Molecular targets on the horizon for kidney and urothelial cancer

NATURE REVIEWS | CLINICAL ONCOLOGY ADVANCE ONLINE PUBLICATION | 1

Dana-Farber and Brigham and Women’s Cancer Center, Harvard Medical School, 450 Brookline Avenue, Boston, MA 02215, USA and Hospital del Mar-IMIM-IMAS, Doctor Aiguader 88, 08003 Barcelona, Spain (J. Bellmunt). National Cancer Centre Singapore, National Cancer Centre, 11 Hospital Drive, Singapore 169610, Singapore (B. T. Teh). Medical Oncology, Azienda Ospedaliera Universitaria Integrata, University of Verona, Piazzale Luduvico Antonio Scuro 10, 37134 Verona, Italy (G. Tortora). Non-Prostate Genitourinary Malignancies, Memorial Sloan-Kettering Cancer Center, Weill-Cornell Medical College, 1275 York Avenue, New York, NY 10065, USA (J. E. Rosenberg).

Correspondence to: J. Bellmunt [email protected]

Molecular targets on the horizon for kidney and urothelial cancerJoaquim Bellmunt, Bin T. Teh, Giampaolo Tortora and Jonathan E. Rosenberg

Abstract | As whole-genome sequencing technology rapidly advances, the insights gained from deciphering cancer genomes are shifting the paradigm in the diagnosis and treatment of cancer with the promise of individualized treatment for each patient. Information gained in this way is extensive for certain cancers, but fairly limited in renal cell carcinomas and urothelial carcinoma. Mutations in multiple, potentially druggable genes have been identified in urothelial carcinomas; however, the association between molecular alterations and clinical outcome has not yet been robustly demonstrated. Data in this area are emerging in renal cell carcinoma, leading to the development of targeted agents that have improved overall survival. Unfortunately, these treatments rarely yield complete responses, are not curative, and development of resistance ensues. This Review will focus on the biology of non-hormonally driven urological cancers. We discuss how approaches using whole-genome sequencing can facilitate the discovery of biomarkers of drug sensitivity in both renal cell carcinomas and urothelial carcinomas. For renal cell carcinomas, we will describe how genomic and epigenomic mining has uncovered novel genes and pathways involved in tumorigenesis, tumour classification and mechanisms of resistance in the various subsets of this disease and the potential for exploiting these discoveries in the clinic.

Bellmunt, J. et al. Nat. Rev. Clin. Oncol. advance online publication 27 August 2013; doi:10.1038/nrclinonc.2013.155

IntroductionRenal cell carcinomas (RCCs) account for over 80% of renal malignancies. More importantly, the incidence of RCC has increased at a rate of 2.6% annually in the USA since 1997, and only a part of this increase can be explained by increased detection.1 Approximately 2% of RCCs are associated with inherited syndromes, with the remaining 98% occurring sporadically. The most- common forms of sporadic RCC can be classified into three main subtypes: clear cell, papillary and chromo-phobe RCC. Surgery is currently the primary treatment for RCC. However, one third of patients who undergo surgical resection have a recurrence.1 Accurately deter-mining the risk of relapse after nephrectomy is a critical issue for RCC treatment.

Adjuvant therapy is continuously evolving, with advances in novel markers and therapeutic targets. Before 2005, patients with RCC who received immuno-modulatory therapy or traditional chemotherapy showed modest survival benefits, at the expense of considerable adverse effects. Since then, seven targeted therapeutic agents—bevacizumab, sorafenib, sunitinib, pazopanib, temsirolimus, axitinib and everolimus—have been approved by the FDA for the treatment of RCC. Genomic

classification of the tumour allows clinicians to determine the risk–benefit ratio for adjuvant therapy in patients with RCC.

RCC is heterogeneous and each histological subtype shows variations in genetics and behaviour. Recent large-scale studies have provided insight into pathways and mechanisms that contribute to the pathogenesis of RCC. Yet, despite our understanding of the molecu-lar character istics, personalized and tumour-specific t reatment has yet to be fully realized in RCC.

Whereas many common malignancies, such as breast, prostate and lung cancer, have been extensively studied using genomic approaches, urothelial carcinoma remains fairly understudied, despite its high prevalence.2,3 Urothelial carcinoma—also called transitional cell carci-noma, most commonly of the bladder—is the fourth leading cause of new cancers in men in the USA, and accounts for approximately 15,000 deaths per year in the USA.1 For patients with muscle-invasive disease, 5-year survival is below 50%.4,5 Both genetic mutations and epi-genetic events are important in urothelial cancer patho-genesis and prognosis, and environmental triggers such as Schistosoma infection and tobacco have been identified.6–8 Although many patients’ tumours recur in the urinary tract only, this relapsing non-invasive disease is usually not life-threatening. By contrast, other patients develop invasive and/or metastatic disease, the lethal phenotype of urothelial carcinoma.

Our understanding of the molecular changes in urothelial carcinoma has rapidly evolved over the past

Competing interests J. Bellmunt declares an association with the following companies: Novartis, OncoGenex, Pfizer. J. E. Rosenberg declares an association with the following companies: Boehringer-Ingelheim, Bristol–Myers Squibb, Dendreon, OncoGenex. See the article online for full details of the relationships. The other authors declare no competing interests.

REVIEWS

© 2013 Macmillan Publishers Limited. All rights reserved

mailto:[email protected]

mailto:[email protected]

http://www.nature.com/doifinder/10.1038/nrclinonc.2013.155

2 | ADVANCE ONLINE PUBLICATION www.nature.com/nrclinonc

few decades; however, the therapeutic arsenal has not. First-line treatment for advanced-stage disease is platinum-based combination chemotherapy, and no FDA-approved second-line treatment exists. Attempts to improve current therapies have focused on cytotoxic chemotherapy dose intensity and combination doublet and triplet regimens, none of which has led to substantial improvements in survival. Metastatic urothelial carcinoma remains incurable in the vast majority of patients, with a median survival of approximately 8 months without treat-ment, and 14 months with treatment.9 Unlike treatment of other solid tumours, targeted therapies have failed to advance the standard of care for urothelial carcinoma. Furthermore, there is no clear molecular understanding of what defines the lethal phenotype of urothelial carci-noma. Thus, there is an urgent need for new biomarkers and treatment approaches.

Currently, urothelial carcinoma prognosis is primar-ily determined by grade and stage. For patients with metastatic disease, clinical prognostic variables such as performance status, visceral metastases, haemo globin level, or liver metastases are able to identify broad risk groups.10,11 However, the variability of outcomes even within these groups is significant, and the ability to accu-rately prognosti cate is limited. Although multiple potential tissue-based biomarkers have been proposed in urothelial carcinoma, none so far has been validated.12

In this Review, we will describe how genomic and epi-genomic mining has uncovered novel genes and pathways involved in genomic regulation, classification and mecha-nisms of resistance in the various subtypes of RCC and the potential for these discoveries in the clinic. Focusing on the biology of non-hormonally driven urological cancers, since the biology of prostate cancer is quite different and centres on hormonal axes, we will discusses how recent approaches using whole-genome sequencing has facili-tated the discovery of biomarkers of drug sensitivity in both RCC and urothelial tumours.

Molecular targets for RCCGenetics of clear cell RCCVHL and hypoxia-inducible factorIn the early 1990s, a germline mutation in the VHL gene, a tumour-suppressor gene located on the distal tip of the short arm of chromosome 3, was shown to cause von Hippel-Lindau (VHL) disease.13 This autosomal domi nantly inherited neoplastic disorder is associated

with the development of clear-cell renal cell carcinoma (ccRCC) and a range of other tumours, including retinal and CNS haemangioblastomas, phaeochromo-cytoma, pancreatic islet tumours and endolymphatic sac tumours.14 In individuals with VHL disease, the germline mutation in VHL is coupled with a deletion of the short arm of chromosome 3, deleting the remaining wild-type VHL allele. Inactivation of the VHL gene is also associated with the majority of sporadic ccRCCs.14 The product of VHL, pVHL, is the recognition component of an ubiqui tin ligase complex that facilitates degradation of many cellular proteins, including the α-subunit of the hypoxia- inducible factor (HIF) transcription factor, which mediates the cellu lar response to hypoxia.15 Deregulation of these key hypoxia and angiogenesis mediators is observed in both ccRCC tumours in VHL disease and sporadic ccRCC tumours that arise due to somatic VHL mutations, indi-cating that functional inactivation of these genes is a domi-nant feature of ccRCC.15 However, VHL mutational status has not been shown to correlate with clinical outcome in this disease.16,17

HIFs form heterodimeric complexes, composed of an O2-labile α-subunit and a stable β-subunit, which are instrumental in the adaptation of cancer cells to hypoxic tumour microenvironments; two paralogues of HIFα subunits exist in cells: HIF-1α and HIF-2α.18 Differences between these two isoforms are central to RCC biology and the development of novel therapeutics. Both proteins and their downstream targets are the focus of targeted therapy in ccRCC.18 HIF-1α is located on chromosome 14q, a region that is commonly deleted in ccRCC, and loss of this region is associated with poorer outcomes in RCC.19 However, most human cells express both HIF-1α and HIF-2α and evidence indicates that HIF-2α is the critical driver in pVHL-defective ccRCC.20–23 Although both para-logues activate proangiogenic factors, they differ markedly in their ability to promote tumour growth, and also inhibit expression of one another. In vivo animal experiments support HIF-2α as the main driver of tumour growth in RCC,24 and HIF-1α has been considered to be a tumour suppressor in this disease.20 Short-hairpin RNA-mediated knockdown of HIF-2α in human RCC cells (engineered 786-O-derived clones) is sufficient to prevent tumour growth in nude mice xenograft assays.25 Stable expres-sion of HIF-2α, but not HIF-1α, is necessary to restore the tumorigenic phenotype of VHL-reconstituted 786-O cells.26 Furthermore, HIF-2α but not HIF-1α activates Oct-4, a transcription factor critical for stem cell growth.27 Similarly, HIF-2α promotes MYC activation, and HIF-1α opposes it.22,28

Genome-wide association studies have also identified two single-nucleotide polymorphisms (SNPs) associated with RCC susceptibility. These SNPs map to the gene EPAS1 on chromosome 2p21, which encodes HIF-2α.11 These observations are consistent with the unique and sometimes opposing activities of these HIFα isoforms. However, there is also evidence that HIF-1α rather than HIF-2α is more important for tumour development. Fu et al.29 found that HIF-1α transgenic mice develop renal cancers whereas HIF-2α transgenic mice did not. Based

Key points

■ Whole-genome sequencing is helping to facilitate the discovery of biomarkers of drug sensitivity in both renal cell carcinoma (RCC) and urothelial tumours

■ Urothelial carcinomas contain mutations in multiple genes that are potentially druggable, although clinical data supporting the use of agents targeting these mutations in this disease is still in its infancy

■ Some data linking molecular alterations and clinical outcome is emerging in RCC; overall survival in RCC has improved and patients are being treated for increasingly longer periods of time

■ Genomic and epigenomic mining in RCC has uncovered novel genes and pathways involved in tumorigenesis, genomic regulation, tumour classification and mechanisms of resistance in the various forms of RCC

REVIEWS



on these observations, the researchers argue against the idea that HIF-2α activation is critical for ccRCC tumori-genesis. A number of other tumour models also implicate HIF-1α as an oncoprotein and not a tumour-suppressor protein, adding to the controversy surrounding the role of HIF in RCC.30–32

The downstream targets of pVHL are being evaluated as novel therapeutic targets for ccRCC. In one example, a high-throughput chemical synthetic-lethal screen identi-fied three compounds that selectively kill ccRCC by target ing glucose uptake through the HIF-target GLUT-1 (Solute carrier family 2, facilitated glucose transporter member 1).33 Treatment with these chemical agents inhib-ited the growth of ccRCCs in vivo by binding GLUT-1 directly and impeding glucose uptake without toxicity to normal tissue.33 Therefore, there is still a need for thera-peutic agents that directly target HIF-2α and a number of HIF-2α inhibitors are now in preclinical developmental.34

SWI/SNF complex and the ubiquitin pathwayNext-generation sequencing efforts have uncovered several additional recurrent gene mutations associated with ccRCC. Recurrent mutations have been identified in PBRM1,35 SETD2, KDM5C,36 BAP1 and ARID1A.37,38 ccRCC mutation rates in these genes vary in frequency and the aggressiveness of the tumours that harbour these mutations are also variable (Table 1).

