Genotypic and Histological Evolution of Lung Cancers ... · John C. Wain,1,2,7 Thomas J. Lynch,5...

R E S EARCH ART I C L E

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Genotypic and Histological Evolution of Lung CancersAcquiring Resistance to EGFR InhibitorsLecia V. Sequist,1,2*† Belinda A. Waltman,2* Dora Dias-Santagata,2,3* Subba Digumarthy,2,4

Alexa B. Turke,1,2 Panos Fidias,1,2 Kristin Bergethon,3 Alice T. Shaw,1,2 Scott Gettinger,5

Arjola K. Cosper,1 Sara Akhavanfard,2,3 Rebecca S. Heist,1,2 Jennifer Temel,1,2 James G. Christensen,6

John C. Wain,1,2,7 Thomas J. Lynch,5 Kathy Vernovsky,1 Eugene J. Mark,2,3 Michael Lanuti,1,2,7

A. John Iafrate,2,3 Mari Mino-Kenudson,2,3 Jeffrey A. Engelman1,2†

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Lung cancers harboring mutations in the epidermal growth factor receptor (EGFR) respond to EGFR tyrosinekinase inhibitors, but drug resistance invariably emerges. To elucidate mechanisms of acquired drug resistance,we performed systematic genetic and histological analyses of tumor biopsies from 37 patients with drug-resistantnon–small cell lung cancers (NSCLCs) carrying EGFR mutations. All drug-resistant tumors retained their original ac-tivating EGFR mutations, and some acquired known mechanisms of resistance including the EGFR T790M mutationor MET gene amplification. Some resistant cancers showed unexpected genetic changes including EGFRamplification andmutations in the PIK3CA gene, whereas others underwent a pronounced epithelial-to-mesenchymaltransition. Surprisingly, five resistant tumors (14%) transformed from NSCLC into small cell lung cancer (SCLC) andwere sensitive to standard SCLC treatments. In three patients, serial biopsies revealed that genetic mechanisms ofresistance were lost in the absence of the continued selective pressure of EGFR inhibitor treatment, and suchcancers were sensitive to a second round of treatment with EGFR inhibitors. Collectively, these results deepenour understanding of resistance to EGFR inhibitors and underscore the importance of repeatedly assessingcancers throughout the course of the disease.

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INTRODUCTION

Non–small cell lung cancer (NSCLC) is the leading cause of cancerdeath in the world, and traditional chemotherapeutic drugs are onlymodestly effective. Recent advances with targeted therapies haveprovided a marked benefit to subsets of patients whose tumors harborspecific genetic abnormalities. In particular, NSCLCs with mutations inthe gene encoding the epidermal growth factor receptor (EGFR) areuniquely sensitive to EGFRblockadewith specific tyrosine kinase inhib-itors (TKIs) (1–3). Most cancers with EGFRmutations achieve markedand durable responses to treatment with the EGFR TKIs gefinitib orerlotinib. However, despite this initial response, patients with NSCLCscontaining EGFRmutations acquire resistance to EGFR inhibitors, andthe median time to disease progression is about 12 months (4, 5).

To date, twomechanisms of acquired drug resistance have been con-firmed in patients. About half of cancers that acquire resistance to EGFRTKIs develop a secondarymutation inEGFR (T790M), which abrogatesthe inhibitory activity of the TKIs (6, 7). Another 15 to 20% undergoamplification of theMET receptor tyrosine kinase, which activates down-stream intracellular signaling independent of EGFR (8, 9). Additionally,clinical experience has revealed that, after a drug-free interval, resistantcancers can respond again to EGFR TKIs (10, 11). However, the molec-ular basis for this phenomenon remains poorly understood. To increase

1Massachusetts General Hospital Cancer Center, Boston, MA 02114, USA. 2Harvard MedicalSchool, Boston, MA 02115, USA. 3Department of Pathology, Massachusetts General Hospital,Boston, MA 02114, USA. 4Department of Radiology, Massachusetts General Hospital, Boston,MA 02114, USA. 5Yale University School of Medicine and Yale Cancer Center, New Haven, CT06520, USA. 6 Translational Pharmacology, Pfizer Oncology, La Jolla, CA 92121, USA.7Department of Surgery, Massachusetts General Hospital, Boston, MA 02114, USA.*These authors contributed equally to this work.†To whom correspondence should be addressed. E-mail: [email protected] (L.V.S.);[email protected] (J.A.E.)

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our understanding of the full spectrumof acquired resistance byNSCLCsto EGFR TKIs, we rebiopsied recurrent disease sites in patients withEGFR mutations who developed resistance to EGFR TKIs. Molecularanalyses were performed to assess the prevalence of known resistancemechanisms and to validate or refute potential mechanisms based onlaboratory studies, with the aim of identifying new molecular mecha-nisms of resistance to EGFR TKIs. These investigations identified sub-stantial histological and genetic changes in NSCLCs resistant to EGFRTKIs. In a few patients whose cancers were assessed at multiple pointsalong their treatment course, we observed that genetic resistance mech-anisms were “lost” without continued TKI treatment, thereby providinga molecular basis for the retreatment responses observed in the clinic.These resultsmayprovide abasis for developingnew therapeutic strategiesto overcome resistance and potentially to thwart its emergence. Addition-ally, our findings point to the value of repeat tumor biopsies throughoutthe course of a patient’s disease to determine the best treatment regimen.

RESULTS

Biopsies of resistant cancersTo identify how EGFR-mutantNSCLCs develop resistance to EGFR in-hibitors, we performed biopsies on patients at the time that drugresistance was acquired. All patients had EGFR-mutant NSCLC andhad achieved a clinical response to EGFRTKI therapy but subsequentlydeveloped progressive disease. They underwent repeat tumor tissuebiopsies as part of routine clinical care. Clinical and pathologicalinformation was abstracted retrospectively under an Institutional Re-view Board (IRB)–approved protocol.

Thirty-seven patients had tumor tissue available both before and af-ter TKI treatment. They included 15 men and 22 women (Table 1 and

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Table 1. Thirty-seven paired lung tumor biopsies resistant to EGFR in-hibitors. EGFR, epidermal growth factor receptor; amp, amplification; del, de-letion; Adeno, adenocarcinoma; Adenosquam, adenosquamous; NSCLC,

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non–small cell lung cancer not otherwise specified; SCLC, small cell lungcancer; CA, carcinoma; EMT, epithelial to mesenchymal transition; TKI, tyro-sine kinase inhibitor; Erlo, erlotinib; Gef, gefitinib.

ID#
Age Sex EGFR mutation Baseline histology Summary of changes
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Primary TKI (time on TKI)

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TKI status at repeat biopsy

T790M

1
66 M L858R Adeno T790M Erlo (6 months) Off (2 months)
2
74 F Exon 19 del Adeno T790M Erlo (12 months) On
3
47 F Exon 19 del Adeno T790M Gef (15 months) On
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60 F Exon 19 del Adeno T790M Erlo (7 months) On
5
57 M L858R Adeno T790M Gef (5+ years) On
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47 M Exon 19 del Adeno T790M Erlo (12 months) Off (14 months)
7
58 F Exon 19 del Adeno T790M Erlo (3+ years) On
8
69 M L858R Adeno T790M* Erlo (2 years) On
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58 F G719C, S768I Adeno T790M Erlo (2+ years) On
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10 46 F Exon 19 del Adeno T790M Erlo (3 years) Off (3 months)
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11 53 F Exon 19 del Adeno T790M Erlo (16 months) On
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12 59 F L858R Adeno T790M Erlo (8 months) Off (5 months)
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T790M + EGFR amp
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13 42 M Exon 19 del Adeno T790M, EGFR amp Erlo (5 months) On
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55 M Exon 19 del Adeno T790M, EGFR amp Erlo (10 months) On
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15 37 F Exon 19 del Adeno T790M, EGFR amp Erlo (6 months) On
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T790M + new, additional mutations
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16 88 F Exon 19 del Adeno T790M, b-catenin Erlo (2+ years) On
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17 85 M Exon 19 del Adeno T790M, b-catenin Erlo (22 months) On
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75 F Exon 19 del Adeno T790M, APC† Erlo (18 months) On
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MET amplification
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19 61 M L858R Adenosquam MET amp, loss EGFR amp Erlo (15 months) On
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20 76 M L858R Adeno MET amp Erlo (13 months) Off (5 months)
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Acquired PIK3CA mutation
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21 65 M Exon 19 del Adeno PIK3CA acquisition Erlo (21 months) On
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Histologic transformation (one with acquired PIK3CA mutation)
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22 67 F L858R Adeno SCLC transformation Erlo (22 months) On
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23 54 F Exon 19 del Adeno SCLC transformation Erlo (3+ years) On
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24 56 F L858R Adeno SCLC transformation, PIK3CA Erlo (14 months) On 0
25
40 F Exon 19 del Adeno SCLC transformation Erlo (2+ years) Off (2 months)
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61 F L858R Adeno SCLC transformation Erlo (18 months) On
27
66 M L858R Adeno EMT Erlo (11 months) On
28
59 M Exon 20 ins‡ Adeno EMT Gef (11 months) On
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64 M L858R Adeno Sa rcomatoid CA, loss of b-catenin Erlo (11 months) Off (2 weeks)
No histological or genetic changes identified

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62 F L858R Adeno None Erlo (6 months) On
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52 F Exon 19 del Adeno None Gef (17 months) On
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58 F Exon 19 del Adeno None Erlo (14 months) On
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61 F L858R Adeno None Erlo (13 months) On
34
35
62 M L858R NSCLC None Gef (3+ years) On
36
56 M L858R Adeno None Erlo (5 months) Off (<2 weeks)
37
*TP53 mutation suspected to be present, but not confirmed. †APC mutation confirmed in resistant specimen, but not confirmed to be present in initial biopsy. ‡Exon 20 insertion assayadded to SNaPshot for this patient given direct sequencing result from pretreatment sample.

