Targeted therapies in urothelial carcinoma

Abstract

Purpose of review

Greater understanding of the biology and genetics of urothelial carcinoma is helping to identify and define the role of molecules and pathways appropriate for novel-targeted therapies. Here, we review the targeted therapies that have been reported or are in ongoing urothelial carcinoma clinical trials, and highlight molecular targets characterized in preclinical and clinical studies.

Recent findings

Trials in nonmuscle-invasive bladder cancer are evaluating the role of immunotherapy and agents targeting vascular endothelial growth factor (VEGF) or fibroblast growth factor receptor-3. In muscle-invasive bladder cancer, neoadjuvant studies have focused on combining VEGF agents with chemotherapy; adjuvant studies are testing vaccines and agents targeting the human epidermal growth factor receptor 2, p53, and Hsp27. In the first-line treatment of metastatic urothelial carcinoma, tubulin, cytotoxic T-lymphocyte antigen 4, Hsp27, and p53 are novel targets in clinical trials. The majority of targeted agents studied in urothelial carcinoma are in the second-line setting; new targets include CD105, polo-like kinase-1, phosphatidylinositide 3-kinases (PI3K), transforming growth factor β receptor/activin receptor-like kinase β, estrogen receptor, and the hepatocyte growth factor receptor (HGFR or MET).

Summary

Development of targeted therapies for urothelial carcinoma is still in early stages, consequently there have been no major therapeutic advances to date. However, greater understanding of urothelial carcinoma and solid tumor biology has resulted in a proliferation of clinical trials that could lead to significant advances in treatment strategies.

INTRODUCTION

In the United States in 2013, there were an estimated 72500 new cases of, and 15 210 deaths from, urothelial carcinoma. Although urothelial carcinoma is considered chemotherapy-sensitive, very few patients with metastatic disease are cured. Patients with a good prognosis can expect a median survival of 12–14 months [1]. Approximately 70–80% of urothelial carcinoma is nonmuscle-invasive bladder cancer (NMIBC); the remaining 20–30% is muscle-invasive bladder cancer (MIBC) or metastatic. Almost 70% of patients with NMIBC will have disease recurrence within 2 years of diagnosis. Chemotherapy regimens for MIBC and first-line metastatic urothelial carcinoma contain cisplatin, including methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC), dose-dense MVAC, and gemcitabine and cisplatin. However, there is currently no standard second-line therapy for urothelial carcinoma [2,3^■,4]. Several trials evaluating single chemotherapy agents, such as docetaxel, pemetrexed, gemcitabine, and paclitaxel have shown response rates of 9–27%, with 2–3-month progression-free survival (PFS) and no improvement in overall survival (OS). Multidrug regimens have shown higher response rates, but no OS benefit [5–9].

A better understanding of the tumor biology and molecular patterns of urothelial carcinoma has led to clinical trials of several targeted therapies, including agents targeting angiogenesis, epidermal growth factor receptor (EGFR), and immunomodulatory agents. Research in immunotherapy and vaccines for urothelial carcinoma is in very early stages. Multiple tumor-associated antigens for vaccine targeting have been identified, but data on these agents are limited to safety, as their efficacy has not been demonstrated. Still, many targeted agents have been studied in all stages of urothelial carcinoma and many more are in ongoing clinical trials (Table 1).

Table 1.

Targeted agents studied in urothelial carcinoma

Agent	Target	Agent	Target
Angiogenesis inhibitors		EGFR inhibitors
Aflibercept	VEGF, PDGF	Cetuximab	EGFR
Bevacizumab	VEGF	Erlotinib	EGFR
Cabozantinib	VEGFR/cMET	Gefitinib	EGFR
Everolimus	PI3K/Akt/mTOR	Lapatinib	HER2/EGFR
Lenalidomide	Apoptosis, antiangiogenesis, immune modulation	Trastuzumab	HER2
Pazopanib	VEGFR1/2/3, PDGFR a/b, c-Kit	Panitumumab	EGFR
PF-03446962	TGFbR/ALK1	Vandetanib	VEGFR, EGFR, RET
rAD-IFN/Syn3	Antiangiogenesis and immune modulation
Ramucirumab	VEGFR2	Hormone-associated
Sorafenib	B-Raf, c-Raf, VEGFR-2/3, PDGFR-b	AEZS-108	LHRH linked to doxorubicin
Sunitinib	VEGFR-1/2, C-KIT, PDGFR a/b, FLT3, RET	Tamoxifen	ER-B
Temsirolimus	mTOR
TRC105	CD105
Trebananib	Ang1, Ang2
Immune therapy		Other targets
ALT-801	p53	Apaziquone	Mitomycin-C analogue, apoptosis
ASG-15ME	SLITRK6	BKM120	PI3K
CDX-1307	Dendritic-cell vaccine hCG-B	Bortezomib	Proteasome inhibitor
Dendritic-cell vaccine	Ad/HER2/neu	Dasatinib	BCR/ABL and Src
DN24–02	HER2 vaccine	Dovitinib	FGFR3
DTA-H19/PEI	DNA plasmid, DT-A gene, H19 transcription factors	Eribulin	Tubulin
HS-410	gp96-chaperoned peptides	Lonafarnib	Farnesyl transferase
Ipilimumab	CTLA-4	OGX-427	HSP27
PANVAC	MUC-1, CEA	Oportuzumab monatox	EpCAM
recMAGE-A3 þ AS15	MAGE-A3 vaccine	Valproic acid	Thrombospondin-1
Rituximab	CD20	Volasertib	Polo-like kinase 1
TMX-101	TLR-7	Vorinostat (SAHA)	SAHA: histone deacetylase

CTLA-4, cytotoxic T-lymphocyte antigen 4; DT-A, diphtheria toxin A; EGFR, epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; ER, estrogen receptor; FGFR, fibroblast growth factor receptor; hCG, human chorionic gonadotropin; HER, human epidermal growth factor receptor; HSP27, heat shock protein 27; LHRH, leutinizing hormone-releasing hormone; mTOR, mammalian target of rapamycin; P13K, phosphatidylinositide 3-kinases; PR, progesterone receptor; SAHA, suberanilohydroxamic acid; SLITRK6, SLIT and TRK-like family member 6; TLR, toll-like receptor; VEGFR, vascular endothelial growth factor receptor

TARGETED AGENTS IN NONMUSCLE-INVASIVE BLADDER CANCER

The current standard of care for patients with newly diagnosed high-grade NMIBC is transurethral resection, followed by intravesical Bacillus Calmette-Guerin (BCG) over 6 weeks, followed by maintenance therapy. BCG has an initial failure rate of approximately 35% in terms of disease recurrence or progression. For the 20–35% of patients who fail an initial induction of BCG, a second 6-week cycle of BCG may be beneficial. However, patients who fail two courses often proceed to radical cystectomy [10,11]. The majority of targeted therapies for NMIBC have been studied in patients who have failed intravesical BCG therapy.

