heterogeneous mechanisms of primary and acquired...

1

Heterogeneous mechanisms of primary and acquired resistance to third-

generation EGFR inhibitors

Sandra 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 König8, Hans-Ulrich Schildhaus9, Jana Fassunke4,

Michaela A. Ihle4, Helen Pasternack4,10, Carina Heydt4, Christian Becker11, Janine

Altmüller11, Hongbin Ji12,13, Christian Müller1, Alexandra Florin4, Johannes M.

Heuckmann14, Peter Nuernberg11, Sascha Ansén2, Lukas C. Heukamp4,14, Johannes

Berg15, William Pao6,Ψ, Martin Peifer1,16, Reinhard Buettner4,*, Jürgen Wolf2,*, Roman K.

Thomas1,4,*, Martin L. Sos3,*

1 Department of Translational Genomics, Center of Integrated Oncology Cologne–Bonn,

Medical Faculty, University of Cologne, 50931, Cologne, Germany.

2 Lung Cancer Group Cologne and Network Genomic Medicine (Lung Cancer),

Department I of Internal Medicine, Center for Integrated Oncology Cologne-Bonn,

University Hospital Cologne, Cologne, 50931, Cologne, Germany.

3 Molecular Pathology, Center of Integrated Oncology, University Hospital Cologne,

50937, Cologne, Germany.

4 Institute of Pathology, Center of Integrated Oncology, University Hospital Cologne,

50937, Cologne, Germany.

Research. on May 19, 2018. © 2016 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 1, 2016; DOI: 10.1158/1078-0432.CCR-15-1915

http://clincancerres.aacrjournals.org/

2

5 Genetic Cancer Susceptibility Group, Section of Genetics, International Agency for

Research on Cancer (IARC-WHO), 69008 Lyon, France.

6 Department of Medicine, Vanderbilt University, Nashville, Tennessee 37212, USA.

7 Radiology Department, University Hospital Cologne, 50937, Cologne, Germany.

8 Labor Dr. Quade und Kollegen GmbH, Aachener Str. 338, 50933 Cologne, Germany.

9 Institute of Pathology, University Hospital Goettingen. Goettingen, Germany.

10 Pathology of the University Hospital of Luebeck and Leibniz Research Center Borstel,

Lübeck and Borstel, Germany.

11Cologne Center for Genomics (CCG), University of Cologne, 50931 Cologne,

Germany.

12 Key Laboratory of Systems Biology, CAS Center for Excellence in Molecular Cell

Science, Innovation Center for Cell Signaling Network, Institute of Biochemistry and Cell

Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science,

Shanghai, 200031, China.

13School of Life Science and Technology, Shanghai Tech University, Shanghai, 200120,

China.

14 NEO New Oncology AG, 51105, Cologne, Germany.

15 Institute for Theoretical Physics. University of Cologne. 50931 Cologne, Germany.

16 Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne,

Germany.

†These authors contributed equally to this work.




3

ψ Current address: Roche Innovation Center Basel, Pharma Research and Early

Development, Roche, Basel, Switzerland.

Running Title

Resistant mechanisms to AZD9291 and rociletinib.

Keywords

Resistance, EGFR, adenocarcinoma, AZD9291, rociletinib

Financial Support

This work was supported by the German federal state North Rhine Westphalia (NRW)

and by the European Union (European Regional Development Fund: Investing In Your

Future) as part of the PerMed.NRW initiative (grant 005-1111-0025 to R.K.T., J.W. and

R.B) and by the German Ministry of Science and Education (BMBF) as part of the

e:Med program (grant no. 01ZX1303 to R.K.T., J.W., R.B. and M.P. and grant no.

01ZX1406 to M.L.S and M.P.). Additional funding was provided by the German Cancer

Aid (Deutsche Krebshilfe) as part of the small cell lung cancer genome sequencing

consortium (grant ID: 109679 to R.K.T., M.P., R.B.), by SFB832 (TP6 to R.K.T.), by the

Deutsche Forschungsgemeinschaft (DFG; through TH1386/3-1 to R.K.T and M.L.S.), by

the Deutsche Krebshilfe as part of the Oncology Centers of Excellence funding program

(to R.B., R.K.T. and J.W) by the EU-Framework program CURELUNG (HEALTH-F2-

2010-258677 to R.K.T. and J.W.), by a Stand Up to Cancer Innovative Research Grant




4

(SU2C-AACR-IR60109 to R.K.T.) Stand Up To Cancer is a program of the

Entertainment Industry Foundation administered by the American Association for

Cancer Research, by the German Consortium for Translational Cancer Research

(DKTK) Joint Funding program. This study was supported in part by the National

Institutes of Health (NIH) and National Cancer Institute (NCI) R01CA121210 (C.M.L.)

and P01CA129243 (C.M.L.). C.M.L. was additionally supported by a Damon Runyon

Clinical Investigator Award a LUNGevity Career Development Award, a V Foundation

Scholar in Training Award, and an AACR-Genentech Career Development Award.

Corresponding authors (*)

Martin L. Sos

[email protected]

Roman K. Thomas

[email protected]

Jürgen Wolf

[email protected]

Reinhard Buettner

[email protected]

Conflicts of Interest:

R.K.T. is a founder and shareholder of NEO New Oncology AG, received commercial

research grants from AstraZeneca, EOS and Merck KgaA and honoraria from




5

AstraZeneca, Bayer, NEO New Oncology AG, Boehringer Ingelheim, Clovis Oncology,

Daiichi-Sankyo, Eli Lilly, Johnson & Johnson, Merck KgaA, MSD, Puma, Roche and

Sanofi. M.P. is a founder and shareholder of NEO New Oncology AG and receives

consultation fees from NEO New Oncology AG. J.W. Consultant or Advisory Role and

Honoraria from Roche, Novartis, Pfizer, Boehringer-Ingelheim, AstraZeneca, Bayer

Pharmaceuticals, Eli Lilly, MSD and Clovis Oncology. C.M.L. has served as a consultant

for Pfizer, Novartis, Genoptix, Sequenom, and Ariad and has been an invited speaker

for Abbott and Qiagen. M.S. Consultant or Advisory role for Novartis, Boehringer

Ingelheim, and BMS, honoraria (lectures) from Novartis, AstraZeneca, Boehringer

Ingelheim, Roche, BMS. J.M.H. is a founder, employee and shareholder of NEO New

Oncology AG. S.M.B: received grants from Novartis and Merck Serono and honoraria

from Roche, AstraZeneca and Pfizer. M.L.S received a commercial research grant from

Novartis. R.B is a co-founder and CSO of Targos Molecular Pathology Inc, Kassel,

Germany. He has received lecture honoraria and participated in scientific advisory

boards with AstraZeneca, Roche, Pfizer, Novartis, Qiagen, Lilly, Boehringer, MSD, BMS

and Merck-Serono. H.U.S. has received research grants by Novartis Oncology and

ZytoVision, and honoraria and reimbursements for advisory membership and lectures

by Pfizer, Novartis, Roche, Zytomed, ZytoVision and Abbott Molecular. J.F received

honoraria from Roche.

