Emergence of Multiple EGFR Extracellular Mutations during ... · Biology of Human Tumors Emergence...

Biology of Human Tumors

Emergence of Multiple EGFR ExtracellularMutations during Cetuximab Treatment inColorectal CancerSabrina Arena1,2,3, Beatriz Bellosillo4,5, Giulia Siravegna1,2, Alejandro Martínez4,6,Israel Ca~nadas4, Luca Lazzari1,2, Noelia Ferruz7, Mariangela Russo1,2, Sandra Misale1,Iria Gonz�alez6, Mar Iglesias5, Elena Gavilan4, Giorgio Corti1, Sebastijan Hobor1,Giovanni Crisafulli1, Marta Salido5, Juan S�anchez8, Alba Dalmases4,5, Joaquim Bellmunt4,Gianni De Fabritiis7,9, Ana Rovira4, Federica Di Nicolantonio1,2, Joan Albanell4,6,10,Alberto Bardelli1,2, and Clara Montagut4,6

Abstract

Purpose: Patients with colorectal cancer who respond to theanti-EGFR antibody cetuximab often develop resistance withinseveral months of initiating therapy. To design new lines oftreatment, the molecular landscape of resistant tumors must beascertained. We investigated the role of mutations in the EGFRsignaling axis on the acquisition of resistance to cetuximab inpatients and cellular models.

Experimental Design: Tissue samples were obtained from 37patients with colorectal cancer who became refractory to cetux-imab. Colorectal cancer cells sensitive to cetuximab were treateduntil resistant derivatives emerged. Mutational profiling of biop-sies and cell lines was performed. Structural modeling and func-tional analyses were performed to causally associate the alleles toresistance.

Results: The genetic profile of tumor specimens obtained aftercetuximab treatment revealed the emergence of a complex pattern

of mutations in EGFR, KRAS, NRAS, BRAF, and PIK3CA genes,including two novel EGFR ectodomain mutations (R451C andK467T). Mutational profiling of cetuximab-resistant cells recapit-ulated the molecular landscape observed in clinical samples andrevealed three additional EGFR alleles: S464L, G465R, andI491M. Structurally, these mutations are located in the cetuxi-mab-binding region, except for the R451C mutant. Functionally,EGFR ectodomain mutations prevent binding to cetuximab but asubset is permissive for interaction with panitumumab.

Conclusions: Colorectal tumors evade EGFR blockade byconstitutive activation of downstream signaling effectors andthrough mutations affecting receptor–antibody binding. Bothmechanisms of resistance may occur concomitantly. Our datahave implications for designing additional lines of therapy forpatients with colorectal cancer who relapse upon treatment withanti-EGFR antibodies. Clin Cancer Res; 21(9); 2157–66. �2015 AACR.

IntroductionMonoclonal antibodies (mAb) directed against the EGFR,

cetuximab, and panitumumab, provide significant survivalbenefit to patients with RAS wild-type metastatic colorectalcancer (mCRC) and are now standard components of treat-ment regimens for these patients, either alone or in combina-tion with chemotherapy. However, the duration of thisresponse is only transient and does not last more than 3 to12 months, after which secondary resistance occurs (1–5).Definition of the molecular changes underlying acquired resis-tance to anti-EGFR antibodies is needed to improve clinicalbenefit and devise further lines of treatment. Molecular pro-filing of patient samples and preclinical models has previouslydefined a number of mechanisms of secondary resistance. Themost common event is the emergence of KRAS and NRASmutations which occurs in approximately 50% to 80% of thecases (6–10). Other escape routes include acquisition of theS492R mutation in the ectodomain of EGFR (10–13), as well asamplification of tyrosine kinase receptor genes HER2 or MET(14, 15). Interestingly, recent data indicate the coexistence ofseveral molecular mechanisms of secondary resistance withinan individual patient (6–10, 12, 16).

1Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Italy. 2Departmentof Oncology, University of Torino, Candiolo, Italy. 3FIRC Institute ofMolecular Oncology (IFOM), Milano, Italy. 4Cancer Research Program,IMIM (Institut Hospital del Mar Investigacions Mediques), Hospital delMar, Barcelona, Spain. 5Department of Pathology, Hospital del Mar,Barcelona, Spain. 6Department of Medical Oncology, Hospital del Mar,Barcelona, Spain. 7Computational Biophysics Laboratory (GRIB-IMIM), Pompeu Fabra University, PRBB, Barcelona, Spain. 8Depart-ment of Radiology, Hospital del Mar, Barcelona, Spain. 9Instituci�oCatalana de Recerca i Estudis Avancats, Barcelona, Spain. 10PompeuFabra University, Barcelona, Spain.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Current address for S. Hobor: Cancer Research UK London Research Institute,London.

S. Arena and B. Bellosillo share first authorship.

A. Bardelli and C. Montagut share senior authorship.

Corresponding Authors: Clara Montagut, Department of Medical Oncology,Hospital del Mar, Passeig Maritim 25-29, Barcelona 08003, Spain. Phone:349-3248-3137; Fax: 349-3248-3366; E-mail: [email protected];and Alberto Bardelli, Department of Oncology, University of Torino, Str prov 142Km 3.95, Candiolo 10060, Torino, Italy. Phone: 3901-1993-3235; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-14-2821

�2015 American Association for Cancer Research.