Reported studies of targeted agents in nonmuscle-invasive bladder cancer

Several clinical trials using targeted therapy in NMIBC have been reported (Table 2a) [12,13,14^■■,15]. Two were phase I dose-escalation studies using intravesical oportuzumab monatox [13] and TMX-101 [14^■■]. Preliminary results from these trials show that the drugs are well tolerated, with no grade III-V adverse events. One phase II study was in patients who had failed BCG treatment and who were then treated with intravesical oportuzumab monatox targeting epithelial cell adhesion (EpCAM) [15]. A complete response was seen in 20 patients (44%) at the 3-month evaluation. In another phase II trial evaluating the use of intravesical apaziquone in the setting of intermediate-risk and high-risk NMIBC, the observed recurrence rate at 1 year was 34.7% [12]. The clinical impact of these early-phase trials will require further investigation.

Table 2.

Reported and ongoing trials of targeted agents in nonmuscle-invasive bladder cancer

Table 2a. Reported trials of targeted agents in NMIBC
Author	Year	Agent	Target	Intravesical therapy	Study type	n	RR %
Kowalski [15]	2010	Oportuzumab monatox	EpCAM	None	Phase I	64	NR
Hendricksen [12]	2012	Apaziquone	Mitomycin-C analogue, apoptosis	Apaziquone	Phase II	53	NR
Falke [14■■]	2012	TMX-101	TLR-7	None	Phase I	16	NR
Kowalski [13]	2012	Oportuzumab monatox	EpCAM	None	Two-arm, phase II	46	44
Table 2b. Ongoing trials of targeted agents in nonmuscle-invasive bladder cancer
PI	Start year	Agent	Target	Intravesicaltherapy	Study type	Target enrollment	Clinicaltrials.gov ID
Lamm [17]	2008	DTA-H19/PEI	DNA plasmid, DT-A gene, H19 transcription factors	None	Phase IIb intermediate risk with prior intravesical induction	47	NCT00595088
Weizer [18]	2009	Sunitinib	VEGFR-1/2, C-KIT, PDGFR a/b, FLT3, RET	BCG	Phase II high grade	36	NCT00794950
Dalbagni [19]	2010	Everolimus	PI3K/Akt/mTOR	Gemcitabine	Phase I/II BCG failure	45	NCT01259063
Fishman [20]	2011	Lenalidomide	Apoptosis, antiangiogenesis, and immune modulation	BCG	Phase II with prior BCG induction	70	NCT01373294
Fishman [16]	2011	Rituximab	CD20	BCG	Phase II high grade	70	NCT01373294
Sun [21]	2011	Apaziquone	Mitomycin-C analogue, apoptosis	None	Randomized phase III low risk	658	NCT01469221
Dinney [22]	2012	rAD-IFN/Syn3	Antiangiogenesis and immune modulation	None	Randomized phase II high-grade BCG failure	40	NCT01687244
Rosser [23]	2012	ALT-801	p53	Gemcitabine	Phase Ib/II BCG failure	52	NCT01625260
Jichlinski [24]	2012	recMAGE-A3+AS-15	MAGE-A3 vaccine	BCG	Phase I high grade	24	NCT01498172
Shapiro [25]	2012	Valproic acid	Thrombospondin-1	BCG/MMC	Phase 0	50	NCT01738815
Lamm [26]	2013	TMX-101	TLR-7	None	Phase II with CIS	12	NCT01731652
Hahn [27]	2013	Dovitinib	FGFR3	None	Phase II BCG failure with FGFR3 mutation overexpression	50	NCT01732107
Steinberg [28]	2013	HS-410	gp96-chaperoned peptides	BCG	Phase I/II high grade with prior BCG induction phase I/II high grade with prior BCG induction	93	NCT02010203
Agarwal [29]	2013	PANVAC	MUC-1, CEA	BCG	Randomized phase II BCG failure	54	NCT02015104

BCG, Bacillus Calmette-Guerin; CIS, carcinoma in situ; EpCAM, epithelial cell adhesion molecule; FGFR, fibroblast growth factor receptor; MMC, mitomycin C; mTOR, mammalian target of rapamycin; n, number of patients enrolled; NR, not reported; PDGFR, platelet-derived growth factor receptor; PI, principal investigator; RR, response rate; TLR, toll-like receptor; VEGFR, vascular endothelial growth factor receptor.