Word count: 3502

Figures: 3 main, 4 supplementary




6

Tables: 1 main

Translational relevance

Although the majority of EGFRT790M-mutant adenocarcinomas of the lung respond well

to third-generation EGFR inhibitors, resistance to these inhibitors remains a major

challenge in the field. We uncover ERBB2 and MET activation as potential bypass-track

mechanisms to third-generation EGFR inhibitors and suggest that treatment with third-

generation EGFR inhibitors may select for EGFR-mutant subclones that tolerate co-

occuring mutations in the MAPK pathway. Together with previous preclinical studies our

data supports the notion that activation of MAPK signaling might play a role in

resistance to these drugs. Our findings might be of broad interest to basic cancer

scientists and medical oncologists alike, as they provide insight related to the biology of

EGFR-mutant adenocarcinoma and have immediate implications for genetically

stratified treatment of these patients.




7

Abstract

Purpose: To identify novel mechanisms of resistance to third-generation EGFR

inhibitors in lung adenocarcinoma patients that progressed under therapy with either

AZD9291 or rociletinib (CO-1686).

Experimental Design: We analyzed tumor biopsies from seven patients obtained

before, during and/or after treatment with AZD9291or rociletinib (CO-1686). Targeted

sequencing and FISH analyses were performed and the relevance of candidate genes

was functionally assessed in in vitro models.

Results: We found recurrent amplification of either MET or ERBB2 in tumors that were

resistant or developed resistance to third-generation EGFR inhibitors and show that

ERBB2 and MET activation can confer resistance to these compounds. Furthermore,

we identified a KRASG12S mutation in a patient with acquired resistance to AZD9291 as

a potential driver of acquired resistance. Finally, we show that dual inhibition of

EGFR/MEK might be a viable strategy to overcome resistance in EGFR-mutant cells

expressing mutant KRAS.

Conclusions: Our data suggests that heterogeneous mechanisms of resistance can

drive primary and acquired resistance to third-generation EGFR inhibitors and provides

a rationale for potential combination strategies.




8

Introduction

In EGFR-mutant lung adenocarcinoma, targeted therapy with EGFR tyrosine

kinase inhibitors (TKIs) performs substantially better than 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 PFS of 7 to

16 months.

The major cause of resistance is the EGFR mutation T790M, at the gatekeeper

site (50-60%) (2–4). A third generation of covalent EGFR TKIs that specifically target

EGFR T790M as well as the activating EGFR mutations (e.g., L858R or exon 19

deletion), while sparing wild-type EGFR, have been evaluated clinically (5,6). In phase I

trials, rociletinib and AZD9291 have shown impressive clinical activity in patients with

EGFRT790M-mediated acquired resistance to reversible EGFR-TKIs with response rates

of 61% and 59%, and PFS of 13.5 and 13.1 months, respectively (5–9).

The acquisition of a secondary EGFR mutation, C797S, has been recently

reported in patients with relapse under therapy with AZD9291 (10,11). In preclinical

models, resistance to these inhibitors can also be driven by activation of the MAPK

pathway (12–14). These observations are of particular interest since EGFR mutations

are known to mainly occur in mutually exclusive manner with oncogenic mutations

within the MAPK pathway (15,16).

We performed genetic analyses on biopsies obtained from patients treated with

third-generation EGFR inhibitors in order to identify additional mechanisms of resistance

to these drugs.




9

Patients and Methods

Study population

Whenever possible biopsies were obtained before treatment and after radiographic

progression under TKI therapy (with the exception of P2 and P3), at the times and with

the methods described in Table 1. Standard histopathology was performed to confirm

the histological subtype. Patients were treated in trials NCT01802632 and

NCT01526928. The Institutional Review Board (IRB) and the responsible ethics

committee approved collection and use of all patient specimens in this study. Written

informed consent was obtained from all subjects.

FISH analyses

Fluorescence in situ hybridization (FISH) for MET and ERBB2 was performed on

formalin-fixed paraffin-embedded tissue using labeled dual-color probes. Sections of

1.5µm were cut and hybridized with labeled probes for MET or ERBB2 and the

respective centromeric reference probe (ZytoLight Spec MET/CEN7 probes; ZytoLight

Spec ERBB2/CEN17 probes by ZytoVision, Bremerhaven, Germany). Slides were

reviewed at 630x and scored according to appropriate guidelines (17). For ERBB2 the

gastric cancer scoring system was used since no lung-specific recommendations are

available (18) .

DNA extraction and sequencing

Total DNA was obtained from formalin-fixed paraffin-embedded tumor tissue. DNA from

sections was extracted using the Puregene Extraction kit (Qiagen) according to the

manufacturer's instructions. DNA was eluted in 1× TE buffer (Qiagen), diluted to a

working concentration of 150 ng/µl and stored at –80 °C.




10

Targeted sequencing

Targeted sequencing analysis was performed as described previously (19) using a

custom-made lung cancer panel consisting of 102 amplicons for the detection of hotspot

mutations in 14 lung cancer related genes. Isolated DNA (up to 50 ng) was amplified

with two customized Ion AmpliSeq™ Primer Pools. Library products were quantified

using Qubit® 2.0 Fluorometer (Qubit®ds DNA HS Kit, Life Technologies™), diluted and

pooled in equal amounts. 6-8 pM were spiked with 5% PhiX DNA (Illumina® San Diego,

CA, USA) and sequenced with the MiSeq™ reagent Kit V2 (300-cycles, Illumina®). Data

were exported as FASTQ files and analyzed using the in-house pipeline.

Generation of stably transduced cell lines

H1975 (CRL-5908) and HCC827 (CRL-2868) cells were obtained from the American

Type Culture Collection (ATCC). PC9, PC9GR and HCC827GR cells were kindly

provided by Dr. Hongbin Ji (Shanghai Institutes for Biological Sciences, China), Dr.

Passi Jänne (Broad Institute, USA), and Dr. Jeffrey Engelman (MGH, USA).

respectively. Cells were cultured with RMPI medium supplemented with 10% FBS and

1% penicillin/streptomycin at 37ºC under 5% CO2.

Generation of cells harboring pBabe-puro empty vector, EGFRT790M, ERBB2WT, KRASWT

or KRASG12S was done by stable transduction as described previously (20) using a

pBabe-puro retroviral vector (Addgene). Cells expressing the corresponding mutants

were generated by retroviral transduction and subsequent puromycin selection. The

expression of the mutant was verified by RT-PCR and further Sanger sequencing and

by Western blot analysis.




11

cDNA transcription

RNA was isolated from PC9KRAS-G12S and HCC827KRAS-G12S cells using TRIZOL reagent

(Invitrogen) and cleaned up using the RNeasy MinElute Cleanup Kit (Qiagen) following

the manufacturers’ protocols. Finally, 1 μg of RNA was transcribed into cDNA using

Superscript III reverse transcriptase (Invitrogen, #18064).

Reagents

AZD9291, rociletinib, afatinib, crizotinib, trametinib and selumetinib were synthesized

according to published methods. For cell culture experiments, each compound was

dissolved in dimethyl sulfoxide (DMSO), aliquoted and stored as 10mM stocks at -80°C.

