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ORIGINAL ARTICLE

A Sensitive Method for Detecting EGFR Mutations in


Non-small Cell Lung Cancer Samples with Few
Tumor Cells
Miguel A. Molina-Vila, PhD,*† Jordi Bertran-Alamillo, MSc,*† Noemí Reguart, MD, PhD,*
Miquel Taron, PhD,*† Eva Castellà, MD,‡ Mariona Llatjós, MD,‡ Carlota Costa, PhD,*†
Clara Mayo, PhD,* Anna Pradas, MSc,* Cristina Queralt, MSc,* Mónica Botia,* María Pérez-Cano,*
Esther Carrasco, MSc,* Mireia Tomàs, 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)
M utations 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).1–5 The
patients has relied on DNA purification from biopsies, amplification, most common oncogenic mutations are small in-frame dele-
and sequencing. However, the number of tumor cells in a sample is tions in exon 19 and a point mutation (L858R) in exon 21.
often insufficient for EGFR assessment. These mutations likely cause constitutive activation of the
Methods: We prospectively screened 1380 NSCLC patients for EGFR kinase6 and confer dramatic sensitivity to TK inhibitors
mutations but found that 268 were not evaluable because of insufficient (TKIs) gefitinib and erlotinib.7,8 Clinically, the efficacy of
tumor tissue. We therefore developed and validated a method of these TKIs has been demonstrated in numerous studies,9 –13
detecting EGFR mutations in these samples. Tumor cells were micro- and clinical trials of first-line gefitinib13 and erlotinib14 are
dissected into polymerase chain reaction buffer and amplified. EGFR being carried out in patients whose tumors harbor EGFR
mutations were detected by length analysis of fluorescently labeled mutations. Unfortunately, the effect of TKIs is limited in time
polymerase chain reaction products and TaqMan assay. because of the emergence of drug resistance. A second
Results: We determined EGFR status in 217 (81%) of the 268 mutation, a substitution T790M in exon 20,15–17 appears in
primary NSCLC samples not evaluable in our original study—fresh about half of all patients with acquired resistance to TKIs.18,19
and paraffin-embedded with less than 150 cells. Exon 19 deletions However, the T790M kinase remains sensitive to irreversible
were detected in 11.5% of patients and exon 21 L858R mutations in inhibitors.16,17,20 –24
5.5%. In addition, the exon 20 T790M mutation was detected in 6 of Screening of EGFR mutations, both for selecting
15 (40%) patients at the time of progression to erlotinib. The patients for treatment with TKIs and for detecting the
primary, sensitive mutation was present in all tumor cells, whereas resistance mutation, is thus extremely important. However,
the T790M mutation was absent in some groups. at present, the most common method of mutation detec-
Conclusions: The method presented here eliminates the need for
tion—involving DNA purification from the whole tumor
sample, polymerase chain reaction (PCR)-based amplifi-
DNA purification and allows for detection of EGFR mutations in
cation, and sequencing— has several limitations, the most
samples containing as few as eight cancer cells.
important of which is the need for large-sized samples.
Key Words: Cytologic samples, EGFR mutations, erlotinib, Non- Most of stage IV NSCLC patients have limited tumor
small cell lung cancer, T790M. tissue available from biopsies, and the number of cells
present is often insufficient for DNA purification. In addi-
(J Thorac Oncol. 2008;3: 1224 –1235) tion, cytologic samples—such as fine-needle biopsies,
bronchoalveolar aspirates, bronchoalveolar lavages, pleu-
From the *Catalan Institute of Oncology, Medical Oncology Service, Hosp- ral effusions, and sputa—are frequently used to diagnose
tial Germans Trias i Pujol, Badalona, Spain; †Pangaea Biotech, USP NSCLC and may constitute the only available sample.
Dexeus University Institute, Barcelona, Spain; and ‡Pathology Depart- New methods are therefore required to detect EGFR mu-
ment, Hospital Germans Trias i Pujol, Badalona, Spain.
Disclosure: The authors declare no conflicts of interest. tations in biopsies with a limited number of tumor cells
Address for correspondence: Rafael Rosell, MD, Medical Oncology Service, and in cytologic specimens.
Catalan Institute of Oncology, Hospital Germans Trias i Pujol, Ctra Canyet, A second drawback of sequencing techniques is that
s/n, 08916 Badalona (Barcelona), Spain. E-mail: rrosell@ico.scs.es the total DNA needs to contain a large proportion of
The first four authors contributed equally to the article.
Copyright © 2008 by the International Association for the Study of Lung mutant DNA, which does not occur in many samples
Cancer containing only a small fraction of tumor cells. Sensitive
ISSN: 1556-0864/08/0311-1224 assays are currently being developed to detect mutations in

