Molecular Analysis-Based Treatment Strategies for the ...opment of technology such as the circulating tumor cell (CTC) chip, a microﬂuidic device that can isolate rare CTCs from

SUPPLEMENT

Molecular Analysis-Based Treatment Strategies for theManagement of Non-small Cell Lung Cancer

Howard West, MD,* Rogerio Lilenbaum, MD,* David Harpole, MD,† Antoinette Wozniak, MD,‡and Lecia Sequist, MD, MPH§

Abstract: Even with the introduction of targeted agents and theestablishment of multiple lines of therapy, the median survival forpatients with advanced non-small cell lung cancer (NSCLC) doesnot considerably extend beyond 1 year. Emerging research suggeststhat clinical characteristics alone are insufficient for selecting pa-tients for therapies that may confer significant survival benefit. Thediscovery of predictive and prognostic molecular markers such asgene mutations in EGFR and KRAS as well as high tumor expressionlevels of DNA repair pathway components ribonucleotide reductasesubunit 1 and excision repair cross-complementing group 1 hassparked an interest in the development of individualized therapy asa strategy for increasing survival in patients with NSCLC. Tech-niques to analyze molecular biomarkers, such as immunohistochem-istry, fluorescence in situ hybridization, polymerase chain reaction,and, more recently, gene microarray techniques, are being investi-gated for their potential to accurately predict an individual patient’sresponse to therapy. Many prospective trials are still needed toclarify and confirm the utility of molecular biomarkers for guidingtreatment selection, and continued participation in clinical trials iscritical for the development of tools to provide customized treatmentplans for patients with NSCLC.

Key Words: Molecular biomarkers, Non-small cell lung cancer,Epidermal growth factor receptor.

(J Thorac Oncol. 2009;4: S1029–S1039)

Lung cancer is the leading cause of cancer death in theUnited States, accounting for an estimated 161,840 deaths

during 2008.1 Despite several decades of extensive research,the median survival for patients with non-small cell lungcancer (NSCLC) is approximately 1 year.2 Recent studieshave confirmed the efficacy of platinum doublets for thetreatment of advanced or metastatic NSCLC; however, anefficacy plateau has been reached with existing cytotoxicagents.3–5 To increase efficacy and reduce treatment-relatedtoxicities, investigators have focused on the development ofstrategies to individualize therapy on the basis of tumormolecular characteristics. Recently, molecular biomarkershave been identified, which may be a predictive of responseto therapy or prognostic of survival. Predictive markers canbe used for therapeutic decision making, whereas prognosticmarkers have the ability to estimate patient outcome inde-pendent of treatment modality.6 On November 12, 2008, apanel of medical and surgical oncologists convened in Chi-cago, IL, to assess the status of molecular biomarkers incurrent clinical practice. This article summarizes their discus-sion on recent advances in biomarker development and thepresent clinical applicability of predictive markers as tools forselecting patients for chemotherapy and targeted agents.

ASSAY TECHNOLOGIES FOR MOLECULARBIOMARKER ANALYSIS

The advent of targeted therapy and the discovery ofpotentially predictive and prognostic markers have sparkedan interest in the development of individualized therapy forpatients with NSCLC. Techniques such as immunohisto-chemistry (IHC), fluorescence in situ hybridization (FISH),reverse-transcription polymerase chain reaction, and microar-rays have been used to characterize molecular biomarkersthat may indicate the likelihood of patient response to aspecific therapy.

IHC is a semiquantitative method for estimating proteinexpression that can be conducted at the same time as histo-logic classification.7 However, antibody-based detectionmethods can yield inconsistent results because of variation inantibody type, production batch, staining protocol, scoring

*Thoracic Oncology Program, Swedish Cancer Institute, Seattle, WA; †De-partment of Surgery, Duke University Medical Center, Durham, NorthCarolina; ‡Department of Medicine, Barbara Ann Karmanos CancerInstitute, Detroit, Michigan; and §Department of Medicine, HarvardMedical School, Massachusetts General Hospital Cancer Center, Boston,Massachusetts.

Disclosure: Dr. Lilenbaum is a consultant for AstraZeneca PharmaceuticalsLP, Genentech Inc., and Lilly USA, LLC. Dr. West is a consultant forImCone Systems Incorporated and Lilly USA, LLC; he has receivedhonorarium from Lilly USA, LLC. Dr. Wozniak is on the advisory boardfor AstraZeneca Pharmaceuticals LP, Bristol-Myers Squibb Company,Genentech, Inc., and Lilly USA, LLC; she is a consultant for AstraZenecaPharmaceuticals LP, Bristol-Myers Squibb Company, and Genentech, Inc.,and Lilly USA, LLC; she has received grant/research support from LillyUSA, LLC; she is on the speaker’s bureau and has received honorarium fromGenentech, Inc., and Lilly USA, LLC. Dr. Sequist and Dr. Harpole report nodisclosures.

CME Credit: This article is being submitted by the CBCE™ (The Center forBiomedical Continuing Education) as a continuing medical education(CME)-accredited activity. Corresponding CME information accompa-nies this submission.