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To identify yet unknownmolecular events leading to anti-EGFRtreatment failure, we investigated the role of mutations in theEGFR signaling pathway on the acquisition of resistance tocetuximab in patients with colorectal cancer and cellular models.Our findings suggest that we have yet to fully characterize thecomplete spectrum of genetic variants through which colorectaltumors evade EGFR blockade. The data presented here will helpdesign additional therapeutic strategies to circumvent resistanceof patients with colorectal cancer who receive EGFR-targetedantibodies.

Materials and MethodsTumor samples and patients

All mCRC consenting patients treated with anti-EGFR mAb atHospital del Mar (Barcelona, Spain) between January 2010 andJune 2013 were included in this study. In the analysis, we onlyincluded patients who had good quality paired pre- and post-treatment biopsies and who acquired resistance to anti-EGFR–based therapy defined as progression disease following (i) com-plete response or partial response or (ii) stable disease for morethan 16weeks. Responsewas evaluated according to the ResponseEvaluation Criteria in Solid Tumors (RECIST; ref. 17). Our studyincluded re-biopsy following treatment failure in patients whoconsented to this extra procedure. Re-biopsies at the time ofprogression were obtained from the most accessible lesion withless potential risk of related complications for the patient accord-ing to ethical considerations. Pretreatment sample was obtainedfrom the regular diagnosis procedure. In this study, we included 9cases (patients #21–#28 and patient #36) that had been previ-ously assessed for EGFR S492R, KRAS exon 2, BRAF V600E, andPIK3CA mutations by Sanger sequencing (12) and that in thecurrent work were analyzed for a broader panel of mutations asspecified below using more sensitive sequencing technologies.Biologic samples were obtained from Parc de Salut Mar Biobank(MARBiobanc). This studywas approved by the local Ethics Board(CEIC-2012/4741/I). All participating patients signed writteninformed consent.

Mutational analysis in tissue samplesDNA extraction from tumoral samples was performed as

previously described (12). Mutational analysis of KRAS (exons

2, 3, and 4), BRAF (exon 15), NRAS (exons 2 and 3), PIK3CA(exons 9 and 20), and EGFR (exon 12) was performed bySanger sequencing using BigDye v3.1 (Applied Biosystems)following manufacturer's instructions and analyzed on a3500Dx Genetic Analyzer (Applied Biosystems). KRAS (exons3 and 4) and NRAS (exons 2, 3, and 4) were also analyzed bypyrosequencing (Qiagen) and KRAS (exon 2) was also assessedby Therascreen real-time PCR (Qiagen) following the manu-facturer's instructions. All cases were also screened by pyrose-quencing using a next generation sequencing (NGS) 454 GSJunior platform (Roche Applied Science). Processed and qual-ity-filtered reads were analyzed using the GS Amplicon VariantAnalyzer software version 2.5p1 (Roche). Mutations detectedby NGS were confirmed by competitive allele-specific TaqManPCR (CAST-PCR, Applied Biosystems) when specific assayswere available.

Cell culture and generation of resistant cellsDiFi cells were cultured in F12 medium (Invitrogen) supple-

mented with 5% FBS; OXCO-2 cells were cultured in Iscovemedium (Invitrogen) supplemented with 5% FBS; LIM1215cells were cultured in RPMI1640 medium (Invitrogen) supple-mented with 5% FBS and insulin (1 mg/mL); NCIH508 cellswere cultured in RPMI1640 medium (Invitrogen) supplemen-ted with 5% FBS; HCA-46 cells were cultured in DMEM (Invi-trogen) supplemented with 5% FBS and CCK81 cells werecultured in MEM (Invitrogen) supplemented with 5% FBS. Allmedia also contained 2 mmol/L L-glutamine and antibiotics(100 U/mL penicillin and 100 mg/mL streptomycin), and cellswere grown in a 37�C and 5% CO2 air incubator. The DiFi andOXCO-2 cell lines were a kind gift from Dr. J. Baselga inNovember 2004 (Oncology Department of Vall d'HebronUniversity Hospital, Barcelona, Spain) and Dr V. Cerundoloin March 2010 (Weatherall Institute of Molecular Medicine,University of Oxford, Oxford, United Kingdom), respectively.The LIM1215 parental cell line has been described previously(18) and was obtained from Prof. Robert Whitehead (Vander-bilt University, Nashville, TN) with permission from the Lud-wig Institute for Cancer Research (Zurich, Switzerland). TheNCIH508 cell line was purchased from ATCC (LGC StandardsS.r.l). HCA-46 cell lines were obtained from European Collec-tion of Animal Cell Cultures (distributed by Sigma-Aldrich Srl).CCK81 cell line was obtained from Health Science ResearchResources Bank. The identity of each cell line was tested andauthenticated by Cell ID System and by Gene Print 10 System(Promega), through short tandem repeats (STR) at 10 differentloci (D5S818, D13S317, D7S820, D16S539, D21S11, vWA,TH01, TPOX, CSF1PO, and amelogenin). Amplicons frommultiplex PCRs were separated by capillary electrophoresis(3730 DNA Analyzer, Applied Biosystems) and analyzed usingGeneMapperID software from Life Technologies. Resulting cellline STR profiles were cross-compared and matched with theavailable STR from ATCC, ECACC, and CellBank Australiarepositories online databases. All cell lines were tested andresulted negative for mycoplasma contamination with VenorGeM Classic Kit (Minerva Biolabs).

