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Figure 1.
Flowchart Showing Inclusion and Results of the Present Study of Epidermal Growth Factor (EGFR) Mutations in Circulating Free DNA
Flowchart Showing Inclusion and Results of the Present Study of Epidermal Growth Factor (EGFR) Mutations in Circulating Free DNA
Figure 2.
Overall Survival According to Epidermal Growth Factor (EGFR) Mutation Status in Tissue and in Circulating Free DNA (cfDNA)
Overall Survival According to Epidermal Growth Factor (EGFR) Mutation Status in Tissue and in Circulating Free DNA (cfDNA)

A, In all 97 patients according to type of mutation in tissue. B, In all 97 patients according to type of mutation in cfDNA. C, In the 40 patients with mutations detected in cfDNA who received erlotinib, according to type of mutation in cfDNA. D, In the 41 patients with the L858R mutation in tissue according to the EGFR L858R mutation in cfDNA (detected vs not detected). E, In the 56 patients with the exon 19 deletion in tissue according to the exon 19 deletion in cfDNA (detected vs not detected).

Table 1.  
Patient Characteristics
Patient Characteristics
Table 2.  
Overall Survival According to Epidermal Growth Factor (EGFR) Mutation Status in Tissue and in Circulating Free DNA (cfDNA)
Overall Survival According to Epidermal Growth Factor (EGFR) Mutation Status in Tissue and in Circulating Free DNA (cfDNA)
Table 3.  
Univariate Analyses of Progression-Free Survival (PFS) and Overall Survival (OS)
Univariate Analyses of Progression-Free Survival (PFS) and Overall Survival (OS)
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    Karachaliou N, Mayo-de las Casas C, Queralt C, de Aguirre I, Melloni B, Cardenal F, Garcia-Gomez R, Massuti B, Sánchez JM, Porta R, Ponce-Aix S, Moran T, Carcereny E, Felip E, Bover I, Insa A, Reguart N, Isla D, Vergnenegre A, de Marinis F, Gervais R, Corre R, Paz-Ares L, Morales-Espinosa D, Viteri S, Drozdowskyj A, Jordana-Ariza N, Ramirez-Serrano JL, Molina-Vila MA, Rosell R, for the Spanish Lung Cancer Group. Association of EGFR L858R Mutation in Circulating Free DNA With Survival in the EURTAC Trial. JAMA Oncol. 2015;1(2):149-157. doi:10.1001/jamaoncol.2014.257

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Original Investigation
May 2015

Association of EGFR L858R Mutation in Circulating Free DNA With Survival in the EURTAC Trial

Author Affiliations
  • 1Instituto Oncológico Dr Rosell, Quiron-Dexeus University Hospital, Barcelona, Spain
  • 2Laboratory of Molecular Biology, Pangaea Biotech, Barcelona, Spain
  • 3Catalan Institute of Oncology, Hospital Germans Trias i Pujol, Badalona, Spain
  • 4Service de Pathologie Respiratoire et d’Allergologie, Hôpital du Cluzeau, Limoges, France
  • 5Medical Oncology Service, Catalan Institute of Oncology, Hospital Duran i Reynals, L’Hospitalet, Spain
  • 6Medical Oncology Service, Hospital General Universitario Gregorio Marañón, Madrid, Spain
  • 7Medical Oncology Service, Hospital General de Alicante, Alicante, Spain
  • 8Medical Oncology Service, Hospital 12 de Octubre, Madrid, Spain
  • 9Medical Oncology Service, Catalan Institute of Oncology, Hospital Dr Josep Trueta, Girona, Spain
  • 10Medical Oncology Service, Hospital La Paz, Madrid, Spain
  • 11Medical Oncology Service, Hospital Vall d’Hebron, Barcelona, Spain
  • 12Medical Oncology Service, Hospital Son Llatzer, Palma de Mallorca, Spain
  • 13Medical Oncology Service, Hospital Clínic de Valencia, Valencia, Spain
  • 14Medical Oncology Service, Hospital Clínic, Barcelona, Spain
  • 15Medical Oncology Service, Hospital Lozano Blesa, Zaragoza, Spain
  • 16Azienda Ospedaliera San Camillo-Forlanini, Rome, Italy
  • 17European Institute of Oncology, Milan, Italy
  • 18Medical Oncology Service, Centre François Baclesse, Caen, France
  • 19CHU Rennes Hopital Ponchaillou, Rennes, France
  • 20Medical Oncology Service, Hospital Universitario Virgen del Rocío, Sevilla, Spain
  • 21Pivotal, Madrid, Spain
  • 22MORe Foundation, Barcelona, Spain
  • 23Cancer Therapeutic Innovation Group, New York, New York
JAMA Oncol. 2015;1(2):149-157. doi:10.1001/jamaoncol.2014.257
Abstract