1224 Journal of Thoracic Oncology • Volume 3, Number 11, November 2008


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

samples containing less than 10% of mutant DNA.25–31 second PCR, forward 5⬘-GCTCAGAGCCTGGCATGAA-3⬘
However, most of these assays also have limitations: they and reverse 5⬘-CATCCTCCCCTGCATGTGT-3⬘); exon 20
require fresh-frozen tumor tissue samples, a large number (first PCR, forward 5⬘-ACTTCACAGCCCTGCGTAAAC-3⬘
of cells, or refined molecular biology techniques. and reverse 5⬘-ATGGGACAGGCACTGATTTGT-3⬘; nested
In the present study, we have developed and vali- PCR, forward 5⬘-AGGCAGCCGAAGGGCA-3⬘ and reverse
dated a method to detect EGFR mutations that can be 5⬘-CCTCACCTCCACCGTGCA-3⬘). The first PCR was per-
applied to all types of samples: fresh or paraffin-embedded formed in 50-␮l volumes adding 2 ␮l of sample, 2 U of
biopsies and cytologic specimens. With this method, Ecotaq Polimerase (Ecogen, Barcelona, Spain), 7.5 ␮l of
EGFR mutations can be detected in samples containing as PCR buffer ⫻10, 250 ␮M dNTPs, 3.5 mM MgCl2, and 0.5
few as eight cancer cells, in microscopic areas of the pmol of each primer. Amplification was as follows: 25 cycles
tumors, and in separate clumps of cancer cells within of 30 seconds at 94°C, 30 seconds at 64°C, and 1 minute at
cytologic specimens. 72°C (exons 19 and 21), or 35 cycles of 30 seconds at 94°C,
30 seconds at 58°C, and 1 minute at 72°C (exon 20). For the
MATERIALS AND METHODS nested PCR, amplification was done using 2 ␮l (for exons 19
and 20) or 4 ␮l (for exon 21) of first PCR product, 1.25 U of
Cell Culture Ecotaq Polymerase, 250 ␮M dNTPs, 1.5 mM MgCl2, and 0.5
The PC-9 lung tumor cell line cell line was kindly pmol of each primer. Cycles were as follows: for exon 19, 35
provided by Roche (Basel, Switzerland); the H1975 cell cycles of 30 seconds at 94°C, 30 seconds at 64°C, and 1
line was purchased from the American Type Culture Col- minute at 72°C; for exon 21, 40 cycles of 30 seconds at 94°C,
lection (Manassas, VA). All tissue culture materials were 30 seconds at 64°C, and 1 minute at 72°C; and for exon 21,
obtained from Biologic Industries (Kibbutz Beit Haemek, 20 cycles of 30 seconds at 94°C, 30 seconds at 59°C, and 1
Israel) or Invitrogen (Paisley, Scotland, United Kingdom). minute at 72°C.
PCR products were visualized on a 2% agarose gel.
Clinical Samples and Microdissection of Tumor Sequencing was performed using forward and reverse nested
Cells primers with the ABI Prism 3100 DNA Analyzer (Applied
A total of 268 NSCLC samples were analyzed: 223 Biosystems, Foster City, CA).
paraffin-embedded and 45 fresh specimens. All 268 samples
contained less than 150 tumor cells and had previously been Length Analysis of Fluorescently Labeled PCR
deemed unevaluable for EGFR mutations in our laboratory. Products for EGFR Deletions in Exon 19