Generation of resistant cells utilized in this article has alreadybeen previously described (6, 9). CCK81 cetuximab-resistantderivatives were obtained by increasing the cetuximab dosagestepwise from 680 nmol/L to 1.4 mmol/L during the course of 6months.

Translational Relevance

The anti-EGFR monoclonal antibody cetuximab is effectivefor the treatment of patients with "RAS" wild-type metastaticcolorectal cancer (mCRC). Unfortunately, responses are tran-sient, and most patients develop acquired resistance. In thecurrent study, we analyzed the genetic profile of clinical speci-mens and preclinical models of acquired resistance to cetux-imab, revealing a complex pattern of mutations in both EGFRand its downstream effectors, KRAS, NRAS, BRAF, andPIK3CA. We discovered five novel point mutations in theectodomain of EGFR, which confer resistance to cetuximab.Of note, only a subset of these EGFR variants remains sensitiveto panitumumab. Accordingly, the identification of patient-specific mechanisms of resistance to EGFR blockade will beparamount to design additional lines of therapy.

Arena et al.

Clin Cancer Res; 21(9) May 1, 2015 Clinical Cancer Research2158




Mutational analysis in cell linesGenomic DNA samples were extracted by Wizard SV Genomic

DNA Purification System (Promega). For Sanger sequencing, allsamples were subjected to automated sequencing by ABI PRISM3730 (Applied Biosystems). Primer sequences are listed elsewhere(6, 9). The following genes and exonswere analyzed:KRAS (exons2, 3, and 4), NRAS (exons 2 and 3), PIK3CA (exons 9 and 20),BRAF (exon 15), EGFR (exon 12). All mutations were confirmedtwice, starting from independent PCR reactions.

Drug assaysCetuximab was obtained from the Pharmacy at Niguarda Ca'

Granda Hospital, Milan, Italy. Cell lines were seeded in 100 mLmedium at the following densities (2� 103 for DiFi, 1.5� 103 forLIM1215, HCA-46, NCIH508, and OXCO-2, 3� 103 for CCK81)in 96-well culture plates. After serial dilutions, cetuximab inserum-free medium was added to cells, and medium-only wellswere included as controls. Plates were incubated at 37�C in 5%CO2 for 6 days, after which cell viability was assessed by ATPcontent using the CellTiter-Glo Luminescent Assay (Promega).

DNA constructs and mutagenesisThe pLX301-EGFR WT construct was a generous gift from Dr.

C. Sun and Prof R. Bernards (NKI, Amsterdam, the Netherlands).EGFRmutants containing the 6 point mutations (R451C, S464L,G465R, K467T, I491M, and S492R) were constructed using theQuikChange II site-directed mutagenesis kits from Agilent Tech-nologies with pLX301-EGFR WT plasmid as the template DNA.The presence of mutations was confirmed by DNA sequencing.

Droplet digital PCRIsolated gDNA was amplified using ddPCR Supermix for

Probes (Bio-Rad) using KRAS, NRAS, BRAF, and EGFR assay(PrimePCR ddPCR Mutation Assay, Bio-Rad and customdesigned).Droplet digital PCR (ddPCR)was performed accordingto manufacturer's protocol and the results reported as percentageor fractional abundance of mutant DNA alleles to total (mutantplus wild-type) DNA alleles. Eight to 10 mL of DNA template wasadded to 10 mL of ddPCR Supermix for Probes (Bio-Rad) and 2 mLof the primer/probe mixture. This 20 mL sample was added to70mLofDropletGenerationOil for Probes (Bio-Rad) andused fordroplet generation. Droplets were then thermal cycled with thefollowing conditions: 5 minutes at 95�C, 40 cycles of 94�C for 30seconds, 55�C for 1 minute followed by 98�C for 10 minutes(ramp rate 2�C/sec). Samples were then transferred to a QX200Droplet Reader (Bio-Rad) for fluorescent measurement of FAMand HEX probes. Gating was performed on the basis of positiveand negative controls, and mutant populations were identified.Fractional abundances of the mutant DNA in the wild-type DNAbackground were calculated for each sample using QuantaSoftsoftware (Bio-Rad). Multiple replicates (minimum of four) wereperformed for each sample. ddPCR analysis of normal controlgDNA from cell lines and noDNA template (water) controls wereperformed in parallel with all the samples, including againmultiple replicates as a contamination-free control.

EGFRprobes andprimers sequences are available upon request.

Molecular simulationsInput coordinates for the structure of cetuximabbound towild-

type sEGFR (extracellular part) were taken from PDB:1YY9 (19).The systemwas parameterized using the Amber12 force field (20)

and solvated and neutralized in a TIP3P water box. Energyminimization was conducted under NPT conditions at 1 atm,298K, and a cutoff of 9Å,with rigid bonds andPME for long-rangeelectrostatics. Potential energy minimization was run for 2 ps.During minimization, the heavy protein atoms were notrestrained. The simulation was run using ACEMD (21) on a localGraphics Processing Unit (GPU)-equipped workstation.