Importance  The EURTAC trial demonstrated the greater efficacy of erlotinib compared with chemotherapy for the first-line treatment of European patients with advanced non–small-cell lung cancer (NSCLC) harboring oncogenic epidermal growth factor receptor (EGFR) mutations (exon 19 deletion or L858R mutation in exon 21) in tumor tissue.

Objective  To assess the feasibility of using circulating free DNA (cfDNA) from blood samples as a surrogate for tumor biopsy for determining EGFR mutation status and to correlate EGFR mutations in cfDNA with outcome.

Design, Setting, and Participants  This prespecified analysis was a secondary objective of the EURTAC trial using patients included in the EURTAC trial from 2007 to 2011 with available baseline serum or plasma samples. Patients had advanced NSCLC, oncogenic EGFR mutations in the tumor, and no prior chemotherapy for metastatic disease and were treated with erlotinib or chemotherapy. EGFR mutations were examined in cfDNA isolated from 97 baseline blood samples by our novel peptide nucleic acid–mediated 5´ nuclease real-time polymerase chain reaction (TaqMan) assay.

Main Outcomes and Measures  Overall survival (OS), progression-free survival (PFS), and response to therapy were correlated with type of EGFR mutations in cfDNA.

Results  In samples from 76 of 97 (78%) patients with usable blood samples, EGFR mutations in cfDNA were detected. Median OS was shorter in patients with the L858R mutation in cfDNA than in those with the exon 19 deletion (13.7 [95% CI, 7.1-17.7] vs 30.0 [95% CI, 19.3-37.7] months; P < .001). Univariate analyses of patients with EGFR mutations in cfDNA identified the L858R mutation in tumor tissue or in cfDNA as a marker of shorter OS (hazard ratio [HR], 2.70 [95% CI, 1.60-4.56]; P < .001) and PFS (HR, 2.04 [95% CI, 1.20-3.48]; P = .008). For patients with the L858R mutation in tissue, median OS was 13.7 (95% CI, 7.1-17.7) months for patients with the L858R mutation in cfDNA and 27.7 (95% CI, 16.1-46.2) months for those in whom the mutation was not detected in cfDNA (HR, 2.22 [95% CI, 1.09-4.52]; P = .03). In the multivariate analysis of the 76 patients with EGFR mutations in cfDNA, only erlotinib treatment remained an independent predictor of longer PFS (HR, 0.41 [95% CI, 0.23-0.74]; P = .003).

Conclusions and Relevance  The peptide nucleic acid–mediated 5´ nuclease real-time polymerase chain reaction (TaqMan) assay used in this study can be used to efficiently assess EGFR mutations in cfDNA. The L858R mutation in cfDNA may be a novel surrogate prognostic marker.

Trial Registration  clinicaltrials.gov Identifier: NCT00446225

Introduction

The epidermal growth factor receptor (EGFR) is a transmembrane protein that transduces growth factor signaling from the extracellular milieu to the cell.1 Oncogenic EGFR (OMIM 131550) mutations—either a deletion in exon 19 or an amino acid substitution at codon 858 in exon 21 (L858R)2,3—induce constant phosphorylation of EGFR and activation of downstream signals that induce carcinogenesis.1 Mutations in EGFR—present in 16.6% of Spanish patients with non–small-cell lung cancer (NSCLC)4—confer sensitivity to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib hydrochloride.57 In 2 randomized trials based on clinical—but not molecular—selection of patients, comparing chemotherapy with gefitinib, a significant improvement in progression-free survival (PFS) with gefitinib therapy was observed in patients harboring EGFR mutations.8,9 Several randomized clinical trials of patients with EGFR-mutant NSCLC comparing chemotherapy with gefitinib,10,11 erlotinib,12,13 or afatinib dimaleate (a second-generation EGFR TKI)14,15 also identified a significant improvement in PFS with EGFR TKI therapy. However, no differences in overall survival (OS) were observed, likely due to crossover at the time of progression. We had previously reported no differences in PFS with erlotinib as first-line or second-line therapy in patients with EGFR mutations.4

