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Pharmacogenomics
MET: a new promising biomarker in non-small-cell lung carcinoma
Non-small-cell lung cancer (NSCLC) leads cancer-related deaths worldwide. Mutations in the kinase domain of the EGFR gene provide sensitivity to tyrosine kinase inhibitors (TKI) drugs. TKI show initial response rates over 75% in mutant EGFR-NSCLC patients, although most of these patients acquire resistance to EGFR inhibitors after therapy. EGFR-TKI resistance mechanisms include amplification in MET and its ligand, and also MET mutations. MET signaling dysregulation has been involved in tumor cell growth, survival, migration and invasion, angiogenesis and activation of several pathways, therefore representing an attractive target for anticancer drug development. In this review, we will discuss MET-related mechanisms of EGFR-TKI resistance in NSCLC, as well as the main drugs targeted to inhibit MET pathway. Keywords: EGFR • MET • pharmacogenetics • resistance • targeted therapies • TKI
Background Lung cancer currently constitutes a serious health problem. According to the latest statistics published, more than 224,210 cases are diagnosed each year only in the USA [1] . In 2013, lung cancer ranked as the second tumor in incidence for men and women (after prostate and breast cancer, respectively) [1] . The incidence is similar in both genders, being approximately 14% in men (116,000) and women (108,210) [1] . This tumor presents a very high mortality, since the relative survival rate at 5 years does not exceed 10% in most countries, and therefore it is generally considered that the overall mortality figures are close to those of incidence. It represents the leading cause of cancer death in both genders in the USA, up to 28% in males (86,930) and 26% in women (72,330) [1] . Lung cancer is classified in two types, small-cell-lung carcinoma (SCLC), which accounts for approximately 15% of cases, and non-small-cell lung carcinoma (NSCLC), which represents the remaining 85%, and presents different subtypes: squamous cell or epidermoid carcinoma, adenocarcinoma and large cell carcinoma. At the time of diag-
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nosis, approximately 70% of patients with NSCLC are metastatic, staged IV according to the AJCC, therefore incurable [2–4] . The standard treatment for advanced or recurrent NSCLC is platinum-based chemotherapy, however, its results are unsatisfactory with a response rate (RR) of 17–32%, progression-free survival (PFS) of 3.1–3.5 months and overall survival (OS) of 7.4–11.3 months [5–8] . The identification of mutations that contribute to the pathogenesis of NSCLC has contributed to improved prognosis, classification and management of this disease. The EGFR is the most frequently mutated and overexpressed gene in NSCLC. EGFR mutations are more common in women, Asians and nonsmokers with the adenocarcinoma subtype [9–12] . The most common EGFR mutations are deletions in exon 19 and a point mutation in exon 21, located in the kinase domain, which replaces an arginine for a leucine at codon 858 (L858R) [9–12] . The kinase domain also hosts a rare mutation in exon 18, consisting in a substitution of glycine for alanine, cysteine or serine at codon 719 (G719X) [9–12] .
Pharmacogenomics (Epub ahead of print)
Cristina Pérez-Ramírez1,2, Marisa Cañadas-Garre*,1, Enrique Jiménez-Varo1,3, María José Faus-Dáder2 & Miguel Ángel CallejaHernández1,3 Pharmacogenetics Unit, UGC Provincial de Farmacia de Granada, Instituto de Investigación Biosanitaria de Granada, Complejo Hospitalario Universitario de Granada, Avda Fuerzas Armadas, 2, 18014 Granada, Spain 2 Department of Biochemistry, Faculty of Pharmacy, University of Granada, Campus Universitario de Cartuja, s/n, 18071 Granada, Spain 3 Department of Pharmacology, Faculty of Pharmacy, University of Granada, Campus Universitario de Cartuja, s/n, 18071 Granada, Spain *Author for correspondence: Tel.: +34 9580 20108 Fax: +34 9010 21804 marisacgarre@ gmail.com 1
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Review Pérez-Ramírez, Cañadas-Garre, Jiménez-Varo, Faus-Dáder & Calleja-Hernández The clinical interest of the determination of mutations in the kinase domain of the EGFR gene lies on the prediction of sensitivity of these tumors to tyrosine kinase inhibitors (TKI), such as erlotinib (Tarceva®, Roche) and gefitinib (Iressa®, AstraZeneca), drugs which have shown 75% response rates in NSCLC patients [13–15] . Despite this initial good response, about 75% of NSCLC patients with mutations in EGFR acquire resistance to EGFR-TKIs after a mean period of 12 months [16–20] . Many causes of resistance to EGFR-TKIs have been described. The most studied are a secondary point mutation in exon 20 of EGFR (T790M), which replaces methionine for threonine at amino acid position 790, MET protooncogene (MET) amplification and protein overexpression of its ligand, HGF, PTEN loss, KRAS mutations, BRAF mutations, HER-2 mutations, epithelial-to-mesenchymal transition (EMT), AXL overexpression and small-cell transformation [21–32] . Among them, one of the most investigated causes for EGFR-TKI resistance is MET amplification and HGF overexpression, whose potential role in several processes as cell survival, proliferation, motility, invasiveness and angiogenesis makes them attractive therapeutic targets [33] . In NSCLC, abnormalities in MET signaling pathway have been associated with poor clinical outcome and drug resistance [28,29,34–37] . Therefore, single or combined MET inhibition by targeted drugs represents a promising strategy to approach resistance in tumors with dysregulated MET signaling. MET structure & functions MET gene, located on chromosome 7q21-q31, was discovered in 1984 as one of the components of the translocated promoter region (TPR)-MET fusion oncoprotein in an osteosarcoma immortalized cell line [38] . MET gene has a length of 120 kb and comprises 21 exons separated by 20 introns [39,40] . MET protein shares 80% homology with Ron and Sea tyrosine kinases family [41,42] and is a heterodimer composed by a 50 kD extracellular α-chain and a 140 kD transmembrane β-chain, bonded by disulfide bridges [39,40] . The extracellular portion, which encompasses the whole α-chain and part of the β-chain, is constituted by a semaphorin (SEMA) and four IPT repeats (immunoglobin-like fold shared by plexins and transcription factors) domains separated by a plexinsemaphorin-integrin (PSI) domain. The intracellular portion involves the β-chain and contains the domains transmembrane, juxtamembrane (JM), and tyrosine kinase (TK) (Figure 1) [43–46] . MET activity can be modulated by the phosphorylation of several amino acids, mainly located in JM and TK domains. MET kinase activity is regulated