After VHL mutations, PBRM1 mutations are the second-most-frequent mutation found in ccRCC tumours.35,37 Mutations in PBRM1 are observed in 41% of ccRCCs; however, more than 85% of these mutations are indels or nonsense mutations.35,37 In a cohort of 117 Asian patients who developed ccRCC, 30% of the tumour samples were found to harbour PBRM1 mutations (B. Teh, unpublished data). PBRM1 encodes protein polybromo- 1, the chromatin-remodelling subunit of the SWI/SNF chromatin-remodelling complex, which is implicated in transcription, DNA replication, DNA repair, and control of proliferation and differentiation.39 Generally, SWI/SNF activity is thought to protect against tumour progres-sion,40 but few studies have analysed the role of SWI/SNF in tumour invasion and metastasis. Interestingly, PBRM1 maps to chromosome 3p and is located within the region commonly deleted in ccRCC.41

Mutations in ARID1A, a gene that encodes ARID1A protein—another member of the SWI/SNF complex—are found in 3% of ccRCC tumours, and 60–70% of ccRCC tumours exhibit significantly lower ARID1A mRNA and protein-expression levels than the matched normal kidney cortex.42 ARID1A protein associates with protein polybromo-1 and BAF250B (AT-rich interactive domain-containing protein 1B) to form the PBAF subunit of the SWI/SNF chromatin-remodelling complex.42 ARID1A mutations have previously been linked to ovarian clear cell carcinoma, occurring at a frequency of almost 50% in this malignancy.43

Whole-exome sequencing has identified 23 genes mutated at a high frequency in ccRCC, including genes encoding proteins involved in the ubiquitin- mediated proteo lysis pathway.44 Defects in this pathway

are associ ated with overexpression of HIF proteins, even in the absence of VHL mutations.38 A positive correla-tion was found between the alterations in the ubiquitin– proteasome pathway and overexpression of HIF-1α and HIF-2α in ccRCC tumours, suggesting that alterations in this pathway might contribute to ccRCC via HIF-1α.44 In these exome-sequencing studies, the genes found to be mutated in ccRCC were VHL (altered in 27% of tumour samples), BAP1 (altered in 8-15%), CUL7 (altered in 3%), and BTRC (altered in 2%).38,44

BAP1, located in close proximity to VHL within the commonly lost short arm of chromosome 3, was originally discovered as a gene that codes for ubiquitin carboxyl- terminal hydrolase BAP1 (BAP1), an inter action partner of BRCA1, which regulates the DNA damage response.38 BAP1 also binds to transcription factor host cell factor 1 (HCF1) via a UCH37-like domain and facilitates the formation of complexes between histone modifiers and transcription factors and regulates cell-cycle progres-sion.45 In addition to the interactions with BRCA1 and HCF1, BAP1 also interacts with ASXL1 to form the poly-comb repressive deubiquitinase complex (PR-DUB). This complex deubiqui nates histone H2A and represses HOX gene expression.46 BAP1 has been identified as a tumour suppressor in ccRCC, requiring loss or inactivation of both copies of the gene.38 In addition, BAP1 loss is associ-ated with high tumour grade in ccRCC and activation of mTOR.38 This association might have potential predictive value for the response to targeted therapies.

Mutations in BAP1 and PBRM1 tend to be mutually exclusive in ccRCC.37 In a retrospective analysis of 145 patients with primary ccRCC, overall survival was found to be significantly shorter for patients with BAP1-mutant tumours than for patients with PBRM1-mutant tumours.37 The worst overall survival was observed in an extremely small subset of patients whose tumours harboured muta-tions in both PBRM1 and BAP1. In the two cohorts of patients included in this study, patients with mutations in both PBRM1 and BAP1 had a median overall survival of 2.1 years (95% CI 0.3–3.8) and 0.2 years (95% CI 0.0–1.2). Clinically, tumours with mutations in PBRM1 or BAP1 rep-resent two biologically distinct entities with distinct prog-noses. Molecular classifi cation of tumours based on the presence or absence of the BAP1 and/or PBMR1 m utations might lead to a new generation of targeted therapies.

Table 1 | Recurrent ccRCC gene mutations and their impact on tumour prognosis

Gene RCC mutation frequency (%)

Impact on prognosis

Reference

VHL 55 No correlation Yao et al. (2002)16

PBRM1 41 Poor Varela et al. (2011)35

KDM5C 9 ND Dalgliesh et al. (2010)36

BAP1 8 Poor Kapur et al. (2013)37 and Peña-Llopis et al. (2012)38

SETD2 4 ND Dalgliesh et al. (2010)36

ARID1A 3 Poor Kapur et al. (2013)37 and Peña-Llopis et al. (2012)38

Abbreviations: ccRCC, clear-cell renal cell carcinoma; ND, not determined; VHL, von Hippel-Lindau.

REVIEWS



Histone modificationSeveral genes associated with histone modification, including the histone methylases SETD2, MLL, MLL2, and MLL4 and the histone demethylases JARID1C, JARID1D, and UTX, are implicated in ccRCC, with mutations in these genes found in 1–4% of ccRCC tumours.36 The MLL, MLL2, and MLL4 gene loci are large and it is possible that mutations in these genes are ‘passenger’ mutations that arise owing to random background mutation frequency. In VHL-deficient ccRCC cells, histone H3 lysine 4 tri-methylation (H3K4Me3) levels are decreased through increased JARID1C activity.47 SETD2 encodes a histone H3 lysine methyltransferase that maps to chromosome 3p21.3, a region noted in loss-of-heterozygosity studies as being associated with development of ccRCC.48 As such, most renal tumours are haploinsufficient for SETD2 and have decreased expression of SETD2.48 Mutations found in either SET2 or JARID1C in ccRCC lead to decreased expression of the respective gene compared to normal kidney tissue.48 Furthermore, multiregional genetic analy-sis has revealed that SETD2 and JARID1C harbour multi-ple distinct and spatially separated inactivating mutations within a single tumour, supporting their potential roles as tumour suppressors in ccRCC.48

Genetics of papillary RCCPapillary RCC (pRCC) occurs in about 10–15% of RCC patients.49 Based on histological criteria, sporadic pRCCs are divided into type 1 and type 2.50 Type 1 pRCCs are fairly indolent and are associated with overall survival rates of approximately 90%. By contrast, up to 50% of i ndividuals with type 2 pRCC die within 10 years of diagnosis.51

Hereditary type 1 pRCC is associated with germline missense MET mutations, and overexpression of MET is found in nearly all sporadic type 1 pRCC tumours.52 In addition to amplification of the MET locus, LRRK2 coding for leucine-rich repeat serine/threonine-protein kinase 2 and required for oncogenic MET signalling, is also ampli-fied and overexpressed in type 1 pRCC tumours.53 MET and LRRK2 cooperate during tumour growth via the mTOR and STAT3 pathways to promote cell growth and survival.53 LRRK2 might be a valuable therapeutic target in tumours driven by ligand-independent activation of MET.

Sporadic type 2 pRCCs do not harbour MET muta-tions and amplification of the MET oncogene is less fre-quently observed.54 Hereditary leiomyomatosis and renal cell cancer (HLRCC) occurs when individuals inherit a germline mutation in the FH gene, leading to a high fre-quency of renal tumours, uterine fibroids, and c utaneous leiomyomatosis (fibroid skin tumours).55 Mutations of the FH gene are found in over 90% of HLRCC fami-lies.56 Sequencing of the genomes of families suspected or proven to have HLRCC has identified 21 novel muta-tions in the FH gene, which results in reduced fumarate hydratase (FH) activity.57 Although FH mutations are less commonly found in sporadic type 2 pRCC tumours, genes associated with FH activity are more significantly deregu-lated in the type 2 sporadic pRCC tumours compared with other common subtypes of RCC.57,58 Activation of the

MYC oncogene, owing to the amplification of chromo-some 8q, has also been reported in a subset of samples in type 2 pRCC.58 However, despite the moderate incidence of pRCC, we have limited knowledge about the molecu-lar pathology underlying development and progression of this disease.

Importance of epigenetics and RCCAberrant DNA methylation has an important role in the development of RCC. In inherited RCC, as in other inherited cancer syndromes, de novo VHL promoter hypermethylation can provide the ‘second hit’ that initi ates tumour development, as has been observed in VHL disease.59 In sporadic cases of RCC, VHL promo-ter methyl ation has been detected in both ccRCC and pRCC.60 In addition to VHL, promoter methylation of the RASSF1 gene, also located at 3p21, has been reported in 23% of primary ccRCC tumours and 44% of pRCC tumours.61 Interestingly, RASSF1A methylation has been detected in kidney tissue surrounding excised tumours, suggesting that inactivation of this gene might have a role in early tumorigenesis.62 Other frequently methy lated genes in RCC are TU3A (42% in ccRCC, 25% in pRCC),63 FHIT (53% in both ccRCC and pRCC),62,64 and members of the WNT signalling pathway.65

Histone modifications are another major epigenetic event involved in RCC. In general, lower levels of global histone methylation and acetylation are observed in both ccRCC and pRCC. Importantly, the decrease of histone modifications is associated with higher tumour grade, poor prognosis or more-aggressive phenotype.66 A number of histone demethylases such as JARID1C/KDM5A and KDM6A/UTX, are reported to have inacti-vating mutations in ccRCC. However, these mutations do not result in decreased histone modifications, suggesting the role of these histone modifiers in RCC needs to be further investigated.36

Targeting pathways and mechanisms of resistanceMany angiogenesis-related signalling pathways, including HIF-1α, VEGF and mTOR pathways, are hyper active in RCC (Figure 1), particularly in ccRCC.15 The vast major-ity of novel therapeutic strategies are based on the target-ing of VEGF and its receptors (VEGFR-1, VEGFR-2, VEGFR-3) or of mTOR.67 Notably, VEGFRs, HIF-1α and mTOR are present not only in renal endo thelial cells, but also in tumour cells, providing the basis for dual targeting by therapeutic agents.68 Sunitinib, sorafenib, pazopanib, axitinib and tivozanib are tyrosine kinase inhibitors (TKIs) that share the ability to inhibit all three VEGFRs and have additional targets including the PDGF recep-tors,69 and everolimus and temsirolimus mainly inhibit mTOR.67,70 Bevacizumab targets the VEGF ligand, rather than the receptor.