A finding common to many of these studies is that patients with exon 19 deletions have longer PFS with erlotinib or afatinib therapy than those with L858R mutations,1216 although this differential effect was not observed in the 2 studies of gefitinib.10,11 Moreover, in our previous study,4 the presence of the L858R mutation in circulating free DNA (cfDNA) had an adverse effect on PFS with erlotinib therapy.

The interim analysis of the randomized phase III European Tarceva vs Chemotherapy (EURTAC) trial13 showed that EGFR mutations were associated with longer PFS with Tarceva (erlotinib) therapy (hazard ratio [HR], 0.37; P < .001), and on the basis of these results, in May 2013, erlotinib was approved by the US Food and Drug Administration for the first-line treatment of patients with NSCLC whose tumors harbor oncogenic EGFR mutations. One of the secondary objectives in the EURTAC trial was the assessment of EGFR mutations in cfDNA13 (Supplement 1).

The rate of detection of EGFR mutations in cfDNA varies according to the method used.1729 A large meta-analysis including 1591 cases tested with different methods reported a pooled sensitivity rate of 64.5% but found that results were influenced by the type of assay (P = .04).30 In the EURTAC interim analysis,13 we assessed EGFR mutations in cfDNA using a fragment analysis–derived method with low sensitivity (53%). We have since developed a novel peptide nucleic acid (PNA)31–mediated 5´ nuclease real-time polymerase chain reaction (PCR) (TaqMan) assay, which yields 78% sensitivity and 100% specificity. Using this assay, we have assessed EGFR mutations in cfDNA isolated from baseline blood samples from patients included in the EURTAC trial and correlated EGFR mutation status with OS, PFS, and response to therapy.

Box Section Ref ID

At a Glance

  • In the EURTAC trial, circulating tumor free DNA (cfDNA) could be obtained in 57% of patients.

  • Epidermal growth factor receptor (EGFR) mutations were identified in cfDNA in 78% of patients who had documented EGFR mutations in their tumors.

  • In univariate analysis, both overall and progression-free survival was shorter in patients with L858R mutation in tissue or cfDNA.

  • Epidermal growth factor receptor mutations in cfDNA did not predict response in either arm (erlotinib vs chemotherapy).

  • In multivariate analysis, only erlotinib treatment was an independent predictor of progression-free survival.

Methods
Study Design

From 2007 to 2011, 173 patients with advanced NSCLC were enrolled in the EURTAC trial.13 Inclusion criteria included the presence of oncogenic EGFR mutations (deletion in exon 19 or L858R in exon 21) in the tumor. Patients were randomized to receive platinum-based chemotherapy or erlotinib (eMethods in Supplement 2). The primary end point was PFS; secondary end points were OS, response to therapy, and EGFR mutation analysis in blood. The protocol was approved by the institutional review board of each participating center, and all patients provided their signed informed consent.

Tumor and Serum Samples

Provision of baseline tumor and blood samples for EGFR mutation testing was mandatory in the EURTAC trial.13 Paraffin-embedded tumor samples were obtained by means of standard procedures as previously reported.13 All tissue samples were analyzed by an independent technique: length analysis of fluorescently labeled PCR products (for exon 19) and TaqMan assay (for exon 21), using from 1 to 3 μL of the DNA solution. All mutants were confirmed by PCR followed by DNA Sanger sequencing using a nested PCR with 3 to 4 μL of sample.32