Ongoing studies of targeted agents in nonmuscle-invasive bladder cancer

Targeted agents being studied in high-grade NMIBC include sunitinib (VEGFR-1,2 inhibitor), lenalido-mide (immunomodulatory, antiangiogenic, and cytotoxic), recombinant melanoma-associated antigen (recMAGE)-A3 + AS-15 (vaccine MAGE), and rituximab (CD20 antibody) in combination with intravesical BCG (Table 2b) [16–29]. TMX-101 (toll-like receptor 7 agonist) is also being evaluated as a first-line intravesical therapy in patients with NMIBC with carcinoma in situ. Two clinical trials are evaluating apaziquone (cytotoxic) and DTA-H19/PEI (H19 transcription factor) in low-risk and intermediate-risk NMIBC, respectively. A phase 0 study is analyzing the use of valproic acid in the 4 weeks between tumor resection and initiation of intravesical therapy. Four ongoing trials in BCG-refractory patients are assessing ALT-801 (Altor Bioscience) [interleukin 2 (IL-2)/T-cell receptor fusion protein that targets p53] combined with intravesical gemcitabine, everolimus [mammalian target of rapamycin (mTOR) inhibitor] in combination with intravesical gemcitabine, recombinant adenovirus mediated interferon (antiangiogenic and immunomodulatory), and dovitinib [fibroblast growth factor receptor 3 (FGFR-3) inhibitor] in tumors positive for FGFR-3 mutation or overexpression. HS-410 (Heat Biologics) is a clone of live urothelial carcinoma cells from a single tumor source that continuously secretes gp96, a chaperone protein. In turn, gp96 chaperones tumor antigens to T cells designed to induce a pan-tumor antigen immune response mediated by T cells. This is now being evaluated in a phase I/II study. The National Cancer Institute has initiated a study with an induction course of intravesical BCG in combination with a ‘priming vaccine’ called PANVAC [PAN (all) VAC (vaccine)], a subcutaneous immunomodulatory vaccine, in patients who have failed prior BCG therapy. PANVAC is a poxviral cancer vaccine that has demonstrated therapeutic efficacy against a variety of carcinomas. PANVAC consists of a primary vaccination with a replication-competent recombinant vacciniavector, followed by multiple boosts with a replication-incompetent recombinant fowlpox (rF) vector. These vectors contain transgenes for both human T-cell costimulatory molecules and tumor-associated antigens against mucin 1 (MUC-1) [30,31] and (carcinoembryonic antigen) CEA [32]. MUC-1 and CEA are expressed in approximately 93 and 76% of high-grade bladder tumors, respectively.

TARGETED AGENTS IN MUSCLE-INVASIVE BLADDER CANCER

The primary therapy for MIBC is radical cystectomy and lymph node dissection. In randomized phase III trials, cisplatin-based neoadjuvant chemotherapy has demonstrated a survival benefit [33–35] over cystectomy alone [36,37] by increasing the rate at which MIBC tumors (stage T2-T4a) are downstaged to NMIBC tumors (< T2). Several targeted agents for MIBC have been reported and are currently under investigation (Table 3). Trials in the neoadjuvant setting have focused on combining VEGF agents with cisplatin-based chemotherapy. The data for adjuvant chemotherapy in MIBC are less compelling than for neoadjuvant chemotherapy, and there are limited data in the setting of persistent disease after cisplatin-based neoadjuvant chemotherapy, making the adjuvant setting ideal for novel-targeted agents. Ongoing or planned adjuvant studies are testing several vaccines and agents targeting human epidermal growth factor receptor 2 (HER2), p53, and Hsp27.

Table 3.

Reported and ongoing trials of targeted agents in muscle-invasive bladder cancer

Table 3a. Reported trials of targeted agents with chemotherapy in muscle-invasive bladder cancer
Author	Year	Agent	Target	Chemotherapy	Study type	n	Path <T2 RR%
Chaudhary [40]	2011	Bevacizumab	VEGF	Gemcitabine and cisplatin	Phase II neoadjuvant	15	31
Siefker-Radtke [38]	2012	Bevacizumab	VEGF	Dose-dense MVAC	Phase II neoadjuvant	60	53
Balar [41]	2012	Sunitinib	VEGFR-1/2, C-KIT, PDGFR a/b, FLT3, RET	Gemcitabine and cisplatin	Phase II neoadjuvant	18	33
Galsky [39]	2013	Sunitinib	VEGFR-1/2, C-KIT, PDGFR a/b, FLT3, RET	Gemcitabine and cisplatin	Phase II neoadjuvant	9	22
Table 3b. Reported trials of targeted agents as single-agent therapy in muscle-invasive bladder cancer
Author	Year	Agent	Target	Chemotherapy	Study type	n	Path <T2 RR%
Pruthi [42]	2010	Erlotinib	EGFR	None	Phase II neoadjuvant	20	35
Hahn [43]	2012	Dasatinib	BCR/ABL and Src	None	Phase II neoadjuvant	25	14
Table 3c. Ongoing trials of targeted agents with chemotherapy in muscle-invasive bladder cancer
PI/group	Start Year	Agent	Target	Chemotherapy	Study type	Target enrollment	Clinicaltrials.gov ID
Michaelson RTOG [54]	2005	Trastuzumab	HER2	Paclitaxel and radiation	Phase I/II adjuvant	88	NCT00238420
Kraft [52]	2005	Bevacizumab	VEGF	Neoadjuvant gemcitabine and cisplatin adjuvant paclitaxel	Phase II neoadjuvant and adjuvant	25	NCT00268450
Bradley [55]	2010	CDX-1307	Dendritic-cell vaccine hCG-B	Gemcitabine and cisplatin	Phase II neoadjuvant and adjuvant	30	NCT01094496
Wong [56]	2011	ALT-801	p53	Gemcitabine and cisplatin; gemcitabine alone	Phase Ib/II neoadjuvant or metastatic	76	NCT01326871
Table 3d. Ongoing trials of targeted agents as single therapy in muscle-invasive bladder cancer
PI/group	Start Year	Agent	Target	Chemotherapy	Study type	Target enrollment	Clinicaltrials.gov ID
So [57]	2009	OGX-427	Hsp27	None	Phase I neoadjuvant	36	NCT00959868
Bajorin [58]	2011	DN24–02	HER-2 vaccine	None	Randomized phase II adjuvant	180	NCT01353222
Mulders [59]	2011	recMAGE-A3þAS-15	MAGE-A3 vaccine	None	Randomized phase II adjuvant	273	NCT01435356
Wood [53]	2013	Dendritic-cell vaccine	Ad/HER2/neu	None	Phase I adjuvant	65	NCT01730118

EGFR, epidermal growth factor receptor; HER, human epidermal growth factor receptor; n, number of patients enrolled; PI, principal investigator; RR, pathologic response rate to <pT2; VEGF, vascular endothelial growth factor.

Reported studies of targeted agents in muscle-invasive bladder cancer

Several clinical trials of targeted agents in MIBC have been reported (Table 3a and 3b) [38–43]. Two studies have used tyrosine kinase inhibitors (TKIs) as monotherapy in the neoadjuvant setting. Studies of erlotinib, which targets EGFR [44], and dasatinib, which targets breakpoint cluster region/abelson murine leukemia [45], reported pathologic response rates (RRs) to less than pT2 of 35 and 14%, respectively. Two studies used bevacizumab (VEGF inhibitor and monoclonal antibody) with chemotherapy. One of those studies used dose-dense MVAC and reported a pathologic RR (< pT2) of 53% [38]. Two studies used sunitinib with chemotherapy in the neoadjuvant setting. One study was stopped early due to excess toxicity. The second study did not report excess toxicity, although cisplatin was split between days 1 and 8. Nevertheless, this study was stopped early as well due to lack of interest from the sponsoring drug company [46].

