[

Translating Basic Research Into Clinical Practice

]

Advances in Molecular Biology of Lung Disease Aiming for Precision Therapy in Non-small Cell Lung Cancer Claire Rooney, MBChB; and Tariq Sethi, MD, PhD

Lung cancer is the principal cause of cancer-related mortality in the developed world, accounting for almost one-quarter of all cancer deaths. Traditional treatment algorithms have largely relied on histologic subtype and have comprised pragmatic chemotherapy regimens with limited efficacy. However, because our understanding of the molecular basis of disease in non-small cell lung cancer (NSCLC) has improved exponentially, it has become apparent that NSCLC can be radically subdivided, or molecularly characterized, based on recurrent driver mutations occurring in specific oncogenes. We know that the presence of such mutations leads to constitutive activation of aberrant signaling proteins that initiate, progress, and sustain tumorigenesis. This persistence of the malignant phenotype is referred to as “oncogene addiction.” On this basis, a paradigm shift in treatment approach has occurred. Rational, targeted therapies have been developed, the first being tyrosine kinase inhibitors (TKIs), which entered the clinical arena . 10 years ago. These were tremendously successful, significantly affecting the natural history of NSCLC and improving patient outcomes. However, the benefits of these drugs are somewhat limited by the emergence of adaptive resistance mechanisms, and efforts to tackle this phenomenon are ongoing. A better understanding of all types of oncogene-driven NSCLC and the occurrence of TKI resistance will help us to further develop second- and third-generation small molecule inhibitors and will expand our range of CHEST 2015; 148(4):1063-1072

precision therapies for this disease.

ABBREVIATIONS: AKT1 5 protein kinase B-a; ALK 5 anaplastic lymphoma kinase; ATP 5 adenosine triphosphate; BRAF 5 protein kinase B-raf; EGFR 5 epidermal growth factor receptor; ERK 5 extracellular signal-regulated kinase; HER 5 human epidermal growth factor receptor; HGF 5 hepatocyte growth factor; Hsp90 5 heat shock protein 90; JAK 5 Janus kinase; KRAS 5 Kirsten rat sarcoma viral oncogene homolog; MAPK 5 mitogen-activated protein kinase; MEK 5 dual-specificity mitogen-activated protein kinase kinase; MET 5 hepatocyte growth factor receptor; mTOR 5 mammalian target of rapamycin; NSCLC 5 non-small cell lung cancer; PI3K 5 phosphoinositide-3-kinase; PI3KCA 5 phosphoinositide3-kinase catalytic a-polypeptide; RET 5 “rearranged during transfection” protooncogene; ROS1 5 protooncogene receptor tyrosine kinase; SCC 5 squamous cell carcinoma; STAT 5 signal transducer and activator of transcription; TKI 5 tyrosine kinase inhibitor

In the past few years, increasingly refined methods of tissue sampling in combination with new insights in molecular biology

Manuscript received October 26, 2014; revision accepted June 16, 2015; originally published Online First July 16, 2015. AFFILIATIONS: From the Division of Asthma, Allergy and Lung Biology (Drs Rooney and Sethi), King’s College London; and Department of Respiratory Medicine (Dr Sethi), King’s Health Partners, London, England. FUNDING/SUPPORT: The research was supported by the National Institute for Health Research Biomedical Research Centre based at Guy’s and St. Thomas’ NHS Foundation Trust and King’s College London.

journal.publications.chestnet.org

have swiftly altered the diagnostic and therapeutic landscape of non-small cell lung cancer (NSCLC), allowing us the

Tariq Sethi, MD, PhD, Division of Asthma, Allergy and Lung Biology, King’s College London, Guy’s Hospital, Great Maze Pond, London, SE1 9RT, England; e-mail: [email protected] © 2015 AMERICAN COLLEGE OF CHEST PHYSICIANS. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details. DOI: 10.1378/chest.14-2663 CORRESPONDENCE TO:

1063

opportunity to develop treatments for a subset of molecularly well-defined patients. Large-scale genomic, mutational, and proteomic profiling studies of NSCLC have succeeded in identifying the presence of mutually exclusive driver mutations throughout the full gamut of NSCLC histology and smoker status. For example, in approximately 60% of lung adenocarcinomas, mutations in multiple oncogenes are readily apparent, including protein kinase B-a (AKT1), anaplastic lymphoma kinase (ALK), protein kinase B-raf (BRAF), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER) 2, Kirsten rat sarcoma viral oncogene homolog (KRAS), dualspecificity mitogen-activated protein kinase kinase (MEK) 1, hepatocyte growth factor receptor (MET), NRAS (neuroblastoma RAS viral oncogene homolog), phosphoinositide-3-kinase catalytic a-polypeptide (PI3KCA), “rearranged during transfection” protooncogene (RET), and protooncogene receptor tyrosine kinase (ROS1). However, within this population, never smokers with adenocarcinoma are identified as having the highest likelihood of EGFR, HER2, ALK, RET, and ROS1 mutations.1 Conversely, characterization of squamous cell carcinoma (SCC) has proven much more difficult. SCC generally has higher mutation rates and copy number alterations than the other histologic subtypes of lung cancer, and the most frequent driver mutations found in adenocarcinoma are only rarely found in SCC. Currently, there are no clinically approved target agents for SCC in use; however, newly discovered mutations that may be linked to outcomes with targeted therapies in SCC are emerging, among them PI3KCA mutations, fibroblast growth factor receptor 1 amplifıcation, and discoidin domain receptor tyrosine kinase 2 mutations.2 However, many of these gene alterations are relatively rare, as illustrated in Figures 1 and 2, and as such, their specific testing and identification is not yet advocated. However, sequential testing for EGFR, KRAS, and ALK is recommended, beginning with either KRAS or EGFR analysis, with ALK analysis reserved for KRAS- and EGFR-negative specimens.13 Treatment targeted to these mutations can dramatically affect patient outcome, as has been illustrated with the use of tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib, and afatinib in patients with EGFR mutations14-17 and crizotinib in patients with ALK rearrangements.18 Tyrosine kinase receptor inhibition in this context has shown superiority to traditional platinum-based chemotherapy in terms