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negatively by protein kinase C or calcium-calmodulindependent kinases phosphorylation of Ser-985, whereas internalization and degradation of MET is triggered by the recruitment of c-Cbl, after Tyr-1003 phosphorylation; both residues are located in the JM domain [47–50] . The TK domain is involved in PI3K activation after p85 recruitment by phosphorylated Tyr-1313 [51] and also participates in cell morphogenesis through Tyr1365 (Figure 1) [52] . Other important activities also lie on the TK domain, such as the initiation of TK activity after autophosphorylation of Tyr-1230, 1234, 1235, located in the activation loop [46,53,54] and interaction with proteins which contain SRC homology-2 (SH2), phosphotyrosine binding and methyl-CpG binding domains [55,56] . The ligand for MET is the HGF, which is secreted by fibroblasts and smooth muscle cells [57] . HGF gene is also located on chromosome 7, in the 7q21.1 band and spans 70 kb [58] . It is initially produced and secreted as pro-HGF and is subsequently fractionated extracellularly to a bioactive form: a heterodimer composed of an α-chain and a β-chain linked by a disulfide bond [58] . Several proteases, including pro-urokinase and a plasminogen activator, have been shown to bind HGF, generating the mature HGF [56,59,60] . The α-chain comprises an N-terminal domain and four kringle domains, whereas the β-chain has a serine protease domain (Figure 1) [58] . The HGF residues conforming the receptor binding site are unknown, and different roles for the α- and β-chains have been proposed [58,61,62] . MET dimerization, autophosphorylation and activation require the binding with a lowaffinity site, accessible only when HGF is fully mature. However, a high-affinity site located in the α-chain would probably bind to the receptor regardless of the HGF maturation status [63] . MET and HGF are expressed in numerous tissues. Although both are essentially involved in development, MET is limited to epithelial tissues whereas HGF is confined to cells from mesenchymal origin [64–74] . MET pathway activation participates in several embryogenic processes, such as motogenesis [64,66,68,72] , angiogenesis [67,73] , mitogenesis [65] and morphogenesis [71,74] , and in the generation of several tissues, like skeletal muscles, neurons, diaphragm, tongue, kidney, placenta, ovary and testis [64–68,71–74] . In particular, MET signaling leads to PI3K/AKT pathway activation, which increases the translation of MDM2 to avoid cell apoptosis mediated by p53 during liver development [70] . In adult cells, survival and proliferative actions mediated by MET activation are required in tissue regeneration processes, and is upregulated in injured tissue [69,70,75,76] . MET expression also plays a role in hematopoiesis, including the development of B cells during antigen selection [77] .
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MET in non-small-cell lung carcinoma
N-Terminal HL domain
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Figure 1. MET and HGF structures.
Therefore, MET participation in these many physiological and pathological processes, makes it an important therapeutic target to inhibit cell survival [68] . MET signaling & regulation Numerous interactions have been described for MET, in the activation of multiple pathways, including the GRB2-RAS-RAF-ERK1/2 pathway, the PI3K pathway and STAT3 pathway [54] .
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MET activation is triggered by the binding of its ligand. HGF induces dimerization and transphosphorylation of the receptor on Tyr-1230, 1234, 1235, as explained above. The C-terminal end of MET contains a conserved sequence referred as multisubstrate signal transducer docking site, Tyr-1349-VHVXXX-Tyr1356VNV, which is an absolute requirement for MET signaling [56] . Unlike most tyrosine kinase recep-
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Review Pérez-Ramírez, Cañadas-Garre, Jiménez-Varo, Faus-Dáder & Calleja-Hernández tors, which use different phosphotyrosines to recruit distinct signal effectors, this sequence recruits all the signal transducers required for invasive growth. It is capable to bind proteins containing sites for SH2, phosphotyrosine domains and MBD domain, which is a domain of 13 amino acids rich in proline, found in the adapter protein GAB1 [55,56,78–84] . The MBD allows GAB1 to directly interact with MET, thus providing a level of specificity to this signaling pathway (Figure 2) [82] . Among the molecules interacting with MET, GAB1 is the most important and is directly binded through MBD [85,86] . Met activation induces GAB1 phosphorylation [85] . Phosphorylated GAB1 can attract further docking molecules and enzymes such as PI3K, SHP2 (also known as PTPN11), PLC-γ and CRKL, that together activate various downstream signaling cascades [53,56,82,87–92] . Binding of SHP2 leads to activation of the RAS-RAF-ERK1/2 pathway [89,90] . Furthermore, GRB2, which is recruited either to the SH2 domain of MET multisubstrate docking site,
α
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Y1356VNV or to GAB1, also leads to RAS and ERK1/2 pathways activation (Figure 2) [87,90] . MET is involved in cell survival and proliferation through the PI3K/AKT, RAS-RAF-ERK1/2 and STAT3 pathways (Figure 2) [54,78] . STAT3 activation can be induced by the recruitment and activation of SRC to MET multisubstrate docking site or directly binding to MET [45] , and may also be involved in anchorage-independent growth and formation of branch tubules [79] . The RAS-RAF-ERK1/2 pathway mediates proliferation and cell cycle progression, and together with PI3K/AKT pathway have been shown to be involved in spreading and cell motility [93–97] . MET is also involved in cell migration and invasion mediated by CRKL and PCL-γ; morphogenesis also requires STAT3 and PLC-γ pathways [79,88,98–106] . Likewise, activation of RAS-RAC1/CDC42/RHOPAK regulates cell adhesion and cytoskeletal proteins (Figure 2) [78] . The juxtamembrane domain of MET also plays a regulatory role. Phosphorylation of Tyr 1003 in this
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Figure 2. MET signaling pathway.