Address for correspondence: Howard West, MD, Thoracic Oncology Pro-gram, Swedish Cancer Institute, 1221 Madison St, Suite 1020, Seattle,WA 98104. E-mail: [email protected]

Copyright © 2009 by the International Association for the Study of LungCancerISSN: 1556-0864/09/0409-1029

Journal of Thoracic Oncology • Volume 4, Number 9, Supplement 2, September 2009 S1029

method, and cutoff criteria.8 For example, in a study of theIressa Survival Evaluation in Lung Cancer trial, which inves-tigated the efficacy of gefitinib versus placebo, two differentantibodies were compared for IHC detection of epidermalgrowth factor receptor (EGFR) to predict survival among 736patients; different antibody staining cutoff points were re-quired to predict benefit from gefitinib, which suggests vari-ations in antibody sensitivity.9

FISH detects genomic and chromosomal changes intissue block preparations by hybridizing labeled DNA probesto a sample.10 Proper tissue handling and processing of FISHsamples are crucial to minimize background signal and en-sure test accuracy, but standard protocols do not yet exist.8,10

Direct sequencing is the current standard method fordetecting gene mutations, but new techniques that providehigher sensitivity and use smaller samples are being investi-gated.8 Given the heterogeneity of lung tumors, it can bedifficult to determine mutation status throughout an entiretumor, and for this reason, assay technology development ismoving toward global analysis to characterize tumor hetero-geneity, tumor microenvironment, and individual geneticfactors that may influence disease management.

Gene expression profiles derived from microarrays mayreveal genomic signatures that more accurately predict pa-tient outcomes or responses to therapy compared with tech-niques based on individual gene characterization. Moreover,advances in miniaturization and automation aid in the devel-opment of technology such as the circulating tumor cell(CTC) chip, a microfluidic device that can isolate rare CTCsfrom whole blood (Figure 1).11 One major advantage of thistool is that cancer cells captured against microposts on theCTC chip maintain cell viability, and isolated cells canundergo molecular analysis or feasible functional assays.

The integration of new technologies and molecularmarkers into clinical practice will require protocol standard-ization, marker validation, and a commitment to collectadequate tissue. Although the platforms for molecular assaysare still in development, techniques for biomarker analysiswill be invaluable to understanding the biology and treatmentof NSCLC.

MOLECULAR PREDICTORS OF RESPONSE TOCHEMOTHERAPY

Increased understanding in the molecular biology andgenetics of lung tumorigenesis has resulted in the identifica-tion of genes that may be useful for clinical decision making,which has led to investigations of using molecular markers asa strategy to overcome the platinum-doublet chemotherapytreatment plateau and reduce treatment-related toxicities. Inan effort to better select patients for chemotherapy, translationalresearchers are examining molecular markers, including exci-sion repair cross-complementing group 1 gene (ERCC1), ribo-nucleotide reductase subunit 1 (RRM1), and thymidylate syn-thase (TS), as well as gene expression profiling as tools to guidechemotherapy selection for patients with NSCLC.12–16

Ribonucleotide Reductase Subunit 1 (RRM1)RRM1 functions as the regulatory subunit of ribonu-

clease reductase, an enzyme that catalyzes the deoxynucle-

otide production required for DNA repair.12,17 The implica-tion of RRM1 in the DNA repair process suggests thatdifferential RRM1 gene expression could influence the effi-cacy of chemotherapeutic nucleoside analogue agents such asgemcitabine.12 Although preliminary preclinical and clinicalevidence supports the observation that the response to gem-citabine is inversely correlated with RRM1 gene expres-sion,17–19 the correlation of tumoral RRM1 expression with astatistically significant improvement in survival from chemo-therapy has yet to be demonstrated consistently. In a retro-spective study of a large, randomized trial investigating theefficacy of frontline chemotherapy in 100 patients with ad-

FIGURE 1. A, SEM image of an array of microposts etchedin silicon. B, SEM image of an NCI-H1650 lung cancer cellspiked into blood. It has been captured on a micropost thathas been chemically functionalized with antibodies againstEpCAM. Inset image shows the cell at high magnification.EpCAM, epithelial cell adhesion molecule; SEM, scanningelectron micrograph. Part A is reproduced with permissionfrom Sunitha Nagrath, PhD and part B is reproduced withpermission from Macmillan Publishers Ltd: Nature 2007;450:1235–1239.

West et al. Journal of Thoracic Oncology • Volume 4, Number 9, Supplement 2, September 2009

Copyright © 2009 by the International Association for the Study of Lung CancerS1030

vanced NSCLC, it was observed among patients who re-ceived gemcitabine/cisplatin that those with low RRM1 mes-senger RNA (mRNA) expression levels showed significantlyincreased overall survival (OS) compared with patients whohad high RRM1 expression levels (13.7 months versus 3.6months, p � 0.009).20 However, RRM1 mRNA expressionlevel did not correlate with increased OS in patients whoreceived other gemcitabine-based chemotherapeutic regimensin this trial. Results from a prospective phase II trial inpatients with advanced, unresectable NSCLC demonstratethat tumors with low RRM1 gene expression respond signif-icantly better to induction therapy with carboplatin and gem-citabine than tumors with high levels of RRM1 expression(p � 0.002).6,17 In contrast, early results from a phase III trialof gemcitabine/carboplatin versus gemcitabine alone demon-strate no significant correlation between RRM1 expressionand survival among patients who are receiving carboplatin/gemcitabine compared with those receiving gemcitabinealone (6.7 months versus 5.1 months, p � 0.28).21

