MET amplification occurs with or without T790M
mutations in EGFR mutant lung tumors with
acquired resistance to gefitinib or erlotinib
James Beana, Cameron Brennanb, Jin-Yuan Shihc, Gregory Rielyd,e, Agnes Vialef, Lu Wangg, Dhananjay Chitaleg,
Noriko Motoig,h, Janos Szokeg,i, Stephen Broderickj, Marissa Balaka, Wen-Cheng Changk, Chong-Jen Yuc, Adi Gazdarl,
Harvey Passm, Valerie Ruschj, William Geralda,g, Shiu-Feng Huangn, Pan-Chyr Yangc, Vincent Millerd,e, Marc Ladanyia,g,
Chih-Hsin Yango, and William Paoa,d,e,p
aHuman Oncology and Pathogenesis Program,bDepartment of Neurosurgery,jThoracic Surgery Service, Department of Surgery,dThoracic Oncology Service,
Division of Solid Tumor Oncology, Department of Medicine,fGenomics Core Laboratory,gDepartment of Pathology, Memorial Sloan–Kettering Cancer
Center, New York, NY 10021;cDepartment of Internal Medicine, College of Medicine andoDepartment of Oncology, National Taiwan University Hospital
and Graduate Institute of Clinical Medicine, National Taiwan University Hospital, Taipei 100, Taiwan;eDepartment of Medicine, Weill Medical College of
Cornell University, New York, NY 10021;hDepartment of Pathology, Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550,
Japan;iDepartment of Molecular Pathology, National Institute of Oncology, Rath Gy. u. 7-9, 1122, Budapest, Hungary;kDepartment of Hematology–
Oncology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan;lHamon Center for Therapeutic Oncology Research, University of Texas Southwestern
Medical Center, Dallas, TX 75390;mDepartment of Cardiothoracic Surgery, New York University Medical Center, New York, NY 10016; andnDivision of
Molecular and Genomic Medicine, National Health Research Institutes, Miaoli 350, Taiwan
Communicated by Harold E. Varmus, Memorial Sloan–Kettering Cancer Center, New York, NY, November 2, 2007 (received for review June 22, 2007)
In human lung adenocarcinomas harboring EGFR mutations, a
second-site point mutation that substitutes methionine for threo-
nine at position 790 (T790M) is associated with approximately half
of cases of acquired resistance to the EGFR kinase inhibitors,
contribute to disease progression, we used array-based compara-
tive genomic hybridization (aCGH) to compare genomic profiles of
EGFR mutant tumors from untreated patients with those from
patients with acquired resistance. Among three loci demonstrating
recurrent copy number alterations (CNAs) specific to the acquired
resistance set, one contained the MET proto-oncogene. Collec-
tively, analysis of tumor samples from multiple independent pa-
tient cohorts revealed that MET was amplified in tumors from 9 of
43 (21%) patients with acquired resistance but in only two tumors
from 62 untreated patients (3%) (P ? 0.007, Fisher’s Exact test).
Among 10 resistant tumors from the nine patients with MET
amplification, 4 also harbored the EGFRT790Mmutation. We also
found that an existing EGFR mutant lung adenocarcinoma cell line,
EGFR mutation and the T790M change. Growth inhibition studies
demonstrate that these cells are resistant to both erlotinib and an
irreversible EGFR inhibitor (CL-387,785) but sensitive to a multiki-
nase inhibitor (XL880) with potent activity against MET. Taken
together, these data suggest that MET amplification occurs inde-
pendently of EGFRT790Mmutations and that MET may be a clinically
relevant therapeutic target for some patients with acquired resis-
tance to gefitinib or erlotinib.
