR3, is of particular interest. It encodes for a transmembrane protein, fibroblast growth factor receptor-3, involved in regulating cell proliferation and angiogenesis; putative downstream signaling pathways include the PI3K-AKT, PKC, and Ras/MAPK pathways (Figure 4). The most common FGFR3 alterations in UC are activating point mutations. Dysregulation of FGFR3 signaling occurs in approximately 80% of non-invasive and 50% of invasive bladder cancers, through mutation, overexpression, or both, and over-expression is also common in tumors with no detectable mutations, including in muscle-invasive tumors [Tomlinson et al. 2007]. FGFR3 alterations have been reported in up to 21% of patients with high-grade and advanced stage bladder UC. Activating FGFR3 fusions have also been described in UC [Glaser et al. 2017] [CGARN 2014]. Urothelial cell lines harboring these fusions are highly sensitive to FGFRselective agents and responses to FGFR-directed inhibitors have been observed in patients with FGFR3 fusion positive UC. Thus, these alterations may serve as predictive biomarkers that could aid patient selection for FGFR-targeted therapy [Williams et al. 2013; Costa et al. 2016].

Although dovitinib, a multi-targeted inhibitor of tyrosine kinases including FGFR3, has not shown significant activity in UC (due in part to challenges with appropriate patient selection using the available sequencing platforms at the time), other agents targeting FGFR3-mutant UC remain under investigation. BGJ398 is an FGFR1–3 inhibitor that showed promising anti-tumor activity in patients with advanced solid tumors, including those with UC [Nogova et al. 2017]. These data led to evaluation of drug efficacy in an extended cohort of patients with advanced/metastatic UC and

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activating FGFR3 mutations/fusions after platinum-based chemotherapy [Pal et al. 2016]. Seventy-six percent of patients had received 2 or more prior treatments. The ORR was 36%, including 8 partial responses, suggesting clinical benefit with FGFR3 signaling blockade in this patient population. Clinical activity has also been observed with erdafitinib, a pan-FGFR inhibitor, in patients with metastatic UC and this drug recently received FDA breakthrough designation for this disease. In a phase 2 study of erdafitinib in patients with metastatic UC (BLC2001) and pre-specified FGFR alterations, the overall response rate was 42% (3% CR rate) [Siefker-Radtke et al. 2018]. Notably, the response rate was 59% in the subset of patients who had received prior immune checkpoint blockade.

Inhibition of mechanistic target of rapamycin (mTOR) is also a target for therapy in UC under investigation. Everolimus, which blocks mTOR signaling, was tested in two phase 2 studies in patients with advanced UC [Seront et al. 2012; Milowsky et al. 2013]. The study by Milowsky et al did not reach the primary endpoint of an improvement in 2-month progression-free survival compared to historical rates. In the study by Seront et al, a disease control rate of 27% was seen, with 2 partial responses. Everolimus in combination with paclitaxel was not effective as a second-line treatment for UC in the German AUO Trial AB 35/09 [Niegisch et al. 2015], showing a 13% overall response rate. In addition, a regimen combining everolimus with gemcitabine and split-dose cisplatin in advanced UC was not feasible due to dose-limiting hematologic toxicities [Abida et al. 2016]. Despite these outcomes, evidence to support the use of everolimus in specific molecular contexts exists, with some patients harboring specific mutations in the mTOR pathway experiencing exceptional responses to everolimus-containing therapy. The activating mutations mTORE2419K and mTORE2014K were found in a patient tumor that was exquisitely sensitive to mTOR inhibition; the patient had platinum- and taxane-refractory UC and experienced a complete radiologic response that lasted for 14 months on pazopanib plus everolimus [Wagle et al. 2014]. Loss-of-function mutations in TSC1 and NF2, both regulators of mTOR pathway activation, have been correlated with durable everolimus sensitivity in an exceptional responder that has been ongoing for greater than 6 years [Iyer et al. 2012]; additionally, NF2 mutations have been identified as conferring significant response to everolimus-containing regimens in patients with urothelial malignancies [Ali et al. 2015]. Studies to investigate the clinical benefit of everolimus targeted to cancer patients with inactivating TSC1 or TSC2 mutations or activating MTOR mutations (NCT02201212) and NF2 mutations (NCT02352844) are on-going.

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Analysis of TCGA has also shown that epidermal growth factor receptor (EGFR) signaling pathways are upregulated in selected advanced UC tumors, with EGFR amplification in 11% of UC and ERBB2 amplification and mutation in 9% [TCGA 2014]. Lapatinib is a dual tyrosine kinase inhibitor of EGFR and HER2 that showed anti-tumor activity in a phase 2 study in a subset of 34 patients with EGFR/HER2-overexpressing tumors [Wülfing et al. 2009]. Conversely, in a study of 232 patients with metastatic bladder cancer who had clinical benefit from first-line chemotherapy and HER1/HER2positive status confirmed by centralized immunohistochemistry (IHC), no improvement in progression-free survival was observed with maintenance lapatinib compared to placebo [Powles et al. 2015]. Targeting mutant/amplified tumors may still be a useful strategy, however, and negative results may reflect a failure of proper patient selection due to the assay used to determine gene amplification or protein overexpression.