The development of resistance to therapeutic agents represents a major problem in the management of RCC. Alterations in the previously mentioned angiogenic path-ways and in the production of alternative proangiogenic factors have a critical role in this context. Resistance can be either primary, occurring within 2–3 months from the

REVIEWS



start of treatment, or secondary occurring 6–12 months after initially successful treatment.71 Primary resistance, also defined as intrinsic resistance, is a problem affecting about 20% of patients with RCC and its causes are still under investigation.71 Key factors involved in the acquisi-tion of resistance to treatment in RCC are tumour hypoxia, production of alternative proangiogenic factors—such as IL-8, FGF, HGF or MET; and activation of alternative s ignalling pathways.72

HypoxiaHypoxia has a critical role in angiogenesis through the activation of HIF genes. In hypoxic conditions, pVHL is inactive, leading to accumulation of HIF-1α and HIF-2α proteins. In ccRCC, the same accumulation of HIF-1α and HIF-2α proteins occurs because of loss of VHL expression.73 HIF transcription factors affect an array of cellular functions, including cellular metabolism, apop-tosis, proliferation, epithelial-to-mesenchymal transition, genomic instability, and resistance to radiotherapy and chemotherapy.74 Paradoxically, an effective anti angiogenic therapy can cause collapse of the vasculature and produce hypoxic conditions, which, in turn, results in so-called ‘evasive resistance’, which might encourage tumour growth by inducing revascularization, increased invasiveness and induction of metastasis.75

Activation of alternative proangiogenic factorsSeveral proteins with proangiogenic functions, such as IL-8, FGFs, HGF and its receptor MET, might be acti-vated in RCC to bypass the blockade of the VEGFRs. In xenograft models, IL-8 has been shown to be associ ated with sunitinib resistance, and neutralization of IL-8 by a specific antibody causes 786-O ccRCC xenografts to become re-sensitized to sunitinib.76 FGF2 exerts its pro-angiogenic activity by interacting with the FGF receptors, heparan-sulfate proteoglycans, and integrins expressed on the endothelial cell surface. VEGFR-2 blockade transi ently stops tumour growth and decreases vascularity, but reacti-vation of angiogenesis can occur through FGF activation leading to tumour progression.72,77 Preclinical data show that inhibition of FGF2 recovers sensitivity to sunitinib in the presence of FGF2.77 Targeting both FGFRs and VEGFRs with drugs such as dovitinib is a strategy that is now being tested in a phase III trial conducted in the third-line setting.78

MET activating mutations and amplifications are among the most frequently genetically altered receptor tyrosine kinases (RTKs) in human cancers.73 Polymorphisms in the VEGFA gene have been associated with decreased objec-tive response rate in patients treated with pazopanib.79 Drugs targeting the MET pathway are being tested in treatment of pRCC and ccRCC.80,81

MEK

S6-kinase

VHL

Raf

EGFRMET FGFRs

MAPK

mTOR

CAIX

Ras

AKT PTEN

EGFHGF FGF

Fibroblastsand stroma

SDF1α

BEZ235PI3K/Akt inhibitors

Cell proliferationand survival

Growth factor receptors

CXCR4

Other proangiogenicfactors

IL-8PDGFTGFα

AngiopoietinFGF

TemsirolimusEverolimus

TSC2TSC1

Invasion metastasis

PI3K

PDGFRVEGFRs

Dovitinib

Dovitinib

VEGF

SunitinibPazopanibSorafenibAxitinib

Tivozanib

Bevacizumab

HIF-1 Angiogenesis

Figure 1 | Signalling pathways involved in cell growth, angiogenesis and metastasis in RCC. The inhibitors acting on single or multiple signalling proteins are shown. Abbreviations: CAIX, carbonic anhydrase IX; CXCR4, C-X-C chemokine receptor type 4; EGF, epidermal growth factor; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; HIF, hypoxia-inducible factor; IL, interleukin; PDGF, platelet-derived growth factor; PTEN, phosphatase and tensin homologue; RCC, renal cell carcinoma; SDF1α, stromal-cell-derived factor 1α; TGFα, transforming growth factor α; TSC, tuberous sclerosis protein; VHL, von Hippel-Lindau.

REVIEWS



Biomarkers predictive of efficacyA large variety of angiogenic factors and receptors, cytokines and adhesion molecules, oncogenes and other signalling proteins are currently under evaluation as potential biomarkers for use in predicting treatment response in RCC (Table 2).82 None of these are used in clinical practice yet, although further exploration may lead to clinically relevant predictive biomarkers.

Molecular targets for urothelial carcinomaGenetics of urothelial carcinomaUrothelial carcinoma is an attractive target for biomarker discovery and translational science. These tumours fre-quently recur within the bladder, and resection is routine, leading to abundant tissue available in pathology depart-ments for research purposes. Nonmuscle-invasive uro-thelial carcinoma is the predominant phenotype, affecting more than 70% of patients.83 Patients with muscle-invasive urothelial carcinoma routinely undergo radical cystec-tomy,84 and bulky tumours are frequently available for genomic analysis. Unfortunately, limited improvements in the treatment of urothelial carcinoma have been achieved in the past 20 years. The Bacillus Calmette–Guérin vaccine and intravesical chemotherapy continue to be standards of care for nonmuscle-invasive urothelial carcinoma, and cisplatin-based combination chemotherapy remains the standard of care for locally advanced-stage and metastatic urothelial carcinoma.85,86

Even though clinical and pathological variables provide certain prognostic and predictive information for chemo-therapy selection, they are not helpful at guiding the choice of the best therapeutic option for patients with urothelial

cancer. Active research is ongoing into several candidate biomarkers to define platinum resistance and sensitiv-ity and select patients who are most likely to respond to platinum- based chemotherapy.87 However, it is now becoming clear that prognostic and predictive models that are based on a single parameter are generally inadequate. To optimize treatment individualization, complex genetic signatures obtained from gene-expression microarray analysis have the potential to provide reliable prognostic and predictive value, not only for chemotherapy selection, but also for future target discovery.88

Molecular analyses have identified many altered genes that have potential as therapeutic targets in patients with advanced urothelial carcinoma (Figure 2), includ-ing PIK3CA, HER2, and FGFR3.89–92 Understanding the linkage between genotype and outcome is critical to validate therapeutic targets and advance the treatment of this disease. Targeted agents can often induce dramatic responses, but only in a small minority of patients.93 Whole-genome sequencing in those outlier patients to investigate the genetic basis of durable remissions or response failures can lead to useful and potentially trans-formative knowledge. This approach has been applied in bladder cancer to identify a previously occult biomarker of sensitivity to everolimus.94 A strategy like this can help to identify patients most likely to respond to targeted anticancer drugs and might be of use in patients with u rothelial carcinoma.

Cell-cycle deregulation frequently occurs in advanced urothelial carcinoma, with TP53 mutations observed in approximately 34% of tissue samples and RB1 mutated in 15% of samples.95 Amplification of MDM2, which encodes

Table 2 | Potential biomarkers predictive of response to treatment in RCC

Description Sample or data collection method Outcome of biomarkers evaluation

VHL and HIF deletion or inactivation

Collection and assay of tumour biopsies (frameshift, nonsense, splice and in-frame deletions or insertions)

No relationship was found between VHL deletion or inactivation and outcome of patients with RCC treated with either TKIs or mTOR inhibitors178

HIF expression

HIF levels measured in frozen tumour samples (by western blot)

Increased HIF-1α and HIF-2α expression might predict response to sunitinib179

Carbonic anhydrase IX (CAIX)

Collection and assay of tumour biopsies A potential predictive role of CAIX was not demonstrated with IL-2.180 No correlation between CAIX and outcome after treatment with TKIs and mTOR inhibitors178

VEGF and VEGFRs

Collection and assay of serum samples at regular intervals. Collection of tumour tissue, serum samples (for measurement of soluble VEGFR-2) and plasma at regular intervals

Correlation found between lower baseline levels of VEGF and response to sunitinib and between lower baseline levels of VEGFR-3 or its ligand VEGFC and longer PFS in patients treated with sunitinib.181 Decrease of soluble VEGFR-2 correlates with PFS and tumour response to pazopanib182

Cytokines and angiogenic factors

Collection of plasma samples at regular intervals. Concentrations of plasma cytokine and angiogenic factors

IL-6, IL-8, VEGF, HGF, osteopontin and TIMP1 correlated with outcome in patients receiving pazopanib. In particular, in the phase III trial, IL-6, IL-8, HGF and osteopontin correlated with PFS.183 A signature was identified using six biomarkers (soluble CAIX, osteopontin, VEGF, TRAIL, collagen V, and soluble VEGFR-2) with a strong correlation with PFS in patients treated with sorafenib184

SNPs Collection and assay of samples at regular intervals (germline DNA extracted from peripheral blood)

Combined presence of SNPs 889 and 1416 correlated with improved outcome in patients treated with sunitinib.185 SNPs in the IL-8, FGFR2, VEGFA and VEGFR-3 are associated with overall survival in patients treated with pazopanib.79 Relationship identified between SNPs in genes in the VEGF pathway and PFS and blood pressure in patients treated with axitinib186

Abbreviations: CAIX, carbonic anhydrase IX; HIF, hypoxia-inducible factor; mTOR, mammalian target of rapamycin; PFS, progression-free survival; RCC, renal cell carcinoma; SNP, single-nucleotide polymorphism; TIMP1, TIMP metallopeptidase inhibitor 1; TKI, tyrosine kinase inhibitor; TRAIL, TNF-related apoptosis inducing ligand; VHL, von Hippel-Lindau.

REVIEWS



the p53 regulatory molecule MDM2, has been found in 5% of urothelial carcinoma samples, and is mutually exclusive from TP53 mutations.95 Other members of the Rb pathway are either amplified or deleted in advanced-stage urothelial carcinoma: CDKN2A has been found to be deleted in 24% of urothelial carcinoma samples, and CCND1 and E2F3 have been found to be amplified in 14% and 21% of samples, respectively.95 Similarly, tumours con-taining MDM2 amplifications and wild-type TP53 might be targeted by inhibitors of the MDM2–p53 interaction, leading to restored p53 function.96 Inactivating mutations in genes encoding epigenomic regulatory proteins, such as ARID1A, MLL, MLL3, and KDM6A, have also been identified in urothelial carcinoma, although the functional effects of these mutations have yet to be elucidated.97

Data from The Cancer Genome Atlas Project uro-thelial carcinoma cohort are expected to provide a com-prehensive multiplatform analysis of genomic alterations in this type of cancer. This study is enrolling patients with muscle-invasive bladder urothelial carcinoma who have not received chemotherapy, and should be the most- comprehensive molecular annotation of urothelial c arcinoma to date.98

Targeting the EGFR familyTargeting EGFRThe EGFR family was one of the first treatment targets in urothelial tumours.99 EGFR is overexpressed as a function of progression in muscle-invasive urothelial carcinoma, making it an attractive therapeutic target.99 Early results for small-molecule and antibody-based inhibitors of the EGFR family showed promising anti-tumour activity in certain preclinical models through induction of tumour growth arrest and inhibition of angio genesis.100–102 Unfortunately, targeted agents against these kinases, such as trastuzumab, lapatinib, erlotinib, have demonstrated disappointing activity in the clinic for urothelial carcinoma.103 For example, gefitinib, a TKI of EGFR, was tested in 31 patients with metastatic trans itional cell carcinoma of the urothelial tract.104 Despite increased EGFR expression being detected in nearly half of the pretreatment biopsy specimens, only one patient responded to gefitinib treatment.104 A single trial of 20 patients with cT2 (muscle invasive) urothelial carcinoma treated with neoadjuvant erlotinib prior to cystectomy resulted in 25% of patients being downstaged to pT0, suggesting some activity of this strategy.105 These