Flow cytometryTo measure cetuximab and panitumumab binding to cells

expressing mutant EGFR, we harvested by trypsinization andwashed the cells twice with PBS. We incubated the cells with Fcblocking solution for 15 minutes on ice to block nonspecific Fcbinding of immunoglobulins. We then washed the cells andincubated them with the monoclonal antibodies for EGFR bind-ing during 30minutes on ice. To visualize the primary antibody agoat anti-human IgGg phycoerythrin-conjugated (Invitrogen)was used as a secondary antibody. EGFR binding was analyzedusing the FACScan flow Cytometer.

Protein detectionWe subjected total cell lysates to Western blot analysis as

previously reported (12). The phospho-EGFR antibody(Y1068) was purchased from Cell Signaling Technology.

ResultsEmergence of EGFR ectodomain mutations in patients treatedwith cetuximab

Thirty-seven consecutive patients with mCRC who hadacquired resistance to cetuximab after an initial response to thetreatment and had good quality paired pre- and posttreatmentspecimens were included in this study. Tumor biopsy obtainedduring the regular diagnosis procedure was used as the pretreat-ment sample. In most cases, this sample was obtained from theprimary tumor during routine colonoscopy. Biopsies at progres-sion were taken from liver (21 samples), lung (6), bone (2),peritoneum (3), colon-rectum (3), retroperitoneal lymph node(1), and subcutaneous node (1) with ultrasound guidance, CTscan guidance, or colonoscopy in the case of colon-rectum lesions.There were no major biopsy-related complications. Clinical char-acteristics of the patients are showed in Supplementary Table S1.

All pretreatment biopsies were screened formutations inKRAS,NRAS, and BRAF by Sanger and pyrosequencing as part of routineclinical practice atHospital delMar (Barcelona, Spain).Mutationsin PIK3CA and EGFR were also assessed as part of this study. Allpretreatment biopsieswerewild-type except for three samples thatharbored mutations in PIK3CA. As PIK3CA mutations do notpreclude response to anti-EGFR therapy, these patients weretreated with cetuximab-based regimens. Posttreatment tissuesamples were analyzed for the same mutations using the samesequencing platforms. In total, we detected the emergence of 31mutations in posttreatment biopsies from 20 patients (Table 1).The additional 17 posttreatment samples did not reveal variantsin any of the genes we analyzed. In 7 cases, mutations in differentgeneswere detected in the same tissue sample (medianof detectedmutations within the same specimen 2, range 1–5; Table 1).

Acquired mutations were found in NRAS (9 events) and KRASgenes (8 events) followed bymutations in PIK3CA (6 events) andBRAF (3 events). Interestingly, mutations in RAS frequentlyoccurred in exons 3–4 (67% of NRAS and 50% of KRAS

EGFR Mutations and Resistance to Cetuximab

www.aacrjournals.org Clin Cancer Res; 21(9) May 1, 2015 2159




mutations). Of note, mutations in PIK3CA always coexisted withother mutations within the same sample.

Notably, in addition to "RAS" gene mutations the molecularanalysis revealed the emergence of multiple alleles in the EGFRextracellular domain. The previously reported EGFR S492R var-iant was detected in 3 patients. Of note, EGFR mutations wereaccompanied by gene amplification in both the pre- and theposttreatment specimen from two of these patients (data notshown); in the third patient, EGFR gene copy number was notassessed due to lack of sufficient tissue for FISH analysis.

Two novel mutations located in exon 12 of the EGFR gene werediscovered by the analysis of posttreatment samples. Patient #31harbored an A ! C substitution at codon 1400 that caused asubstitution of a lysine to threonine at amino acid 467 (p.K467T).The postcetuximab biopsy from patient #35 harbored a C ! Tsubstitution at codon 1351, resulting in an arginine to cysteinesubstitution at amino acid 451 (p.R451C; Table 1; SupplementaryFig. S1).

EGFR ectodomain mutations and acquired resistance tocetuximab in colorectal cancer cell models

We previously reported that acquisition of resistance in colo-rectal cancer cells is associated with emergence of KRAS-, BRAF-,andNRAS-activating mutations (6, 9, 12). To discover additionalmechanisms of resistance to EGFR blockade, we exploited 5colorectal cancer cell lines (DiFi, LIM1215, HCA-46, NCIH508,OXCO-2, and CCK81), which are highly sensitive to cetuximab(Supplementary Fig. S2). These cell lines are wild-type for KRAS,NRAS, BRAF, and PIK3CAwith the exception of NCIH508, whichdisplays the p.E545K PIK3CA mutation. Altogether, these cell

