Review For reprint orders, please contact: [email protected]
EGFR and KRAS mutations, and ALK fusions: current developments and personalized therapies for patients with advanced non-small-cell lung cancer Personalized therapy has significantly developed in lung cancer treatment over recent years. VEGF and EGF play a major role in non-small-cell lung cancer (NSCLC) tumor angiogenesis and aggressiveness. EGFR mutation as well as KRAS and ALK rearrangements are important biomarkers in the field owing to potential targeted therapies involved in clinical practice: erlotinib, geftinib, cetuximab and crizotinib. More recently, regulation of tumor immunity through CTLA4 and PD1/L1 has emerged as a promising field in NSCLC management. This review will focus on the current and future biomarkers in the advanced NSCLC field and also address potential related targeted therapies for these patients. KEYWORDS: afatinib n ALK–EML4 n bevacizumab n cetuximab n crizotinib n EGFR mutation n erlotinib n gefitinib n lung cancer
Currently, it is estimated that approximately 11 million people worldwide have cancer. Lung cancer is the leading cause of cancer-related death and accounts for approximately 30% of all cancer deaths [1,2]. In the USA, lung cancer has a global incidence of approximately 70 cases per 100,000 inhabitants. In Europe, lung cancer incidence is approximately 52.5 cases per 100,000 inhabitants (82.5 in 100,000 in males and 23.9 in 100,000 in females) and mortality is approximately 48.7 out of 100,000 (77 in 100,000 in males and 23.9 in 100,000 in females) [3–6]. Lung cancer can be generally divided into small-cell lung cancer and non-small-cell lung cancer (NSCLC) [7,8]. NSCLC accounts for approximately 85% of all lung cancers [1,9,10]. NSCLC originates in lung epithelial cells, and comprises diverse histological subtypes including adenocarcinoma, as well as squamous, anaplastic and large-cell carcinomas [8,11]. Most patients with NSCLC have advanced disease at diagnosis, present with metastatic disease and, if left untreated, have a median survival time after diagnosis of 4–5 months and a 1‑year survival of less than 10% [8,10]. The advanced stages (IIIB and IV; according to the tumor node metastasis classification system by the American Joint Cancer Committee [AJCC] 2010) account for approximately 75% of all diagnosed NSCLC cases [9,12]. Even with the advent of personalized therapy the prognosis in NSCLC is very poor, with an overall survival (OS) of approximately 1% in 5 years [13]. Risk factors known to be associated with the development of NSCLC are smoking, passive smoke, silicosis and radon exposure [12].
Advances in cytotoxic chemotherapy use for advanced NSCLC no longer seems to significantly improve median survival of these patients over recent years, indicating that the effectiveness of chemotherapy has perhaps reached its limit. Surgery is the best curative therapeutic approach for early stage disease (I and II). However, even in these patients, the 5-year mean OS is less than 70% [8]. In the last decade, several studies [5,14] have contributed to a better understanding of lung cancer biology, leading to the development of new personalized therapies. A hallmark study, in 2004, highlighted the clinical value of a targeted therapy against EGF receptor (EGFR) in patients with NSCLC [15]. Those patients harboring hotspot mutations in exons 19 and 21 of EGFR showed improved outcome when treated with EGFR tyrosine kinase inhibitors (TKIs) [15–17]. Shepherd et al. first showed that erlotinib, an EGFR TKI, was effective as a second- and third-line therapy to prolong survival in unselected refractory NSCLC IIIB and IV stages [16]. TKI’s are generally less toxic to normal cells than cytotoxic chemotherapy and have improved tolerability. Nevertheless, these targeted therapies have modest activity when given to unselected patient populations. Female patients, nonsmokers and patients with adenocarcinomas have been observed more frequently to respond to EGFR TKIs [15,18]. These clinical characteristics are strongly associated with somatic mutations of EGFR [19]. In addition, new oncogenic driver mutations are continuously being assessed and reported as influencing NSCLC development or to dictate clinical management. In this review we
10.2217/PGS.13.177 © 2013 Future Medicine Ltd
Pharmacogenomics (2013) 14(14), 1765–1777
Ramon Andrade de Mello*1,2,3, Pedro Madureira4,5, Liliana S Carvalho5,6, António Araújo1,4, Mary O’Brien3 & Sanjay Popat3,7 Department of Medical Oncology, Portuguese Oncology Institute, Rua Dr António Bernardino de Almeida, 4200-072, Porto, Portugal 2 Department of Medicine, Faculty of Medicine, University of Porto (FMUP), Alameda Prof Hernani, Monteiro, 4200-319, Porto, Portugal 3 Department of Medicine, Royal Marsden NHS Foundation Trust, Fulham Road, London SW3 6JJ, UK 4 ICBAS – Instituto de Ciências Biomédicas Abel Salazar, University of Porto, Porto, Portugal 5 IBMC – Instituto de Biologia Molecular e Celular, Porto, Portugal 6 Department of Pharmacology & Therapeutics, FMUP – Faculty of Medicine, University of Porto, Alameda Prof Hernani Monteiro, 4200-319, Porto, Portugal 7 Molecular Genetics & Genomics Group, Imperial College, London, UK *Author for correspondence: Tel.: +351 912 040 770 Fax: +351 225 084 010 [email protected] 1
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ISSN 1462-2416
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will focus on specific genetic alterations that may contribute to the development of NSCLC novel personalized therapy.