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MET in non-small-cell lung carcinoma
domain is involved in MET downregulation, as it binds proteins such as E3 ubiquitin ligase and CBL [107,108] . Binding of CBL also leads to recruitment of the endophilin–CIN85 complex, resulting in MET internalization and degradation [45] . Phosphorylation of Ser 975 in the juxtamembrane domain is also involved in the internalization of MET [45] . Several protein tyrosine phosphatases (PTPs), as the receptor-type PTPs dEP1 (or PTPRJ), LAR (or PTPRF) [109,110] , PTPLB and TCPTP [111] , are also involved in the regulation of MET signaling. Modulation of MET activity by PTPs can be exerted on tyrosines located in the TK domain or in the docking sites [109–111] . The binding of PLC-γ to MET leads to activation of protein PKC, which also downregulates MET [48,112] . Finally, increased intracellular calcium levels may also lead to downregulation of MET pathway (Figure 2) [113] . MET signaling can also be modulated through interactions with other proteins and enzymes at the cell surface, including Ron, EGFR, integrin α6β4, plexin B1, CD44 and FAS. [70,114] Although the exact mechanisms are still unknown, the complex network for MET receptor interaction with many proteins, many of which are known to have important roles in cancer, evidences the HGF/MET axis is a key regulator in cancer. Consequently, MET activation promotes tumor cell growth, survival, migration and invasion, as well as tumor angiogenesis (Figure 2) . Furthermore, cells may suffer EMT and dissociation of cell mass after HGF stimulation [70] . In the course of normal growth, proliferative activity is inhibited after EMT [115] , whereas in tumor cells the ability to proliferate may be preserved [116] . MET also plays an important role in EMT, leading to activation of small GTPases [117–120] , internalizing jointly occupied plasma membrane domains after removing E-cadherin from cell–cell adhesion sites, and modulating integrins activity [121–124] .
Review
MET gene amplification and overexpression in NSCLC has been shown in several cell lines [126] . In particular, the HCC827 lung cancer cell line, shows activating mutations in EGFR, sensitivity to gefitinib and resistance to gefitinib by amplification of MET, as demonstrated after being exposed to rising concentrations of gefitinib, a tyrosine kinase inhibitor [28] . To assess the significance of this finding, the number of copies of MET in patients with NSCLC and mutant EGFR who had become resistant after initial TKI response was examined [28] . MET gene amplification was detected in 21% (13/61) of patients with gefitinib or erlotinib-resistant lung adenocarcinoma, compared with only 3% (2/62) of patients who were not TKI treated [28,34] . Patients with MET amplification also presented the resistance mutation T790M in EGFR in 44% (4/9) of the cases, suggesting that both alterations were involved in EGFR-TKI resistance [34] . However, these frequencies of MET high gene copy number have not been confirmed in the two most recent studies based on molecular analysis of rebiopsied tissue from NSCLC patients with acquired EGFR-TKI-resistant NSCLC, which have yielded considerably lower percentages (4/37; 11% and 2/37; 5%, respectively) [127,128] . MET gene amplification is associated to disease progression and decreased survival in NSCLC patients treated with gefitinib or erlotinib, therefore plays an essential role in acquired EGFR-TKI resistant in NSCLC [28,34,127–130] . MET activated by amplification phosphorylates ERBB3, leading to the activation of the PI3K/AKT pathway, which results in a cell survival signal [28,129] . ERBB3 phosphorylation is able to keep the PI3K/AKT pathway activated even when EGFR is inhibited by TKI [28,129] . Co-administration of EGFRTKI along with MET inhibitors could potentially block the survival signal in such cases (Figure 3) . Overexpression of HGF
Dysregulated MET activity: EGFR-TKI resistance MET signaling is a complex system that activates a great diversity of cellular processes; HGF/MET alterations contribute to resistance to EGFR-TKI therapy by several mechanisms, like MET gene amplification, overexpression of HGF or MET mutations. MET gene amplification
Amplification of MET gene has been described in 20% of lung cancer patients with acquired resistance to EGFR-TKIs, mostly in adenocarcinoma [28,34] . This alteration has also been described in 33% of treatmentnaive adeno- and squamous cell carcinomas of the lung [125] .
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HGF regulation can be altered through upregulation of HGF and increased circulating HGF, originated from tumor and stromal cells [131] . HGF is overexpressed in lung cancer cells from patients with smoking history, and HGF production is stimulated by nicotine in NSCLC cell lines [132] . Nonsmokers with lung cancer exhibit EGFR mutations more frequently and therefore show a better response to EGFR-TKIs than smokers [9–12] . In smokers, the induced HGF production may also contribute to resistance to gefitinib [132] . Overexpression of HGF was proposed as a mechanism of resistance to EGFR-TKIs, since it was able to induce dose-dependent resistance to gefitinib in adenocarcinoma cell lines harboring EGFR mutations in
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Review Pérez-Ramírez, Cañadas-Garre, Jiménez-Varo, Faus-Dáder & Calleja-Hernández
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Figure 3. Mechanisms of resistance to tyrosine kinase inhibitors. R: Resistance mutations; S: Sensitive mutations; TKI: Tyrosine kinase inhibitor.