Paraffin-embedded samples and slides were obtained The products of the first PCR for exon 19 were ampli-
by standard procedures.11 Fresh specimens were extended fied with the following primers: forward 5⬘-ACTCTGGATC-
over an appropriate slide, fixed with 96% ethyl-alcohol and CCAGAAGGTGAG-3⬘ and reverse 5⬘-FAM-CCACACAG-
stained with Harris hematoxylin for 1 minute. Once the CAAAGCAGAAACTC-3⬘. Amplification was done for 32
specimen was stained and rinsed in running water, a cover cycles (30 seconds at 94°C, 30 seconds at 58°C, and 1 minute
slide was placed over it to observe and mark the presence of at 72°C) in 50-␮l volumes using 1 U of Ecotaq Polymerase,
malignant cells. Later, the cover slide could be removed and 250 ␮M dNTPs, 1 mM MgCl2, and 0.5 pmol of each primer.
the sample kept in this stage for not more than 2 or 3 days. One microliter of a 1/50 to 1/200 dilution of each PCR
Tumor cells were identified by a pathologist. product was mixed with 0.5 ␮l of size standard (Applied
For both fresh and paraffin-embedded samples, tumor Biosystems) and denatured in 9 ␮l formamide at 90°C for 5
cells (8 –150) were captured by laser microdissection (Palm, minutes. Separation was done with a four-color laser-induced
Oberlensheim, Germany) into 10 ␮l of PCR buffer (Ecogen, fluorescence capillary electrophoresis system (ABI Prism
Barcelona, Spain) plus proteinase K and incubated 4 hours to 3130 Genetic Analyzer, Applied Biosystems). The collected
overnight at 60°C. Proteinase was inactivated at 95°C for 10 data were evaluated with the GeneScan Analysis Software
minutes, and the cell extract submitted to PCR. DNA from (Applera, Norwalk, CT).
the 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 TaqMan Assay for EGFR Mutation in Exons 20
H1975 cell line was used as a wild-type control for exon 19, (T790M) and 21 (L858R)
and mutated control for exons 21/20. The products of the first PCR for exons 20 and 21 were
analyzed by TaqMan. Primers and probes were as follows: exon
PCR Analysis and EGFR Gene Sequencing 21 (forward primer, 5⬘-AACACCGCAGCATGTCAAGA-3⬘,
Exons 19, 20, and 21 of the EGFR gene were reverse primer 5⬘-TTCTCTTCCGCACCCAGC-3⬘; probes
amplified by a nested PCR as described.11 Primers were as 5⬘-FAM-CAGATTTTGGGCGGGCCAAAC-TAMRA-3⬘; and
follows: exon 19 (first PCR, forward 5⬘-GCAATAT- 5⬘-VIC-TCACAGATTTTGGGCTGGCCAAAC-TAMRA-3⬘)
CAGCCTTAGGTGCGGCTC-3⬘, and reverse 5⬘-CATA- and exon 20 (forward primer, 5⬘-AGGCAGCCGAAGGGCA-
GAAAGTGAACATTTAGGATGTG-3⬘; second PCR, forward 3⬘, reverse primer 5⬘-CCTCACCTCCACCGTGCA-3⬘; probes
5⬘-GTGCATCGCTGGTAACATCC-3⬘ and reverse 5⬘-TG- 5⬘ VIC-TGAGCTGCGTGATGA-MGB-3⬘; and 5⬘-FAM-
TGGAGATGAGCAGGGTCT-3⬘); exon 21 (first PCR, TGAGCTGCATGATGA-MGB-3). Amplification was per-
forward 5⬘-CTAACGTTCGCCAGCCATAAGTCC-3⬘ and formed in 25-␮l volumes using 2 ␮l of first PCR product, 12.5
reverse 5⬘-GCTGCGAGCTCACCCAGAATGTCTGG-3⬘, ␮l of Ampli Taq Gold PCR Master Mix (Applied Biosystems),