Molecular pathways Carcinogenesis is characterized by changes at the cellular and molecular level that induce uncontrolled cell division, which may be explained by cell intrinsic mutations in tumor suppressor genes and/or oncogenes (Figure 1). Nevertheless, carcinomas are heterogeneous and structurally complex tumors, and recent evidence suggests that additional cell types also contribute to the carcinogenesis and pathophysiological properties of these tumors [20]. Here, transformed cells do not result merely from autonomous processes but can be affected by reciprocal interactions between parenchymal and stromal cells [21]. Thus, there are several lines of evidence suggesting that distinct molecules, participating in different metabolic processes may be implicated in tumor genesis and/or tumor progression. EGFR pathway & NSCLC EGFR is the first characterized of a family of four ErbB receptor tyrosine kinases that comprise EGFR (also known as ErbB1 or HER1), ErbB2 (also known as HER3), ErbB3 (also known as HER3) and ErbB4 (also known as HER4) [22]. EGFR has been a focus of intense research in the last two decades as a therapeutic target. Downstream signaling of EGFR is implicated in tumor cell growth, local invasion, angiogenesis, metastasis, protein translation and cell metabolism (Figure 1) [23,24]. Binding of ligands or growth factors, such as EGF, amphiregulin and TGF-a, to the extracellular domain of ErbB receptors stabilizes them in a conformation that allows dimerization, an essential requirement for activation of the kinase portion of the dimer moiety, leading to phosphorylation and downstream signaling [25,26]. The dimerization of ErbB receptors can lead to homo- or hetero-dimer formation, resulting in different signaling events [27,28]. The most important pathways activated in solid tumors are RAS/RAF/MEK/ERK and PI3K/AKT/mTOR. Interestingly, EGFR mainly activates the MEK pathway and signaling through ErbB3 heterodimers strongly activates the PI3K component [29,30]. The two most common somatic EGFR mutations associated with EGFR TKI responses in advanced NSCLC are short inframe deletions of exon 19 and a point mutation (CTG to CGG) in exon 21 at nucleotide 2573 that results in substitution of leucine by arginine at codon 858 (L858R) [31–33]. 1766
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KRAS & NSCLC KRAS is an important downstream molecule of the EGFR signaling pathway [34,35]. Mutations that cause the loss of KRAS GTPase activity render the protein constitutively GTPbound, resulting in sustained activation of downstream effectors of the RAF/MAPK/MEK/ERK cascade and persistent proliferation signal [36,37]. Gain-of-signal mutations in KRAS also promote hyperactivation of NF-kB activation, resulting in excess of proinflammatory molecules and chronic inflammatory conditions [38]. Prevalence of KRAS mutation is approximately 20% in the overall lung cancer patient population [39,40]. Mutations in the EGFR kinase domain occur early in the development of adenoc arcinomas that are generally unrelated to smoking, while KRAS mutations occur early in the development of smoking-related adenocarcinoma [40,41]. Direct inhibition of EGFR downstream pathways was shown to be a promising alternative for overcoming EGFR TKI resistance mainly in KR AS- and BR AF-mutated tumors [42]. MEK inhibition was shown to be an interesting strategy in this regard owing to the fact that it blocked upstream signaling pathways involved in cancer cell growth and metastasis. Furthermore, MEK activation is found mostly in BRAF mutant cell lines and in a subgroup of KRAS mutant cell lines [42]. Thus, these cells seem to be more sensitive to MEK inhibitors. Notwithstanding, activating mutations of PI3KCA and hyperactivity of the PTEN/PI3K/AKT pathway are associated with reduced activity or resistance to MEK inhibitors in human cancer cells. In this regard, potential strategies focusing on MEK inhibition (selumetinib [43] and pimasertib) and PI3K/AKT/mTOR pathways inhibition (everolimus) are promising, although still premature [43,44]. In preclinical models, targeting the MEK pathway in combination with selective inhibitors of PTEN/PI3K/AKT/mTOR or in combination with multitarget kinase inhibitors including BRAF, such as sorafenib and regorafenib, could overcome primary resistance to MEK inhibitors and may enhance antitumor efficacy [42]. ALK gene rearrangements The EML4-ALK fusion-type tyrosine kinase is an oncoprotein found in 4–5% of NSCLCs and is generated as a result of a small inversion within the short arm of human chromosome 2 [45–47]. The resulting ALK fusion protein undergoes constitutive dimerization through inter action between the coiled-coil domains within the EML4 region of each monomer, thereby future science group
EGFR & KRAS mutations, & ALK fusions: current developments & personalizing therapies
Review
ErbB family
p85 PIP2 p110
GRB2 SOS GTP
PIP3
RAF
AKT
EML4–ALK fusion
RAS
GDP GAP
MEK
ERK
Figure 1. Molecular pathways concerning EGF receptor intracellular signaling and EML4–ALK fusion interaction in non-small-cell lung cancer carcinogenesis. Presented are changes at the cellular and molecular level that induce uncontrolled cell division, which may be explained by cell intrinsic mutations in tumor suppressor genes and/or oncogenes. Interactions between ligands, such as EGF or TGF-a, and extracellular ErbB family domain leads to conformational interactions and hetero-/homo-dimer formation, resulting in different signaling events, such as RAS/RAF/MEK/ERK and PI3K/AKT/mTOR.
activating ALK and generating oncogenic activity [48]. In transgenic mice that express EML4-ALK specifically in lung epithelial cells, there is spontaneous development of adenocarcinoma nodules in both lungs soon after birth, but these are rapidly eradicated after administration of ALK TKIs [48,49]. These observations demonstrate the essential role of EML4-ALK in the carcinogenesis of non-small-cell lung cancer harboring this fusion kinase. ALK rearrangements are generally associated with a younger age of diagnosis, and are typically observed in never-smokers. ALK fusions as well as EGFR and KRAS mutations are all generally mutually exclusive, implicating ALK rearrangement as a potential therapeutic in EGFR wild-type and KRAS wild-type lung cancer [50,51]. Angiogenic-related factors & NSCLC As tumors require an adequate blood supply to maintain viability and metastatic potential, sustained angiogenesis is a hallmark of cancer and is well described in NSCLC pathogenesis [52,53]. Increased lung tumor microvessel density correlates with metastatic potential and reduced survival [54,55]. VEGF is a well-characterized angiogenic factor and mediates angiogenesis future science group
through activation of endothelial cells, predominantly through binding to VEGF receptor-2 (VEGFR-2) [56]. Endogenously produced VEGF from platelets, muscle cells or the tumor stroma contribute to signaling. Autocrine, paracrine and intracrine signaling have also been described. Because of its dominant role in angiogenesis, the VEGF/VEGFR pathway is an obvious interesting therapeutic target [57,58]. Targeting blood vessel formation with either monoclonal antibodies directed against the VEGF ligand or small-molecule TKIs directed against VEGFRs has corroborated VEGF pathway-directed therapy in a number of different tumors, as well as in NSCLC [59–62]. Notwithstanding that, caution should be taken with VEGF/VEGFR targeted therapies, owing to potential toxicities [63]. Other molecules other than VEGF, such as IP-10, interferons, angiopoietin/TIE-2 inter action, Notch/d-like ligand 4 and FGFs, also have a strong influence on angiogenesis [64–66]. These factors may drive angiogenesis directly in tumors refractory to prior VEGF/VEGFRdirected therapies or may contribute to acquired resistance via selection pressures following VEGF/VEGFR-directed therapy [67]. www.futuremedicine.com
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Activating mutations, overexpression or gene amplification associated with the FGF signaling pathway has also been reported in a number of human malignancies, including lung cancer, myeloproliferative disorders and lymphomas [68,69]. The ligand FGF-2 is directly associated with neovascularization [70,71]. Autocrine and paracrine signaling independent of somatic mutations have also been implicated in FGFrelated tumorigenesis [53]. Marek and colleagues described frequent coexpression of distinct FGFs (FGF-2 and FGF-9) and FGFRs, suggesting a potential autocrine loop in NSCLC cell lines [72]. The FGF/FGFR pathway prevails among mesenchymal NSCLC histologies, and activation seems to be associated with resistance to EGFR TKIs [72,73]. Also, von Willebrand factor (vWF) is a multi meric plasma glycoprotein synthesized in megakaryocytes/platelets and in endothelial cells [74]. The plasma concentration of vWF is used as a marker of endothelial cell activation and reduced plasma levels of vWF are observed in NSCLC [74]. Studies conducted in mice indicate that the decreased plasma levels of vWF in NSCLC patients could result from the activity of ADAM28 [75]. Inhibition of ADAM28 expression or activity showed a substantial reduction in lung metastasis and increased apoptosis of cancer cells in the blood vessels [75]. Notwithstanding that, further studies are needed to ascertain the importance of ADAM28 as an appropriate target for intervention in lung cancer. Nuclear molecular events associated with NSCLC In normal physiological conditions, DNA damage, such as that caused by genotoxic chemotherapeutic agents, induces apoptosis through double stranded break associated kinases, a process mediated through p53 [76,77]. Bcl-2 family members regulated by p53 are central to the activation of apoptosis [78]. The antiapoptotic Bcl-2 family member Bcl-xL protects cancer cells from p53-induced apoptosis and acts through the binding and inactivation of Bax and binding of proteins that recruit Bax to the mitochondrial membrane [78]. Bcl-xL is frequently over expressed in lung tumors and plays an important role in resistance to genotoxic chemotherapeutic agents in lung and other cancer types [79]. BclxL overexpression in tumors is also associated with poor prognosis [80,81]. Other enzymes involved in cell cycle regulation such as TERT, which control telomer length and stability, and CLPTM1L are also overexpressed in lung 1768