exon 19, probably through activation of PI3K/AKT pathway after MET phosphorylation [29] . Downstream pathway activation seems to be different between MET gene amplification and HGF overexpression. MET gene amplification leads to ERBB3 phosphorylation, whereas HGF causes downstream signal activation through MET, independently of ERBB3 or EGFR (Figure 3) [133] . In this case, as for MET amplification, combination of drugs inhibiting HGF/MET and EGFR should be necessary to overcome the resistance. To confirm this hypothesis, several trials have investigated the combination between an EGFR-TKIs and an HGF/MET inhibitor (tivantinib, onartuzumab) in EGFR TKI-naive advanced NSCLC patients, with suggested greater benefit in PFS and OS in patients with KRAS mutations [134,135] . MET mutations
MET mutations were first identified in hereditary papillary renal carcinoma cells [136,137] , but they have also been described in many other cancers, including gastric, head and neck, liver, ovarian, NSCLC and thyroid
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cancer and even in metastases from some of these types of cancer [35–37,138–144] . There have been identified over 20 mutations in MET, mainly missense mutations located in the TK domain [37,136–138,140–142] . However, in lung cancer, mutations are clustered in the JM and SEMA domains [35,36,139,144] . HGF binding may be dysrupted by mutations in the SEMA domain, whereas mutations in the JM domain alter processes related to the catalytic activity and the actin cytoskeleton, like cell motility and migration. Oligomerization and HGFindependent MET activation is altered by mutations in the TK domain [35–37,138–142,144] . MET mutations have been described in approximately 5% in NSCLC, especially in exons 1 and 2, without a marked difference between different histological groups (Box 1) . Unlike renal and gastric carcinoma, mutations in the TK domain of MET are rare in NSCLC [35–37,139,140,144] , although some, like have G1242C in exon 18, have been identified [35] . Mutations in the JM domain have been described in 1.3% of adenocarcinoma tissues (4/315) and in one out of four
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MET in non-small-cell lung carcinoma
NSCLC cell lines [37] . Three of the point mutations (T1010I, S1058P, Y1003N) were identified in tissue and one (R988C) in both tissue and a cell line [37] . An in-frame alternative splice variant lacking the whole exon 14 from the JM domain, consequently resulting in the loss of the c-Cbl-binding site, was also identified in 0.63% of adenocarcinomas (2/315) [35,37] . Six mutations have been reported in the SEMA domain (E168D, L229F, S323G, N375S, S178, I377), all in adenocarcinoma tissue, being N375S the more frequent [37] . The type and distribution of MET mutations have been shown to be different according to the ethnicity and histology, being the highest frequency in East Asians. Particularly, the N375S mutation was associated to squamous cell carcinoma in East Asians. Since this mutation has demonstrated loss of affinity for HGF and less apoptosis rate induced by MET kinase inhibitors, lung cancer patients harboring MET mutations could have altered response or resistance to specific MET kinase inhibitors [36] . Squamous cells in African American and adenocarcinoma in Caucasian were less prone to present MET mutations [36] . MET mutations were more common in smokers. Similarly, two synonymous mutations were observed in a relatively high frequency. S178 was described mainly in East Asians and I377 mostly in Afro-Americans [36] . Somatic MET mutations are quite rare in the primary tumor, however they are more associated with tumor progression [145] . Metastatic tumors have shown a significant number of mutations identified in metastatic foci, probably conferring them the ability to spread and invade cells [145] . MET as a therapeutic target The HGF/MET signaling pathway is being currently investigated as a potential target to overcome resistance in NSCLC [134,135,146–150] . Table 1 shows the main drugs currently under investigation, which are monoclonal antibodies (binding HGF or competing with HGF for binding to MET) or MET tyrosine kinase inhibitors (selective and nonselective). Monoclonal antibodies Rilotumumab
Rilotumumab (AMG 102, Amgen, CA, USA) is a fully humanized IgG2 monoclonal antibody, which avoids MET activation by HGF binding [146] . Rilotumumab has shown to improved PFS (6.9 vs 4.6 months; HR: 0.51; p = 0.09) and OS (11.1 vs 5.7 months; HR: 0.29; p = 0.01) among patients with high MET levels when added in combination with a scheme based on epirubicin, cisplatin and capecitabine in patients with locally advanced or metastatic gastric or esophagogastric junction (EGJ) cancer (NCT00719550) [151–153] . These
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Review
promising results have impulsed the investigation of rilotumumab associated with erlotinib in recurrent or advanced NSCLC patients (NCT01233687). Ficlatuzumab
Ficlatuzumab (AV-299, AVEO, MA, USA), is a humanized IgG1 antibody which inhibits MET/HGF by binding the HGF ligand [154] . The potent antitumor activity observed for ficlatuzumab in combination with EGFR inhibitors like cetuximab in preclinical models prompted further investigation in NSCLC patients [147,155] . In a Phase Ib–II trial conducted in NSCLC Asian patients, combination therapy of ficlatuzumab with gefitinib versus gefitinb alone showed a trend for overall response rate (ORR) (70 vs 44%) and PFS (11.1 vs 5.5 months), favoring ficlatuzumab plus gefitinib, in patients with low MET expression and activating mutations in EGFR, providing a potential mechanism to overcome EGFR-TKI resistance (NCT01039948) [147] . In addition, ficlatuzumab prolongs survival in patients with high stromal HGF (p = 0.03) [147] . Onartuzumab
Onartuzumab (MetMAb, Genentech-Roche) is a humanized monovalent antibody against MET SEMA domain which blocks MET dimerization and consequent activation of the intracellular tyrosine kinase domain, thereby inhibiting the downstream cellular response [63,156] . A Phase II trial has investigated onartuzumab combined with erlotinib versus placebo plus erlotinib in NSCLC patients, yielding improved PFS (2.9 vs 1.5 months; HR: 0.53; p = 0.04) and OS (12.6 vs 3.8 months; HR: 0.37; p = 0.002) in patients with high MET expression (NCT00854308) [135] . However, despite these encouraging data, the initial promising outcomes of onartuzumab have been challenged by a Box 1. MET mutations identified in non-small-cell lung cancer. SEMA • E168D • L229F • S323G • N375S • S178 • I377
Juxtamembrane • R988C • T1010I • S1058P • Alternative splice variant (47 AA. Exon 14 skipped) • Y1003N
Tyrosine kinase • G1242C
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Review Pérez-Ramírez, Cañadas-Garre, Jiménez-Varo, Faus-Dáder & Calleja-Hernández recent Phase III trial in 490 previously treated stage IIIb/IV NSCLC patients comparing onartuzumaberlotinib versus erlotinib alone in MET 2+/3+ tumors (NTC01456325), which has failed to confirm the benefit in terms of OS (6.8 months vs 9.1 months; HR: 1.27; p = 0.068), PFS (2.7 months vs 2.6 months; HR: 0.99; p = 0.92) or ORR (8.4 vs 9.6%; p = 0.63) [157] . MET kinase inhibitors Tivantinib