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table S1). All patients had activating EGFRmutations; 20 (54%) had anexon 19 deletion mutation and 15 (41%) had the exon 21 point muta-tion L858R.All patients had responded clinically to either gefitinib (n=5)or erlotinib (n = 32). Radiographs were obtained and robust treatmentresponseswere confirmedwith theResponse EvaluationCriteria in SolidTumors (RECIST) method in 14 of 17 patients with available scans(fig. S1) (12). The median duration of primary TKI therapy was 14.1months (range, 4 to 69 months) and the 1- or 2-year progression-freerates were 64 or 30%, respectively.Most patients (78%)were still takingan EGFRTKI at the time of repeat biopsy, and biopsieswere performedamedian of 30months (range, 5 to 99months) after original diagnosis.Only four patients received chemotherapy between the development ofresistance and the repeat biopsy. Anatomic sites of repeat biopsy mostcommonly included lung lesions (38%), liver lesions (16%), and medi-astinal or cervical lymph nodes (16%). Most biopsies (68%) were per-cutaneouswith either computed tomographyor ultrasoundguidance, butsome were performed via bronchoscopy, mediastinoscopy, or anothersurgical procedure. There were no major biopsy-related complications,including no cases of clinically significant bleeding, pneumothorax, orunanticipated hospital admission.

Genotypic mechanisms of acquired drug resistanceThe 37 paired pre- and post-EGFR TKI tumor samples were analyzedfor the presence of genetic alterations with our standard clinical geno-typing platform, the SNaPshot assay. SNaPshot is a multiplex platformthat is used at Massachusetts General Hospital (MGH) to genotype

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cancers at specific genetic loci across 13 genes, as previously reported(table S2) (13). In addition, samples were analyzed for EGFR andMET amplification with fluorescence in situ hybridization (FISH). Thepretreatment activating EGFR mutation was present in each drug-resistant specimen (Table 1 and table S1). As predicted, we observedmechanisms of TKI resistance that were previously validated in clinicalspecimens. Eighteen (49%) patients acquired the exon 20 EGFRmuta-tion T790M, and two (5%) patients developedMET amplification (Fig. 1).In one case of an L858R EGFR-mutant cancer that subsequently devel-opedMET amplification, the pretreatment specimenhadmarkedEGFRamplification but noMET amplification (Fig. 2A). After resistance de-veloped,MET amplification was abundant, but the EGFR amplificationwas lost (Fig. 2A). Given that the resistant lesion biopsied had initiallyresponded to the TKI and harbored the same activating EGFRmutationas the treatment-naïve cancer, it seems most likely that the resistant tu-mor was derived from a distinct MET-amplified subpopulation ofEGFR-mutant cells (that did not harbor EGFR amplification) that wereselectively enriched during EGFR TKI administration, consistent withprevious observations (14).

We also observed acquired resistance mechanisms previously as-sessed only in in vitro studies and not previously identified in patients.These included two (5%) patients with acquired PIK3CA mutations(15). In addition, three (8%) patients acquired EGFR amplifications intheir resistant specimens (Fig. 2B), all of which also acquired the classicT790M EGFRmutation. Moreover, in two cases with high-level EGFRamplification (>10-fold), it was clear by comparison of the peak heightson the SNaPshot chromatogram that the T790M allele was the amplified

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Fig. 1. The frequency of observed drug resistance mechanisms. The piechart depicts the prevalence of observed mechanisms of resistance to EGFR
TKIs in 37 patients with NSCLC biopsied at the time that resistance wasacquired. Pre- and posttreatment specimens were compared and onlyacquiredmechanisms of resistance are depicted. The bluewedge representsresistant cancers that developed the EGFR T790M resistance mutation in-cluding a subset that developed concomitant EGFR amplification. The greenwedge represents cancers that developed MET amplification, and the redwedge represents cancers that underwent transformation to SCLC. The yel-low wedge represents cancers that developed PIK3CA mutations, and theorange wedge represents one patient who had both SCLC transformationand acquisition of a PIK3CA mutation.
Fig. 2. Acquired genetic amplifications in drug-resistant lung tumors. Am-plification of MET and EGFR genes was observed in biopsies of tumors from
patients who had acquired resistance to EGFR TKIs. Shown are FISH analysesthat detect the MET gene (green), EGFR gene (red), and the control CEP7gene (aqua). (A) The pretreatment specimen from patient 19 (left panel)shows a normal MET copy number but significant EGFR amplification; thedrug-resistant posttreatment specimen (right panel) from the samepatientexhibits acquiredMET amplification but normal EGFR copy number. (B) Pa-tient 13 demonstrated an acquired EGFR amplification in the drug-resistantposttreatment specimen (right panel) compared to the pretreatment spec-imen (left panel).



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allele (fig. S2). In the third case, we were unable to make a definitive de-termination. Other cases with acquired mutations of uncertain signifi-cance included two (5%) cancers with b-catenin mutations, both ofwhich occurred concomitantly with the EGFR T790Mmutation. Fifteen(41%) posttreatment biopsies did not reveal any new mutations as as-sessed by the SNaPshot assay, norMET or EGFR amplification. Two pa-tients in this group had insufficient posttreatment tissue for EGFR andMET gene copy number analyses. Among the 15 patients without anidentified genetic resistance mechanism, only 2 patients had stoppedEGFR TKI therapy for more than 2 weeks at the time of biopsy.

Phenotypic changes in tumors with acquired resistanceAll of the drug-resistant tumor specimens underwent routine patholog-ical analyses, and in some cases, significant alterations in the predom-inant histology of the resistant tumors were observed. To our surprise,five patients (14%) were found to have a diagnosis of small cell lungcancer (SCLC) in their drug-resistant tumor biopsies (Table 2). All ofthese cases were lung adenocarcinoma before EGFR TKI treatment.The transformation to SCLC at the time of clinical TKI resistancewas validated by histological examination and confirmed by expressionof neuroendocrine markers (Fig. 3, A and B). The original EGFR mu-tation was maintained during the histological transformation in all fivecases. One patient also acquired a PIK3CAmutation accompanying theSCLC transformation. Clinically, these five patients ranged in their dis-ease courses. Two patients had relatively indolent disease immediatelyafter the SCLC transformation, whereas the other three patients showedamarked progression thatwas reminiscent of classic SCLC. Four patientswere treated with a classic SCLC treatment, platinum-etoposide–basedchemotherapy, which induced marked responses in three cases (Fig.3C). The fourth treated patient had an initial response to radiation ther-apy, but declined quickly upon salvage chemotherapy. Autopsy of thiscase revealed extensive metastatic disease in the lung, thoracic lymphnodes, liver, and nodules along the diaphragm, all consisting entirelyof SCLC and all maintaining the original EGFR L858R mutation withno additional mutations (table S3). However, brain metastases still re-tained the appearance of lung adenocarcinoma, consistent with theoriginal diagnosis.

In the laboratory,we observed a different phenotypic transformationwhen using the H1975 (L858R/T790M) lung adenocarcinoma cell lineto model acquired resistance to an EGFR inhibitor. The cell line was

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made resistant to the irreversible EGFR inhibitor, PF00299804, towhichit was initially sensitive, as previously described (Fig. 4A) (8, 14–16). Theresistant cell line (H1975 Resistant) did not acquireMET amplification,but did show an increased copy number of the EGFR T790M allele,consistent with previous reports (17). In addition, it underwent amarkedhistological change and developed a spindle-like morphology (Fig. 4B).Assessment of E-cadherin and vimentin expression confirmed that theresistant cell line had undergone an epithelial-to-mesenchymal transi-tion (EMT) (Fig. 4C). EMT describes a cancer cell that loses its epithelialmorphology and develops a more spindle-like mesenchymal morphol-ogy; this histological change is often associated with a shift in expres-sion of specific proteins (for example, loss of E-cadherin and gain ofvimentin) and amore invasive phenotype. In contrast, HCC827GR cellsthat had developed MET amplification upon resistance to an EGFRTKI (8) did not undergo an EMT (Fig. 4C).

This finding supported previous observations that cancer cell linesundergoing an EMT have intrinsic resistance to EGFR inhibitors(18–22). This prompted us to analyze paired tissue samples from sevenpatients with unknownmechanisms of resistance and five patients withthe T790M EGFR mutation (who had sufficient remaining tissue) forthe development of mesenchymal features and changes in vimentinand E-cadherin expression. Three of the 12 resistant specimens hadphenotypic changes consistent with a mesenchymal appearance at thetime of TKI resistance; all 3 cases were among the 7 without anotheridentified resistance mechanism. Further analyses confirmed that twoof these three posttreatment specimens had acquired vimentin expres-sion and lost E-cadherin expression compared to their pretreatmentcounterparts, supporting an EMT (Fig. 4D). Both cancers that under-went this transition retained their originalEGFRmutation. Furthermore,one of these patients subsequently underwent autopsy, and phenotypicheterogeneity was observed among the differing sites of metastatic dis-ease (table S4). A left bronchial lymph node exhibited adenocarcinomaand did not have immunohistochemical evidence of EMT.However, an-other specimen from the right lower lobe with sarcomatoidmorphologyhadmarked evidence of EMT(Fig. 4Dand table S4). Both of these tissuesretained the original EGFR mutation, an exon 20 insertion. Notably,although exon 20 insertions are not uniformly activating and have beenassociated with TKI resistance, this patient had achieved stable diseaseand symptom improvement on gefitinib treatment lasting 11months,whichis consistent with the clinical criteria of acquired resistance to EGFR TKIs

Table 2. Patients with lung tumors showing an NSCLC to SCLCtransformation. ID number refers to the patient number in Table 1.Table 2. Patients with lung tumors showing an NSCLC to SCLC transftreatment; Post-, posttreatment/drug-resistant.

Pre-, pretreatment; Post-, posttreatment/drug-resistant.mation. ID number refers to the patient number in Table 1. Pre-, pre-

Age (years)
Gender ID no. Biopsy Histology
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Female 22 Pre- Adenocarcinoma − − − L858R
Post-
SCLC + + + L858R
54
Female 23 Pre- Adenocarcinoma − − Weak+ Exon 19 deletion
Post-
SCLC Strong+ Exon 19 deletion
56
Female 24 Pre- Adenocarcinoma − − L858R
Post-
SCLC + + L858R, PIK3CA
40
Female 25 Pre- Adenocarcinoma − − − Exon 19 deletion
Post-
SCLC + − Exon 19 deletion
61
Female 26 Pre- Adenocarcinoma − − − L858R
Post-
SCLC + + L858R
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(23). In contrast to these cases that underwent an EMT upon the devel-opment of resistance, we failed to observe this transition in all five casesexamined that had developed T790M as their resistance mechanism.