lung adenocarcinoma ? XL880
found in a proportion of lung adenocarcinomas (1). Nearly 90%
of these mutations occur as either multinucleotide in-frame
deletions in exon 19 that eliminate four amino acids (LREA), or
at position 858 (L858R). Both genetic lesions are associated with
increased sensitivity of lung adenocarcinomas to the selective
EGFR kinase inhibitors, gefitinib (Iressa) and erlotinib
(Tarceva) (2–4). Multiple prospective trials have demonstrated
an ?75% response rate for patients whose tumors harbor these
Unfortunately, lung cancers with drug-sensitive EGFR muta-
tions that initially respond to gefitinib or erlotinib eventually
omatic mutations in exons encoding the tyrosine kinase
domain of the epidermal growth factor receptor (EGFR) are
tumor cells obtained after disease progression contain a second-
site mutation in the EGFR kinase domain (8–12). The most
common (?90%) lesion involves a C 3 T change at nucleotide
2369 in exon 20, which substitutes methionine for threonine at
position 790 (T790M). Other mechanisms that contribute to
resistance to EGFR inhibitors, either in the absence or presence
of the T790M mutation, remain to be established.
To determine whether lung cancers that acquire resistance to
either gefitinib or erlotinib display additional and/or specific
genetic alterations that might play a role in disease progression,
we performed high-resolution genomic analysis (aCGH) of
tissue samples from 12 patients whose tumors initially responded
but subsequently progressed while on these drugs. We compared
these results with those obtained from genomic analysis of lung
adenocarcinomas with EGFR mutations resected from 38 pa-
tients who were never treated with kinase inhibitors. Among
three genomic loci with recurrent differences in CNAs between
the two groups, we focused on one that encompasses the gene
encoding the MET tyrosine kinase. Using several molecular and
cellular techniques, we verified the aCGH findings and then
extended our studies to additional EGFR mutant tumors. We
also examined the activity of MET protein in available EGFR
mutant lung adenocarcinoma cell lines and studied drug re-
sponses in one cell line (NCI-H820) found to contain an EGFR
drug-sensitive mutation (an exon 19 deletion), an EGFR drug-
resistance mutation (T790M), and MET amplification.
Characterization of the Cancer Genome in Lung Adenocarcinomas
from Patients with Acquired Resistance to EGFR Kinase Inhibitors.We
obtained 12 tumor DNA samples from 12 patients with lung
adenocarcinomas containing EGFR mutations and documented
Author contributions: J.B., C.B., A.V., W.G., M.L., and W.P. designed research; J.B., C.B.,
analytic tools; J.B., C.B., J.-Y.S., A.V., J.S., W.G., M.L., C.-H.Y., and W.P. analyzed data; and
J.B. and W.P. wrote the paper.
Conflict of interest statement: V.M. and W.P. are part of a pending patent application on
EGFR T790M mutations.
Freely available online through the PNAS open access option.
pTo whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
December 26, 2007 ?
vol. 104 ?
disease progression after prolonged treatment on gefitinib or
erlotinib. We then subjected the DNAs to aCGH, using a 60-mer
oligonucleotide array platform (Agilent). We analyzed fluores-
cence ratios of scanned images of the arrays to identify statis-
tically significant changes in copy number using a version of the
circular binary segmentation algorithm (13). The overall pattern
of large-scale genomic events was consistent with previous
high-resolution genomic profiles of human lung cancer (14, 15)
(Fig. 1 Upper).
Specific Recurrent CNAs Identified in Tumor Samples from Patients
with Acquired Resistance vs. Those from Untreated Resected EGFR
Mutant Tumors. We next compared results from tumors with
acquired resistance to those obtained from a separate aCGH
analysis of 38 mutant EGFR lung adenocarcinomas resected
from patients who had never received treatment with kinase
inhibitors. DNA from the untreated tumors was analyzed by
using 44K Agilent chips (16). The recurrent genomic gains and
losses in these samples appeared grossly similar to the acquired
resistance set (Fig. 1 Lower).
After mode-centering, comparison of the two sets (at the
location of each of the 44K probes; see Materials and Methods)
revealed three major loci of recurrent CNAs unique to samples
from patients with acquired resistance (Fig. 1 Upper and Table
1). One locus, at 7p11-12, includes EGFR and was amplified
compared with the untreated set in 3 of the 12 tumor samples
(nos. 5, 6, and 10a). These results are consistent with the notion
that EGFR mutation and amplification occur frequently in
tumors from patients with acquired resistance (11). A second
locus, at an interval encompassing 7q31.2, was found in two
samples (nos. 6 and 10a) [supporting information (SI) Fig. 4].