VEGFR-2VEGFR-1

Ras

PIP2 PIP3

VEGF

PNDRGI

HSP27

Ge�tinibErlotinibLapatinib

HER2 HER3 HER4FGFR1

FGFR2FGFR3

AKT Gsk

PDKPI3K

PTEN

mTOR

mTORC1

mTORC2

Protein translationCell growth

mLSTB

S6K

S6

4E-BP

PRAS40Raptor

RictorSINI mTOR

mLSTB

EGFR

Apoptosis

Nucleus

Vandetanib

HER2vaccine

(DN24-02)

BKM120

TSC2

TSC1

mut

INK128

EverolimusINK128

Bevacizumab

Cetuximab

HER1

DovitinibLapatinib

OGX-427

Sora�nibSunitinibPazopanib

Figure 2 | Therapeutic targets in urothelial tumours. A number of targeted agents have already been tested, but without proven efficacy. These include sorafenib, sunitinib, pazopanib, everolimus, cetuximab, lapatinib, trastuzumab, dovitinib, and vandetanib. Testing of bevacizumab, OGX-427, everolimus, INK128, BKM120 and lapatinib is ongoing (some in selected patient subgroups). Abbreviations: 4E-BP, IF4E-binding protein; HSP27, Heat shock 27 kDa protein; mTOR, mammalian target of rapamycin; mTORC1/2, mammalian target of rapamycin complex 1/2; PDK, phosphoinositide-dependent kinase; PI3K, phosphatidylinositol-3-kinase; PRAS40, proline-rich AKT1 substrate 1 40 kDa; S6K, S6 kinase; SIN1, stress-activated protein kinase interaction protein 1; TSC1/2, tuberous sclerosis protein 1/2.

REVIEWS



small studies have not consistently shown a benefit with single-agent inhibition of EGFR.

Attempts to combine anti-EGFR agents with chemo-therapy in urothelial carcinoma have been unsuccessful, as has been observed in lung cancer.106 In a Cancer and Leukemia Group B trial, the addition of gefitinib did not improve response rate or overall survival in compari-son to historical controls of patients with advanced-stage urothelial carcinoma treated with cisplatin and gemcita bine alone.107 Cetuximab, a monoclonal anti-body targeting the extracellular domain of EGFR, has also been tested in combination with chemotherapy in urothelial tumours. In a randomized phase II trial of gemcitabine and cisplatin with or without cetuximab as a first-line treatment in patients with advanced-stage urothelial carcinoma, gemcitabine and cisplatin plus cetuximab was associated with a higher rate of adverse events and did not lead to any survival advantage over chemo therapy alone.108 It should be noted that this trial was conducted in an unselected population without any type of b iomarker-based enrichment.

In vivo studies have shown that EGFR-targeted anti-bodies have a potential synergistic effect when combined with taxanes in urothelial carcinoma.109 This observa-tion has been tested in patients in a randomized non-comparative phase II study that assessed the efficacy of cetuximab with or without paclitaxel in 36 previously treated patients with metastatic urothelial carcinoma.110 In this study, single-agent cetuximab had limited activ-ity with the arm closing after nine of the first 11 patients experienced disease progression by week 8.110 However, cetuximab seemed to augment the antitumour activity of paclitaxel. Of the 28 patients receiving the combination treatment, 12 had progression-free survival greater than 16 weeks. The overall response rate was 25% (95% CI 11%–45%) and included three complete responses and four partial responses.110

Some potential hypotheses to explain the failure of targeting EGFR in urothelial carcinoma have been pro-posed. A study using a panel of 20 human urothelial carcinoma cell lines showed that EGFR inhibitors only inhibit cell proliferation and angiogenesis in a subset of cell lines.111 Molecular profiling and biochemical analyses have linked EGFR resistance to expression of markers of the epithelial-to-mesenchymal transition, a develop mental process that has been implicated in tumour invasion and metastasis.112 Further under-standing of predictive biomarkers of EGFR targeting in urothelial carcinoma are required to guide future therapeutic developments.

HER2-directed therapyThe cell surface signalling molecule HER2 might have a role in the progression of selected urothelial tumours. The HER2 gene has been reported to be amplified in approximately 5% of tumours in urothelial carcinoma,113 although other studies indicate the frequency of amplifi-cation is even higher.114,115 The safety and efficacy of adding trastuzumab to chemotherapy has been tested in 44 patients with advanced-stage urothelial carcinoma in

a single-arm phase II study.116 Patient eligibility required overexpression of HER2, measured by immunohisto-chemistry, gene amplification and/or elevated serum HER2. A 70% objective response rate was observed with the addition of trastuzumab to paclitaxel, gemcitabine, and carboplatin chemotherapy; the regimen was gener-ally well tolerated, although low-grade cardiac toxicity was more frequent than expected.116 Another phase II study comparing platinum-based chemotherapy with or without trastuzumab in patients with HER2-positive metastatic urothelial carcinoma was underpowered and, unfortunately, no conclusions could be drawn.117

Owing to the lack of definitive studies, HER2 is still considered as a potential therapeutic target in urothelial carcinoma. Using HER2 as a target, a novel approach (based on the same platform as Sipuleucel-T in prostate cancer) is being conducted using dendritic cell immuno-therapy in the adjuvant setting in an ongoing randomized phase II study.118 This study will accrue 180 patients who have previously been treated with cisplatin neoadjuvant therapy or who were ineligible for cisplatin adjuvant chemotherapy with high-risk resected muscle-invasive or node-positive urothelial carcinoma. These patients will be randomly assigned to observation or treatment with this autologous cellular immunotherapy; the primary end point of this study is overall survival.118

Dual targeting of EGFR and HER2Both EGFR and HER2 have been implicated in the pro-gression of urothelial carcinoma, therefore, dual target-ing of these receptors would seem to be an attractive therapeutic option. Dual targeting of EGFR and HER2 has been attempted using lapatinib, an oral bifunctional EGFR and HER2 kinase inhibitor, as a second-line therapy in patients with locally advanced or metastatic urothelial carcinoma.119 In this study of 59 platinum-refractory patients, 25 patients could not be evaluated for response owing to dropout prior to the first evalu-ation. This high dropout rate was due to early disease progression before the first evaluation (13 patients), serious adverse events (seven patients), withdrawal of consent (one patient), protocol violation (one patient) and incomplete baseline and follow-up imaging (three patients). Objective response was observed in only 1.7% of patients (95% CI 0.0–9.1%) and 31% of patients achieved stable disease (95% CI 19–44%). The median time-to-disease progression and overall survival were 8.6 weeks (95% CI 8.0–11.3 weeks) and 17.9 weeks (95% CI 13.1–30.3 weeks), respectively. Although these results suggest that lapatinib has little activity in unselected patients with urothelial carcinoma, further analysis demonstrated an improvement in overall survival in a subset of patients with tumours overexpressing EGFR and/or HER2.119 Unsurprisingly, this indicates that dual targeting of EGFR and HER2 will only be efficacious in patients overexpressing these receptors and that this therapeutic area warrants further investigation. Another ongoing phase II–III study is evaluating maintenance lapatinib versus placebo in patients with advanced-stage urothelial carcinoma overexpressing EGFR and/or

REVIEWS



HER2.120 Patients enrolled on this trial have been treated with first-line chemotherapy and have either stable disease or have achieved an objective response.120 Results from this study might help to define if dual targeting of EGFR and HER2 in urothelial carcinoma is a promising treatment strategy that should be pursued further.

Targeting FGFR3FGFR3 is a transmembrane RTK that transduces cellu-lar growth signals in response to external stimuli, and is an oncogene in urothelial carcinoma. Activating FGFR3 point mutations are frequently found in urothelial carci-noma,121 and inhibition of mutant FGFR3 protein leads to cell-cycle arrest and/or apoptosis in vitro and in vivo.122 Mutations in FGFR3 are most frequent in nonmuscle-invasive urothelial carcinoma, detected in up to 84% of Ta tumours and 21% of T1 tumours,123,124 although the frequency in muscle-invasive urothelial carcinoma is less than 15%.97 FGFR3 mutations are associated with an improved prognosis124,125 and are less common in patients with advanced-stage urothelial carcinoma, although in this setting they are more frequently seen in con-junction with deletion of CDKN2A.126 Most urothelial carci noma tumours show mutual exclusivity of FGFR3 mutations with TP53 mutations, the latter of which is more common in invasive and metastatic tumours.127 Dovitinib, a small-molecule FGFR3 and VEGFR inhibi-tor, has been tested in 44 patients with metastatic uro-thelial carcinoma, with and without FGFR3 mutations, who relapsed after first-line chemotherapy.128 In this study, treatment with dovitinib resulted in an overall response rate of 3% in patients with wild-type FGFR3 and no response in patients with FGFR3 mutations.128 A phase II trial of dovitinib in patients with refractory nonmuscle-invasive urothelial carcinoma with tumours harbouring FGFR3 mutations or overexpressing FGFR is ongoing.129Additional trials are planned to target FGFR3 with alternative therapeutic agents, both in nonmuscle- invasive urothelial carcinoma and advanced-stage urothelial carcinoma. Whether some or all urothelial carci noma tumours with FGFR3 activating point muta-tions, gene amplifications, gene rearrangements, or FGFR overexpression will respond to pathway inhibition remains to be determined. A recent report has described genetic translocations and re-arrangements as an alter-native mechanism of pathway activation of FGFR3 in urothelial carcinoma.130

Targeting the PI3K/AKT/mTOR pathwayThe PI3K family are serine–threonine kinases, of which class IA seems to be involved in signal transduction of external growth signals and is critical for cancer cell proliferation.131 The PI3K α-subunit (PIK3CA) is fre-quently mutated in both non-muscle invasive bladder cancer (15–25%) and muscle-invasive bladder cancer (15–20%).132,133 In addition, 30% of muscle-invasive urothelial carcinoma tumours demonstrate evi-dence of PTEN inactivation, a suppressor of the PI3K pathway.134,135 Mutations in PIK3CA tend to occur in the helical domain, resulting in activation of the kinase.136

PI3K-directed therapy is being tested in patients with previously treated metastatic urothelial carcinoma in an ongoing phase II trial of BKM120, an oral pan-PI3K class IA inhibitor.137 This drug is an inhibitor of the wild-type and mutant PI3Kα isoform, as well the PI3Kβ, PI3Kγ, and PI3Kδ isoforms.138 Preliminary results from a phase I study of the PI3K inhibitor GSK2126458 in 170 patients with advanced solid tumours showed objective responses in one third of patients with PIK3CA mutant urothelial carcinoma and two out of 15 patients with PIK3CA wild-type urothelial carcinoma.139 Although these results are preliminary, they raise the possibil-ity that other PI3K pathway alterations might predict anti tumour response, and that the anticancer activ-ity of agents targeting the PI3K pathway might not be restricted to PIK3CA mutant tumours.