Ongoing studies of targeted agents in muscle-invasive bladder cancer

The neoadjuvant setting is ideal for studying targeted therapy, as tissue can be analyzed for changes in protein expression and tumor morphology before and after treatment. Vaccine immunotherapy is a promising therapeutic approach for patients with MIBC, especially in the adjuvant setting wherein patients are at high risk of recurrence and have limited therapeutic choices after neoadjuvant chemotherapy. Agents being studied in the neoadjuvant and adjuvant settings are described in Table 3c and 3d [47,48,49^■,50–59].

TARGETED AGENTS IN METASTATIC UROTHELIAL CARCINOMA

Although first-line treatment of metastatic urothelial carcinoma with cisplatin-based chemotherapy may have RRs as high as 70%, duration of response is short (median PFS is about 7 months) [60], highlighting the urgent need to improve treatment options in this setting. Multiple targeted agents used alone or in combination with chemotherapy have been studied in the first-line setting (Table 4a and 4b) [39,45,61,62^■■,63–69]. Cumulative toxicities in a patient population that is frail at baseline have limited the success of first-line combination studies with targeted agents, particularly TKIs. Nevertheless, there are many ongoing trials of targeted agents as first-line therapy with chemotherapy in metastatic bladder cancer for cisplatin eligible (Table 4c) [70–87] and cisplatin ineligible (Table 4d). In the second-line setting, vinflunine, a microtubule-inhibiting vinca alkaloid, has been extensively investigated and was approved as a second-line therapy by the European Medicines Agency based on a modest survival benefit compared with best supportive care (6.9 vs. 4.3 months, P = 0.04) [88]. In the United States, where the majority of targeted agents are studied (Table 5), there is no approved second-line therapy for metastatic urothelial carcinoma.

Table 4.

Reported and ongoing trials of targeted agents as first-line therapy in metastatic bladder cancer

Table 4a. Reported trials of targeted agents with chemotherapy as first-line therapy in metastatic bladder cancer
Author	Year	Agent	Target	Chemotherapy	Study type	n	RR%	OS (months)
Hussain [63]	2007	Trastuzumab	HER2	Paclitaxel, gemcitabine, carboplatin	Phase II	44	70	14.1
Philips [64]	2009	Gefitinib	EGFR	Gemcitabine and cisplatin	Phase II	54	43	15.1
Hahn [45]	2011	Bevacizumab	VEGF	Gemcitabine and cisplatin	Phase II	43	72	19.1
Menhert [65]	2011	Sorafenib	B-Raf,c-Raf, VEGFR-2/3, PDGFR-b	Gemcitabine and cisplatin	Phase II	17	30	NR
Krege [66]	2011	Sorafenib	B-Raf,c-Raf, VEGFR-2/3, PDGFR-b	Gemcitabine and cisplatin	Phase II	40	53	11.3
Grivas [67]	2012	Cetuximab	EGFR	Gemcitabine and cisplatin + cetuximab; gemcitabine and cisplatin alone	Randomized phase II	88	57	14
Balar [62■■]	2013	Bevacizumab	VEGF	Gemcitabine and carboplatin	Randomized phase II (1 : 2)	51	63	14
Galsky [39]	2013	Sunitinib	VEGFR-1/2, C-KIT, PDGFR a/b,FLT3, RET	Gemcitabine and cisplatin	Phase II	36	49	13.9
Table 4b. Reported trials of targeted agents as single-agent first-line therapy in metastatic bladder cancer
Author	Year	Agent	Target	Chemotherapy	Study type	n	RR%	OS (months)
Bellmunt [68]	2010	Sunitinib	VEGFR-1/2, C-KIT, PDGFR a/b, FLT3, RET	None	Phase II	38	8	8.1
Quinn [61]	2010	Eribulin	Tubulin	None	Phase II	37	38	9.4
Sridhar [69]	2011	Sorafenib	B-Raf,c-Raf, VEGFR-2/3, PDGFR-b	None	Phase II	17	0	5.9

Table 4c. Ongoing trials of targeted agents as first-line therapy with chemotherapy in metastatic bladder cancer
PI/group	Start year	Agent	Target	Chemotherapy	Study type	Target enrollment	Clinicaltrials.gov ID
Hoffman [73]	2001	Trastuzumab	HER2	Gemcitabine and cisplatin	Phase II	13	NCT02006667
Oudard [74]	2004	Trastuzumab	HER2	Gemcitabine and cisplatin or carboplatin	Randomized phase II	61	NCT01828736
Chester [75]	2008	Temsirolimus	mTOR	Gemcitabine and cisplatin	Phase I/II	99	NCT01090466
EORTC [76]	2008	Lapatinib	HER2/EGFR	Gemcitabine and cisplatin	Phase I	25	NCT00623064
Rosenberg [77]	2009	Bevacizumab	VEGF	Gemcitabine and cisplatin	Randomized phase III	500	NCT00942331
Eisai INC [78]	2010	Eribulin	Tubulin	Gemcitabine and cisplatin	Phase Ib/II	95	NCT01126749
Miller [79]	2010	Panitumumab	EGFR	Gemcitabine and cisplatin	Randomized phase II	124	NCT01374789
Salvioni [80]	2010	Sorafenib	B-Raf,c-Raf, VEGFR-2/3, PDGFR-b	Gemcitabine and cisplatin	Phase II	45	NCT01222676
Bajorin [81]	2010	Everolimus	PI3K/Akt/mTOR	Gemcitabine and split-dose cisplatin	Phase I	30	NCT01182168
Petrylak [71]	2011	OGX-427	HSP27	Gemcitabine and cisplatin	Randomized phase II	180	NCT01454089
Wong [82]	2011	ALT-801	p53	Gemcitabine and cisplatin	Phase I/II	76	NCT01326871
Galsky [72]	2012	Ipilimumab	CTLA-4	Gemcitabine and cisplatin	Phase II	36	NCT01524991
Ullen [83]	2012	Sorafenib	B-Raf,c-Raf, VEGFR-2/3, PDGFR-b	Vinflunine	Phase I	24	NCT01844947
Flag [84]	2013	Trebananib	Ang1, Ang2	Docetaxel	Phase II	38	NCT01907308
Table 4d. Ongoing trials of targeted agents for cisplatin-ineligible patients as first-line therapy in metastatic bladder cancer
PI/group	Start year	Agent	Target	Chemotherapy	Study type	Target enrollment	Clinicaltrials.gov ID
Oudard [74]	2004	Trastuzumab	HER2	Gemcitabine and cisplatin or carboplatina	Randomized phase II	61	NCT01828736
Jones [70]	2010	Lapatinib	HER2/EGFR	Gemcitabine and carboplatin	Phase II	122	NCT01191892
Galsky [85]	2010	Everolimus	PI3K/Akt/mTOR	Paclitaxel	Phase II	68	NCT01215136
Apolo [86]	2011	Lenalidomide	Apoptosis, antiangiogenesis and immune modulation	Gemcitabine and carboplatin	Phase I	42	NCT01352962
Bajorin [87]	2012	Pazopanib	VEGFR1/2/3, PDGFR a/b,c-Kit	Gemcitabine	Phase II	45	NCT01622660