The gene encoding MET lies in this interval and encodes a
receptor tyrosine kinase implicated in the development, main-
tenance, and progression of cancers in both animals and humans
(reviewed in ref. 17). The third CNA occurred on 5p15.2–15.3
and was found in two samples (nos. 5 and 10a); candidate genes
in this region remain to be identified (Table 1). We did not
observe any genomic deletions that were significantly overrep-
resented in either treated or untreated groups.
Genomic Gains on Chromosome 7 in Tumor Cells from Patients with
Acquired Resistance. We next examined the individual aCGH
profiles of the region of interest on chromosome 7 at higher
resolution in all of the samples. Eleven of the samples showed
broad gains of chromosome 7, including the region of EGFR
(data not shown). Samples 5, 6, and 10a displayed further focal
amplification at the locus encompassing EGFR, and samples 6
and 10a had additional focal amplifications at the locus, includ-
ing MET (SI Fig. 4). None of these samples had focal amplifi-
cation of the gene encoding hepatocyte growth factor (HGF),
the ligand for MET, located at 7q21.1. An additional tumor
sample from patient number 10 (10b, a metastatic lymph node)
also displayed focal amplifications at both EGFR and MET.
To determine the proportion of drug-resistant tumor cells
with amplified MET, we assessed MET gene copy number per
cell by dual-color fluorescent in situ hybridization (FISH) in the
one tumor sample (no. 6) for which we had sufficient viable
tumor cells for analysis. Cells were labeled with probes that
hybridized to the centromere of chromosome 7 (CEP7; green) or
to MET (red). We found that the tumor sample comprised a
mixed population of cells. All were polysomic for chromosome
7. However, although some cells displayed equal numbers of
copies of CEP7 and MET (averaging 4–6 copies), others har-
bored greater numbers of copies of MET than CEP7 (SI Fig. 5).
Taken together with the aCGH results on the same tumor
sample, these data suggest that tumor cells in patient no. 6 have
?4–6 copies of chromosome 7 with additional focal amplifica-
tions of the region containing MET in approximately half of the
Confirmation of MET Status by Quantitative PCR and Analysis of
Additional Patient Tumor Samples. To confirm further the results
from the aCGH studies, we next performed quantitative ‘‘real-
time’’ PCR (qPCR) to determine the status of MET in DNA
samples from four independent cohorts of tumor samples. As a
control gene, we selected one [(MTHFR (5,10-methylenetetra-
that showed no CNA in any sample by aCGH and is not subject
to germ-line copy number polymorphism.
In the first cohort—the tumor samples already analyzed by
aCGH—quantitative PCR results confirmed the results (Table
2, nos. 1–12). We then tested four additional tumors from our
own patients with acquired resistance (Table 2, nos. 13–16) and
three additional drug-resistant tumors from Taiwan (Table 2,
nos. 17–19). In total, in these tumor samples from 19 patients,
four displayed MET amplification (e.g., a fold change relative to
MTHFR ? 1.5). Here, we chose a ratio of MET:MTHFR ? 1.5
to define MET amplification based on corresponding data from
FISH and aCGH analysis of tumor cells from patient no. 6 (SI
Fig. 5) and of cell lines (see below and Table 2).
To extend these findings to another independent cohort, we
performed qPCR analysis on DNA from EGFR mutant tumors
obtained from 24 Taiwanese patients with acquired resistance to
Table 1. Genomic loci with significant copy number changes in
12 EGFR mutant tumor samples from patients with acquired
resistance compared with 38 EGFR mutant tumor samples from
Data were obtained from aCGH chips as described in the Materials and
Methods. Loci were listed if they displayed a log2ratio ?1, corresponding to
(Mb); CNA, maximum copy number alteration; RefSeq, reference sequence
according to the National Center for Biotechnology Information.
adenocarcinomas from patients with acquired resistance to EGFR tyrosine
kinase inhibitors (n ? 12) or from untreated patients (n ? 38). Shown is the
percentage of samples with CNAs after data segmentation (y axis) plotted for
each probe evenly aligned along the x axis in chromosome order. The gray
Amplifications or deletions having ?2-fold change in copy number, defined
by log2ratios ?1.0, are shown by bright red and bright green lines, respec-
tively. Asterisks denote amplifications that occurred in more than one sample
in the acquired resistance cohort.