Copyright © 2008 by the International Association for the Study of Lung Cancer 1225
Molina-Vila et al. Journal of Thoracic Oncology • Volume 3, Number 11, November 2008

0.6 pmol of each primer and 0.2 pmol of probes. Samples were results were identical to those obtained by length analysis. In
submitted to 40 cycles of 15 seconds at 94°C and 1 minute at 170 of the 217 samples, the L858R mutation was assessed by
60°C in an Applied Biosystems 7000 real-time cycler. sequencing, and the results were identical to those obtained
with the TaqMan assay. The method was further validated in
RESULTS 30 additional NSCLC tumor samples analyzed by standard
The method described here involves 3 steps: (1) direct procedures in a previous study.32 The results obtained with
microdissection of tumor cells into PCR buffer; (2) a first round our method were identical to the original results.
of PCR for each EGFR exon; and (3) determination of EGFR status EGFR mutations were detected in 37 of the 217 sam-
by length analysis (exon 19) or TaqMan Assay (exons 20, 21) using ples successfully analyzed (17%). Exon 19 deletions were
the first PCR product as a template. This method was comple- found in 11.5% of samples, and L858R mutations in 5.5%
mented by further analysis using nested PCR and sequencing. (Table 2 and Figure 1). The frequency of mutations was
To evaluate the sensitivity of our assay, we used seri- higher in females, never-smokers, and adenocarcinoma pa-
ally diluted genomic DNA from the cell lines PC-9 (harbor- tients (Table 2). Eighteen exon 19 deletions were delE746-
ing a deletion in exon 19) and H1975 (harboring both the A750, four were delL747-S752 (two of them with an addi-
T790M and the L858R mutations). Ten pg of DNA were tional P753S, and one with P753S⫹A755S), one was
successfully amplified and the corresponding mutations de- delL747-E749 ⫹ A750P and one was delA750-E758 plus
tected. Finally, we trypsinized PC9 and H1975 tumor cells in two additional silent point mutations. Finally, a case with a
culture, extended them on a slide and microdissected them in single somatic point mutation (A750P) was also detected.
different quantities (1–20 cells). Four tumor cells were suf- Eleven samples had the L858R mutation and only one had the
ficient to determine EGFR mutation status (supplementary L861Q mutation at exon 21.
Figure S1). Thirty-five patients with EGFR mutations were treated
with erlotinib (Table 3). Survival data is available for 20 of
Exon 19 Deletion and L858R Mutation them. Overall median survival has not been reached; and
From May 2006 to December 2007, we prospectively some have obtained dramatic, long lasting responses (Figure
screened EGFR mutations in 1380 NSCLC patients for the 2). Of 14 patients evaluable for response, two attained com-
purpose of customizing erlotinib treatment. Of these, 268
were not evaluable with the standard procedures used (DNA
extraction with phenol:chloroform, followed by ethanol pre- TABLE 2. Patient Characteristics. Percentages Refer to the
cipitation, PCR, and sequencing of PCR products) because of No. of Patients with Complete Data Recorded
insufficient tumor tissue (fewer than 150 tumor cells). In the Patients with Patients without
present study, we have reexamined these 268 samples (Table Mutations Mutations
1), that included: fresh and paraffin-embedded biopsies (192 N Percent N Percent
samples), cytologic specimens from bronchoalveolar lavages
Total number 37 180
and aspirates (9 samples), fine-needle aspirates (31 samples),
Patients with complete data 37 145
pleural and pericardial fluids (33 samples), and others (3 recorded
samples). EGFR mutation status was successfully determined Exon 19 25 11.5 — —
in 217 (81%) of the 268 samples: 45 of 45 fresh and 172 of Deletion 24
223 paraffin-embedded specimens (Table 1). Although sam- Point mutation 1
ples with as little as eight tumor cells were successfully Exon 21 12 5.5 — —
amplified, a few containing as many as 100 tumor cells were L858R 11
not, because of poor quality. L861Q 1
These results were then validated by sequencing. In 187 Gender
of the 217 samples successfully analyzed, exon 19 deletion Male 10 27 87 60
was assessed by nested PCR followed by sequencing, and the Female 27 73 58 40
Age, median (range) 62.3 (39–89) 65.0 (35–89)
Smoking history
TABLE 1. Baseline Assessment of EGFR Mutations at Current 7 19 36 25
Exons 19 and 21 in Samples with Fewer Than 150 Tumor Ex 4 11 24 17
Cells
Never 24 65 66 45
No. of EGFR Status No. of Tumor Cells Not reported 2 5 19 13
Type of Sample Samples Determined (%) per Sample (range) Histology
Paraffin-embedded 223 172 (77) 8–150 Adenocarcinoma 34 92 118 81
Ctologies 32 28 (88) Undifferentiated 1 3 16 11
Biopsies 191 144 (74) Other 2 5 11 8
Fresh samples 45 45 (100) 25–150 Stage
Cytologies 44 44 (100) I-II 2 5 8 6
Biopsies 1 1 (100) III–IV 34 92 128 88
Total 268 217 (81) Not reported 1 3 9 6

