Download High MET Receptor Expression But Not Gene Amplification in ALK

Original Article
High MET Receptor Expression But Not Gene
Amplification in ALK 2p23 Rearrangement Positive
Non–Small-Cell Lung Cancer
Yan Feng, MD,*†‡ Eugen C. Minca, MD,§‖ Christopher Lanigan, MS,§ Angen Liu, MD, PhD,‡‖
Wei Zhang, MS,† Lihong Yin, PhD,† Nathan A. Pennell,MD, PhD,*‡ Carol Farver, MD,†
Raymond Tubbs, DO,§ and Patrick C. Ma, MD, MSc*†‡
Introduction: Overexpression of MET receptor tyrosine kinase and
its ligand hepatocyte growth factor (HGF) and MET gene amplification have been well-documented in non–small-cell lung cancer (NSCLC). Activated MET signaling plays an important role
in human cancer tumorigenesis, metastasis, and drug resistance.
However, the deregulation of MET/HGF pathway in NSCLC harboring ALK gene rearrangement (ALK[+]), which is sensitive to dual
ALK and MET inhibitor Crizotinib, has not been reported.
Methods: We performed systematic analysis of MET/HGF expression by immunohistochemistry (IHC) and MET gene amplification
by dual color, dual hapten bright field in situ hybridization in 19
ALK(+) and 73 ALK(−) NSCLC tumor tissues from those who had
clinical ALK rearrangement test done at the Cleveland Clinic from
August 2010 to January 2013. IHC scoring was interpreted on a standard four-tier system.
Results: The percentage of MET IHC score 0, 1+, 2+, and 3+ were
5.5%, 27.8%, 50.0%, and 16.7% in ALK(+) group, compared with
28.8%, 33.9%, 23.7%, and 13.6% in ALK(−) group, respectively.
The MET high expression (IHC score 2 or 3) was significantly
higher in ALK(+) group statistically (66.7% versus 37.3%, p = 0.03).
­HGF-high expression (IHC score 2 or 3) was 33.3% in ALK(+) and
15.8% in ALK(−) (p = 0.17). We identified eight cases in ALK(−) and
one case in ALK(+) tumor who had MET gene amplification (18.4%
versus 7.1%, p = 0.43) by dual color, dual hapten bright field in
situ hybridization. No significant correlation between MET protein
receptor expression and gene amplification was identified.
Conclusions: Our study demonstrated for the first time that
MET receptor expression, but not MET gene amplification, is
*Departments of Solid Tumor Oncology; †Translational Hematology and
Oncology Research, Cleveland Clinic Taussig Cancer Institute; ‡Case
Comprehensive Cancer Center; §Departments of Molecular Pathology;
and ‖Anatomic Pathology, Cleveland Clinic Robert J. Tomsich Pathology
and Laboratory Medicine Institute, Cleveland, OH.
Disclosure: The authors declare no conflict of interest.
Address for correspondence: Patrick C. Ma, MD, MSc, Director, Aerodigestive
Oncology Translational Research, Staff, Translational Hematology and
Oncology Research, and Solid Tumor Oncology, Cleveland Clinic Taussig
Cancer Institute, 9500 Euclid Avenue, R40, Cleveland, OH 44195. E-mail:
[email protected]
Copyright © 2014 by the International Association for the Study of Lung
Cancer
ISSN: 1556-0864/14/0905-0646
646
significantly increased in ALK(+) NSCLC. MET gene amplification
is a relatively rare event in this unique population compared with
ALK(−) NSCLC.
Key Words: MET, HGF, ALK Gene rearrangement, Non–small-cell
lung cancer, Crizotinib.
(J Thorac Oncol. 2014;9: 646–653)
M
ET is a human tyrosine kinase that functions as the
receptor for its natural ligand hepatocyte growth factor
(HGF), also known as the scatter factor. The deregulation of
MET/HGF pathway plays an important role in tumorigenesis
and tumor progression by promoting cell proliferation, survival,
scattering, motility, and migration, invasion, and metastasis.1
Studies have shown that MET pathway is activated in many
solid and hematological malignancies (http://www.vai.org/
met), including lung cancer, and can be altered through ligand
or receptor overexpression, genomic amplification, MET mutations, and alternative splicing.2,3 MET gene amplification has
been found as one of the mechanisms of acquired epidermal
growth factor receptor (EGFR) tyrosine kinase inhibitors resistance in non–small-cell lung cancer (NSCLC).4–7 MET receptor kinase has been under intensive preclinical investigation for
over 25 years. MET is now known to be a “druggable” target
within the human kinome. Early phase clinical investigations
have shown promising results of MET inhibitors in NSCLC
and a few of them have been further evaluated in phase 3 lung
cancer clinical trials recently.1,8 Clinical trial studies now begin
to shed more insight to suggest the potential predictive utility
of MET receptor expression level as assayed in IHC as clinical
biomarker for companion diagnostics development. Moreover,
there have been case reports documenting tumor response in
MET amplified NSCLC under treatment by crizotinib.9
Rearrangements of the anaplastic lymphoma kinase (ALK)
gene was discovered as a driver oncogene in NSCLC in 2007.10
Although it only represents 5 to 7% NSCLC,10,11 it has made significant impact in the treatment of advanced NSCLC as precision
targeted therapy. ALK rearrangement is found to be primarily
mutually exclusive with EGFR mutation and KRAS mutation.12,13
However, the relationship between MET/HGF expression
Journal of Thoracic Oncology ® • Volume 9, Number 5, May 2014
Journal of Thoracic Oncology ® • Volume 9, Number 5, May 2014
levels and ALK rearrangement in lung cancer is not yet known.