Recurrence of chromosomal alterations found in EGFR mutant lung
Bean et al.
December 26, 2007 ?
vol. 104 ?
no. 52 ?
gefitinib (SI Table 3, nos. 20–43). Matched pretreatment tumor
DNA samples were available for comparison. We detected MET
amplification in tumors from five patients. In four of these
samples, MET amplification was found in only posttreatment
samples, suggesting that selection for cells with MET amplifica-
tion occurred while these patients were on gefitinib. Note that
here, we used a more stringent criterion for MET amplification
(MET:MTHFR ratio ? 5) because the samples were tested with
an independent protocol (see Materials and Methods and SI
Table 3 legend) for which concurrent aCGH or FISH data were
of these patients (no. 32), the untreated sample was a surgically
resected lung tumor. Disease recurred more than three years
later, and the posttreatment specimen was derived from omen-
tum 15 months after an initial response to gefitinib. The other
patient (no. 30) was diagnosed with a CT-guided lung biopsy and
had a confirmed partial radiographic response on gefitinib. The
‘‘acquired resistance’’ specimen was obtained when pleural fluid
developed 8 months after starting gefitinib. These results could
be due to genetic heterogeneity within individual tumors and/or
tumor heterogeneity within individual patients. The observed
amplification of MET in some tumors after treatment with TKIs
could be attributed to selection of subpopulations of cells with
When data (using qPCR, aCGH, and/or FISH) from the
multiple independent cohorts were combined, we found MET
amplification in 9 of 43 patients with acquired resistance,
compared with 2 of 62 untreated patients (P ? 0.007, Fisher’s
Exact test). The common EGFRT790Mresistance mutation was
found in 20 of 43 (46.5%) patients with acquired resistance.
MET amplification harbored the EGFRT790Mmutation as well.
Thus, tumors with acquired resistance to gefitinib or erlotinib
may exhibit amplification of MET in the absence or presence of
a second-site drug-resistant mutation in EGFR. There was no
correlation between increased copies of MET and type of
primary drug-sensitive EGFR mutation (exon 19 deletion vs.
exon 21 point mutation) or duration of drug treatment (data not
An Established Lung Adenocarcinoma Cell Line Contains an Exon 19
Deletion Associated with Drug-Sensitivity, an Exon 20 Point Mutation
Associated with Drug-Resistance, and Increased Copies of MET. We
performed qPCR analysis of MET in lung adenocarcinoma cell
lines with EGFR mutations. Surprisingly, we found that one cell
line—H820—contained not only drug-sensitive (del E746-
E749) and drug-resistant (T790M) EGFR kinase domain muta-
tions (data not shown) but also MET amplification (Table 2).
Consistent with these results, we found by using FISH that the
majority of H820 cells harbored 4–6 copies of chromosome 7
(CEP7) and 7–9 copies of MET (Fig. 2A). As judged by aCGH,
but no specific focal amplifications were observed at loci con-
taining either EGFR or MET (data not shown).