It appears that an EMT and a histological change to SCLC may beenriched specifically in EGFR-mutant cancers acquiring resistance toTKI therapy, because we failed to observe EMT in 10 available biopsyspecimens from EGFR wild-type tumors that developed resistance tochemotherapy. Additionally, we failed to identify a changeover to SCLCin these 10 samples and in an additional 69 cases of stage IIINSCLC thatwere resected after preoperative chemotherapy and radiation. The

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overlap of the genotypic and phenotypic changes observed in the entirecohort of EGFR-mutant TKI-resistant specimens is shown in fig. S3.

Longitudinal genotypic and phenotypic changes in responseto EGFR TKIThree patients underwent multiple repeat biopsies over the course oftheir disease (Fig. 5). The first patient (Fig. 5A) had adenocarcinomathat harbored the L858R EGFRmutation and a mutation in the tumorsuppressorTP53. As expected, this patient experienced a substantial ini-tial response to erlotinib lasting 8 months, at which time a lung core

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Fig. 3. Drug resistance and transformation of NSCLC to SCLC. The SCLChistological phenotype was observed in five (14%) NSCLC patients whohad acquired resistance. Two examples are shown. (A) Patient 23 had anexon 19 deletion in EGFR. (B) Patient 22 carried the L858R mutation in EGFR.The presence of the original activating mutation was confirmed in bothpretreatment (pre-Rx) specimens (upper panels) and drug-resistant speci-mens (lower panels). Hematoxylin and eosin (H&E) (left) and synaptophysin(right) staining are shown for each case. Notably, the pretreatment bi-opsies (A and B, upper panels) demonstrate adenocarcinoma consist-

ing of cellular growths of atypical glands with (A) or without (B) a cribriform pattern that are negative for synaptophysin. The post-resistancebiopsies (A and B, lower panels) demonstrate a SCLC phenotype consisting of nests of small cells with a high nuclear-to-cytoplasmic ratio with(A) or without (B) the classic SCLC-associated finding of “crush artifact,” with positive immunohistochemical staining for synaptophysin. (C) Com-puted tomography scans of a representative patient (patient 25) with SCLC in the acquired resistance specimen before (above) and after (below)chemotherapy with cisplatin and etoposide (the standard regimen for treating SCLC). Yellow arrows denote right third rib metastases, and whitearrows denote left axillary adenopathy.

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biopsy revealed adenocarcinoma with the same L858R and p53 muta-tions, as well as an acquired T790M EGFRmutation. After a 10-monthinterval without any EGFR TKI exposure, a second repeat biopsy per-formed on the same lung lesion as the first repeat biopsy revealed thatthe T790M mutation could no longer be detected. The patient subse-quently responded to treatment in a clinical trial of erlotinib plus aninvestigational agent that does not target T790M. A second patient withan exon 19 deletion had a similar clinical course involving gain and lossof the T790Mmutation in multiple biopsies from the same anatomicallocation during periods of erlotinib and chemotherapy treatment,respectively.

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The lung core biopsy from the drug-resistant tumor of a third pa-tient (Fig. 5B) demonstrated SCLC with the original EGFR L858R mu-tation plus an acquired PIK3CA mutation (Table 2). This patient wastreatedwith chemotherapy and radiation for SCLC and her cancerwentinto a partial remission. After a 7-month interval without any erlotinibexposure, she developed a symptomatic pleural effusion and a thoracen-tesis revealed adenocarcinoma (negative for neuroendocrine markers)with the L858R EGFR mutation only; the PIK3CA mutation was notdetectable. Erlotinibwas readministeredwith a second clinical response.When this patient developed resistance once again, a soft tissue metas-tasis originating from bone revealed SCLC with the EGFR L858R and


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Fig. 4. EMT and acquired resistance. (A)H1975 cells were cultured in the presenceof the irreversible EGFR inhibitor PF00299804until drug resistance developed, as demon-strated by Syto60 viability assays. (B) Im-ages of the parental and drug-resistantH1975 cells by bright-fieldmicroscopy dem-onstrate that the resistant cells have de-veloped a spindle-like morphology. (C)Parental and resistant H1975 cells werelysed and probed with antibodies againstE-cadherin, vimentin, and actin, revealingloss of E-cadherin expression and gain of vi-mentin expression among drug-resistantH1975 cells. For comparison, HCC827 cellsand the derived HCC827 GR6 cell line(HCC827 cells that acquired resistance togefitinib via MET amplification), which donot undergo an EMT, are shown. (D) Ex-ample of a case (patient 28) whose drug-resistant tumor shows evidence of an EMT(top, pretreatment specimens; bottom, drug-resistant posttreatment specimens). Left,H&E staining; middle, staining for vimentin;right, staining for E-cadherin. Notably, thepretreatment cancer had an adenocarcino-ma histology (panel 1), does not stain forvimentin (panel 2), and shows preservedmembranous stainingwith E-cadherin (pan-el 3). The vimentin-positive areas in panel 2include alveolar macrophages (red circles),inflammatory and stromal cells in fibro-vascular cores (black arrows), but not tumorcells lining papillary structures (yellow ar-rows). The drug-resistant posttreatmentspecimen has sarcomatoid histology (panel4), is positive for vimentin (panel 5), and isnegative for E-cadherin (panel 6), consistentwith an EMT.




the PIK3CAmutation. In total, these findings provide a molecular linkto the clinical observation that patients with EGFR-mutant NSCLC tu-mors will often respond to erlotinib after a TKI-free interval (10, 11).Without the continued selective pressure of the TKI, the genetic re-sistance mechanisms and potentially the phenotypic resistance mech-anisms are lost.


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DISCUSSION

Here, we have performed in-depth genetic and histological analyses oncancers that acquired resistance to EGFR inhibitors. We observed bothknown molecular mechanisms of acquired resistance and also severalgenotypic and phenotypic changes that we believe broaden the con-ceptual model of acquired drug resistance. Notably, we observed a sur-prisingly high frequency of conversion of NSCLC to SCLC, markedEGFR amplification in a subset of cases with the T790M EGFR muta-tion, the development of PIK3CAmutations, EMT, and the loss of ge-netic resistancemechanisms in the absence of continuous TKI treatment.These findings provide new insights into our understanding of drugresistance and emphasize the need to perform tumor biopsies afterthe development of resistance to identify the best treatment optionsfor patients.

The development of drug resistance that invariably occurs afterabout 12months of initiating treatment (4, 5, 24–26) has spurred effortsto understand the biology underlying resistance and to identify thera-peutic strategies to overcome or prevent it. These laboratory studieshave primarily focused on exposing EGFR-mutant, TKI-sensitive celllines to EGFR TKIs until resistance develops. They have identified sev-eral resistance mechanisms, two of which—EGFR mutation T790M(6, 7) andMET amplification (8, 9)—have been validated in the clinic.Other acquired resistancemechanisms identified by studying the devel-opment of resistance to EGFR TKIs in vitro include loss of PTEN(27, 28) and activation of the insulin growth factor receptor (in celllines addicted to wild-type EGFR) (16). However, these resistancemech-anisms have not yet been validated in the clinic. Activation ofMET byhepatocyte growth factor (HGF) has been shown to drive resistance toEGFR TKIs, but these experiments were performed by adding exoge-nous HGF or HGF-secreting tumor-derived fibroblasts (14, 29–31),not by selecting cells after chronic exposure toTKIs. Analyses of resistantspecimens support, but do not prove, that HGF may be a resistancemechanism in patients. To date, the various EGFRTKI resistancemech-anisms share the sameunderlying concept: They enable the cancer cell tomaintain its intracellular growth signaling pathways, especially the phos-phatidylinositol 3-kinase (PI3K)–AKT pathway, in the presence of theEGFR TKI (32–37).

In our cohort of patients with EGFRmutation–positive NSCLC andacquired EGFR TKI resistance, we observed known mechanisms ofresistance, the EGFR T790M mutation and MET amplification. Forty-nine percent developed the T790Mmutation, consistent with the previ-ously reported incidence of this mutation in patients with acquiredresistance (8, 38–40). A subset of these patients also developed pro-nounced EGFR amplification, and it appears that the T790M allele isselectively amplified. To the best of our knowledge, amplification ofEGFRT790Mhas not been previously appreciated inTKI-resistant spec-imens of NSCLC tumors. Balak et al. (40) reported one patient withabout twofold increase in EGFR copy number in a drug-resistant spec-imen, but that case did not harbor the T790M mutation in EGFR. De-

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spite the promising activity of newer, irreversible EGFR inhibitors inpatients with EGFR mutations (41), their efficacy has been minimal inpatients with acquired resistance to gefitinib and erlotinib (42, 43). Therecent report by Ercan and colleagues (17) that amplified T790Mmuta-tions may promote resistance to irreversible EGFR inhibitors suggeststhat these patients may not respond to the current irreversible EGFR in-hibitors and should be directed to other potential therapeutic strategiessuch as combined PI3K and MEK (mitogen-activated or extracellularsignal–regulated protein kinase kinase) inhibition (44), newer, morepotent T790M-specific EGFR inhibitors (45), or combinations of anti-EGFR therapies (46). In addition, we observed that a subset of the

Fig. 5. Longitudinal evaluation of patients treated repeatedlywith erlotinib.The color-coded boxes to the left of each panel describe the data displayed
across the timeline. The tumor burden depicted is not quantitative but rep-resents tumor growth and shrinkage. (A) Patient 12 with a lung adenocar-cinoma carrying the L858R EGFR mutation and a mutation in the tumorsuppressor p53 had a modest response to first-line chemotherapy. The pa-tient then achieved a more robust and durable response to second-lineerlotinib, with near-complete resolution of her lung nodules. After 8 monthson an EGFR TKI, she developed resistance with growing bilateral pulmonarynodules. A lung core biopsy revealed an acquired T790Mmutation in EGFR.There was no response to chemotherapy and she subsequently developedbone and liver metastases. At that time (after not taking the EGFR inhibitorfor 8months), a second lung core biopsy revealed the L858R EGFRmutation,but no detectable T790MEGFR resistacemutation. The patient responded toerlotinib (in combination with an investigational agent that did not targetT790M). (B) Patient 24 had an L858R EGFR-mutant adenocarcinoma thatresponded markedly to first-line erlotinib for 12 months with resolution ofher pleural effusion and pulmonary nodules. After 1 year, there was progres-sion of the largest nodule. Core biopsy of this lesion revealed histologicaltransformation to SCLC that harbored the EGFR L858R mutation and ac-quired a PIK3CA mutation. She was treated with radiation and chemo-therapy. After a 6-month break from all treatment, her pleural effusionreaccumulated and small bilateral pulmonary nodules reappeared. Pleuraleffusion analysis revealed adenocarcinoma with the L858R EGFR mutationonly (no PIK3CA mutation). Her disease responded to a second-line courseof erlotinib. After 6 months, bony metastases and an adrenal lesion devel-oped. Assessment of a growing bone metastasis revealed SCLC with boththe L858R EGFR and PIK3CA mutations.