Afatinib, another dual EGFR and HER2 inhibitor, has demonstrated clinical activity in patients with platinum-refractory UC with ERBB2 or ERBB3 genetic alterations detected by quantitative polymerase chain reaction or fluorescent in situ hybridization [Choudhury et al. 2016]. Five of the 6 patients with ERBB2 and/or ERBB3 alterations achieved the primary endpoint of 3-month progression-free survival compared with none of the 15 patients without such alterations (P < 0.001); median time to progression/therapy discontinuation was 6.6 months in patients with ERBB2/ERBB3 alterations versus 1.4 months in those without alterations (P < 0.001). This study suggests that selection by genomic alterations may be more relevant to the development of urothelial carcinoma than protein expression as detected by IHC.

Overall mutational load is also emerging as an additional biomarker of response in advanced UC, at least in patients treated with PD-1-blocking agents (Table 1). The phase 2 studies of atezolizumab in patients with locally advanced and metastatic UC who have progressed following treatment with platinum-based chemotherapy [Rosenberg et al. 2016a] and as first-line treatment in cisplatinineligible patients with locally advanced and metastatic UC [Balar et al. 2017a] showed that mutation load was consistently associated with improved response to atezolizumab therapy.

Combining various immunotherapies may also yield benefits surpassing those of monotherapy, and several combinations are currently being examined, including combination immune checkpoint blockade plus chemotherapy as well as with novel agents (both targeted therapies and immune modulating agents). A number of trials are examining the utility of combining platinum-based chemotherapy with checkpoint blockade in the first-line setting for metastatic urothelial carcinoma

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in an attempt to leverage the response rates observed with platinum chemotherapy in this disease with the durability seen with checkpoint blockade. A study of durvalumab as first-line monotherapy or in combination with tremelimumab, an anti-CTLA-4 antibody, in patients with unresectable stage IV urothelial bladder cancer (the phase 3 DANUBE study) is recruiting patients [Powles et al. 2016b], with primary outcome results expected in 2018. The BISCAY trial, an open-label, randomized, multidrug, biomarker-directed, phase 1b study in patients with muscle-invasive bladder cancer who have failed at least one prior platinum regimen, will assess immunotherapy with durvalumab as monotherapy and in combination with select targeted therapies. Treatment assignment is based upon the tumor biomarker profile, and includes: olaparib, a PARP inhibitor; vistusertib, an mTOR inhibitor; AZD4547, an FGFR inhibitor; and AZD1775, a WEE1 inhibitor. The incorporation of prospective molecular analysis of tumors to inform assignment to a specific therapy could optimize the efficacy of small molecule inhibitors and possibly enhance responses to checkpoint blockade through the generation of neoantigens in this setting. Multiple checkpoint inhibitors are currently being investigated in combination with novel immunotherapies to enhance or overcome resistance to PD-1 blockade in patients with advanced solid tumors. Some of these include combinations with GSK3174998, an agonist of OX40, a potent costimulatory tumor necrosis factor receptor expressed on activated CD4+ and CD8+ T cells; MK-4280, targeting the lymphocyte-activation gene 3 (LAG3) receptor; NKTR-214, a CD122 agonist; TSR-022, an anti-T cell immunoglobulin and mucin containing protein-3 (TIM-3) antibody; BMS-98625, an IDO1 inhibitor; and CDX-1127, targeting CD27, a lymphocyte-specific member of the tumor necrosis factor receptor.

Re-conceptualizing targeted therapy Platinum chemotherapy as targeted therapy As discussed above, platinum-based combinations remain the standard-of-care for neoadjuvant chemotherapy (NAC). Cisplatin-based NAC is associated with improved survival, with a 14%–25% relative risk reduction for death from muscle-invasive UC [International collaboration of trialists 1999; Grossman et al. 2003; ABC Collaboration 2005]. The benefits of this approach are most apparent in patients with pathologic down-staging at the time of surgical resection, but the molecular determinants of response to cisplatin are unproven. Identifying extreme responders to platinum chemotherapy would therefore allow targeting of this treatment to those who would benefit most, and spare those who would not from the side effects of treatment.