HGF (R&D Systems) was re-suspended in PBS +0.1 % BSA and aliquots stored at -

20ºC.

Cell viability assays

Cells were plated into 96-well culture plates in RMPI medium supplemented with 10%

FBS and 1% penicillin/streptomycin, at a density of 1500 cells/well, cultured overnight

and treated the following day. Cell viability was determined after 4 to 6 days by

measuring cellular ATP content (CellTiter-Glo, Promega) as described previously (20).

GI50 values were calculated by plotting luminescence intensity against drug

concentration in nonlinear curves using GraphPad Prism (GraphPad). Synergy scores

were calculated as previously described (21). For long-term viability assays, the

reported “Cell number change (%)” was determined as: 100*(Value/Baseline)/Baseline.

Each point was performed with three replicates. Unless otherwise noted, three

independent experiments were performed and a representative result is presented.

Error bars are mean -/+ standard deviation (SD).




12

Crystal Violet Staining

Cells were plated at a density of one million cells/well and incubated overnight. Cells

were treated with AZD9291 the following day and then again 72 hours after. After 6

days of drug exposure, cells were fixed with 1% paraformaldehyde. For staining, a

solution of 0.1% crystal violet was added and incubated for 20 minutes. Plates were

inverted and dried overnight and images were taken the next day.

Western blot analyses

Cells lysates were blotted as described previously (20). The following antibodies were

used: phospho-EGFR (Y1068), total EGFR, 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 Technology) and conjugated

antibodies to rabbit and mouse (Millipore).

Statistical analyses

Statistical analyses were performed using Graph Pad Prism 5.0 (USA). We used

student’s t-test (unpaired, 2-sided). A p-value of <0.05 was used as a threshold

considered to call statistical significance. PFS was assessed from start of the respective

therapy until progression or death. Survival analyses were performed using Kaplan

Meier statistics, providing median and 95% confidence interval (CI). For response

evaluation, the Response Evaluation Criteria in Solid Tumors (RECIST V1.1) (22) were

used.




13

Results

Mechanisms of primary and acquired resistance to third generation EGFR inhibitors

We obtained specimens from seven patients treated with third-generation EGFR

inhibitors AZD9291 (n=5) or rociletinib (n=2), within two clinical trials (clinicaltrials.gov,

NCT01802632 and NCT02147990) after acquired EGFRT790M positive resistance to first-

or second-generation EGFR kinase inhibitors. The PFS for third-generation EGFR TKI

therapy ranged from 1.2 to 19.4 months (median 4.2 months, CI 3.7 - 4.7 months,

Figure 1A). The best reduction of the sum of the largest tumor diameters under

treatment, according to RECIST V1.1 (22), ranged between 0.1% and 79.2%, with three

patients presenting growth of non-target lesions (P1, P3 and P5, Figure 1B).

The pattern of response varied across these patients: two patients (P1, P3)

experienced primary resistance to rociletinib (defined as progression on the first

restaging scan), while two patients had stable disease (P2, P4) under treatment with

AZD9291. Finally, three cases (P5, P6 and P7) presented with partial responses to

treatment with AZD9291, with reductions in diameters of more than 50% (Figure 1A and

1B). For these patients, unfortunately, disease recurred after 16.5, 19.4 and 7 months,

respectively.

In order to characterize the molecular mechanisms that may be linked with such

heterogeneous response patterns, we performed fluorescence in-situ hybridization

(FISH) for known EGFR resistance markers (ERBB2 and MET) and targeted next-

generation sequencing on tumor tissue from these patients (see Methods). FISH

analyses of a massively progressive pleural effusion (non-target lesion) collected after

three weeks of treatment initiation from P1 (PD, 625 mg rociletinib twice daily) and a




14

lung biopsy sample collected before treatment from P2 (SD, 180mg AZD9291 daily),

revealed ERBB2 amplification (7.3 and 3.7 gene copies, respectively) (Figure 1C).

Since ERBB2 amplification has been shown to confer resistance to first

generation EGFR TKIs (23), we hypothesized that amplified ERBB2 might substitute for

EGFR signaling in this context and explain the lack of response to of AZD9291 and

rociletinib in P1 and P2. To test this hypothesis, we evaluated the impact of ERBB2

overexpression on the sensitivity to AZD9291 and rociletinib in PC9GR cells

(EGFRT790M). Despite relatively a relatively low overexpression of ERBB2 in PC9GR

cells we observed decreased sensitivity to both drugs at nanomolar concentrations

(Figure 1D and Supplementary Figure 1A). Thus, our results indicate that ERBB2

activation may contribute to resistance against rociletinib and AZD9291.

The tumor biopsy collected before treatment of P3 (PD, 625mg rociletinib twice

daily) showed high-level amplification of MET (MET/CEN7 ratios 2.10, 4.42 MET

copies) (Figure 2A). Unfortunately, no baseline comparative sample (prior to erlotinib

treatment) was available for this patient. In the case of P4 (SD, 80mg AZD9291 daily)

an initial tumor reduction (20.8%) was followed by an increase in the tumor volume after

4.1 months of treatment (Figure 2B). In this patient too, the biopsy taken after the initial

response exhibited high-level amplification of MET (MET/CEN7 ratio 5.35, 11.42 MET

copies) that was not observed before therapy with AZD9291 (Figure 2C). P5 (PR, 80

mg AZD9291 daily) had an initial tumor response of 79% and presented a new lesion in

the liver that harbored a high-level MET amplification (MET/CEN7 ratios 1.88, 21.92

MET copies, Figure 2D, left panel) and ERBB2 amplification (4.25 gene copies, Figure




15

2D, right panel). Of note, targeted sequencing of these samples did not show additional

clinically relevant mutations (Table 1).

To functionally test whether MET amplification might reduce the sensitivity to

AZD9291 or rociletinib, we stably overexpressed EGFRT790M-mutant constructs in

HCC827 cells (MET wild type) and their gefitinib-resistant derivate, HCC827GR cells

(MET amplified) (Supplementary Figure 1B). In line with previous reports (24), the

presence of MET amplification decreased the sensitivity to both third-generation EGFR

inhibitors (Figure 2E) and led to sustained phosphorylation of ERK and AKT (Figure 2F).

Furthermore, concomitant inhibition with either AZD9291 or rociletinib and the MET

inhibitor, crizotinib, resulted in pronounced cytotoxicity and reduced AKT and ERK

activation in HCC827GREGFR-T790M cells (Figure 2E and 2G). These results were

confirmed in EGFRT790M expressing cells (H1975 and PC9GR) where MET was

activated through addition of exogenous HGF (Supplementary Figure 1 C-F).

Thus, our clinical observations are in line with previous reports where both

ERBB2 and MET amplification has been found to be associate with loss of efficacy of

third-generation EGFR inhibitors in EGFR-mutant tumors (25). More importantly, we

provide functional evidence that activation of ERBB2 and MET signaling may induce

resistance to this new class of EGFR inhibitors.

