The prospect of precision therapy for renal cell carcinoma

Cancer Treatment Reviews 49 (2016) 37–44

Contents lists available at ScienceDirect

Cancer Treatment Reviews journal homepage: www.elsevierhealth.com/journals/ctrv

Anti-Tumour Treatment

The prospect of precision therapy for renal cell carcinoma Chiara Ciccarese a, Matteo Brunelli b, Rodolfo Montironi c, Michelangelo Fiorentino d, Roberto Iacovelli a, Daniel Heng e, Giampaolo Tortora a, Francesco Massari f,⇑ a

Medical Oncology, Azienda Ospedaliera Universitaria Integrata, University of Verona, Verona, Italy Department of Pathology and Diagnostic, Azienda Ospedaliera Universitaria Integrata (AOUI), University of Verona, Verona, Italy c Section of Pathological Anatomy, Polytechnic University of the Marche Region, School of Medicine, AOU Ospedali Riuniti, Ancona, Italy d Pathology Service, Addarii Institute of Oncology, S-Orsola-Malpighi Hospital, Bologna, Italy e Department of Medical Oncology, Tom Baker Cancer Centre, University of Calgary, Calgary, AB, Canada f Division of Oncology, S.Orsola-Malpighi Hospital, Bologna, Italy b

a r t i c l e

i n f o

Article history: Received 9 May 2016 Received in revised form 1 July 2016 Accepted 4 July 2016

Keywords: Metastatic renal cell carcinoma Clear cell renal cell carcinoma Non-clear cell renal cell carcinoma Precision medicine Personalized medicine Targeted therapy TKI mTOR PD-1 PD-L1

a b s t r a c t The therapeutic landscape of renal cell carcinoma (RCC) has greatly expanded in the last decade. From being a malignancy orphan of effective therapies, kidney cancer has become today a tumor with several treatment options. Renal cell carcinoma (RCC) is a metabolic disease, being characterized by the dysregulation of metabolic pathways involved in oxygen sensing (VHL/HIF pathway alterations and the subsequent up-regulation of HIF-responsive genes such as VEGF, PDGF, EGF, and glucose transporters GLUT1 and GLUT4, which justify the RCC reliance on aerobic glycolysis), energy sensing (fumarate hydratasedeficient, succinate dehydrogenase-deficient RCC, mutations of HGF/MET pathway resulting in the metabolic Warburg shift marked by RCC increased dependence on aerobic glycolysis and the pentose phosphate shunt, augmented lipogenesis, and reduced AMPK and Krebs cycle activity) and/or nutrient sensing cascade (deregulation of AMPK-TSC1/2-mTOR and PI3 K-Akt-mTOR pathways). In this complex scenario it is important to find prognostic and predictive factors that can help in decision making in the treatment of mRCC. Ó 2016 Elsevier Ltd. All rights reserved.

Introduction The therapeutic landscape of renal cell carcinoma (RCC) has greatly expanded in the last decade. From being a malignancy orphan of effective therapies, kidney cancer has become today a tumor with several treatment options. Angiogenesis – a dynamic process required to sustain tumor cells growth and metastatic spread, mediated by multiple proangiogenic factors (of which VEGF is certainly the most important) and influenced by the tumor microenvironment – is the hallmark of ccRCC. Therefore, angiogenesis represents one of the key target for therapy, explaining the antitumor activity of anti-VEGF targeted agents (VEGFR tyrosine-kinase inhibitors – sunitinib [1], pazopanib [2,3], axitinib [4], and sorafenib [5] –, and the antiVEGF monoclonal antibody – bevacizumab [6]). In addiction, tumor growth relies on the mTOR pathway hyperactivation, justifying the

⇑ Corresponding author. E-mail address: [email protected] (F. Massari). http://dx.doi.org/10.1016/j.ctrv.2016.07.003 0305-7372/Ó 2016 Elsevier Ltd. All rights reserved.

efficacy of mTOR-inhibitors (temsirolimus [7] and everolimus [8]). More recently, it has awakened an old interest in the role of the immune system in controlling RCC cancerogenesis and progression. The immune checkpoint inhibitor targeting the PD-1/PD-L1 axis – nivolumab –, by stimulating the hosts’ antitumor immunity, represents one of the main oncological breakthroughs, causing impressive long-lasting responses and significantly prolonging overall survival (OS) of RCC patients [9]. Choosing from the available therapies maximizing the efficacy and minimizing the toxicity is the future challenge. Identifying predictors of response leading to a personalized therapy represents the main goal of cancer research. What drives decision making in the treatment of RCC patients?

