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ORIGINAL ARTICLE

A Sensitive Method for Detecting EGFR Mutations inNon-small Cell Lung Cancer Samples with Few

Tumor Cells

Miguel A. Molina-Vila, PhD,* Jordi Bertran-Alamillo, MSc,* Noem Reguart, MD, PhD,*

Miquel Taron, PhD,* Eva Castella, MD, Mariona Llatjos, MD, Carlota Costa, PhD,*

Clara Mayo, PhD,* Anna Pradas, MSc,* Cristina Queralt, MSc,* Monica Botia,* Mara Perez-Cano,*

Esther Carrasco, MSc,* Mireia Tomas, MSc,* Jose Luis Mate, MD, Teresa Moran, MD,*

and Rafael Rosell, MD, PhD*

Background: Detection of epidermal growth factor receptor(EGFR) mutations in advanced non-small cell lung cancer (NSCLC)

patients has relied on DNA purification from biopsies, amplification,

and sequencing. However, the number of tumor cells in a sample is

often insufficient for EGFR assessment.

Methods: We prospectively screened 1380 NSCLC patients for EGFR

mutations but found that 268 were not evaluable because of insufficient

tumor tissue. We therefore developed and validated a method of

detecting EGFR mutations in these samples. Tumor cells were micro-

dissected into polymerase chain reaction buffer and amplified. EGFR

mutations were detected by length analysis of fluorescently labeled

polymerase chain reaction products and TaqMan assay.

Results: We determined EGFR status in 217 (81%) of the 268

primary NSCLC samples not evaluable in our original studyfresh

and paraffin-embedded with less than 150 cells. Exon 19 deletions

were detected in 11.5% of patients and exon 21 L858R mutations in

5.5%. In addition, the exon 20 T790M mutation was detected in 6 of

15 (40%) patients at the time of progression to erlotinib. The

primary, sensitive mutation was present in all tumor cells, whereas

the T790M mutation was absent in some groups.

Conclusions: The method presented here eliminates the need for

DNA purification and allows for detection of EGFR mutations in

samples containing as few as eight cancer cells.

Key Words: Cytologic samples, EGFR mutations, erlotinib, Non-

small cell lung cancer, T790M.

(J Thorac Oncol. 2008;3: 12241235)

Mutations in the tyrosine kinase (TK) domain of the epi-dermal growth factor receptor (EGFR) have been identi-

fied as a cause of non-small cell lung cancer (NSCLC).15 Themost common oncogenic mutations are small in-frame dele-tions in exon 19 and a point mutation (L858R) in exon 21.These mutations likely cause constitutive activation of thekinase6 and confer dramatic sensitivity to TK inhibitors(TKIs) gefitinib and erlotinib.7,8 Clinically, the efficacy ofthese TKIs has been demonstrated in numerous studies,913

and clinical trials of first-line gefitinib13 and erlotinib14 are being carried out in patients whose tumors harbor EGFRmutations. Unfortunately, the effect of TKIs is limited in time because of the emergence of drug resistance. A secondmutation, a substitution T790M in exon 20,1517 appears in

about half of all patients with acquired resistance to TKIs.18,19

However, the T790M kinase remains sensitive to irreversibleinhibitors.16,17,2024

Screening of EGFR mutations, both for selecting patients for treatment with TKIs and for detecting theresistance mutation, is thus extremely important. However,at present, the most common method of mutation detec-tioninvolving DNA purification from the whole tumorsample, polymerase chain reaction (PCR)-based amplifi-cation, and sequencinghas several limitations, the mostimportant of which is the need for large-sized samples.Most of stage IV NSCLC patients have limited tumortissue available from biopsies, and the number of cells

present is often insufficient for DNA purification. In addi-tion, cytologic samplessuch as fine-needle biopsies,bronchoalveolar aspirates, bronchoalveolar lavages, pleu-ral effusions, and sputaare frequently used to diagnose NSCLC and may constitute the only available sample. New methods are therefore required to detect EGFR mu-tations in biopsies with a limited number of tumor cellsand in cytologic specimens.