Excision Repair Cross-Complementing Group 1(ERCC1)

ERCC1 is an excision nuclease that participates in thenucleotide excision repair process responsible for the repairof platinum-induced damage to DNA.14,22 Preclinical studieshave shown that expression levels of ERCC1 mRNA directlycorrelate with platinum resistance.22–25 At this time, it re-mains unclear whether ERCC1 expression can be consistentlycorrelated with survival benefit from chemotherapy. In theInternational Adjuvant Lung Trial, which compared the effi-cacy of cisplatin-based adjuvant chemotherapy with postop-erative observation in patients with completely resected stageI to III NSCLC, a significant improvement in the 5-yearsurvival rate with cisplatin-based adjuvant chemotherapycompared with observation was observed in the ERCC1protein-negative but not ERCC1 protein-positive subgroup

(hazard ratio [HR] � 0.65, p � 0.002 versus HR � 1.14, p �0.40; Figure 2).26 Nevertheless, in a phase III trial investigat-ing the efficacy of ERCC1 mRNA expression-based customi-zation of cisplatin-based chemotherapy in patients with stageIV NSCLC, there was no significant difference in OS be-tween the control arm (cisplatin/docetaxel) and the genotypicarm (9.8 months versus 9.9 months, p � 0.59), even thoughpatients in the genotypic arm were stratified such that thosewith low ERCC1 expression received cisplatin/docetaxel andpatients with high ERCC1 expression received gemcitabine/docetaxel.27

The presence of single nucleotide polymorphisms inERCC1 may account for inconsistency in the correlationbetween ERCC1 expression and survival benefit from che-motherapy.28,29 For example, a retrospective study of 130patients with advanced NSCLC reported that patients withcodon 118 C/T or 118 T/T polymorphisms in ERCC1 hadsignificantly longer survival than patients with 118 C/Cpolymorphisms after treatment with platinum-based chemo-therapy (17.5 months versus 13.5 months, p � 0.003).28

Evaluating the combined status of ERCC1 expression andpolymorphism may be a better predictor of response andbenefit from platinum-based chemotherapy.

RRM1 and ERCC1Some studies have investigated the predictive value of

measuring both RRM1 and ERCC1 expression to guide se-lection of chemotherapy for patients with NSCLC. For ex-ample, a retrospective analysis of 100 patients from a largertrial, which investigated the efficacy of frontline chemother-apy in patients with advanced NSCLC, observed that lowlevels of both RRM1 and ERCC1 mRNA expressions weresignificant predictors of increased OS compared with highlevels after treatment with gemcitabine/cisplatin (median OSnot reached versus 6.8 months, p � 0.02) but not gemcitab-ine/cisplatin/vinorelbine (11.1 months versus 18.9 months, p �

FIGURE 2. Kaplan-Meier curve estimates of survival probability from the International Adjuvant Lung Trial. A significant im-provement in the 5-year survival rate with cisplatin-based adjuvant chemotherapy compared with observation (control) oc-curred in the ERCC1-negative but not ERCC1-positive subgroup (HR � 0.65 and p � 0.002 versus HR � 1.14 and p � 0.4).ERCC1, excision repair cross-complementing group; HR, hazard ratio; OS, overall survival. Reproduced with permission fromN Engl J Med 2006;355:983–991.

Journal of Thoracic Oncology • Volume 4, Number 9, Supplement 2, September 2009 Molecular Analysis-Based Strategies for NSCLC

Copyright © 2009 by the International Association for the Study of Lung Cancer S1031

0.29) or gemcitabine/vinorelbine followed by vinorelbine/ifosf-amide (8.8 months versus 5.9 months, p � 0.74).20 To investi-gate the feasibility of chemotherapy selection based on RRM1and ERCC1 gene expression, the prospective Molecular Anal-yses–Directed Individualized Therapy for Advanced NSCLCphase II study was conducted at a single institution.30 Intheory, patients with low ERCC1 levels were more likely toreceive benefit from platinum-based regimens, whereas pa-tients with low RRM1 levels were more likely to receivebenefit from gemcitabine-based regimens. Thus, patients withlow RRM1 and ERCC1 levels were treated with gemcitabine/carboplatin, and patients with high ERCC1 and RRM1 levelswere treated with docetaxel/vinorelbine. Patients with lowRRM1 and high ERCC1 levels were treated with gemcitabine/docetaxel, and patients with high RRM1 and low ERCC1levels were treated with docetaxel/carboplatin (Figure 3).Results from this trial indicate that chemotherapy selectionbased on tumor expression of RRM1 and ERCC1 seemsfeasible and may confer patient benefit (response rate [RR],44%; median OS, 13.3 months; and progression-free survival[PFS], 6.6 months). These results are being confirmed in anongoing, multicenter, phase III trial of customized chemo-therapy based on both RRM1 and ERCC1 expression inpatients with advanced NSCLC. The strategy of RRM1 andERCC1-directed customization of chemotherapy is also beingexplored in the adjuvant setting in the Southwest OncologyGroup (SWOG) 0720 phase II trial, with the hope thatpotential advances in biomarker development will translateinto broadly applicable and clinically meaningful improve-ments in patient outcomes.