^aChemotherapy is gemcitabine and cisplatin or gemcitabine and carboplatin based on creatinine clearance.

EGFR, epidermal growth factor receptor; EORTC, European Organisation for Research and Treatment of Cancer; HER, human epidermal growth factor receptor; mTOR, mammalian target of rapamycin; n, number of patients enrolled; NR, not reported; OS, overall survival; PDGFR, platelet-derived growth factor receptor; PI, principal investigator; RR, response rate; SLITRK6, SLIT and TRK-like family member 6; VEGFR, vascular endothelial growth factor receptor.

Table 5.

Reported and ongoing trials of targeted agents as second-line therapy in metastatic bladder cancer

Table 5a. Reported trials of targeted agents with chemotherapy as second-line therapy in metastatic bladder cancer
Author	Year	Agent	Target	Chemotherapy	Study type	n	RR%	OS (months)
Theodore [93]	2005	Lonafarnib	Farnesyltransferase	Gemcitabine	Phase II	31	32	NR
Choueiri [91]	2012	Vandetanib	VEGFR, EGFR, RET	Docetaxel and vandetanib	Randomized phase II	142	7	5.56
				Docetaxel and placebo			11	7.39
Wong [92]	2012	Cetuximab	EGFR	Pactilaxel and cetuximab alone	Randomized phase II	39	25	10.5
							0	1.3
Table 5b. Reported trials of targeted agents with single-agent second-line therapy in metastatic bladder cancer
Author	Year	Agent	Target	Chemotherapy	Study type	n	RR%	OS (months)
Gomez-Abuin [94]	2007	Bortezomib	Proteasome inhibitor	None	Phase II	20	0	NR
Rosenberg [96]	2008	Bortezomib	Proteasome inhibitor	None	Phase II	25	0	5.7
Wulfing [97]	2009	Lapatinib	HER2/EGFR	None	Phase II	59	3	4.5
Petrylak [98]	2009	Gefitinib	EGFR	None	Phase II	31	3	3
Dreicer [90]	2009	Sorafenib	B-Raf,c-Raf, VEGFR-2/3, PDGFR-b	None	Phase II	27	0	6.8
Gallagher [89]	2010	Sunitinib	VEGFR-1/2, C-KIT, PDGFR a/b, FLT3, RET	None	Phase II	45	7	6.9
Twardowski [99]	2010	Aflibercept	VEGF, PDGF	None	Phase II	22	4.5	NR
Cheung [100]	2010	Vorinostat	SAHA: histone deacetylase	None	Phase II	14	0	4.3
Stadler [95]	2011	Volasertib	Polo-like kinase 1	None	Phase II	31	19	NR
Milowsky [101]	2011	Everolimus	PI3K/Akt/mTOR	None	Phase II	45	5	10.5
Pili [102]	2011	Pazopanib	VEGFR1/2/3, PDGFR a/b,c-Kit	None	Phase II	19	0	NR
Necchi [103]	2012	Pazopanib	VEGFR1/2/3, PDGFR a/b,c-Kit	None	Phase II	41	17	4.7
Lerner [104]	2012	Tamoxifen	ER-B	None	Phase II	28	NR	NR
Milowsky [105]	2013	Dovitinib	FGFR3	None	Phase II	44	0	NR

Table 5c. Ongoing trials of targeted agents with chemotherapy as second-line therapy in metastatic bladder cancer
PI/group	Start year	Agent	Target	Chemotherapy	Study type	Target enrollment	Clinicaltrials.gov ID
Quinn [107]	2011	Lapatinib	HER2/EGFR	Docetaxel	Phase II	40	NCT01382706
ImClone [109]	2011	Ramucirumab	VEGFR2	Docetaxel	Three-arm randomized phase II	138	NCT01282463
Otto [110]	2011	Pazopanib	VEGFR1/2/3, PDGFR a/b,c-Kit	Vinflunine	Phase I/II	NR	NCT01265940
Hahn [111]	2013	OGX-427	Hsp27	Docetaxel	Randomized phase II	200	NCT01780545
Table 5d. Ongoing trials of targeted agents as single-agent second-line therapy in metastatic bladder cancer
PI/group	Start year	Agent	Target	Chemotherapy	Study type	Target enrollment	Clinicaltrials.gov ID
Hoffman [112]	2001	Trastuzumab	HER2	None	Phase II	5	NCT02013765
Quinn [113]	2006	Eribulin	Tubulin	None	Phase I/II, renal dysfunction	82	NCT00365157
Fowl es [106]	2009	Lapatinib	HER2/EGFR	None	Randomized phase II/III	204	NCT00949455
Fernandez [114]	2010	AEZS-108	LHRH linked to doxorubicin	None	Phase I/II	64	NCT01234519
Apolo [115]	2011	TRC105	CD 105	None	Phase II	45	NCT01328574
Bajorin [108]	2012	BKM120	PI3K	None	Phase II	35	NCT01551030
Apolo [116]	2012	Cabozantinib	VEGFR/cMET	None	Phase II	55	NCT01688999
Necchi [117]	2012	PF-03446962	TGFβR/ALKl	None	Phase II	45	NCT01620970
Wood [53]	2013	Dendritic-cell vaccine	Ad/HER2/neu	None	Phase I	65	NCT01730118
Agensys [118]	2013	ASG-15ME	SLITRK6	None	Phase I	45	NCT01963052