A second drawback of sequencing techniques is thatthe total DNA needs to contain a large proportion ofmutant DNA, which does not occur in many samplescontaining only a small fraction of tumor cells. Sensitiveassays are currently being developed to detect mutations in

From the *Catalan Institute of Oncology, Medical Oncology Service, Hosp-tial Germans Trias i Pujol, Badalona, Spain; Pangaea Biotech, USPDexeus University Institute, Barcelona, Spain; and Pathology Depart-ment, Hospital Germans Trias i Pujol, Badalona, Spain.

Disclosure: The authors declare no conflicts of interest.Address for correspondence: Rafael Rosell, MD, Medical Oncology Service,

Catalan Institute of Oncology, Hospital Germans Trias i Pujol, Ctra Canyet,s/n, 08916 Badalona (Barcelona), Spain. E-mail: [email protected]

The first four authors contributed equally to the article.Copyright 2008 by the International Association for the Study of LungCancerISSN: 1556-0864/08/0311-1224

Journal of Thoracic Oncology Volume 3, Number 11, November 20081224


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samples containing less than 10% of mutant DNA.2531

However, most of these assays also have limitations: theyrequire fresh-frozen tumor tissue samples, a large numberof cells, or refined molecular biology techniques.

In the present study, we have developed and vali-dated a method to detect EGFR mutations that can be

applied to all types of samples: fresh or paraffin-embedded biopsies and cytologic specimens. With this method,EGFR mutations can be detected in samples containing asfew as eight cancer cells, in microscopic areas of thetumors, and in separate clumps of cancer cells withincytologic specimens.

MATERIALS AND METHODS

Cell CultureThe PC-9 lung tumor cell line cell line was kindly

provided by Roche (Basel, Switzerland); the H1975 cellline was purchased from the American Type Culture Col-

lection (Manassas, VA). All tissue culture materials wereobtained from Biologic Industries (Kibbutz Beit Haemek,Israel) or Invitrogen (Paisley, Scotland, United Kingdom).

Clinical Samples and Microdissection of TumorCells

A total of 268 NSCLC samples were analyzed: 223paraffin-embedded and 45 fresh specimens. All 268 samplescontained less than 150 tumor cells and had previously beendeemed unevaluable for EGFR mutations in our laboratory.

Paraffin-embedded samples and slides were obtained by standard procedures.11 Fresh specimens were extendedover an appropriate slide, fixed with 96% ethyl-alcohol andstained with Harris hematoxylin for 1 minute. Once the

specimen was stained and rinsed in running water, a coverslide was placed over it to observe and mark the presence ofmalignant cells. Later, the cover slide could be removed andthe sample kept in this stage for not more than 2 or 3 days.Tumor cells were identified by a pathologist.

For both fresh and paraffin-embedded samples, tumorcells (8150) were captured by laser microdissection (Palm,Oberlensheim, Germany) into 10 l of PCR buffer (Ecogen,Barcelona, Spain) plus proteinase K and incubated 4 hours toovernight at 60C. Proteinase was inactivated at 95C for 10minutes, and the cell extract submitted to PCR. DNA fromthe cell line PC-9 was used as a mutated control for exon 19,and wild-type control for exons 20 and 21. DNA from the

H1975 cell line was used as a wild-type control for exon 19,and mutated control for exons 21/20.

PCR Analysis and EGFR Gene SequencingExons 19, 20, and 21 of the EGFR gene were

amplified by a nested PCR as described.11 Primers were asfollows: exon 19 (first PCR, forward 5-GCAATAT-CAGCCTTAGGTGCGGCTC-3, and reverse 5-CATA-GAAAGTGAACATTTAGGATGTG-3; second PCR, forward5-GTGCATCGCTGGTAACATCC-3 and reverse 5-TG-TGGAGATGAGCAGGGTCT-3); exon 21 (first PCR,forward 5-CTAACGTTCGCCAGCCATAAGTCC-3 andreverse 5-GCTGCGAGCTCACCCAGAATGTCTGG-3,