1064 Translating Basic Research Into Clinical Practice

Figure 1 – Target oncogenes in adenocarcinoma.3-10 ALK 5 anaplastic lymphoma kinase; BRAF 5 protein kinase B-raf; EGFR 5 epidermal growth factor receptor; FGFR 5 fibroblast growth factor receptor; KRAS 5 Kirsten rat sarcoma viral oncogene homolog; MET 5 hepatocyte growth factor receptor; PI3KCA 5 phosphoinositide-3 kinase catalytic alpha polypeptide; PTEN 5 phosphatase and tensin homolog; RET 5 “rearranged during transfection” protooncogene.

of response rate, progression-free survival, and quality of life. This review explores the growing population of targetable oncogenes in NSCLC, with a focus on the most well-defined subtypes in lung adenocarcinoma, namely EGFR mutations and ALK-rearranged NSCLC. We discuss strategies to tackle the problem of resistance to first-line TKI therapy. Epidermal Growth Factor Receptor

An EGFR kinase domain-activating mutation is one of the most common and, arguably, robustly treatable subtypes of NSCLC. It is observed in 9% to 15% of NSCLC adenocarcinomas and occurs most frequently in women,

Figure 2 – Target oncogenes in squamous cell carcinoma.8,11,12 DDR2 5 discoidin domain receptor tyrosine kinase 2. See Figure 1 legend for expansion of other abbreviations.

[

148#4 CHEST OCTOBER 2015

]

nonsmokers, and people of East Asian descent, where the incidence is significantly higher, approaching 62%.19 EGFR belongs to the ErbB family of transmembrane receptor tyrosine kinases, which recognize a number of ligands, including EGF, transforming growth factor-a, and amphiregulin. Ligand binding induces a conformational change, dimerization, and autophosphorylation of tyrosine residues within the intracellular domain, creating binding sites for transduction proteins containing Src-homology 2 or phosphotyrosine-binding domains. This mediates cell growth, invasion, metastatic spread, apoptosis, and tumor angiogenesis through downstream targets, including PI3K/AKT/mammalian target of rapamycin (mTOR), RAS/RAF/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways.20 The degree of overlap among these paths

means that a functional EGFR is necessary for survival. Most mutations of EGFR occur in exons 18 to 21 of the adenosine triphosphate (ATP)-binding pocket of the kinase domain, of which 90% are exon 19 deletions or exon 21 point mutations of L858R, leading to increased receptor kinase activity and constitutive activation of the aforementioned downstream pathways.4,21 The effect of tyrosine kinase inhibition in this scenario leads to the initiation of the intrinsic mitochondrial apoptosis pathway, enhancement of BIM expression and subsequent cellular apoptosis (Figs 3A, 3B).21,22 Clinically, EGFR mutations (mostly exon 19 deletions and L858R mutations in exon 21) are associated with benefit from gefitinib and erlotinib treatment. Initially, before the discovery of specific activating mutations, targeting of EGFR in phase III trials of unselected patients with NSCLC demonstrated disappointingly low radiographic response rates with short (, 3 months)

Figure 3 – A, Downstream EGFR signaling. B, Location and effect of most common EGFR mutations. AKT 5 protein kinase B; ATP 5 adenosine triphosphate; ERK 5 extracellular signal-regulated kinase; MEK 5 dual specificity mitogenactivated protein kinase; PI3K 5 phosphoinositide-3 kinase; PIP3 5 phosphatidylinositol 3,4,5 trisphosphate; TKI 5 tyrosine kinase inhibitor. See Figure 1 legend for expansion of other abbreviations. (Illustrations by Haderer & Muller Biomedical Art, LLC.)

journal.publications.chestnet.org

1065

progression-free survival and overall survival rate (6.7 months).23

occur in 0.5% of patients with adenocarcinoma35 and may be associated with familial cancer risk.36

However, in light of the discovery of EGFR mutations as a predictive biomarker, several large prospective phase III first-line trials directly compared an EGFR TKI with platinum doublet chemotherapy in patients with NSCLC harboring EGFR mutations. These trials strongly confirmed the benefit of gefitinib or erlotinib in EGFR-mutant lung cancer, irrespective of ethnic background.14,15,24 Based on trial treatment where platinum doublet chemotherapy in combination with erlotinib is nonsuperior to erlotinib alone in the management of patients with EGFR-mutated NSCLC,25 EGFR TKIs are currently recommended as single therapy in a first-line setting where a classic mutation (eg, exon 19 deletions, L858R mutations) is known. Some large clinical trial data to date are summarized in Table 1.