Thymidylate Synthase (TS)TS is an enzyme involved in DNA biosynthesis that has

been investigated as a potential predictor of response topemetrexed.31 TS is responsible for maintaining intracellularlevels of thymidine, which is important for DNA synthesisand repair.32 In preclinical studies, TS overexpression hasbeen correlated with resistance to pemetrexed.15,33 In addi-tion, median TS mRNA expression seems to be significantlyhigher in squamous cell carcinoma compared with adenocar-cinoma (2.17 versus 1.16; p � 0.0001), which suggests thatpemetrexed has decreased efficacy in patients with squamouscell carcinoma compared with adenocarcinoma histologies.31

This hypothesis is supported by the results from a phase IIItrial that compared the efficacy of cisplatin/gemcitabine withcisplatin/pemetrexed in patients with advanced NSCLC.Among the intent-to-treat population, the OS of patients whoreceived cisplatin/pemetrexed was similar to those who re-ceived cisplatin/gemcitabine (10.3 months versus 10.3months, HR � 0.94).34 However, among patients withnonsquamous histologies, cisplatin/pemetrexed significantlyimproved OS compared with cisplatin/gemcitabine (11.8months versus 10.4 months; p � 0.005; Figure 4). In contrast,patients with squamous cell carcinoma derived significantlyimproved OS with cisplatin/gemcitabine compared with cis-platin/pemetrexed (10.8 months versus 9.4 months; p �0.05). Although these results are hypothesis generating andprovide evidence that the expression of TS in various tumorhistologies could be used to guide the use of pemetrexed,prospective studies must be conducted to define the potentialrole of TS as a predictive molecular marker.

Gene Expression ProfilingAn increased understanding in the molecular biology

and the genetics of lung tumorigenesis has resulted in theidentification of prognostic or predictive gene expressionprofiles that may inform treatment decisions. For example,risk assessment models are being developed from gene ex-pression profiles to predict the probability of tumor recur-rence and potential for benefit from adjuvant chemotherapy.Molecular signatures derived from composites of as few asthree to as many as 64 genes have been used to stratifypatients into high-risk and low-risk groups that differ insurvival.35–37 One study of 101 patients that developed afive-gene signature predictor reported that patients with ahigh-risk gene signature had a shorter OS than patients witha low-risk gene signature (20 months versus 40 months; p �0.001).36 Models predicting risk of tumor recurrence havealso been developed from diverse data sets, including agenome-wide expression profile, a microRNA expressionprofile, and an integrated profile of clinical factors and geneexpression.38–40 The lung metagene model based on geneexpression profiles was able to identify a subgroup of stageIA patients at a high risk for recurrence.38 Validation usingclinical sample sets from two trials, the American College ofSurgical Oncologists Group Z0030 study (n � 25) and theCancer and Leukemia Group B (CALGB) 9761 study (n �84), also showed that the lung metagene model was a signif-icantly more accurate predictor of tumor recurrence thandisease stage, tumor diameter, nodal status, age, sex, histo-

FIGURE 3. The phase II MADeIT trial selected chemother-apy regimens on the basis of tumor expression levels ofERCC1 and RRM1 in patients with advanced NSCLC. ERCC1,excision repair cross-complementing group 1; MADeIT, Mo-lecular Analyses–Directed Individualized Therapy for Ad-vanced NSCLC; NSCLC, non-small cell lung cancer; RRM1,ribonucleotide reductase subunit 1; RT-PCR, reverse-tran-scriptase polymerase chain reaction.



logic classification, or smoking history (p � 0.001; Figure 5).By applying the lung metagene model to 68 patients withstage IA disease, a subgroup of patients was identified whohad a high risk for recurrence and may benefit from adjuvantchemotherapy. On the basis of these results, the CALGB30506 phase III trial has been activated to evaluate whetherthe lung metagene score can direct adjuvant chemotherapyselection for patients with stage I NSCLC (expected enroll-ment � 1525 patients).