second PCR, forward 5-GCTCAGAGCCTGGCATGAA-3and reverse 5-CATCCTCCCCTGCATGTGT-3 ); exon 20(first PCR, forward 5-ACTTCACAGCCCTGCGTAAAC-3 and reverse 5-ATGGGACAGGCACTGATTTGT-3 ; nestedPCR, forward 5-AGGCAGCCGAAGGGCA-3 and reverse5-CCTCACCTCCACCGTGCA-3 ). The first PCR was per-

formed in 50-l volumes adding 2 l of sample, 2 U ofEcotaq Polimerase (Ecogen, Barcelona, Spain), 7.5 l ofPCR buffer10, 250 M dNTPs, 3.5 mM MgCl2, and 0.5pmol of each primer. Amplification was as follows: 25 cyclesof 30 seconds at 94C, 30 seconds at 64C, and 1 minute at72C (exons 19 and 21), or 35 cycles of 30 seconds at 94C,30 seconds at 58C, and 1 minute at 72C (exon 20). For thenested PCR, amplification was done using 2 l (for exons 19and 20) or 4 l (for exon 21) of first PCR product, 1.25 U ofEcotaq Polymerase, 250 M dNTPs, 1.5 mM MgCl2, and 0.5pmol of each primer. Cycles were as follows: for exon 19, 35cycles of 30 seconds at 94C, 30 seconds at 64C, and 1minute at 72C; for exon 21, 40 cycles of 30 seconds at 94C,

30 seconds at 64C, and 1 minute at 72C; and for exon 21,20 cycles of 30 seconds at 94C, 30 seconds at 59C, and 1minute at 72C.

PCR products were visualized on a 2% agarose gel.Sequencing was performed using forward and reverse nestedprimers with the ABI Prism 3100 DNA Analyzer (AppliedBiosystems, Foster City, CA).

Length Analysis of Fluorescently Labeled PCRProducts for EGFR Deletions in Exon 19

The products of the first PCR for exon 19 were ampli-fied with the following primers: forward 5-ACTCTGGATC-CCAGAAGGTGAG-3 and reverse 5-FAM-CCACACAG-CAAAGCAGAAACTC-3. Amplification was done for 32

cycles (30 seconds at 94C, 30 seconds at 58C, and 1 minuteat 72C) in 50-l volumes using 1 U of Ecotaq Polymerase,250 M dNTPs, 1 mM MgCl2, and 0.5 pmol of each primer.One microliter of a 1/50 to 1/200 dilution of each PCR product was mixed with 0.5 l of size standard (AppliedBiosystems) and denatured in 9 l formamide at 90C for 5minutes. Separation was done with a four-color laser-inducedfluorescence capillary electrophoresis system (ABI Prism3130 Genetic Analyzer, Applied Biosystems). The collecteddata were evaluated with the GeneScan Analysis Software(Applera, Norwalk, CT).

TaqMan Assay for EGFR Mutation in Exons 20

(T790M) and 21 (L858R)The products of the first PCR for exons 20 and 21 were

analyzed by TaqMan. Primers and probes were as follows: exon21 (forward primer, 5-AACACCGCAGCATGTCAAGA-3,reverse primer 5-TTCTCTTCCGCACCCAGC-3; probes5-FAM-CAGATTTTGGGCGGGCCAAAC-TAMRA-3; and5-VIC-TCACAGATTTTGGGCTGGCCAAAC-TAMRA-3)and exon 20 (forward primer, 5-AGGCAGCCGAAGGGCA-3, reverse primer 5-CCTCACCTCCACCGTGCA-3; probes5 VIC-TGAGCTGCGTGATGA-MGB-3; and 5-FAM-TGAGCTGCATGATGA-MGB-3). Amplification was per-formed in 25-l volumes using 2 l of first PCR product, 12.5l of Ampli Taq Gold PCR Master Mix (Applied Biosystems),

Journal of Thoracic Oncology Volume 3, Number 11, November 2008 Sensitive Method for Detecting EGFR Mutations

Copyright 2008 by the International Association for the Study of Lung Cancer 1225


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0.6 pmol of each primer and 0.2 pmol of probes. Samples weresubmitted to 40 cycles of 15 seconds at 94C and 1 minute at60C in an Applied Biosystems 7000 real-time cycler.