Other mechanisms of resistance have been described in individual cases, such as drug-drug interactions where induction of cytochrome P450 3A4 leads to increased metabolism of TKIs.37 Similarly, one study has demonstrated that smoking leads to upregulation of cytochrome P450 1A1, causing increased metabolic clearance of erlotinib.38

Yet, as many as 90% of NSCLC tested are wild type or have no detectable EGFR mutation, and these are less sensitive to standard EGFR TKIs. A phase III study comparing docetaxel and erlotinib found an improved response rate (13.9% vs 2.2%, P , .004), overall enhanced progression-free survival in the docetaxel arm (3.4 months vs 2.4 months; hazard ratio, 0.69; P , .014), and median overall survival (8.2 months vs 5.4 months; hazard ratio, 0.73; P 5 .05).31 On the basis of the BR.21 trial,32 where a 2-month survival benefit was apparent in an unselected patient group with NSCLC treated with erlotinib vs placebo, erlotinib is in fact approved for use in scenarios where the EGFR mutation status is unknown and may, therefore, be appropriate in circumstances where mutation status testing is unavailable or clinically inadvisable. EGFR TKI Resistance

Primary resistance is seen in approximately 30% of patients with EGFR-mutated NSCLC who do not respond to appropriately targeted therapy; moreover, a full response is seen in , 5% of cases. The most common culprit mutations conferring de novo resistance in this circumstance are EGFR exon 20 insertions (4%-9.2% of EGFR-mutated NSCLC) where in preclinical and clinical studies, progressive disease despite treatment is demonstrated.33 This is believed to reflect an unaltered ATP-binding pocket, which activates EGFR without any increased affinity for EGFR TKI binding, leading to subsequent drug insensitivity.34 A further mutation in exon 20 where methionine is substituted for threonine at position 790 (T790M) confers primary resistance to TKI treatment when it occurs as a germline mutation. The mutation is believed to 1066 Translating Basic Research Into Clinical Practice

Examining further downstream of the tyrosine kinase receptors themselves, failure of apoptosis initiation also limits TKI efficacy. The proapoptotic molecule BIM, a member of the Bcl-2 family of proteins, is essential for apoptosis triggered by EGFR kinase inhibitors in mutant EGFR-dependent NSCLC. Consequently, low expression of BIM in primary tumors has been associated with shorter progression-free survival in patients treated with EGFR TKIs.39 Additionally, genetic polymorphisms resulting in BIM isoforms lacking the proapoptotic Bcl-2 homology domain 3 may also explain unpredictability of response in NSCLC.40 Secondary resistance generally occurs in the context of second-site EGFR mutations. This is the most frequent mechanism of acquired resistance to EGFR TKIs in lung cancer. Described mutations include L747S, D761Y, T854A, and T790M of which . 90% comprise the T790M gatekeeper mutation.41 The somatic T790M methionine substitution (as in the case of the T790M germline mutation) restores the affinity for ATP vs drug back to the level of wild-type EGFR by impeding ATP pocket drug binding.42 Acquisition of resistance by T790M may occur in several ways: (1) a de novo mutation arises and persists during treatment with a TKI, (2) the T790M substitution exists already in tandem with a primary activating mutation, and (3) selection bias due to TKI treatment promotes the expansion of this cell population. In addition, lung adenocarcinoma cell lines exhibiting both a sensitizing EGFR mutation and a T790M mutation show a diminished upregulation of BIM in the context of gefitinib treatment,43 suggesting that BIM is involved in EGFR inhibitor treatment resistance caused by the T790M mutation. Acquired resistance may also be gained where other anomalous molecule expression activates EGFR signaling cascades. This is seen where amplification of the gene encoding MET is present, which occurs in up to 5% to 10% of patients.41 Overexpression of MET activates downstream PI3K/AKT signaling through interaction

[

148#4 CHEST OCTOBER 2015

]

journal.publications.chestnet.org

1067

Carboplatinpaclitaxel

71.2 vs 47.3

Chemotherapy

ORR (RECIST), %

1 (.99)

21.6 vs 21.9

0.48 (, .001)

9.5 vs 6.3

1.64 (.21)

30.0 vs not reached

0.49 (, .001)

9.2 vs 6.3

, .001

62.1 vs 32.2

Cisplatin-docetaxel

Gefitinib 250 mg OD

177

2010

WJTOG 340526 2010

NR

30.5 vs 23.6

0.3 (, .001)

10.8 vs 5.4

, .001

73.7 vs 30.7

Carboplatinpaclitaxel

Gefitinib 250 mg OD

230

NEJ 00227 2011

1.06 (.68)

22.6 vs 28.8

0.16 (, .001)

13.1 vs 4.6

, .001

83 vs 36

Carboplatingemcitabine

Erlotinib 150 mg OD

154

OPTIMAL28 2012

1.04 (.87)

19.3 vs 19.5

0.37 (, .001)

9.7 vs 5.2

, .001

58 vs 15

Platinum doublet

Erlotinib 150 mg OD

173

EURTAC15

1.12 (.60)

NR

0.58 (, .001)

11.1 vs 6.9

.001

56 vs 23

Cisplatinpemetrexed

Afatinib 40 mg OD

345

2013

LUX-Lung 324

0.95 (.76)

22.1 vs 22.2

0.28 (, .001)

11 vs 5.6

, .001

66.9 vs 23

Cisplatingemcitabine

Afatinib 40 mg OD

366

2014

LUX-Lung 629

EGFR 5 epidermal growth factor receptor; EURTAC 5 Erlotinib Versus Standard Chemotherapy as First-Line Treatment for European Patients With Advanced EGFR Mutation-Positive Non-Small-Cell Lung Cancer; HR 5 hazard ratio; IPASS 5 Iressa Pan-Asian Study; LUX-Lung 5 Afatinib Versus Cisplatin Plus Gemcitabine for First-Line Treatment of Asian Patients With Advanced Non-Small-Cell Lung Cancer Harbouring EGFR Mutations; NEJ 002 5 North East Japan 002 Study; NR 5 not reported; OD 5 oral dose; OPTIMAL 5 A Randomized, Open-Label, Multi-center Phase III Study of Erlotinib Versus Gemcitabine/Carboplatin in ChemoNaive Stage IIIB/IV Non-Small Cell Lung Cancer Patients With EGFR Exon 19 or 21 Mutation; ORR 5 objective response rate; OS 5 overall survival; PFS 5 progression-free survival; RECIST 5 Response Evaluation Criteria in Solid Tumors; TKI 5 tyrosine kinase inhibitor; WJTOG 5 West Japan Thoracic Oncology Group. (Adapted with permission from Gerber et al.30)

