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Cancer Therapy: Clinical

Heterogeneous Mechanisms of Primary andAcquired Resistance to Third-Generation EGFRInhibitorsSandra Ortiz-Cuaran1, Matthias Scheffler2, Dennis Plenker1,3, llona Dahmen1,Andreas H. Scheel4, Lynnette Fernandez-Cuesta1,5, Lydia Meder4, Christine M. Lovly6,Thorsten Persigehl7, Sabine Merkelbach-Bruse4, Marc Bos1, Sebastian Michels2,Rieke Fischer2, Kerstin Albus4, Katharina K€onig8, Hans-Ulrich Schildhaus9,Jana Fassunke4, Michaela A. Ihle4, Helen Pasternack4,10, Carina Heydt4, Christian Becker11,Janine Altm€uller11, Hongbin Ji12,13, Christian M€uller1, Alexandra Florin4,Johannes M. Heuckmann14, Peter Nuernberg11, Sascha Ans�en2, Lukas C. Heukamp4,14,Johannes Berg15,William Pao6, Martin Peifer1,16, Reinhard Buettner4, J€urgen Wolf2,Roman K. Thomas1,4, and Martin L. Sos3

Abstract

Purpose: To identify novel mechanisms of resistance to third-generation EGFR inhibitors in patients with lung adenocarci-noma that progressed under therapy with either AZD9291 orrociletinib (CO-1686).

Experimental Design: We analyzed tumor biopsies fromseven patients obtained before, during, and/or after treatmentwith AZD9291 or rociletinib (CO-1686). Targeted sequencingand FISH analyses were performed, and the relevance ofcandidate genes was functionally assessed in in vitro models.

Results:We found recurrent amplification of either MET orERBB2 in tumors that were resistant or developed resistanceto third-generation EGFR inhibitors and show that ERBB2

and MET activation can confer resistance to these com-pounds. Furthermore, we identified a KRASG12S mutation ina patient with acquired resistance to AZD9291 as a potentialdriver of acquired resistance. Finally, we show that dualinhibition of EGFR/MEK might be a viable strategy to over-come resistance in EGFR-mutant cells expressing mutantKRAS.

Conclusions: Our data suggest that heterogeneous mechan-isms of resistance can drive primary and acquired resistance tothird-generation EGFR inhibitors and provide a rationale forpotential combination strategies. Clin Cancer Res; 22(19); 4837–47.�2016 AACR.

Introduction

In EGFR-mutant lung adenocarcinoma, targeted therapy withEGFR tyrosine kinase inhibitors (TKI) performs substantially betterthan standard chemotherapy in terms of response rate (RR),progression-free survival (PFS), and tolerability (1).Unfortunately,

all patients will ultimately experience relapse with a median PFSof 7 to 16 months.

The major cause of resistance is the EGFRmutation T790M, atthe gatekeeper site (50%–60%; refs. 2–4). A third generation ofcovalent EGFR TKIs that specifically target EGFR T790M as well asthe activating EGFRmutations (e.g., L858R or exon 19 deletion),

1Department of Translational Genomics, Center of IntegratedOncology Cologne–Bonn, Medical Faculty, University of Cologne,Cologne, Germany. 2Department I of Internal Medicine, Lung CancerGroup Cologne and Network Genomic Medicine (Lung Cancer),Center for Integrated Oncology Cologne-Bonn, University HospitalCologne, Cologne, Cologne, Germany. 3Molecular Pathology, Centerof Integrated Oncology, University Hospital Cologne, Cologne, Ger-many. 4Institute of Pathology, Center of Integrated Oncology, Uni-versity Hospital Cologne, Cologne, Germany. 5Genetic Cancer Sus-ceptibility Group, Section of Genetics, International Agency forResearch on Cancer (IARC-WHO), Lyon, France. 6Department ofMedicine, Vanderbilt University, Nashville, Tennessee. 7RadiologyDepartment, University Hospital Cologne, Cologne, Germany.8Labor Dr. Quade und Kollegen GmbH, Cologne, Germany. 9Instituteof Pathology, University Hospital Goettingen, Goettingen, Germany.10Pathology of the University Hospital of Luebeck and LeibnizResearch Center Borstel, L€ubeck and Borstel, Germany. 11CologneCenter for Genomics (CCG), University of Cologne, Cologne, Ger-many. 12Key Laboratory of Systems Biology, CAS Center for Excel-lence in Molecular Cell Science, Innovation Center for Cell SignalingNetwork, Institute of Biochemistry and Cell Biology, Shanghai Insti-tutes for Biological Sciences, Chinese Academy of Science, Shang-

hai, China. 13School of Life Science and Technology, Shanghai TechUniversity, Shanghai, China. 14NEO New Oncology AG, Cologne,Germany. 15Institute for Theoretical Physics. University of Cologne,Cologne, Germany. 16Center for Molecular Medicine Cologne(CMMC), University of Cologne, Cologne, Germany.

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

S. Ortiz-Cuaran and M. Scheffler contributed equally to this article.

Current address for W. Pao: Roche Innovation Center Basel, Pharma Researchand Early Development, Roche, Basel, Switzerland.

Corresponding Authors: Martin L. Sos, University Hospital Cologne, Weyertal115b, Cologne 50931, Germany. Phone: 49-221-4789-8771; Fax: 49-221-4789-8779; E-mail: [email protected]; Roman K. Thomas,[email protected]; J€urgen Wolf, [email protected]; andReinhard Buettner, [email protected]

doi: 10.1158/1078-0432.CCR-15-1915

�2016 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org 4837

on July 31, 2020. © 2016 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst June 1, 2016; DOI: 10.1158/1078-0432.CCR-15-1915

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while sparing wild-type EGFR have been evaluated clinically (5,6). In phase I trials, rociletinib and AZD9291 have shown impres-sive clinical activity in patients with EGFRT790M-mediatedacquired resistance to reversible EGFR-TKIs with response ratesof 61% and 59%, and PFS of 13.5 and 13.1 months, respectively(5–9).