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tumors and are thought to confer resistance to apoptosis caused by genotoxic agents, in association with Bcl-xL [82]. Interestingly, TERT and CLPTM1L genes are colocalized on chromosome 5p15 where polymorphisms in rs402710, rs401681 and rs31489 genes have been associated with NSCLC susceptibility [83–85]. TS, an enzyme associated with de novo synthesis of DNA that also plays an important role in DNA repair, is highly expressed in NSCLC. Moreover, a deletional polymorphisms at the TS 3´-UTR (1494del 6 bp) has been associated with sensitivity of lung adenocarcinoma to pemetrexed therapy, indicating that TS polymorphisms should be further evaluated as candidate predicitive markers for personalized therapy in lung adenocarcinoma [86]. CTLA-4 & tumor immunity Important aspects of tumor biology are the active mechanisms employed by cancer cells to avoid killing by the immune system. The immune system of vertebrates is composed by a multitude of cellular and molecular mechanisms that are crucial for the elimination of cancer cells. Nevertheless, once a tumor is well histologically differentiated; the immune system seems to be incapable of directing a specific response towards it. Despite extensive research efforts, a suitable explanation for this phenomenon is still missing. Eradication of tumor cells is dependent on the activation of T cells, which requires not only stimulation of the T cell receptor by peptide–MHCs on antigen-presenting cells, but also an orchestrated balance of costimulatory and inhibitory signals that modulate the magnitude and effectiveness of the immune response. Thus, in normal physiological conditions, T-cell activation results from a signal transduction cascade downstream of cell-surface receptors. Nevertheless, this activation signal, which is delivered by cell-surface receptors, needs to be tightly regulated in order to avoid excess of inflammation [87]. In that sense, negative regulatory proteins on T cells, such as CTLA-4, PD-1, B7 family member B7-H4, Tim-3 and LAG-3, interact with their ligands on various cells types, including antigen-presenting cells, regulatory T cells, and nonhematopoietic cells, resulting in reduced T-cell proliferation and functional activity [88,89]. Interestingly, tumor-infiltrating T cells have a marked upregulation of cell-surface molecules that block efficient activation [90]. Hence, tumor environment drives a sustained expression of CTLA-4, PD-1 and LAG-3 on antigen-specific future science group
EGFR & KRAS mutations, & ALK fusions: current developments & personalizing therapies
lymphocytes, which culminates in peripheral cell tolerance to the tumor-specific antigens [91]. In fact, several studies in mice, demonstrate that antibody-mediated blockade of CTLA-4 increases endogenous responses to tumor cells, inducing tumor cell death [90]. Preclinical findings have translated into clinical development of a fully human, IgG1 monoclonal antibody, ipilimumab, and a fully human, IgG2 monoclonal antibody, tremelimumab, both of which bind CTLA-4. Of interest, ipilimumab was recently approved in several countries for the treatment of advanced or metastatic melanoma [90,92]. Also, many trials in lung cancer and ipilimumab are still in process, but its role remains experimental in NSCLC.
Integrating EGFR mutation, & K-RAS & ALK fusions into NSCLC clinical management A hallmark of modern pharmacogenomics is the possibility to develop personalized therapies based of the genetic background of each individual. The presence of specific point mutations or polymorphisms in key genes may dictate the response rate toward a specific drug [66]. A promising strategy for the future development of therapeutic interventions is based on the discovery that distinct subsets of cancers are strictly dependent on specific driver mutations for cellular proliferation and survival. Usually, these driver mutations result in the altered expression of proteins involved in signaling pathways crucial for cellular proliferation and survival [6]. Hence, targeting the activity of these mutant proteins can lead to cell death and therapeutic benefit [66]. This phenomenon is referred to as ‘oncogene addiction’ [6]. EGFR-mutant NSCLC was first recognized in 2004 as a distinct, clinically relevant molecular subset of lung cancer and currently represents the best-studied example of oncogene addiction in lung cancer. EGFR-mutant tumors are usually adenocarcinomas and are associated with a better prognosis than EGFR wild-type tumors [6]. NSCLC is associated with EGFR overexpression in up to 80% of the patients, and a high EGFR gene copy number is found in nearly 60% of the cases. Mutation of EGFR is observed in 10–20% of lung carcinomas, and nearly 80% of lung cancer-specific EGFR mutations comprise a leucine-to-arginine substitution at position 858 (L858R) and deletion mutations in exon 19 [6]. Importantly, these mutations are of extreme clinical relevance since they increase the sensitivity of NSCLC to TKIs, such as erlotinib and gefitinib (Table 1). Both these drugs are reversible future science group
Review
inhibitors of the EGFR tyrosine kinase, designed to act as competitive inhibitors of ATP binding at the active site of the EGFR tyrosine kinase. EGFR mutations are associated with the marked responses observed in patients treated with EGFR TKIs, and are more common in neversmokers, women, individuals of Asian ethnic background and those with adenocarcinoma histology. In addition to providing a genetic marker for a highly EGFR-TKI-responsive subset of NSCLCs, this correlation has also highlighted the crucial importance of mutational activated kinases as anticancer drug targets [8]. In addition to EGFR overexpression, its cognate ligands, EGF and TGF-a are also frequently expressed in NSCLCs, and can establish positive feedback loops that promote receptor hyperactivity. The disruption of these autocrine loops is the rationale for the use of EGFR-specific antibody cetuximab in patients with NSCLC [8]. An important downstream signaling target of EGFR, KRAS has also been implicated in the development and prognosis of NSCLC [6]. Mutations that cause the loss of KRAS GTPase activity render the protein constitutively GTPbound, resulting in a persistent proliferation signal [8]. In lung cancers, the most common KRAS mutations occur primarily at codons 12 and 13; the most common KRAS mutation in smoking patients with NSCLC is a G-to-T transition (84%) resulting in substitution of cysteine (47%), valine (24%), aspartate (15%) or alanine (7%) for wild-type glycine [6]. As with EGFR mutations, KRAS mutations are detected mainly in lung adenocarcinomas and are less frequently observed in squamous cell carcinomas of the lung [6]. By contrast with lung adenocarcinomas harboring EGFR mutations, KRAS mutant tumors are seen at a higher frequency (20–30%) in Caucasian patients than in east Asian patients (5%) [6]. Also, compared with EGFR mutations, KRAS mutations are more common in current or former smokers than in never-smokers, although the absence of a history of tobacco use does not eliminate the possibility of such abnormalities [8]. KRAS mutations are associated with primary resistance to EGFR TKIs [6]. Phase II trials have shown very small or absent response rates to erlotinib in patients with KRAS mutations [10]. The use of chemotherapy with or without erlotinib in previously untreated patients with NSCLC (TRIBUTE trial) suggested that, in KRAS mutated patients, OS and response rate (RR) might be even worse with the addition of EGFR TKIs [6]. www.futuremedicine.com
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[105]
2.9 IIIB and IV
‡
†
EGFR mutation in exon 19 and exon 21. Response rate, complete plus partial response. –: No significant data; EGFR: EGF receptor; OS: Overall survival; PFS: Progression-free survival.
464 No France Erlotinib maintenance Pérol et al. (2012)
1770
Interestingly, the presence of KRAS or EGFR mutations seem to be mutually exclusive with ALK fusions [8]. Patients positive for the ALK fusion protein frequently present at an advanced clinical stage, and the tumors pattern tends to be a solid adenocarcinoma pattern with signet ring cell morphology [45]. As above, the ALK fusion oncoprotein is associated with never-smokers or light smokers and is more frequent in younger patients [45,93]. The US FDA and the EMA have approved crizotinib, a small-molecule inhibitor of ALK tyrosine kinase, as a treatment for patients with locally advanced or metastatic NSCLC expressing the ALK fusion [5].