Crizotinib, an oral ALK inhibitor, has demonstrated dramatic clinical benefit with minimal toxicity in ALK(+) advanced NSCLC.11
Interestingly, crizotinib was initially developed as a MET inhibitor in preclinical studies and the phase 1 clinical trial.14 The MET/
HGF pathway alterations, such as protein expression and gene
amplification, have not been reported in NSCLC harboring ALK
gene rearrangement (ALK[+]). It conceivably may impact the
efficacy and resistance of crizotinib therapy.
Here, we report the MET and HGF expression by
immunohistochemistry (IHC) and MET gene amplification
by dual color, dual hapten bright field in situ hybridization
(DDISH) in ALK(+) NSCLC compared with ALK(−) controls. Our study demonstrated that MET receptor expression
but not MET gene amplification is significantly increased in
ALK(+) NSCLC. We also found that MET gene amplification
is a relatively rare event in this unique population compared
with ALK(−) NSCLC.
PATIENTS AND METHODS
Patient Population and Tumor Tissue
This retrospective study was conducted in a cohort of
92 patients with NSCLC who were seen at the Cleveland
Clinic (Cleveland, OH) with their tumors clinically tested
for ALK 2p23 rearrangement by fluorescence in situ hybridization (FISH; AMV ALK Break Apart FISH Probe Kit,
Abbott Molecular, Des Plaines, IL) or IHC at the Cleveland
Clinic Molecular Pathology Laboratory from August 2010 to
January 2013. All 19 patients whose tumors were found to
be ALK FISH (+) and had available formalin-fixed paraffinembedded (FFPE) tumor tissues were included in this study.
Forty-seven (47) NSCLC FFPE tumor tissues that had ALK
rearrangement tested by FISH to be negative during the same
period were randomly selected and included in the study
as controls. We also screened 26 tumors of young NSCLC
patients who were diagnosed at age of 45 years or younger, to
be included. Our recent published study demonstrated 100%
sensitivity and specificity of ALK IHC using D5F3 rabbit
monoclonal antibody (Cell Signaling Technology) linked to
ultrasensitive bright field detection (OptiView AMP; Ventana
Medical Systems, Inc., Tucson, AZ) compared with FISH.15
Hence, the screening of ALK 2p23 translocation in this young
cohort was conducted using ALK IHC, which was uniformly
negative. Thus, all of these 21 FFPE samples were included in
the ALK(−) control group. All FFPE tissue were cut at 5 μm
and mounted on Superfrost Plus treated slides and the whole
tissue section on the slide was used for the following studies. Each case also had one slide stained for hematoxylin and
eosin to confirm the presence of adequate tumor tissue. The
clinical data were derived from patients’ electronic medical
records. Tumor histology was assessed by pathologists (C.F.
and A.F.) and staging were classified based on American Joint
Committee on Cancer 7th edition of tumor, node, metastasis staging criteria. None of the patients received any MET
or HGF inhibitor before the tumor samples were collected.
This study was conducted in accordance with the Institutional
Review Board approved study protocol.
High MET Expression in ALK(+) NSCLC
Immunohistochemistry
MET IHC
Staining was performed on a Ventana Benchmark XT
automated immunostainer (Ventana Medical Systems) using
a Ventana Optiview DAB IHC Detection Kit with CC1 antigen retrieval. Primary antibody, CONFIRM monoclonal
rabbit anti-Total c-MET (Ventana Medical Systems, clone
SP44, catalog 790–4430) was used. Lung cancer cell line
HCC827 which is known to express MET was used as positive control. MET IHC scoring was interpreted on a four-tier
system based on the membranous or cytoplasmic staining
in greater than 10% tumor cells, which has been previously
reported in other studies16,17 as follows: none or staining in
less than 10% tumor cells (0), weak (1+), moderate (2+), and
strong (3+; Fig. 1). Score 0 and 1+ were considered as low
MET expression, and score of 2+ and 3+ were considered as
high MET expression. An additional high MET expression
criteria of 2+ or 3+ in ≥50% tumor cells, which was used
in some MET inhibitor clinical trial and study,8,18,19 was also
evaluated in our study. A H-score that is the sum of each
intensity multiple its percentage was also calculated for each
case. Each IHC slide was reviewed and scored independently
by two pathologists (E.M. and A.L.) who were unaware of
the clinical details of individual patients. Discordant cases
were reviewed, discussed and resolved by the two pathologists in a consensus review microscopy session.