models recapitulate the molecular features of tumors frompatients with colorectal cancer likely to respond to anti EGFRtherapies. For each line, at least five million cells were exposedcontinuously to cetuximab until resistant populations emerged(Supplementary Fig. S2). To definemolecularmechanisms under-lying acquisition of resistance, we initially performed Sangersequencing of genes involved in regulation of the EGFR signalingpathway (EGFR, KRAS, BRAF,NRAS, and PIK3CA). In accordancewith our previous reports, resistant populations often displayedKRAS, BRAF, and NRAS mutations (Table 2; ref. 9). All of thesealleles were detected in the resistant cells but not in the corre-sponding parental population from which they originated.Importantly, in several occasionsmultiple genetic alterationswereconcomitantly present in the resistant cell population (Table 2)indicating their polyclonal status. To assess themolecular featuresof individual clones, we performed limited cell dilutions ofLIM1215 and CCK81 as these cell lines are amenable to thisprocedure. We then subjected single clones to Sanger sequencingfor candidate genes (EGFR, KRAS, BRAF, NRAS, and PIK3CA).Notably, mutation profiling of clones identified three novel EGFRvariants: S464L, G465R, and I491M (Supplementary Fig. S3).Considering that the resistant derivatives are polyclonal, and inlight of the limited sensitivity of the Sanger sequencing method,we postulated that variants present in less than 20% of the cellpopulations might have remained undetected. To identify muta-tions present at low frequency, we employed ddPCR, which isknown to have amutant/wild-type sensitivity of 1:20,000. ddPCRprobes were designed and individually validated using controlmutant DNA to detect EGFR variants previously identified intumor biopsy or cell lines (Supplementary Table S2). This analysis

Table 1. EGFR pathway mutations in tissue samples from patients with mCRC treated with cetuximab

Pre-treatment Post-treatment

Patie

nt#

KRA

S ex

on 2

KRA

S ex

on 3

KRA

S ex

on 4

NRA

S ex

on 2

NRA

S ex

on 3

NRA

S ex

on 4

BRA

F ex

on 15

PIK3

CA e

xon

9

PIK3

CA e

xon

20

EGFR

exo

n 12

KRA

S ex

on 2

KRA

S ex

on 3

KRA

S ex

on 4

NRA

S ex

on 2

NRA

S ex

on 3

NRA

S ex

on 4

BRA

F ex

on 1

5

PIK3

CA e

xon

9

PIK3

CA e

xon

20

EGFR exon 12

R45

1C

S464

L

G46

5R

K46

7T

I491

M

S492

R

458

1113151617182021232627313334353637

NOTE: KRAS, NRAS, BRAF, PIK3CA, and EGFR mutations were analyzed in paired tissue samples obtained at diagnosis (pre-treatment) and at progression(post-treatment). Black boxes indicate the presence of mutations detected by clinical routine sequencing procedures.

Arena et al.





unveiled the presence 3 new EGFR variants (S464L, G465R, andI491M) that were not detected by Sanger sequencing in resistantcell populations (Table 2 and Supplementary Table S3). TheddPCR approach could not be performed in tissue samples, asthere was no sufficient material available. Overall, themutationallandscape of cell lines with acquired resistance to cetuximabrecapitulates the molecular profiles of tumors that relapsed uponcetuximab treatment.

Structural model analysis of EGFR ectodomain mutationsTo understand how the EGFR ectodomain mutations detected

in tumor samples and cell lines could drive resistance to EGFRblockade, we performed computational structure-based analyses.With the exception of R451C, the EGFR mutations are located inthe receptor region, which has been shown to interact withcetuximab (Fig. 1A). More specifically, the S464L, G465R,K467T, I491M, and S492R mutations lie in the middle of thesurface recognized by the antibody (Fig. 1B) and therefore mod-ifications of this interface have the potential to affect complexformation. Of these five mutations, three appear to disruptfavorable interactions, namely S464L, K467T, and I491M. Thepolar amino acid S464 is within H-bond distance from thecarbonyl backbone of Y102 and the phenolic OH-group ofY104 (Fig. 1C). Replacement by a hydrophobic bulky amino acidas leucine (S464L) has the potential to disrupt the network.Analogously, the positively charged amino acid K467 is involvedin a stabilizing salt bridge with E58 (Fig. 1C). Insertion of a polarbut neutral amino acid as threonine K467T, although still per-mitting the H-bond E58, would affect the favorable electrostaticinteraction. Residue I491 is a rather large, aliphatic residue locatedin a hydrophobic cavity mainly formed by aromatic amino acids(Fig. 1C). Although the side chains present similar size, methio-

nine (I491M) is a polar amino acid and would be unfavorablylocated in a nonpolar environment. The other two mutations(G465R and S492R) involve the change from rather small, polar,and uncharged side chain to a large and electrically charged sidechain in arginine (Fig. 1C; ref. 13).

R451C is the only mutation not located in the cetuximab-binding site; however, the mutation could lead to critical struc-tural changes. As the domain IV is formed by a sequence ofdisulphide bonds,whichpreserve its tertiary structure, the replace-ment by a cysteine at that position could perturb one of thedisulphide bonds in domain III (C475-C462) possibly forming anew one between domains III and IV (C451-C475). We verifiedthat the distance in the crystal structure between these residues(C451-C475, carbon alpha distance 6.8 Å) is compatible with theformation of a disulphide bond. Indeed, a system setup in thisnew configuration successfully minimizes to a C451-C475 dis-tance of 5.2 Å. Overall, all identified mutations in EGFR (exceptR451C) were located in the cetuximab-binding epitope.