Table 2. EGFR mutation and MET status of lung adenocarcinoma cell lines and tumor samples from patients with
acquired resistance to EGFR inhibitors
Patient1° EGFR mutation
T790M MET F.C.Drug
Del L747-E749; A750P
Del L747-T751; K754E
Del L747-T751; K754E
not amp. by aCGH
not amp. by aCGH
not amp. by aCGH
not amp. by aCGH
not amp. by aCGH
not amp. by aCGH
not amp. by aCGH
EGFR mutation status was determined as described in the Materials and Methods; the absence or presence of the drug-resistance
EGFRT790Mmutation is indicated by a Y (yes) or N (no). For patient no. 10, two individual samples (10a and 10b) were examined. For MET
sample (Ref.) and H820 cells included in each set. Samples with MET amplification are in boldface. None of the seven samples for which
only aCGH was performed showed MET amplification [?not amplified (amp.) by aCGH?]. Samples for which both qPCR and aCGH were
performed are marked with an asterisk. Tumor samples 6, 10a, and 10b all showed focal MET amplification by aCGH (see SI Fig. 4) and
ins; insertion; erl., erlotinib; gef., gefitinib. Time in months patient was on kinase inhibitor treatment when re-biopsy was performed.
www.pnas.org?cgi?doi?10.1073?pnas.0710370104Bean et al.
To determine whether the high number of copies of MET
caused increased MET enzymatic activity, we used a surrogate
kinase assay in which immunoblots of H820 cell lysates were
probed with polyclonal antibodies that recognize MET phos-
phorylated at tyrosine residues (Y1234/5). These phosphorylation
sites are located in the activation loop and are indicative of
kinase activation (Fig. 2B). Total MET protein was measured by
immunoblotting with an anti-MET monoclonal antibody. Both
antibodies recognize the 170-kDa precursor form of the enzyme
and the mature 140-kDa beta subunit. The smaller protein is the
product of proteolytic cleavage of the larger precursor and also
contains the tyrosine kinase catalytic domain (18). We com-
pared results with H820 cells to those with PC-9 cells, a lung
cancer line that harbors an exon 19 deletion (E746-A750) of
EGFR but no T790M mutation and no MET amplification (Table
2). Extracts from PC-9 cells contained relatively small amounts
of total and phosphorylated MET protein. By contrast, H820 cell
extracts displayed much greater amounts of MET protein, much
of which was phosphorylated at Y1234/5(Fig. 2B).
Sensitivity of H820 Cells to a MET Inhibitor but Not to EGFR Inhibitors.
To explore further the functional consequences of MET ampli-
fication in EGFR mutant tumor cells, we used fluorescence-
cells to erlotinib, CL-387,785 [an irreversible EGFR inhibitor
known to overcome T790M-mediated resistance (10, 19, 20)],
and XL880. The third compound abolishes MET and VEGFR2
tyrosine kinase activity at subnanomolar levels and also inhibits
the PDGFR?, KIT, FLT3, TIE-2, and RON kinases in vitro with
low nM potency (21, 22).
H820 cells were relatively insensitive to erlotinib, because the
concentration of drug required to inhibit the growth of 50% of
the cells (IC50) was ?10 micromolar (Fig. 2C Top). The IC50of
CL-387,785 was also in the micromolar range (Fig. 2C Top and
Middle). By contrast, H820 cells displayed sensitivity to XL880
in the nanomolar range (Fig. 2C Middle). At these concentra-
tions of XL880, the kinase activity of MET was inhibited, as
measured by examination of H820 cell lysates from XL880-
treated cells using the surrogate kinase assays described above
(Fig. 3 Left). By comparison, PC-9 cells were insensitive to
XL880 but were much more sensitive to CL-387,785 (Fig. 2C
Bottom). These results suggest that MET inhibitors like XL880
may be able to overcome MET-mediated resistance to EGFR
kinase inhibitors, even in cells that harbor the T790M mutation.
Dependence of ERBB3 Signaling on MET in H820 Cells. During the
course of the work described here, others reported that MET
amplification leads to gefitinib resistance in lung cancer by
activating ERBB3 signaling (23). ERBB3 is a heterodimeric
partner for EGFR that mediates phosphoinositide 3-kinase
that XL880 treatment strongly inhibited the phosphorylation of
with CL-387,785 strongly inhibited phosphorylation of ERBB3
in PC-9 cells but not in H820 cells (Fig. 3).