Tivantinib (ARQ-197; ArQule, MA, USA) is an oral, non-ATP-dependent selective allosteric MET inhibitor, which blocks MET receptor in its inactive state conformation [158] . A Phase II trial in EGFR inhibitornaive patients previously treated with locally advanced or metastatic NSCLC showed a significant improvement in PFS (HR: 0.61; p = 0.04) and OS (HR: 0.58; p = 0.04) for the addition of tivantinib to erlotinib (NCT00777309) [134] . PFS was particularly improved by the combination of both drugs in patients positive for KRAS mutations (2.3 vs. 1 months; HR: 0.18; p = 0.006) [134] . Based on the above results, a Phase III trial of tivantinib in combination with erlotinib versus erlotinib plus placebo was initiated and enrolled in 1048 advanced nonsquamous NSCLC patients previously treated (MARQUEE, NTC01244191). KRAS, EGFR and MET status was analyzed before randomization and EGFR and KRAS mutational status were considered for the stratification of patients, among other factors, like the number of previous therapies, sex and smoking history [149] . Despite the study confirmed a notable benefit in terms of PFS (3.6 vs 1.9 months; HR: 0.74; p
Pharmacogenomics
MET: a new promising biomarker in non-small-cell lung carcinoma
Non-small-cell lung cancer (NSCLC) leads cancer-related deaths worldwide. Mutations in the kinase domain of the EGFR gene provide sensitivity to tyrosine kinase inhibitors (TKI) drugs. TKI show initial response rates over 75% in mutant EGFR-NSCLC patients, although most of these patients acquire resistance to EGFR inhibitors after therapy. EGFR-TKI resistance mechanisms include amplification in MET and its ligand, and also MET mutations. MET signaling dysregulation has been involved in tumor cell growth, survival, migration and invasion, angiogenesis and activation of several pathways, therefore representing an attractive target for anticancer drug development. In this review, we will discuss MET-related mechanisms of EGFR-TKI resistance in NSCLC, as well as the main drugs targeted to inhibit MET pathway. Keywords: EGFR • MET • pharmacogenetics • resistance • targeted therapies • TKI
Background Lung cancer currently constitutes a serious health problem. According to the latest statistics published, more than 224,210 cases are diagnosed each year only in the USA [1] . In 2013, lung cancer ranked as the second tumor in incidence for men and women (after prostate and breast cancer, respectively) [1] . The incidence is similar in both genders, being approximately 14% in men (116,000) and women (108,210) [1] . This tumor presents a very high mortality, since the relative survival rate at 5 years does not exceed 10% in most countries, and therefore it is generally considered that the overall mortality figures are close to those of incidence. It represents the leading cause of cancer death in both genders in the USA, up to 28% in males (86,930) and 26% in women (72,330) [1] . Lung cancer is classified in two types, small-cell-lung carcinoma (SCLC), which accounts for approximately 15% of cases, and non-small-cell lung carcinoma (NSCLC), which represents the remaining 85%, and presents different subtypes: squamous cell or epidermoid carcinoma, adenocarcinoma and large cell carcinoma. At the time of diag-
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nosis, approximately 70% of patients with NSCLC are metastatic, staged IV according to the AJCC, therefore incurable [2–4] . The standard treatment for advanced or recurrent NSCLC is platinum-based chemotherapy, however, its results are unsatisfactory with a response rate (RR) of 17–32%, progression-free survival (PFS) of 3.1–3.5 months and overall survival (OS) of 7.4–11.3 months [5–8] . The identification of mutations that contribute to the pathogenesis of NSCLC has contributed to improved prognosis, classification and management of this disease. The EGFR is the most frequently mutated and overexpressed gene in NSCLC. EGFR mutations are more common in women, Asians and nonsmokers with the adenocarcinoma subtype [9–12] . The most common EGFR mutations are deletions in exon 19 and a point mutation in exon 21, located in the kinase domain, which replaces an arginine for a leucine at codon 858 (L858R) [9–12] . The kinase domain also hosts a rare mutation in exon 18, consisting in a substitution of glycine for alanine, cysteine or serine at codon 719 (G719X) [9–12] .
Pharmacogenomics (Epub ahead of print)
Cristina Pérez-Ramírez1,2, Marisa Cañadas-Garre*,1, Enrique Jiménez-Varo1,3, María José Faus-Dáder2 & Miguel Ángel CallejaHernández1,3 Pharmacogenetics Unit, UGC Provincial de Farmacia de Granada, Instituto de Investigación Biosanitaria de Granada, Complejo Hospitalario Universitario de Granada, Avda Fuerzas Armadas, 2, 18014 Granada, Spain 2 Department of Biochemistry, Faculty of Pharmacy, University of Granada, Campus Universitario de Cartuja, s/n, 18071 Granada, Spain 3 Department of Pharmacology, Faculty of Pharmacy, University of Granada, Campus Universitario de Cartuja, s/n, 18071 Granada, Spain *Author for correspondence: Tel.: +34 9580 20108 Fax: +34 9010 21804 marisacgarre@ gmail.com 1
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ISSN 1462-2416
Review Pérez-Ramírez, Cañadas-Garre, Jiménez-Varo, Faus-Dáder & Calleja-Hernández The clinical interest of the determination of mutations in the kinase domain of the EGFR gene lies on the prediction of sensitivity of these tumors to tyrosine kinase inhibitors (TKI), such as erlotinib (Tarceva®, Roche) and gefitinib (Iressa®, AstraZeneca), drugs which have shown 75% response rates in NSCLC patients [13–15] . Despite this initial good response, about 75% of NSCLC patients with mutations in EGFR acquire resistance to EGFR-TKIs after a mean period of 12 months [16–20] . Many causes of resistance to EGFR-TKIs have been described. The most studied are a secondary point mutation in exon 20 of EGFR (T790M), which replaces methionine for threonine at amino acid position 790, MET protooncogene (MET) amplification and protein overexpression of its ligand, HGF, PTEN loss, KRAS mutations, BRAF mutations, HER-2 mutations, epithelial-to-mesenchymal transition (EMT), AXL overexpression and small-cell transformation [21–32] . Among them, one of the most investigated causes for EGFR-TKI resistance is MET amplification and HGF overexpression, whose potential role in several processes as cell survival, proliferation, motility, invasiveness and angiogenesis makes them attractive therapeutic targets [33] . In NSCLC, abnormalities in MET signaling pathway have been associated with poor clinical outcome and drug resistance [28,29,34–37] . Therefore, single or combined MET inhibition by targeted drugs represents a promising strategy to approach resistance in tumors with dysregulated MET signaling. MET structure & functions MET gene, located on chromosome 7q21-q31, was discovered in 1984 as one of the components of the translocated promoter region (TPR)-MET fusion oncoprotein in an osteosarcoma immortalized cell line [38] . MET gene has a length of 120 kb and comprises 21 exons separated by 20 introns [39,40] . MET protein shares 80% homology with Ron and Sea tyrosine kinases family [41,42] and is a heterodimer composed by a 50 kD extracellular α-chain and a 140 kD transmembrane β-chain, bonded by disulfide bridges [39,40] . The extracellular portion, which encompasses the whole α-chain and part of the β-chain, is constituted by a semaphorin (SEMA) and four IPT repeats (immunoglobin-like fold shared by plexins and transcription factors) domains separated by a plexinsemaphorin-integrin (PSI) domain. The intracellular portion involves the β-chain and contains the domains transmembrane, juxtamembrane (JM), and tyrosine kinase (TK) (Figure 1) [43–46] . MET activity can be modulated by the phosphorylation of several amino acids, mainly located in JM and TK domains. MET kinase activity is regulated