Ultimately, risk models from gene signatures may beused to drive the selection of adjuvant chemotherapy. Usingrisk assessment models, patients with resected tumors andlow-risk gene signatures could be spared additional cytotoxicchemotherapy, whereas patients with resected tumors andhigh-risk gene signatures would be given adjuvant chemo-therapy.36 A recently reported study identified a 15-geneexpression profile that could classify patients with resectedstage IB or II NSCLC from the JBR.10 trial of vinorelbine/cisplatin versus observation into groups with a high or lowrisk for death.41,42 Adjuvant chemotherapy significantly re-duced the risk of death for 67 high-risk patients (HR � 0.33;p � 0.0005), but this benefit was not observed among the 66low-risk patients (HR � 3.67; p � 0.0133).41 Furthermore,

the interaction between chemotherapy and gene expressionsignature was highly significant (p � 0.0001). Genomicsignatures are also being investigated for their ability topredict sensitivity to individual and multidrug chemothera-peutic regimens.16

To use gene expression-based classifiers in the clinicalsetting, prospective trials will be needed to develop andconfirm the utility of predictive models for guiding treatmentselection. Gene expression profiling as a single mechanismfor model development may be inadequate, because theaddition of clinical covariates may improve predictive accu-racy.40,43 It will also be necessary to determine the feasibilityof coordinating clinical and pathologic data collection acrossmultiple institutions. Despite significant challenges, the po-tential for improving patient care and increasing survivalprovides strong motivation for the continued development ofgenomic tools for the treatment of patients with NSCLC.

MOLECULAR PREDICTORS OF RESPONSE TOTARGETED THERAPY

Monoclonal antibodies and small-molecule tyrosine ki-nase inhibitors (TKIs) targeting EGFR and the vascular

FIGURE 4. Kaplan-Meier OS curves for patients with nonsquamous and squamous histologies. In patients with nonsqua-mous histologies, cisplatin/pemetrexed significantly improved OS compared with cisplatin/gemcitabine (11.8 months versus10.4 months; p � 0.005), whereas patients with squamous cell carcinoma derived significantly improved OS with cisplatin/gemcitabine compared with cisplatin/pemetrexed (10.8 months versus 9.4 months; p � 0.05). CG, cisplatin/gemcitabine; CP,cisplatin/pemetrexed; HR, hazard ratio; OS, overall survival; SCC, squamous cell carcinoma. Reproduced with permission fromJ Clin Oncol 2008;26:3543–3551.

FIGURE 5. Kaplan-Meier survivalcurve estimates for the Duke Train-ing Cohort from the lung met-agene model and a model basedon combined clinical variables. Thelung metagene model more clearlystratifies high-risk and low-risk pa-tients with respect to survival whencompared with the clinical model.Reproduced with permission fromN Engl J Med. 2006;355:570–580.



endothelial growth factor have recently emerged as effectiveagents for the treatment of patients with advanced NSCLC.44

A number of clinical characteristics have been associatedwith response to EGFR TKIs, including female gender,45

Asian ethnicity,46,47 never-smoker history,47–49 and patientswith adenocarcinoma, bronchioloalveolar carcinoma (BAC),or papillary histologies.50–53 However, it is important to notethat male smokers and patients with squamous cell carcinomamay still derive benefit from EGFR TKIs despite low RRs.54

Clinical characteristics alone may be insufficient for selectingpatients to receive targeted therapy, and because of theheterogeneity of lung cancer biology, individual tumors maybe diverse at the molecular level within clinically selectedpatient populations. Therefore, tumor molecular characteris-tics are also being investigated as potential predictors ofsensitivity or resistance to targeted agents.

Predictors of Sensitivity to EGFR InhibitorsEGFR protein overexpression, increased gene copy

number, and gene mutations have all been examined aspotential biomarkers of response to EGFR inhibitors.

EGFR MutationsMutations in EGFR have been correlated with RRs

more than 70% in multiple prospective studies of patientswith EGFR mutations who received treatment with EGFRTKIs, and increasing clinical evidence suggests that patientswith EGFR mutations may receive more benefit from EGFRTKI therapies than patients with wild-type EGFR (Table1).2,47,53,55–60 For example, in the ongoing, phase III trial,Iressa Pan-Asian Study, gefitinib and carboplatin/paclitaxelchemotherapy are being compared in never or light smokerswith adenocarcinoma, and recent reports indicate that the PFSin the intent-to-treat population was significantly increasedamong patients who received gefitinib compared with carbo-platin/paclitaxel (HR � 0.74, p � 0.0001).47 When patientoutcomes were stratified by the presence or absence of EGFRmutation, patients with EGFR mutations had significantlylonger PFS if they received gefitinib compared with chemo-therapy (HR � 0.48, p � 0.0001), whereas patients without

EGFR mutations had significantly longer PFS if they receivedchemotherapy compared with gefitinib (gefitinib HR � 2.85,p � 0.0001). In addition, the preliminary HR for OS amongpatients with EGFR mutations who received gefitinib was0.78 compared with an HR of 1.38 for patients without EGFRmutations who received gefitinib, which indicates that ge-fitinib may be detrimental to patients without EGFR muta-tions, even in a clinically defined subset of never or lightsmokers of Asian ethnicity who would likely be selected forearly EGFR-TKI therapy in current oncology practice.