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T790Mpatients also acquired additionalmutations, including two (11%ofthe T790M cohort) with acquired mutations in b-catenin. To our knowl-edge, b-catenin has not been postulated as an EGFRTKI resistancemech-anism. Anecdotally, in our clinic, we have three patients with concurrentEGFR and b-cateninmutations at baseline, all of whom responded well toerlotinib without evidence of early-onset resistance.

MET amplificationwas identified in only two (5%) patients, which isless than the 15 to 20% frequency reported by our group and others(8, 9, 38). We cannot easily explain this lower than expected frequency.Possible contributing reasons include the lack of sufficient tissue forMET testing in two patients in the “unknown mechanism” category,the fairly conservative (high) threshold used for designating amplifica-tion used by our pathologists, and the sample size of our cohort. In ad-dition, we failed to identify any acquired genetic resistance mechanismin several cases. Although we were unable to test for all potentialresistance mechanisms because of tissue exhaustion and inadequate re-agents, it does seem likely that further analyses with more sophisticatedtechniques such as deep sequencingwill lead to the identification of newmechanisms of resistance to EGFR TKIs.

In addition to these two well-described mechanisms of TKI re-sistance, we observed acquired PIK3CA mutations in two patients. Toour knowledge, this represents the first documentation of PIK3CAmu-tations leading to drug resistance in cancer patients. This finding issupported by our previous laboratory findings that introduction of aPIK3CA mutation in EGFR-mutant HCC827 cells confers resistanceto gefitinib (15). This has important therapeutic implications becausethere are several ongoing early-phase clinical trials combining EGFRand PI3K pathway inhibitors that are attractive targeted therapy strate-gies to overcome this mode of resistance. We also hypothesize that pa-tients who have EGFR and PIK3CA mutations in the original primarytumor (baseline) might experience an abbreviated duration of benefitfrom EGFR TKI therapy compared with patients lacking PIK3CAmu-tations, and could be considered for enrollment in a first-line clinicaltrial combining an EGFR and PI3K inhibitor. Indeed, we have observedtwo patients with EGFR and PIK3CA mutations at baseline who bothresponded to first-line erlotinib therapy, but the responses lasted only 5and 7 months. Neither of these cases is included in this cohort of pa-tients who received repeat biopsies; one underwent a repeat biopsy butthe tissue was nondiagnostic, and the other was not offered a repeatbiopsy.

Perhaps, one of the more surprising findings from our study is theobservation that 5 (14%) of the 37 patients experienced a fundamentalhistology transformation from NSCLC to SCLC at the time of TKIresistance. The original EGFR mutation was maintained in all five pa-tients, disputing the rare possibility that these patients developed a sec-ond primary cancer. One patient also acquired a PIK3CA mutation inthe SCLC specimen, but none of the patients demonstrated EGFRT790M orMET amplification. The pre- and posttreatment tissues weresubjected to neuroendocrine immunohistochemical analyses includingstaining for synaptophysin, chromogranin, and/or CD56. Although theposttreatment (SCLC) specimens were all positive for neuroendocrinemarkers, most consistently synaptophysin, the pretreatment (NSCLC)samples were uniformly negative for neuroendocrine markers. Wespeculate that the high frequency of recognizing this unusual histolog-ical phenomenon may have been partly because of the implementationof thorough pathological evaluation of drug-resistant specimens as partof routine clinical care. These findings directly affected patient caredecisions, and four of the five patients received SCLC chemotherapy

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regimens with a response obtained in three patients. This unequivocallysuggests that the posttreatment biopsies provided useful clinical in-formation in addition to research information, and that repeat biopsiesat the time that clinical resistance to EGFR TKIs develops can directlybenefit patients. The transition fromNSCLC to SCLCappears to be spe-cific for resistance to EGFR TKIs. We observed no evidence of SCLC in10 cases of EGFR wild-type chemotherapy-resistant NSCLC and in 69resected stage III lung cancers, where the patients had received chemo-therapy and radiation.

Previous case reports have described patients with biopsy-provenSCLC and EGFR mutations (47–51). The individual cases reported byZakowski et al. (47) and by Morinaga et al. (48) are most similar to ourpatients, and each describes a never-smoking female that presentedwithEGFR-mutant metastatic adenocarcinoma that transformed into SCLCafter developing resistance. Okamoto et al. (49) describe a never-smoking female diagnosed with CD56-positive advanced SCLC har-boring an exon 19 deletion in EGFR, who had a good partial responseto first-line gefitinib. Fukui et al. (50) identified 6 patients with com-bined NSCLC-SCLC histology from a cohort of 64 SCLC patients un-dergoing surgical resection; one was a never-smoking female with anL858R EGFR mutation in both the SCLC and adenocarcinoma com-ponents. The final report is a case series arising from an analysis of 122Asian patientswith SCLCormixed histology tumors that were screenedfor EGFRmutations, of which 5 (4%) samples were found to bemutation-positive (3 L858R, 1 exon 19 deletion, and 1G719A) including a never-smoker and 4 smokers with tobacco histories ranging from 3 to 68pack-years (51). In this series, only one patient had a pretreatment ad-enocarcinoma that transformed into a combined SCLC-adenocarcinomaafter developing clinical resistance to an EGFR TKI. The other four pa-tients had EGFR-mutant SCLC or mixed histology tumors at baseline.

The biological underpinnings of the SCLC transformation are un-known and are of great interest. The finding that the sameEGFR-mutantcancer can manifest both as an adenocarcinoma and as a SCLC hints atthe existence of a pluripotent population of EGFR-mutant cancer cells orcancer stem cells (52, 53) that are the source of resistance. The cause ofthe phenotypic switch to SCLC and concordant development of re-sistance remain to be determined. Perhaps, these patients developeddrug resistance through a genetic or epigenetic event that concurrentlyled to a shift in phenotypic appearance. One of the marked moleculardifferences between NSCLC and SCLC is that most SCLCs exhibit lossof expression of the retinoblastoma protein (54–56), a tumor suppressor.We attempted to determine whether the resistant specimens had loss ofretinoblastoma protein expression by immunohistochemistry, but stain-ing was not of sufficient quality for interpretation.

In addition, we clearly observed the EMT in two cases of acquiredTKI resistance. Neither case had another identified resistance mecha-nism, but more cases will be required to determine whether this mutualexclusivity can be generalized. Similarly, we observed an EMT in anEGFR-mutant cell line rendered resistant to an EGFR inhibitor in vitro.Several groups have noted that cell lines undergoing EMT are intrinsi-cally resistant to EGFR inhibitors (18–22). However, those cancermodels do not have EGFRmutations and many have KRASmutations,so the relevance of those findings to acquired TKI resistance is lessstraightforward. Two case reports just published support our observa-tion of an EMT in EGFR-mutant NSCLC at the time of TKI resistance(57, 58). The molecular mechanisms connecting the resistance of thecancer cells to themesenchymal phenotype remain unknown. However,the recent findings that KRAS-mutant lung cancers with mesenchymal





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features are resistant to bothKRASknockdown (59) and combinedPI3Kand MEK inhibition (60) suggest that mesenchymal cells may have anintrinsic lack of sensitivity to the intracellular signaling pathway down-regulation that is normally the hallmark of sensitivity to EGFR TKIs.

Evidence from three patients with multiple biopsies over the courseof their disease suggests that both tumor genotype and phenotype mayevolve dynamically under the selective pressure of targeted therapies.Two patients developed T790M EGFR mutations at the time of TKIresistance and subsequently lost evidence of that resistance mutationin the same anatomic tumor after a period free from TKI treatment.These patients both responded to a challenge with an EGFR inhibitorsubsequent to losing the T790Mmutation. The third patient underwenta SCLC transformation with acquisition of a PIK3CA mutation at thetime of resistance and, after a TKI-free interval, was found to have adeno-carcinoma without a detectable PIK3CA mutation. This cycle wasrepeated when, after a second response to erlotinib, the cancer ultimatelydeveloped resistance again and the biopsy of the resistant cancer againrevealed the SCLC phenotype with the EGFR L858R and PIK3CAmutations. The mechanisms underlying these fluctuations remain tobe proven, but it is tempting to speculate that the baseline heterogeneityof the cancersmay contribute to these findings. Indeed, it is possible thatsubstantial populations of “sensitive” cancer cells may remain dormantwhile subjected to TKI treatment, as recently suggested by laboratorystudies (61). Withdrawal of the TKI may permit their rapid expansionto a degree that overtakes the bulk of the tumor burden. Such a mech-anism could also provide insight into the pronounced tumor flare thatis often clinically observed when the TKI is removed from slowly pro-gressing cancers (62). Indeed, these findings confirm that even “genetic”mechanisms of resistance are potentially reversible. Therefore, a static di-agnostic biopsy may be insufficient to guide therapeutic decision-making throughout the course of a patient’s disease. Moreover, all ofour patients experienced a second response to erlotinib when theirresistance mechanism was no longer detectable, suggesting that repeatbiopsies can provide molecular guidance about the likely benefit of asecond treatment regimen with EGFR TKI therapy.