One of the signalling pathways downstream of PI3K is the mTOR pathway. mTOR is a central regulator of metabolism, cell proliferation, and growth and inhibi-tion of this protein has proven useful in RCC and other solid tumours.140 Testing of the mTOR inhibi-tor everolimus as a salvage therapy in advanced-stage urothelial carcinoma has demonstrated limited activity in unselected patients. In a phase II study of 37 patients with previously treated locally advanced or metastatic urothelial carcinoma, everolimus showed disappointing results.141 Confirmed partial responses were observed in two patients and stable disease in eight patients, result-ing in a disease control rate of 27% at 8 weeks.141 In another phase II trial of everolimus in 45 patients with metastatic urothelial carcinoma, progression-free sur-vival of 3.3 months was observed and 5.4% of patients demonstrated a partial response.142 Therefore, single-agent everolimus therapy in unselected patients was not considered promising. Whole-genome sequencing of a patient with metastatic urothelial carcinoma who had a durable response when treated with everolimus revealed the presence of a tuberous sclerosis complex 1 (TSC1) inactivating mutation.94 TSC1 is a regulator of mTOR pathway activation and this mutation led to upregulation of mTOR kinase activity. In the same study, retrospective analysis of urothelial carcinoma samples showed a very strong association between TSC1 mutation and tumour shrinkage and prolonged progression-free survival in patients treated with everolimus.94 These findings suggest that TSC1 might mediate everolimus sensitivity and raise the possibility that mTOR inhibition could be an effective therapeutic strategy for patients with metastatic urothelial carci-noma whose tumours harbour TSC1 mutations. In a study of 41 bladder tumours, this mutation was present in 4.9% of tumours, although loss of heterozygosity was evident in 54% of tumours.143 Everolimus is also being tested in combination with paclitaxel in patients with advanced-stage urothelial carcinoma who are ineligi-ble for treatment with cisplatin as first-line therapy,144 and also in the second-line setting.145 Other trials are exploring everolimus in combination with gemcitabine or with the oral a ntiangiogenic agent pazopanib in urothelial carcinoma.146,147

REVIEWS



Targeting angiogenesisA greater understanding of the central role that angio-genesis has in urothelial carcinoma, and the availabil-ity of antiangiogenic agents has prompted their testing in metastatic urothelial carcinoma. Increased levels of VEGF, bFGF and IL-8 have been shown to correlate with stage and poorer disease-free survival in advanced uro thelial carcinoma.148–150 In addition, mean vessel density is a predictor of progression, vascular invasion, positive nodes, recurrence, and survival in invasive urothelial carcinoma.151

Sorafenib, which inhibits a number of targets including VEGF, has been tested as a monotherapy for advanced-stage urothelial carcinoma in phase II trials, both as first-line and second-line therapy.152,153 In the second-line setting, no objective responses were observed in 27 patients and the median overall survival was 6.8 months.152 In a study of sorafenib as a first-line treat-ment in urothelial carcinoma, 17 patients were treated and no objective responses were observed; only one patient had stable disease by RECIST criteria and remained on treatment for more than 3 months.153

In a preclinical model, sunitinib, another multi- targeted RTK inhibitor that inhibits VEGF, has demon-strated activity against urothelial carcinoma, both as a single agent and in combination with cisplatin.154 Modest clinical activity of sunitinib as a second-line treatment has been reported in a phase II study of 77 patients with meta static urothelial carcinoma.155 The study did not achieve the pre-planned response rate, but 29% of patients achieved disease stabilization of longer than 3 months.155 A phase II trial of sunitinib as a first-line treatment in

38 patients with urothelial carcinoma unsuitable for cispla tin treatment has reported 8% of patients with objective responses and 45% with disease stabilization greater than 3 months.156 A phase II trial evaluating suni-tinib combined with chemotherapy in 36 patients with metastatic urothelial carcinoma showed that the combi-nation of sunitinib with gemcitabine and cisplatin in the first-line setting was associated with severe haemato-logical toxic effects.157 Maintenance sunitinib therapy after response to chemotherapy has also been tested in comparison with placebo in a randomized phase II trial of patients with advanced-stage urothelial carcinoma.158 This study closed early due to slow accrual after 54 patients were randomly assigned to treatment. Results from the study indicated that maintenance therapy with sunitinib was feasible, but did not improve the 6-month progres-sion rate; sunitinib had limited activity when used after progression in patients previously receiving placebo.158

Pazopanib, a VEGFR-targeted TKI, demonstrated a confirmed objective response in 17% of the 41 patients in a phase II study of chemorefractory advanced-stage urothelial carcinoma.159 However, the non-standard evalu ation schedule of imaging every 4-weeks used in this study should be considered when assessing the results. Pazopanib in combination with gemcitabine is being tested in a phase II trial in patients with advanced-stage or metastatic urothelial carcinoma who are ineligible for cisplatin- based chemotherapy,160 and as second-line therapy in combination with paclitaxel.161 Vandetanib, an oral TKI of VGFR2 and EGFR, in combination with docetaxel has been assessed in a phase II trial of 142 platinum- pretreated patients with metastatic urothelial carcinoma.162 The addition of vandetanib to docetaxel did not result in a significant improvement in progression-free survival, overall response rate, or overall survival and was also associated with increased toxic effects.162

The combination of bevacizumab, an anti-VEGF mono-clonal antibody, with chemotherapy has been assessed in a phase II trial as a first-line treatment in meta static urothelial carcinoma.163 Cisplatin and gemcita bine were combined with bevacizumab in 43 patients, resulting in an encouraging overall response rate of 72% (95% CI 56– 85%) and an overall survival of 19.1 months (95% CI 12.4–22.7 months).163 This treatment regimen is now being tested in a randomized phase III trial comparing cisplatin and gemcitabine plus bevacizumab with cisplatin and gemcitabine plus placebo in patients with advanced-stage urothelial carcinoma.164 In a phase II study of 51 patients with advanced-stage unresectable or metastatic urothelial carcinoma ineligible for cisplatin treatment, gemcitabine, carboplatin, and bevacizumab demonstrated higher than expected activity, with an overall survival of 13.9 months in a patient population where the median survival is expected to be approximately 9 months.165 Studies of other antiangiogenic t reatments such as VEGF-trap aflibercept are ongoing.166–168

Targeting the stress responseHeat-shock proteins (HSP) are a group of proteins that are upregulated during cellular stress. They have numerous

Table 3 | Selected targets and trials in urothelial carcinoma

Target Agent Setting

EGFR GefitinibGefitinib + chemotherapyErlotinibGemcitabine/cisplatin ± cetuximabCetuximab ± paclitaxel

Second-line104

First-line107

Neoadjuvant105

First-line108

Second-line158

HER2 Gemcitabine/carboplatin + trastuzumabTrastuzumab ± chemotherapyDN24-02 versus observation

First-line116

First-line117

Adjuvant118

EGFR + HER2 Lapatinib maintenance versus placeboLapatinib

First-line120

Second-line187

FGFR3 + VEGFR Dovitinib Second-line128

VEGFR SunitinibSunitinibGemcitabine/cisplatin + sunitinibSunitinib maintenance versus placeboSorafenibSorafenibPazopanibPazopanib + paclitaxelGemcitabine/cisplatin + bevacizumab

First-line156

Second-line155

Second-line157

First-line158

First-line153

Second-line152

Second-line159

Second-line161

First-line164

VEGFR + EGFR Docetaxel + vandetanib Second-line162

mTOR Everolimus Second-line141

HSP27 Gemcitabine/cisplatin + OGX-427Docetaxel ± OGX-427

First-line176

Second-line177

Abbreviations: HSP27, heat shock protein 27; mTOR, mammalian target of rapamycin.

REVIEWS



functions, one of which is to act as molecular chaperones to stabilize signalling proteins, which can include oncogenic proteins.169 HSP27 is a stress-activated ATP-independent cytoprotective chaperone that mediates treatment resist-ance in cancer.170 HSP27 prevents cancer cell death by pro-tecting against apoptosis, and promotes cell prolifer ation in an AKT-dependent manner.171–173 HSP27 over expression results in activation of AKT, ultimately leading to enhanced ERK translocation to the nucleus and increased cell prolifer ation.174 HSP27 has been shown to be expressed in urothelial tumours, and knockdown of its expression in a HTB-1 urothelial carcinoma cell xenograft model sup-pressed tumour growth.175 In addition, HSP27 has been implicated in urothelial carcinoma chemo resistance.170 HSP27 is not a druggable target at this time, but its mRNA could be targeted by an antisense oligo nucleotide. Targeting of HSP27 mRNA expression by an antisense oligonucleotide is under active investigation in urothelial carcinoma.125,126 OGX-427, a second-generation antisense oligonucleotide targeting HSP27A mRNA, is being tested in an ongoing phase II randomized.176 A phase II study of docetaxel with or without OGX-427 in the second-line setting in patients with relapsed or r efractory metastatic urothelial carcinoma is also ongoing.177

As these various examples demonstrate, a wide-range of targeted agents is being tested, alone or in combina-tion with cytotoxic drugs, in clinical trials for urothelial carcinoma (Table 3). These agents have the potential to provide promising new treatment options for patients with this disease.

ConclusionsGenomic advances have allowed researchers to uncover novel genetic mutations, improve classification of tumours, and have revealed tumour subsets with their

own shared genetic, expression and therapeutic target profile. The knowledge gained from sequencing efforts are finally moving into the clinic, helping to make personal-ized medicine a reality. In RCC, several factors involved in signalling and therapeutic resistance have been identi-fied that might also have potential roles as predictive bio-markers. These markers encompass a range of molecules and tumour-specific genetic alterations that can be found in tumours, serum or plasma. Validation of such markers is ongoing, with several noteworthy results. It is hoped that the advances in understanding the genetic background of urothelial tumours, will lead to the identification of useful predictive and prognostic biomarkers, as well as novel therapeutic targets. However, as for many cancers, an underlying limitation is the heterogeneity observed in these tumours, such that a single biopsy result might be inaccurate to predict a prognostic profile. Critical genomic alterations might not be present in every site of metastases, and, thus, biopsies may not capture all genetic aberrations.48 Despite the challenges, breakthroughs in genetic data mining and improved mechanistic under-standing of resistance are accelerating progress and should soon be reflected in improved survival rates in RCC and urothelial carcinoma.

Review criteria

The articles on which this Review is based were selected by searching the PubMed database for full-text articles published in English since 2000 using the search terms “genome sequencing, renal cell carcinoma”, “genome sequencing, urothelial carcinoma”, “genome sequencing, bladder cancer”, “molecular targets, renal cell carcinoma”, “molecular targets, urothelial carcinoma”, and “molecular targets, bladder cancer”. Conference abstracts were obtained by conference attendance.

1. Siegel, R., Naishadham, D. & Jemal, A. Cancer statistics, 2013. CA Cancer J. Clin. 63, 11–30 (2013).

2. Shah, S. P. et al. The clonal and mutational evolution spectrum of primary triple-negative breast cancers. Nature 486, 395–399 (2012).

3. Ding, L. et al. Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing. Nature 481, 506–510 (2012).

4. Lehmann, J. et al. Adjuvant cisplatin plus methotrexate versus methotrexate, vinblastine, epirubicin, and cisplatin in locally advanced bladder cancer: results of a randomized, multicenter, phase III trial (AUO-AB 05/95). J. Clin. Oncol. 23, 4963–4974 (2005).

5. Parekh, D. J., Bochner, B. H. & Dalbagni, G. Superficial and muscle-invasive bladder cancer: principles of management for outcomes assessments. J. Clin. Oncol. 24, 5519–5527 (2006).

6. McConkey, D. J. et al. Molecular genetics of bladder cancer: emerging mechanisms of tumor initiation and progression. Urol. Oncol. 28, 429–440 (2010).