Tumor histology Clinicians are used to classify RCC based on tumor histology, distinguishing the most frequent clear cell RCC type (accounting singly for about 70–85% of renal tumors) from the other RCC sub-

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types, which are simplistically grouped as non-clear cell RCC (nccRCC). Actually, the bulk container of nccRCC tumors includes multiple different malignancies profoundly diverse in terms of morphological and immunohistochemical features, molecular genetic profile, clinical behavior and prognosis [10–12]. Current treatment recommendations for nccRCC patients are borrowed from evidence available for ccRCC, lacking robust direct evidence of effective therapies for nccRCC patients that are generally excluded or underrepresented in pivotal clinical trials testing the novel compounds [13]. To date, the two most recent and large prospective studies (ESPN and ASPEN) conducted in patients with non-clear-cell RCC compared the use of sunitinib to everolimus, failed in demonstrating the superiority of the mTOR-inhibitor over the VEGFR-targeted agent [14,15]. Both trials demonstrated a prolonged PFS for first-line sunitinib (mPFS 8.3 vs 5.6 months and 6.1 vs 4.1 months in the ASPN and ESPN trials respectively), with everolimus therapy providing benefit only in certain subgroups of patients (poor risk and chromophobe subtypes), but this evidence is not sufficient to recommend everolimus as preferable option in poor risk and chromofobe subtypes. It is important to point out that, regardless of the type of therapy (sunitinib or everolimus), non-clear cell RCC histotypes display shorter PFS times and lower RRs than the clear cell RCC counterpart, reinforcing the acknowledge worse outcome of nccRCC patients treated with VEGF- and mTOR-targeted therapies compared to ccRCC patients (mOS 22.3 vs 12.8 months; p < .0001) [16]. Clinical prognostic factors To date, the only validated systems for prognostically stratifying patients with metastatic renal cell carcinoma rely on the evaluation of clinical factors, since no molecular biomarkers with a prognostic or predictive value have been identified so far. The Memorial Sloan-Kettering Cancer Center (MSKCC) risk model categorizes patients with metastatic RCC treated with interferon-alfa as first-line systemic therapy into three risk groups based on five pretreatment clinical factors prognostic of short survival (Karnofsky performance status <80%, serum lactate dehydrogenase >1.5 times upper limit of normal [ULN], low serum hemoglobin, ‘‘corrected” serum calcium >10 mg/dL, and time from initial RCC diagnosis to start of systemic therapy of less than one year) [17,18]. The median survival times range from approximately 5 months for poor risk patients (with 3 or more risk factors) to more than 29 months for patients with a good prognosis (with no risk factors) [18]. An independent group at the Cleveland clinic subsequently validated the MSKCC criteria, by using a data set of 353 patients enrolled on clinical trials involving immunotherapy [19]. Of note, the MSKCC prognostic risk model was developed in the era of immunotherapy, and limited to patients eligible for participation in immunotherapy clinical trials. The approval of VEGFR-targeted agents had subsequently required a novel prognostic system capable of better stratifying patients in clinical trials, providing clinical information to patients receiving therapy, and helping risk-directed treatment selection in daily clinical practice in the era of targeted therapy. Heng et al. identified a prognostic model (the IMDC risk score) composed of two clinical (Karnofsky performance status less than 80%, time from diagnosis to treatment of less than one year) and four laboratory values (hemoglobin less than lower limit of normal, corrected calcium greater than ULN, neutrophils greater than ULN, and platelets greater than ULN) able to stratify patients into favorable (43.2 mOS months), intermediate (22.5 mOS months), and poor prognosis groups (7.8 mOS months) [20,21]. Of note, the IMDC prognostic model reliably predicts OS not only in ccRCC patients, but also in non-clear cell RCC histology [16] (Table 1).

Table 1 Heng and MSKCC prognostic factors. Modified MSKCC prognostic factors  LDH > 1.5  upper limit of normal  Corrected Calcium > 10 mg/dL  Time from diagnosis to first treatment < 1 year  Karnofsky performance status 60–70  Multiple organ sites metatsasis

Heng prognostic factors  Hemoglobin less than lower limit of normal  Corrected calcium above the upper limit of normal  Time from diagnosis to treatment < 1 year  Karnovsky performance status < 80%  Platelets greater than the upper limit of normal  Neutrophils greater than the upper limit of normal

LDH: lactate dehydrogenase; MSKCC: Memorial Sloan Kettering Cancer Center.