To determine whether the inhibition of ERBB3 phosphory-
lation in H820 cells treated with XL880 was due specifically to
Methods), ?4–6 copies of chromosome 7 (CEP7), with additional focal am-
plifications at loci containing MET. The cells shown are representative of the
whole population. Scale bar: 10-?m. CEP7 probe, green; MET probe, red;
nuclei, blue (DAPI). (B) Lysates from H820 and PC-9 cells were immunoblotted
by using anti-phospho-(p)-MET (Y1234/5), anti-total MET, and anti-actin anti-
and amplified no MET, whereas PC-9 cells harbor an exon 19 deletion (E746-
A750), no T790M mutation, and no MET amplification. (C) Growth inhibition
curves of H820 and PC-9 cells treated with MET and EGFR inhibitors at various
MET status and sensitivity of H820 cells to the MET inhibitor, XL880.
H820 and PC-9 cells, either untreated (DMSO-only control; 0 ?M) or treated
with 0.3 ?M XL880 or 0.3 ?M CL-387,785, were immunoblotted by using
anti-phospho-(p)-MET (Y1234/5), anti-total MET, anti-phospho-(p)-ERBB3
(Y1289), anti-total ERBB3, anti-phospho-(p)-EGFR (Y1092), anti-total EGFR, and
ERBB3 signaling in H820 cells depends on MET status. Lysates from
Bean et al.
December 26, 2007 ?
vol. 104 ?
no. 52 ?
inhibition of the MET kinase, we transfected MET-specific
siRNAs into H820 and PC-9 cells to knockdown expression of
the protein. After transfection with MET siRNAs, both phos-
pho-MET and total-MET were virtually undetectable by immu-
noblotting of H820 cell extracts (SI Fig. 6). In concordance with
the results obtained with XL880, phospho-ERBB3 levels were
By contrast, we observed no effect on phospho-ERBB3 status in
similarly treated PC-9 cells and no effect on MET or ERBB3
status in cells treated with siRNAs against a control gene
(GAPD) (SI Fig. 6). Thus, ERBB3 signaling appears to depend
on MET protein in H820 cells, even though these cells harbor
We also measured the effect of siRNA knockdown of MET on
the growth of H820 cells. In multiple experiments, the number
of viable cells remaining 72 h after treatment with MET siRNAs
was reduced, whereas treatment with GAPD siRNA had no
effect. However, the effect of MET siRNA was modest (between
80 and 90% of controls in both total cell count and growth
inhibition assays; data not shown), less than that seen with the
kinase inhibitor, XL880. Thus, in H820 cells, XL880 may inhibit
other kinases in addition to MET that affect cell viability.
Alternatively, the effect of kinase inhibition by XL880 is differ-
ent from the effect of siRNA-mediated knockdown of MET,
because the latter probably diminishes MET activity more
Tumor Samples with MET Amplification Lack MET Mutations. Gain-
of-function mutations of MET have been discovered in both
sporadic and inherited forms of human renal papillary carcino-
mas (25–27). The majority of mutations are located in exons that
encode the kinase domain of the receptor (17). In the samples
with MET amplification and adequate DNA for analysis (sam-
ples 2, 6, 10a, 10b, and H820 cells), we sequenced coding regions
for the MET kinase domain (exons 15–21) and did not find any
somatic mutations (data not shown). In addition, we did not
identify any somatic mutations outside the MET kinase domain
(exons 3–14) in H820 cells (data not shown).
Mutations that substitute methionine for threonine at position
790 in the EGFR kinase domain have been found in ?50% of
lung adenocarcinomas from patients with acquired resistance to
the EGFR inhibitors, gefitinib and erlotinib (refs. 8–12 and this
article). This knowledge has led to the identification of alterna-
tive EGFR inhibitors that can overcome T790M-mediated re-
sistance in vitro and potentially in patients (19, 20). However,
other mechanisms could additionally contribute to disease pro-
gression in these patients.