7. Rothman, N. et al. A multi-stage genome-wide association study of bladder cancer identifies multiple susceptibility loci. Nat. Genet. 42, 978–984 (2010).

8. Sidransky, D. et al. Clonal origin bladder cancer. N. Engl. J. Med. 326, 737–740 (1992).

9. Loehrer, P. J. et al. A randomized comparison of cisplatin alone or in combination with methotrexate, vinblastine, and doxorubicin in patients with metastatic urothelial carcinoma: a cooperative group study. J. Clin. Oncol. 10, 1066–1073 (1992).

10. Bajorin, D. F. et al. Long-term survival in metastatic transitional-cell carcinoma and prognostic factors predicting outcome of therapy. J. Clin. Oncol. 17, 3173–3181 (1999).

11. Bellmunt, J. et al. Prognostic factors in patients with advanced transitional cell carcinoma of the urothelial tract experiencing treatment failure with platinum-containing regimens. J. Clin. Oncol. 28, 1850–1855 (2010).

12. Kamat, A. M. et al. ICUD-EAU International Consultation on Bladder Cancer 2012: screening, diagnosis, and molecular markers. Eur. Urol. 63, 4–15 (2013).

13. Latif, F. et al. Identification of the von Hippel-Lindau disease tumor suppressor gene. Science 260, 1317–1320 (1993).

14. Maher, E. R., Neumann, H. P. & Richard, S. von Hippel-Lindau disease: a clinical and scientific review. Eur. J. Hum. Genet. 19, 617–623 (2011).

15. Beroukhim, R. et al. Patterns of gene expression and copy-number alterations in von-Hippel Lindau disease-associated and sporadic clear cell carcinoma of the kidney. Cancer Res. 69, 4674–4681 (2009).

16. Yao, M. et al. VHL tumor suppressor gene alterations associated with good prognosis in sporadic clear-cell renal carcinoma. J. Natl Cancer Inst. 94, 1569–1575 (2002).

17. Choueiri, T. K. et al. von Hippel-Lindau gene status and response to vascular endothelial growth factor targeted therapy for metastatic clear cell renal cell carcinoma. J. Urol. 180, 860–865 (2008).

18. Zimmer, M. et al. Small-molecule inhibitors of HIF-2a translation link its 5’UTR iron-responsive element to oxygen sensing. Mol. Cell 32, 838–848 (2008).

19. Alimov, A., Sundelin, B., Wang, N., Larsson, C. & Bergerheim, U. Loss of 14q31-q32.2 in renal cell carcinoma is associated with high malignancy grade and poor survival. Int. J. Oncol. 25, 179–185 (2004).

20. Shen, C. et al. Genetic and functional studies implicate HIF1α as a 14q kidney cancer suppressor gene. Cancer Discov. 1, 222–235 (2011).

21. Maxwell, P. H. et al. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399, 271–275 (1999).

22. Gordan, J. D. et al. HIF-alpha effects on c-Myc distinguish two subtypes of sporadic VHL-deficient clear cell renal carcinoma. Cancer Cell 14, 435–446 (2008).

REVIEWS



23. Mandriota, S. J. et al. HIF activation identifies early lesions in VHL kidneys: evidence for site-specific tumor suppressor function in the nephron. Cancer Cell 1, 459–468 (2002).

24. Yan, Q., Bartz, S., Mao, M., Li, L. & Kaelin, W. G. Jr. The hypoxia-inducible factor 2alpha N-terminal and C-terminal transactivation domains cooperate to promote renal tumorigenesis in vivo. Mol. Cell. Biol. 27, 2092–2102 (2007).

25. Zimmer, M., Doucette, D., Siddiqui, N. & Iliopoulos, O. Inhibition of hypoxia-inducible factor is sufficient for growth suppression of VHL–/– tumors. Mol. Cancer Res. 2, 89–95 (2004).

26. Maranchie, J. K. et al. The contribution of VHL substrate binding and HIF1-alpha to the phenotype of VHL loss in renal cell carcinoma. Cancer Cell 1, 247–255 (2002).

27. Covello, K. L. et al. HIF-2α regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth. Genes Dev. 20, 557–570 (2006).

28. Keith, B., Johnson, R. S. & Simon, M. C. HIF1α and HIF2α: sibling rivalry in hypoxic tumour growth and progression. Nat. Rev. Cancer 12, 9–22 (2012).

29. Fu, L., Wang, G., Shevchuk, M. M., Nanus, D. M. & Gudas, L. J. Activation of HIF2α in kidney proximal tubule cells causes abnormal glycogen deposition but not tumorigenesis. Cancer Res. 73, 2916–2925 (2013).

30. Kondo, K., Kim, W. Y., Lechpammer, M. & Kaelin, W. G. Jr. Inhibition of HIF2α is sufficient to suppress pVHL-defective tumor growth. PLoS Biol. 1, E83 (2003).

31. Kondo, K., Klco, J., Nakamura, E., Lechpammer, M. & Kaelin, W. G. Jr. Inhibition of HIF is necessary for tumor suppression by the von Hippel-Lindau protein. Cancer Cell 1, 237–246 (2002).

32. Raval, R. R. et al. Contrasting properties of hypoxia-inducible factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-associated renal cell carcinoma. Mol. Cell Biol. 25, 5675–5686 (2005).

33. Chan, D. A. et al. Targeting GLUT1 and the Warburg effect in renal cell carcinoma by chemical synthetic lethality. Sci. Transl. Med. 3, 94ra70 (2011).

34. Maru, S. et al. Inhibition of mTORC2 but not mTORC1 up-regulates E-cadherin expression and inhibits cell motility by blocking HIF-2α expression in human renal cell carcinoma. J. Urol. 189, 1921–1929 (2013).

35. Varela, I. et al. Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature 469, 539–542 (2011).

36. Dalgliesh, G. L. et al. Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes. Nature 463, 360–363 (2010).

37. Kapur, P. et al. Effects on survival of BAP1 and PBRM1 mutations in sporadic clear-cell renal-cell carcinoma: a retrospective analysis with independent validation. Lancet Oncol. 14, 159–167 (2013).

38. Peña-Llopis, S. et al. BAP1 loss defines a new class of renal cell carcinoma. Nat. Genet. 44, 751–759 (2012).

39. Reisman, D., Glaros, S. & Thompson, E. A. The SWI/SNF complex and cancer. Oncogene 28, 1653–1668 (2009).

40. Weissman, B. & Knudsen, K. E. Hijacking the chromatin remodeling machinery: impact of SWI/SNF perturbations in cancer. Cancer Res. 69, 8223–8230 (2009).

41. Cancer Genome Atlas Research Network. Comprehensive molecular characterization

of clear cell renal cell carcinoma. Nature 499, 43–49 (2013).

42. Lichner, Z. et al. The chromatin remodeling gene ARID1A is a new prognostic marker in clear cell renal cell carcinoma. Am. J. Pathol. 182, 1163–1170 (2013).

43. Jones, S. et al. Frequent mutations of chromatin remodeling gene ARID1A in ovarian clear cell carcinoma. Science 330, 228–231 (2010).

44. Guo, G. et al. Frequent mutations of genes encoding ubiquitin-mediated proteolysis pathway components in clear cell renal cell carcinoma. Nat. Genet. 44, 17–19 (2011).

45. Misaghi, S. et al. Association of C-terminal ubiquitin hydrolase BRCA1-associated protein 1 with cell cycle regulator host cell factor 1. Mol. Cell Biol. 29, 2181–2192 (2009).

46. Scheuermann, J. C. et al. Histone H2A deubiquitinase activity of the polycomb repressive complex PR-DUB. Nature 465, 243–247 (2010).

47. Niu, X. et al. The von Hippel-Lindau tumor suppressor protein regulates gene expression and tumor growth through histone demethylase JARID1C. Oncogene 31, 776–786 (2012).

48. Gerlinger, M. et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N. Engl. J. Med. 366, 883–892 (2012).

49. Lopez-Beltran, A. et al. Pathology of renal cell carcinoma: an update. Anal. Quant. Cytol. Histol. 35, 61–76 (2013).

50. Delahunt, B. et al. Morphologic typing of papillary renal cell carcinoma: comparison of growth kinetics and patient survival in 66 cases. Hum. Pathol. 32, 590–595 (2001).

51. Waldert, M. et al. Comparison of type I and II papillary renal cell carcinoma (RCC) and clear cell RCC. BJU Int. 102, 1381–1384 (2008).

52. Bellon, S. F. et al. c-Met inhibitors with novel binding mode show activity against several hereditary papillary renal cell carcinoma-related mutations. J. Biol. Chem. 283, 2675–2683 (2008).

53. Looyenga, B. D. et al. Chromosomal amplification of leucine-rich repeat kinase-2 (LRRK2) is required for oncogenic MET signaling in papillary renal and thyroid carcinomas. Proc. Natl Acad. Sci. USA 108, 1439–1444 (2011).

54. Sanders, M. E., Mick, R., Tomaszewski, J. E. & Barr, F. G. Unique patterns of allelic imbalance distinguish type 1 from type 2 sporadic papillary renal cell carcinoma. Am. J. Pathol. 161, 997–1005 (2002).

55. Refae, M. A., Wong, N., Patenaude, F., Bégin, L. R. & Foulkes, W. D. Hereditary leiomyomatosis and renal cell cancer: an unusual and aggressive form of hereditary renal carcinoma. Nat. Clin. Pract. Oncol. 4, 256–261 (2007).

56. Toro, J. R. et al. BHD mutations, clinical and molecular genetic investigations of Birt-Hogg-Dubé syndrome: a new series of 50 families and a review of published reports. J. Med. Genet. 45, 321–331 (2008).

57. Gardie, B. et al. Novel FH mutations in families with hereditary leiomyomatosis and renal cell cancer (HLRCC) and patients with isolated type 2 papillary renal cell carcinoma. J. Med. Genet. 48, 226–234 (2011).

58. Furge, K. A. et al. Detection of DNA copy number changes and oncogenic signaling abnormalities from gene expression data reveals MYC activation in high-grade papillary renal cell carcinoma. Cancer Res. 67, 3171–3176 (2007).

59. Prowse, A. H. et al. Somatic inactivation of the VHL gene in Von Hippel-Lindau disease tumors. Am. J. Hum. Genet. 60, 765–771 (1997).

60. Hori, Y. et al. Oxidative stress and DNA hypermethylation status in renal cell carcinoma

arising in patients on dialysis. J. Pathol. 212, 218–226 (2007).

61. Morrissey, C. et al. Epigenetic inactivation of the RASSF1A 3p21.3 tumor suppressor gene in both clear cell and papillary renal cell carcinoma. Cancer Res. 61, 7277–7281 (2001).

62. Costa, V. L. et al. Quantitative promoter methylation analysis of multiple cancer-related genes in renal cell tumors. BMC Cancer 7, 133 (2007).

63. Awakura, Y., Nakamura, E., Ito, N., Kamoto, T. & Ogawa, O. Methylation-associated silencing of TU3A in human cancers. Int. J. Oncol. 33, 893–899 (2008).

64. Kvasha, S. et al. Hypermethylation of the 5’CpG island of the FHIT gene in clear cell renal carcinomas. Cancer Lett. 265, 250–257 (2008).

65. Hirata, H. et al. Wnt antagonist DKK1 acts as a tumor suppressor gene that induces apoptosis and inhibits proliferation in human renal cell carcinoma. Int. J. Cancer 128, 1793–1803 (2011).