Molecular-based classification of renal cell carcinoma Efforts are directed at delineating signaling pathways underlying clear cell and non-clear cell RCC carcinogenesis, possibly identifying driven-mutations as potential targets for therapy. The comprehensive molecular characterization of clear cell RCC conducted by the cancer genome Atlas research network represented a fundamental step towards the deep understanding of RCC carcinogenesis [22]. In particular, the whole exome sequencing identified 19 significantly mutated genes, with VHL, PBRM1, SETD2, KDM5C, PTEN, BAP1, MTOR and TP53 representing the 8 most extreme members. As regard the DNA methylation profiles, epigenetic silencing involved VHL in 7% of cases and the tumor suppressor UQCRH gene in 36%. Moreover, mutations in the specific epigenetic modifier SETD2 (H3K36 methyltransferase) determined widespread DNA hypomethylation. Unsupervised clustering methods identified four stable subsets in both mRNA (m1–m4) and miRNA (mi1–mi4) expression datasets: the m1-subtype with PBRM1 mutations, the m3-subtype with deletion of CDKN2A and mutations of PTEN, and the m4-subtype with mutations of BAP1 and mTOR. Integrative pathway analysis supported the importance of the VHL/HIF pathway, the key role of PI3K/AKT in tumor progression, and the role of chromatin modifier genes in renal tumorigenesis. In particular, alterations in SWI/SNF chromatin remodeling complex (PBRM1, ARID1A, SMARCA4) could have far-reaching effects on other pathways. Chromosome 3p-encoded chromatin remodeling tumor suppressor genes, SETD2, PBRM1 and BAP1 are frequently mutated in ccRCC (respectively in about 15%, 40%, and 15% of cases) [22,23]. Of note, BAP1 and PBRM1 mutations, which are mutually exclusive [24], identify new distinctive classes of RCC with different clinical behavior: a poor-prognosis BAP1-mutant group and a favorable PBRM1-mutant group [25]. Finally, the ATLAS ccRCC characterization identified a specific subtype of ccRCC, with aggressive behavior and poor prognosis, marked by a metabolic shift (Warburg-effect – tumor dependence on aerobic glycolysis) characterized by increased dependence on the pentose phosphate shunt, increased glutamine transport, decreased AMPK and Krebs cycle activity, and increased fatty acid production [22,26]. Dysregulation of cellular metabolic pathways involved in oxygen, energy and/or nutrient sensing is a peculiar feature of ccRCC, offering new opportunities for disease treatment. A substantial contribution in understanding the genetic basis of nccRCC comes from familiar studies of hereditary tumors, marked by germline mutations in specific oncogenes or onco-suppressors. These hereditary cancer syndromes are paradigmatic circumstances in which a specific gene mutation, pathognomonic of a definite histotype, can translate into a definite therapeutic target:

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– MET in Hereditary Papillary Renal Carcinoma (HPRC) type 1: autosomal dominant hereditary syndrome marked by germline activating missense mutations in the tyrosine kinase domain of the MET proto-oncogene (the receptor for hepatocyte growth factor – HGF) at chromosome 7q31. HPRC is clinically characterized by a predisposition to multiple, bilateral papillary type 1 renal tumors [27,28] (Fig. 1). – FH in Hereditary Leiomyomatosis and Renal Cell Carcinoma (HLRCC) cancer syndrome: germ-line inactivating mutations (missense, frameshift, or complete or partial deletion) of the fumarate hydratase (FH) gene, mapped to chromosome 1q42.3-q43, characterize the HLRCC syndrome that is associated with a predisposition to cutaneous and uterine leiomyomas and a particularly form of type II papillary renal cancer with an aggressive behavior despite small primary tumor size [29] (Fig. 2). – SDH in succinate dehydrogenase deficient kidney cancer (SDHRCC): germ-line mutations of multiple subunits (SDHB/C/D) of the Krebs cycle enzyme, succinate dehydrogenase, mark the SDH-RCC hereditary cancer syndrome – a novel form of aggressive kidney cancer. SDH mutation, resulting in increased succinate levels causing prolyl hydroxylase inhibition and HIF accumulation, represents a peculiar example of the Warburgeffect in RCC [30,31]. – FLCN in Birt-Hogg-Dubé (BHD) syndrome: an autosomal dominant inherited genodermatosis, determined by germ-line mutations in the folliculin (FLCN) tumor suppressor gene, and clinically characterized by high risk for developing cutaneous fibrofolliculomas, pulmonary cysts, spontaneous pneumothorax, and kidney tumors (including the benign neoplasm oncocytoma, its malignant variant – the eosinophilic chromophobe RCC, and hybrid oncocytic tumors) [32–34]. – PTEN in Cowden disease: germ-line mutation of the tumor suppressor gene phosphatase and tensin homolog (PTEN), mapped to chromosome 10q23.3, is the hallmark of the Cowden disease, a hereditary autosomal dominant cancer syndrome associated with dermatologic manifestations and multiple types of cancers including RCC of different histotypes (clear cell, chromophobe and papillary RCC) [35]. – TSC1 and TSC2 in tuberous sclerosis complex (TSC) syndrome: autosomal dominant syndrome caused by germ-line mutations in TSC1 and TSC2 genes, with subsequent constitutive activation of the LKB1/AMPK/TSC/mTOR signaling pathway. Clinically, it manifests as cutaneous angiofibroma, pulmonary lymphangiomyomatosis and renal tumors of different histotypes, includ-

Fig. 1. Renal cell carcinoma: papillary type 1.