Finally, patient 6 (P6) presented with a tumor response of 72.6% followed by

emergence of new liver metastases 19.4 months after start of therapy. The liver biopsy

revealed the retention of the initial EGFR exon19 deletion (allelic fraction, AF: 42.6%,

Supplementary Figure 2A) and the EGFRT790M mutation (AF: 27.66%) together with the

presence of a secondary EGFRC797S mutation (Figure 2H) that was not present in the




16

pre-treatment biopsy (Supplementary Figure 2B). Of note, the liver biopsy obtained at

relapse also harbored an intermediate-level MET amplification (MET/CEN7 ratios 1.14,

5.67 MET copies, Figure 2I). The EGFRC797S mutation abrogates the irreversible binding

of third-generation EGFR inhibitors and has been described to confer resistance to

AZD9291 thus providing a rational for the observed resistance phenotype in P6

(10,26,27).

KRAS mutation and resistance to third-generation EGFR inhibition

In our cohort, a forth patient (P7) relapsed after having initially responded to

treatment with AZD9291. This patient received different lines of treatment before

developing resistance through EGFRT790M under combination therapy with afatinib and

cetuximab. After enrollment into the AURA trial (NCT01802632) the tumor responded to

AZD9291 treatment (160 mg/d) with a PR (best response: 54.3%, Figure 3A) for 8

months. However, the fifth follow-up revealed the appearance of a new lesion in a fast

growing cervical lymph node. Targeted sequencing of the specimens obtained before

and after treatment with AZD9291 (both from the cervical lymph nodes) showed a slight

decrease in the fraction of reads containing the original activating EGFR mutation (43%

before treatment with AZD9291 vs. 32% after treatment, Supplementary Figure 2C) and

the disappearance of EGFRT790M (25% before treatment with AZD9291, Supplementary

Figure 2D). Finally, in two independent sequencing runs we found a novel KRASG12S

mutation (38% of the reads), which had not been detected before therapy with AZD9291

(Figure 3B). In general, the disproportionate levels of C>T/G>A changes that occur as a

consequence of formalin fixation are more apparent at low allelic fractions (1-10%) (28).




17

In the relapse sample of P7, the C>T/G>A changes where comparable to other patient

samples or freshly prepared DNA from cell lines sequenced in the same batch,

suggesting that the KRAS mutation detected is not a formalin-fixation artifact

(Supplementary Figure 2E). The EGFRC797S mutation had also been found in the

plasma of this patient (10) but was not observed in the lymph node biopsy

(Supplementary Figure 2F). Moreover, the KRASG12S mutation was not detected in the

plasma of this patient. These observations suggest, that heterogeneous mechanisms of

acquired resistance might be operant in this patient.

To investigate whether expression of an activated KRAS allele might contribute

to resistance to third-generation EGFR inhibitors we stably transduced EGFR-mutant

PC9 cells with the KRASG12S-mutant. PC9 cells that expressed the KRASG12S-mutant

were selected under treatment with puromycin for at least two weeks. The resulting

PC9KRAS-G12S cells expressed both the original EGFR exon19 deletion and the KRASG12S

mutation (Supplementary Figure 3A). Concomitant expression of both mutants was also

observed in a PC9KRAS-G12S clone obtained by serial dilution (Supplementary Figure 3B).

When compared to the KRAS wild type transduced cells (PC9KRAS-wt), PC9KRAS-

G12S were less sensitive to AZD9291 (Figure 3C and 3D). Furthermore, introduction of

KRASG12S resulted in increased KRAS expression and sustained ERK phosphorylation

under treatment with AZD9291 (Figure 3E). Similar results were observed in

HCC827KRAS-G12S cells (Supplementary Figures 3C and 3D) where the introduction of

the KRAS mutation also resulted in decreased sensitivity to rociletinib (Supplementary

Figure 3C). Confirming a role of MAPK signaling in inducing resistance in PC9KRAS-G12S

cells, combined treatment with AZD9291 and the MEK inhibitor, trametinib, was highly




18

synergistic (synergy score: 5.48 (21), p-value 4.14E-08) (Figure 3F and Supplementary

Figure 4A). Consistent with these results, AZD9291 failed to fully inhibit downstream

MAPK signaling except in the presence of trametinib (Figure 3G). Similar results were

observed with the combination of AZD9291 and selumetinib (Figure 3F and

Supplementary Figure 4B). Finally, concomitant EGFR and MEK inhibition was also

synergistic in HCC827KRAS-G12S cells (Supplementary Figures 4C and 4D). These

findings demonstrate that mutant KRAS may induce acquired resistance to third-

generation EGFR inhibitors.

Discussion

Here, we provide clinical and functional evidence for the role of ERBB2 and MET

amplifications as well as KRAS mutations as possible mechanisms of resistance to

third-generation EGFR inhibitors. The role of ERBB2 activation in resistance has been

previously reported for first-generation EGFR inhibitors (23). The activity of AZD9291

against ERBB2 is known to be limited and despite the presence of a metabolite AZ5104

that shows inhibition of ERBB2 it is conceivable that ERBB2 signaling may reduce the

overall activity of third generation inhibitors in patients (6). Since the potency against

ERBB2 was greater with the AZ5104 metabolite than with AZD9291, it would be

interesting to determine the metabolites levels in the plasma of patients to predict the

emergence of ERBB2 as a potential bypass mechanism of resistance to AZD921. Our

functional in vitro results suggest that the activity of both AZD9291 and rociletinib may

decrease through specific activation of ERBB2 signaling. These observations go in line

with the studies reporting ERBB2 as a mechanism of resistance to first-generation




19

EGFR inhibitors (29). The use of patient-derived EGFRT790M-mutant cell lines may help

defining the activity of these inhibitors on ERBB2 and characterizing the molecular

context of the role of ERBB2 as a mechanism of resistance to third-generation EGFR

inhibitors.

The role of MET amplification in acquired resistance to first-generation EGFR

inhibitors has been studied extensively (30–32). According to our results MET activation

may similarly play a role in resistance to third-generation EGFR inhibitors since MET is

not a relevant off-target for these drugs. Our observations also confirm previous reports

on the efficacy of the combination of rociletinib with crizotinib in these tumors (33).

Combination therapies have proven successful in the setting of resistance to first-

generation EGFR inhibitors (34) and early phase clinical trials are evaluating the

combination of rociletinib with crizotinib (33), or AZD9291 with the MET inhibitor,

savolitinib, in EGFR-mutant lung cancer (35). It might therefore be important to

determine the presence of these alterations before therapy with any EGFR inhibitor.

Interestingly, we identify an activating KRASG12S mutation in a tumor rebiopsy

obtained at the time of acquired resistance to AZD9291. This mutation is of particular

interest since EGFR and KRAS mutations typically occur in a mutually exclusive fashion

in lung cancer (16,36). Indeed, a recent report evidenced that the expression of

inducible KRASG12V constructs in EGFR-mutant PC9 cells results in decreased cell

viability after seven days of selection with doxycyclin (37). Anecdotal reports show

evidence of co-occurrence of KRAS and EGFR mutations in lung cancer patients

(38,39). More recently Hata et al. described two distinct evolutionary paths for the

development of resistance to EGFR inhibition (14). In this study cell lines that developed




20

resistance at late stages were enriched for NRAS and KRAS mutations suggesting that

under prolonged selection a distinct population of EGFR mutant cells may well tolerate

the presence of oncogenic MAPK pathway activation. In our functional experiments,

decreased cell viability was observed in cells expressing both EGFR and KRAS

mutants; however, this phenotype was overcome after about 10 passages under

antibiotic selection. In line with our results, in the study performed by Unni et al. (37) the

induction of mutant KRAS rescued PC9 cells from the lethal effects of erlotinib, thus

implying that the toxicity of co-expression of mutant KRAS and EGFR depend on the

kinase activity of mutant EGFR. We speculate that EGFR inhibition through AZD9291

may functionally deplete oncogenic EGFR signaling to a degree that would allow the

emergence of cells that harbor EGFR and KRAS mutations. Unfortunately, our analyses

do not permit distinguishing the individual clones that harbor each individual lesion.