RESULTSThe method described here involves 3 steps: (1) direct

microdissection of tumor cells into PCR buffer; (2) a first roundof PCR for each EGFR exon; and (3) determination of EGFR statusby length analysis (exon 19) or TaqMan Assay (exons 20, 21) usingthe first PCR product as a template. This method was comple-mented by further analysis using nested PCR and sequencing.

To evaluate the sensitivity of our assay, we used seri-ally diluted genomic DNA from the cell lines PC-9 (harbor-ing a deletion in exon 19) and H1975 (harboring both theT790M and the L858R mutations). Ten pg of DNA weresuccessfully amplified and the corresponding mutations de-tected. Finally, we trypsinized PC9 and H1975 tumor cells inculture, extended them on a slide and microdissected them indifferent quantities (120 cells). Four tumor cells were suf-

ficient to determine EGFR mutation status (supplementaryFigure S1).

Exon 19 Deletion and L858R MutationFrom May 2006 to December 2007, we prospectively

screened EGFR mutations in 1380 NSCLC patients for the purpose of customizing erlotinib treatment. Of these, 268were not evaluable with the standard procedures used (DNAextraction with phenol:chloroform, followed by ethanol pre-cipitation, PCR, and sequencing of PCR products) because ofinsufficient tumor tissue (fewer than 150 tumor cells). In thepresent study, we have reexamined these 268 samples (Table1), that included: fresh and paraffin-embedded biopsies (192samples), cytologic specimens from bronchoalveolar lavages

and aspirates (9 samples), fine-needle aspirates (31 samples), pleural and pericardial fluids (33 samples), and others (3samples). EGFR mutation status was successfully determinedin 217 (81%) of the 268 samples: 45 of 45 fresh and 172 of223 paraffin-embedded specimens (Table 1). Although samples with as little as eight tumor cells were successfullyamplified, a few containing as many as 100 tumor cells werenot, because of poor quality.

These results were then validated by sequencing. In 187of the 217 samples successfully analyzed, exon 19 deletionwas assessed by nested PCR followed by sequencing, and the

results were identical to those obtained by length analysis. In170 of the 217 samples, the L858R mutation was assessed bysequencing, and the results were identical to those obtainedwith the TaqMan assay. The method was further validated in30 additional NSCLC tumor samples analyzed by standardprocedures in a previous study.32 The results obtained with

our method were identical to the original results.EGFR mutations were detected in 37 of the 217 sam-

ples successfully analyzed (17%). Exon 19 deletions werefound in 11.5% of samples, and L858R mutations in 5.5%(Table 2 and Figure 1). The frequency of mutations washigher in females, never-smokers, and adenocarcinoma pa-tients (Table 2). Eighteen exon 19 deletions were delE746-A750, four were delL747-S752 (two of them with an addi-tional P753S, and one with P753SA755S), one wasdelL747-E749 A750P and one was delA750-E758 plustwo additional silent point mutations. Finally, a case with asingle somatic point mutation (A750P) was also detected.Eleven samples had the L858R mutation and only one had the

L861Q mutation at exon 21.Thirty-five patients with EGFR mutations were treatedwith erlotinib (Table 3). Survival data is available for 20 ofthem. Overall median survival has not been reached; andsome have obtained dramatic, long lasting responses (Figure2). Of 14 patients evaluable for response, two attained com-

TABLE 1. Baseline Assessment of EGFR Mutations atExons 19 and 21 in Samples with Fewer Than 150 TumorCells

Type of SampleNo. of

SamplesEGFR Status

Determined (%)No. of Tumor Cellsper Sample (range)

Paraffin-embedded 223 172 (77) 8150

Ctologies 32 28 (88)

Biopsies 191 144 (74)

Fresh samples 45 45 (100) 25150

Cytologies 44 44 (100)

Biopsies 1 1 (100)

Total 268 217 (81)

TABLE 2. Patient Characteristics. Percentages Refer to theNo. of Patients with Complete Data Recorded