The acquisition of a secondary EGFRmutation, C797S, has beenrecently reported in patients with relapse under therapy withAZD9291 (10, 11). In preclinical models, resistance to theseinhibitors can also be driven by activation of the MAPK pathway(12–14). Theseobservations are of particular interest becauseEGFRmutations are known to mainly occur in mutually exclusive man-ner with oncogenic mutations within the MAPK pathway (15, 16).

We performed genetic analyses on biopsies obtained frompatients treated with third-generation EGFR inhibitors to identifyadditional mechanisms of resistance to these drugs.

Patients and MethodsStudy population

Whenever possible, biopsies were obtained before treatmentand after radiographic progression under TKI therapy (with theexception of P2 and P3), at the times and with the methodsdescribed in Table 1. Standard histopathology was performed toconfirm the histologic subtype. Patients were treated in trialsNCT01802632 and NCT01526928. The Institutional ReviewBoard (IRB) and the responsible ethics committee approvedcollection and use of all patient specimens in this study. Writteninformed consent was obtained from all subjects.

FISH analysesFISH for MET and ERBB2 was performed on formalin-fixed

paraffin-embedded tissue using labeled dual-color probes. Sec-tions of 1.5 mm were cut and hybridized with labeled probes forMET or ERBB2 and the respective centromeric reference probe(ZytoLight Spec MET/CEN7 probes; ZytoLight Spec ERBB2/CEN17 probes by ZytoVision). Slides were reviewed at�630 andscored according to appropriate guidelines (17). For ERBB2, thegastric cancer scoring system was used because no lung-specificrecommendations are available (18).

DNA extraction and sequencingTotal DNA was obtained from formalin-fixed paraffin-embed-

ded tumor tissue. DNA from sections was extracted using thePuregene Extraction Kit (Qiagen) according to themanufacturer'sinstructions. DNAwas eluted in 1� TE buffer (Qiagen), diluted toa working concentration of 150 ng/mL and stored at �80�C.

Targeted sequencingTargeted sequencing analysis was performed as described pre-

viously (19) using a custom-made lung cancer panel consisting of102 amplicons for the detection of hotspot mutations in 14 lungcancer–related genes. Isolated DNA (up to 50 ng) was amplifiedwith two customized Ion AmpliSeq Primer Pools. Library pro-ducts were quantified using Qubit 2.0 Fluorometer (Qubit dsDNA HS Kit; Life Technologies), diluted, and pooled in equalamounts. Six to 8 pmol/L was spiked with 5% PhiX DNA (Illu-mina) and sequenced with the MiSeq reagent Kit V2 (300-cycles;Illumina). Data were exported as FASTQ files and analyzed usingthe in-house pipeline.

Generation of stably transduced cell linesH1975 (CRL-5908) and HCC827 (CRL-2868) cells were

obtained from the ATCC. PC9, PC9GR and HCC827GR cellswere kindly provided by Dr. Hongbin Ji (Shanghai Institutes forBiological Sciences, China), Dr. Passi J€anne (Broad Institute,Cambridge, MA), and Dr. Jeffrey Engelman (Massachusetts Gen-eral Hospital, Boston, MA), respectively. Cells were cultured withRPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37�C under 5% CO2.

Generation of cells harboring pBabe-puro empty vector,EGFRT790M, ERBB2WT, KRASWT, or KRASG12S was done by stabletransduction as described previously (20) using a pBabe-puroretroviral vector (Addgene). Cells expressing the correspondingmutants were generated by retroviral transduction and subse-quent puromycin selection. The expression of the mutant wasverified by RT-PCR and further Sanger sequencing and byWesternblot analysis.

cDNA transcriptionRNA was isolated from PC9KRAS-G12S and HCC827KRAS-G12S

cells using TRIzol reagent (Invitrogen) and cleaned up using theRNeasy MinElute Cleanup Kit (Qiagen) following the manufac-turers' protocols. Finally, 1 mg of RNA was transcribed into cDNAusing Superscript III reverse transcriptase (Invitrogen, #18064).

ReagentsAZD9291, rociletinib, afatinib, crizotinib, trametinib, and

selumetinib were synthesized according to published methods.For cell culture experiments, each compound was dissolved indimethyl sulfoxide (DMSO), aliquoted, and stored as 10mmol/Lstocks at �80�C. HGF (R&D Systems) was resuspended in PBSþ0.1 % BSA and aliquots stored at �20�C.

Cell viability assaysCells were plated into 96-well culture plates in RPMI medium

supplementedwith 10%FBS and1%penicillin/streptomycin, at adensity of 1,500 cells per well, cultured overnight, and treated thefollowing day. Cell viability was determined after 4 to 6 days bymeasuring cellular ATP content (CellTiter-Glo, Promega) asdescribed previously (20). GI50 values were calculated by plotting

Translational Relevance

Although themajority of EGFRT790M-mutant adenocarcino-mas of the lung respond well to third-generation EGFR inhi-bitors, resistance to these inhibitors remains amajor challengein the field. We uncover ERBB2 and MET activation as poten-tial bypass-track mechanism to third-generation EGFR inhi-bitors and suggest that treatment with third-generation EGFRinhibitors may select for EGFR-mutant subclones that toleratecooccuring mutations in the MAPK pathway. Together withprevious preclinical studies, our data support the notion thatactivation of MAPK signaling might play a role in resistance tothese drugs. Our findings might be of broad interest to basiccancer scientists andmedical oncologists alike, as they provideinsight related to the biology of EGFR-mutant adenocarcino-ma and have immediate implications for genetically stratifiedtreatment of these patients.

Ortiz-Cuaran et al.