The primary limitations of our study are its retrospective natureand the heterogeneity among practice patterns that led to patients un-dergoing repeat biopsies at various times during their disease (Table 1).Although all of these treatment variations could have affected theresistance mechanisms observed, the most direct confounder is likelyto be whether the patient was “on” or “off ” of the primary TKI at thetime of biopsy. All of our patients except one were on TKI at the time ofbiopsy, or had been off drug treatment for≤5months (table S1). Anotherlimitation is that in many cases, because of safety and feasibilityconcerns or because of the predominant radiographic progression inone anatomic area over another, the repeat biopsies were obtainedfrom different tumor locations compared to the original biopsies. Al-though distinct mechanisms of resistance in different anatomic locationswithin the same patient have been described (8), we observed that theprimary resistance mechanism was often consistent throughout differentmetastatic sites both in our autopsy cases and in patients with multiplesites biopsied over time. Larger studies will be helpful in further clarify-ing the impact of these variables.

In conclusion, this study provides further impetus for the utility ofreassessing cancers after they acquire resistance to targeted therapies.As our study shows, there is tremendous heterogeneity in resistancemechanisms, each of which may require its own therapeutic strategy.A recent report suggests that cancers with various resistance mecha-

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nismsmay have distinct prognoses (63). Although invasive biopsies haveassociated risks, we did not encounter any significant complications. Weanticipate that technologies to assess cancers via noninvasivemeasuressuch as circulating tumor cell analyses, plasma DNA analyses, or mo-lecular radiology may eventually obviate the need for invasive proce-dures. The knowledge gained from our repeat biopsy program directlyaffected treatment decisions and outcomes, and we were better equippedto rationally treat patients (for example, those with SCLC) as their tu-mors evolved. Several patients in our cohort were enrolled in clinicaltrials specifically targeting T790M,MET, or the PI3K signaling pathwayafter biopsies of their drug-resistant tumors, and several had disease sta-bilization or response to those therapies. Indeed, it is becoming increas-ingly clear, from experiences with both chronic myelogenous leukemiatreated with ABL kinase inhibitors and EGFR-mutant lung cancerstreated with EGFR kinase inhibitors, that the era of targeted therapieswill mandate continual assessment of each cancer’s evolution over thecourse of treatment to determine how it became resistant to therapyand to identify the optimal strategies to prevent or overcome it.

MATERIALS AND METHODS

PatientsAll 43 consecutiveEGFR-mutantNSCLCpatients with acquired EGFRTKI resistance undergoing standard post-resistance biopsy of their tu-mor from January 2007 to May 2010 at the MGH were considered forinclusion in the study cohort. Patients included in the final analysis hadto have both pre- and posttreatment tumor specimens available fortesting at MGH. To ensure sufficient tissue for molecular analysis, weobtained core biopsies whenever possible, and all fine-needle aspirationsamples (except one) undertook multiple passes, which were prospec-tively combined and spun down into a cell block. Six patients did notmeet criteria andwere excluded, including onewhose repeat biopsy wasnondiagnostic formalignancy, one bone biopsywith poor-qualityDNAfor molecular testing, one with a concomitant thyroid cancer in whichthe resistant biopsy showed malignant cells that were inconclusive re-garding bronchogenic or thyroid origin, one fine-needle aspiration withinsufficient DNA, one with a medical contraindication to biopsy, andone pretreatment biopsy that could not be located for molecular anal-ysis. Thirty-seven patients were included in the study cohort; the fea-sibility of repeat biopsy and comparative molecular analysis in ourclinic was therefore 37/43 or 86%. The electronic medical record wasreviewed retrospectively to obtain all demographic and clinical infor-mation under an IRB-approved protocol.

Genetic analysesOur group recently developed amultiplexed polymerase chain reaction(PCR)-based assay, based on the commercially available SNaPshot plat-form (Applied Biosystems), to detect mutations in tumor DNA fromformalin-fixed, paraffin-embedded tissue (13). Our SNaPshot tumorgenotyping assay detects multiple mutations in 13 key cancer genes in-cluding EGFR, KRAS, BRAF, PI3KCA, b-catenin, APC, and TP53 (tableS2); these genes were selected on the basis of clinical relevance, withpotential therapeutic agents either already available or with multiplepipeline drugs under development. TheDNAof interest is amplifiedwithmultiplexedPCR.Genotypes are determinedwith a single-base extensionsequencing reaction, in which allele-specific probes interrogate lociof interest and are extended by fluorescently labeled dideoxynucleotides.





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The allele-specific probes have different sizes and are subsequently re-solved by electrophoresis and analyzed by an automatedDNAsequencer.The sensitivity of the SNaPshot assay ranges from 94 to 99% per al-lele, with an average sensitivity of 95%. The average specificity is >95%.The SNaPshot assay has been validated for use in a Clinical LaboratoryImprovement Act (CLIA)–certified lab and is performed as a clinicalroutine test, with results included in the medical record (13).

In our study, all pre- and posttreatment tumor specimens under-went genotyping with SNaPshot. Some pretreatment samples had alsobeen analyzed via direct sequencing ofEGFR at the time of diagnosis, asthat was our standard clinical analysis up until 2009. Paired tumor sam-ples also underwent FISH of both MET and EGFR using standardprotocols (24). Tumor content by hematoxylin and eosin (H&E) wasalways confirmed before FISH slides were prepared.When tumor tissuewas limited or at risk of becoming exhausted, the genetic testswere prior-itized in the following order: (i) SNaPshot testing to confirm EGFRmu-tation, (ii) the remaining SNaPshot assays, (iii)MET FISH testing, and(iv) EGFR FISH testing.

Histological analysesAll biopsy specimens were reviewed at MGH to confirm diagnoses.Histology was confirmed by H&E staining, and tissue-specific mark-ers such as TTF-1 (thyroid transcription factor 1) were included atthe discretion of the pathologist. More tissue-specific markers wereincluded for metastatic specimens when the primary site was in ques-tion. Neuroendocrine immunohistochemistry with synaptophysin,chromogranin, and/or CD56 was performed on both the pre- andposttreatment samples that were suggestive of SCLC transformationon H&E staining. Vimentin and E-cadherin immunohistochemistrywas also performed on selected patient samples under an IRB-approvedprotocol. All immunohistochemical staining was performed on rep-resentative tissue sections from formalin-fixed and paraffin-embeddedtissue blocks. A Ventana autostainer (Benchmark XT) and the com-pany’s prediluted antibodies (Ventana) were used for synaptophysin,chromogranin, CD56, and vimentin immunostaining, following themanufacturer’s instructions. For E-cadherin immunohistochemistry,the antibody from a different vendor (M3612, dilution 1:50, Dako) wasapplied. HGF was not tested because of a lack of sufficient tissue innearly all cases and is therefore not included in this article.

Analyses of H1975 cells made resistant to PF00299804To generate a resistant cell line, we maintained H1975 cells (L858R/T790M) in RPMI 1640 supplemented with 10% fetal bovine serum(FBS) and exposed them to increasing concentrations of PF00299804similar to our previously described methods (8, 15). PF00299804 wasprovided by J. Christensen at Pfizer. PF00299804 concentrations wereincreased stepwise from 1 nM to 2 mM when the cells resumed growthkinetics similar to that of the untreated parental cells. The developmentof the resistant cell line took ~3 months. To confirm the emergence of aresistant clone, we performed survival assays after growth at each concen-tration after allowing the cells to grow in drug-free conditions for at least4 days.

Western blots were performed as previously described (44). TheE-cadherin antibody was from BD Biosciences, the vimentin antibodywas from Cell Signaling, and the actin antibody was from Sigma.

Growth and inhibition of growth were assessed by Syto60 staining(Invitrogen). Cells were fixed with 4% formaldehyde for 20 min at37°C and incubated with a 1:5000 dilution of Syto60 stain for 60 min.

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Cell density in each well was determined with an Odyssey Infrared Im-ager (LI-CORBiosciences), corrected for background fluorescence fromempty wells, and normalized to untreated wells, as described previ-ously (64).

SUPPLEMENTARY MATERIAL

www.sciencetranslationalmedicine.org/cgi/content/full/3/75/75ra26/DC1Fig. S1. Percent change in measurable disease, by patient.Fig. S2. Amplification of the T790M allele in tumors with EGFR amplification.Fig. S3. The overall distribution of the genetic and histological findings in the resistant biopsies.Table S1. Detailed results for 37 paired specimens with TKI resistance.Table S2. Mutations tested in SNaPshot version I.Table S3. Results from first autopsy case.Table S4. Results from second autopsy case.

REFERENCES AND NOTES

1. T. J. Lynch, D. W. Bell, R. Sordella, S. Gurubhagavatula, R. A. Okimoto, B. W. Brannigan, P. L.Harris, S. M. Haserlat, J. G. Supko, F. G. Haluska, D. N. Louis, D. C. Christiani, J. Settleman,D. A. Haber, Activating mutations in the epidermal growth factor receptor underlying respon-siveness of non–small-cell lung cancer to gefitinib. N. Engl. J. Med. 350, 2129–2139 (2004).

2. J. G. Paez, P. A. Jänne, J. C. Lee, S. Tracy, H. Greulich, S. Gabriel, P. Herman, F. J. Kaye,N. Lindeman, T. J. Boggon, K. Naoki, H. Sasaki, Y. Fujii, M. J. Eck, W. R. Sellers, B. E. Johnson,M. Meyerson, EGFRmutations in lung cancer: Correlation with clinical response to gefitinibtherapy. Science 304, 1497–1500 (2004).

3. W. Pao, V. Miller, M. Zakowski, J. Doherty, K. Politi, I. Sarkaria, B. Singh, R. Heelan, V. Rusch,L. Fulton, E. Mardis, D. Kupfer, R. Wilson, M. Kris, H. Varmus, EGF receptor gene mutationsare common in lung cancers from “never smokers” and are associated with sensitivity oftumors to gefitinib and erlotinib. Proc. Natl. Acad. Sci. U.S.A. 101, 13306–13311 (2004).

4. T. S. Mok, Y. L. Wu, S. Thongprasert, C. H. Yang, D. T. Chu, N. Saijo, P. Sunpaweravong, B. Han,B. Margono, Y. Ichinose, Y. Nishiwaki, Y. Ohe, J. J. Yang, B. Chewaskulyong, H. Jiang, E. L. Duffield,C. L. Watkins, A. A. Armour, M. Fukuoka, Gefitinib or carboplatin–paclitaxel in pulmonaryadenocarcinoma. N. Engl. J. Med. 361, 947–957 (2009).