1226 Copyright © 2008 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology • Volume 3, Number 11, November 2008 Sensitive Method for Detecting EGFR Mutations

FIGURE 1. A, Example of length analysis for a wild-type (top panel) and a mutated patient (bottom panel) for exon 19
of 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 of
patients. The two green triangles correspond to the H1975 cell line and to a tumor harboring the L858R mutation. The
red circles correspond to the PC-9 cell line and to several wild-type tumors. The gray squares are negative and extraction
controls.

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

Copyright © 2008 by the International Association for the Study of Lung Cancer 1227
Molina-Vila et al. Journal of Thoracic Oncology • Volume 3, Number 11, November 2008

FIGURE 2. Example of response to


erlotinib (patient 13 in Table 3) harbor-
ing exon 19 delL746 –A750, detected in
pleural fluid. Top panel: CT scan prior
to erlotinib treatment (A) and after 1
year of erlotinib treatment (B). Bottom
panel: plain chest radiograph before
erlotinib treatment (C) and after 1 year
of erlotinib treatment (D).

plete and five partial response, six stable disease, and one tion was detected in all four areas, but the T790M mutation
progressive disease. All responses were observed in patients was detected in only three (Figure 3). In patient E, three
with exon 19 deletions, whereas all L858R patients had stable clumps of tumor cells from pleural fluid and three micro-
disease. Progressive disease was observed in the only patient scopic areas of the rebiopsy at the site of progression were
with the rare L861Q mutation. In contrast, the patient har- studied; the primary exon 19 deletion was present in all
boring the exon 19 delL747-E749 ⫹ A750P attained a com- areas, but the T790M mutation could not be detected in
plete response. two separate groups of cells, one from the rebiopsy and the
other from the pleural fluid.
EGFR T790M Mutations in Patients Progressing
to Erlotinib
EGFR T790M mutation status was evaluated in 15 DISCUSSION
patients at the time of progression in cytologic specimens or Since the discovery of its clinical relevance, the detec-
tumor samples obtained from rebiopsy. The T790M mutation tion of EGFR mutations has relied on direct sequencing of
was detected in six patients (40%), a frequency within the PCR products, a method still widely used. However, there is
range reported in other studies, that is also around 40 a growing interest in new methods that can overcome some of
to50%.33,34 Exons 19 and 21 were also analyzed; the mutation its drawbacks. Most of these novel methods include steps
detected in the primary tumor sample was also present in the designed to amplify mutant DNA when a large amount of
rebiopsy or recytology sample in all 15 cases. The T790M wild-type, nontumor DNA is present or to avoid sequencing
mutation was more frequent in patients with exon 19 dele- and standard PCR to accelerate the assay. For example, the
tions (five of nine) than in those with the L858R mutation SMart-Amplification Process33 can detect a mutation in
(one of six). mixed-cell populations in just 30 minutes. However, this
In 7 of the 15 patients progressing to erlotinib, the method has only been tested in fresh biopsies containing 5 mg
size and cellularity of the samples were sufficient to of tissue, a kind of sample rarely available in advanced
microdissect and analyze separate areas of the tumor NSCLC. The mutant-enriched PCR27 and the Scorpions Am-
sample or clumps of tumor cells in the cytologic specimens plified Refractory Mutation System28 have been used in other