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Fig. 2. Renal cell carcinoma: papillary type 2.

ing angiomyolipoma (and its potentially malignant mesenchymal counterpart – epithelioid angiomyolipoma, EAML), clear cell RCC, papillary RCC and unclassifiable RCCs [36]. – MITF in MiT tumors: a specific MITF germline mutation (MiE318 K) encoding for the MITF transcription, leading to the up-regulation of key proto-oncogenes like MET and HIF1a, seems to predispose to co-occurence of melanoma and kidney cancer [37]. Recently, a key step towards the molecular characterization of the different RCC histotypes comes from the results of comprehensive genomic analyses of human primary nccRCC samples using next-generation sequencing technologies. nccRCC carries a higher average of protein-coding alterations compared to ccRCC. Moreover, among nccRCC, chomophobe RCC (chRCC) and renal oncocytoma tumors have, on average, a significantly lower rate of somatic mutations than papillary RCC (pRCC) tumors [38]. Moreover, using RNA sequencing (RNA-seq)-based gene expression analysis a set of five genes (ASB1, GLYAT, PDZK1IP1, PLCG2 and SDCBP2) that enable the molecular classification of chRCC, renal oncocytoma and pRCC has been identified [38], paving the way towards a possible classification and molecular genetic diagnosis of kidney cancer. Five recurrently mutated genes (MET, NF2, SLC5A3, PNKD and CPQ) and six additional significantly mutated genes (AT1, BAP1, PBRM1, STAG2, NFE2L2, and TP53) have been identified in pRCC [39]. The alteration peculiar to type I pRCC involves c-MET. The direct activation of c-MET caused by mutations in the c-MET gene has been identified both in hereditary and sporadic pRCC. In particular, the majority of type I papillary tumors carries an altered MET status (mutations, amplification, overexpression, gene fusion, splice variants), or increased chromosome 7 copy number, reaching 80% of cases, with sporadic somatic MET mutations occurring in about 15–17% of pRCC [39]. The hyper-activation of HGF/MET pathway promotes tumor cell growth, invasion and angiogenesis through a cascade of downstream intracellular effectors: the activation of RAS-MAPK and PI3 K/Akt signaling pathways, the phosphorylation of STAT3 and JNK, and a cross-talk with EGFR and several membrane proteins (like Plexin-B, CD44, a6b4 integrin) that mediates a continuous integration with extracellular environment signals [40]. Therefore, a strong rational supports the investigation of compounds targeting cMET-pathway for the treatment of pRCC (espe-

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cially type I pRCC carrying MET activating mutations). Several MET-pathway antagonists have been developed [41]. Foretinib, a multi-kinase inhibitor potently targeting MET, VEGFR, RON, AXL, and TIE-2 receptors demonstrated antitumor activity in a phase II trial enrolling 74 patients with pRCC, with response rate of 13.5% and median PFS of 9.3 months. The presence of a germ-line MET mutation was highly predictive of response [42]. Of note, a significant deletion has been identified in pRCC tumors – the loss of 1p136 – which includes the tumor suppressor ERRFI1 (negative regulator of EGFR), and co-occurs significantly with gain of Chr 7 and EGFR amplification [39]. This represents the rational supporting the expected augmented activity of combining a MET-inhibitor with an anti-EGFR agent. A randomized phase II trial evaluates tivantinib (an oral, non-ATP competitive, selective inhibitor of c-MET) alone or in combination with erlotinib hydrochloride in treating patients with metastatic or locally advanced pRCC (NCT01688973). On the other hand, specific alterations of type 2 pRCC include: – CDKN2A: about 13% of pRCC display loss of CDKN2A, which is related to focal loss of 9p21 or mutations or promoter hypermethylation of the CDKN2A gene [39]. The CDKN2A gene encodes two proteins, p16 (or p16INK4a) and p14ARF. Both act as tumor suppressors by negatively regulating the cell cycle. p16 inhibits cyclin dependent kinases 4 and 6 (CDK4 and CDK6), thereby activating the family of retinoblastoma (Rb) protein, which blocks crossing from G1 to S-phase. p14ARF activates the p53 tumor suppressor [43]. CDKN2A-alterated tumors are mainly type 2 pRCC, with a significantly lower rate of OS (in a univariate analysis) than those without CDKN2A-altererations [39]. – Chromatin-modifying genes (BAP1, SETD2, PBRM1): papillary type 2 RCCs are associated with alterations of chromatin remodeling tumor suppressor genes [39]. BAP1 is a deubiquitinating enzyme that regulates G1/S phase transition by binding to BRCA1 and BARD1 and inhibiting their ability to mediate ubiquitination and auto-ubiquitination [24]. SETD2 encodes for a histone methyltransferase. PBRM1 encodes for BAF180, a component of the SWI/SNF chromatin-remodeling complex, which plays a fundamental role in the regulation of DNA accessibility for transcription, cell-cycle regulation, cell differentiation and proliferation and TP53-mediated replicative senescence [44]. Altered chromatin-modifying genes in pRCC type 2 can result from point mutations (mutations of SETD2 and PBRM1 frequently co-occur, while BAP1 and PBRM1 mutations are mutually exclusive) or heterozygous or mild deletion of chromosome 3p (haploinsufficiency of these genes may be sufficient to promote tumorigenesis); no homozygous loss of chromosome 3p or promoter hypermethylation have been observed [39]. Therapeutic perspective under evaluation for BAP1-mutated tumors include: (1) Histone deacetylase (HDAC) inhibitors, given the accumulation of histone H2A ubiquitinated due to the loss of BAP1, masking transcription of various genes promoters. LBH589 (panobinostat), a pan-DAC inhibitor, was tested in a phase II trial enrolling refractory renal carcinoma patients previously treated with TKI and mTOR inhibitors, with disappointing results (no objective responses, with all patients progressed or stopped treatment prior to the 16-week revaluation) [45]. Analogously, no significant activity has been demonstrated by FK228 (depsipeptide), an HDAC inhibitor, tested in a phase II trial in refractory metastatic renal cell cancer [46]. Several trials are currently testing different HDAC inhibitors in RCC patients (Vorinostat [NCT00278395], MS-275 [NCT00020579], Belinostat [NCT00413075], Entinostat [NCT01038778]). (2) PARP-inhibitors, since the loss of BAP1 sensitizes tumor cells to DNA repair defects to induce tumor cells lethality. An ongoing phase I study is evaluating the com-