The EURTAC protocol specified that EGFR mutations would be assessed in serum samples collected before the study (Supplement 1). Blood samples (15 mL) were collected from patients in 2 Vacutainer tubes (Becton, Dickinson), 1 for plasma and 1 for serum isolation. Tubes were centrifuged twice at 2300 rpm for 10 minutes and the supernatant (serum or plasma) aliquoted. Circulating free DNA was purified from 1.2 mL of serum and plasma by means of standard procedures, using the QIAamp DNA Blood Mini Kit (Qiagen), and resuspended in 40 μL of water. We developed a multiplex 5´ nuclease real-time PCR (Taqman) assay to be used in the presence of a PNA clamp (Eurogentec) designed to inhibit the amplification of the wild-type DNA allele. Mix 1 was designed to detect the following mutations: p.Glu746_Ala750del, p.Leu747_Thr751del, and p.Glu746_Thr751delinsAla. Mix 2 was designed to detect the following mutations: p.Glu746_Ser752delinsVal, p.Leu747_Pro753delinsSer, p.Leu747_Ser752del, p.Leu747_Ala750delinsPro, and p.Leu747_Thr751delinsPro. Mix 3 was targeted to the p.L858R mutation in exon 21. Amplification was performed in 12.5-μL volumes using 1 μL of DNA sample, 6.25 μL of Genotyping Master Mix (Applied Biosystems), 0.96 pmol of primers, 1.2 pmol of probes, and 62.4 pmol of PNA. The specificity of our assay was determined by analyzing cfDNA purified from the blood of 55 patients whose tumors did not harbor EGFR mutations; all samples had negative results, indicating 100% specificity (eMethods in Supplement 2).

Statistical Analyses

All patients were censored at crossover for the analysis of PFS but not for that of OS. Kaplan-Meier curves were drawn and compared with the log-rank test. Hazard ratios with their 95% confidence intervals were calculated with a Cox proportional-hazards analysis. Prespecified adjustment factors included Eastern Cooperative Oncology Group performance status and type of EGFR mutation. Response rates were compared between the 2 groups with the χ2 test. All analyses were 2 sided with a 5% significance level and were performed with SAS, version 9.3; SPSS, version 17.0; or S-PLUS, version 6.1 (eMethods in Supplement 2).

Results
Patients

Of the 173 patients included in the EURTAC trial, 50 blood samples were damaged or lost during shipment and an additional 26 were of insufficient quality or quantity for molecular analyses. Of the 97 remaining patients, 56 harbored the exon 19 deletion and 41 the L858R mutation in exon 21 in tissue. Epidermal growth factor receptor mutations in cfDNA were detected in 76 patients (78%) including 47 with the exon 19 deletion and 29 with the L858R mutation (Figure 1). Baseline characteristics were well balanced between those with and without EGFR mutations detected in cfDNA (Table 1).

Overall Survival

As of December 9, 2013, with a median follow-up of 49.4 months, median OS for all 173 patients in the EURTAC study was 22.9 (95% CI, 17.0-29.5) months in the erlotinib arm vs 22.1 (95% CI, 16.5-28.4) months in the chemotherapy arm (P = .97). Overall survival for the 115 patients with the exon 19 deletion in tissue was significantly longer than for the 58 with the L858R mutation (25.0 [95% CI, 18.9-30.9] vs 17.7 [95% CI, 13.5-23.5] months; P = .001) (eFigure 1A and B in Supplement 2).

Details on OS in the subset of 97 patients in the present study are shown in Table 2. Overall survival was longer in the erlotinib arm, both overall (eFigure 1C in Supplement 2) and within subsets of patients according to EGFR mutation status (Table 2), but these differences were not significant. In contrast, median OS was significantly longer for those with the exon 19 deletion than for those with the L858R mutation—both in tissue (24.9 [95% CI, 18.8-36.2] vs 17.7 [95% CI, 10.0-23.5] months; P = .006) and when detected in cfDNA (30.0 [95% CI, 19.3-37.7] vs 13.7 [95% CI, 7.1-17.7] months; P < .001) (Figure 2A and B and Table 2). Moreover, among the 40 patients in the erlotinib arm with mutations detected in cfDNA, median OS was 34.4 (95% CI, 22.9 to not reached) months for those with the exon 19 deletion, compared with 13.7 (95% CI, 2.6-21.9) months for those with the L858R mutation (P = .001) (Figure 2C and Table 2). For the 41 patients with the L858R mutation in tissue, median OS was longer in those in whom the mutation was not detected in cfDNA (27.7 [95% CI, 16.1-46.2] vs 13.7 [95% CI, 7.1-17.7] months; HR, 2.22 [95% CI, 1.09-4.52]; P = .03) (Figure 2D and Table 2). In contrast, among the 56 patients with the exon 19 deletion in tissue, median OS was longer in those in whom the deletion was also detected in cfDNA (30.0 [95% CI, 19.3-37.7] vs 14.2 [95% CI, 3.0-19.8] months; HR, 0.39 [95% CI, 0.17-0.89]; P = .02) (Figure 2E and Table 2).