HR (P value)

OS, mo

HR (P value)

PFS, mo

, .001

Gefitinib 250 mg OD

TKI

P value

2009

437

Date

IPASS14

] Summary of Clinical Outcomes: EGFR TKIs Vs Doublet Chemotherapy

No. patients

Trial

TABLE 1

with HER3/ERBB3, resulting in cells that are less reliant on mutant EGFR for survival.44 In particular tumors, the MET ligand hepatocyte growth factor (HGF) (which autophosphorylates and activates MET) may help to multiply preexisting MET-amplified populations of cells.45 Other mutant signaling proteins may also support secondary resistance. It has been shown that PI3KCA mutations in the context of EGFR TKI therapy developed in approximately 5% of patients with acquired resistance examined in one study.41 Similarly, another group demonstrated that 1% of patients with acquired resistance have BRAF mutations.46 Other studies have suggested that ERBB2, CRKL, or ERK amplification may be responsible for disease progression.19,47,48 Second-Generation EGFR TKIs

Following on from the first-generation reversible TKIs like gefitinib and erlotinib, the drug development field is rapidly expanding, with second-generation inhibitors emerging, such as afatinib, an irreversible inhibitor, more potent than gefitinib, or erlotinib, which acts against both EGFR and other ErbB receptor tyrosine kinases. A phase III trial for TKI-naive patients with EGFR mutant tumors demonstrated afatinib superiority to pemetrexed/cisplatin treatment in terms of response rate and progression-free survival.17 In addition, a separate phase IIb/III trial showed an improvement in response rate and progression-free survival (but not overall survival) with afatinib vs placebo in patients with disease progression despite first-generation TKI treatment.49 Furthermore, in development are third-generation TKIs , which are irreversible and specifically targeted against the T790M mutant as opposed to wild-type EGFR. Although at an early stage, trials of these compounds have demonstrated clinical improvement in patients with EGFR-mutant NSCLC who had previously progressed to first-line TKI treatment. In a phase I trial of third-generation TKI CO-1686, the overall response rate was 58% in 40 patients with T790M-positive tumors.50 Similarly, results from the phase I trial of another compound, AZD9291, showed a response rate of 64% in 107 patients with T790Mpositive tumors.51

ALK-Rearranged Mutations ALK rearrangements were first described in 2007, are found in approximately 3% to 7% of all cases of NSCLC, and are predominantly seen in younger patients and

1068 Translating Basic Research Into Clinical Practice

never smokers.52,53 Described initially as fusions to echinoderm microtubule-associated protein-like 4,52 and more recently less commonly to other fusion partners, including transforming growth factor and KIF5B,54 the net result is the creation of a novel oncogene that promotes dimerization and, therefore, constitutive ALK tyrosine kinase activation. Downstream effects are believed to be mediated by STAT-3 and ERK activation, promoting proliferation and a reduction in BIM-mediated apoptosis (Fig 4).55 Clinically, initial phase I trials of treatment with crizotinib, a MET, ROS1, and ALK inhibitor, showed a 60.8% radiographic objective response rate and median progression-free survival of 9.7 months.18 Since, a phase III trial randomizing patients with advanced lung cancer with ALK fusions to crizotinib vs standard chemotherapy (docetaxel or pemetrexed) after disease progression on first-line treatment has confirmed progression-free benefit with crizotinib in this patient population (7.7 months vs 3.0 months with chemotherapy), albeit with no overall survival benefit at interim analysis.56 Interestingly, it transpires that crizotinib, despite convincing trial data, is a pharmacologically relatively weaker ALK TKI in NSCLC compared with other ALK-rearranged cancers, including lymphoma. Resistance to ALK TKIs

Unfortunately, as with EGFR TKI-targeted therapy, the period of clinical benefit of crizotinib in ALK-positive NSCLC is limited. Progression is frequently seen in the CNS, and this is one significant constraint. Although the mechanisms underlying crizotinib resistance are not wholly understood, multiple different point mutations within the ALK tyrosine kinase domain have been reported in patients and are believed to drive this phenomenon, including L1152R, 1151Tins, G1269A, C1156Y, and the gatekeeper mutation L1196M.57 Furthermore, although some of the identifıed mutations, such as L1196M and G1269A, are found within the active site and, therefore, most likely hinder crizotinib binding, in other mutations (ie, L1152R, C1156Y), the mechanism of resistance is not understood.58,59 In fact, in many resistant cases, no ALK secondary mutation is identifıed. However, evidence of HER family pathway activation (including EGFR), ALK amplification, KIT amplifıcation, and KRAS mutations in crizotinibresistant patients illustrate that hitherto unsuspected additional genetic alterations may be an underlying factor in resistance.58-60

[

148#4 CHEST OCTOBER 2015

]

Figure 4 – ALK mutations and downstream signaling. JAK 5 Janus kinase; mTOR 5 mammalian target of rapamycin; STAT 5 signal transducer and activator of transcription. See Figure 1 and 3 legends for expansion of other abbreviations. (Illustrations by Haderer & Muller Biomedical Art, LLC.)