5. R. Rosell, T. Moran, C. Queralt, R. Porta, F. Cardenal, C. Camps, M. Majem, G. Lopez-Vivanco,D. Isla, M. Provencio, A. Insa, B. Massuti, J. L. Gonzalez-Larriba, L. Paz-Ares, I. Bover, R. Garcia-Campelo, M. A. Moreno, S. Catot, C. Rolfo, N. Reguart, R. Palmero, J. M. Sánchez, R. Bastus,C. Mayo, J. Bertran-Alamillo, M. A. Molina, J. J. Sanchez, M. Taron; Spanish Lung Cancer Group,Screening for epidermal growth factor receptor mutations in lung cancer. N. Engl. J. Med. 361,958–967 (2009).

6. S. Kobayashi, T. J. Boggon, T. Dayaram, P. A. Jänne, O. Kocher, M. Meyerson, B. E. Johnson,M. J. Eck, D. G. Tenen, B. Halmos, EGFR mutation and resistance of non–small-cell lungcancer to gefitinib. N. Engl. J. Med. 352, 786–792 (2005).

7. W. Pao, V. A. Miller, K. A. Politi, G. J. Riely, R. Somwar, M. F. Zakowski, M. G. Kris, H. Varmus,Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with asecond mutation in the EGFR kinase domain. PLoS Med. 2, e73 (2005).

8. J. A. Engelman, K. Zejnullahu, T. Mitsudomi, Y. Song, C. Hyland, J. O. Park, N. Lindeman,C. M. Gale, X. Zhao, J. Christensen, T. Kosaka, A. J. Holmes, A. M. Rogers, F. Cappuzzo, T. Mok,C. Lee, B. E. Johnson, L. C. Cantley, P. A. Jänne, MET amplification leads to gefitinib resistancein lung cancer by activating ERBB3 signaling. Science 316, 1039–1043 (2007).

9. J. Bean, C. Brennan, J. Y. Shih, G. Riely, A. Viale, L. Wang, D. Chitale, N. Motoi, J. Szoke, S. Broderick,M. Balak, W. C. Chang, C. J. Yu, A. Gazdar, H. Pass, V. Rusch, W. Gerald, S. F. Huang, P. C. Yang,V. Miller, M. Ladanyi, C. H. Yang, W. Pao, MET amplification occurs with or without T790Mmutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib.Proc. Natl. Acad. Sci. U.S.A. 104, 20932–20937 (2007).

10. S. Yano, E. Nakataki, S. Ohtsuka, M. Inayama, H. Tomimoto, N. Edakuni, S. Kakiuchi, N. Nishikubo,H. Muguruma, S. Sone, Retreatment of lung adenocarcinoma patients with gefitinib who hadexperienced favorable results from their initial treatment with this selective epidermal growthfactor receptor inhibitor: A report of three cases. Oncol. Res. 15, 107–111 (2005).

11. A. Yoshimoto, K. Inuzuka, T. Kita, A. Kawashima, K. Kasahara, Remarkable effect of gefitinibretreatment in a patient with nonsmall cell lung cancer who had a complete response toinitial gefitinib. Am. J. Med. Sci. 333, 221–225 (2007).

12. P. Therasse, S. G. Arbuck, E. A. Eisenhauer, J. Wanders, R. S. Kaplan, L. Rubinstein, J. Verweij,M. Van Glabbeke, A. T. van Oosterom, M. C. Christian, S. G. Gwyther, New guidelines toevaluate the response to treatment in solid tumors. European Organization for Researchand Treatment of Cancer, National Cancer Institute of the United States, National CancerInstitute of Canada. J. Natl. Cancer Inst. 92, 205–216 (2000).

nceTranslationalMedicine.org 23 March 2011 Vol 3 Issue 75 75ra26 10




.sciencemag.org/

Dow

nloaded from

13. D. Dias-Santagata, S. Akhavanfard, S. S. David, K. Vernovsky, G. Kuhlmann, S. L. Boisvert,H. Stubbs, U. McDermott, J. Settleman, E. L. Kwak, J. W. Clark, S. J. Isakoff, L. V. Sequist,J. A. Engelman, T. J. Lynch, D. A. Haber, D. N. Louis, L. W. Ellisen, D. R. Borger, A. J. Iafrate,Rapid targeted mutational analysis of human tumours: A clinical platform to guide perso-nalized cancer medicine. EMBO Mol. Med. 2, 146–158 (2010).

14. A. B. Turke, K. Zejnullahu, Y. L. Wu, Y. Song, D. Dias-Santagata, E. Lifshits, L. Toschi, A. Rogers,T. Mok, L. Sequist, N. I. Lindeman, C. Murphy, S. Akhavanfard, B. Y. Yeap, Y. Xiao, M. Capelletti,A. J. Iafrate, C. Lee, J. G. Christensen, J. A. Engelman, P. A. Jänne, Preexistence and clonalselection of MET amplification in EGFR mutant NSCLC. Cancer Cell 17, 77–88 (2010).

15. J. A. Engelman, T. Mukohara, K. Zejnullahu, E. Lifshits, A. M. Borrás, C. M. Gale, G. N. Naumov,B. Y. Yeap, E. Jarrell, J. Sun, S. Tracy, X. Zhao, J. V. Heymach, B. E. Johnson, L. C. Cantley, P. A.Jänne, Allelic dilution obscures detection of a biologically significant resistance mutation inEGFR-amplified lung cancer. J. Clin. Invest. 116, 2695–2706 (2006).

16. M. Guix, A. C. Faber, S. E. Wang, M. G. Olivares, Y. Song, S. Qu, C. Rinehart, B. Seidel, D. Yee,C. L. Arteaga, J. A. Engelman, Acquired resistance to EGFR tyrosine kinase inhibitors in cancercells is mediated by loss of IGF-binding proteins. J. Clin. Invest. 118, 2609–2619 (2008).

17. D. Ercan, K. Zejnullahu, K. Yonesaka, Y. Xiao, M. Capelletti, A. Rogers, E. Lifshits, A. Brown,C. Lee, J. G. Christensen, D. J. Kwiatkowski, J. A. Engelman, P. A. Jänne, Amplification ofEGFR T790M causes resistance to an irreversible EGFR inhibitor. Oncogene 29, 2346–2356(2010).

18. S. Thomson, E. Buck, F. Petti, G. Griffin, E. Brown, N. Ramnarine, K. K. Iwata, N. Gibson, J. D.Haley, Epithelial to mesenchymal transition is a determinant of sensitivity of non–small-celllung carcinoma cell lines and xenografts to epidermal growth factor receptor inhibition.Cancer Res. 65, 9455–9462 (2005).

19. B. A. Frederick, B. A. Helfrich, C. D. Coldren, D. Zheng, D. Chan, P. A. Bunn Jr., D. Raben,Epithelial to mesenchymal transition predicts gefitinib resistance in cell lines of head andneck squamous cell carcinoma and non–small cell lung carcinoma. Mol. Cancer Ther. 6,1683–1691 (2007).

20. B. C. Fuchs, T. Fujii, J. D. Dorfman, J. M. Goodwin, A. X. Zhu, M. Lanuti, K. K. Tanabe, Epithelial-to-mesenchymal transition and integrin-linked kinase mediate sensitivity to epidermalgrowth factor receptor inhibition in human hepatoma cells. Cancer Res. 68, 2391–2399(2008).

21. C. D. Coldren, B. A. Helfrich, S. E. Witta, M. Sugita, R. Lapadat, C. Zeng, A. Barón, W. A.Franklin, F. R. Hirsch, M. W. Geraci, P. A. Bunn Jr., Baseline gene expression predicts sensitivityto gefitinib in non–small cell lung cancer cell lines. Mol. Cancer Res. 4, 521–528 (2006).

22. J. K. Rho, Y. J. Choi, J. K. Lee, B. Y. Ryoo, I. I. Na, S. H. Yang, C. H. Kim, J. C. Lee, Epithelial tomesenchymal transition derived from repeated exposure to gefitinib determines the sen-sitivity to EGFR inhibitors in A549, a non-small cell lung cancer cell line. Lung Cancer 63,219–226 (2009).

23. D. Jackman, W. Pao, G. J. Riely, J. A. Engelman, M. G. Kris, P. A. Jänne, T. Lynch, B. E. Johnson,V. A. Miller, Clinical definition of acquired resistance to epidermal growth factor receptortyrosine kinase inhibitors in non–small-cell lung cancer. J. Clin. Oncol. 28, 357–360 (2010).

24. L. V. Sequist, R. G. Martins, D. Spigel, S. M. Grunberg, A. Spira, P. A. Jänne, V. A. Joshi,D. McCollum, T. L. Evans, A. Muzikansky, G. L. Kuhlmann, M. Han, J. S. Goldberg, J. Settleman,A. J. Iafrate, J. A. Engelman, D. A. Haber, B. E. Johnson, T. J. Lynch, First-line gefitinib in patientswith advanced non–small-cell lung cancer harboring somatic EGFR mutations. J. Clin. Oncol.26, 2442–2449 (2008).

25. T. Mitsudomi, S. Morita, Y. Yatabe, S. Negoro, I. Okamoto, J. Tsurutani, T. Seto, M. Satouchi,H. Tada, T. Hirashima, K. Asami, N. Katakami, M. Takada, H. Yoshioka, K. Shibata, S. Kudoh,E. Shimizu, H. Saito, S. Toyooka, K. Nakagawa, M. Fukuoka; West Japan Oncology Group,Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer har-bouring mutations of the epidermal growth factor receptor (WJTOG3405): An open label,randomised phase 3 trial. Lancet Oncol. 11, 121–128 (2010).

26. M. Maemondo, A. Inoue, K. Kobayashi, S. Sugawara, S. Oizumi, H. Isobe, A. Gemma, M. Harada,H. Yoshizawa, I. Kinoshita, Y. Fujita, S. Okinaga, H. Hirano, K. Yoshimori, T. Harada, T. Ogura,M. Ando, H. Miyazawa, T. Tanaka, Y. Saijo, K. Hagiwara, S. Morita, T. Nukiwa; North-East JapanStudy Group, Gefitinib or chemotherapy for non–small-cell lung cancer with mutated EGFR.N. Engl. J. Med. 362, 2380–2388 (2010).