In the univariate analysis of all 97 patients, the L858R mutation in tissue (HR, 1.85 [95% CI, 1.18-2.90]; P = .007) or in cfDNA (HR, 2.80 [95% CI, 1.60-4.72]; P < .001) was associated with shorter OS (Table 3). When the univariate analysis was limited to only the 76 patients with EGFR mutations detected in cfDNA, the L858R mutation remained a marker of shorter OS (HR, 2.70 [95% CI, 1.60-4.56]; P < .001) (Table 3).

Progression-Free Survival

As of 9 December 2013, median PFS for all 173 patients in the EURTAC study was 10.4 (95% CI, 8.4-12.9) months for those in the erlotinib arm and 5.1 (95% CI, 4.5-5.8) months for those in the chemotherapy arm (HR, 0.34 [95% CI, 0.23-0.49]; P < .001). The PFS for the subset of 97 patients included in the present study was also longer in the erlotinib arm (12.3 [95% CI, 8.4-14.7] vs 5.5 [95% CI, 4.5-6.7] months; P < .001) (eFigure 2 in Supplement 2).

Among the 49 patients treated with erlotinib, PFS was longer for those with the exon 19 deletion than for those with the L858R mutation—both in tissue (14.7 [95% CI, 10.4-17.3] vs 8.4 [95% CI, 2.6-12.3] months; P = .07) and in cfDNA (15.5 [95% CI, 10.4-18.8] vs 6.9 [95% CI, 1.3-10.8] months; P = .03) (eFigure 3 in Supplement 2). For the 48 patients treated with chemotherapy, no significant differences were observed according to the type of EGFR mutation in tissue or in cfDNA (eFigure 4 in Supplement 2).

In the univariate analysis of all 97 patients, treatment with erlotinib (HR, 0.33 [95% CI, 0.21-0.54]; P < .001) was associated with longer PFS (Table 3), whereas the L858R mutation in cfDNA (HR, 2.09 [95% CI, 1.23-3.54]; P = .02) was associated with shorter PFS. When the univariate analysis was restricted to the 76 patients with EGFR mutations detected in cfDNA, the L858R mutation in tissue or in cfDNA (HR, 2.04 [95% CI, 1.20-3.48]; P = .008) and erlotinib treatment (HR, 0.36 [95% CI, 0.21-0.62]; P < .001) were associated with shorter and longer PFS, respectively (Table 3). In the multivariate analysis of the 76 patients with EGFR mutations in cfDNA, only erlotinib treatment remained an independent predictor of longer PFS (HR, 0.41 [95% CI, 0.23-0.74]; P = .003).

Response

The overall response rates in the EURTAC study were 65.1% in the erlotinib group and 16.1% in the chemotherapy group (P < .001). For the 97 patients included in the present study, response rates were 65.3% (32 of 49) and 14.6% (7 of 48), respectively (P < .001) (eTable 1 in Supplement 2). No differences in response to therapy were observed according to the presence of EGFR mutations in cfDNA (eTable 2 in Supplement 2).