Second-line ALK TKIs

At present, several novel ALK TKIs are in clinical development. One such drug is ceritinib, a second-generation TKI of ALK that is more potent than crizotinib. Following the results from an expanded trial cohort, ceritinib has recently undergone accelerated approval by the US Food and Drug Administration for patients with disease progression despite, or with intolerance to, crizotinib. The results demonstrated an objective response rate of 58% overall, 55% in those with prior crizotinib treatment, and 66% in ALK inhibitor-naive patients. The median duration of response was 10 months in the entire cohort and 7.4 months in those with prior crizotinib treatment. The median progression-free survival for the entire cohort was 8.2 months, including 6.9 months for those previously treated with an ALK inhibitor and 8.3 months for those who had not been treated with an ALK inhibitor.61 Importantly, ceritinib and other second-generation ALK inhibitors such as alectinib have also shown substantial CNS activity in both the brain and the cerebrospinal fluid. Given their potential for both effective systemic and CNS disease control, these second-line agents may dramatically alter long-term prognosis for patients with ALK-positive NSCLC. Adjuncts to ALK inhibition are being explored at present, with one notable target in heat shock protein 90 (Hsp90), a molecular chaperone that protects folding, stability, and function in a number of client proteins,

journal.publications.chestnet.org

including ALK. Blockade of Hsp90 downregulates ALK, reducing tumor growth through downstream ERK, AKT, and mTOR signaling and inducing apoptosis in crizotinib-naive and resistant tumors.63 Tandem treatment with crizotinib and Hsp90 inhibitors, such as ganetespib, may, therefore, represent a potential approach for ALK-driven cancers and overcome the multiple mechanisms of resistance to direct tyrosine kinase inhibition seen in patients with ALK-positive NSCLC.

Future Directions and Further Treatment Strategies Far from being a clear-cut entity, some unexpected traits of EGFR TKI-resistant NSCLC have recently come to light. Conventionally, the emergence of resistance to treatment means withdrawal of that specific chemotherapeutic agent. However, in vitro we see that cell lines that acquire EGFR T790M demonstrate a reduction in proliferation compared with the parental cohort and, indeed, become resensitized to treatment when passaged without TKI exposure.63 This is demonstrated in the clinical arena where case reports have described patients who acquired resistance regaining EGFR TKI sensitivity after a drug-free window64; in fact, patients who acquire the somatic EGFR T790M mutation may fare better clinically than those who do not.65 The assumption that resistant NSCLC comprises a mixed clonal population of cells disposed to selective pressure in the context of treatment might infer benefit in 1069

continuing EGFR TKI administration even after resistance occurs. This is borne out in some clinical scenarios66; however, recent randomized trial data from IMPRESS (A Study of IRESSA Treatment Beyond Progression in Addition to Chemotherapy Versus Chemotherapy Alone) provide contrary evidence.67 Additionally, the manipulation of acquired resistance pathways to EGFR tyrosine kinase inhibition as an adjunct to treatment seems a promising avenue, and on this basis, amplified cMET is a potential candidate for intervention. Multiple means to targeting MET and HGF signaling exist, including agents that block the binding of HGF to MET, anti-MET monoclonal antibodies, and small-molecule MET kinase inhibitors. In one such example, a phase II trial assessed the use of onartuzumab (MetMAb; Hoffmann-La Roche Ltd) in tandem with erlotinib vs erlotinib only in a cohort of 128 patients with NSCLC who had undergone previous treatment. The researchers examined progression-free survival in the whole cohort and in a subgroup of high MET-expressing patients; the latter group showed a significant reduction in progression risk and overall survival (12.6 months vs 3.8 months).68 However promising as this may seem, a subsequent phase III trial in MET-positive patients to explore this further failed to demonstrate superiority,69 albeit with further analyses based on molecular subgroups still pending. How else may we expand our molecular armory to improve clinical outcomes? Other novel oncogenic drivers have proven targetable, and recent early stage trials are exploring receptor tyrosine kinase-mediated inhibition of targets such as ROS1 and BRAF with promising initial results. ROS1 is a receptor tyrosine kinase of the insulin receptor superfamily, with structural similarities to ALK, and is present in 1% to 2% of NSCLCs. ROS1 fusions promote activation of several oncogenic pathways, including PI3K/AKT/mTOR, JAK/STAT, and MAPK/ERK.70 As yet, there are no specific inhibitors to ROS1, but multitarget inhibitors against ALK/MET/ROS1, such as crizotinib, have proven some effect. Indeed the firstin-human crizotinib study (PROFILE 1001 [A Study of Oral PF-02341066, a c-Met/Hepatocyte Growth Factor Tyrosine Kinase Inhibitor, in Patients With Advanced Cancer]) revealed a preliminary response rate of 57% and disease control rate of 79% within an expanded cohort of ROS1-positive patients, with a duration of response and median progression-free survival almost double that seen in ALK-positive patients.71

1070 Translating Basic Research Into Clinical Practice

BRAF is a serine-threonine kinase that links RAS GTPases to downstream proteins of the MAPK family, predominantly MEK, which control cell proliferation. Approximately 50% of mutations correspond to a transversion in position T1799A on exon 15 where glutamine/valine substitution occurs at residue 600 (V600E). This mutation causes the inactive form of the enzyme to destabilize, with constitutive kinase activation and downstream target activation.72 A large retrospective analysis demonstrated the V600E phenotype to be common in female patients, not influenced by smoking history, and of an aggressive subtype. These patients have shorter disease-free and overall survival than those with wild-type tumors.73 A small phase II study of 20 patients with V600E BRAF mutation reported a 40% response rate using dabrafenib, a reversible, small molecular BRAF inhibitor, which has previously shown strong clinical benefit in BRAF-mutant melanoma. To our knowledge, this study represents the first example of clinical efficacy of a BRAF inhibitor in patients with BRAF V600E NSCLC.74 In fact, it has been awarded breakthrough status by the Food and Drug Administration as of January 2014 for the treatment of metastatic BRAF V600E mutation-positive NSCLC in patients who have received at least one prior line of platinum-containing chemotherapy.