Patients with

Mutations

Patients without

Mutations

N Percent N Percent

Total number 37 180Patients with complete data

recorded37 145

Exon 19 25 11.5

Deletion 24

Point mutation 1

Exon 21 12 5.5

L858R 11

L861Q 1

Gender

Male 10 27 87 60

Female 27 73 58 40

Age, median (range) 62.3 (3989) 65.0 (3589)

Smoking history

Current 7 19 36 25

Ex 4 11 24 17

Never 24 65 66 45

Not reported 2 5 19 13

Histology

Adenocarcinoma 34 92 118 81

Undifferentiated 1 3 16 11

Other 2 5 11 8

Stage

I-II 2 5 8 6

IIIIV 34 92 128 88

Not reported 1 3 9 6

Molina-Vila et al. Journal of Thoracic Oncology Volume 3, Number 11, November 2008

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FIGURE 1. A, Example of length analysis for a wild-type (top panel) and a mutated patient (bottom panel) for exon 19of EGFR. The peaks corresponding to the samples are represented in blue, the molecular weight markers (75, 100, 139,150, and 160 bp) in orange. The mutated patient harbors a 15-bp deletion. B, Example of Taqman assay for a subset ofpatients. The two green triangles correspond to the H1975 cell line and to a tumor harboring the L858R mutation. Thered circles correspond to the PC-9 cell line and to several wild-type tumors. The gray squares are negative and extractioncontrols.

TABLE 3. Clinical Outcome of Patients with EGFR Mutations Treated with Erlotinib

Patient EGFR Mutati on Treatment Started (date) Response to Erlotinib Survival Status

1 A750P June 30, 2006 NE Da (after 1 mo)

2 delE746-A750 October 26, 2006 PR A

3 L858R November 15, 2006 SD A

4 delE746-A750 April 1, 2007 PR A

5 L858R February 08, 2007 NE A

6 L861R December 5, 2006 PD D (after 1 mo)

7 delE746-A750 January 24, 2007 SD A

8 L858R February 20, 2007 SD A

9 delE746-A750 April 11, 2007 PR A

10 delE746-A750 August 18, 2007 NE A

11 delL747-E749 A750P May 24, 2007 CR A

12 delE746-A750 May 23, 2007 SD A

13 delE746-A750 December 8, 2006 PR A

14 L858R June 29, 2007 NE A

15 delE746-A750 July 30, 2007 NE A

16 delE746-A750 July 1, 2007 NE A

17 delE746-A750 October 1, 2007 SD A

18 delE746-A750 February 12, 2007 CR A

19 delE746-A750 April 15, 2007 PR A

20 L858R June 8, 2007 SD D (after 6 mo)

a The cause of death was a bacterial infection in the lungs.

NE, not evaluated; PD, progressive disease; SD, stable disease; PR, partial response; CR, complete response; D, dead; A, alive.




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plete and five partial response, six stable disease, and oneprogressive disease. All responses were observed in patientswith exon 19 deletions, whereas all L858R patients had stabledisease. Progressive disease was observed in the only patientwith the rare L861Q mutation. In contrast, the patient har-boring the exon 19 delL747-E749 A750P attained a com-plete response.

EGFR T790M Mutations in Patients Progressingto Erlotinib

EGFR T790M mutation status was evaluated in 15patients at the time of progression in cytologic specimens ortumor samples obtained from rebiopsy. The T790M mutationwas detected in six patients (40%), a frequency within therange reported in other studies, that is also around 40to50%.33,34 Exons 19 and 21 were also analyzed; the mutationdetected in the primary tumor sample was also present in therebiopsy or recytology sample in all 15 cases. The T790Mmutation was more frequent in patients with exon 19 dele-

tions (five of nine) than in those with the L858R mutation(one of six).