Discussion

Testing of tumor tissue remains the recommended method for detecting the presence of oncogenic EGFR mutations; however, the amount of tumor tissue obtained by biopsy is often insufficient, especially in advanced NSCLC, raising the question of whether cfDNA may be used as a surrogate liquid biopsy for the noninvasive assessment of EGFR mutations. We have assessed EGFR mutations in cfDNA from blood samples obtained at baseline from 97 patients included in the EURTAC trial.13 Epidermal growth factor receptor mutations in cfDNA were detected in 78% of patients. The L858R mutation in tumor tissue (HR, 1.85; P = .007) and in cfDNA (HR, 2.70; P < .001) was related to shorter OS. Importantly, among the 41 patients with the L858R mutation in tissue, those in whom the L858R mutation was detected in cfDNA had notably shorter median survival than those in whom the mutation was not detected in cfDNA (13.7 vs 27.7 months; HR, 2.22; P = .03).

In our previous study,4 a deleterious effect on PFS and OS was observed for the L858R mutation in tissue as compared with the exon 19 deletion (HR for risk of progression, 1.92; P = .02; HR for risk of death, 2.98; P = .002). In addition, a recent pooled analysis of the LUX-Lung 3 and 6 trials demonstrated a differential activity for afatinib according to the type of EGFR mutation (exon 19 deletions: HR, 0.59; P < .001; L858R mutations: HR, 1.25; P = .16).16 The shorter OS—both for all patients and for those receiving erlotinib therapy—observed in the present study among patients with L858R mutations in tissue or cfDNA reinforces this concept of the existence of more than 1 clinical phenotype of EGFR-mutant NSCLC and suggests that in future analyses, outcomes of patients with exon 19 deletions and those with L858R mutations should not be pooled. The exon-specific activity of EGFR TKIs warrants further exploration.1

Several studies have assessed EGFR mutations in serum or plasma, using different methods, with varying results20,23,29,3336 (eTable 3 in Supplement 2). The first of these studies used the Scorpion amplified refractory mutation system and found a high concordance between EGFR mutation status in tissue and serum (72.7%).25 Other early studies, however, used techniques that were often too complicated to be introduced into routine clinical practice.20,22 Although more recent studies included larger numbers of patients and used more accessible methods, their clinical usefulness is still limited by a relatively low sensitivity (43%-68%).24,28,3739 The 78% sensitivity and 100% specificity of our assay compares favorably with these previous studies and indicates that cfDNA may be a promising method for screening for EGFR mutations in clinical studies, minimizing the need for invasive tumor biopsies. Moreover, although several studies have related EGFR mutations to clinical outcome,22,24,33,37 to the best of our knowledge, none has compared outcome according to EGFR mutations in tissue vs cfDNA or according to the type of EGFR mutation. Moreover, although EGFR mutation assessment in peripheral blood has been performed as a post hoc exploratory analysis in several trials,24 only the EURTAC trial13 has prespecified this analysis as a secondary objective.

Our findings should be interpreted in the light of several limitations. The fact that only slightly more than half of the patients in the EURTAC trial had blood samples of sufficient quality and quantity for molecular analysis may have had some impact on the analysis. In addition, whereas our results and those of others16 show a greater survival benefit for erlotinib therapy in patients harboring the exon 19 deletion, this finding has yet to be confirmed in a randomized trial and integrated into clinical care algorithms. Our finding that the L858R mutation detected in cfDNA exerts a negative influence on survival indicates that a prospective study is warranted to examine the use of EGFR mutations in cfDNA as a viable surrogate biomarker. The Spanish Lung Cancer Group is participating in 2 ongoing clinical trials in patients with advanced-stage EGFR-mutant NSCLC, in which plasma and serum are being collected for EGFR mutation testing in cfDNA at baseline, at response, and at progression: the European phase II BELIEF trial (NCT01562028) of erlotinib plus bevacizumab and the Spanish Lung Cancer Group phase II GOAL trial (NCT01513174) comparing gefitinib plus olaparib vs gefitinib alone. These trials will shed additional light on EGFR mutation assessment in cfDNA and pave the way for its implementation in clinical care.

Conclusions

Our results validate previous findings indicating the existence of more than 1 clinical phenotype of EGFR-mutant NSCLC based on the type of EGFR mutation and on detection in tissue vs cfDNA. In addition, using the PNA-mediated 5´ nuclease real-time polymerase chain reaction (TaqMan) assay, we have shown that the EGFR L858R mutation in cfDNA is a negative prognostic biomarker. Although EGFR TKI therapy improves PFS over chemotherapy and still remains the standard of care for patients with L858R mutations, our results indicate the need for new combination therapies in this subgroup of patients.