Biochemical and functional analyses of EGFR ectodomainmutations

To experimentally assess the impact of the EGFR ectodomainmutations on the ability of the receptor to interact with cetux-imab, we performed forward genetic experiments on the newlydiscovered mutations. Wild-type and mutant EGFR cDNAs wereectopically expressed in NIH 3T3 cells that lack endogenousEGFR. Flow cytometry was used to establish the extent of cetux-imab binding to cells expressing themutants; wild-type EGFR andS492R served as positive and negative controls, respectively. Theseexperiments clearly showed that the newly discovered EGFRK467T, R451C, S464L, G465R, and I491M mutations were notpermissive for binding to cetuximab thus providing functional

Table 2. EGFR pathway mutations in parental and cetuximab-resistant cell lines

Pre-treatment Post-treatment

Pare

ntal

cel

l Li

nes

KRA

S ex

on 2

KRA

S ex

on 3

KRA

S ex

on 4

NRA

S ex

on 2

NRA

S ex

on 3

NRA

S ex

on 4

BRA

F ex

on 15

PIK3

CA e

xon

9

PIK3

CA e

xon

20

EGFR

exo

n 12

Res

ista

nt c

ell

Line

s

KRA

S ex

on 2

KRA

S ex

on 3

KRA

S ex

on 4

NRA

S ex

on 2

NRA

S ex

on 3

NRA

S ex

on 4

BRA

F ex

on 15

PIK3

CA e

xon

9

PIK3

CA e

xon

20 EGFR exon 12

R45

1C

S464

L

G46

5R

K46

7T

I491

M

S492

R

CCK81 CCK81 R1 CetuxCCK81 R2 CetuxCCK81 R3 Cetux

DiFi DiFi R1 CetuxDiFi R2 Cetux

HCA-46 HCA -46 R1 CetuxHCA -46 R2 Cetux

LIM1215

LIM1215 R1 CetuxLIM1215 R2 CetuxLIM1215 R3 CetuxLIM1215 R4 CetuxLIM1215 R5 Cetux

NCIH508 NCIH508 R1 CetuxNCIH508 R2 Cetux

OXCO-2 OXCO -2 R1 CetuxOXCO -2 R2 CetuxOXCO -2 R3 Cetux

NOTE: Parental and correspondent cetuximab-resistant derivatives of DiFi, LIM1215, HCA-46, NCIH508, OXCO-2, and CCK81 cell lineswere analyzed for KRAS,NRAS,BRAF,PIK3CA, andEGFRmutations. Black boxes indicate the presence of themutations (no specified alleles); in resistant cell lines,mutational analysis for EGFRexon12 was performed by ddPCR and black boxes indicate which EGFR ectodomain alleles emerged.Abbreviation: Cetux, cetuximab.






evidence of their role in driving acquired resistance to EGFRblockade.Of note, the effect of EGFRR451Con cetuximab bindingwas less prominent compared with the other mutants (Fig. 2).

To further characterize the functional properties of the EGFRmutations we performed biochemical studies in cells expressingindividual mutations. As expected, cetuximab abrogated ligand-mediated activation of the wild-type receptor, while had no orvery limited impact in cells carrying mutated EGFR (S464L,G465R, K467T, I491M, and S492R; Fig. 3). Notably, in R451C-mutant cells, cetuximab was still capable of inhibiting EGFRphosphorylation (Fig. 3).

We then examined whether panitumumab, the other anti-EGFR drug approved to treat colorectal cancer, was active in cellsoverexpressing EGFR mutations. As previously described, S492R-mutant cells efficiently bound to panitumumab (12). We foundthat the K467T and R451C mutants were to some extent permis-sive for panitumumab binding, whereas S464L, G465R, andI491M mutants did not bind to this antibody (Fig. 2). Accord-ingly, biochemical analyses showed that panitumumab preventsEGFR activation in S492R, K467, and R451C mutants (Fig. 3).

DiscussionWe present a comprehensive analysis of mutational changes

affecting keymembers of the EGFR signaling pathway emerging intumor biopsies of patients treated with cetuximab and in cellmodels, which acquired resistance to cetuximab in vitro.We foundthat colorectal cancer cells evadeEGFRblockade through twomainstrategies. Themainmechanismof resistance involves downstream

pathway reactivation that occurs in 43% of patients' samples and58.8% of the cells, respectively. The second entails EGFR extracel-lular domain mutations that were detected in 10.8% of patients'samples and 29% of the cells. Although the specific mutationspartially differ among cell lines and patients, they overlap in termsof activated cellular pathways observed in preclinical models andpatients samples (22). The cell-based findings may be translatedback into the clinic. For example, the EGFR ectodomain alleles,initially discovered in cells, might reasonably also be present inpatients who relapse upon EGFR blockade and this could beverified using tissue and liquid biopsies.