Clin Cancer Res; 22(19) October 1, 2016 Clinical Cancer Research4838




Table

1.Patient

Inform

ation

Patient

inform

ation

Pre-treatmen

tsample

Drug-resistant

sample

Reb

iopsy

inform

ation

Seque

ncingdatarebiopsy

Patient

number

Age

Sex

Initial

biopsy

site

FISH

Primary

mutation

TKI

resistan

ce

PFS

(mo)

Startof

trea

tmen

t

Timeon

trea

tmen

t

(mo)

Trea

tmen

thistory

(tim

e

ontrea

tmen

tin

mo)

OS

(y)

Dateof

rebiopsy

Reb

iopsy

site

Typeof

rebiopsy

Typeof

sample

FISH

Metho

d

Mea

n

cove

rage

Other

mutations

171

FNA

Notdone

EGFR

Exo

n

19del;

EGFR

T79

0M

Rociletinihb

1.9Decem

ber

2014

1.9CIS/PEM

(5),gap

(6),

Erlotinib(11),CO-1686

(2)

2.1b

Janu

ary

2015

Pleural

effusionc

.FFPE

ERBB2am

pmiSeq

4,975

EGFR

Exo

n

19del,E

GFR

T79

0M

264

FLu

ngERBB22

amp

EGFR

L861Q

;

EGFR

T79

0M

AZD929

14.2

October

2014

7.4a

CIS/V

INO/R

TX(3),

Afatinib(4),AZD929

1

(7a)

1.2b

..

..

.miSeq

9,560

EGFR

L861Q

,

EGFR

T79

0M

354

FLive

rMETam

pEGFR

Exo

n

19del;

EGFR

T79

0M

Rociletinib

1.2Nove

mber

2014

3.2

CIS/PEM

(1),Erlotinib/

RTX(2),Gefi

tinib(9),

Rociletinib(3),

Rociletinib/C

rizo

tinib

(1)

1.3.

..

..

..

.

457

MLive

rNoMET

amp

EGFR

L858

R;

EGFR

T79

0M

AZD929

14.0

July

2014

4.6

Gefi

tinib(2),Carbo/PEM

(2),mainten

ance

PEM

(3),CARBO/

DOCE(4),AZD929

1

(4),EMD1214063(3),

Erlotinib/C

rizo

tinib

(1)

1.9Nove

mber

2014

Live

rCT-guided

biopsy

FFPE

METam

pmiSeq

4,561

EGFR

L858

R,

EGFR

T79

0M

560

MLive

rBlock

not

available

EGFR

Exo

n

19del,

EGFR

T79

0M

AZD929

116.5

May

2014

17.5

Erlotinib(7),CARBO/

PACLI

(2),

interm

ittent

RTX(13),

AZD929

1(18)

3.2b

October

2015

Live

rCT-guided

biopsy

FFPE

METam

p,

ERBB2am

p

miSeq

1,217

EGFR

Exo

n

19del,E

GFR

T79

0M

656

FLive

r

(2x)

Notdone

EGFR

Exo

n

19del,

EGFR

T79

0M

AZD929

119.4

October

2013

22.3

Gefi

tinib(9),RTX(3),

Erlotinib(7),Afatinib

(15),A

fatinib/

Cetux

imab

(3),PEM/

Erlotinib(9),

AZD929

1(22)

5.9b

July 20

15

Live

rCT-guided

biopsy

FFPE

METam

pmiSeq

2,176

EGFR

Exo

n

19del,E

GFR

T79

0M,

EGFR

C79

7S

751

FLy

mph

node

NoMET

amp,n

o

ERBB2am

p

EGFR

Exo

n

19del,

EGFR

T79

0M

AZD929

17.5

Nove

mber

2013

8.9

Gefi

tinib(17),C

IS/PEM/

RTX(4),Afatinib(6),

Afatinib/C

etux

imab

(11),A

ZD929

1(9)

4.5

May 20

14

Lymph

node

End

oscopic

biopsy

via

esopha

gus

FFPE

NoMETam

p,

noERBB2

amp

miSeq

540

EGFR

Exo

n

19del,

KRASG12S

Abbreviations:B

EVA,b

evacizum

ab;C

ARBO,carboplatin;

CIS,cisplatin;

DOCE,d

ocetaxel;GEMCI,gem

citabine;

Het,h

eterogen

eity;O

P,surgery;

OS,o

verallsurvival;P

ACLI,p

aclitaxel;P

EM,p

emetrexed;R

TX,rad

iotherap

y;TRO,trofosfam

ide;

VINO,v

inorelbine.

aTreatmen

tong

oing.

bStillalive.

c Obtained

duringtherap

y.

Resistant Mechanisms to AZD9291 and Rociletinib

www.aacrjournals.org Clin Cancer Res; 22(19) October 1, 2016 4839




luminescence intensity against drug concentration in nonlinearcurves using GraphPad Prism (GraphPad). Synergy scores werecalculated as described previously (21). For long-term viabilityassays, the reported "Cell number change (%)" was determinedas: 100 � (Value/Baseline)/Baseline. Each point was performedwith three replicates. Unless otherwise noted, three independentexperiments were performed, and a representative result is pre-sented. Error bars are mean � SD.

Crystal violet stainingCells were plated at a density of 1 million cells per well and

incubated overnight. Cells were treated with AZD9291 the nextday and then again 72 hours after. After 6 days of drug exposure,cells were fixed with 1% paraformaldehyde. For staining, a solu-tion of 0.1% crystal violet was added and incubated for 20minutes. Plates were inverted and dried overnight, and imageswere taken the next day.

Western blot analysesCells lysates were blotted as described previously (20). The

following antibodies were used: phospho-EGFR (Y1068), totalEGFR, phospho-ERBB2 (Y1248), total ERBB2, phospho-MET(Y1234/Y1235), total MET, phospho-AKT (Ser473), total AKT,phospho-ERK (T202/Y204), total ERK, actin (Cell Signaling Tech-nology), and conjugated antibodies to rabbit and mouse(Millipore).

Statistical analysesStatistical analyses were performed using GraphPad Prism 5.0.

We used the Student t test (unpaired, two-sided). A P value of<0.05 was used as a threshold considered to call statistical signif-icance. PFS was assessed from start of the respective therapy untilprogression or death. Survival analyses were performed usingKaplan–Meier statistics, providing median and 95% confidenceinterval (CI). For response evaluation, the Response EvaluationCriteria in Solid Tumors (RECIST V1.1; ref. 22) were used.