RAS activation is an alternative bypass pathway of resistance in lung

adenocarcinomas. Reactivation of the MAPK pathway via either KRAS copy number

gain or decreased expression of the MAPK phosphatase DUSP6 was associated with

resistance to ALK inhibitors in patients with EML4-ALK–rearranged lung

adenocarcinoma (40). Furthermore, activation of members of the RAS family was

shown to confer resistance to ROS1 inhibitors (41). In EGFR-mutant lung cancers,

resistance to EGFR TKIs may be associated with increased dependency on RAS-MAPK

signaling, including ERK activation, loss of NF1 and CRKL amplification (13,42–44).

Amplification of MAPK1 was reported as a resistance mechanism of WZ4002 and has

been observed in AZD9291 (12). Moreover, NRAS mutations as well as copy number

gain of KRAS and NRAS (12,13) were recently identified as mechanisms of resistance




21

to AZD9291 in preclinical models. Our data therefore add to these findings and provide

clinical evidence for a possible role of MAPK pathway activation in the context of

acquired resistance to third-generation EGFR inhibitors.

Our findings also highlight the role of dual EGFR/MEK inhibition in cells that

express EGFR and KRAS mutations and are in line with previous reports that identified

that this combination may be effective in cell that display EGFR-TKI resistance through

activation of MAPK signaling (13). The combination of AZD9291 and selumetinib is

currently being evaluated in a phase I trial (NCT02143466) and may thus help to further

delineate the role of MAPK signaling in modulating the response to third-generation

EGFR inhibitors.

Of note, a plasma sample collected from P7 one month after the cervical lymph

node had been obtained for these studies, revealed the presence of a EGFR C797S

mutation (AF: 1.3%), as well as EGFR T790M (AF: 0.7%), yet no KRAS mutation

(Subject 2, (10)). Neither the EGFRC797S nor the EGFRT790M mutations were detected in

the lymph node lesion after progression (Supplementary Figures 2D and 2F), thus

suggesting that the KRAS mutation might not necessarily be the unique driver of

resistance in this patient and likely reflecting heterogeneity in the acquisition of

resistance to third-generation EGFR inhibitors. Our data provides further evidence

multiple clones with unique resistance mechanisms may give rise to the overall

resistance observed in patients treated with third-generation tyrosine kinase inhibitors

(45).

The analysis of a larger cohort of patients would be key to confirm our

observations and perform a more comprehensive profile of primary and/or acquired




22

resistance to AZD9291 and rociletinib. Moreover, a longitudinal analysis of the

emergence of multiple mechanisms of resistance using liquid biopsies could provide

evidence of the prevalence of each mechanism and help defining their individual role in

resistance to third-generation EGFR inhibitors.




23

References

1. Ohashi K, Maruvka YE, Michor F, Pao W. Epidermal growth factor receptor tyrosine kinase inhibitor-resistant disease. J Clin Oncol. 2013;31:1070–80.

2. Pao W, Miller V a, Politi K a, Riely GJ, Somwar R, Zakowski MF, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2005;2:e73.

3. Kobayashi S, Boggon TJ, Dayaram T, Jänne PA, Kocher O, Meyerson M, et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med. 2005;352:786–92.

4. Yun C-H, Mengwasser KE, Toms A V, Woo MS, Greulich H, Wong K-K, et al. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc Natl Acad Sci U S A. 2008;105:2070–5.

5. Walter AO, Sjin RTT, Haringsma HJ, Ohashi K, Sun J, Lee K, et al. Discovery of a mutant-selective covalent inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC. Cancer Discov. 2013;3:1404–15.

6. Cross DAE, Ashton SE, Ghiorghiu S, Eberlein C, Nebhan CA, Spitzler PJ, et al. AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer. Cancer Discov. 2014;4:1046–61.

7. Jänne PA, Yang JC-H, Kim D-W, Planchard D, Ohe Y, Ramalingam SS, et al. AZD9291 in EGFR Inhibitor-Resistant Non-Small-Cell Lung Cancer. N Engl J Med. 2015;372:1689–99.

8. Sequist L V, Soria J-C, Goldman JW, Wakelee HA, Gadgeel SM, Varga A, et al. Rociletinib in EGFR-Mutated Non-Small-Cell Lung Cancer. N Engl J Med. 2015;372:1700–9.

9. Jänne PA, Ahn M, Kim D, Kim S, Planchard D, Ramalingam S., et al. A phase I study of AZD9291 in patients with EGFR-TKI-resistant advanced NSCLC – updated progression free survival and duration of response data. Geneva: European Lung Cancer Conference; 2015.

10. Thress KS, Paweletz CP, Felip E, Cho BC, Stetson D, Dougherty B, et al. Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M. Nat Med. 2015;21:560–2.

11. Yu HA, Tian SK, Drilon AE, Borsu L, Riely GJ, Arcila ME, et al. Acquired Resistance of EGFR- Mutant Lung Cancer to a T790M-Specific EGFR Inhibitor. JAMA Oncol. 2015;1:982–4.

12. Eberlein CA, Stetson D, Markovets AA, Al-Kadhimi KJ, Lai Z, Fisher PR, et al. Acquired resistance to mutant-selective EGFR inhibitor AZD9291 is associated with increased dependence on RAS signaling in preclinical models. Cancer Res. 2015; 75:2489-500




24

13. Ercan D, Xu C, Yanagita M, Monast CS, Pratilas C a, Montero J, et al. Reactivation of ERK signaling causes resistance to EGFR kinase inhibitors. Cancer Discov. 2012;2:934–47.

14. Hata AN, Niederst MJ, Archibald HL, Gomez-Caraballo M, Siddiqui FM, Mulvey HE, et al. Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition. Nat Med; 2016;22:262–9.

15. Tam IYS, Chung LP, Suen WS, Wang E, Wong MCM, Ho KK, et al. Distinct epidermal growth factor receptor and KRAS mutation patterns in non-small cell lung cancer patients with different tobacco exposure and clinicopathologic features. Clin Cancer Res. 2006 ;12:1647–53.

16. Unni AM, Lockwood WW, Zejnullahu K, Lee-Lin S-Q, Varmus H. Evidence that synthetic lethality underlies the mutual exclusivity of oncogenic KRAS and EGFR mutations in lung adenocarcinoma. Elife; 2015;4:e06907.