Of note, somatic genomic alterations in DNA damage repair (DDR)-associated genes have been associated with benefit from both NAC and PD1/PD-L1 blockade. An initial extreme responder

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analysis of patients with complete responses or residual carcinoma in situ within the bladder following neoadjuvant cisplatin-based chemotherapy and radical cystectomy for muscle-invasive disease versus patients with residual muscle-invasive or higher stage disease identified point mutations within the DNA helicase ERCC2 as strongly associated with response (9 of 25 responders harbored ERCC2 mutations versus 0 of 25 non-responders). These results have since been validated in a separate cohort of patients receiving neoadjuvant chemotherapy for muscle-invasive bladder cancer as part of a clinical trial in which ERCC2 mutations were also associated with improved survival (Liu et al, 2016). A subsequent retrospective analysis of 100 patients with locally advanced or metastatic UC who underwent exon capture sequencing revealed an association between the presence of DDR gene alterations and improved progression-free and overall survival [Teo et al, 2017a]. In this analysis, a higher mutation load was observed in DDR gene altered tumors. Defects in other DDR genes, including RB1, ATM, and FANCC, were detected in patients who responded to neoadjuvant chemotherapy [Plimack 2015]. Emerging evidence also suggests an association between overall response to PD1/PD-L1 blockade and the presence of somatic DDR alterations [Teo et al. 2017b].

A high frequency of patients with germline DDR mutations has also been reported. In a recent study, 12/25 pathogenic or likely pathogenic mutations in 22 patients with urothelial cancer originating from all sites within the urinary tract had heritable DDR gene alterations in CHEK2, BRCA1, BRCA2, ATM, BRIP1, and NBN [Carlo et al. 2017]. The role of these germline alterations will require validation.

Recent research suggests that RNA expression of non-coding microRNAs may contribute to the control of the gene expression patterns and can identify basal and luminal muscle-invasive bladder cancer subtypes. These could act as biomarkers for tumors with higher infiltration rates and provide candidate therapeutic targets [Ochoa et al. 2016]. Molecular subtyping to predict response to NAC in muscle-invasive bladder cancers is also an area which may allow prediction of outcomes with cisplatin-based combination chemotherapy. The benefit of NAC varies between molecular subtypes (as identified by transcriptome-wide microarray analysis), with patients with basal tumors achieving the best outcomes in a recent investigation: luminal/TCGA cluster I tumors could not be improved with NAC while claudin-low tumors appeared resistant to cisplatin-based chemotherapy [Seiler et al. 2017].

Immune-delivery of cytotoxic chemotherapy

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Antibodies can be used for the targeted delivery of chemotherapeutic molecules to tumor cells in the form of antibody-drug conjugates (ADCs); the monoclonal antibody targets a tumor antigen and cleavage of the linker leads to internalization and liberation of the cytotoxic component. Promising results from early phase studies have been seen with some ADCs in advanced UC, which are therefore in further evaluation. Enfortumab vedotin (ASG-22ME) is an ADC comprising a fully human antibody targeting nectin-4 and the potent microtubule-disrupting agent monomethyl auristatin E (MMAE). Moderate-to-strong staining for nectin-4 has been observed in 60% of bladder tumor specimens, with positive pre-clinical results in vitro and in vivo in several cancer models [Challita-Eid et al. 2016]. Initial interim clinical results show encouraging antitumor activity in patients with metastatic UC treated with enfortumab vedotin [Rosenberg et al. 2016b]. ASG-15ME is an ADC comprising an antibody targeting the bladder tumor antigen SLITRK6 and MMAE. It is in phase 1 investigation as monotherapy in patients with metastatic UC, with positive results from an exploratory analysis reported [Petrylak et al. 2016]. In addition, sacituzumab govitecan (IMMU-132), an ADC comprising an anti-TROP-2 antibody and the active metabolite of irinotecan (SN-38). TROP2 is a cell-surface receptor over-expressed by many human tumors, including those of the urinary bladder [Stepan et al. 2011], and in 6 patients with metastatic, platinum-resistant UC, 3 had a clinically significant response to sacituzumab govitecan [Faltas et al. 2016].

Conclusions Currently, platinum-based chemotherapy is standard for first-line treatment of metastatic UC; this has been the case for some time despite its limitations and attempts to re-imagine platinumcontaining management strategies. Although treatment options after failure of platinum-based therapy are improving, they remain suboptimal; new approaches are therefore needed. Novel biomarkers may allow rational patient selection and the range of targeted agents being investigated is promising, with particular interest in those targeting FGFR3. Despite promise in other areas, the validity of EGFR and HER2 alterations as predictive biomarkers for patient selection remains unclear in UC management. Reconceptualizing chemotherapy is also an area in which targeting of patients to maximize outcomes is being evaluated. The variety of novel therapies in development for UC and progress in biomarker-driven approaches is encouraging for enabling future shifts in the treatment paradigm to optimize individual patient management.