–
11.4
[104]
8.9 No Erlotinib Gridelli et al. (2012)
Italy
760
IIIB and IV
20.3
11.6
[101]
13.1 Yes Erlotinib Zhou et al. (2011)
China
83
IIIB and IV
82.9
–
[103]
12.3 Yes Erlotinib Capuzzo et al. (2010)
Italy
437
IIIB and IV
11.9
12.3
[102]
6 No Erlotinib Herbst et al. (2005)
USA
526
IIIB and IV
30
10.6
[16]
2.2 6.7 No Erlotinib Shepherd et al. (2005)
America, Europe and Asia
731
IIIB and IV
8.9
9.2 Yes Geftinib Mitsudomi et al. (2010)
Japan
177
IIIB and IV
62.1
30.9
[97]
[100]
5.7 No Geftinib Mok et al. (2009)
Asia
609
III and IV
71.2
18.6
[96]
10.8 Yes Gefitinib Maemondo et al. (2010)
Asia
230
IIIB and IV
73.7
30.5
[99]
– No Gefitinib Perez-Soler et al. (2004)
USA
57
IIIB and IV
12.3
8.4
[98]
– 6–7 No Gefitinib Kris et al. (2003)
USA
221
IIIB and IV
22
Median PFS (months) EGFR†-positive selected mutations Molecule
Place of study
Patients (n)
Clinical stage
Response rate‡ (%)
Median OS (months)
Ref.
de Mello, Madureira, Carvalho, Araújo, O’Brien & Popat
Study
Table 1. Summary of the studies that address the current main EGF receptor tyrosine kinase inhibitor drugs in the non-small-cell lung cancer field.
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Pharmacogenomics (2013) 14(14)
Targeted therapies for NSCLC: current & future perspectives In 2004, a new therapeutic era for NSCLC began with the emergence of targeted therapies against EGFR [8,9,94,95]. Gefitinib [96–100] and erlotinib [16,101–105], two small-molecule drugs that specifically target the tyrosine kinase activity of EGFR TKIs, were promptly approved by the FDA for patients with advanced NSCLC in specific subsettings previous described (Table 1) [6]. Unfortunately, patients start to develop resistance against these EGFR TKIs within 6–12 months after the initiation of therapy. Acquired resistance to gefitinib and erlotinib is predominantly due to the appearance of the T790M allele, which affects the gatekeeper residue in the catalytic domain of the kinase that weakens the interaction of the inhibitor with its target, and results in a markedly elevated IC50. Nevertheless, this acquired resistance has led to the development of new drugs as alternative therapies to NSCLC [6]. Table 2 summarizes the current targeted therapies and clinical trials involved in the NSCLC setting discussed below. Cetuximab Cetuximab is a chimeric monoclonal antibody that binds to the extracellular domain of EGFR to block ligand binding, which results in inhibition of autophosphorylation of EGFR [106]. EGFR binding by cetuximab results in cell cycle arrest, increased expression of p27, and increased expression of proapoptotic proteins, including Bax, caspase 3, caspase 8 and caspase 9, or decreased expression of antiapoptotic proteins such as Bcl-2 [107,108]. Cetuximab has also been shown to decrease the production of VEGF and basic FGF in orthotopic tumor models. Preclinical studies indicated synergism between cetuximab and a number of chemotherapeutic future science group
EGFR & KRAS mutations, & ALK fusions: current developments & personalizing therapies
Review
Table 2. Summary of clinical trials evaluating current target therapies in advanced non-small-cell lung cancer patients. Study
Phase
Treatment
Patients (n)
OS (month)
RR (%)
Ref.
III
CT + cetuximab 400 mg/m2 D1 >250 mg/m2
557
11.3
36
CT: CDDP 80 mg/m2 D1 + vinorelbine 25 kg/m2
568
10.1
29
Afatinib 50 mg per day
390
10.8
7
Placebo
195
12
11
Crizotinib 250 mg/day in 28-day cycle
82
NR
57
[114]
Crizotinib 250 mg b.i.d. in 28-day cycle
143
NR
60.8
[117]
CBDCA + paclitaxel
427
10.3
35
[118]
CBDCA + paclitaxel + bevacizumab 15 mg/kg
440
12.3
15
CDDP + gemcitabine + placebo
347
NR
20.1
CDDP + gemcitabine + bevacizumab 7.5 mg/kg
345
NR
34.1
CDDP + gemcitabine + bevacizumab 15 mg/kg
351
NR
30.4
Cetuximab FLEX (2009)
[110,111]
Afatinib LUX-Lung 1 (2012)
IIb/III
[112]
Crizotinib Kwak et al. (2010)
I
Camidge et al. (2012) I Bevacizumab ECOG 4599 (2010) AVAiL (2009)
III III
[60]
Sunitinib Socinski et al. (2008)
II
Sunitinib 50 mg daily†
63
5.7
11.1
[120]
Gervais et al. (2011)
III
Sunitinib 37.5 mg/day + erlotinib 150 mg/day
480
9.0
10.6
[121]
Placebo + erlotinib 150 mg/day
480
8.5
6.9
Sorafenib (400 mg b.i.d.) + gemcitabine 385 1250 mg/m2 per day on days 1 and 8 and cisplatin 75 mg/m2 on day 1 for up to six 21‑day cycles
12.4
27.8
Sorafenib (400 mg twice a day) + placebo
12.5
25.8
Sorafenib Paz-Ares et al. (2012)
III
387
[128]
Sunitinib administered daily for 4 weeks of 6 weeks cycle. b.i.d.: Twice daily; CT: Chemotherapy; NR: Not reported; OS: Overall survival; RR: Response rate. †
agents, including cisplatin and paclitaxel, in a wide variety of cell lines [109]. In a Phase III trial study (FLEX), 1125 patients were randomly assigned to cisplatin/vinorelbine chemotherapy plus cetuximab (n = 557) or chemotherapy alone (n = 568) [110,111]. Whilst patients given chemotherapy plus cetuximab survived statistically longer than those in the chemotherapy-alone group (median 11.3 months vs 10.1 months; hazard ratio for death 0.871 [95% CI: 0.762–0.996]; p = 0.044), the clinical significance of this has been debated, and cetuximab was not approved for use by the FDA. The main cetuximab-related adverse event was acne-like rash (57 [10%] of 548, grade 3). Afatinib Afatinib is an irreversible ErbB-family kinase inhibitor [112]. Afatinib shows preclinical activity when tested in EGFR mutant models with future science group
mutations that confer resistance to EGFR TKIs, including the most common mutations, L858R and deletion-19, and the exon 20 gatekeeper T790M mutations. Afitinib was investigated in a randomized Phase IIb/III trial (LUX-Lung 1) of 585 patients (390 to afatinib and 195 to placebo) with stage IIIB or IV adenocarcinoma who had received one or two previous chemotherapy regimens and had disease progression after at least 12 weeks of treatment with erlotinib or gefitinib [112]. This study demonstrated no benefit in terms of OS with afatinib (the primary end point). Nonetheless, the median progression-free survival (PFS) was longer in the afatinib group (3.3 months, 95% CI: 2·79–4·40) than it was in the placebo group (1.1 months, 0.95–1.68; hazard ratio: 0.38; 95% CI: 0.31–0.48; p