Conclusions EGFR mutations and echinoderm microtubule-associated protein-like-ALK rearrangements are presently the most clinically relevant alterations that determine personalized treatment of patients with NSCLC. The growing number of other promising target oncogenes makes it probable that other markers may also aid in decision-making regarding treatment in the near future. It is amply clear that no panacea in NSCLC exists given the histologic and molecular heterogeneity of NSCLC. To deliver truly personalized and effective therapy, specific drug combinations must be directed against individualized tumor genetic signatures. We believe that for the future, this will necessitate sophisticated initial molecular assessment as a standard of care, with broader access to techniques such as multiplex or next-generation sequencing of tumor or blood to allow cancer typing. Perhaps in the future a dynamic approach to repeat screening of molecular targets may also allow us to exploit changes in patterns of resistance and sensitivity to chemotherapeutic regimens. Ultimately, the goal should be to become as resourceful as possible with new strategies and rationally guided therapies to reduce the burden of targetable oncogene-driven lung cancer.

[

148#4 CHEST OCTOBER 2015

]

Acknowledgments Conflict of interest: None declared. Role of sponsors: The sponsors had no role in the design of the study, the collection and analysis of the data, or the preparation of the manuscript. Other contributions: The views expressed are those of the authors and not necessarily those of the National Health Service, the National Institute of Health Research, or the Department of Health.

References 1. Kris MG, Johnson BE, Kwiatkowski DJ, et al. Identification of driver mutations in tumor specimens from 1,000 patients with lung adenocarcinoma: the NCI’s Lung Cancer Mutation Consortium (LCMC) [abstract]. J Clin Oncol. 2011;29(suppl):CRA7506. 2. Cancer Genome Atlas Research Network. Comprehensive genomic characterization of squamous cell lung cancers [published correction in Nature. 2012;491(7426):288]. Nature. 2012;489(7417):519-525.

adenocarcinoma of the lung harboring EGFR-activating mutations [abstract]. J Clin Oncol. 2012;30(suppl):LBA7500. 18. Kwak EL, Bang YJ, Camidge DR, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med. 2010;363(18): 1693-1703. 19. Shigematsu H, Lin L, Takahashi T, et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst. 2005;97(5):339-346. 20. Shaw RJ, Cantley LC. Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature. 2006;441(7092):424-430. 21. Sordella R, Bell DW, Haber DA, Settleman J. Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science. 2004;305(5687):1163-1167. 22. Tracy S, Mukohara T, Hansen M, Meyerson M, Johnson BE, Jänne PA. Gefitinib induces apoptosis in the EGFRL858R non-smallcell lung cancer cell line H3255. Cancer Res. 2004;64(20):7241-7244.

3. Riely GJ, Kris MG, Rosenbaum D, et al. Frequency and distinctive spectrum of KRAS mutations in never smokers with lung adenocarcinoma. Clin Cancer Res. 2008;14(18):5731-5734.

23. Thatcher N, Chang A, Parikh P, et al. Gefitinib plus best supportive care in previously treated patients with refractory advanced non-small-cell lung cancer: results from a randomised, placebocontrolled, multicentre study (Iressa Survival Evaluation in Lung Cancer). Lancet. 2005;366(9496):1527-1537.

4. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 2004;350(21): 2129-2139.

24. Sequist LV, Yang JC, Yamamoto N, et al. Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations. J Clin Oncol. 2013;31(27): 3327-3334.

5. Lee SY, Kim MJ, Jin G, et al. Somatic mutations in epidermal growth factor receptor signaling pathway genes in non-small cell lung cancers. J Thorac Oncol. 2010;5(11):1734-1740.

25. Jänne PA, Wang X, Socinski MA, et al. Randomized phase II trial of erlotinib alone or with carboplatin and paclitaxel in patients who were never or light former smokers with advanced lung adenocarcinoma: CALGB 30406 trial. J Clin Oncol. 2012;30(17): 2063-2069.

6. Rodig SJ, Mino-Kenudson M, Dacic S, et al. Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population. Clin Cancer Res. 2009;15(16):5216-5223. 7. Bean J, Brennan C, Shih JY, et al. MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci U S A. 2007;104(52):20932-20937. 8. Kawano O, Sasaki H, Endo K, et al. PIK3CA mutation status in Japanese lung cancer patients. Lung Cancer. 2006;54(2):209-215. 9. Kohno T, Ichikawa H, Totoki Y, et al. KIF5B-RET fusions in lung adenocarcinoma. Nat Med. 2012;18(3):375-377. 10. Bergethon K, Shaw AT, Ou SH, et al. ROS1 rearrangements define a unique molecular class of lung cancers. J Clin Oncol. 2012;30(8): 863-870. 11. Cardarella S, Ogino A, Nishino M, et al. Clinical, pathologic, and biologic features associated with BRAF mutations in non-small cell lung cancer. Clin Cancer Res. 2013;19(16):4532-4540. 12. Dutt A, Ramos AH, Hammerman PS, et al. Inhibitor-sensitive FGFR1 amplification in human non-small cell lung cancer. PLoS One. 2011;6(6):e20351. 13. Socinski MA, Evans T, Gettinger S, et al. Treatment of stage IV non-small cell lung cancer: diagnosis and management of lung cancer, 3rd ed: American College of Chest Physicians evidencebased clinical practice guidelines. Chest. 2013;143(5_suppl): e341S-e368S. 14. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatinpaclitaxel in pulmonary adenocarcinoma. N Engl J Med. 2009; 361(10):947-957. 15. Rosell R, Carcereny E, Gervais R, et al; Spanish Lung Cancer Group in collaboration with Groupe Français de Pneumo-Cancérologie and Associazione Italiana Oncologia Toracica. Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial. Lancet Oncol. 2012;13(3):239-246. 16. Yang JC, Shih JY, Su WC, et al. Afatinib for patients with lung adenocarcinoma and epidermal growth factor receptor mutations (LUX-Lung 2): a phase 2 trial. Lancet Oncol. 2012;13(5):539-548. 17. Yang JC-H, Schuler MH, Yamamoto N, et al. LUX-Lung 3: a randomized, open-label, phase III study of afatinib versus pemetrexed and cisplatin as first-line treatment for patients with advanced