ResultsMechanisms of primary and acquired resistance to third-generation EGFR inhibitors

We obtained specimens from seven patients treated with third-generationEGFR inhibitorsAZD9291(n¼5)or rociletinib (n¼2),within two clinical trials (clinicaltrials.gov, NCT01802632 andNCT02147990) after acquired EGFRT790M-positive resistance tofirst- or second-generation EGFR kinase inhibitors. The PFS forthird-generationEGFRTKI therapy ranged from1.2 to19.4months(median 4.2 months; 95% CI, 3.7–4.7 months, Fig. 1A). The bestreduction of the sum of the largest tumor diameters under treat-ment, according to RECIST V1.1 (22), ranged between 0.1% and79.2%,with3patients presenting growthofnon-target lesions (P1,P3, and P5, Fig. 1B).

The pattern of response varied across these patients: twopatients (P1 and P3) experienced primary resistance to rociletinib(defined as progression on the first restaging scan), whereas twopatients had stable disease (P2 and P4) under treatment withAZD9291. Finally, three cases (P5, P6, and P7) presented withpartial responses to treatment with AZD9291, with reductions indiameters of more than 50% (Fig. 1A and B). For these patients,unfortunately, disease recurred after 16.5, 19.4, and 7 months,respectively.

To characterize the molecular mechanisms that may belinked with such heterogeneous response patterns, we per-formed FISH for known EGFR resistance markers (ERBB2 andMET) and targeted next-generation sequencing on tumor tissuefrom these patients (see Patients and Methods). FISH analysesof a massively progressive pleural effusion (nontarget lesion)collected after 3 weeks of treatment initiation from P1 (PD, 625mg rociletinib twice daily) and a lung biopsy sample collectedbefore treatment from P2 (SD, 180 mg AZD9291 daily),revealed ERBB2 amplification (7.3 and 3.7 gene copies,respectively; Fig. 1C).

Because ERBB2 amplification has been shown to confer resis-tance to first-generation EGFR TKIs (23), we hypothesized thatamplified ERBB2 might substitute for EGFR signaling in thiscontext and explain the lack of response to of AZD9291 androciletinib in P1 and P2. To test this hypothesis, we evaluated theimpact of ERBB2 overexpression on the sensitivity to AZD9291and rociletinib in PC9GR cells (EGFRT790M). Despite a relativelylow overexpression of ERBB2 in PC9GR cells, we observeddecreased sensitivity to both drugs at nanomolar concentrations(Fig. 1D and Supplementary Fig. S1A). Thus, our results indicatethat ERBB2 activation may contribute to resistance against roci-letinib and AZD9291.

The tumor biopsy collected before treatment of P3 (PD, 625mgrociletinib twice daily) showed high-level amplification of MET(MET/CEN7 ratios 2.10, 4.42MET copies; Fig. 2A). Unfortunate-ly, no baseline comparative sample (before erlotinib treatment)was available for this patient. In the case of P4 (SD, 80 mgAZD9291 daily), an initial tumor reduction (20.8%) was fol-lowed by an increase in the tumor volume after 4.1 months oftreatment (Fig. 2B). In this patient too, the biopsy taken after theinitial response exhibited high-level amplification ofMET (MET/CEN7 ratio 5.35, 11.42MET copies) that was not observed beforetherapy with AZD9291 (Fig. 2C). P5 (PR, 80 mg AZD9291 daily)had an initial tumor response of 79% and presented a new lesionin the liver that harbored a high-level MET amplification (MET/CEN7 ratios 1.88, 21.92 MET copies, Fig. 2D, left) and ERBB2amplification (4.25 gene copies, Fig. 2D, right). Of note, targetedsequencing of these samples did not show additional clinicallyrelevant mutations (Table 1).

To functionally test whetherMET amplification might reducethe sensitivity to AZD9291 or rociletinib, we stably overex-pressed EGFRT790M-mutant constructs in HCC827 cells (METwild-type) and their gefitinib-resistant derivate, HCC827GRcells (MET amplified; Supplementary Fig. S1B). In line withprevious reports (24), the presence of MET amplificationdecreased the sensitivity to both third-generation EGFR inhi-bitors (Fig. 2E) and led to sustained phosphorylation of ERKand AKT (Fig. 2F). Furthermore, concomitant inhibition witheither AZD9291 or rociletinib and the MET inhibitor, crizoti-nib, resulted in pronounced cytotoxicity and reduced AKT andERK activation in HCC827GREGFR-T790M cells (Fig. 2E and G).These results were confirmed in EGFRT790M-expressing cells(H1975 and PC9GR) where MET was activated through addi-tion of exogenous HGF (Supplementary Fig. S1C–S1F).

Thus, our clinical observations are in line with previous reportswhere both ERBB2 and MET amplification has been found to beassociate with loss of efficacy of third-generation EGFR inhibitorsin EGFR-mutant tumors (25). More importantly, we providefunctional evidence that activation of ERBB2 and MET signalingmay induce resistance to this new class of EGFR inhibitors.

Ortiz-Cuaran et al.





Finally, patient 6 (P6) presented with a tumor response of72.6% followed by emergence of new liver metastases 19.4months after start of therapy. The liver biopsy revealed theretention of the initial EGFR exon19 deletion (allelic fraction,AF: 42.6%, Supplementary Fig. S2A) and the EGFRT790Mmutation(AF: 27.66%) togetherwith thepresence of a secondaryEGFRC797S

mutation (Fig. 2H) that was not present in the pretreatmentbiopsy (Supplementary Fig. S2B). Of note, the liver biopsyobtained at relapse also harbored an intermediate-level METamplification (MET/CEN7 ratios 1.14, 5.67 MET copies, Fig.2I). The EGFRC797S mutation abrogates the irreversible bindingof third-generation EGFR inhibitors and has been described toconfer resistance to AZD9291 thus providing a rational for theobserved resistance phenotype in P6 (10, 26, 27).