17. Schildhaus H-U, Schultheis AM, Rüschoff J, Binot E, Merkelbach-Bruse S, Fassunke J, et al. MET amplification status in therapy-naïve adeno- and squamous cell carcinomas of the lung. Clin Cancer Res. 2015;21:907–15.

18. Rüschoff J, Hanna W, Bilous M, Hofmann M, Osamura RY, Penault-Llorca F, et al. HER2 testing in gastric cancer: a practical approach. Mod Pathol. 2012;25:637–50.

19. König K, Peifer M, Fassunke J, Ihle MA, Künstlinger H, Heydt C, et al. Implementation of Amplicon Parallel Sequencing Leads to Improvement of Diagnosis and Therapy of Lung Cancer Patients. J Thorac Oncol. 2015;10:1049–57.

20. Sos ML, Michel K, Zander T, Weiss J, Frommolt P, Peifer M, et al. Predicting drug susceptibility of non-small cell lung cancers based on genetic lesions. J Clin Invest. 2009;119:1727–40.

21. Peifer M, Weiss J, Sos ML, Koker M, Heynck S, Netzer C, et al. Analysis of compound synergy in high-throughput cellular screens by population-based lifetime modeling. PLoS One. 2010;5:e8919.

22. Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, Rubinstein L, et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst. 2000;92:205–16.

23. Takezawa K, Pirazzoli V, Arcila ME, Nebhan CA, Song X, de Stanchina E, et al. HER2 amplification: a potential mechanism of acquired resistance to EGFR inhibition in EGFR-mutant lung cancers that lack the second-site EGFRT790M mutation. Cancer Discov. 2012;2:922–33.

24. Wilson TR, Fridlyand J, Yan Y, Penuel E, Burton L, Chan E, et al. Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors. Nature. 2012;487:505–9.




25

25. Planchard D, Loriot Y, André F, Gobert A, Auger N, Lacroix L, et al. EGFR-independent mechanisms of acquired resistance to AZD9291 in EGFR T790M-positive NSCLC patients. Ann Oncol.; 2015;26:2073–8.

26. Ercan D, Choi HG, Yun C-H, Capelletti M, Xie T, Eck MJ, et al. EGFR mutations and resistance to Irreversible pyrimidine based EGFR inhibitors. Clin Cancer Res. 2015; 21:3913-23

27. Niederst MJ, Hu H, Mulvey HE, Lockerman EL, Garcia AR, Piotrowska Z, et al. The allelic context of the C797S mutation acquired upon treatment with third generation EGFR inhibitors impacts sensitivity to subsequent treatment strategies. Clin Cancer Res. 2015;1078–0432.CCR – 15–0560

28. Wong SQ, Li J, Tan AY-C, Vedururu R, Pang J-MB, Do H, et al. Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing. BMC Med Genomics. 2014;7:23.

29. Takezawa K, Pirazzoli V, Arcila ME, Nebhan CA, Song X, de Stanchina E, et al. HER2 amplification: a potential mechanism of acquired resistance to EGFR inhibition in EGFR-mutant lung cancers that lack the second-site EGFRT790M mutation. Cancer Discov.; 2012;2:922–33.

30. Engelman J, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO, et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science. 2007;316:1039–43.

31. Turke AB, Zejnullahu K, Wu Y-L, Song Y, Dias-Santagata D, Lifshits E, et al. Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC. Cancer Cell.; 2010;17:77–88.

32. Bean J, Brennan C, Shih J-Y, Riely G, Viale A, Wang L, et al. MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci U S A. 2007;104:20932–7.

33. Haringsma HJ. Annual Meeting of the American Association of Cancer Research, Philadelphia PA, 18-22 April 2015.

34. Scheffler M, Merkelbach-Bruse S, Bos M, Fassunke J, Gardizi M, Michels S, et al. Spatial Tumor Heterogeneity in Lung Cancer with Acquired Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor Resistance: Targeting High-Level MET-Amplification and EGFR T790M Mutation Occurring at Different Sites in the Same Patient. J Thorac Oncol. 2015;10:e40–3.

35. Oxnard GR. Annual Meeting of the American Society of Clinical Oncology, Chicago IL, 29 May to 2 June 2015.

36. Pao W, Miller VA. Epidermal growth factor receptor mutations, small-molecule kinase inhibitors, and non-small-cell lung cancer: current knowledge and future directions. J Clin Oncol. 2005;23:2556–68.




26

37. Unni AM, Lockwood WW, Zejnullahu K, Lee-Lin S-Q, Varmus H. Evidence that synthetic lethality underlies the mutual exclusivity of oncogenic KRAS and EGFR mutations in lung adenocarcinoma. Elife. 2015;4: e06907.

38. Gainor JF, Varghese AM, Ou S-HI, Kabraji S, Awad MM, Katayama R, et al. ALK rearrangements are mutually exclusive with mutations in EGFR or KRAS: an analysis of 1,683 patients with non-small cell lung cancer. Clin Cancer Res. 2013;19:4273–81.

39. Choughule A, Sharma R, Trivedi V, Thavamani A, Noronha V, Joshi A, et al. Coexistence of KRAS mutation with mutant but not wild-type EGFR predicts response to tyrosine-kinase inhibitors in human lung cancer. Br J Cancer. Cancer Research UK; 2014;111:2203–4.

40. Hrustanovic G, Olivas V, Pazarentzos E, Tulpule A, Asthana S, Blakely CM, et al. RAS-MAPK dependence underlies a rational polytherapy strategy in EML4-ALK-positive lung cancer. Nat Med; 2015;21:1038–47.

41. Cargnelutti M, Corso S, Pergolizzi M, Mévellec L, Aisner DL, Dziadziuszko R, et al. Activation of RAS family members confers resistance to ROS1 targeting drugs. Oncotarget. 2015;6:5182–94.

42. de Bruin EC, Cowell C, Warne PH, Jiang M, Saunders RE, Melnick MA, et al. Reduced NF1 expression confers resistance to EGFR inhibition in lung cancer. Cancer Discov. 2014;4:606–19.

43. Suda K, Mizuuchi H, Murakami I, Uramoto H, Tanaka F, Sato K, et al. CRKL amplification is rare as a mechanism for acquired resistance to kinase inhibitors in lung cancers with epidermal growth factor receptor mutation. Lung Cancer. 201485:147-51.

44. Suda K, Mizuuchi H, Sato K, Takemoto T, Iwasaki T, Mitsudomi T. The insulin-like growth factor 1 receptor causes acquired resistance to erlotinib in lung cancer cells with the wild-type epidermal growth factor receptor. Int J Cancer. 2014135:1002-6.

45. Piotrowska Z, Niederst MJ, Karlovich CA, Wakelee HA, Neal JW, Mino-Kenudson M, et al. Heterogeneity Underlies the Emergence of EGFR T790 Wild-Type Clones Following Treatment of T790M-Positive Cancers with a Third Generation EGFR Inhibitor. Cancer Discov. 2015; 5:713-22.




27

Acknowledgments:

We are grateful to all the patients who contributed their tumor specimens. We thank

Lead Discovery GmbH for synthetizing the compounds used for this study, as well as

the computing center of the University of Cologne (RRZK) for providing the CPU time on

the DFG-funded supercomputer ‘CHEOPS’. We thank Nike Bahlmann, Seda Ercanoglu,

Graziella Bosco, Gladys Cuarán and Ximena Ortiz for their technical assistance.