journal.publications.chestnet.org

26. Mitsudomi T, Morita S, Yatabe Y, et al; West Japan Oncology Group. Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol. 2010;11(2):121-128. 27. Maemondo M, Inoue A, Kobayashi K, et al; North-East Japan Study Group. Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med. 2010;362(25):2380-2388. 28. Zhou C, Wu YL, Chen G, et al. Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutationpositive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study. Lancet Oncol. 2011;12(8):735-742. 29. Wu Y-L, Zhou C, Hu CP, et al. Afatinib versus cisplatin plus gemcitabine for first-line treatment of Asian patients with advanced non-small-cell lung cancer harbouring EGFR mutations (LUX-Lung 6): an open-label, randomised phase 3 trial. Lancet Oncol. 2014;15(2):213-222. 30. Gerber D, Gandhi L, Costa DB. Management and future directions in non-small cell lung cancer with known activating mutations. Am Soc Clin Oncol Educ Book. 2014:e353-e365. 31. Garassino MC, Martelli O, Broggini M, et al; TAILOR Trialists. Erlotinib versus docetaxel as second-line treatment of patients with advanced non-small-cell lung cancer and wild-type EGFR tumours (TAILOR): a randomised controlled trial. Lancet Oncol. 2013;14(10):981-988. 32. Shepherd FA, Rodrigues Pereira J, Ciuleanu T, et al; National Cancer Institute of Canada Clinical Trials Group. Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med. 2005;353(2):123-132. 33. Yasuda H, Kobayashi S, Costa DB. EGFR exon 20 insertion mutations in non-small-cell lung cancer: preclinical data and clinical implications. Lancet Oncol. 2012;13(1):e23-e31. 34. Eck MJ, Yun CH. Structural and mechanistic underpinnings of the differential drug sensitivity of EGFR mutations in non-small cell lung cancer. Biochim Biophys Acta. 2010;1804(3):559-566. 35. Girard N, Lou E, Azzoli CG, et al. Analysis of genetic variants in never-smokers with lung cancer facilitated by an Internet-based blood collection protocol: a preliminary report. Clin Cancer Res. 2010;16(2):755-763.

1071

36. Bell DW, Gore I, Okimoto RA, et al. Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR. Nat Genet. 2005;37(12):1315-1316.

56. Shaw AT, Kim DW, Nakagawa K, et al. Crizotinib versus chemotherapy in advanced ALK-positive lung cancer. N Engl J Med. 2013;368(25):2385-2394.

37. Mir O, Blanchet B, Goldwasser F. Drug-induced effects on erlotinib metabolism. N Engl J Med. 2011;365(4):379-380.

57. Katayama R, Shaw AT, Khan TM, et al. Mechanisms of acquired crizotinib resistance in ALK-rearranged lung cancers. Sci Transl Med. 2012;4(120):120ra17.

38. Hamilton M, Wolf JL, Rusk J, et al. Effects of smoking on the pharmacokinetics of erlotinib. Clin Cancer Res. 2006;12(7):2166-2171. 39. Faber AC, Corcoran RB, Ebi H, et al. BIM expression in treatmentnaive cancers predicts responsiveness to kinase inhibitors. Cancer Discov. 2011;1(4):352-365. 40. Ng KP, Hillmer AM, Chuah CT, et al. A common BIM deletion polymorphism mediates intrinsic resistance and inferior responses to tyrosine kinase inhibitors in cancer. Nat Med. 2012;18(4):521-528. 41. Sequist LV, Waltman BA, Dias-Santagata D, et al. Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. Sci Transl Med. 2011;3(75):75ra26. 42. Yun CH, Mengwasser KE, Toms AV, et al. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc Natl Acad Sci U S A. 2008;105(6):2070-2075. 43. Costa DB, Halmos B, Kumar A, et al. BIM mediates EGFR tyrosine kinase inhibitor-induced apoptosis in lung cancers with oncogenic EGFR mutations. PLoS Med. 2007;4(10):1669-1679. 44. Engelman JA, Zejnullahu K, Mitsudomi T, et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science. 2007;316(5827):1039-1043. 45. Yano S, Wang W, Li Q, et al. Hepatocyte growth factor induces gefitinib resistance of lung adenocarcinoma with epidermal growth factor receptor-activating mutations. Cancer Res. 2008;68(22): 9479-9487. 46. Ohashi K, Sequist LV, Arcila ME, et al. Lung cancers with acquired resistance to EGFR inhibitors occasionally harbor BRAF gene mutations but lack mutations in KRAS, NRAS, or MEK1. Proc Natl Acad Sci U S A. 2012;109(31):E2127-E2133. 47. Ercan D, Xu C, Yanagita M, et al. Reactivation of ERK signaling causes resistance to EGFR kinase inhibitors. Cancer Discov. 2012; 2(10):934-947. 48. Cheung HW, Du J, Boehm JS, et al. Amplification of CRKL induces transformation and epidermal growth factor receptor inhibitor resistance in human non-small cell lung cancers. Cancer Discov. 2011;1(7):608-625. 49. Miller VA, Hirsh V, Cadranel J, et al. Afatinib versus placebo for patients with advanced, metastatic non-small-cell lung cancer after failure of erlotinib, gefitinib, or both, and one or two lines of chemotherapy (LUX-Lung 1): a phase 2b/3 randomised trial. Lancet Oncol. 2012;13(5):528-538. 50. Sequist LV, Soria J-C, Gadgeel SM, et al. First-in-human evaluation of CO-1686, an irreversible, highly selective tyrosine kinase inhibitor of mutations of EGFR (activating and T790M) [abstract]. J Clin Oncol. 2014;32(suppl 5):8010. 51. Janne PA, Ramalingam SS, Yang JC-H, et al. Clinical activity of the mutant-selective EGFR inhibitor AZD9291 in patients (pts) with EGFR inhibitor–resistant non-small cell lung cancer (NSCLC) [abstract]. J Clin Oncol. 2014;32(suppl 5):8009.