(Table 4). In all seven cases, the primary exon 19 deletions types of fresh samples but not in paraffin-embedded speci-
or exon 21 L858R mutations were detected in all the cells mens, where successful EGFR analysis is more difficult.34 In
analyzed. The T790M mutation was found in only two addition, these methods include several DNA purification
patients (Table 4, patients A and E), where it was detected steps (phenol: chloroform extraction, ethanol precipitation)
in many but not all of the cells. In patient A, four that prevent their use in samples containing a limited number
microscopic areas of the tumor sample (rebiopsy at the site of cells. Finally, the loop-hybrid mobility shift assay30 and
of progression) were analyzed; the primary exon 19 dele- the SURVEYOR analysis26 have been used in paraffin-

1228 Copyright © 2008 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology • Volume 3, Number 11, November 2008 Sensitive Method for Detecting EGFR Mutations

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


Groups of Tumor Exon 19/21 Exon 20
Patient Primary Mutation Cells Analyzed Status Status
A Exon 19 (delE746-A750) PB1 delE746-A750 wt
PB2 delE746-A750 T790M
PB3 delE746-A750 T790M
PB4 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
PB2 delE746-A750 T790M
PB3 delE746-A750 T790M
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.

embedded samples, but they also require prior DNA purifi- Because there is no need for prior DNA purification,
cation. our method is highly sensitive; EGFR mutations at exons 19,
In this study, we have developed and validated a 20, and 21 can be detected in samples containing as few as
method to determine EGFR status in samples containing less eight cells (in 10 ␮l of buffer), approximately 5 pg of DNA
than 150 tumor cells that can be used in both fresh and per microliter of crude extract. This compares favorably to
paraffin-embedded biopsies and cytologic samples. In our the sensitivity of the SMart-Amplification Process (210 pg, or
experience, at least one third of NSCLC primary diagnoses 30 copies, of mutant, purified DNA per microliter) or the
are made solely on the basis of cytologic specimens, where Scorpions Amplified Refractory Mutation System (⬎100 pg
the number of tumor cells is very limited. In addition, even of purified DNA per microliter). Other methods are even less
when a biopsy is available, the number of tumor cells is less sensitive; mutant enriched PCR requires 5 to 100 ng of DNA,
than 150 in more than 15% of samples. Our method thus the loop-hybrid mobility shift assay needs two complete
significantly increases the number of NSCLC patients that 10-␮m sections of a paraffin-embedded biopsy, and the
can be screened for EGFR mutations. SURVEYOR assay requires ten 5-␮m sections or four 10-␮m
This method is based on microdissection of tumor cells sections of a paraffin-embedded biopsy to obtain 3 to 30 ␮g
directly into PCR buffer, followed by amplification and of DNA. Finally, slide scrape with no prior purification
determination of EGFR status by length analysis of fluores- requires at least 10 mm2 of tumor area to obtain a minimum
cently-labeled products (exon 19 deletion) or TaqMan assay of 79 ng DNA per microliter.29
(exon 20 T790M and exon 21 L858R). This method can be The method described here also allows for analysis of
applied to any other exon, however, our focus was on dele- separate, microscopic groups of cells within a tumor mass
tions in exon 19 and the L858R in exon 21, because they and clumps of cells in cytologic specimens. We have used it
account for the great majority of sensitive EGFR mutations. successfully to analyze more than three groups of cells in six