EGFR and KRAS mutations, and ALK fusions: current developments and personalized therapies for patients with advanced non-small-cell lung cancer Personalized therapy has significantly developed in lung cancer treatment over recent years. VEGF and EGF play a major role in non-small-cell lung cancer (NSCLC) tumor angiogenesis and aggressiveness. EGFR mutation as well as KRAS and ALK rearrangements are important biomarkers in the field owing to potential targeted therapies involved in clinical practice: erlotinib, geftinib, cetuximab and crizotinib. More recently, regulation of tumor immunity through CTLA4 and PD1/L1 has emerged as a promising field in NSCLC management. This review will focus on the current and future biomarkers in the advanced NSCLC field and also address potential related targeted therapies for these patients. KEYWORDS: afatinib n ALK–EML4 n bevacizumab n cetuximab n crizotinib n EGFR mutation n erlotinib n gefitinib n lung cancer
Currently, it is estimated that approximately 11 million people worldwide have cancer. Lung cancer is the leading cause of cancer-related death and accounts for approximately 30% of all cancer deaths [1,2]. In the USA, lung cancer has a global incidence of approximately 70 cases per 100,000 inhabitants. In Europe, lung cancer incidence is approximately 52.5 cases per 100,000 inhabitants (82.5 in 100,000 in males and 23.9 in 100,000 in females) and mortality is approximately 48.7 out of 100,000 (77 in 100,000 in males and 23.9 in 100,000 in females) [3–6]. Lung cancer can be generally divided into small-cell lung cancer and non-small-cell lung cancer (NSCLC) [7,8]. NSCLC accounts for approximately 85% of all lung cancers [1,9,10]. NSCLC originates in lung epithelial cells, and comprises diverse histological subtypes including adenocarcinoma, as well as squamous, anaplastic and large-cell carcinomas [8,11]. Most patients with NSCLC have advanced disease at diagnosis, present with metastatic disease and, if left untreated, have a median survival time after diagnosis of 4–5 months and a 1‑year survival of less than 10% [8,10]. The advanced stages (IIIB and IV; according to the tumor node metastasis classification system by the American Joint Cancer Committee [AJCC] 2010) account for approximately 75% of all diagnosed NSCLC cases [9,12]. Even with the advent of personalized therapy the prognosis in NSCLC is very poor, with an overall survival (OS) of approximately 1% in 5 years [13]. Risk factors known to be associated with the development of NSCLC are smoking, passive smoke, silicosis and radon exposure [12].
Advances in cytotoxic chemotherapy use for advanced NSCLC no longer seems to significantly improve median survival of these patients over recent years, indicating that the effectiveness of chemotherapy has perhaps reached its limit. Surgery is the best curative therapeutic approach for early stage disease (I and II). However, even in these patients, the 5-year mean OS is less than 70% [8]. In the last decade, several studies [5,14] have contributed to a better understanding of lung cancer biology, leading to the development of new personalized therapies. A hallmark study, in 2004, highlighted the clinical value of a targeted therapy against EGF receptor (EGFR) in patients with NSCLC [15]. Those patients harboring hotspot mutations in exons 19 and 21 of EGFR showed improved outcome when treated with EGFR tyrosine kinase inhibitors (TKIs) [15–17]. Shepherd et al. first showed that erlotinib, an EGFR TKI, was effective as a second- and third-line therapy to prolong survival in unselected refractory NSCLC IIIB and IV stages [16]. TKI’s are generally less toxic to normal cells than cytotoxic chemotherapy and have improved tolerability. Nevertheless, these targeted therapies have modest activity when given to unselected patient populations. Female patients, nonsmokers and patients with adenocarcinomas have been observed more frequently to respond to EGFR TKIs [15,18]. These clinical characteristics are strongly associated with somatic mutations of EGFR [19]. In addition, new oncogenic driver mutations are continuously being assessed and reported as influencing NSCLC development or to dictate clinical management. In this review we
10.2217/PGS.13.177 © 2013 Future Medicine Ltd
Pharmacogenomics (2013) 14(14), 1765–1777
Ramon Andrade de Mello*1,2,3, Pedro Madureira4,5, Liliana S Carvalho5,6, António Araújo1,4, Mary O’Brien3 & Sanjay Popat3,7 Department of Medical Oncology, Portuguese Oncology Institute, Rua Dr António Bernardino de Almeida, 4200-072, Porto, Portugal 2 Department of Medicine, Faculty of Medicine, University of Porto (FMUP), Alameda Prof Hernani, Monteiro, 4200-319, Porto, Portugal 3 Department of Medicine, Royal Marsden NHS Foundation Trust, Fulham Road, London SW3 6JJ, UK 4 ICBAS – Instituto de Ciências Biomédicas Abel Salazar, University of Porto, Porto, Portugal 5 IBMC – Instituto de Biologia Molecular e Celular, Porto, Portugal 6 Department of Pharmacology & Therapeutics, FMUP – Faculty of Medicine, University of Porto, Alameda Prof Hernani Monteiro, 4200-319, Porto, Portugal 7 Molecular Genetics & Genomics Group, Imperial College, London, UK *Author for correspondence: Tel.: +351 912 040 770 Fax: +351 225 084 010 [email protected] 1
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will focus on specific genetic alterations that may contribute to the development of NSCLC novel personalized therapy.