ALK1, activin receptor-like kinase 1; EGFR, epidermal growth factor receptor; ER, estrogen receptor; FGFR, fibroblast growth factor receptor; HER, human epidermal growth factor receptor; mTOR, mammalian target of rapamycin; n, number of patients enrolled; NR, not reported; OS, overall survival; PDGFR, platelet-derived growth factor receptor; PI, principal investigator; RR, response rate; SAHA, suberanilohydroxamic acid; TGFβR, transforming growth factor β receptor; VEGFR, vascular endothelial growth factor receptor.

Reported studies of targeted agents as first-line therapy in metastatic urothelial carcinoma

From 2007 to 2013, eight trials were reported using targeted agents in combination with chemotherapy for first-line treatment of metastatic urothelial carcinoma (Table 4a). The agents studied included antiangiogenic agents (bevacizumab, sunitinib, and sorafenib) and EGFR-targeted agents (trastuzumab, gefitinib, and cetuximab). The addition of targeted agents to standard platinum-based regimens (with the exception of bevacizumab) has not demonstrated an improvement in OS compared with historical data. Three trials of targeted single agents as first-line treatment of metastatic urothelial carcinoma have been reported since 2010 (Table 4b). Single-agent sunitinib in cisplatin-ineligible patients and single-agent sorafenib had less than 10% RRs in the first-line setting [89,90]. Single-agent eribulin (microtubule inhibitor) reported a RR of 38% in the first-line setting [61].

Reported and ongoing studies of targeted agents as second-line therapy in metastatic urothelial carcinoma

Patients who progress after platinum-based combination therapy have urothelial carcinoma tumors that are often resistant to other available therapies. In these patients, tumor shrinkage with targeted single agents has been low in reported studies with the majority of RR of less than 10% (Table 5b) [89–105]. Similarly, the combination of targeted agents with chemotherapy has not shown significant improved RRs in this setting (Table 5a) [91–93]. A randomized phase II study of docetaxel and vandetanib (EGFR, VEGFR, and RET-TKI) compared with docetaxel alone did not show significant improvement in PFS, RR, or OS. Among 37 patients who crossed over to single-agent vandetanib, the overall RR was 3% [91]. A randomized phase II study of cetuximab (EGFR inhibitor) alone or in combination with paclitaxel reported a RR of 0% for cetuximab alone and 25% for the combination [92]. Even with the low activity of targeted agents reported in the second-line setting, the number of ongoing studies continues to rapidly grow (Table 5c and 5d) [53,106–118].

ANGIOGENESIS INHIBITION IN UROTHELIAL CARCINOMA

VEGF, one of the most important factors in angiogenesis, is increased in the tumor tissue, serum, and urine of patients with urothelial carcinoma and correlates with tumor stage and survival [9,119–121]. Higher levels of VEGF in the tumors and serum of patients with urothelial carcinoma correlate with worse prognosis and higher likelihood of disease recurrence. Angiogenesis inhibitors have been shown in vitro to be active in urothelial carcinoma cells. VEGF signaling occurs through binding of its receptors VEGFR1 (fms-like tyrosine kinase-1) and VEGFR2 (fetal liver kinase-1). In a preclinical study, six of 13 urothelial carcinoma cell lines examined expressed VEGFR2. Further analysis of the T24 uro-thelial carcinoma cell line revealed a functional autocrine loop involving VEGF and VEGFR2 [122]. One study revealed that VEGF and VEGFR1 expression was higher in NMIBC compared with MIBC specimens (P < 0.001), possibly due to a higher rate of cell growth in early stages of disease, whereas VEGFR2 was significantly higher in MIBC, as compared with NMIBC (P< 0.001) [123^■■]. VEGFR inhibition has been shown to decrease proliferation and invasion of urothelial carcinoma cells, leading to the investigation of VEGF-targeted agents in urothelial carcinoma.

Bevacizumab in urothelial carcinoma

Phase II trials of bevacizumab in combination with chemotherapy have shown promising results in urothelial carcinoma compared with historical data. A phase II study of first-line gemcitabine, cisplatin, and bevacizumab showed a RR of 72% and an OS of 19.1 months in metastatic urothelial carcinoma [45]. Another phase II study of first-line gemcitabine, carboplatin, and bevacizumab in cisplatin-ineligible patients with metastatic urothelial carcinoma showed a RR of 49% and an OS of 13.9 months [62^■■]. This study compared survival in patients who received bevacizumab with gemcitabine and carbo-platin to a contemporary control population that received gemcitabine and carboplatin alone during the same time the study was conducted. The study group that received bevacizumab had an OS of 13.9 vs. 10.3 months in the contemporary population that did not receive bevacizumab. A third phase II study of neoadjuvant dose-dense MVAC and bevacizumab in MIBC had a pathologic RR of 53% [38], which is higher than the pathologic RR reported with neoadjuvant MVAC. An ongoing study in MIBC of neoadjuvant gemcitabine and cisplatin with bevacizumab followed by adjuvant paclitaxel with bevacizumab has completed accrual [52]. An ongoing Cancer And Leukemia Group B/Alliance phase III study of first-line gemcitabine and cisplatin with or without bevacizumab in patients with meta-static urothelial carcinoma is nearing its anticipated accrual of 500 patients.