In this study, we used high-resolution genome-wide profiling
of EGFR mutant tumor samples before and after treatment to
implicate the MET proto-oncogene as an additional therapeutic
target in patients with acquired resistance to gefitinib or erlo-
tinib. MET encodes a heterodimeric transmembrane receptor
tyrosine kinase composed of an extracellular alpha-chain disul-
fide-bonded to a membrane spanning beta-chain (18, 28). Bind-
ing of the receptor to its ligand, hepatocyte growth factor/scatter
factor, induces receptor dimerization, triggering conformational
changes that activate MET tyrosine kinase activity. MET acti-
vation can have profound effects on cell growth, survival,
motility, invasion, and angiogenesis (17). Dysregulation of MET
signaling has been shown to contribute to tumorigenesis in a
number of malignancies. For example, activating mutations have
been associated with both sporadic and inherited forms of
human papillary renal carcinomas (25–27). In addition, gastric
carcinomas have high-level MET amplification (29), and some
other cancers display aberrant transcriptional up-regulation of
In our studies of various EGFR mutant lung adenocarcinoma
samples, we found MET to be amplified in 9 of 43 (21%) patients
with acquired resistance vs. 2 of 62 (3%) patients unexposed to
EGFR kinase inhibitors. In a separate genomic analysis of 371
primary lung adenocarcinoma samples and 242 matched normal
controls, MET amplification was not identified as a significant
recurrent focal event (31). Thus, although MET amplification
can be found in lung cancers (32, 33), it does appear to be a rare
event in lung adenocarcinomas never treated with EGFR kinase
The presence of MET amplification in combination with
gain-of-function drug-sensitive EGFR mutations could together
lead to cellular changes that confer enhanced fitness to cells
bearing both alterations. The EGFR T790M resistance mutation
could further potentiate the growth properties of such tumor
cells, because the oncogenic activity of EGFR kinase mutant
alleles is enhanced by the T790M change (34). Consistent with
this notion, 40% of the samples with MET amplification in this
study harbored the T790M mutation. Furthermore, we found
that an existing lung adenocarcinoma cell line—H820 cells—
harbored an EGFR mutation associated with drug-sensitivity
(E746-E749), an EGFR mutation associated with drug-
resistance (T790M), and MET amplification. Notably, these cells
were isolated from a patient who did not undergo any prior
treatment with gefitinib or erlotinib. Thus, these cells may not
represent ‘‘acquired resistance’’ per se; however, their existence
does demonstrate that all these genetic lesions can occur within
the same cells.
MET amplification could lead to EGFR inhibitor resistance by
activating ERBB3 signaling (23). Our data using XL880, a small
molecule that inhibits MET kinase activity, and siRNAs that
knockdown MET expression, suggest that in H820 cells, ERBB3
signaling depends highly on MET and not EGFR activity. This
interaction between EGFR, MET, and ERBB3 in H820 cells
appears to be different from that observed in an EGFR mutant
lung adenocarcinoma cell line (HCC827 GR) selected for ge-
fitinib-resistance in vitro (23). In those resistant cells, treatment
with a MET inhibitor alone did not affect ERBB3 phosphory-
lation. Such discrepancies may be explained by the highly
different ways in which the cell lines were derived.
Recently, small-molecule MET inhibitors have shown promise
as anti-cancer therapy in phase I trials (22). Our in vitro data
demonstrate that the MET inhibitor, XL880, is more effective at
inhibiting the viability of lung adenocarcinoma cells with
EGFRT790Mand MET amplification than either reversible (er-
lotinib) or irreversible (CL-387,785) EGFR inhibitors. Collec-
tively, these findings suggest that compounds like XL880 could
play a significant role in the treatment of patients whose EGFR
mutant lung adenocarcinomas have developed acquired resis-
tance to existing EGFR inhibitors as a result of increased copy
numbers of MET.
Materials and Methods
Tissue Procurement. Tumor specimens were obtained through protocols ap-
proved by the Institutional Review Boards of Memorial Sloan–Kettering Can-
Center, Chang-Gung Memorial Hospital, and National Taiwan University Hos-
pital. All patients gave informed consent.