Molecular pathways Carcinogenesis is characterized by changes at the cellular and molecular level that induce uncontrolled cell division, which may be explained by cell intrinsic mutations in tumor suppressor genes and/or oncogenes (Figure 1). Nevertheless, carcinomas are heterogeneous and structurally complex tumors, and recent evidence suggests that additional cell types also contribute to the carcinogenesis and pathophysiological properties of these tumors [20]. Here, transformed cells do not result merely from autonomous processes but can be affected by reciprocal interactions between parenchymal and stromal cells [21]. Thus, there are several lines of evidence suggesting that distinct molecules, participating in different metabolic processes may be implicated in tumor genesis and/or tumor progression. EGFR pathway & NSCLC EGFR is the first characterized of a family of four ErbB receptor tyrosine kinases that comprise EGFR (also known as ErbB1 or HER1), ErbB2 (also known as HER3), ErbB3 (also known as HER3) and ErbB4 (also known as HER4) [22]. EGFR has been a focus of intense research in the last two decades as a therapeutic target. Downstream signaling of EGFR is implicated in tumor cell growth, local invasion, angiogenesis, metastasis, protein translation and cell metabolism (Figure 1) [23,24]. Binding of ligands or growth factors, such as EGF, amphiregulin and TGF-a, to the extracellular domain of ErbB receptors stabilizes them in a conformation that allows dimerization, an essential requirement for activation of the kinase portion of the dimer moiety, leading to phosphorylation and downstream signaling [25,26]. The dimerization of ErbB receptors can lead to homo- or hetero-dimer formation, resulting in different signaling events [27,28]. The most important pathways activated in solid tumors are RAS/RAF/MEK/ERK and PI3K/AKT/mTOR. Interestingly, EGFR mainly activates the MEK pathway and signaling through ErbB3 heterodimers strongly activates the PI3K component [29,30]. The two most common somatic EGFR mutations associated with EGFR TKI responses in advanced NSCLC are short inframe deletions of exon 19 and a point mutation (CTG to CGG) in exon 21 at nucleotide 2573 that results in substitution of leucine by arginine at codon 858 (L858R) [31–33]. 1766
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KRAS & NSCLC KRAS is an important downstream molecule of the EGFR signaling pathway [34,35]. Mutations that cause the loss of KRAS GTPase activity render the protein constitutively GTPbound, resulting in sustained activation of downstream effectors of the RAF/MAPK/MEK/ERK cascade and persistent proliferation signal [36,37]. Gain-of-signal mutations in KRAS also promote hyperactivation of NF-kB activation, resulting in excess of proinflammatory molecules and chronic inflammatory conditions [38]. Prevalence of KRAS mutation is approximately 20% in the overall lung cancer patient population [39,40]. Mutations in the EGFR kinase domain occur early in the development of adenoc arcinomas that are generally unrelated to smoking, while KRAS mutations occur early in the development of smoking-related adenocarcinoma [40,41]. Direct inhibition of EGFR downstream pathways was shown to be a promising alternative for overcoming EGFR TKI resistance mainly in KR AS- and BR AF-mutated tumors [42]. MEK inhibition was shown to be an interesting strategy in this regard owing to the fact that it blocked upstream signaling pathways involved in cancer cell growth and metastasis. Furthermore, MEK activation is found mostly in BRAF mutant cell lines and in a subgroup of KRAS mutant cell lines [42]. Thus, these cells seem to be more sensitive to MEK inhibitors. Notwithstanding, activating mutations of PI3KCA and hyperactivity of the PTEN/PI3K/AKT pathway are associated with reduced activity or resistance to MEK inhibitors in human cancer cells. In this regard, potential strategies focusing on MEK inhibition (selumetinib [43] and pimasertib) and PI3K/AKT/mTOR pathways inhibition (everolimus) are promising, although still premature [43,44]. In preclinical models, targeting the MEK pathway in combination with selective inhibitors of PTEN/PI3K/AKT/mTOR or in combination with multitarget kinase inhibitors including BRAF, such as sorafenib and regorafenib, could overcome primary resistance to MEK inhibitors and may enhance antitumor efficacy [42]. ALK gene rearrangements The EML4-ALK fusion-type tyrosine kinase is an oncoprotein found in 4–5% of NSCLCs and is generated as a result of a small inversion within the short arm of human chromosome 2 [45–47]. The resulting ALK fusion protein undergoes constitutive dimerization through inter action between the coiled-coil domains within the EML4 region of each monomer, thereby future science group
EGFR & KRAS mutations, & ALK fusions: current developments & personalizing therapies
Review
ErbB family
p85 PIP2 p110
GRB2 SOS GTP
PIP3
RAF
AKT
EML4–ALK fusion
RAS
GDP GAP
MEK
ERK
Figure 1. Molecular pathways concerning EGF receptor intracellular signaling and EML4–ALK fusion interaction in non-small-cell lung cancer carcinogenesis. Presented are changes at the cellular and molecular level that induce uncontrolled cell division, which may be explained by cell intrinsic mutations in tumor suppressor genes and/or oncogenes. Interactions between ligands, such as EGF or TGF-a, and extracellular ErbB family domain leads to conformational interactions and hetero-/homo-dimer formation, resulting in different signaling events, such as RAS/RAF/MEK/ERK and PI3K/AKT/mTOR.
activating ALK and generating oncogenic activity [48]. In transgenic mice that express EML4-ALK specifically in lung epithelial cells, there is spontaneous development of adenocarcinoma nodules in both lungs soon after birth, but these are rapidly eradicated after administration of ALK TKIs [48,49]. These observations demonstrate the essential role of EML4-ALK in the carcinogenesis of non-small-cell lung cancer harboring this fusion kinase. ALK rearrangements are generally associated with a younger age of diagnosis, and are typically observed in never-smokers. ALK fusions as well as EGFR and KRAS mutations are all generally mutually exclusive, implicating ALK rearrangement as a potential therapeutic in EGFR wild-type and KRAS wild-type lung cancer [50,51]. Angiogenic-related factors & NSCLC As tumors require an adequate blood supply to maintain viability and metastatic potential, sustained angiogenesis is a hallmark of cancer and is well described in NSCLC pathogenesis [52,53]. Increased lung tumor microvessel density correlates with metastatic potential and reduced survival [54,55]. VEGF is a well-characterized angiogenic factor and mediates angiogenesis future science group
through activation of endothelial cells, predominantly through binding to VEGF receptor-2 (VEGFR-2) [56]. Endogenously produced VEGF from platelets, muscle cells or the tumor stroma contribute to signaling. Autocrine, paracrine and intracrine signaling have also been described. Because of its dominant role in angiogenesis, the VEGF/VEGFR pathway is an obvious interesting therapeutic target [57,58]. Targeting blood vessel formation with either monoclonal antibodies directed against the VEGF ligand or small-molecule TKIs directed against VEGFRs has corroborated VEGF pathway-directed therapy in a number of different tumors, as well as in NSCLC [59–62]. Notwithstanding that, caution should be taken with VEGF/VEGFR targeted therapies, owing to potential toxicities [63]. Other molecules other than VEGF, such as IP-10, interferons, angiopoietin/TIE-2 inter action, Notch/d-like ligand 4 and FGFs, also have a strong influence on angiogenesis [64–66]. These factors may drive angiogenesis directly in tumors refractory to prior VEGF/VEGFRdirected therapies or may contribute to acquired resistance via selection pressures following VEGF/VEGFR-directed therapy [67]. www.futuremedicine.com
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Activating mutations, overexpression or gene amplification associated with the FGF signaling pathway has also been reported in a number of human malignancies, including lung cancer, myeloproliferative disorders and lymphomas [68,69]. The ligand FGF-2 is directly associated with neovascularization [70,71]. Autocrine and paracrine signaling independent of somatic mutations have also been implicated in FGFrelated tumorigenesis [53]. Marek and colleagues described frequent coexpression of distinct FGFs (FGF-2 and FGF-9) and FGFRs, suggesting a potential autocrine loop in NSCLC cell lines [72]. The FGF/FGFR pathway prevails among mesenchymal NSCLC histologies, and activation seems to be associated with resistance to EGFR TKIs [72,73]. Also, von Willebrand factor (vWF) is a multi meric plasma glycoprotein synthesized in megakaryocytes/platelets and in endothelial cells [74]. The plasma concentration of vWF is used as a marker of endothelial cell activation and reduced plasma levels of vWF are observed in NSCLC [74]. Studies conducted in mice indicate that the decreased plasma levels of vWF in NSCLC patients could result from the activity of ADAM28 [75]. Inhibition of ADAM28 expression or activity showed a substantial reduction in lung metastasis and increased apoptosis of cancer cells in the blood vessels [75]. Notwithstanding that, further studies are needed to ascertain the importance of ADAM28 as an appropriate target for intervention in lung cancer. Nuclear molecular events associated with NSCLC In normal physiological conditions, DNA damage, such as that caused by genotoxic chemotherapeutic agents, induces apoptosis through double stranded break associated kinases, a process mediated through p53 [76,77]. Bcl-2 family members regulated by p53 are central to the activation of apoptosis [78]. The antiapoptotic Bcl-2 family member Bcl-xL protects cancer cells from p53-induced apoptosis and acts through the binding and inactivation of Bax and binding of proteins that recruit Bax to the mitochondrial membrane [78]. Bcl-xL is frequently over expressed in lung tumors and plays an important role in resistance to genotoxic chemotherapeutic agents in lung and other cancer types [79]. BclxL overexpression in tumors is also associated with poor prognosis [80,81]. Other enzymes involved in cell cycle regulation such as TERT, which control telomer length and stability, and CLPTM1L are also overexpressed in lung 1768