27. F. Yamasaki, M. J. Johansen, D. Zhang, S. Krishnamurthy, E. Felix, C. Bartholomeusz, R. J.Aguilar, K. Kurisu, G. B. Mills, G. N. Hortobagyi, N. T. Ueno, Acquired resistance to erlotinib inA-431 epidermoid cancer cells requires down-regulation of MMAC1/PTEN and up-regulationof phosphorylated Akt. Cancer Res. 67, 5779–5788 (2007).

28. Y. Kokubo, A. Gemma, R. Noro, M. Seike, K. Kataoka, K. Matsuda, T. Okano, Y. Minegishi,A. Yoshimura, M. Shibuya, S. Kudoh, Reduction of PTEN protein and loss of epidermal growthfactor receptor gene mutation in lung cancer with natural resistance to gefitinib (IRESSA).Br. J. Cancer 92, 1711–1719 (2005).

29. T. Yamada, K. Matsumoto, W. Wang, Q. Li, Y. Nishioka, Y. Sekido, S. Sone, S. Yano, Hepa-tocyte growth factor reduces susceptibility to an irreversible epidermal growth factor re-ceptor inhibitor in EGFR-T790M mutant lung cancer. Clin. Cancer Res. 16, 174–183 (2010).

30. W. Wang, Q. Li, T. Yamada, K. Matsumoto, I. Matsumoto, M. Oda, G. Watanabe, Y. Kayano,Y. Nishioka, S. Sone, S. Yano, Crosstalk to stromal fibroblasts induces resistance of lung

www.Scie

cancer to epidermal growth factor receptor tyrosine kinase inhibitors. Clin. Cancer Res.15, 6630–6638 (2009).

31. S. Yano, W. Wang, Q. Li, K. Matsumoto, H. Sakurama, T. Nakamura, H. Ogino, S. Kakiuchi,M. Hanibuchi, Y. Nishioka, H. Uehara, T. Mitsudomi, Y. Yatabe, T. Nakamura, S. Sone, Hepato-cyte growth factor induces gefitinib resistance of lung adenocarcinoma with epidermalgrowth factor receptor–activating mutations. Cancer Res. 68, 9479–9487 (2008).

32. J. A. Engelman, P. A. Jänne, C. Mermel, J. Pearlberg, T. Mukohara, C. Fleet, K. Cichowski, B. E.Johnson, L. C. Cantley, ErbB-3mediates phosphoinositide 3-kinase activity in gefitinib-sensitivenon-small cell lung cancer cell lines. Proc. Natl. Acad. Sci. U.S.A. 102, 3788–3793 (2005).

33. R. Sordella, D. W. Bell, D. A. Haber, J. Settleman, Gefitinib-sensitizing EGFR mutations inlung cancer activate anti-apoptotic pathways. Science 305, 1163–1167 (2004).

34. S. Tracy, T. Mukohara, M. Hansen, M. Meyerson, B. E. Johnson, P. A. Jänne, Gefitinib inducesapoptosis in the EGFRL858R non–small-cell lung cancer cell line H3255. Cancer Res. 64,7241–7244 (2004).

35. T. Mukohara, J. A. Engelman, N. H. Hanna, B. Y. Yeap, S. Kobayashi, N. Lindeman, B. Halmos,J. Pearlberg, Z. Tsuchihashi, L. C. Cantley, D. G. Tenen, B. E. Johnson, P. A. Jänne, Differentialeffects of gefitinib and cetuximab on non–small-cell lung cancers bearing epidermalgrowth factor receptor mutations. J. Natl. Cancer Inst. 97, 1185–1194 (2005).

36. J. Amann, S. Kalyankrishna, P. P. Massion, J. E. Ohm, L. Girard, H. Shigematsu, M. Peyton,D. Juroske, Y. Huang, J. Stuart Salmon, Y. H. Kim, J. R. Pollack, K. Yanagisawa, A. Gazdar,J. D. Minna, J. M. Kurie, D. P. Carbone, Aberrant epidermal growth factor receptor signalingand enhanced sensitivity to EGFR inhibitors in lung cancer. Cancer Res. 65, 226–235 (2005).

37. I. B. Weinstein, Cancer. Addiction to oncogenes—the Achilles heal of cancer. Science 297,63–64 (2002).

38. J. A. Engelman, P. A. Jänne, Mechanisms of acquired resistance to epidermal growth factorreceptor tyrosine kinase inhibitors in non–small cell lung cancer. Clin. Cancer Res. 14,2895–2899 (2008).

39. T. Kosaka, Y. Yatabe, H. Endoh, K. Yoshida, T. Hida, M. Tsuboi, H. Tada, H. Kuwano, T. Mitsudomi,Analysis of epidermal growth factor receptorgenemutation in patientswith non–small cell lungcancer and acquired resistance to gefitinib. Clin. Cancer Res. 12, 5764–5769 (2006).

40. M. N. Balak, Y. Gong, G. J. Riely, R. Somwar, A. R. Li, M. F. Zakowski, A. Chiang, G. Yang,O. Ouerfelli, M. G. Kris, M. Ladanyi, V. A. Miller, W. Pao, Novel D761Y and common secondaryT790M mutations in epidermal growth factor receptor–mutant lung adenocarcinomaswith acquired resistance to kinase inhibitors. Clin. Cancer Res. 12, 6494–6501 (2006).

41. T. Mok, D. R. Spigel, K. Park, M. A. Socinski, S. Y. Tung, D. Kim, G. Borzillo, H. Zhang, J. P.O’Connell, P. A. Jänne, Efficacy and safety of PF-00299804 (PF299), an oral, irreversible,pan-human epidermal growth factor receptor (pan-HER) tyrosine kinase inhibitor (TKI),as first-line treatment (tx) of selected patients (pts) with advanced (adv) non-small celllung cancer (NSCLC). J. Clin. Oncol. 28, abstract 7537 (2010).

42. L. V. Sequist, B. Besse, T. J. Lynch, V. A. Miller, K. K. Wong, B. Gitlitz, K. Eaton, C. Zacharchuk,A. Freyman, C. Powell, R. Ananthakrishnan, S. Quinn, J. C. Soria, Neratinib, an irreversiblepan-ErbB receptor tyrosine kinase inhibitor: Results of a phase II trial in patients with ad-vanced non–small-cell lung cancer. J. Clin. Oncol. 28, 3076–3083 (2010).

43. P. A. Janne, K. Reckamp, M. Koczywas, J. A. Engelman, D. R. Camidge, A. Rajan, F. Khuri, J. Q.Liang, J. O’Connell, G. Giaccone, Efficacy and safety of PF-00299804 (PF299) in patients (pt)with advanced NSCLC after failure of at least one prior chemotherapy regimen and priortreatment with erlotinib (E): A two-arm, phase II trial. J. Clin. Oncol. 27, abstract 8063(2009).

44. A. C. Faber, D. Li, Y. Song, M. C. Liang, B. Y. Yeap, R. T. Bronson, E. Lifshits, Z. Chen, S. M.Maira, C. García-Echeverría, K. K. Wong, J. A. Engelman, Differential induction of apoptosisin HER2 and EGFR addicted cancers following PI3K inhibition. Proc. Natl. Acad. Sci. U.S.A.106, 19503–19508 (2009).

45. W. Zhou, D. Ercan, L. Chen, C. H. Yun, D. Li, M. Capelletti, A. B. Cortot, L. Chirieac, R. E. Iacob,R. Padera, J. R. Engen, K. K. Wong, M. J. Eck, N. S. Gray, P. A. Jänne, Novel mutant-selectiveEGFR kinase inhibitors against EGFR T790M. Nature 462, 1070–1074 (2009).

46. L. Regales, Y. Gong, R. Shen, E. de Stanchina, I. Vivanco, A. Goel, J. A. Koutcher, M. Spassova,O. Ouerfelli, I. K. Mellinghoff, M. F. Zakowski, K. A. Politi, W. Pao, Dual targeting of EGFR canovercome a major drug resistance mutation in mouse models of EGFRmutant lung cancer.J. Clin. Invest. 119, 3000–3010 (2009).

47. M. F. Zakowski, M. Ladanyi, M. G. Kris; Memorial Sloan-Kettering Cancer Center LungCancer OncoGenome Group, EGFR mutations in small-cell lung cancers in patients whohave never smoked. N. Engl. J. Med. 355, 213–215 (2006).

48. R. Morinaga, I. Okamoto, K. Furuta, Y. Kawano, M. Sekijima, K. Dote, T. Satou, K. Nishio,M. Fukuoka, K. Nakagawa, Sequential occurrence of non-small cell and small cell lungcancer with the same EGFR mutation. Lung Cancer 58, 411–413 (2007).

49. I. Okamoto, J. Araki, R. Suto, M. Shimada, K. Nakagawa, M. Fukuoka, EGFR mutation ingefitinib-responsive small-cell lung cancer. Ann. Oncol. 17, 1028–1029 (2006).

50. T. Fukui, K. Tsuta, K. Furuta, S. Watanabe, H. Asamura, Y. Ohe, A. M. Maeshima, T. Shibata,N. Masuda, Y. Matsuno, Epidermal growth factor receptor mutation status and clinico-pathological features of combined small cell carcinoma with adenocarcinoma of the lung.Cancer Sci. 98, 1714–1719 (2007).




by guhttp://stm

.sciencemag.org/

Dow

nloaded from

51. A. Tatematsu, J. Shimizu, Y. Murakami, Y. Horio, S. Nakamura, T. Hida, T. Mitsudomi, Y. Yatabe,Epidermal growth factor receptor mutations in small cell lung cancer. Clin. Cancer Res. 14,6092–6096 (2008).

52. J. E. Visvader, G. J. Lindeman, Cancer stem cells in solid tumours: Accumulating evidenceand unresolved questions. Nat. Rev. Cancer 8, 755–768 (2008).

53. J. M. Rosen, C. T. Jordan, The increasing complexity of the cancer stem cell paradigm.Science 324, 1670–1673 (2009).

54. H. Dosaka-Akita, P. T. Cagle, H. Hiroumi, M. Fujita, M. Yamashita, A. Sharma, Y. Kawakami, W. F.Benedict, Differential retinoblastoma and p16INK4A protein expression in neuroendocrine tu-mors of the lung. Cancer 88, 550–556 (2000).