KRAS mutation and resistance to third-generation EGFRinhibition

In our cohort, a forth patient (P7) relapsed after having initiallyresponded to treatment with AZD9291. This patient received

different lines of treatment before developing resistance throughEGFRT790M under combination therapy with afatinib and cetux-imab. After enrollment into the AURA trial (NCT01802632) thetumor responded to AZD9291 treatment (160 mg/d) with a PR(best response: 54.3%, Fig. 3A) for 8 months. However, the fifthfollow-up revealed the appearance of a new lesion in a fastgrowing cervical lymph node. Targeted sequencing of the speci-mens obtained before and after treatment with AZD9291 (bothfrom the cervical lymph nodes) showed a slight decrease in thefraction of reads containing the original activatingEGFRmutation(43% before treatment with AZD9291 vs. 32% after treatment,Supplementary Fig. S2C) and the disappearance of EGFRT790M

(25% before treatment with AZD9291, Supplementary Fig. S2D).Finally, in two independent sequencing runs we found a novelKRASG12S mutation (38% of the reads), which had not beendetected before therapy with AZD9291 (Fig. 3B). In general, thedisproportionate levels of C>T/G>A changes that occur as aconsequence of formalin fixation are more apparent at lowallelic fractions (1%–10%; ref. 28). In the relapse sample of

A

C

B

D

P7P6P5P4P3P2P1–100

–80

–60

–40

–20

0

Bes

t res

pons

e un

der t

reat

men

t

*

*

* PD of nontarget lesions*

DMSO1mmol/L

1mmol/L

100nmol/L

100nmol/L

10nmol/L

10nmol/L

DMSO–200

0

200

400

600

Cha

nge

in c

ell n

umbe

r (%

) PC9GRe.v PC9GRERBB2

RociletinibAZD9291

**

***

***

** ** ***

2520151050

P7

P6

P5

P4

P3

P2

P1

Time of treatment (months)

MET

am

pER

BB

2 am

p

AZD9291Rociletinib

PD

SD

PD

SD

PR

PR

PR

P1 (PD)ERBB2 amp

EGFR p.T790M

P2 (SD)ERBB2 amp

EGFR p.T790M Before AZD9291 Before AZD9291

Figure 1.

Primary and acquired resistance to third-generation EGFR inhibitors. A, duration of treatment of patients that underwent therapy with AZD9291 (blue) or rociletinib(green). PD, progressive disease; SD, stable disease; PR, partial response. MET and/or ERBB2 amplification were detected before treatment (gray) orat relapse (black) under AZD9291 or rociletinib. B, waterfall plots showing best percentage change in the size of target or non-target (�) lesions. C, FISH analyseswith probes for chromosomal loci containing ERBB2 (P1 and P2, green signal) and the respective centromeric reference probe (red signal). D, viabilityassays of PC9GRe.v and PC9GRERBB2 cells indicating the change in cell number after 6 days of treatmentwith DMSOor increasing concentrations of either AZD9291 orrociletinib, in comparison with the number of cells at the initiation of drug exposure. �� , P < 0.01; �� , P < 0.00.






A

F

H

B

AZD9291 (1µmol/L) Rociletinib (1µmol/L)

HCC827GR EGFR T790M

EGFR

pEGFRY1068

pAKTS473

AKT

ERK

BIM

pERKT202/Y204

Actin

Crizotinib (1µmol/L)

MET

pMETY1234/Y1235

---

+--

--+

+-+

-+-

-++

DM

SO

A (1

mm

ol/L

)

R (1

mm

ol/L

)

C (1

mm

ol/L

)

Com

bo A

+C

Com

bo R

+C

0

50

100

150

200

250

Cha

nge

in c

ell n

umbe

r (%

)

******

HCC827GREGFR T790M GE

EGFR

pEGFRY1068

pAKTS473

AKT

ERK

pERKT202/Y204

MET pMETY1234/Y1235

HCC827EGFR T790M

DMSOAZD

9291

Rocile

tinib

HCC827GREGFR T790M

DMSOAZD

9291

Rocile

tinib

BIM

Actin

D

I

Baseline 1 2 3Follow-ups

300 mm

250 mm

200 mm

150 mm

100 mm 50 mm

0 mm June 14’

*Aug 14’ Sep 14’

*Nov 14’

*

Pleura

Liver S8 Liver S2/3

Kidney left

C

T790M

Patient 6

C797S

P3 (PD)MET amp

EGFR p.T790M Before AZD9291

P4 (SD)MET amp and EGFR p.T790M

Before AZD9291 Under AZD9291

P5 (PR)EGFR p.T790M

MET amp ERBB2 amp Under AZD9291 Under AZD9291

P6 (PR)MET amp, EGFR p.T790M

Under AZD9291

EGFR

Figure 2.

Activation of MET confers resistance to third-generation EGFR inhibitors. A, FISH analyses with probes for chromosomal loci containing MET (P3, green signal)and the respective centromeric reference probe (red signal). B, tumor response analysis for P4 using the quantitative imaging tool mint Lesion. Here, thevolumes of the target lesions are shown and added up, according to RECIST. This representation shows how the different lesions respond individually as well as thesum of the changes in the target lesions. Follow-up scans (�) were performed every 8 weeks. C, FISH analyses assessing the presence of MET amplificationin the liver lesion collected before AZD9291 and after relapse. (Continued on the following page.)

Ortiz-Cuaran et al.





P7, the C>T/G>A changes were comparable with other patientsamples or freshly prepared DNA from cell lines sequenced in thesame batch, suggesting that the KRAS mutation detected is not aformalin-fixation artifact (Supplementary Fig. S2E). TheEGFRC797S mutation had also been found in the plasma of thispatient (10) but was not observed in the lymph node biopsy(Supplementary Fig. S2F). Moreover, the KRASG12S mutation wasnot detected in the plasma of this patient. These observationssuggest that heterogeneous mechanisms of acquired resistancemight be operant in this patient.

To investigate whether expression of an activated KRAS allelemight contribute to resistance to third-generation EGFR inhibi-tors we stably transduced EGFR-mutant PC9 cells with theKRASG12S-mutant. PC9 cells that expressed the KRASG12S-mutantwere selectedunder treatmentwithpuromycin for at least 2weeks.The resulting PC9KRAS-G12S cells expressed both the original EGFRexon19 deletion and theKRASG12Smutation (Supplementary Fig.S3A). Concomitant expression of bothmutants was also observedin a PC9KRAS-G12S clone obtained by serial dilution (Supplemen-tary Fig. S3B).