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tumors and are thought to confer resistance to apoptosis caused by genotoxic agents, in association with Bcl-xL [82]. Interestingly, TERT and CLPTM1L genes are colocalized on chromosome 5p15 where polymorphisms in rs402710, rs401681 and rs31489 genes have been associated with NSCLC susceptibility [83–85]. TS, an enzyme associated with de novo synthesis of DNA that also plays an important role in DNA repair, is highly expressed in NSCLC. Moreover, a deletional polymorphisms at the TS 3´-UTR (1494del 6 bp) has been associated with sensitivity of lung adenocarcinoma to pemetrexed therapy, indicating that TS polymorphisms should be further evaluated as candidate predicitive markers for personalized therapy in lung adenocarcinoma [86]. CTLA-4 & tumor immunity Important aspects of tumor biology are the active mechanisms employed by cancer cells to avoid killing by the immune system. The immune system of vertebrates is composed by a multitude of cellular and molecular mechanisms that are crucial for the elimination of cancer cells. Nevertheless, once a tumor is well histologically differentiated; the immune system seems to be incapable of directing a specific response towards it. Despite extensive research efforts, a suitable explanation for this phenomenon is still missing. Eradication of tumor cells is dependent on the activation of T cells, which requires not only stimulation of the T cell receptor by peptide–MHCs on antigen-presenting cells, but also an orchestrated balance of costimulatory and inhibitory signals that modulate the magnitude and effectiveness of the immune response. Thus, in normal physiological conditions, T-cell activation results from a signal transduction cascade downstream of cell-surface receptors. Nevertheless, this activation signal, which is delivered by cell-surface receptors, needs to be tightly regulated in order to avoid excess of inflammation [87]. In that sense, negative regulatory proteins on T cells, such as CTLA-4, PD-1, B7 family member B7-H4, Tim-3 and LAG-3, interact with their ligands on various cells types, including antigen-presenting cells, regulatory T cells, and nonhematopoietic cells, resulting in reduced T-cell proliferation and functional activity [88,89]. Interestingly, tumor-infiltrating T cells have a marked upregulation of cell-surface molecules that block efficient activation [90]. Hence, tumor environment drives a sustained expression of CTLA-4, PD-1 and LAG-3 on antigen-specific future science group
EGFR & KRAS mutations, & ALK fusions: current developments & personalizing therapies
lymphocytes, which culminates in peripheral cell tolerance to the tumor-specific antigens [91]. In fact, several studies in mice, demonstrate that antibody-mediated blockade of CTLA-4 increases endogenous responses to tumor cells, inducing tumor cell death [90]. Preclinical findings have translated into clinical development of a fully human, IgG1 monoclonal antibody, ipilimumab, and a fully human, IgG2 monoclonal antibody, tremelimumab, both of which bind CTLA-4. Of interest, ipilimumab was recently approved in several countries for the treatment of advanced or metastatic melanoma [90,92]. Also, many trials in lung cancer and ipilimumab are still in process, but its role remains experimental in NSCLC.
Integrating EGFR mutation, & K-RAS & ALK fusions into NSCLC clinical management A hallmark of modern pharmacogenomics is the possibility to develop personalized therapies based of the genetic background of each individual. The presence of specific point mutations or polymorphisms in key genes may dictate the response rate toward a specific drug [66]. A promising strategy for the future development of therapeutic interventions is based on the discovery that distinct subsets of cancers are strictly dependent on specific driver mutations for cellular proliferation and survival. Usually, these driver mutations result in the altered expression of proteins involved in signaling pathways crucial for cellular proliferation and survival [6]. Hence, targeting the activity of these mutant proteins can lead to cell death and therapeutic benefit [66]. This phenomenon is referred to as ‘oncogene addiction’ [6]. EGFR-mutant NSCLC was first recognized in 2004 as a distinct, clinically relevant molecular subset of lung cancer and currently represents the best-studied example of oncogene addiction in lung cancer. EGFR-mutant tumors are usually adenocarcinomas and are associated with a better prognosis than EGFR wild-type tumors [6]. NSCLC is associated with EGFR overexpression in up to 80% of the patients, and a high EGFR gene copy number is found in nearly 60% of the cases. Mutation of EGFR is observed in 10–20% of lung carcinomas, and nearly 80% of lung cancer-specific EGFR mutations comprise a leucine-to-arginine substitution at position 858 (L858R) and deletion mutations in exon 19 [6]. Importantly, these mutations are of extreme clinical relevance since they increase the sensitivity of NSCLC to TKIs, such as erlotinib and gefitinib (Table 1). Both these drugs are reversible future science group
Review
inhibitors of the EGFR tyrosine kinase, designed to act as competitive inhibitors of ATP binding at the active site of the EGFR tyrosine kinase. EGFR mutations are associated with the marked responses observed in patients treated with EGFR TKIs, and are more common in neversmokers, women, individuals of Asian ethnic background and those with adenocarcinoma histology. In addition to providing a genetic marker for a highly EGFR-TKI-responsive subset of NSCLCs, this correlation has also highlighted the crucial importance of mutational activated kinases as anticancer drug targets [8]. In addition to EGFR overexpression, its cognate ligands, EGF and TGF-a are also frequently expressed in NSCLCs, and can establish positive feedback loops that promote receptor hyperactivity. The disruption of these autocrine loops is the rationale for the use of EGFR-specific antibody cetuximab in patients with NSCLC [8]. An important downstream signaling target of EGFR, KRAS has also been implicated in the development and prognosis of NSCLC [6]. Mutations that cause the loss of KRAS GTPase activity render the protein constitutively GTPbound, resulting in a persistent proliferation signal [8]. In lung cancers, the most common KRAS mutations occur primarily at codons 12 and 13; the most common KRAS mutation in smoking patients with NSCLC is a G-to-T transition (84%) resulting in substitution of cysteine (47%), valine (24%), aspartate (15%) or alanine (7%) for wild-type glycine [6]. As with EGFR mutations, KRAS mutations are detected mainly in lung adenocarcinomas and are less frequently observed in squamous cell carcinomas of the lung [6]. By contrast with lung adenocarcinomas harboring EGFR mutations, KRAS mutant tumors are seen at a higher frequency (20–30%) in Caucasian patients than in east Asian patients (5%) [6]. Also, compared with EGFR mutations, KRAS mutations are more common in current or former smokers than in never-smokers, although the absence of a history of tobacco use does not eliminate the possibility of such abnormalities [8]. KRAS mutations are associated with primary resistance to EGFR TKIs [6]. Phase II trials have shown very small or absent response rates to erlotinib in patients with KRAS mutations [10]. The use of chemotherapy with or without erlotinib in previously untreated patients with NSCLC (TRIBUTE trial) suggested that, in KRAS mutated patients, OS and response rate (RR) might be even worse with the addition of EGFR TKIs [6]. www.futuremedicine.com
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[105]
2.9 IIIB and IV
‡
†
EGFR mutation in exon 19 and exon 21. Response rate, complete plus partial response. –: No significant data; EGFR: EGF receptor; OS: Overall survival; PFS: Progression-free survival.
464 No France Erlotinib maintenance Pérol et al. (2012)
1770
Interestingly, the presence of KRAS or EGFR mutations seem to be mutually exclusive with ALK fusions [8]. Patients positive for the ALK fusion protein frequently present at an advanced clinical stage, and the tumors pattern tends to be a solid adenocarcinoma pattern with signet ring cell morphology [45]. As above, the ALK fusion oncoprotein is associated with never-smokers or light smokers and is more frequent in younger patients [45,93]. The US FDA and the EMA have approved crizotinib, a small-molecule inhibitor of ALK tyrosine kinase, as a treatment for patients with locally advanced or metastatic NSCLC expressing the ALK fusion [5].