bination of olaparib and AZD5363, an AKT inhibitor, in patients with advanced solid tumors including RCC refractory to standard therapy (NCT02338622). Interestingly, PARP-inhibitors seem to exert a remarkable anti-tumor activity in a specific histopathologic variant of pRCC – labeled oncocytic pRCC (opRCC) – that carries mutations on the Ataxia Telangiectasia Mutated (ATM) tumor suppressor gene [47]. Since about 3% of kidney cancer has ATM mutations [48], PARP inhibitors could represent a therapeutic strategy selected to this specific molecular subgroup of tumors. – NRF2-ARE: the transcription factor NFE2-related factor 2 (NRF2) directly controls several transcriptional programs after integrating cellular stress signals. Under basal conditions, NRF2 is kept transcriptionally inactive by binding to its inhibitor complex, KEAP1 and CUL3 that mediate its proteasomal degradation. In response to cellular stressors (ROS, H2O2, NO), the conformational change of KEAP1 impairs the degradation of NRF2, which binds to antioxidant response elements (AREs) and induces the transcription of several genes involved in cell differentiation and proliferation, inflammation, apoptosis, and tissue regeneration (NOTCH1, NF-jB, p53, mTOR, AP1, AhR). Activating mutations of NRF2 (or loss-of-function mutations of KEAP1, KRAS, BRAF and MYC genes), by increasing NRF2 activity, promote carcinogenesis and mediate chemotherapy resistance [49]. The activation of NRF2-ARE pathway, and the subsequent increased expression of NQO1 gene, is a distinguishing feature of type 2 pRCC tumors, being associated with decreased survival [39]. A promising therapeutic approach developed for targeting this pathway concerns the inhibition of ABL1, given the stimulating effects of ABL1 on NRF2-dependent antioxidant response pathway in oxidatively stressed tumors (i.e. FH-deficient tumors) [50]. – NF2 and the Hippo pathway: mutations of neurofibromin 2 (NF2), occurring in 10% of pRCC type 2, result in aberrant signal of the Hippo pathway that is involved in the proliferation, invasion and metastatic spread of RCC [39]. In fact, NF2 encodes the MERLIN tumor suppressor protein that regulates the Hippo pathway, whose principal function is to negatively regulate the activity of two homologous transcriptional co-activators downstream effectors YAP and TAZ. Therefore, pharmacological inhibition of YAP and TAZ activity represents an attractive anticancer strategy [51]. – CpG Island Methylator Phenotype (CIMP): about 6% of Type 2 pRCC have increased DNA methylation at loci unmethylated in corresponding normal tissue, including the universal hypermethylation of the CDKN2A promoter. This peculiar phenotype is associated with germ-line or somatic mutation of FH gene, and a consequent Warburg-like metabolic shift to glycolysisdependent metabolism (increased expression of key genes involved in glycolysis [HK1, LDHA, and PDK1], pentose phosphate pathway [G6PD], and fatty-acid synthesis [FASN], decreased expression of Krebs cycle genes and AMPK complex, an inhibitor of fatty-acid synthesis) [39]. Therapeutic perspectives are directed toward the glycolysis inhibition: (1) Inhibition of the critic glycolytic enzyme lactate dehydrogenase-A (LDHA), which promotes fermentative glycolysis by converting pyruvate to lactate, resulting in ROS-mediated apoptosis of FHdeficient cells [26]. (2) Activation of AMPK, leading to increased fatty acid and protein synthesis in vitro and in vivo, by using metformin [52] or 5-aminoimidazole-4-carboxamide-1-b-D-ri bofuranoside (AICAR) [53]. (3) Up-regulation of ROS levels through the use of pro-oxidant molecules like the proteasome inhibitor bortezomib alone [54] or in combination with other anti-angiogenic agents. Although bortezomib has demonstrated a synergistic cytotoxic effect in vitro when combined to sorafenib [55], it failed in proving increased activity in vivo in a phase