66. Rogenhofer, S. et al. Global histone H3 lysine 27 (H3K27) methylation levels and their prognostic relevance in renal cell carcinoma. BJU Int. 109, 459–465 (2012).

67. Escudier, B., Szczylik, C., Porta, C. & Gore, M. Treatment selection in metastatic renal cell carcinoma: expert consensus. Nat. Rev. Clin. Oncol. 9, 327–337 (2012).

68. Kelly, R. J., Billemont, B. & Rixe, O. Renal toxicity of targeted therapies. Target. Oncol. 4, 121–133 (2009).

69. Karaman, M. W. et al. A quantitative analysis of kinase inhibitor selectivity. Nat. Biotechnol. 26, 127–132 (2008).

70. Hu-Lowe, D. D. et al. Nonclinical antiangiogenesis and antitumor activities of axitinib (AG-013736), an oral, potent, and selective inhibitor of vascular endothelial growth factor receptor tyrosine kinases 1, 2, 3. Clin. Cancer Res. 14, 7272–7283 (2008).

71. Heng, D. Y. et al. Primary anti-vascular endothelial growth factor (VEGF)-refractory metastatic renal cell carcinoma: clinical characteristics, risk factors, and subsequent therapy. Ann. Oncol. 23, 1549–1555 (2012).

72. Casanovas, O., Hicklin, D. J., Bergers, G. & Hanahan, D. Drug resistance by evasion of antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet tumors. Cancer Cell 8, 299–309 (2005).

73. Linehan, W. M., Srinivasan, R. & Schmidt, L. S. The genetic basis of kidney cancer: a metabolic disease. Nat. Rev. Urol. 7, 277–285 (2010).

74. Rapisarda, A. & Melillo, G. Overcoming disappointing results with antiangiogenic therapy by targeting hypoxia. Nat. Rev. Clin. Oncol. 9, 378–390 (2012).

75. Pàez-Ribes, M. et al. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell 15, 220–231 (2009).

76. Huang, D. et al. Interleukin-8 mediates resistance to antiangiogenic agent sunitinib in renal cell carcinoma. Cancer Res. 70, 1063–1071 (2010).

77. Welti, J. C. et al. Fibroblast growth factor 2 regulates endothelial cell sensitivity to sunitinib. Oncogene 30, 1183–1193 (2011).

78. US National Library of Medicine. ClinicalTrials.gov [online], http://clinicaltrials.gov/show/NCT01223027 (2013).

79. Xu, C. F. et al. Pazopanib efficacy in renal cell carcinoma: evidence for predictive genetic markers in angiogenesis-related and exposure-related genes. J. Clin. Oncol. 29, 2557–2564 (2011).

REVIEWS


http://clinicaltrials.gov/show/NCT01223027



80. Choueiri, T. K. et al. Phase II and biomarker study of the dual MET/VEGFR2 inhibitor foretinib in patients with papillary renal cell carcinoma. J. Clin. Oncol. 31, 181–186 (2013).

81. Choueiri, T. K. et al. Activity of cabozantinib (XL184) in patients (pts) with metastatic, refractory renal cell carcinoma (RCC) [abstract]. J. Clin. Oncol. 30 (Suppl. 5), a364 (2012).

82. Carbone, C. et al. Anti-VEGF treatment-resistant pancreatic cancers secrete proinflammatory factors that contribute to malignant progression by inducing an EMT cell phenotype. Clin. Cancer Res. 17, 5822–5832 (2011).

83. Sievert, K. et al. Economic aspects of bladder cancer: what are the benefits and costs? World J. Urol. 27, 295–300 (2009).

84. Gakis, G. et al. ICUD-EAU International Consultation on Bladder Cancer 2012: Radical cystectomy and bladder preservation for muscle-invasive urothelial carcinoma of the bladder. Eur. Urol. 63, 45–57 (2013).

85. Burger, M. et al. ICUD-EAU International Consultation on Bladder Cancer 2012: non-muscle-invasive urothelial carcinoma of the bladder. Eur. Urol. 63, 36–44 (2013).

86. Sternberg, C. N. et al. ICUD-EAU International Consultation on Bladder Cancer 2012: Chemotherapy for urothelial carcinoma-neoadjuvant and adjuvant settings. Eur. Urol. 63, 58–66 (2013).

87. Bellmunt, J. & Petrylak, D. P. New therapeutic challenges in advanced bladder cancer. Semin. Oncol. 39, 598–607 (2012).

88. Sanchez-Carbayo, M. & Cordon-Cardo, C. Applications of array technology: identification of molecular targets in bladder cancer. Br. J. Cancer 89, 2172–2177 (2003).

89. Sjodahl, G. et al. A molecular taxonomy for urothelial carcinoma. Clin. Cancer Res. 18, 3377–3386 (2012).

90. Sjodahl, G. et al. A systematic study of gene mutations in urothelial carcinoma; inactivating mutations in TSC2 and PIK3R1. PLoS ONE 6, e18583 (2011).

91. Lindgren, D. et al. Integrated genomic and gene expression profiling identifies two major genomic circuits in urothelial carcinoma. PLoS ONE 7, e38863 (2012).

92. Lindgren, D. et al. Combined gene expression and genomic profiling define two intrinsic molecular subtypes of urothelial carcinoma and gene signatures for molecular grading and outcome. Cancer Res. 70, 3463–3472 (2010).

93. Milowsky, M. I. et al. Phase II study of everolimus in metastatic urothelial cancer. BJU Int. 112, 462–470 (2013).

94. Iyer, G. et al. Genome sequencing identifies a basis for everolimus sensitivity. Science 338, 221 (2012).

95. Iyer, G. et al. Prevalence and co-occurrence of actionable genomic alterations in high-grade bladder cancer. J. Clin. Oncol. http:// dx.doi.org/10.1200/JCO.2012.46.5740.

96. Wang, Z. & Sun, Y. Targeting p53 for novel anticancer therapy. Transl. Oncol. 3, 1–12 (2010).

97. Gui, Y. et al. Frequent mutations of chromatin remodeling genes in transitional cell carcinoma of the bladder. Nat. Genet. 43, 875–878 (2011).

98. National Cancer Institute. The Cancer Genome Atlas [online], http://cancergenome.nih.gov/ (2012).

99. Bellmunt, J., Hussain, M. & Dinney, C. P. Novel approaches with targeted therapies in bladder cancer. Therapy of bladder cancer by blockade of the epidermal growth factor receptor family. Crit. Rev. Oncol. Hematol. 46 (Suppl.), S85–S104 (2003).

100. Nutt, J. E., Lazarowicz, H. P., Mellon, J. K. & Lunec, J. Gefitinib (‘Iressa’, ZD1839) inhibits the growth response of bladder tumour cell lines to epidermal growth factor and induces TIMP2. Br. J. Cancer 90, 1679–1685 (2004).

101. Ruck, A. & Paulie, S. EGF, TGF alpha, AR and HB-EGF are autocrine growth factors for human bladder carcinoma cell lines. Anticancer Res. 18, 1447–1452 (1998).

102. Perrotte, P. et al. Anti-epidermal growth factor receptor antibody C225 inhibits angiogenesis in human transitional cell carcinoma growing orthotopically in nude mice. Clin. Cancer Res. 5, 257–265 (1999).

103. Bambury, R. M. & Rosenberg, J. E. Advanced urothelial carcinoma: overcoming treatment resistance through novel treatment approaches. Front. Pharmacol. 4, 3 (2013).

104. Petrylak, D. P. et al. Results of the Southwest Oncology Group phase II evaluation (study S0031) of ZD1839 for advanced transitional cell carcinoma of the urothelium. BJU Int. 105, 317–321 (2010).

105. Pruthi, R. S. et al. A phase II trial of neoadjuvant erlotinib in patients with muscle-invasive bladder cancer undergoing radical cystectomy: clinical and pathological results. BJU Int. 106, 349–354 (2010).

106. Pirker, R. et al. Cetuximab plus chemotherapy in patients with advanced non-small-cell lung cancer (FLEX): an open-label randomised phase III trial. Lancet 373, 1525–1531 (2009).

107. Philips, G. K. et al. A phase II trial of cisplatin (C), gemcitabine (G) and gefitinib for advanced urothelial tract carcinoma: results of Cancer and Leukemia Group B (CALGB) 90102. Ann. Oncol. 20, 1074–1079 (2009).

108. Grivas, P. et al. Randomized phase II trial of gemcitabine/cisplatin (GC) with or without cetuximab (CET) in patients (pts) with advanced urothelial carcinoma (UC) [abstract]. J. Clin. Oncol. 30 (Suppl.), a4506 (2012).

109. Inoue, K. et al. Paclitaxel enhances the effects of the anti-epidermal growth factor receptor monoclonal antibody ImClone C225 in mice with metastatic human bladder transitional cell carcinoma. Clin. Cancer Res. 6, 4874–4884 (2000).

110. Wong, Y. N. et al. Phase II trial of cetuximab with or without paclitaxel in patients with advanced urothelial tract carcinoma. J. Clin. Oncol. 30, 3545–3551 (2012).

111. Black, P. C. et al. Sensitivity to epidermal growth factor receptor inhibitor requires E-cadherin expression in urothelial carcinoma cells. Clin. Cancer Res. 14, 1478–1486 (2008).

112. McConkey, D. J. et al. HDAC inhibitors reverse epithelial-to-mesenchymal transition (EMT) and restore sensitivity to EGFR inhibitors in urothelial carcinoma cells [abstract]. ASCO Genitourinary Cancers Symp. a267 (2009).

113. Laé, M. et al. Assessing HER2 gene amplification as a potential target for therapy in invasive urothelial bladder cancer with a standardized methodology: results in patients. Ann. Oncol. 21, 815–819 (2010).

114. Fleischmann, A., Rotzer, D., Seiler, R., Studer, U. E. & Thalmann, G. N. HER2 amplification is significantly more frequent in lymph node metastases from urothelial bladder cancer than in the primary tumours. Eur. Urol. 60, 350–357 (2011).

115. Galsky, M. D. et al. Target-specific, histology-independent, randomized discontinuation study of lapatinib in patients with HER2-amplified solid tumors. Invest. New Drugs 30, 695–701 (2012).

116. Hussain, M. H. et al. Trastuzumab, paclitaxel, carboplatin, and gemcitabine in advanced

human epidermal growth factor receptor-2/neu-positive urothelial carcinoma: results of a multicenter phase II National Cancer Institute trial. J. Clin. Oncol. 25, 2218–2224 (2007).

117. Beuzeboc, P. et al. Trastuzumab (T) combined with standard chemotherapy in HER+ metastatic bladder cancer (BC) patients: interim safety results of a prospective randomized phase II study [abstract]. J. Clin. Oncol. 25 (Suppl.), a15565 (2007).


119. Wülfing, C. et al. A single-arm, multicenter, open-label phase 2 study of lapatinib as the second-line treatment of patients with locally advanced or metastatic transitional cell carcinoma. Cancer 115, 2881–2890 (2009).


121. Iyer, G. & Milowsky, M. I. Fibroblast growth factor receptor-3 in urothelial tumorigenesis. Urol. Oncol. 31, 303–311, (2013).

122. Lamont, F. R. et al. Small molecule FGF receptor inhibitors block FGFR-dependent urothelial carcinoma growth in vitro and in vivo. Br. J. Cancer 104, 75–82 (2011).