When compared with the KRAS wild-type transduced cells(PC9KRAS-wt), PC9KRAS-G12S were less sensitive to AZD9291 (Fig.3C and D). Furthermore, introduction of KRASG12S resulted inincreased KRAS expression and sustained ERK phosphorylationunder treatment with AZD9291 (Fig. 3E). Similar results wereobserved in HCC827KRAS-G12S cells (Supplementary Fig. S3C andS3D) where the introduction of the KRASmutation also resultedin decreased sensitivity to rociletinib (Supplementary Fig. S3C).Confirming a role of MAPK signaling in inducing resistance inPC9KRAS-G12S cells, combined treatment with AZD9291 and theMEK inhibitor, trametinib, was highly synergistic [synergy score,5.48 (ref. 21); P value of 4.14E�08; Fig. 3F and SupplementaryFig. S4A]. Consistent with these results, AZD9291 failed to fullyinhibit downstream MAPK signaling except in the presence oftrametinib (Fig. 3G). Similar results were observed with thecombination of AZD9291 and selumetinib (Fig. 3F and Supple-mentary Fig. S4B). Finally, concomitant EGFR and MEK inhibi-tion was also synergistic in HCC827KRAS-G12S cells (Supplemen-tary Fig. S4C and S4D). These findings demonstrate that mutantKRAS may induce acquired resistance to third-generation EGFRinhibitors.

DiscussionHere, we provide clinical and functional evidence for the role of

ERBB2 and MET amplifications as well as KRAS mutations aspossible mechanisms of resistance to third-generation EGFRinhibitors. The role of ERBB2 activation in resistance has beenpreviously reported for first-generation EGFR inhibitors (23). Theactivity of AZD9291 against ERBB2 is known to be limited anddespite the presence of a metabolite AZ5104 that shows inhibi-tion of ERBB2 it is conceivable that ERBB2 signaling may reduce

the overall activity of third-generation inhibitors in patients (6).Because the potency against ERBB2 was greater with the AZ5104metabolite than with AZD9291, it would be interesting to deter-mine themetabolites levels in the plasmaof patients to predict theemergence of ERBB2 as a potential bypass mechanism of resis-tance to AZD921. Our functional in vitro results suggest that theactivity of both AZD9291 and rociletinib may decrease throughspecific activation of ERBB2 signaling. These observations go inline with the studies reporting ERBB2 as a mechanism of resis-tance to first-generation EGFR inhibitors (23). The use of patient-derived EGFRT790M-mutant cell lines may help defining the activ-ity of these inhibitors on ERBB2 and characterizing the molecularcontext of the role of ERBB2 as amechanism of resistance to third-generation EGFR inhibitors.

The role of MET amplification in acquired resistance to first-generation EGFR inhibitors has been studied extensively (29–31).According to our results MET activation may similarly play a rolein resistance to third-generation EGFR inhibitors as MET is not arelevant off-target for these drugs. Our observations also confirmprevious reports on the efficacy of the combination of rociletinibwith crizotinib in these tumors (32). Combination therapies haveproven successful in the setting of resistance to first-generationEGFR inhibitors (33) and early-phase clinical trials are evaluatingthe combination of rociletinib with crizotinib (32), or AZD9291with the MET inhibitor, savolitinib, in EGFR-mutant lung cancer(34). It might therefore be important to determine the presence ofthese alterations before therapy with any EGFR inhibitor.

Interestingly, we identify an activating KRASG12S mutation in atumor rebiopsy obtained at the time of acquired resistance toAZD9291. This mutation is of particular interest because EGFRand KRAS mutations typically occur in a mutually exclusivefashion in lung cancer (16, 35). Indeed, a recent report evidencedthat the expression of inducible KRASG12V constructs in EGFR-mutant PC9 cells results in decreased cell viability after 7 days ofselection with doxycyclin (16). Anecdotal reports show evidenceof co-occurrence of KRAS and EGFR mutations in lung cancerpatients (36, 37). More recently Hata and colleagues (14)described two distinct evolutionary paths for the developmentof resistance to EGFR inhibition. In this study, cell lines thatdeveloped resistance at late stages were enriched for NRAS andKRAS mutations suggesting that under prolonged selection adistinct population of EGFR-mutant cells may well tolerate thepresence of oncogenic MAPK pathway activation. In our func-tional experiments, decreased cell viability was observed in cellsexpressing both EGFR and KRAS mutants; however, this pheno-type was overcome after about 10 passages under antibioticselection. In line with our results, in the study performed by Unniand colleagues (16), the induction of mutant KRAS rescued PC9cells from the lethal effects of erlotinib, thus implying that thetoxicity of coexpression ofmutant KRAS and EGFR depend on thekinase activity of mutant EGFR. We speculate that EGFR inhibi-tion throughAZD9291may functionally deplete oncogenic EGFR

(Continued.) D, evaluation of MET (left) and ERBB2 amplification (right) by FISH in a new liver lesion presented by P5. E, viability assay of HCC827GREGFR-T790M

cells after treatment with AZD9291 (A) or rociletinib (R), alone or in combination with crizotinib (C). Presented is the change in cell number in comparison tothe number of cells at baseline. After 6 days of treatment, cell viability was determined by measuring intracellular ATP content; ��, P < 0.001.F, HCC827EGFR-T790M and HCC827GREGFR-T790M cells treated with AZD9291 (1 mmol/L) or rociletinib (1 mmol/L) for 24 hours and lysed. Protein expressionand phosphorylation of the MET, EGFR, AKT, and ERK, as well as expression of BIM and actin was monitored by immunoblotting. G, HCC827GREGFR-T790M cellstreated with AZD9291 (1 mmol/L) or rociletinib (1 mmol/L) alone or in combination with crizotinib (1 mmol/L) for 24 hours. Expression of the indicatedproteins actin was monitored by immunoblotting. H, Integrative Genomics Viewer (IGV)—based visualization of reads of targeted NGS that identify theEGFRT790M and EGFRC797Smutations in P6. I, FISH analyses with probes for chromosomal loci containingMET (green signal) and the corresponding centromericreference probe (red signal).