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2 study [56]. 4) Inhibition of the glutaminase enzyme, key protein involved in the amino acid glutamine utilization to produce energy. An ongoing phase I trial is evaluating the role of CB-839, a potent and selective glutaminase inhibitor in patients with solid tumors (NCT02071862). Chromophobe RCC is associated with a peculiar aneuploidy pattern, resulting from specific loss of chromosomes 1, 2, 6, 10, 13, 17, and 21. TP53, PTEN, FAAH2, PDHB, PDXDC1 and ZNF765 have been found significantly mutated in ChRCC [38,58]. In addiction, a multidimensional and comprehensive characterization of chRCC, including mtDNA and whole-genome sequencing, revealed recurrent genomic structural rearrangements within TERT promoter region and elevated TERT expression [58]. Certainly, the recent RCC genomic classification represents the first window open on the future. Based on multidimensional and comprehensive molecular characterization (including DNA methylation and copy number, and RNA and protein expression) RCC of different histology were classified into 9 genomic subtypes: three different subtypes of predominantly clear cell RCC, four different subtypes of predominantly papillary RCC, one subtype of predominantly chromophobe tumor, and one subtype of mixed tumor. Notably, the site of tumor origin within the nephron was one of the main determinants. Specific molecular features distinguished the RCC genomic subtypes from each other. For example, within the three clear cell RCC subtypes, the loss of CDKN2A, higher expression of cell cycle genes, hypoxia-related genes, markers of epithelial-mesenchymal transition, immune checkpoint markers, and molecular signatures of T cell infiltrates characterized the more aggressive subtype. Moreover, alteration of specific pathways — hypoxia, metabolism, MAP kinase, NRF2-ARE, Hippo, immune checkpoint, PI3K/AKT/mTOR, chromatin modifier genes — could further discriminate the diverse molecular subtypes of kidney cancer [59]. Novel molecular targets for therapy? MET Activation of the cMET-pathway plays a crucial role also in nonpapillary RCC invasive growth and progression, being overexpressed in many clear cell RCC. Pre-clinical in vitro studies demonstrated an up-regulation of c-MET expression induced by loss of VHL expression and hypoxia in ccRCC [60,61]. Moreover, c-MET up-regulation has been implicated in the development of resistance to VEGFR inhibition [62]. In addiction, c-MET seems to be associated with poor pathologic features, aggressive tumor behavior and worse survival in RCC [63–65]. Therefore, a strong molecular rationale justifies the investigation of agents that recognize MET as target for the treatment of RCCs. Recently, Choueiri et al. demonstrated significant progression-free survival (mPFS 7.4 vs 3.8 months; HR 0.58, 95% CI 0.45–0.75, p < 0.001) and response rate (ORR 21 vs 5%, p < 0.001) advantage of cabozantinib – a tyrosine kinases inhibitor of MET, VEGF receptors, and AXL – compared to everolimus in previously treated ccRCC patients in a large randomized phase 3 trial (METEOR study) [66]. At the 2016 ASCO Genitourinary Cancers Symposium, Escudier presented subgroup analysis of this trial, establishing the persistent benefit of cabozantinib compared to everolimus in term of PFS across key subgroups, including MSKCC risk groups and patients ECOG performance status (greater impact of cabozantinib in ECOG PS 0 vs 1, and in good/ intermediate vs poor MSKCC risk factors patients), metastatic organ involvement and tumor burden (higher activity of cabozantinib in high compared to low tumor burden disease), and prior therapies (increased PFS with cabozantinib after a longer – >6 months – period of first VEGFR-TKI) [67]. The planned interim analysis showed a prolongation – not statistically significant – of