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negatively by protein kinase C or calcium-calmodulindependent kinases phosphorylation of Ser-985, whereas internalization and degradation of MET is triggered by the recruitment of c-Cbl, after Tyr-1003 phosphorylation; both residues are located in the JM domain [47–50] . The TK domain is involved in PI3K activation after p85 recruitment by phosphorylated Tyr-1313 [51] and also participates in cell morphogenesis through Tyr1365 (Figure 1) [52] . Other important activities also lie on the TK domain, such as the initiation of TK activity after autophosphorylation of Tyr-1230, 1234, 1235, located in the activation loop [46,53,54] and interaction with proteins which contain SRC homology-2 (SH2), phosphotyrosine binding and methyl-CpG binding domains [55,56] . The ligand for MET is the HGF, which is secreted by fibroblasts and smooth muscle cells [57] . HGF gene is also located on chromosome 7, in the 7q21.1 band and spans 70 kb [58] . It is initially produced and secreted as pro-HGF and is subsequently fractionated extracellularly to a bioactive form: a heterodimer composed of an α-chain and a β-chain linked by a disulfide bond [58] . Several proteases, including pro-urokinase and a plasminogen activator, have been shown to bind HGF, generating the mature HGF [56,59,60] . The α-chain comprises an N-terminal domain and four kringle domains, whereas the β-chain has a serine protease domain (Figure 1) [58] . The HGF residues conforming the receptor binding site are unknown, and different roles for the α- and β-chains have been proposed [58,61,62] . MET dimerization, autophosphorylation and activation require the binding with a lowaffinity site, accessible only when HGF is fully mature. However, a high-affinity site located in the α-chain would probably bind to the receptor regardless of the HGF maturation status [63] . MET and HGF are expressed in numerous tissues. Although both are essentially involved in development, MET is limited to epithelial tissues whereas HGF is confined to cells from mesenchymal origin [64–74] . MET pathway activation participates in several embryogenic processes, such as motogenesis [64,66,68,72] , angiogenesis [67,73] , mitogenesis [65] and morphogenesis [71,74] , and in the generation of several tissues, like skeletal muscles, neurons, diaphragm, tongue, kidney, placenta, ovary and testis [64–68,71–74] . In particular, MET signaling leads to PI3K/AKT pathway activation, which increases the translation of MDM2 to avoid cell apoptosis mediated by p53 during liver development [70] . In adult cells, survival and proliferative actions mediated by MET activation are required in tissue regeneration processes, and is upregulated in injured tissue [69,70,75,76] . MET expression also plays a role in hematopoiesis, including the development of B cells during antigen selection [77] .
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MET in non-small-cell lung carcinoma
N-Terminal HL domain
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Figure 1. MET and HGF structures.
Therefore, MET participation in these many physiological and pathological processes, makes it an important therapeutic target to inhibit cell survival [68] . MET signaling & regulation Numerous interactions have been described for MET, in the activation of multiple pathways, including the GRB2-RAS-RAF-ERK1/2 pathway, the PI3K pathway and STAT3 pathway [54] .
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MET activation is triggered by the binding of its ligand. HGF induces dimerization and transphosphorylation of the receptor on Tyr-1230, 1234, 1235, as explained above. The C-terminal end of MET contains a conserved sequence referred as multisubstrate signal transducer docking site, Tyr-1349-VHVXXX-Tyr1356VNV, which is an absolute requirement for MET signaling [56] . Unlike most tyrosine kinase recep-
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Review Pérez-Ramírez, Cañadas-Garre, Jiménez-Varo, Faus-Dáder & Calleja-Hernández tors, which use different phosphotyrosines to recruit distinct signal effectors, this sequence recruits all the signal transducers required for invasive growth. It is capable to bind proteins containing sites for SH2, phosphotyrosine domains and MBD domain, which is a domain of 13 amino acids rich in proline, found in the adapter protein GAB1 [55,56,78–84] . The MBD allows GAB1 to directly interact with MET, thus providing a level of specificity to this signaling pathway (Figure 2) [82] . Among the molecules interacting with MET, GAB1 is the most important and is directly binded through MBD [85,86] . Met activation induces GAB1 phosphorylation [85] . Phosphorylated GAB1 can attract further docking molecules and enzymes such as PI3K, SHP2 (also known as PTPN11), PLC-γ and CRKL, that together activate various downstream signaling cascades [53,56,82,87–92] . Binding of SHP2 leads to activation of the RAS-RAF-ERK1/2 pathway [89,90] . Furthermore, GRB2, which is recruited either to the SH2 domain of MET multisubstrate docking site,
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Y1356VNV or to GAB1, also leads to RAS and ERK1/2 pathways activation (Figure 2) [87,90] . MET is involved in cell survival and proliferation through the PI3K/AKT, RAS-RAF-ERK1/2 and STAT3 pathways (Figure 2) [54,78] . STAT3 activation can be induced by the recruitment and activation of SRC to MET multisubstrate docking site or directly binding to MET [45] , and may also be involved in anchorage-independent growth and formation of branch tubules [79] . The RAS-RAF-ERK1/2 pathway mediates proliferation and cell cycle progression, and together with PI3K/AKT pathway have been shown to be involved in spreading and cell motility [93–97] . MET is also involved in cell migration and invasion mediated by CRKL and PCL-γ; morphogenesis also requires STAT3 and PLC-γ pathways [79,88,98–106] . Likewise, activation of RAS-RAC1/CDC42/RHOPAK regulates cell adhesion and cytoskeletal proteins (Figure 2) [78] . The juxtamembrane domain of MET also plays a regulatory role. Phosphorylation of Tyr 1003 in this
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Figure 2. MET signaling pathway.