Copyright © 2008 by the International Association for the Study of Lung Cancer 1229
Molina-Vila et al. Journal of Thoracic Oncology • Volume 3, Number 11, November 2008

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-bp
deletion is indicated by an arrow. Bottom panel: sequencing results for exon 20; the presence of the T790M mutation as an
additional T peak in the chromatogram is indicated with an asterisk. While the primary mutation (the 15-bp deletion
delE746 –A750) 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.

samples from patients progressing to erlotinib. In all cases, correlation with clinical response to gefitinib therapy. Science 2004;304:
the primary mutation (in exon 19 or 21) was present in all the 1497–1500.
3. Pao W, Miller V, Zakowski M, et al. EGF receptor gene mutations are
groups of tumor cells. common in lung cancers from “never smokers” and are associated with
Although the primary mutation (exon 19 deletion or L858R) sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci USA
was present in all the groups of tumor cells analyzed, the T790M 2004;101:13306 –13311.
resistance mutation was not. In some tumors, the T790M mutation 4. Gazdar AF, Shigematsu H, Herz J, Minna JD. Mutations and addiction
to EGFR: the Achilles ‘heal’ of lung cancers? Trends Mol Med 2004;
seems to be underrepresented relative to the total number of EGFR 10:481– 486.
alleles,18 leading to the speculation that it might be present in only 5. Johnson BE, Janne PA. Epidermal growth factor receptor mutations in
a subset of resistant cancer cells,35,36 thus increasing the importance patients with non-small cell lung cancer. Cancer Res 2005;65:7525–
of a method able to detect mutations in a small number of cells. 7529.
In conclusion, we have developed and validated a 6. Yun CH, Boggon TJ, Li Y, et al. Structures of lung cancer-derived
EGFR mutants and inhibitor complexes: mechanism of activation and
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taining as few as eight cancer cells, thus widening the range 7. Carey KD, Garton AJ, Romero MS, et al. Kinetic analysis of epidermal
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ACKNOWLEDGMENTS 8. Sordella R, Bell DW, Haber DA, Settleman J. Gefitinib-sensitizing
Support received from Redes Temáticas de Investiga- EGFR mutations in lung cancer activate anti-apoptotic pathways. Sci-
ción Cooperativa (RD06/0020/0056), Ministerio de Sanidad ence 2004;305:1163–1167.
y Consumo (Spain), and from La Fundación Badalona Con- 9. Mitsudomi T, Kosaka T, Endoh H, et al. Mutations of the epidermal
growth factor receptor gene predict prolonged survival after gefitinib
tra el Cáncer (Spain). treatment in patients with non-small-cell lung cancer with postoperative
recurrence. J Clin Oncol 2005;23:2513–2520.
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Molina-Vila et al. Journal of Thoracic Oncology • Volume 3, Number 11, November 2008

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 a
slide and microdissected them in different quantities. Four tumor cells were sufficient to determine EGFR mutation status (B
and D).

1232 Copyright © 2008 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology • Volume 3, Number 11, November 2008 Sensitive Method for Detecting EGFR Mutations

FIGURE S1. (Continued).

Copyright © 2008 by the International Association for the Study of Lung Cancer 1233
Molina-Vila et al. Journal of Thoracic Oncology • Volume 3, Number 11, November 2008

FIGURE S1. (Continued).

1234 Copyright © 2008 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology • Volume 3, Number 11, November 2008 Sensitive Method for Detecting EGFR Mutations

FIGURE S1. (Continued).

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