The molecular landscape of acquired resistance to EGFR block-ade revealed the emergence of multiple point mutations in theectodomain of EGFR. In particular, we detected two novel EGFRexon 12 mutations (EGFR p.R451C and p.K467T) in 2 patients.The previously reported EGFR S492R mutation was detected in 3of 37 postcetuximab tissue samples (8%), whereas a recent studyreports 16% of S492R EGFR mutation detection in 239 postce-tuximab plasma samples (11). Such differences may be explainedby different sensitivity of the detection techniques as well as theability of plasma samples to capture the heterogeneity of solidtumors as compared with single biopsies of one tumoral lesion(8, 16, 23). Plasma samples of the patients included in the currentstudywere not systematically collected and thereforewe could notanalyze the prevalence of EGFR mutations in circulating tumorDNA in the cohort. Accordingly, studies in larger cohorts ofpatients treated with cetuximab or panitumumab are warrantedto define the exact frequency of the other newly identified EGFRectodomain mutations. Of note, most samples harboring the

Figure 1.Structural analysis of EGFR extracellular domain mutants. A, general overview of the EGFR extracellular domain (sEGFR) bound to the antigen-binding fragment ofcetuximab (cetux) as crystallized by Li and colleagues. Four subdomains comprise the sEGFR domain, and domain III contains the six mutations. MutationR451C occurs at the interface of contact with domain IV. B,mutations I491M, S492R, S464L, G465R, and K467T are located at the surface recognized by cetuximab. Aselection of residues locatedwithin 5 Å of the site of eachmutation are shown. C, S464 (indicated in red, top left) is surrounded by tyrosine residues (Y102 and Y104)which favor an H-bond network. G465 (indicated in red, top right) lies in a highly hydrophobic groove formed by Y104, W54 and W52, K467 (indicated inred, central left) defines a salt bridge with E58. I491 (indicated in red, central right) is located in a hydrophobic environment formed by Q91, Y50, and Y104. S492(indicated in red, bottom left) is located in an uncharged cavity formed by Y104, W94, P95, T98, and N91. R451 (indicated in red, bottom right) is not located at thecetuximab-sEGFR interface, but closer to the IV domain. Replacement by a cysteine could form a new disulphide bond between domain III and IV.

Arena et al.





EGFR S492R mutation also exhibited EGFR gene amplification(12), similar to lung cancer tumors harboring the T790M muta-tion of resistance to tyrosine kinase inhibitors, where the T790Mallele appears to be selectively amplified (24). Interestingly, our invitro analysis showed that panitumumab was effective in a subsetof EGFR mutants. As the binding epitopes of cetuximab andpanitumumab overlap but are not identical, it is foreseeable thatmutations arising in EGFR after anti-EGFR treatment will differ-entially disrupt bindingof cetuximaband/or panitumumab to thereceptor (25), with relevant clinical implications for the treatmentof cetuximab-resistant patients.

Activating mutations in EGFR downstream signaling effectorswere the most frequent event in both cell models and patients.Emergence of KRAS and NRAS mutations occurred in 42% ofpatients, similar to what we have previously reported in tissuesamples, but lower than previously reported in plasma samples(6–10). Again, this may be due to the advantage of circulating

DNA in capturing the heterogeneity of solid tumors comparedwith biopsy of one tumoral lesion (8, 16, 23), indicating thatdiagnostic tools such as liquid biopsies are required to capture thecomplexity of the disease. As recently reported, RAS mutationsoften occurred outside of exon 2 in clinical samples and cellmodels (26, 27). The finding that codon 61 and 146 KRASmutations occurmore frequently in the acquired resistance settingthan in the general colorectal cancer population is worth furtherstudies. Of note, all samples harboring a PIK3CA mutation alsodisplayed other mechanisms of resistance. The role of PIK3CAmutations in driving acquired as well as primary resistanceremains controversial and needs to be further characterized. Wedid not identify emergence of molecular alterations in 20% ofpatients, suggesting limitations in the sensitivity of the detectiontechnique, tumor heterogeneity, as well as other mechanisms ofresistance such as ligand overexpression or c-MET amplification(15, 28, 29).

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Figure 2.EGFR mutations differentially affect binding to cetuximab and panitumumab. A, NIH 3T3 cells stably expressing wild-type or the indicated EGFR mutationswere incubated with cetuximab or panitumumab, and antibody binding was analyzed by flow cytometry using a secondary antibody to human IgG conjugatedwith phycoerythrin (PE). NIH 3T3 cells expressing the empty vector were used as a negative control (empty). Graphs show results of one representativeexperiment. B, the percentage of cells binding to the antibody are shown as relative values compared to EGFRwild-type (wt) cells (percentage of EGFRwt cells set to1) and are mean values of two independent experiments. While cetuximab binding was affected in cells expressing EGFR mutants, panitumumab was able tobind to cells expressing the S492R and K467T EGFR mutation. The R451C mutation had a moderate impact on binding to either cetuximab or panitumumab.






Importantly, multiple mechanisms of resistance were oftenpresent in the samples from relapsed tumors and often displayedmore than onemolecular alteration. This was also observed in celllines resistant to cetuximab, strongly suggesting their polyclonalstatus. This likely reflects the heterogeneity of colorectal cancersand supports the role of circulating DNA to comprehensivelycharacterize the molecular landscape of resistance to EGFR block-ade in patients. Activation of EGFR–RAS signaling axis as well asEGFR ectodomain mutations frequently co-occurred. These find-ings suggest the design of clinical trials that include concomitantinhibition of EGFR downstream signaling together with directinhibition of the EGFR receptor. Considering that panitumumabseems to be ineffective on a subset of the newly discoveredmutations, drugs inhibiting EGFR through different mechanismwill also be needed.