Author’s Contributions:

Conception and design: S.Ortiz-Cuaran, M. Scheffler, R. Büttner, J. Wolf, M.L.Sos,

R.K. Thomas.

Development of methodology: S.Ortiz-Cuaran, M. Scheffler, L. Fernandez-Cuesta, W.

Pao, R. Büttner, J. Wolf, M.L.Sos, R.K. Thomas.

Acquisition of data (provided animals, acquired and managed patients, provided

facilities, etc.): S.Ortiz-Cuaran, M. Scheffler, D. Plenker, A. H. Scheel, I. Dahmen, L.

Meder, T. Persigehl, S. Michels, R. Fischer, M.A. Ihle, C. Becker, K. Albus, J. Fassunke,

J. Altmüller, C. Müller, P. Nuernberg, L.C. Heukamp, R. Büttner.

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,

computational analysis): S.Ortiz-Cuaran, M. Scheffler, A. H. Scheel, T. Persigehl, S.

Merkelbach-Bruse, J. Berg, M. Peifer.

Writing, review, and/or revision of the manuscript: S.Ortiz-Cuaran, M. Scheffler, J.

Wolf, M.L.Sos, R.K. Thomas.




28

Administrative, technical, or material support (i.e., reporting or organizing data,

constructing databases): D. Plenker, A. H. Scheel, I. Dahmen, C.M. Lovly, M. Bos, K.

König, S. Merkelbach-Bruse, H-U Schildhaus, H. Künstlinger, C. Heydt, H. Ji, C.

Becker, A. Florin, J. Heuckmann, P. Nuernberg, S. Ansén, R. Büttner.




29

Figure Legends

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) or at relapse

(black) under AZD9291 or rociletinib. B, waterfall plots showing best percentage change

in the size of target or non-target (*) lesions. C, FISH analyses with probes for

chromosomal loci containing ERBB2 (P1 and P2, green signal) and the respective

centromeric reference probe (red signal). D, viability assays of PC9GRe.v and

PC9GRERBB2 cells indicating the change in cell number after 6 days of treatment with

DMSO or increasing concentrations of either AZD9291 or rociletinib, in comparison to

the number of cells at the initiation of drug exposure.

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, the volumes of the target

lesions are shown and added up, according to RECIST. This representation shows how

the different lesions respond individually as well as the sum of the changes in the target

lesions. Follow-up scans (*) were performed every eight weeks. C, FISH analyses

assessing the presence of MET amplification in the liver lesion collected before

AZD9291 and after relapse. D, evaluation of MET (left panel) and ERBB2 amplification




30

(right panel) 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

to the number of cells at baseline. After 6 days of treatment, cell viability was

determined by measuring intracellular ATP content. ***, P < 0.001. HCC827EGFR-T790M

and HCC827GREGFR-T790M cells treated with AZD9291 (1µM) or rociletinib (1µM) for 24

hours and lysed. Protein expression and phosphorylation of the MET, EGFR, AKT and

ERK, as well as expression of BIM and actin was monitored by immunoblotting. G,

HCC827GREGFR-T790M cells treated with AZD9291 (1µM) or rociletinib (1µM) alone or in

combination with crizotinib (1µM) for 24 hours. Expression of the indicated proteins actin

was monitored by immunoblotting. H, Integrative Genomics Viewer (IGV) - based

visualization of reads of targeted NGS that identify the EGFRT790M and EGFRC797S

mutations in P6. I, FISH analyses with probes for chromosomal loci containing MET

(green signal) and the corresponding centromeric reference probe (red signal).

Figure 3. Acquired resistance to AZD9291 mediated by acquired KRASG12S mutation.

A, tumor response analysis for P5 using the quantitative imaging tool mint Lesion™

showing the volumes of the target lesions. This representation shows how the different

lesions respond individually as well as the sum of the changes in the target lesions.

Follow-up scans (*) were performed every eight weeks. B, Integrative Genomics Viewer

(IGV) - based visualization of reads of targeted NGS that identify the wild type KRAS

(baseline, top panel) and an acquired CT mutation in 38% of reads, encoding a

KRASG12S mutation (relapse, bottom panel). C, long-term viability assay of PC9KRASWT




31

and PC9KRAS-G12S cells treated with AZD9291 at the indicated concentrations for 6 days.

Cell viability is presented as the percentage of change in cell number (compared to

baseline). D, long-term proliferation assay of PC9KRAS-WT and PC9KRAS-G12S cells

exposed to increasing concentrations 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 cells treated 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 (100nM)

alone or in combination with either trametinib (1µM) or selumetinib (1µM), in comparison

with the cell number before drug exposure. Cell viability was determined after 6 days of

treatment. G, PC9KRAS-G12S cells were treated with AZD9291 (100nM) alone or in

combination with either trametinib (1µM) for 24 hours and lysed. Protein expression and

phosphorylation levels of EGFR, AKT, ERK and the expression of and KRAS, BIM and

actin were monitored by immunoblotting.




A

C

Figure 1

B

D

P1 P2 P3 P4 P5 P6 P7-100

-80

-60

-40

-20

0

Best

resp

on

se u

nd

er

treatm

en

t

*

*

* PD of non-target lesions

*

DMSO 10nM 100nM 1µM DMSO 10nM 100nM 1µM-200

0

200

400

600

Ch

an

ge in

cell n

um

ber

(%)

PC9GRe.v PC9GRERBB2

AZD9291 Rociletinib

CellNumberHer2Cells_EGFRi_R3

**

***

***

** ** ***

0 5 10 15 20 25

P7

P6

P5

P4

P3

P2

P1

Time of treatment (months)

ME

T a

mp

ER

BB

2 a

mp

AZD9291

Rociletinib

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




A

F

Figure 2

H

B

AZD9291(1µM)

Rociletinib (1µM)

HCC827GR EGFR T790M

EGFR

pEGFRY1068

pAKTS473

AKT

ERK

BIM

pERKT202/Y204

Actin

Crizotinib (1µM)

MET

pMETY1234/Y1235

-

-

-

+

-

-

-

-

+

+

-

+

-

+

-

-

+

+

DM

SO

A (

1µ

M)

R (

1µ

M)

C (

1µ

M)

Co

mb

o A

+C

Co

mb

o R

+C

0

50

100

150

200

250

Ch

an

ge in

cell n

um

ber

(%)

******

HCC827GREGFR T790M E G

EGFR

pEGFRY1068

pAKTS473

AKT

ERK

pERKT202/Y204

MET

pMETY1234/Y1235

HCC827EGFR T790M

DM

SO

A

ZD

9291

R

oci

letinib

HCC827GREGFR T790M

DM

SO

A

ZD

9291

R

oci

letinib

BIM

Actin

D

I

Baseline 1 2 3

Follow-ups

300mm

250mm

200mm

150mm

100mm

50mm

0mm

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




A B

C

F

Figure 3

EGFR

pEGFRY1068

pAKTS473

AKT

ERK

Actin

BIM

pERK/Y204

KRAS wt

AZD9291 (µM)