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Article Information

Accepted for Publication: December 22, 2014.

Corresponding Author: Rafael Rosell, MD, Cancer Biology and Precision Medicine Program, Catalan Institute of Oncology, Hospital Germans Trias i Pujol, Ctra Canyet, s/n, 08916 Badalona, Spain (rrosell@iconcologia.net).

Published Online: February 26, 2015. doi:10.1001/jamaoncol.2014.257.

Author Contributions: Drs Karachaliou and Mayo-de las Casas served as co–first authors, each with equal contribution to the manuscript. Drs Karachaliou and Rosell had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Karachaliou, Garcia-Gomez, Reguart, Isla.

Acquisition, analysis, or interpretation of data: Karachaliou, Mayo-de las Casas, Queralt, de Aguirre, Melloni, Cardenal, Garcia-Gomez, Massuti, Sánchez, Porta, Ponce-Aix, Moran, Carcereny, Felip, Bover, Insa, Isla.

Drafting of the manuscript: Karachaliou, Mayo-de las Casas, Garcia-Gomez.

Critical revision of the manuscript for important intellectual content: Karachaliou, Queralt, de Aguirre, Melloni, Cardenal, Garcia-Gomez, Massuti, Sánchez, Porta, Ponce-Aix, Moran, Carcereny, Felip, Bover, Insa, Reguart, Isla.

Obtained funding: Garcia-Gomez.

Administrative, technical, or material support: Mayo-de las Casas, Queralt, de Aguirre, Massuti, Sánchez, Moran, Carcereny.

Study supervision: Karachaliou, Garcia-Gomez, Sánchez, Ponce-Aix, Felip, Bover, Isla.

Conflict of Interest Disclosures: Dr Massuti is a consultant/advisory board member for Roche. Dr Felip is a consultant/advisory board member for Lilly, Pfizer, Roche, Boehringer Ingelheim, Astra Zeneca, and Novartis. No other disclosures are reported.

Funding/Support: Work in Dr Rosell’s laboratory is partially supported by a grant from the La Caixa Foundation and by a grant from the Red Tematica de Investigacion Cooperativa en Cancer (RTICC; grant RD12/0036/ 0072).

Role of the Funder/Sponsor: The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Group Information: The Spanish Lung Cancer Group members were Felipe Cardenal, MD, Catalan Institute of Oncology, Hospital Duran i Reynals, L’Hospitalet, Spain; Ramon Garcia-Gomez, MD, Hospital General Universitario Gregorio Marañón, Madrid, Spain; Bartomeu Massuti, MD, Hospital General de Alicante, Alicante, Spain; José Miguel Sánchez, MD, Hospital 12 de Octubre, Madrid, Spain; Ruth Porta, MD, Catalan Institute of Oncology, Hospital Dr Josep Trueta, Girona, Spain; Santiago Ponce-Aix, MD, Hospital La Paz, Madrid, Spain; Teresa Moran, MD, Catalan Institute of Oncology, Hospital Germans Trias i Pujol, Badalona, Spain; Enric Carcereny, MD, Catalan Institute of Oncology, Hospital Germans Trias i Pujol, Badalona, Spain; Enriqueta Felip, MD, Hospital Vall d’Hebron, Barcelona, Spain; Isabel Bover, MD, Hospital Son Llatzer, Palma de Mallorca, Spain; Amelia Insa, MD, Hospital Clínic de Valencia, Valencia, Spain; Noemí Reguart, MD, Hospital Clínic, Barcelona, Spain; Dolores Isla, MD, Hospital Lozano Blesa, Zaragoza, Spain; Luis Paz-Ares, MD, PhD, Hospital Universitario Virgen del Rocío, Sevilla, Spain; Rafael Rosell, MD, PhD, Catalan Institute of Oncology, Hospital Germans Trias i Pujol, Badalona, Spain; Santiago Viteri, MD, Instituto Oncológico Dr Rosell, Quirón Dexeus University Hospital, Barcelona, Spain.

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