A B

C

F

EGFR

pEGFRY1068

pAKTS473

AKT

ERK

Actin

BIM

pERK/Y204

KRAS wt AZD9291 (mmol/L)

KRAS

KRAS G12S 0 0.1 1 0 0.1 1

PC9 KRAS G12S

EGFR

pEGFRY1068

pAKTS473

AKT

ERK

BIM

pERKT202/Y204

KRAS

Actin

--

+-

-+

++

AZD9291 (100 nmol/L) Trametinib (1 mmol/L)

ED

G

10 nmol/L

1 mmol/L

100 nmol/L

DMSO–200

0

200

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800

Concentration AZD9291 (mmol/L)

Cha

nge

in c

ell n

umbe

r (%

)

PC9 KRAS wtPC9 KRAS G12S

**

** ***

AZD9291 (mmol/L) 0 0.1 1

PC9 KRAS wt

PC9KRAS G12S

DM

SO

A (1

00 n

mol

/L)

T (1

mm

ol/L

)

Com

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S (1

mm

ol/L

)

Com

bo S

–200

0

200

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Cha

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in c

ell n

umbe

r (%

)

******

Before AZD9291

UnderAZD9291

KRAS G12S

Patient 7

140 mm120 mm

100 mm

80 mm60 mm40 mm10 mm

0 mm

Baseline 1

Kidney left Thorax left Liver S8 Liver S6 Lung left

Oct. 13’ *

Dec. 13’ *

Jan. 14’ Feb. 14’ *

April 14’ *

June 14’ *

2 3 4 5

Follow-ups

Figure 3.

Acquired resistance to AZD9291 mediated by acquired KRASG12S mutation. A, tumor response analysis for P5 using the quantitative imaging tool mint Lesionshowing the volumes of the target lesions. This representation shows how the different lesions respond individually as well as the sum of the changesin the target lesions. Follow-up scans (�) were performed every 8 weeks. B, Integrative Genomics Viewer (IGV)–based visualization of reads of targeted NGS thatidentify the wild type KRAS (baseline, top) and an acquired C!T mutation in 38% of reads, encoding a KRASG12S mutation (relapse, bottom). C, long-termviability assay of PC9KRASWT and PC9KRAS-G12S cells treated with AZD9291 at the indicated concentrations for 6 days. Cell viability is presented as thepercentage of change in cell number (compared with baseline). D, long-term proliferation assay of PC9KRAS-WT and PC9KRAS-G12S cells exposed to increasingconcentrations of AZD9291 for 6 days. Cells were stained using crystal violet; �� , P < 0.01; �� , P < 0.001. E, cellular signaling of PC9KRAS-WT and PC9KRAS-G12S cellstreated with increasing concentrations of AZD9291 for 24 hours. Whole-cell lysates were analyzed by immunoblotting. F, long-term viability assay of PC9KRAS-G12S

cells demonstrating the change in cell number after treatment with AZD9291 (100 nmol/L) alone or in combination with either trametinib (1 mmol/L) orselumetinib (1 mmol/L), in comparison with the cell number before drug exposure. Cell viability was determined after 6 days of treatment. G, PC9KRAS-G12S cellswere treated with AZD9291 (100 nmol/L) alone or in combination with either trametinib (1 mmol/L) for 24 hours and lysed. Protein expression andphosphorylation levels of EGFR, AKT, ERK and the expression of KRAS, BIM, and actin were monitored by immunoblotting.

Ortiz-Cuaran et al.





signaling to a degree that would allow the emergence of cells thatharbor EGFR and KRAS mutations. Unfortunately, our analysesdo not permit distinguishing the individual clones that harboreach individual lesion.

RAS activation is an alternative bypass pathway of resistancein lung adenocarcinomas. Reactivation of the MAPK pathwayvia either KRAS copy number gain or decreased expression ofthe MAPK phosphatase DUSP6 was associated with resistanceto ALK inhibitors in patients with EML4-ALK–rearranged lungadenocarcinoma (38). Furthermore, activation of members ofthe RAS family was shown to confer resistance to ROS1 inhi-bitors (39). In EGFR-mutant lung cancers, resistance to EGFRTKIs may be associated with increased dependency on RAS-MAPK signaling, including ERK activation, loss of NF1, andCRKL amplification (13, 40–42). Amplification of MAPK1 wasreported as a resistance mechanism of WZ4002 and has beenobserved in AZD9291 (12). Moreover, NRAS mutations as wellas copy-number gain of KRAS and NRAS (12, 13) were recentlyidentified as mechanisms of resistance to AZD9291 in preclin-ical models. Our data therefore add to these findings andprovide clinical evidence for a possible role of MAPK pathwayactivation in the context of acquired resistance to third-gener-ation EGFR inhibitors.

Our findings also highlight the role of dual EGFR/MEK inhi-bition in cells that express EGFR and KRAS mutations and are inline with previous reports that identified that this combinationmay be effective in cell that display EGFR-TKI resistance throughactivation ofMAPK signaling (13). The combination of AZD9291and selumetinib is currently being evaluated in a phase I trial(NCT02143466), and may thus help to further delineate the roleof MAPK signaling in modulating the response to third-genera-tion EGFR inhibitors.

Of note, a plasma sample collected fromP7onemonth after thecervical lymph node had been obtained for these studies, revealedthe presence of a EGFR C797S mutation (AF: 1.3%), as well asEGFR T790M (AF: 0.7%), yet no KRAS mutation (Subject 2;ref. 10). Neither the EGFRC797S nor the EGFRT790M mutationswere detected in the lymph node lesion after progression (Sup-plementary Fig. S2D and S2F), thus suggesting that the KRASmutation might not necessarily be the unique driver of resistancein this patient and likely reflecting heterogeneity in the acquisitionof resistance to third-generation EGFR inhibitors. Our data pro-vide further evidence multiple clones with unique resistancemechanisms may give rise to the overall resistance observed inpatients treated with third-generation TKIs (43).

The analysis of a larger cohort of patients would be key toconfirm our observations and perform a more comprehensiveprofile of primary and/or acquired resistance to AZD9291 androciletinib. Moreover, a longitudinal analysis of the emergence ofmultiple mechanisms of resistance using liquid biopsies couldprovide evidence of the prevalence of each mechanism and helpdefining their individual role in resistance to third-generationEGFR inhibitors.