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MET in non-small-cell lung carcinoma
domain is involved in MET downregulation, as it binds proteins such as E3 ubiquitin ligase and CBL [107,108] . Binding of CBL also leads to recruitment of the endophilin–CIN85 complex, resulting in MET internalization and degradation [45] . Phosphorylation of Ser 975 in the juxtamembrane domain is also involved in the internalization of MET [45] . Several protein tyrosine phosphatases (PTPs), as the receptor-type PTPs dEP1 (or PTPRJ), LAR (or PTPRF) [109,110] , PTPLB and TCPTP [111] , are also involved in the regulation of MET signaling. Modulation of MET activity by PTPs can be exerted on tyrosines located in the TK domain or in the docking sites [109–111] . The binding of PLC-γ to MET leads to activation of protein PKC, which also downregulates MET [48,112] . Finally, increased intracellular calcium levels may also lead to downregulation of MET pathway (Figure 2) [113] . MET signaling can also be modulated through interactions with other proteins and enzymes at the cell surface, including Ron, EGFR, integrin α6β4, plexin B1, CD44 and FAS. [70,114] Although the exact mechanisms are still unknown, the complex network for MET receptor interaction with many proteins, many of which are known to have important roles in cancer, evidences the HGF/MET axis is a key regulator in cancer. Consequently, MET activation promotes tumor cell growth, survival, migration and invasion, as well as tumor angiogenesis (Figure 2) . Furthermore, cells may suffer EMT and dissociation of cell mass after HGF stimulation [70] . In the course of normal growth, proliferative activity is inhibited after EMT [115] , whereas in tumor cells the ability to proliferate may be preserved [116] . MET also plays an important role in EMT, leading to activation of small GTPases [117–120] , internalizing jointly occupied plasma membrane domains after removing E-cadherin from cell–cell adhesion sites, and modulating integrins activity [121–124] .
Review
MET gene amplification and overexpression in NSCLC has been shown in several cell lines [126] . In particular, the HCC827 lung cancer cell line, shows activating mutations in EGFR, sensitivity to gefitinib and resistance to gefitinib by amplification of MET, as demonstrated after being exposed to rising concentrations of gefitinib, a tyrosine kinase inhibitor [28] . To assess the significance of this finding, the number of copies of MET in patients with NSCLC and mutant EGFR who had become resistant after initial TKI response was examined [28] . MET gene amplification was detected in 21% (13/61) of patients with gefitinib or erlotinib-resistant lung adenocarcinoma, compared with only 3% (2/62) of patients who were not TKI treated [28,34] . Patients with MET amplification also presented the resistance mutation T790M in EGFR in 44% (4/9) of the cases, suggesting that both alterations were involved in EGFR-TKI resistance [34] . However, these frequencies of MET high gene copy number have not been confirmed in the two most recent studies based on molecular analysis of rebiopsied tissue from NSCLC patients with acquired EGFR-TKI-resistant NSCLC, which have yielded considerably lower percentages (4/37; 11% and 2/37; 5%, respectively) [127,128] . MET gene amplification is associated to disease progression and decreased survival in NSCLC patients treated with gefitinib or erlotinib, therefore plays an essential role in acquired EGFR-TKI resistant in NSCLC [28,34,127–130] . MET activated by amplification phosphorylates ERBB3, leading to the activation of the PI3K/AKT pathway, which results in a cell survival signal [28,129] . ERBB3 phosphorylation is able to keep the PI3K/AKT pathway activated even when EGFR is inhibited by TKI [28,129] . Co-administration of EGFRTKI along with MET inhibitors could potentially block the survival signal in such cases (Figure 3) . Overexpression of HGF
Dysregulated MET activity: EGFR-TKI resistance MET signaling is a complex system that activates a great diversity of cellular processes; HGF/MET alterations contribute to resistance to EGFR-TKI therapy by several mechanisms, like MET gene amplification, overexpression of HGF or MET mutations. MET gene amplification
Amplification of MET gene has been described in 20% of lung cancer patients with acquired resistance to EGFR-TKIs, mostly in adenocarcinoma [28,34] . This alteration has also been described in 33% of treatmentnaive adeno- and squamous cell carcinomas of the lung [125] .
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HGF regulation can be altered through upregulation of HGF and increased circulating HGF, originated from tumor and stromal cells [131] . HGF is overexpressed in lung cancer cells from patients with smoking history, and HGF production is stimulated by nicotine in NSCLC cell lines [132] . Nonsmokers with lung cancer exhibit EGFR mutations more frequently and therefore show a better response to EGFR-TKIs than smokers [9–12] . In smokers, the induced HGF production may also contribute to resistance to gefitinib [132] . Overexpression of HGF was proposed as a mechanism of resistance to EGFR-TKIs, since it was able to induce dose-dependent resistance to gefitinib in adenocarcinoma cell lines harboring EGFR mutations in
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Review Pérez-Ramírez, Cañadas-Garre, Jiménez-Varo, Faus-Dáder & Calleja-Hernández
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Figure 3. Mechanisms of resistance to tyrosine kinase inhibitors. R: Resistance mutations; S: Sensitive mutations; TKI: Tyrosine kinase inhibitor.