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the overall survival with cabozantinib (HR for death 0.67; 95% CI 0.51–0.89, p = 0.005), but final results of the OS analysis are highly expected [66]. MET is a potential therapeutic target also for Microphthalmia transcription factor (MITF)-associated (MiT) tumors. MiT tumors encompass a group of rare tumors (including alveolar soft part sarcoma [ASPS], clear cell sarcoma [CCS], and translocation-associated renal cell carcinoma [tRCC]) that are biologically marked by dysregulated expression of a family of homologous transcription factors including E-box binding transcription factors TFE3, TFEB, TFEC, and MITF2 as a consequence of somatic translocations generating active fusion proteins with MiT transcription factor activity [68]. Frequently, kidney cancer translocations involve the chromosome Xp11.2, causing gene fusions with the TFE3-gene [69]. Molecularly, TFE3-associated kidney cancer is characterized by hyper-expression of phosphorylated S6 (a downstream molecule of the mTOR pathway) and increased levels of stable HIF1a [70]. Therefore, VEGFR-targeted agents and mTOR-inhibitors are a valid option for Xp11 translocation/TFE3 fusion gene RCC patients [71]. Downstream effector genes transcriptionally regulated by MITF family proteins include B cell lymphoma 2 (BCL2), MET, and p21CIP – genes with a key role in regulating cell proliferation and survival [72,73]. Therefore, targeting the MET receptor tyrosine kinase – up-regulated by MITF, TFE3, and TFEB – represents a potential active therapeutic approach for MiT tumors [74]. However, tivantinib, a selective, non ATP-competitive, small-molecule inhibitor of MET, demonstrated modest antitumor activity in patients with advanced MiT tumors (only 6 patients with tRCC) in a single-arm phase 2 trial [75]. FGFR The pro-angiogenic fibroblast growth factor (FGF)/FGF-Receptor pathway plays a crucial role in promoting neo-angiogenesis (vessel assembly, sprouting and branching), lymphangiogenesis and tumor cellular growth [76–78]. Of note, high basic-FGF plasma levels are associated with high tumor grade and stage [79], metastatic diffusion [80] and poor prognosis [81] in RCC patients. Furthermore, FGF serum concentrations significantly increase in mRCC resistant to Sunitinib therapy, supporting the role of FGFpathway in driving VEGF-independent tumor angiogenesis, as a compensatory mechanism to escape VEGF-inhibition [82,83]. Therefore, FGF/FGFR pathway represents an interesting target for therapy after progression on VEGF-inhibitors therapy. Several VEGFR- and FGFR-inhibitors have been developed for the treatment of RCC, with conflicting results [84]:  Dovitinib, a TKI of FGFR, PDGRF and VEGFR, did not demonstrate a PFS and OS advantage compared to sorafenib in mccRCC patients progressing to VEGFR- and mTOR- inhibitors [85]. Probably, the wrong timing of FGF inhibition (immediately after an mTOR inhibitor giving the time to restore a VEGF-driven angiogenesis) – and not the wrong target - is the cause of the GOLD trial failure [86]. A phase 2 study (DILIGENCE-1) is ongoing, testing the activity of dovitinib as first-line therapy for mRCC (NCT01791387).  The importance of blocking FGFR immediately after the occurrence of VEGFR-TKIs resistance has been reinforced by the remarkable results of another FGFR-inhibitor, lenvatinib. Single-agent lenvatinib (and even more the combination of lenvatinib plus everolimus) improved response rate and PFS compared to everolimus (mPFS 5.5 vs 7.4 vs 14.6 months) in 153 mRCC patients who have progressed after one prior VEGFtargeted therapy in a randomized phase 2 study [87]. A phase III randomized trial evaluating this combination is planned to confirm these promising results.

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 Brivanib, a dual VEGFR-2 and FGFR-1 tyrosine kinases inhibitor, demonstrated promising antitumor activity in advanced solid tumor patients [88]. A phase II, open-label trial is assessing the activity of brivanib in refractory mRCC patients (NCT01253668).  Regorafenib (BAY 73-4506), a multikinase inhibitor targeting VEGFR, c-kit, RET, FGFR, PDGFR, RAF and p38MAPK, showed promising antitumor activity as first-line treatment (27% PR and 42% SD) in 49 previously untreated advanced RCC patients, albeit with a significant toxicity (35% of drug-related serious adverse events) [89].  XL999, a small molecule inhibitor of VEGFR, PDGFR, FGFR, FLT3, and Src, was evaluated in mRCC patients after failure of one anti-VEGF therapy (NCT00277316). The study was stopped early due to the cardiac toxicities, impeding further development of this molecule.  E7090 is under evaluation in a phase 1 trial recruiting advanced solid tumor patients, especially with malignancies characterized by genetic abnormalities of FGF/FGFR pathway (NCT02275910). PD-1/PD-L1 RCC is an immunogenic tumor, with a prominent dysfunctional immune cell infiltrate unable to counteract tumor growth [90]. The interaction between programmed death 1 (PD-1) – expressed on Tcells – and its ligand PD-L1 or PD-L2 – aberrantly expressed on cancer cells, results in inhibition of the cellular immune response (Fig. 3). Several studies have identified an interesting correlation between tumor cell expression of PD-L1 and ccRCC advanced grade, stage, and poor prognosis [91,92]. Recently, the monoclonal antibody directed against programmed death 1 (PD-1) nivolumab, by restoring antitumor immunity, significantly improved overall survival (mOS 25.0 vs 19.6 months; HR 0.73 [98.5%CI0.57–0.93]; p = 0.002), objective response rate (ORR 25 vs 5%) with peculiar long-lasting responses, and quality of life compared to everolimus in pre-treated mccRCC patients with an excellent safety profile [9]. Unlike the expectations, the improved overall survival in response to nivolumab therapy was not correlated to PD-L1 expression [9]. Therefore, PD-L1 can be considered a poor prognostic factor in mRCC, but not a biomarker predictive of treatment efficacy. At the 2016 ASCO Genitourinary Cancers Symposium a promising genomic study of whole exome and transcriptome (RNAseq) sequencing of RCC samples from patients treated with nivolumab showed, although in a small cohort (7 mRCC patients), a correlation between the expression of tumor neo-antigens resulted from nonsynonymous somatic mutations and tumor objective response to nivolumab, assuming a predictive value of treatment benefit [93].