Sunitinib in urothelial carcinoma

Sunitinib, an oral multiple-receptor TKI approved for use in many types of solid tumors, was one of the first targeted agents to be studied in advanced urothelial carcinoma. Sunitinib targets VEGF receptors, stem cell factor receptor (KIT), platelet-derived growth factor receptor, RET, Fms-like tyrosine kinase 3, and colony-stimulating factor 1 receptor. It has been shown to be active against urothelial carcinoma cells in vitro and in vivo [124,125]. A phase II second-line study of 77 patients with urothelial carcinoma showed radiographic tumor regression and a clinical benefit (partial response and stable disease) of 43% with sunitinib [89]. However, subsequent studies of sunitinib, including a first-line combination study, two neoadjuvant combinations studies [39], and a maintenance study [46], showed low responses and high toxicity with combined sunitinib and chemotherapy. There is an ongoing study of sunitinib administered with BCG in high-grade NMIBC, but no ongoing studies in MIBC or metastatic urothelial carcinoma. However, there are multiple ongoing studies with other VEGF TKIs, including sorafenib, pazopanib, and cabozantinib in urothelial carcinoma.

Epidermal growth factor receptor/human epidermal growth factor receptor 2 in urothelial carcinoma

EGFR plays a significant role in mediating angiogenesis by regulating the activity of IL-8, VEGF, and basic fibroblast growth factor. Overexpression of EGFR leads to activation of the Ras signaling cascade, which leads to cell proliferation. Similar to VEGFR, EGFR is overexpressed in urothelial carcinoma and correlates with tumor stage, grade, prognosis, and survival [126]. Gefitinib, a selective EGFR inhibitor, was approved by the US Food and Drug Administration (FDA) for use in nonsmall cell lung cancer in 2003. Gefitinib has the potential to be effective in other solid tumors with increased EGFR expression, such as urothelial carcinoma. However, a phase II trial of gefitinib added to standard first-line gemcitabine and cisplatin showed no improvement in OS [127]. Gefitinib and lapatinib (dual TKI-targeting HER2/neu and EGFR) have been tested as single agents in the second-line treatment of metastatic urothelial carcinoma, and both agents demonstrated RRs of less than 5% [97,98].

HER2/neu is the most studied of the four receptors in the EGFR family. HER2/neu regulates multiple processes, including cell proliferation. HER2/neu⁺ urothelial carcinoma patients have more advanced disease and more sites of metastasis [63]. HER2/neu⁺ patients with MIBC have a lower median OS than HER2/neu⁻ patients (33 vs. 50 months, respectively). However, results have been mixed, as a different study found that overexpression of HER2/neu in the primary tumor or any metastatic tumors was not predictive of survival [128]. A phase II study of trastuzumab (HER2/neu receptor-targeted monoclonal antibody), paclitaxel, carboplatin, and gemcitabine in HER2⁺ urothelial carcinoma showed an OS of 14.1 months [63]. Trastuzumab is being evaluated in combination with paclitaxel and radiotherapy for bladder conservation. Multiple ongoing trials are testing the HER2 axis in metastatic urothelial carcinoma, including a study of first-line lapatinib in combination with gemcitabine and cisplatin [70], a randomized phase II/III trial of second-line, single-agent lapatinib [106], a combination study of second-line lapatinib and docetaxel [107], and a vaccine study in HER2⁺ urothelial carcinoma [53].

NOVEL PATHWAYS AND AGENTS BEING STUDIED IN UROTHELIAL CARCINOMA

Multiple novel pathways and molecules that could potentially be targeted in urothelial carcinoma are being explored in preclinical and clinical studies; some of these include multiple receptor tyrosine kinases, MET, phosphatidylinositide 3-kinases (PI3K), heat shock proteins (HSPs), cytotoxic T-lymphocyte antigen 4 (CTLA-4), and prostate stem cell antigen (PSCA).

Cabozantinib in urothelial carcinoma

Cabozantinib, an oral agent that primarily targets MET and VEGFR2, has been shown to prevent angiogenesis and invasive growth by inhibiting hepato-cyte growth factor (HGF)-induced MET signaling in urothelial carcinoma cell lines. MET, a proto-oncogene that encodes for the HGF receptor [129^■■], is usually expressed by cells of epithelial origin. Activation of the MET receptorbyHGF leads to down-stream activation of multiple cytokines involved in motility, matrix remodeling, angiogenesis, survival, and proliferation leading to invasive growth of cancer cells [130]. Soluble urinary MET levels are elevated in urothelial carcinoma and correlate with disease stage. Plasma HGF levels are frequently elevated in advanced urologic malignancies [131].

Urothelial carcinoma cell lines respond to HGF stimulation with increased activation of established intracellular signaling mediators [132]. HGF stimulation also increases invasion, growth rate, and anchorage-independent growth [133]. Cabozantinib reverses these HGF-driven activities and has shown activity in multiple solid tumors in phase I, II, and III clinical trials [134,135]. The efficacy of cabozantinib in advanced urothelial carcinoma is being studied for the first time in an ongoing phase II trial [136], wherein patients receive cabozantinib (60 mg/day) continuously in 28-day cycles. The study has three cohorts: metastatic urothelial carcinoma with measurable disease, bone-only metastatic urothelial carcinoma, and metastatic rare histology of the bladder, urethra, ureter, or renal pelvis.

Buparlisib (BKM120) in advanced urothelial carcinoma

PI3Ks are enzymes involved in regulating multiple cellular functions, including growth, proliferation, motility, and differentiation. PI3K mutations are found in urothelial carcinoma, and this pathway is a potential therapeutic target. BKM120, a PI3K inhibitor, has shown activity in multiple solid tumors [137,138] and is currently undergoing phase III testing in breast cancer. There is also an ongoing phase II study of BKM120 in urothelial carcinoma [108], with the primary objective of PFS.

Apatorsen (OGX-427) in metastatic urothelial carcinoma

HSPs regulate the folding and unfolding of other proteins and are induced by exposure to elevated temperature or stress. OGX-427, an inhibitor of HSP27, inhibits apoptosis via cell death and proteo-toxic stress pathways [47]. Ongoing studies include a multicenter randomized phase II trial [71] of first-line OGX-427 and gemcitabine and cisplatin vs. placebo in advanced urothelial carcinoma, and a multicenter randomized phase II study of second-line OGX-427 and docetaxel vs. docetaxel alone [139].