exon 19, probably through activation of PI3K/AKT pathway after MET phosphorylation [29] . Downstream pathway activation seems to be different between MET gene amplification and HGF overexpression. MET gene amplification leads to ERBB3 phosphorylation, whereas HGF causes downstream signal activation through MET, independently of ERBB3 or EGFR (Figure 3) [133] . In this case, as for MET amplification, combination of drugs inhibiting HGF/MET and EGFR should be necessary to overcome the resistance. To confirm this hypothesis, several trials have investigated the combination between an EGFR-TKIs and an HGF/MET inhibitor (tivantinib, onartuzumab) in EGFR TKI-naive advanced NSCLC patients, with suggested greater benefit in PFS and OS in patients with KRAS mutations [134,135] . MET mutations
MET mutations were first identified in hereditary papillary renal carcinoma cells [136,137] , but they have also been described in many other cancers, including gastric, head and neck, liver, ovarian, NSCLC and thyroid
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cancer and even in metastases from some of these types of cancer [35–37,138–144] . There have been identified over 20 mutations in MET, mainly missense mutations located in the TK domain [37,136–138,140–142] . However, in lung cancer, mutations are clustered in the JM and SEMA domains [35,36,139,144] . HGF binding may be dysrupted by mutations in the SEMA domain, whereas mutations in the JM domain alter processes related to the catalytic activity and the actin cytoskeleton, like cell motility and migration. Oligomerization and HGFindependent MET activation is altered by mutations in the TK domain [35–37,138–142,144] . MET mutations have been described in approximately 5% in NSCLC, especially in exons 1 and 2, without a marked difference between different histological groups (Box 1) . Unlike renal and gastric carcinoma, mutations in the TK domain of MET are rare in NSCLC [35–37,139,140,144] , although some, like have G1242C in exon 18, have been identified [35] . Mutations in the JM domain have been described in 1.3% of adenocarcinoma tissues (4/315) and in one out of four
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MET in non-small-cell lung carcinoma
NSCLC cell lines [37] . Three of the point mutations (T1010I, S1058P, Y1003N) were identified in tissue and one (R988C) in both tissue and a cell line [37] . An in-frame alternative splice variant lacking the whole exon 14 from the JM domain, consequently resulting in the loss of the c-Cbl-binding site, was also identified in 0.63% of adenocarcinomas (2/315) [35,37] . Six mutations have been reported in the SEMA domain (E168D, L229F, S323G, N375S, S178, I377), all in adenocarcinoma tissue, being N375S the more frequent [37] . The type and distribution of MET mutations have been shown to be different according to the ethnicity and histology, being the highest frequency in East Asians. Particularly, the N375S mutation was associated to squamous cell carcinoma in East Asians. Since this mutation has demonstrated loss of affinity for HGF and less apoptosis rate induced by MET kinase inhibitors, lung cancer patients harboring MET mutations could have altered response or resistance to specific MET kinase inhibitors [36] . Squamous cells in African American and adenocarcinoma in Caucasian were less prone to present MET mutations [36] . MET mutations were more common in smokers. Similarly, two synonymous mutations were observed in a relatively high frequency. S178 was described mainly in East Asians and I377 mostly in Afro-Americans [36] . Somatic MET mutations are quite rare in the primary tumor, however they are more associated with tumor progression [145] . Metastatic tumors have shown a significant number of mutations identified in metastatic foci, probably conferring them the ability to spread and invade cells [145] . MET as a therapeutic target The HGF/MET signaling pathway is being currently investigated as a potential target to overcome resistance in NSCLC [134,135,146–150] . Table 1 shows the main drugs currently under investigation, which are monoclonal antibodies (binding HGF or competing with HGF for binding to MET) or MET tyrosine kinase inhibitors (selective and nonselective). Monoclonal antibodies Rilotumumab
Rilotumumab (AMG 102, Amgen, CA, USA) is a fully humanized IgG2 monoclonal antibody, which avoids MET activation by HGF binding [146] . Rilotumumab has shown to improved PFS (6.9 vs 4.6 months; HR: 0.51; p = 0.09) and OS (11.1 vs 5.7 months; HR: 0.29; p = 0.01) among patients with high MET levels when added in combination with a scheme based on epirubicin, cisplatin and capecitabine in patients with locally advanced or metastatic gastric or esophagogastric junction (EGJ) cancer (NCT00719550) [151–153] . These
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Review
promising results have impulsed the investigation of rilotumumab associated with erlotinib in recurrent or advanced NSCLC patients (NCT01233687). Ficlatuzumab
Ficlatuzumab (AV-299, AVEO, MA, USA), is a humanized IgG1 antibody which inhibits MET/HGF by binding the HGF ligand [154] . The potent antitumor activity observed for ficlatuzumab in combination with EGFR inhibitors like cetuximab in preclinical models prompted further investigation in NSCLC patients [147,155] . In a Phase Ib–II trial conducted in NSCLC Asian patients, combination therapy of ficlatuzumab with gefitinib versus gefitinb alone showed a trend for overall response rate (ORR) (70 vs 44%) and PFS (11.1 vs 5.5 months), favoring ficlatuzumab plus gefitinib, in patients with low MET expression and activating mutations in EGFR, providing a potential mechanism to overcome EGFR-TKI resistance (NCT01039948) [147] . In addition, ficlatuzumab prolongs survival in patients with high stromal HGF (p = 0.03) [147] . Onartuzumab
Onartuzumab (MetMAb, Genentech-Roche) is a humanized monovalent antibody against MET SEMA domain which blocks MET dimerization and consequent activation of the intracellular tyrosine kinase domain, thereby inhibiting the downstream cellular response [63,156] . A Phase II trial has investigated onartuzumab combined with erlotinib versus placebo plus erlotinib in NSCLC patients, yielding improved PFS (2.9 vs 1.5 months; HR: 0.53; p = 0.04) and OS (12.6 vs 3.8 months; HR: 0.37; p = 0.002) in patients with high MET expression (NCT00854308) [135] . However, despite these encouraging data, the initial promising outcomes of onartuzumab have been challenged by a Box 1. MET mutations identified in non-small-cell lung cancer. SEMA • E168D • L229F • S323G • N375S • S178 • I377
Juxtamembrane • R988C • T1010I • S1058P • Alternative splice variant (47 AA. Exon 14 skipped) • Y1003N
Tyrosine kinase • G1242C
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Review Pérez-Ramírez, Cañadas-Garre, Jiménez-Varo, Faus-Dáder & Calleja-Hernández recent Phase III trial in 490 previously treated stage IIIb/IV NSCLC patients comparing onartuzumaberlotinib versus erlotinib alone in MET 2+/3+ tumors (NTC01456325), which has failed to confirm the benefit in terms of OS (6.8 months vs 9.1 months; HR: 1.27; p = 0.068), PFS (2.7 months vs 2.6 months; HR: 0.99; p = 0.92) or ORR (8.4 vs 9.6%; p = 0.63) [157] . MET kinase inhibitors Tivantinib
Tivantinib (ARQ-197; ArQule, MA, USA) is an oral, non-ATP-dependent selective allosteric MET inhibitor, which blocks MET receptor in its inactive state conformation [158] . A Phase II trial in EGFR inhibitornaive patients previously treated with locally advanced or metastatic NSCLC showed a significant improvement in PFS (HR: 0.61; p = 0.04) and OS (HR: 0.58; p = 0.04) for the addition of tivantinib to erlotinib (NCT00777309) [134] . PFS was particularly improved by the combination of both drugs in patients positive for KRAS mutations (2.3 vs. 1 months; HR: 0.18; p = 0.006) [134] . Based on the above results, a Phase III trial of tivantinib in combination with erlotinib versus erlotinib plus placebo was initiated and enrolled in 1048 advanced nonsquamous NSCLC patients previously treated (MARQUEE, NTC01244191). KRAS, EGFR and MET status was analyzed before randomization and EGFR and KRAS mutational status were considered for the stratification of patients, among other factors, like the number of previous therapies, sex and smoking history [149] . Despite the study confirmed a notable benefit in terms of PFS (3.6 vs 1.9 months; HR: 0.74; p