Different results were reported by the Iressa NSCLCTrial Evaluating Response and Survival Against Taxotere(INTEREST) trial, which compared the efficacy of gefitiniband docetaxel in previously treated patients with NSCLC andshowed a lack of association between treatment with gefitiniband OS when patients were stratified by EGFR mutations,EGFR gene amplification, or EGFR protein expression.61,62 Itis unclear whether the second-line setting of the INTERESTtrial contributed to the lack of association between the treat-ment with gefitinib and EGFR status because tissue samplesavailable for molecular analysis were most commonly ob-tained before first-line chemotherapy. It is possible that dif-ferential responses to EGFR inhibitors result from the diver-sity of somatic EGFR mutations in patients with NSCLC. Aretrospective study that examined 3303 EGFR mutationsfrom 12,244 patients suggests that RRs to EGFR TKIs maybe specific to mutation type, EGFR exon, and presence ofdual mutations.63

EGFR Protein OverexpressionCompared with EGFR mutation status, EGFR protein

overexpression and increased EGFR gene copy number areless consistent predictors of clinical benefit from EGFR-targeted therapy, and ongoing clinical trials are attempting toclarify the role of EGFR FISH and IHC testing in patientselection. The First-Line Erbitux in Lung Cancer (FLEX)trial (n � 1125) investigated the efficacy of cisplatin andvinorelbine with or without cetuximab in previously un-treated patients with EGFR-positive (by IHC), advanced,NSCLC, and showed significantly increased OS among pa-tients who received cisplatin/vinorelbine and cetuximab com-pared with cisplatin/vinorelbine alone; these results suggestthe potential benefit of cetuximab for preselected patients(11.3 months versus 10.1 months; p � 0.044).64 In the FLEXtrial, EGFR-positive NSCLC was defined as having 1 or morepositively stained tumor cells by IHC. In contrast, the BMS-099 trial (n � 676) compared the efficacy of carboplatin anda taxane with or without cetuximab in previously untreatedpatients with advanced NSCLC and showed that in thisotherwise unselected population, the addition of cetuximabdid not significantly improve OS (9.7 months versus 8.4months; p � 0.17) or PFS (4.4 months versus 4.2 months;p � 0.24).65 On the basis of the results from FLEX andBMS-099, cetuximab in combination with chemotherapy isan increasingly considered option for the frontline treatmentof select patients with NSCLC, and this recommendation wasrecently added to the National Comprehensive Cancer Net-work Clinical Practice Guidelines in Oncology.44

TABLE 1. Prospective Studies of Patients with EGFRMutations Treated with EGFR TKIs

TrialNo. of

PtsEGFR

Mutations Agent RR (%) TTP (mo)

Paz-Ares et al.55 1047 127 Erlotinib 82 13.3

Miller et al.53,a 81 18 Erlotinib 83 13

Inoue et al.2 99 16 Gefitinib 75 9.7

Sutani et al.56 100 38 Gefitinib 78 9.4

Asahina et al.57 82 16 Gefitinib 75 8.9

ONCOBELL58 37 24 Gefitinib 71 3.8

Sunaga et al.59 33 21 Gefitinib 76 12.9

IPASS47 1217 132 Gefitinib 71 NR

Tamura et al.60 118 32 Gefitinib 75 11.5

a Patients with bronchioloalveolar carcinoma.EGFR, epidermal growth factor receptor; IPASS, Iressa Pan-Asian Study; NR, not

reported; RR, response rate; TKI, tyrosine kinase inhibitor; TTP, time to progression.



EGFR Gene Copy NumberHigh EGFR gene copy number has also been associated

with benefit from treatment with EGFR inhibitors (Table2),53,58,66–71 but the predictive significance of EGFR FISHtesting has yet to be confirmed in a prospective phase III trial.A subset analysis (n � 159) of the BR.21 trial investigatingthe efficacy of erlotinib versus placebo in patients withpreviously treated NSCLC reported that significant survivalbenefit from erlotinib was correlated with high but not lowEGFR copy number (p � 0.004 versus p � 0.353).67 Simi-larly, the phase II SWOG 0342 trial of sequential or concur-rent paclitaxel and carboplatin with cetuximab in patientswith advanced NSCLC showed significantly increased OSamong patients with high but not low EGFR gene copynumber (15 months versus 7 months; p � 0.046).71 Theprospective ONCOBELL phase II trial (n � 36) evaluatedgefitinib sensitivity in patients with advanced NSCLC whohad never smoked, had increased EGFR gene copy number,or activated Akt.58 Compared with patients with low EGFRgene copy number, patients with high EGFR gene copynumber had significantly increased time to progression (7.6months versus 2.7 months; p � 0.02) and a trend towardlonger OS that did not reach statistical significance (notreached versus 7.4 months; p � 0.3). EGFR gene copynumber has also been analyzed in patients (n � 101) withBAC or adenocarcinoma with BAC subtype in a phase II trialof erlotinib.53 Patients with high EGFR gene copy numberhad a significantly increased PFS compared with those withlow EGFR gene copy number (9 months versus 2 months;p � 0.01); however, the increase in OS among patients withhigh EGFR gene copy number did not reach statistical sig-nificance (25 months versus 16 months; p � 0.38). Contrast-ing results were reported from the INVITE trial, whichcompared the efficacy of gefitinib with that of vinorelbine inelderly patients (�70 years of age; n � 196) with advancedNSCLC.72 In this trial, patients with high EGFR copy numberwho received treatment with gefitinib had a nonsignificanttrend toward poorer PFS and OS compared with patients whohad low EGFR gene copy number and received treatmentwith gefitinib (HR for PFS � 1.31; HR for OS � 1.61).