In 7 of the 15 patients progressing to erlotinib, thesize and cellularity of the samples were sufficient tomicrodissect and analyze separate areas of the tumorsample or clumps of tumor cells in the cytologic specimens(Table 4). In all seven cases, the primary exon 19 deletionsor exon 21 L858R mutations were detected in all the cellsanalyzed. The T790M mutation was found in only twopatients (Table 4, patients A and E), where it was detectedin many but not all of the cells. In patient A, fourmicroscopic areas of the tumor sample (rebiopsy at the siteof progression) were analyzed; the primary exon 19 dele-

tion was detected in all four areas, but the T790M mutationwas detected in only three (Figure 3). In patient E, threeclumps of tumor cells from pleural fluid and three micro-scopic areas of the rebiopsy at the site of progression werestudied; the primary exon 19 deletion was present in allareas, but the T790M mutation could not be detected intwo separate groups of cells, one from the rebiopsy and theother from the pleural fluid.

DISCUSSIONSince the discovery of its clinical relevance, the detec-

tion of EGFR mutations has relied on direct sequencing ofPCR products, a method still widely used. However, there isa growing interest in new methods that can overcome some ofits drawbacks. Most of these novel methods include stepsdesigned to amplify mutant DNA when a large amount ofwild-type, nontumor DNA is present or to avoid sequencingand standard PCR to accelerate the assay. For example, the

SMart-Amplification Process33 can detect a mutation inmixed-cell populations in just 30 minutes. However, thismethod has only been tested in fresh biopsies containing 5 mgof tissue, a kind of sample rarely available in advancedNSCLC. The mutant-enriched PCR27 and the Scorpions Am-plified Refractory Mutation System28 have been used in othertypes of fresh samples but not in paraffin-embedded speci-mens, where successful EGFR analysis is more difficult.34 Inaddition, these methods include several DNA purificationsteps (phenol: chloroform extraction, ethanol precipitation)that prevent their use in samples containing a limited numberof cells. Finally, the loop-hybrid mobility shift assay30 andthe SURVEYOR analysis26 have been used in paraffin-

FIGURE 2. Example of response toerlotinib (patient 13 in Table 3) harbor-ing exon 19 delL746A750, detected inpleural fluid. Top panel: CT scan priorto erlotinib treatment (A) and after 1

year of erlotinib treatment (B). Bottompanel: plain chest radiograph beforeerlotinib treatment (C) and after 1 yearof erlotinib treatment (D).




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embedded samples, but they also require prior DNA purifi-cation.

In this study, we have developed and validated amethod to determine EGFR status in samples containing lessthan 150 tumor cells that can be used in both fresh and paraffin-embedded biopsies and cytologic samples. In ourexperience, at least one third of NSCLC primary diagnosesare made solely on the basis of cytologic specimens, where

the number of tumor cells is very limited. In addition, evenwhen a biopsy is available, the number of tumor cells is lessthan 150 in more than 15% of samples. Our method thussignificantly increases the number of NSCLC patients thatcan be screened for EGFR mutations.

This method is based on microdissection of tumor cellsdirectly into PCR buffer, followed by amplification anddetermination of EGFR status by length analysis of fluores-cently-labeled products (exon 19 deletion) or TaqMan assay(exon 20 T790M and exon 21 L858R). This method can beapplied to any other exon, however, our focus was on dele-tions in exon 19 and the L858R in exon 21, because theyaccount for the great majority of sensitive EGFR mutations.

Because there is no need for prior DNA purification,our method is highly sensitive; EGFR mutations at exons 19,20, and 21 can be detected in samples containing as few aseight cells (in 10 l of buffer), approximately 5 pg of DNAper microliter of crude extract. This compares favorably tothe sensitivity of the SMart-Amplification Process (210 pg, or30 copies, of mutant, purified DNA per microliter) or theScorpions Amplified Refractory Mutation System (100 pg

of purified DNA per microliter). Other methods are even lesssensitive; mutant enriched PCR requires 5 to 100 ng of DNA,the loop-hybrid mobility shift assay needs two complete10-m sections of a paraffin-embedded biopsy, and theSURVEYOR assay requires ten 5-m sections or four 10-msections of a paraffin-embedded biopsy to obtain 3 to 30 gof DNA. Finally, slide scrape with no prior purificationrequires at least 10 mm2 of tumor area to obtain a minimumof 79 ng DNA per microliter.29