–
11.4
[104]
8.9 No Erlotinib Gridelli et al. (2012)
Italy
760
IIIB and IV
20.3
11.6
[101]
13.1 Yes Erlotinib Zhou et al. (2011)
China
83
IIIB and IV
82.9
–
[103]
12.3 Yes Erlotinib Capuzzo et al. (2010)
Italy
437
IIIB and IV
11.9
12.3
[102]
6 No Erlotinib Herbst et al. (2005)
USA
526
IIIB and IV
30
10.6
[16]
2.2 6.7 No Erlotinib Shepherd et al. (2005)
America, Europe and Asia
731
IIIB and IV
8.9
9.2 Yes Geftinib Mitsudomi et al. (2010)
Japan
177
IIIB and IV
62.1
30.9
[97]
[100]
5.7 No Geftinib Mok et al. (2009)
Asia
609
III and IV
71.2
18.6
[96]
10.8 Yes Gefitinib Maemondo et al. (2010)
Asia
230
IIIB and IV
73.7
30.5
[99]
– No Gefitinib Perez-Soler et al. (2004)
USA
57
IIIB and IV
12.3
8.4
[98]
– 6–7 No Gefitinib Kris et al. (2003)
USA
221
IIIB and IV
22
Median PFS (months) EGFR†-positive selected mutations Molecule
Place of study
Patients (n)
Clinical stage
Response rate‡ (%)
Median OS (months)
Ref.
de Mello, Madureira, Carvalho, Araújo, O’Brien & Popat
Study
Table 1. Summary of the studies that address the current main EGF receptor tyrosine kinase inhibitor drugs in the non-small-cell lung cancer field.
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Pharmacogenomics (2013) 14(14)
Targeted therapies for NSCLC: current & future perspectives In 2004, a new therapeutic era for NSCLC began with the emergence of targeted therapies against EGFR [8,9,94,95]. Gefitinib [96–100] and erlotinib [16,101–105], two small-molecule drugs that specifically target the tyrosine kinase activity of EGFR TKIs, were promptly approved by the FDA for patients with advanced NSCLC in specific subsettings previous described (Table 1) [6]. Unfortunately, patients start to develop resistance against these EGFR TKIs within 6–12 months after the initiation of therapy. Acquired resistance to gefitinib and erlotinib is predominantly due to the appearance of the T790M allele, which affects the gatekeeper residue in the catalytic domain of the kinase that weakens the interaction of the inhibitor with its target, and results in a markedly elevated IC50. Nevertheless, this acquired resistance has led to the development of new drugs as alternative therapies to NSCLC [6]. Table 2 summarizes the current targeted therapies and clinical trials involved in the NSCLC setting discussed below. Cetuximab Cetuximab is a chimeric monoclonal antibody that binds to the extracellular domain of EGFR to block ligand binding, which results in inhibition of autophosphorylation of EGFR [106]. EGFR binding by cetuximab results in cell cycle arrest, increased expression of p27, and increased expression of proapoptotic proteins, including Bax, caspase 3, caspase 8 and caspase 9, or decreased expression of antiapoptotic proteins such as Bcl-2 [107,108]. Cetuximab has also been shown to decrease the production of VEGF and basic FGF in orthotopic tumor models. Preclinical studies indicated synergism between cetuximab and a number of chemotherapeutic future science group
EGFR & KRAS mutations, & ALK fusions: current developments & personalizing therapies
Review
Table 2. Summary of clinical trials evaluating current target therapies in advanced non-small-cell lung cancer patients. Study
Phase
Treatment
Patients (n)
OS (month)
RR (%)
Ref.
III
CT + cetuximab 400 mg/m2 D1 >250 mg/m2
557
11.3
36
CT: CDDP 80 mg/m2 D1 + vinorelbine 25 kg/m2
568
10.1
29
Afatinib 50 mg per day
390
10.8
7
Placebo
195
12
11
Crizotinib 250 mg/day in 28-day cycle
82
NR
57
[114]
Crizotinib 250 mg b.i.d. in 28-day cycle
143
NR
60.8
[117]
CBDCA + paclitaxel
427
10.3
35
[118]
CBDCA + paclitaxel + bevacizumab 15 mg/kg
440
12.3
15
CDDP + gemcitabine + placebo
347
NR
20.1
CDDP + gemcitabine + bevacizumab 7.5 mg/kg
345
NR
34.1
CDDP + gemcitabine + bevacizumab 15 mg/kg
351
NR
30.4
Cetuximab FLEX (2009)
[110,111]
Afatinib LUX-Lung 1 (2012)
IIb/III
[112]
Crizotinib Kwak et al. (2010)
I
Camidge et al. (2012) I Bevacizumab ECOG 4599 (2010) AVAiL (2009)
III III
[60]
Sunitinib Socinski et al. (2008)
II
Sunitinib 50 mg daily†
63
5.7
11.1
[120]
Gervais et al. (2011)
III
Sunitinib 37.5 mg/day + erlotinib 150 mg/day
480
9.0
10.6
[121]
Placebo + erlotinib 150 mg/day
480
8.5
6.9
Sorafenib (400 mg b.i.d.) + gemcitabine 385 1250 mg/m2 per day on days 1 and 8 and cisplatin 75 mg/m2 on day 1 for up to six 21‑day cycles
12.4
27.8
Sorafenib (400 mg twice a day) + placebo
12.5
25.8
Sorafenib Paz-Ares et al. (2012)
III
387
[128]
Sunitinib administered daily for 4 weeks of 6 weeks cycle. b.i.d.: Twice daily; CT: Chemotherapy; NR: Not reported; OS: Overall survival; RR: Response rate. †
agents, including cisplatin and paclitaxel, in a wide variety of cell lines [109]. In a Phase III trial study (FLEX), 1125 patients were randomly assigned to cisplatin/vinorelbine chemotherapy plus cetuximab (n = 557) or chemotherapy alone (n = 568) [110,111]. Whilst patients given chemotherapy plus cetuximab survived statistically longer than those in the chemotherapy-alone group (median 11.3 months vs 10.1 months; hazard ratio for death 0.871 [95% CI: 0.762–0.996]; p = 0.044), the clinical significance of this has been debated, and cetuximab was not approved for use by the FDA. The main cetuximab-related adverse event was acne-like rash (57 [10%] of 548, grade 3). Afatinib Afatinib is an irreversible ErbB-family kinase inhibitor [112]. Afatinib shows preclinical activity when tested in EGFR mutant models with future science group
mutations that confer resistance to EGFR TKIs, including the most common mutations, L858R and deletion-19, and the exon 20 gatekeeper T790M mutations. Afitinib was investigated in a randomized Phase IIb/III trial (LUX-Lung 1) of 585 patients (390 to afatinib and 195 to placebo) with stage IIIB or IV adenocarcinoma who had received one or two previous chemotherapy regimens and had disease progression after at least 12 weeks of treatment with erlotinib or gefitinib [112]. This study demonstrated no benefit in terms of OS with afatinib (the primary end point). Nonetheless, the median progression-free survival (PFS) was longer in the afatinib group (3.3 months, 95% CI: 2·79–4·40) than it was in the placebo group (1.1 months, 0.95–1.68; hazard ratio: 0.38; 95% CI: 0.31–0.48; p