Conversely, patients with high EGFR gene copy number whoreceived vinorelbine had a nonsignificant trend toward im-proved PFS and OS compared with patients who had lowEGFR gene copy number (HR for PFS � 0.77; HR for OS �0.52). Further investigation will be required to determine thepredictive value of EGFR copy number in selecting targetedtherapy for patients with NSCLC. New, adaptive, trial de-signs, such as the one used in the Biomarker-based Ap-proaches of Targeted Therapy for Lung Cancer Elimination(BATTLE) trial, may facilitate and accelerate the validationof molecular biomarkers for clinical practice (Figure 6).73 Inthe BATTLE trial, patients’ biomarker profiles are used toselect treatment, and patients are randomized to receive oneof the four treatments: sorafenib, erlotinib, vandetanib, orerlotinib/bexarotene. Using an adaptive randomization trialdesign, patients are more likely to be randomized to aneffective, targeted-therapy arm.

Predictors of Resistance to EGFR InhibitorsGene mutations have also emerged as potential predic-

tors of resistance to EGFR inhibitors for patients withNSCLC. In particular, mutations in the KRAS gene werereported to confer resistance to gefitinib or erlotinib ther-apy.74 Clinical trials demonstrate that patients with mutantKRAS are unlikely to demonstrate a significant response toEGFR inhibitors, when compared with patients with wild-type KRAS; however, it is unclear whether poor response totherapy correlates with shorter survival (Table 3).53,61,67,75–78

FIGURE 6. Trial schema for the BATTLE trial. BATTLE, Bi-omarker-Based Approaches of Targeted Therapy for LungCancer Elimination.

TABLE 2. Effect of EGFR Gene Copy Number on Response to EGFR Inhibitors

Trial No. of Pts Agent

RR (%) OS (mo)

FISH� FISH� FISH� FISH�

TRIBUTE66 245 Erlotinib 12 22 12 10

Miller et al.53,a 76 Erlotinib 43 13 25 16

BR.2167 91 Erlotinib 21 5 11 6

Bell et al.68 40 Gefitinib 29 15 12 9

Cappuzzo et al.69 102 Gefitinib 36 3 19 7

ONCOBELL58,b 36 Gefitinib 68 9 NR 7

iTARGET70,b 29 Gefitinib 50 43 NR

SWOG 034271,b 76 Cetuximab 45 26 15 7

a Chromogenic in situ hybridization analysis in bronchioloalveolar carcinoma.b Prospective analysis.EGFR, epidermal growth factor receptor; FISH, fluorescence in situ hybridization; NR, not reported; OS, overall survival; RR, response

rate; SWOG, Southwest Oncology Group.



In a retrospective study, patients with advanced NSCLC (n �73) and KRAS mutation who were treated with gefitinib orerlotinib experienced a significantly shorter time to progres-sion (1.7 months versus 2.9 months; p � 0.0025), but not asignificantly shorter OS (5.0 months versus 9.4 months; p �0.62), when compared with patients with wild-type KRAS.75

In a prospective trial of patients (n � 101) with BAC oradenocarcinoma with BAC subtype who were treated witherlotinib, no patient with a KRAS mutation met ResponseEvaluation Criteria in Solid Tumors, when compared withpatients with wild-type KRAS who had an RR of 32% (p �0.01).53 A plot of best response from indicator lesions ac-cording to Response Evaluation Criteria in Solid Tumorsshows that some patients with KRAS mutations achieved aminor response or stable disease (Figure 7), which indicatesthat some patients with KRAS mutations may receive amodest survival benefit from erlotinib. Despite the lack ofresponse to erlotinib among patients with KRAS mutations,the shorter PFS (4 months versus 5 months; p � 0.25) and OS(13 months versus 21 months; p � 0.3) among patients withKRAS mutations did not reach statistical significance, whencompared with patients without KRAS mutations.

A retrospective study of patients (n � 206) from theBR.21 trial (which investigated the efficacy of erlotinibversus placebo) reported that KRAS mutation status did notsignificantly impact survival for patients treated with erlotinib(p � 0.09).67 Similarly, a subgroup analysis of patients (n �114) in the INTEREST trial, which evaluated the efficacy ofgefitinib versus docetaxel, reported that the p value for OS for

interaction between KRAS mutation status and treatment wasnot significant (p � 0.51).61 In contrast, a retrospectivereview of five clinical trials performed in Europe and theUnited States involving patients with NSCLC, who weretreated with erlotinib or gefitinib, suggests that KRAS muta-tions are associated with resistance to EGFR-TKI therapy, atleast when defined by response.76 Additional prospectivetrials are needed to clarify the role of KRAS mutations inpredicting benefit from EGFR-inhibitor therapy for patientswith NSCLC.