Disclosure of Potential Conflicts of InterestM. Scheffler is a consultant/advisory board member for Boehringer Ingel-

heim, and Novartis. C.M. Lovly reports receiving speakers bureau honorariafrom Abbott Molecular, and Qiagen; and is a consultant/advisory boardmember for Ariad, Clovis, Genoptix, Novartis, Pfizer, and Sequenom. S.Merkelbach-Bruse reports receiving speakers bureau honoraria from AstraZe-neca; and is a consultant/advisory board member for AstraZeneca, and Roche

Pharma. Johannes MHeuckmann holds ownership interest (including patents)in NEO NewOncology. L.C. Heukamp is an employee of NEO New Oncology.W. Pao holds ownership interest (including patents) in MolecularMD. MartinPeifer is a consultant/advisory board member for NEO New Oncology GmbH.R. B€uttner reports receiving speakers bureauhonoraria from, and is a consultant/advisory board member for Astrazeneca, Boehringer-Ingelheim, Bristol-MyersSquibb, Lilly, Merck-Serono, MSD, Pfizer, Qiagen, and Roche. J. Wolf reportsreceiving speakers bureau honoraria from AstraZeneca, Clovis, and Novartis.R.K. Thomas reports receiving speakers bureau honoraria from AstraZeneca,Bayer, Boehringer Ingelheim, Clovis, Daiichi-Sankyo, Johnson& Johnson, Lilly,Merck, MSD, Puma, Roche, and Sanofi-Aventis; and is a consultant/advisoryboard member for New Oncology AG. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: S. Ortiz-Cuaran, M. Scheffler, L. Fernandez-Cuesta,R. Buettner, J. Wolf, R.K. Thomas, M.L. SosDevelopment of methodology: S. Ortiz-Cuaran, M. Scheffler, R. Buettner,R.K. Thomas, M.L. SosAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Ortiz-Cuaran, M. Scheffler, D. Plenker, l. Dahmen,A.H. Scheel, L. Meder, C.M. Lovly, T. Persigehl, S. Merkelbach-Bruse, M. Bos,S. Michels, R. Fischer, K. Albus, H.-U. Schildhaus, J. Fassunke, M.A. Ihle,H. Pasternack, C. Becker, J. Altm€uller, J.M. Heuckmann, P. Nuernberg, W. Pao,J. Wolf, R.K. ThomasAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.Ortiz-Cuaran,M. Scheffler, D. Plenker, T. Persigehl,S. Merkelbach-Bruse, K. K€onig, H.-U. Schildhaus, J. Fassunke, H. Pasternack,J.M. Heuckmann, L.C. Heukamp, J. Berg, W. Pao, M. Peifer, R. Buettner, J. Wolf,M.L. SosWriting, review, and/or revision of the manuscript: S. Ortiz-Cuaran,M. Scheffler, L. Fernandez-Cuesta, L. Meder, C.M. Lovly, T. Persigehl,S. Merkelbach-Bruse, R. Fischer, H. Pasternack, J.M. Heuckmann, P. Nuernberg,W. Pao, R. Buettner, J. Wolf, R.K. Thomas, M.L. SosAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Ortiz-Cuaran, A.H. Scheel, T. Persigehl,M.A. Ihle, C. Heydt, H. Ji, C. M€uller, A. Florin, P. Nuernberg, S. Ans�en,L.C. Heukamp, R. BuettnerStudy supervision: S. Ortiz-Cuaran, M. Scheffler, R. Buettner, J. Wolf,R.K. Thomas, M.L. Sos

AcknowledgmentsWeare grateful to all the patientswho contributed their tumor specimens.We

thank Lead Discovery GmbH for synthetizing the compounds used for thisstudy, as well as the computing center of the University of Cologne (RRZK) forproviding the CPU time on the DFG-funded supercomputer "CHEOPS." Wethank Nike Bahlmann, Seda Ercanoglu, Graziella Bosco, Gladys Cuar�an andXimena Ortiz for their technical assistance.

Grant SupportThis work was supported by the German federal state North Rhine West-

phalia (NRW) and by the European Union (European Regional DevelopmentFund: Investing in Your Future) as part of the PerMed. NRW initiative (grant005-1111-0025; to R.K. Thomas, J. Wolf, and R. Buettner) and by the GermanMinistry of Science and Education (BMBF) as part of the e:Med program (grantno. 01ZX1303; to R.K. Thomas, J. Wolf, R. Buettner, andM. Peifer and grant no.01ZX1406; to M.L. Sos and M. Peifer). Additional funding was provided by theGerman Cancer Aid (Deutsche Krebshilfe) as part of the small-cell lung cancergenome sequencing consortium (grant ID: 109679; to R.K. Thomas, M. Peifer,and R. Buettner), by SFB832 (TP6; to R.K. Thomas), by the Deutsche For-schungsgemeinschaft (DFG; through TH1386/3-1; to R.K. Thomas and M.L.Sos), by the Deutsche Krebshilfe as part of the Oncology Centers of Excellencefunding program (to R. Buettner, R.K. Thomas, and J. Wolf) by the EU-Framework program CURELUNG (HEALTH-F2-2010-258677; to R.K. Thomasand J. Wolf), by a Stand Up to Cancer Innovative Research Grant (SU2C-AACR-IR60109; to R.K. Thomas) Stand Up To Cancer is a program of the Entertain-ment Industry Foundation administered by the American Association forCancer Research; and by the German Consortium for Translational CancerResearch (DKTK) Joint Funding program. This study was supported in part bythe National Institutes of Health (NIH) and National Cancer Institute (NCI)






R01CA121210 (to C.M. Lovly) and P01CA129243 (to C.M. Lovly). C.M. Lovlywas additionally supported by a Damon Runyon Clinical Investigator Award aLUNGevity Career Development Award, a V Foundation Scholar in TrainingAward, and an AACR-Genentech Career Development Award.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 8, 2015; revised May 17, 2016; accepted May 21, 2016;published OnlineFirst June 1, 2016.

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2016;22:4837-4847. Published OnlineFirst June 1, 2016.Clin Cancer Res Sandra Ortiz-Cuaran, Matthias Scheffler, Dennis Plenker, et al. Third-Generation EGFR InhibitorsHeterogeneous Mechanisms of Primary and Acquired Resistance to

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