Mutational Analyses. Genomic DNA was extracted from tumor specimens, and
primers for EGFR (exons 18–24) analyses were as published in refs. 4 and 35.
PCR-RFLP assays for exon 19 deletions and L858R and T790M missense muta-
tions were performed as published in refs. 9 and 36. All mutations were
confirmed at least twice from independent PCR isolates, and sequence trac-
Primers for MET sequencing are listed in SI Table 4.
aCGH Profiling. Genomic DNA was extracted from tumor samples, using
www.pnas.org?cgi?doi?10.1073?pnas.0710370104Bean et al.
for all samples. DNA was digested and labeled by random priming using Download full-text
Bioprime reagents (Invitrogen) and Cy3- or Cy5-dUTP. Labeled DNA was
hybridized to Agilent 244K CGH arrays for acquired resistance samples, and
44K CGH arrays for untreated samples. For additional details, see SI Methods.
Quantitative Real-Time PCR. See SI Methods for details.
Cell Lines and Viability Assays. NCI-H820 cells were developed by A. Gazdar
M. Ono (Kyushu University, Fukuoka, Japan). Cells were grown in RPMI
supplemented with FBS. Growth inhibition assays were performed with the
CellTiter-Blue cell viability kit (Promega), per the manufacturer’s instructions.
All assays with H820 cells were performed at least three independent times;
for PC-9 cells, all assays were performed at least two independent times (see
SI Methods). The complete aCGH dataset is available at http://cbio.mskcc.org/
Nomenclature. Two numbering systems are used for EGFR. The first denotes
the initiating methionine in the signal sequence as amino acid ?24. The
second, used here, denotes the methionine as amino acid ?1. There are also
37); because the isoform lacking these 54 nt appears to be the most abundant
isoform (38), we consider full-length MET protein to consist of 1,390 (not
Immunoblotting. See methods and supporting text in ref. 4 for details on cell
lysis and immunoblotting reagents. At least three independent experiments
were performed for all analyses. See SI Methods for a list of the antibodies
siRNA. siGenome ON-TARGETplus SMARTpool MET (L-003156) and ON-
TARGETplus siCONTROL GAPD pool (D-001830) (Dharmacon) were used ac-
cording to the manufacturer’s instructions. All siRNA transfections were per-
formed three independent times (see SI Methods).
Note. During the course of the work described here, others published similar
findings after identifying MET as a candidate resistance gene by using an in
vitro resistance modeling approach (23). In that study, MET amplification was
detected in 4 of 18 (22%) lung cancers that had become resistant to gefitinib
analyzed in that study (i.e., patient 12); results were mostly concordant.
ACKNOWLEDGMENTS. We thank K. Politi, C. Sawyers, and H.E.V. for helpful
discussions; T. Mitsudomi (Aichi Cancer Center Hospital, Nagoya, Japan) for
providing DNA samples; Z. Zeng and M. Weiser (both of Memorial Sloan–
Kettering Cancer Center) for providing the MET BAC probe for FISH; Exelixis
for providing XL880; and all of the patients and/or their family members who
consented for tissue acquisition. This work was supported by University of
Texas Specialized Programs of Research Excellence in Lung Cancer National
Cancer Institute Grants P50CA75907 (to A.G.), U01-CA84999 (to W.G.), and
B-002-113-MY3 (to J.-Y.S.) and NSC95-2314-B-002-227-MY3 (to C.H.Y.);
Chang-Gung Medical Research Fund Grant CMRPG 350031 (to W.-C.C.); Na-
tional Health Research Institutes Grant 96-A1-MG-PP-04-014 (to S.F.H.); the
Doris Duke Charitable Foundation (W.P.), the Thomas G. Labrecque Founda-
tion (W.G. and W.P.), Joan’s Legacy: The Joan Scarangello Foundation to
Conquer Lung Cancer (W.P.); National Insitutes of Health Grants K08-
CA097980 and R01-CA121210 (to W.P.); the Jodi Spiegel Fisher Cancer Foun-
dation (W.P.); the Carmel Hill Fund (W.P.); and funds from the Miner Family.
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