58. Doebele RC, Pilling AB, Aisner DL, et al. Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non-small cell lung cancer. Clin Cancer Res. 2012;18(5):1472-1482. 59. Sasaki T, Koivunen J, Ogino A, et al. A novel ALK secondary mutation and EGFR signaling cause resistance to ALK kinase inhibitors. Cancer Res. 2011;71(18):6051-6060. 60. Zhang S, Wang F, Keats J, et al. Crizotinib-resistant mutants of EML4-ALK identified through an accelerated mutagenesis screen. Chem Biol Drug Des. 2011;78(6):999-1005. 61. Shaw AT, Kim D-W, Mehra R, et al. Ceritinib in ALK-rearranged non-small-cell lung cancer. N Engl J Med. 2014;370(13):1189-1197. 62. Sang J, Acquaviva J, Friedland JC, et al. Targeted inhibition of the molecular chaperone Hsp90 overcomes ALK inhibitor resistance in non-small cell lung cancer. Cancer Discov. 2013;3(4):430-443. 63. Chmielecki J, Foo J, Oxnard GR, et al. Optimization of dosing for EGFR-mutant non-small cell lung cancer with evolutionary cancer modeling. Sci Transl Med. 2011;3(90):90ra59. 64. Watanabe S, Tanaka J, Ota T, et al. Clinical responses to EGFRtyrosine kinase inhibitor retreatment in non-small cell lung cancer patients who benefited from prior effective gefitinib therapy: a retrospective analysis. BMC Cancer. 2011;11:1. 65. Oxnard GR, Arcila ME, Sima CS, et al. Acquired resistance to EGFR tyrosine kinase inhibitors in EGFR-mutant lung cancer: distinct natural history of patients with tumors harboring the T790M mutation. Clin Cancer Res. 2011;17(6):1616-1622. 66. Oxnard GR, Lo P, Jackman DM, et al. Delay of chemotherapy through use of post-progression erlotinib in patients with EGFRmutant lung cancer [abstract]. J Clin Oncol. 2012;30(suppl 491): 7547. 67. Mok TSK, Wu Y, Nakagawa W, et al. LBA2_PR: gefitinib/chemotherapy vs chemotherapy in epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC) after progression on first-line gefitinib: the phase III, randomized IMPRESS study. Ann Oncol. 2014;25(5):1-41. 68. Spigel DR, Ervin TJ, Ramlau R, et al. Final efficacy results from OAM4558g, a randomized phase II study evaluating MetMAb or placebo in combination with erlotinib in advanced NSCLC [abstract]. J Clin Oncol. 2011;29(suppl):7505. 69. Spigel DR, Ervin TJ, O’Byrne K, et al. Onartuzumab plus erlotinib versus erlotinib in previously treated stage IIIb or IV NSCLC: results from the pivotal phase III randomized, multicenter, placebocontrolled METLung (OAM4971g) global trial [abstract]. J Clin Oncol. 2014;32(suppl 5):8000. 70. Rikova K, Guo A, Zeng Q, et al. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell. 2007;131(6):1190-1203.

52. Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature. 2007;448(7153):561-566.

71. Ou SHI, Bang Y-J, Camidge DR, et al. Efficacy and safety of crizotinib in patients with advanced ROS1-rearranged nonsmall cell lung cancer (NSCLC) [abstract]. J Clin Oncol. 2013; 31(suppl):8032.

53. Shaw AT, Yeap BY, Mino-Kenudson M, et al. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK. J Clin Oncol. 2009;27(26):4247-4253.

72. Wan PT, Garnett MJ, Roe SM, et al; Cancer Genome Project. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell. 2004;116(6):855-867.

54. Choi YL, Soda M, Yamashita Y, et al; ALK Lung Cancer Study Group. EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors. N Engl J Med. 2010;363(18):1734-1739.

73. Marchetti A, Felicioni L, Malatesta S, et al. Clinical features and outcome of patients with non-small-cell lung cancer harboring BRAF mutations. J Clin Oncol. 2011;29(26):3574-3579.

55. Takezawa K, Okamoto I, Nishio K, Jänne PA, Nakagawa K. Role of ERK-BIM and STAT3-survivin signaling pathways in ALK inhibitor-induced apoptosis in EML4-ALK-positive lung cancer. Clin Cancer Res. 2011;17(8):2140-2148.

74. Planchard D, Mazieres J, Riely GJ, et al. Interim results of phase II study BRF113928 of dabrafenib in BRAF V600E mutation–positive non-small cell lung cancer (NSCLC) patients [abstract]. J Clin Oncol. 2013;31(suppl):8009.

1072 Translating Basic Research Into Clinical Practice

[

148#4 CHEST OCTOBER 2015

]