55. J. Yuan, J. Knorr, M. Altmannsberger, G. Goeckenjan, A. Ahr, A. Scharl, K. Strebhardt, Expres-sion of p16 and lack of pRB in primary small cell lung cancer. J. Pathol. 189, 358–362 (1999).

56. M. B. Beasley, S. Lantuejoul, S. Abbondanzo, W. S. Chu, P. S. Hasleton, W. D. Travis, E. Brambilla, TheP16/cyclin D1/Rb pathway in neuroendocrine tumors of the lung.Hum. Pathol. 34, 136–142 (2003).

57. J. H. Chung, J. K. Rho, X. Xu, J. S. Lee, H. I. Yoon, C. T. Lee, Y. J. Choi, H. R. Kim, C. H. Kim, J. C.Lee, Clinical and molecular evidences of epithelial to mesenchymal transition in acquiredresistance to EGFR-TKIs. Lung Cancer, 10.1016/j.lungcan.2010.11.011 (2010).

58. H. Uramoto, T. Iwata, T. Onitsuka, H. Shimokawa, T. Hanagiri, T. Oyama, Epithelial–mesenchymaltransition inEGFR-TKI acquired resistant lungadenocarcinoma.AnticancerRes.30, 2513–2517 (2010).

59. A. Singh, P. Greninger, D. Rhodes, L. Koopman, S. Violette, N. Bardeesy, J. Settleman, Agene expression signature associated with “K-Ras addiction” reveals regulators of EMTand tumor cell survival. Cancer Cell 15, 489–500 (2009).

60. J. Carretero, T. Shimamura, K. Rikova, A. L. Jackson, M. D. Wilkerson, C. L. Borgman, M. S.Buttarazzi, B. A. Sanofsky, K. L. McNamara, K. A. Brandstetter, Z. E. Walton, T. L. Gu, J. C.Silva, K. Crosby, G. I. Shapiro, S. M. Maira, H. Ji, D. H. Castrillon, C. F. Kim, C. García-Echeverría,N. Bardeesy, N. E. Sharpless, N. D. Hayes, W. Y. Kim, J. A. Engelman, K. K. Wong, Integrativegenomic and proteomic analyses identify targets for Lkb1-deficient metastatic lung tumors.Cancer Cell 17, 547–559 (2010).

61. S. V. Sharma, D. Y. Lee, B. Li, M. P. Quinlan, F. Takahashi, S. Maheswaran, U. McDermott,N. Azizian, L. Zou, M. A. Fischbach, K. K. Wong, K. Brandstetter, B. Wittner, S. Ramaswamy,M. Classon, J. Settleman, A chromatin-mediated reversible drug-tolerant state in cancercell subpopulations. Cell 141, 69–80 (2010).

62. G. J. Riely, M. G. Kris, B. Zhao, T. Akhurst, D. T. Milton, E. Moore, L. Tyson, W. Pao, N. A. Rizvi,L. H. Schwartz, V. A. Miller, Prospective assessment of discontinuation and reinitiation oferlotinib or gefitinib in patients with acquired resistance to erlotinib or gefitinib followedby the addition of everolimus. Clin. Cancer Res. 13, 5150–5155 (2007).

63. G. R. Oxnard, M. E. Arcila, C. Sima, G. J. Riely, J. Chmielecki, M. G. Kris, W. Pao, M. Ladanyi,V. A. Miller, Acquired resistance to EGFR tyrosine kinase inhibitors in EGFR mutant lungcancer: Distinct natural history of patients with tumors harboring the T790M mutation.Clin. Cancer Res., 10.1158/1078-0432.CCR-10-2692 (2010).

www.Scie

64. S. M. Rothenberg, J. A. Engelman, S. Le, D. J. Riese II, D. A. Haber, J. Settleman, Modelingoncogene addiction using RNA interference. Proc. Natl. Acad. Sci. U.S.A. 105, 12480–12484(2008).

65. Acknowledgments: We thank A. Muzikansky and E. Bast for their help with data analysis.Funding: This study is supported by grants fromUniting Against Lung Cancer, sponsored byUALC: New England and the Marjorie E. Korff Fund (L.V.S.), the NIH Gastrointestinal CancerSPOREP50CA127003 (J.A.E. andM.M.-K.), K08grant CA120060-01 (J.A.E.), R01CA137008-01 (J.A.E.),R01CA140594 (J.A.E.), 1U01CA141457-01 (J.A.E.), National Cancer Institute Lung SPOREP50CA090578 (J.A.E.), Dana-Farber/Harvard Cancer Center, the American Association for CancerResearch (J.A.E.), the VFoundation (J.A.E.), American Cancer Society RSG-06-102-01-CCE (J.A.E.),the Ellison Foundation Scholar (J.A.E.), and the Doris Duke Charitable Foundation (B.A.W.).This work was supported by a gift from the Daniel Crane Foundation. Author contribu-tions: L.V.S., B.A.W., D.D.-S., A.J.I., M.M.-K., and J.A.E. were responsible for the concept anddesign of the study. L.V.S., P.F., A.T.S., S.G., R.S.H., J.T., J.C.W., T.J.L., M.L., and J.A.E. were respon-sible for identifying and providing patients. S.D., J.C.W., and M.L. were responsible forperforming patient biopsies. D.D.-S., A.B.T., K.B., A.K.C., S.A., K.V., E.J.M., A.J.I., M.M.-K., and J.A.E.were responsible for laboratory analyses. S.D. was responsible for radiographic analyses. J.G.C.provided the PF00299804 compound. L.V.S., B.A.W., D.D.-S., M.M.-K., and J.A.E. were primarilyresponsible for writing the manuscript. All authors contributed to manuscript editing andapproval. Competing interests: P.F. is an advisor to Genentech. D.D.-S. has a paid consultingrelationship with Bio-Reference Laboratories Inc. D.D.-S. and A.J.I. are inventors on a patentfiled for SNaPshot genotyping methods (U.S. patent number 12/799415). J.A.E. holds equityin Gatekeeper and is a co-inventor on a patent pertaining to the use of combined MET andEGFR inhibitors (U.S. patent number 12/450826). T.J.L. is an advisor to Boehringer-Ingelheim,Supergen, Merck, AstraZeneca, and Genentech; is on the Board of Directors of Infinity Phar-maceuticals; and is also an inventor on a patent held by Partners Healthcare for EGFRmutationtesting (U.S. patent numbers 11/894135, 11/894159, and 11/894160). Other authors declare nocompeting interests.

Submitted 6 December 2010Accepted 4 March 2011Published 23 March 201110.1126/scitranslmed.3002003

Citation: L. V. Sequist, B. A. Waltman, D. Dias-Santagata, S. Digumarthy, A. B. Turke, P. Fidias,K. Bergethon, A. T. Shaw, S. Gettinger, A. K. Cosper, S. Akhavanfard, R. S. Heist, J. Temel,J. G. Christensen, J. C. Wain, T. J. Lynch, K. Vernovsky, E. J. Mark, M. Lanuti, A. J. Iafrate,M. Mino-Kenudson, J. A. Engelman, Genotypic and histological evolution of lung cancersacquiring resistance to EGFR inhibitors. Sci. Transl. Med. 3, 75ra26 (2011).

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st on June 15, 2020


InhibitorsGenotypic and Histological Evolution of Lung Cancers Acquiring Resistance to EGFR

Iafrate, Mari Mino-Kenudson and Jeffrey A. EngelmanJames G. Christensen, John C. Wain, Thomas J. Lynch, Kathy Vernovsky, Eugene J. Mark, Michael Lanuti, A. JohnBergethon, Alice T. Shaw, Scott Gettinger, Arjola K. Cosper, Sara Akhavanfard, Rebecca S. Heist, Jennifer Temel, Lecia V. Sequist, Belinda A. Waltman, Dora Dias-Santagata, Subba Digumarthy, Alexa B. Turke, Panos Fidias, Kristin

DOI: 10.1126/scitranslmed.3002003, 75ra2675ra26.3Sci Transl Med

may be essential in the quest to reverse or even prevent the development of drug resistance.new insights into the shifting sands of drug resistance evolution in lung cancers and suggests that serial biopsiesor a different EGFR inhibitor. The detailed genetic and histological analysis by Sequist and colleagues provides they lost the resistance mutations and their tumors once again became sensitive to treatment by either the same2 years. They found that when the patients acquired drug resistance and were then taken off the EGFR inhibitor, patients over the course of their disease, the investigators took serial biopsies from three lung cancer patients overresponded well to the typical chemotherapy regimen used to treat SCLCs. To study the evolution of lung tumors in conversion of NSCLCs into small cell lung cancers (SCLCs), which are easier to treat. Indeed, these five patientstype of tumor. In five patients, the authors discovered another type of transition that was even more surprising: the

like appearance, which is associated with a more aggressive−epithelial cell morphology to a mesenchymal cell phosphatidylinositol 3-kinase). In addition, the authors observed that a few lung cancers transitioned from an

gene (which encodes a subunit of the signaling molecule PIK3CA gene itself and mutations in the EGFRpatients acquired drug resistance mechanisms that had not been reported before including amplification of the amplification of a gene encoding the MET tyrosine kinase receptor, which, like EGFR, drives cell growth. Yet otherrendering the tumors resistant. Meanwhile, another group of patients became resistant because they developed

receptor,patients had acquired another mutation in EGFR (T790M), which interferes with binding of the drug to the acquired resistance. All of the lung cancer patients retained their original activating EGFR mutations, but somepatients treated with EGFR inhibitors, the investigators analyzed tumor biopsies from patients at the time they

In an effort to understand the exact mechanism underscoring the acquisition of drug resistance in NSCLCcancer (NSCLC), and they made some surprising discoveries.

small cell lung−colleagues undertook a comprehensive genetic and histological analysis of 37 patients with non inhibitor emerged within 12 months. To better understand how lung cancers develop drug resistance, Sequist and

mutations in EGFR responded clinically to EGFR inhibitors, and even among these patients, resistance to the this deadly disease. However, only a certain subpopulation of lung cancer patients carrying specific activating

(EGFR), which is switched on in many lung cancers, provided hope that targeted therapies would finally combat almost a decade ago of tyrosine kinase inhibitors that specifically block the epidermal growth factor receptor

Lung cancer is the leading cause of death globally and has proven very difficult to treat. The developmentThe Shifting Sands of Lung Cancer

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