Fig. 3. Anti-tumor activity of anti-PD-1 and anti-PD-L1 antibodies.

Immune checkpoint inhibitors could represent a therapeutic promise also in nccRCC even considering the correlation between PD-L1 expression and worse clinical outcome even in non-clear cell histotypes [94]. A promising therapeutic strategy currently under evaluation concerns the possible combination of anti-PD-1 with other different molecules. In particular, encouraging clinical activity and acceptable safety have been reported with combination therapy of nivolumab and ipilimumab (a fully human monoclonal antibody to CTLA-4) in mRCC in a phase I trial [95]. A phase III trial is currently assessing the efficacy of combining nivolumab + ipilimumab in first-line mRCC (NCT02231749). Similarly, the combination of PD-1 inhibition (nivolumab) and VEGF blockade (sunitinib or pazopanib) showed promising antitumor activity, with durable responses and an acceptable toxicity profile (mainly renal and hepatic toxicity) in a phase I open-label study [96]. Interestingly, a significant relationship between von Hippel–Lindau mutations (frequently occurring in RCC), HIF-2a stabilization, and PD-L1 expression in ccRCC reported in a recent study reinforced the hypothesis of a close interplay between angiogenesis and PD-1 pathway, fostering further investigations about the dual VEGF/ PD-1 inhibition and/or the optimal sequence for VEGF and PD-1 inhibition in mRCC patients [97]. A phase 3 randomized trial is evaluating the anti-tumor activity and safety of avelumab (antiPD-1 antibody) in combination with axitinib compared to sunitinib monotherapy as first-line treatment (NCT02684006). Moreover, the combination of PD-L1 and VEGF-inhibition is object of study in a phase III trial comparing atezolizumab plus bevacizumab versus sunitinib in previously untreated mRCC patients (NCT02420821). Conflict of interest All authors declare that they have no conflicts of interest. Financial disclosures None for all authors. References [1] Motzer RJ, Hutson TE, Tomczak P, Michaelson MD, Bukowski RM, Rixe O, et al. Sunitinib versus interferon alfa in metastatic renal-cell carcinoma. N Engl J Med 2007;356(2):115–24. [2] Sternberg CN, Davis ID, Mardiak J, Szczylik C, Lee E, Wagstaff J, et al. Pazopanib in locally advanced or metastatic renal cell carcinoma: results of a randomized phase III trial. J Clin Oncol 2010;28(6):1061–8. [3] Motzer RJ, Hutson TE, Cella D, Reeves J, Hawkins R, Guo J, et al. Pazopanib versus sunitinib in metastatic renal-cell carcinoma. N Engl J Med 2013;369 (8):722–31. [4] Motzer RJ, Escudier B, Tomczak P, Hutson TE, Michaelson MD, Negrier S, et al. Axitinib versus sorafenib as second-line treatment for advanced renal cell carcinoma: overall survival analysis and updated results from a randomised phase 3 trial. Lancet Oncol 2013;14(6):552–62. [5] Escudier B, Eisen T, Stadler WM, Szczylik C, Oudard S, Siebels M, et al. Sorafenib in advanced clear-cell renal-cell carcinoma. N Engl J Med 2007;356(2):125–34. [6] Escudier B, Pluzanska A, Koralewski P, Ravaud A, Bracarda S, Szczylik C, et al. Bevacizumab plus interferon alfa-2a for treatment of metastatic renal cell carcinoma: a randomised, double-blind phase III trial. Lancet 2007;370 (9605):2103–11. [7] Hudes G, Carducci M, Tomczak P, Dutcher J, Figlin R, Kapoor A, et al. Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. N Engl J Med 2007;356(22):2271–81. [8] Motzer RJ, Escudier B, Oudard S, Hutson TE, Porta C, Bracarda S, et al. Efficacy of everolimus in advanced renal cell carcinoma: a double-blind, randomised, placebo-controlled phase III trial. Lancet 2008;372(9637):449–56. [9] Motzer RJ, Escudier B, McDermott DF, George S, Hammers HJ, Srinivas S, et al. Nivolumab versus everolimus in advanced renal-cell carcinoma. N Engl J Med 2015;373(19):1803–13. [10] Moch H, Cubilla AL, Humphrey PA, Reuter VE, Ulbright TM. The 2016 WHO classification of tumours of the urinary system and male genital organs-part A: renal, penile, and testicular tumours. Eur Urol 2016. http://dx.doi.org/10.1016/ j.eururo.2016.02.029. pii: S0302-2838(16)00206-2 [Epub ahead of print].

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