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Acknowledgements JER and GI thank Daniel Salamon for his writing assistance. Funding JER and GI are supported in part by P30 CA008748

Disclosures JER: Consultant for Agensys, Seattle Genetics, Roche/Genentech, Sanofi, BMS, Merck, Bayer, Eli Lilly, AstraZeneca, QED Therapeutics. Holds stock in Illumina. GI: None

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Suggested figures and table [Copyright may be required for use] Figure 1. Immunotherapeutic targets in urothelial malignancies [Gupta et al. 2017]

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Figure 2. Characterization of muscle-invasive bladder cancer [Robertson et al. 2017]

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Figure 3. Application and performance characteristics of the COXEN algorithm for prediction of drug sensitivity in the BLA-40 human urothelial cancer cell lines [Lee et al. 2007]

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Figure 4 FGFR pathway and dysregulation in cancer [Touat et al. 2015]

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Annals of Oncology Review Article – Second Draft

Table 1. Biomarkers of interest in clinical studies of approved agents

Page 27 of 27

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Annals of Oncology Review Article – Second Draft

Tablew 1. Biomarkers of interest in clinical studies of approved agents

Atezolizumab

Study

Biomarkers

Rosenberg et al.

• • • • • • •

PD-L1 status Gene expression Mutation detection Mutation load PD-L1 status Gene expression Mutation load

• • •

Elevated PD-L1 expression did not correlate with objective response rate Response higher in TCGA luminal cluster II subtype Mutation load significantly increased in responders

• • •

Objective responses occurred across all PD-L1 subgroups Responses more frequent in TCGA luminal II subtype Mutation load associated with overall survival

•

PD-L1 expression

• •

Responses in patients with PD-L1–positive and PD-L1–negative tumours at all prespecified PD-L1 expression-level thresholds Trends to higher response rate and longer survival with PD-L1–positive tumors

2016a

Balar 2017

Avelumab

Apolo 2017

Key outcome

Durvalumab

Powles 2017

•

PD-L1 expression

•

Durable response observed regardless of PD-L1 expression

Pembrolizumab

Plimack 2017

•

PD-L1 status

•

PD-L1 positivity seemed to be crucial for detecting responders

Bellmunt 2017

•

PD-L1 status

•

Benefit independent of PD-L1 expression

Balar 2017b

•

PD-L1 expression

•

Responses were observed across all categories of PD-L1 expression

Sharma 2016b

•

PD-L1 expression

•

PD-L1 expression did not correlate with objective responses

Sharma 2017

• •

PD-L1 expression Gene expression

•

Objective responses across PD-L1 subgroups compared favorably with the 10% historical objective response High interferon-γ gene expression and urothelial carcinoma molecular subtype were associated with response CXCL9, CXCL10, CD8, and 12-chemokine signature were highly enriched in responders

Nivolumab

• •

Page 1 of 2

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Annals of Oncology Review Article – Second Draft

Page 2 of 2

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Suggested figures [Copyright may be required for use] Figure 1. Immunotherapeutic targets in urothelial malignancies [Gupta et al. 2017]

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Figure 2. Characterization of muscle-invasive bladder cancer [Robertson et al. 2017]

Alteration landscape for 412 primary tumors. Top to bottom: synonymous and non-synonymous somatic mutation rates, with one ultra-mutated sample with a POLE signature. Mutational signature (MSig) cluster, APOBEC mutation load, and neoantigen load by quartile. Normalized activity of 4 mutational signatures. Combined tumor stage (T1,2 versus T3,4) and node status, papillary histology, gender, and squamous histology. Somatic mutations for significantly mutated genes (SMGs) with frequency R7%. Copy number alterations for selected genes and FGFR3 and PPARG gene fusions.

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Figure 3. Application and performance characteristics of the COXEN algorithm for prediction of drug sensitivity in the BLA-40 human urothelial cancer cell lines [Lee et al. 2007]

Step 1. Experimentally determine the drug's pattern of activity in cells of set 1. Step 2. Experimentally measure molecular characteristics of the cells in set 1. Step 3. Select a subset of those molecular characteristics that most accurately predicts the drug's activity in cell set 1 (“chemosensitivity signature” selection). Step 4. Experimentally measure the same molecular characteristics of the cells in set 2. Step 5. Among the molecular characteristics selected in step 3, identify a subset that shows a strong pattern of co-expression extrapolation between cell sets 1 and 2. Step 6. Use a multivariate algorithm to predict the drug's activity in set 2 cells on the basis of the drug's activity pattern in set 1 and the molecular characteristics of set 2 selected in step 5. The output of the multivariate analysis is a COXEN score.

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Figure 4 FGFR pathway and dysregulation in cancer [Touat et al. 2015]

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