Our results have implications for the care of patients as theysupport the necessity of reassessing the molecular landscape of

tumors after progression to anti-EGFR drugs, which currently isnot routinely done in clinical practice. The plasticity of tumor cellsand their high capacity of adaptation under selective drug pres-sure, emphasizes the need for sequential tumor or plasma biop-sies to better monitor and personalize treatment.

In summary, our study highlights the importance of reasses-sing the molecular profile of the overall disease burden longi-tudinally during therapy, and provides evidence that acquiredresistance to anti-EGFR therapy in patients with mCRC arisesfrom the emergence of heterogeneous and overlapping molec-ular changes. Such complexity converges on two main mechan-isms of resistance: activating mutation in EGFR downstreamsignaling and mutations in EGFR ectodomain that disruptantibody–receptor binding. In light of these findings, pharma-cologic studies combining inhibition of both EGFR and EGFRdownstream signaling effectors to bypass cetuximab resistanceare warranted.

WT

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Figure 3.Ligand-dependent activation of EGFRmutants inthe presence of cetuximab and panitumumab.NIH 3T3 cells expressing WT EGFR or theindicated EGFR mutations were cultured in thepresence of cetuximab (Ctx) or panitumumab(Pnm) for 2 hours and stimulated with EGF(5 ng/mL) for 15 minutes. Immunoblotting wasperformed using antibodies to the indicatedproteins.

Arena et al.





Disclosure of Potential Conflicts of InterestA. Bardelli is a consultant/advisory board member for Biocartis, Horizon

Discovery, and Trovagene. C. Montagut is a consultant/advisory boardmemberfor Merck. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: S. Arena, B. Bellosillo, A. Martinez, I. Canadas,E. Gavilan, F. Di Nicolantonio, A. Bardelli, C. MontagutDevelopment of methodology: B. Bellosillo, G. Siravegna, A. Martinez,I. Canadas, E. Gavilan, A. Dalmases, A. Rovira, F. Di Nicolantonio, A. Bardelli,C. MontagutAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Siravegna, A. Martinez, I. Canadas, S. Misale,I. Gonzalez, M. Iglesias, E. Gavilan, S. Hobor, J. Sanchez, A. Dalmases,J. Bellmunt, J. Albanell, C. MontagutAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Arena, B. Bellosillo, G. Siravegna, A. Martinez,I. Canadas, N. Ferruz, E. Gavilan, G. Corti, S. Hobor, G. Crisafulli, M. Salido,J. Sanchez, A. Dalmases, J. Bellmunt, J. Albanell, A. Bardelli, C. MontagutWriting, review, and/or revision of the manuscript: S. Arena, B. Bellosillo,G. Siravegna, A. Martinez, I. Canadas, S. Misale, E. Gavilan, A. Dalmases,J. Bellmunt, F. Di Nicolantonio, J. Albanell, A. Bardelli, C. MontagutAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Rovira, C. MontagutStudy supervision: A. Dalmases, F. Di Nicolantonio, J. Albanell, A. Bardelli,C. MontagutOther (generation of cetuximab-resistant cell lines): L. Lazzari, M. RussoOther (designed and analyzed the modeling work): G. De Fabritiis

AcknowledgmentsThe authors thank Dr. Salvatore Siena, Dr. Andrea Sartore Bianchi, and

members of the laboratory ofMolecular Genetics for critical reading and editingof this article. The authors also thank Fundaci�oCellex (Barcelona) for a generousdonation to the Hospital del Mar Medical Oncology Service.

Grant SupportThis work was supported by RD12/0036/0051, PI12/00989, PI12/00680,

PT13/0010/0005, 2014 SGR 567 and 2014 SGR 740 grants and by the Xarxa deBanc de Tumors de Catalunya (to C. Montagut). The European Community'sSeventh Framework Programme under grant agreement no. 259015COLTHERES (to A. Bardelli); Associazione Italiana per la Ricerca sul Cancro(AIRC) IG grant no. 12812 (to A. Bardelli); AIRC MFAG no. 11349 (to F.DiNicolantonio); grant ÐFarmacogenomicak—5 per mille 2009 MIUR—Fonda-zione Piemontese per la Ricerca sul Cancro—ONLUS (to F. Di Nicolantonio);AIRC2010Special ProgramMolecular ClinicalOncology 5permille, project no.9970 (to A. Bardelli); FPRC 5permille 2010 and 2011Ministero della Salute (toA. Bardelli); Ministero dell'Istruzione, dell'Universit�a e della Ricerca, progettoPRIN 2010-2011 (to A. Bardelli). J. Albanell is a recipient of intensificationprogram ISCIII/FEDER.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 5, 2014; revised December 19, 2014; accepted January 8,2015; published OnlineFirst January 26, 2015.

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2015;21:2157-2166. Published OnlineFirst January 26, 2015.Clin Cancer Res Sabrina Arena, Beatriz Bellosillo, Giulia Siravegna, et al. Cetuximab Treatment in Colorectal Cancer

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