123. Billerey, C. et al. Frequent FGFR3 mutations in papillary non-invasive bladder (pTa) tumors. Am. J. Pathol. 158, 1955–1959 (2001).

124. van Rhijn, B. W. et al. The FGFR3 mutation is related to favorable pT1 bladder cancer. J. Urol. 187, 310–314 (2012).

125. van Oers, J. M. et al. FGFR3 mutations indicate better survival in invasive upper urinary tract and bladder tumours. Eur. Urol. 55, 650–657 (2009).

126. Rebouissou, S. et al. CDKN2A homozygous deletion is associated with muscle invasion in FGFR3-mutated urothelial bladder carcinoma. J. Pathol. 227, 315–324 (2012).

127. van Rhijn, B. W. et al. FGFR3 and p53 characterize alternative genetic pathways in the pathogenesis of urothelial cell carcinoma. Cancer Res. 64, 1911–1914 (2004).

128. Milowsky, M. et al. Final results of a multicenter, open-label phase II trial of dovitinib (TKI258) in patients with advanced urothelial carcinoma with either mutated or nonmutated FGFR3 [abstract]. J. Clin. Oncol. 31 (Suppl. 6), a255 (2013).


130. Williams, S. V., Hurst, C. D. & Knowles, M. A. Oncogenic FGFR3 gene fusions in bladder cancer. Hum. Mol. Genet. 22, 795–803 (2013).

131. Courtney, K. D., Corcoran, R. B. & Engelman, J. A. The PI3K pathway as drug target in human cancer. J. Clin. Oncol. 28, 1075–1083 (2010).

132. Platt, F. M. et al. Spectrum of phosphatidylinositol 3-kinase pathway gene alterations in bladder cancer. Clin. Cancer Res. 15, 6008–6017 (2009).

133. López-Knowles, E. et al. PIK3CA mutations are an early genetic alteration associated with FGFR3 mutations in superficial papillary bladder tumors. Cancer Res. 66, 7401–7404 (2006).

134. Ching, C. B. & Hansel, D. E. Expanding therapeutic targets in bladder cancer: the PI3K/Akt/mTOR pathway. Lab. Invest. 90, 1406–1414 (2010).

135. Kompier, L. C. et al. FGFR3, HRAS, KRAS, NRAS and PIK3CA mutations in bladder cancer and their potential as biomarkers for surveillance and therapy. PLoS ONE 5, e13821 (2010).

136. Knowles, M. A., Platt, F. M., Ross, R. L. & Hurst, C. D. Phosphatidylinositol 3-kinase (PI3K) pathway activation in bladder cancer. Cancer Metastasis Rev. 28, 305–316 (2009).

REVIEWS


http://dx.doi.org/10.1200/JCO.2012.46.5740

http://dx.doi.org/10.1200/JCO.2012.46.5740

http://cancergenome.nih.gov/









138. Bendell, J. C. et al. Phase I, dose-escalation study of BKM120, an oral pan-class I PI3K inhibitor, in patients with advanced solid tumors. J. Clin. Oncol. 30, 282–290 (2012).

139. Munster, P. et al. PI3K kinase inhibitor GSK2126458 (GSK458): clinical activity in select patient (PT) populations defined by predictive markers (study P3K112826) [abstract]. Ann. Oncol. 23 (Suppl. 9), a4420 (2012).

140. Gomez-Pinillos, A. & Ferrari, A. C. mTOR signaling pathway and mTOR inhibitors in cancer therapy. Hematol. Oncol. Clin. North Am. 26, 483–505 (2012).

141. Seront, E. et al. Phase II study of everolimus in patients with locally advanced or metastatic transitional cell carcinoma of the urothelial tract: clinical activity, molecular response, and biomarkers. Ann. Oncol. 23, 2663–2670 (2012).

142. Milowsky, M. I. et al. Final results of a phase II study of everolimus (RAD001) in metastatic transitional cell carcinoma (TCC) of the urothelium [abstract]. J. Clin. Oncol. 29 (Suppl.), a4606 (2011).

143. Guo, Y. et al. TSC1 involvement in bladder cancer: diverse effects and therapeutic implications. J. Pathol. 230, 17–27 (2013).





148. Kopparapu, P. K. et al. Expression of VEGF and its receptors VEGFR1/VEGFR2 is associated with invasiveness of bladder cancer. Anticancer Res. 33, 2381–2390 (2013).

149. Inoue, K. et al. Interleukin 8 expression regulates tumorigenicity and metastasis in human bladder cancer. Cancer Res. 60, 2290–2299 (2000).

150. Allen, L. E. & Maher, P. A. Expression of basic fibroblast growth factor and its receptor in an invasive bladder carcinoma cell line. J. Cell Physiol. 155, 368–375 (1993).

151. Afonso, J., Santos, L. L., Amaro, T., Lobo, F. & Longatto-Filho, A. The aggressiveness of urothelial carcinoma depends to a large extent on lymphovascular invasion--the prognostic contribution of related molecular markers. Histopathology 55, 514–524 (2009).

152. Dreicer, R. et al. Phase 2 trial of sorafenib in patients with advanced urothelial cancer: a trial of the Eastern Cooperative Oncology Group. Cancer 115, 4090–4095 (2009).

153. Sridhar, S. S. et al. A phase II trial of sorafenib in first-line metastatic urothelial cancer: a study of the PMH Phase II Consortium. Invest. New Drugs 29, 1045–1049 (2011).

154. Sonpavde, G. et al. Sunitinib malate is active against human urothelial carcinoma and enhances the activity of cisplatin in a preclinical model. Urol. Oncol. 27, 391–399 (2009).

155. Gallagher, D. J. et al. Phase II study of sunitinib in patients with metastatic urothelial cancer. J. Clin. Oncol. 28, 1373–1379 (2010).

156. Bellmunt, J. et al. Phase II study of sunitinib as first-line treatment of urothelial cancer patients ineligible to receive cisplatin-based chemotherapy: baseline interleukin-8 and tumor contrast enhancement as potential predictive factors of activity. Ann. Oncol. 22, 2646–2653 (2011).

157. Galsky, M. D. et al. Gemcitabine, cisplatin, and sunitinib for metastatic urothelial carcinoma and as preoperative therapy for muscle-invasive bladder cancer. Clin. Genitourin. Cancer 11, 175–181 (2013).

158. Grivas, P. et al. Randomized phase II trial of maintenance sunitinib versus placebo following response to chemotherapy (CT) for patients (pts) with advanced urothelial carcinoma (UC) [abstract]. J. Clin. Oncol. 30 (Suppl.), a265 (2012).

159. Necchi, A. et al. Pazopanib in advanced and platinum-resistant urothelial cancer: an open-label, single group, phase 2 trial. Lancet Oncol. 13, 810–816 (2012).



162. Choueiri, T. K. et al. Double-blind, randomized trial of docetaxel plus vandetanib versus docetaxel plus placebo in platinum-pretreated metastatic urothelial cancer. J. Clin. Oncol. 30, 507–512 (2012).

163. Hahn, N. M. et al. Phase II trial of cisplatin, gemcitabine, and bevacizumab as first-line therapy for metastatic urothelial carcinoma: Hoosier Oncology Group GU 04–75. J. Clin. Oncol. 29, 1525–1530 (2011).


165. Balar, A. V. et al. Phase II study of gemcitabine, carboplatin, and bevacizumab in patients with advanced unresectable or metastatic urothelial cancer. J. Clin. Oncol. 31, 724–730 (2013).




169. Richardson, P. G. et al. Inhibition of heat shock protein 90 (HSP90) as a therapeutic strategy for the treatment of myeloma and other cancers. Br. J. Haematol. 152, 367–379 (2011).

170. Kamada, M. et al. Hsp27 knockdown using nucleotide-based therapies inhibit tumor growth and enhance chemotherapy in human bladder cancer cells. Mol. Cancer Ther. 6, 299–308 (2007).

171. Kanagasabai, R. et al. Hsp27 protects adenocarcinoma cells from UV-induced apoptosis by Akt and p21-dependent pathways of survival. Mol. Cancer Res. 8, 1399–1412 (2010).

172. Concannon, C. G., Gorman, A. M. & Samali, A. On the role of Hsp27 in regulating apoptosis. Apoptosis 8, 61–70 (2003).

173. Bruey, J. M. et al. Hsp27 negatively regulates cell death by interacting with cytochrome c. Nat. Cell Biol. 2, 645–652 (2000).

174. Hayashi, N. et al. Hsp27 silencing coordinately inhibits proliferation and promotes Fas-induced apoptosis by regulating the PEA-15 molecular switch. Cell Death Differ. 19, 990–1002 (2012).

175. Garg, M. et al. Heat-shock protein 70–72 (HSP70–2) expression in bladder urothelial carcinoma is associated with tumour progression and promotes migration and invasion. Eur. J. Cancer 46, 207–215 (2010).



178. Sonpavde, G. & Choueiri, T. K. Biomarkers: the next therapeutic hurdle in metastatic renal cell carcinoma. Br. J. Cancer 107, 1009–1016 (2012).

179. Patel, P. H. et al. Hypoxia-inducible factor (HIF) 1a and 2a levels in cell lines and human tumor predicts response to sunitinib in renal cell carcinoma (RCC) [abstract]. J. Clin. Oncol. 26 (Suppl. 15), a5008 (2008).

180. McDermott, D. F. et al. The high-dose aldesleukin (HD IL-2) ‘SELECT’ trial in patients with metastatic renal cell carcinoma [abstract]. J. Clin. Oncol. 28 (Suppl.), a4514 (2010).

181. Rini, B. I. et al. Antitumor activity and biomarker analysis of sunitinib in patients with bevacizumab-refractory metastatic renal cell carcinoma. J. Clin. Oncol. 26, 3743–3748 (2008).

182. Hutson, T. E. et al. Biomarker analysis and final efficacy and safety results of a phase II renal cell carcinoma trial with pazopanib (GW786034), a multi-kinase angiogenesis inhibitor [abstract]. J. Clin. Oncol. 26 (Suppl.), a5046 (2008).

183. Tran, H. T. et al. Prognostic or predictive plasma cytokines and angiogenic factors for patients treated with pazopanib for metastatic renal-cell cancer: a retrospective analysis of phase 2 and phase 3 trials. Lancet Oncol. 13, 827–837 (2012).

184. Zurita, A. J. et al. A cytokine and angiogenic factor (CAF) analysis in plasma for selection of sorafenib therapy in patients with metastatic renal cell carcinoma. Ann. Oncol. 23, 46–52 (2012).

185. Kim, J. J. et al. Association of VEGF and VEGFR2 single nucleotide polymorphisms with hypertension and clinical outcome in metastatic clear cell renal cell carcinoma patients treated with sunitinib. Cancer 118, 1946–1954 (2012).

186. Rini, B. I. et al. Diastolic blood pressure as a biomarker of axitinib efficacy in solid tumors. Clin. Cancer Res. 17, 3841–3849 (2011).

187. Wulfing, C. et al. A single-arm, multicenter, open-label phase 2 study of lapatinib as the second-line treatment of patients with locally advanced or metastatic transitional cell carcinoma. Cancer 115, 2881–2890 (2009).

Author contributionsAll the authors researched data for the article, made a substantial contribution to the discussion of the content, wrote the article and reviewed and edited it prior to submission.

REVIEWS




























Documents

Molecular targets on the horizon for kidney and urothelial cancer