The method described here also allows for analysis ofseparate, microscopic groups of cells within a tumor massand clumps of cells in cytologic specimens. We have used itsuccessfully to analyze more than three groups of cells in six

TABLE 4. Patients with Three or More Groups of Tumor Cells Analyzed

Patient Primary Mutation

Groups of Tumor

Cells Analyzed

Exon 19/21

Status

Exon 20

Status

A Exon 19 (delE746-A750) PB1 delE746-A750 wt

PB2 delE746-A750 T790M

PB3 delE746-A750 T790MPB4 delE746-A750 T790M

B Exon 19 (delE746-A750) PB1 delE746-A750 wt

PB2 delE746-A750 wt

PB3 delE746-A750 wt

PB4 delE746-A750 wt

C Exon 19 (delE746-A750) PB1 delE746-A750 wt

PB2 delE746-A750 wt

PB3 delE746-A750 wt

PB4 delE746-A750 wt

PB5 delE746-A750 wt

D Exon 21 (L858R) FP1 L858R wt

PB1 L858R wt

PB2 L858R wt

PB3 L858R wt

E Exon 19 (delE746-A750) PB1 delE746-A750 wt



PE1 delE746-A750 T790M

PE2 delE746-A750 T790M

PE3 delE746-A750 wt

F Exon 21 (L858R) CE1 L858R wt

CE2 L858R wt

CE3 L858R wt

CE4 L858R wt

G Exon 21 (L858R) PP1 L858R wt

PP2 L858R wt

PP3 L858R wt

PB, paraffin-embedded biopsy; FP, fresh biopsy; PE, fresh pleural fluid; CE, fresh pericardial fluid; PP, paraffin-embedded pleural fluid; wt, wild-type.




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samples from patients progressing to erlotinib. In all cases,the primary mutation (in exon 19 or 21) was present in all thegroups of tumor cells.

Although the primary mutation (exon 19 deletion or L858R)was present in all the groups of tumor cells analyzed, the T790Mresistance mutation was not. In some tumors, the T790M mutationseems to be underrepresented relative to the total number of EGFRalleles,18 leading to the speculation that it might be present in onlya subset of resistant cancer cells,35,36 thus increasing the importanceof a method able to detect mutations in a small number of cells.

In conclusion, we have developed and validated amethod to detect EGFR mutations that can be applied to freshor paraffin-embedded biopsies and cytologic specimens con-

taining as few as eight cancer cells, thus widening the rangeof lung tumor patients that can be tested for EGFR mutations.

ACKNOWLEDGMENTSSupport received from Redes Tematicas de Investiga-

cion Cooperativa (RD06/0020/0056), Ministerio de Sanidady Consumo (Spain), and from La Fundacion Badalona Con-tra el Cancer (Spain).

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FIGURE 3. Sequencing chromatogram for EGFR exons 19 and 20 in a patient progressing to erlotinib (patient A in Table 4)showing heterogeneous distribution of the T790M mutation. Four areas of the tumor mass at the site of progression (PB1,PB2, PB3, and PB4) were analyzed. Top panel: length analysis for exon 19; the 104-kb peak showing the presence of a 15-bpdeletion is indicated by an arrow. Bottom panel: sequencing results for exon 20; the presence of the T790M mutation as anadditional T peak in the chromatogram is indicated with an asterisk. While the primary mutation (the 15-bp deletiondelE746A750) was present in all areas, the T790M mutation (C to T transition) was not detected in one area of the tumor(PB1). The result was confirmed by TaqMan assay.




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FIGURE S1. Sensitivity of the assay: Serially diluted genomic DNA from the cell lines PC-9 (harboring a deletion in exon 19)and H1975 (harboring both the T790M and the L858R mutations) was analyzed (A and C). Ten picogram of DNA were suc-cessfully amplified and the corresponding mutations detected. We also trypsinized tumor cells in culture, extended them on aslide and microdissected them in different quantities. Four tumor cells were sufficient to determine EGFR mutation status (Band D).




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FIGURE S1. (Continued).




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