SUMMARY AND CONCLUSIONMolecular markers such as ERCC1, RRM1, and TS, as

well as gene expression profiling may predict response tochemotherapy. Similarly, EGFR protein overexpression, in-creased EGFR gene copy number, and mutations in EGFRand KRAS may predict response to EGFR inhibitors. Theidentification of molecular biomarkers that predict responseto chemotherapy and targeted therapy has the potential totransform the treatment paradigm of NSCLC from relying onclinical characteristics such as tumor stage, performancestatus, and smoking history to utilizing analyses of molecularcharacteristics of individual patients for therapeutic decisionmaking. Additional prospective trials and new clinical trialdesigns are urgently needed to further define and validate thepotential role of these and other novel biomarkers in thetreatment of NSCLC.

FIGURE 7. Plot of best responseof indicator lesions according toRECIST criteria in patients with BACand patients with adenocarcinomawith BAC subtype who receivederlotinib. BAC, bronchioloalveolarcarcinoma; CISH, chromogenic insitu hybridization; EGFR, epidermalgrowth factor receptor; RECIST,Response Evaluation Criteria inSolid Tumors. Reproduced withpermission from J Clin Oncol 2008;26:1472–1478.

TABLE 3. KRAS Mutations and Resistance to EGFR Inhibitors

Trial No. of Pts Agent

RR (%) OS (mo)

Wild-Type KRAS Mutant KRAS Wild-Type KRAS Mutant KRAS

Miller et al.53,a 101 Erlotinib 32 0 21 13

BR.21.67 206 Erlotinib 10.2 5 7.5 3.7

Massarelli et al.75 70 Erlotinib or Gefitinib 10 0 9.4 5

Jackman et al.76 116 Erlotinib or Gefitinib 5 0 11.8 13

INTEREST61 275 Gefitinib 10 0 7.5 7.8

BMS-09977 202 Cetuximab 33 31 10 17

a Patients with bronchioloalveolar carcinoma.INTEREST, Iressa NSCLC Trial Evaluating Response and Survival Against Taxotere; EGFR, epidermal growth factor receptor; OS, overall survival; RR, response rate.



DISCUSSION

Howard West, MDI think that we are moving away from treating NSCLC

as a static entity that has been managed the same way for 10years, to an era where we can personalize treatments and dobetter. New methods are being developed to more accuratelydetect molecular biomarkers. What types of developments orchanges are happening in the field of biomarker detectiontechnology?

Lecia Sequist, MD, MPHIn general, I think that more modern techniques are

moving away from looking at the narrow focus of a singlegene abnormality to a more global type of analysis of whatthe functional implication of a cell’s full genetic makeup is.I think that is a good trend. From a clinician’s standpoint, Ithink the most clinically useful molecular markers will bethose that are clearly paired with some sort of trigger of whatto do—a treatment recommendation. So if you see thismarker, do this, and if you don’t see this marker, do that. Ithink those kinds of markers will move forward into practicethe quickest. The significant obstacle to all of these tech-niques is the availability of tumor tissue, especially in lungcancer, where invasive biopsies can be risky for patients.

Howard West, MDCan you update us on some of the ongoing trials

looking at biomarkers?

Roy S. Herbst, MD, PhDWe have the BATTLE program; the idea behind it is

that you can only do so much with clinical characteristics.We’re getting biopsies on all of our patients, and we do anadaptive randomization, where patients are more likely to berandomized to the arm which has shown benefit for priorpatients with that particular marker profile and a given drug.There are rules to shut down or suspend groups early if we’renot seeing any activity. Basically, here’s the way we do it: anumbrella protocol, equal and then adaptive randomization,and treatment with four drugs. I can tell you we’re nowdeveloping our BATTLE 2 and 3 programs because we’vebeen very happy with this framework. Even if we’re com-pletely wrong about these agents and our 11-marker profile,we’re generating a great deal of information for explorationand discovery in the future.

Antoinette Wozniak, MDThere is actually a trial that is about to start in SWOG.

SWOG 0720 is a feasibility study in patients with stage INSCLC and resected tumors that were larger than 2 cm. Theyare going to use AQUA� technology [HistoRx; New Haven,CT] to look at ERCC1 and RRM1 protein expression. Ifpatients have high expression of both ERCC1 and RRM1,they get observed and will probably not get adjuvant chemo-therapy. If patients have low expression of both ERCC1 andRRM1, they are going to get adjuvant chemotherapy withcisplatin/gemcitabine.

David Harpole, MDWe’re excited about the activation of CALGB 30506.

We’re expecting to randomize about 1500 patients. Our goalis to demonstrate a survival advantage for patients receivingadjuvant chemotherapy compared with observation and tovalidate whether the lung metagene model is prognostic forsurvival in stage I patients. It’s going to be especially inter-esting to see whether there is a highly significant survivalbenefit for patients treated with adjuvant chemotherapy whohave high-risk lung metagene scores. At the end, there’sgoing to be 600 patients for whom we’re going to havecomplete array data as well as chemotherapy data. So I thinkthe opportunity is in this data set to learn a whole lot aboutthese things in the future, which is going to be great forinvestigators.

ACKNOWLEDGMENTSThe CBCE gratefully acknowledges the educational

grants provided by Genentech BioOncology/OSI Pharmaceu-ticals, Inc.; Lilly USA, LLC; Pfizer Oncology; and Sanofi-Aventis.

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