Ipilimumab in metastatic urothelial carcinoma

Ipilimumab is an inhibitor of CTLA-4, a protein receptor on the surface of T cells that regulates T-cell activity by transmitting inhibitory signals. CD28, a costimulatory protein expressed on T cells, transmits stimulatory signals to T cells. Activation of T-cell receptors via CD28 leads to increased CTLA-4 expression. Ipilimumab interrupts the inhibitory process that prevents cytotoxic T lymphocytes from destroying tumor cells. By interrupting the inhibition of T cells mediated by CTLA-4, cytotoxic T lymphocytes are able to recognize and destroy tumor cells [72]. A multicenter phase II trial of ipilimumab with gemcitabine and cisplatin [140] is ongoing in patients with metastatic urothelial carcinoma. In this trial, patients with stable or responding disease continue ipilimumab maintenance therapy after completing six cycles of chemotherapy. There are also several ongoing checkpoint inhibitor phase I studies with bladder cancer expansion cohorts. These studies will help assess the activity of immune checkpoint blockade in metastatic urothelial carcinoma.

Prostate stem cell antigen as a therapeutic target in urothelial carcinoma

PSCA is a novel therapeutic target for urothelial carcinoma in carriers of the rs2294008-T allele [141]. A genetic variant (rs2294008) discovered by urothelial carcinoma genome-wide association studies is a strong predictor of PSCA protein expression in bladder tumors [142^■]. Carriers of the T allele have been shown to be at higher risk of urothelial carcinoma [143], and urothelial carcinoma patients with the T allele have higher expression of PSCA mRNA and protein in tumors. The T allele predicts PSCA expression in tumors of all stages and tumor grades. Therefore, PSCA may be a promising therapeutic target, and genetic testing may be useful in selecting patients for treatment. An anti-PSCA monoclonal antibody is currently being tested in clinical trials [144,145].

THE FUTURE OF TARGETED THERAPIES FOR ADVANCED UROTHELIAL CARCINOMA

Targeted therapies currently being studied in advanced urothelial carcinoma have shown efficacy in other solid tumors. Cancer therapy continues to transition from cytotoxic to targeted therapies. There is a lot of genetic diversity and heterogeneity within tumor types, consequently, specific subsets of patients may have a greater benefit from targeted therapies. In addition, tumor cells can adapt to targeted therapies by developing resistance, usually through mutations in target enzymes and cell-death pathways, or by developing new methods of drug efflux. Therefore, researchers are developing even more selective targets, identified in individual tumors through genomic analysis.

The Bladder Cancer Analysis Working Group of The Cancer Genome Atlas (TCGA) analyzed 131 samples of MIBC and identified 32 significantly mutated genes, the most common of which were P53 (49%), MLL2 (27%), ARID1A (25%), KDM6A (24%), and PIK3CA (20%). The majority (69%) of tumors were found to harbor potential therapeutic targets with a mean and median somatic mutation rate per tumor of 7.7 and 5.5 per megabase, respectively. The analysis also showed that mutations in chromatin-modifying genes were enriched, and that the chromatin-modifying genes MLL2, ARID1A, and KDM6A were inactivated at high frequency in uro-thelial carcinoma. Significantly, ERBB2 (Her-2/neu) was mutated or amplified in 9% of samples, suggesting a possible role for ERBB2 as a therapeutic target in bladder cancer using similar treatment strategies that target ERBB2 amplified breast cancers [146^■■].

Genomic sequencing studies may also be useful in the clinical setting by revealing new potential drug targets as well as mutations that may predict responsiveness to specific targeted therapies. A study that analyzed 97 high-grade bladder tumors identified several genomic alterations in molecular pathways that could be potential drug targets. In this study, 61% of the tumors analyzed had potential actionable drug targets due to mutations in the RTK-RAS-RAF and PI3K/AKT/mTOR pathways. Tumors with high burden of DNA copy number alterations were found to have more mutations in TP53 and RB1 [147^■■]. Another study analyzed 13 bladder cancer patients treated with everolimus, a mTOR-targeted agent. TSC1 (tuberous sclerosis complex 1), which codes for TSC1 protein, a regulator of mTOR signaling, has been shown to be mutated in some bladder cancers [148]. In this study, two of three patients with nonsense mutations in TSC1 gene had some degree of response to everolimus, one patient with missense mutation in TSC1, which is of unknown functional significance, had some tumor response to everolimus, and eight of nine patients with progressive disease were TSC1 wild-type [149^■].

The data obtained from these studies can help target therapies more specifically to tumors based on a large panel of mutational analysis and possibly predict responsiveness to targeted therapies based on the presence of specific mutations - a possible future direction for targeted therapy for urothelial carcinoma.

CONCLUSION

Targeting molecular aberrations has revolutionized the clinical management of multiple cancers. Significant progress has been made in the last few years in testing targeted agents in all stages of urothelial carcinoma, from NMIBC to advanced refractory metastatic urothelial carcinoma. However, clinical trial results of targeted therapies in urothelial carcinoma in unselected patient populations have been negative so far. Therefore, there have been no reported phase III studies nor FDA-approved targeted therapeutic agents for use in urothelial carcinoma. The focus of treatment for NMIBC in ongoing clinical trials is enhancing antitumor immunity with immunotherapeutic agents alone or with concurrent intravesical BCG therapy. Combination trials of TKIs and cytotoxic chemotherapy in MIBC and first-line metastatic urothelial carcinoma have reported significant toxicities. However, targeting angiogenesis via either the VEGF or mTOR pathways has demonstrated activity in a small number of urothelial carcinoma patients [148]. Studies of genomic alterations confirm that urothelial carcinoma is a heterogeneous disease driven by a large array of somatic mutations that may predict responsiveness to specific targeted therapies. In the future, improved understanding of the biology and genetics of urothelial carcinoma may lead to personalized therapies for specific molecular targets that will improve therapeutic outcomes.

KEY POINTS.

• A growing number of targeted agents are being studied in all stages of urothelial carcinoma, especially in the second-line setting.

• Clinical trials of targeted therapies for urothelial carcinoma in an unselected patient population have been negative so far, although agents targeting angiogenesis appear to have some activity in a small number of patients.

• For NMIBC, most ongoing trials are focusing on antitumor immunity using immunotherapeutic agents alone or with concurrent intravesical BCG.

• Combination trials of TKIs and cytotoxic chemotherapy in MIBC and first-line metastatic urothelial carcinoma have reported significant toxicities.

• Genomic studies reveal that urothelial carcinoma is a heterogeneous disease with a large number of somatic mutations that may provide targets for therapy and predict response to specific targeted agents.