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(100nM)

Trametinib (1µM)

D E

G

DMSO 10nM 100nM 1µM-200

0

200

400

600

800

Concentration AZD9291 (µM)

Ch

an

ge in

cell n

um

ber

(%)

PC9 KRAS wt

PC9 KRAS G12S

**

** ***

AZD9291 (µM) 0 0.1 1

PC9

KRAS wt

PC9

KRAS G12S

DM

SO

A (

10

0n

M)

T (

1µ

M)

Co

mb

o T

S (

1µ

M)

Co

mb

o S

-200

0

200

400

600

Ch

an

ge in

cell n

um

ber

(%)

******

Before

AZD9291

Under

AZD9291

KRAS G12S

Patient 7

140mm

120mm

100mm

80mm

60mm

40mm

10mm

0mm

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




Patie

nt

num

ber

Age

Sex

Initi

al

biop

sy

site

FISH

Pr

imar

y m

utat

ion

TKI

resi

stan

cePF

S (m

onth

s)St

art o

f tr

eatm

ent

Tim

e on

tr

eatm

ent

(mon

ths)

Trea

tmen

t his

tory

(tim

e on

trea

tmen

t in

mon

ths)

OS

(yea

rs)

Dat

e of

re

biop

syR

ebio

psy

site

Type

of

rebi

opsy

Type

of

sam

ple

FISH

Met

hod

Mea

n co

vera

geO

ther

m

utat

ions

171

FN

AN

ot d

one

EG

FR E

xon

19 d

el; E

GFR

T7

90M

Roc

iletin

ib1.

9D

ecem

ber

2014

1.9

CIS

/PEM

(5),

gap

(6),

Erlo

tinib

(11)

, CO

-168

6 (2

) 2.

1 ∆

Janu

ary

2015

Pleu

ral

effu

sion

†.

FFPE

ER

BB

2 a

mp

miS

eq49

75E

GFR

Exo

n 19

de

l, E

GFR

T7

90M

264

FLu

ngE

RB

B22

am

p E

GFR

L86

1Q;

EG

FR T

790M

AZD

9291

4.2

Oct

ober

20

147.

4*C

IS/V

INO

/RTX

(3),

Afat

inib

(4

), AZ

D92

91 (7

*)1.

2 ∆

..

..

.m

iSeq

9560

EG

FR L

861Q

, E

GFR

T79

0M

354

FLi

ver

ME

T a

mp

EG

FR E

xon

19 d

el; E

GFR

T7

90M

Roc

iletin

ib1.

2N

ovem

ber

2014

3.2

CIS

/PEM

(1),

Erlo

tinib

/RTX

(2

), G

efiti

nib

(9),

Roc

iletin

ib (3

), R

ocile

tinib

/Criz

otin

ib (1

)

1.3

..

..

..

..

457

MLi

ver

No

ME

T a

mp

EG

FR L

858R

; E

GFR

T79

0MAZ

D92

914.

0Ju

ly 2

014

4.6

Gef

itini

b (2

), C

arbo

/PEM

(2

), m

aint

enan

ce P

EM (3

), C

ARBO

/DO

CE

(4),

AZD

9291

(4),

EMD

12

1406

3 (3

), Er

lotin

ib/C

rizot

inib

(1)

1.9

Nov

embe

r 20

14Li

ver

CT-

guid

ed

biop

syFF

PEM

ET

am

pm

iSeq

4561

EG

FR L

858R

, E

GFR

T79

0M

560

MLi

ver

Bloc

k no

t av

aila

ble

EG

FR E

xon

19 d

el, E

GFR

T7

90M

AZ

D92

9116

.5M

ay 2

014

17.5

Erlo

tinib

(7),

CAR

BO/P

ACLI

(2),

inte

rmitt

end

RTX

(13)

, AZ

D92

91 (1

8)

3.2

∆O

ctob

er

2015

Live

rC

T-gu

ided

bi

opsy

FFPE

ME

T a

mp,

E

RB

B2

am

pm

iSeq

1217

EG

FR E

xon

19

del,

EG

FR

T790

M

656

FLi

ver

(2x)

Not

don

eE

GFR

Exo

n 19

del

, EG

FR

T790

M

AZD

9291

19.4

Oct

ober

20

1322

.3

Gef

itini

b (9

), R

TX (3

), Er

lotin

ib (7

), Af

atin

ib (1

5),

Afat

inib

/Cet

uxim

ab (3

), PE

M/E

rlotin

ib (9

), AZ

D92

91 (2

2)

5.9

∆Ju

ly 2

015

Live

rC

T-gu

ided

bi

opsy

FFPE

ME

T a

mp

miS

eq21

76

EG

FR E

xon

19

del,

EG

FR

T790

M, E

GFR

C

797S

751

FLy

mph

no

de

No

ME

T am

p,

no

ER

BB

2 a

mp

EG

FR E

xon

19 d

el, E

GFR

T7

90M

AZ

D92

917.

5N

ovem

ber

2013

8.9

Gef

itini

b (1

7),

CIS

/PEM

/RTX

(4),

Afat

inib

(6

), Af

atin

ib/C

etux

imab

(1

1), A

ZD92

91 (9

)

4.5

May

201

4Ly

mph

no

de

Endo

scop

ic

biop

sy v

ia

esop

hagu

sFF

PEN

o M

ET

amp,

n

o E

RB

B2

am

pm

iSeq

540

EG

FR E

xon

19

del,

KR

AS

G

12S

* Tre

atm

ent o

ngoi

ng

∆ St

ill al

ive

† O

btai

ned

durin

g th

erap

y

PFS:

Pro

gres

sion

free

sur

viva

l. O

S: O

vera

ll su

rviv

al. H

et: H

eter

ogen

eity

CIS

= C

ispl

atin

, PEM

= p

emet

rexe

d, V

INO

= v

inor

elbi

ne, R

TX =

radi

atio

n th

erap

y, C

ARBO

= c

arbo

plat

in, D

OC

E =

doce

taxe

l, O

P =

surg

ery,

BEV

A =

beva

cizu

mab

, TR

O =

trof

osfa

mid

e, G

EMC

I = g

emci

tabi

ne, P

ACLI

= p

aclit

axel

Patie

nt in

form

atio

nPr

e-tr

eatm

ent a

mpl

eD

rug

resi

stan

t sam

ple

Sequ

enci

ng d

ata

rebi

opsy

Reb

iops

y in

form

atio

n




Published OnlineFirst June 1, 2016.Clin Cancer Res Sandra Ortiz-Cuaran, Matthias Scheffler, Dennis Plenker, et al. to third-generation EGFR inhibitorsHeterogeneous mechanisms of primary and acquired resistance

Updated version

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

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2016/12/20/1078-0432.CCR-15-1915.DC1

Access the most recent supplemental material at:

Manuscript

Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/early/2016/06/01/1078-0432.CCR-15-1915To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-15-1915

http://clincancerres.aacrjournals.org/content/suppl/2016/12/20/1078-0432.CCR-15-1915.DC1

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/early/2016/06/01/1078-0432.CCR-15-1915


heterogeneous mechanisms of primary and acquired...

Documents