HGF IHC
Paraffin slides were dewaxed and rehydrated using
xylene and gradient alcohols. Antigen retrieval was performed using citrate buffer, pH 6.0. The slides were then
incubated in 3% hydrogen peroxide for 30 minutes to block
endogenous peroxidase activity followed by 3% normal rabbit serum block (S-5000, Vector Laboratories, Burlingame,
CA) for another 30 minutes. Endogenous avidin and biotin
were blocked with Avidin/Biotin Blocking Kit (SP-2001,
Vector Laboratories) and then samples were incubated with
primary antibody, goat antihuman HGF (AF-294-NA, R&D
Systems, Minneapolis, MN) diluted 1:20 in 1.5% normal
serum, for one hour at room temperature. After washing,
slides were incubated with rabbit anti-goat biotinylated
secondary antibody (BA-5000, Vector Laboratories) for
30 minutes at room temperature followed by Vector Avidin
Biotin Complex Elite (PK-6100, Vector Laboratories) for
30 minutes at room temperature. Staining was visualized
with Vector ImmPACT 3, ­
3′-diaminobenzidine substrate
(SK-4105, Vector Laboratories) and slides were counterstained with hematoxylin, dehydrated, cleared, and permanently mounted. The protocol was optimized to achieve
best results. We used normal colon tissue and mesothelioma tumor tissue as positive control in the assay. Similar to
MET IHC, HGF IHC scoring was interpreted on a f­ our-tier
system (0, 1+, 2+, 3+; Fig. 2) by the same criteria (both
membranous and cytoplasmic staining in greater than 10%
tumor cells, exclude stromal staining) and scoring process
as described above. Score 0 and 1+ were considered as low
HGF expression, and score of 2+ and 3+ were considered as
high HGF expression.
Copyright © 2014 by the International Association for the Study of Lung Cancer
647
Journal of Thoracic Oncology ® • Volume 9, Number 5, May 2014
Feng et al
FIGURE 1. Representative examples of MET immunohistochemical staining with score 0–3 using SP44 monoclonal rabbit
­anti-Total c-Met (Ventana Medical Systems). The pattern of immunostaining was both cytoplasmic and membranous.
80%
cluster as 12 signals. MET gene amplification was defined as
the ratio of MET/Chromosome 7 equal or greater than 2.0. The
data were interpreted by two independent pathologists (E.M.
and R.T.) and consensus was achieved in all cases.
P=0.03
ALK(-)
70%
ALK(+)
60%
50%
Statistical Analysis
40%
Continuous variables were compared using nonparametric method Wilson Cox test. χ2 or Fisher’s exact tests were
used to compare categorical variables. Significance level
of α ≤ 0.05 was used. All reported p values are two-sided.
Statistical analyses were performed in JMP.
30%
20%
10%
0%
0
1+
2+
3+
0/1+
2+/3+
MET IHC intensity
FIGURE 2. Distribution of MET IHC signal intensity in ALK(−)
and ALK(+) NSCLC.
MET Gene Amplification by DDISH
Fully automated DDISH (Ventana Medical Systems )
was performed on a Ventana BenchMark XT (Ventana Medical
Systems). MET DNA probe and Chromosome 7 reference
alpha-centromeric probe (CEP7; Ventana Medical Systems)
were visualized on the same slides, following the manufacturer’s protocols (some samples were repeated by modified protocols). Signals were enumerated in at least 100 tumor nuclei
per slide, using a light microscope with objectives from ×4 to
×60. Signal patterns were scored using DDISH criteria developed for ERBB2 (HER2) DDISH (http://her2dualish.skillport.
com/skillportfe/custom/login/ ventana/login.action;jsessionid
=EAF883D50616EB46CAAA307601F247A1). A small cluster of multiple signals was counted as six signals and a large
648
RESULTS
Patient characteristics are listed in Table 1. With the
inclusion in the control group of a selective cohort of younger
patients with diagnosed NSCLC (N = 26), the median age
of diagnosis of the two study cohorts between ALK(+) and
ALK(−) patients is well-balanced (51 versus 53 year old,
p = 0.44). The percent of never-smoker in ALK(+) patients is
significantly higher than ALK(−) group (52.6% versus 19.2%,
p = 0.01), which is consistent with literature reports.11,20
The gender distribution is similar in the two groups. All
ALK(+) patients had adenocarcinoma except one large cell
carcinoma (5.3%), whereas 26% of ALK(−) patients had
­non-adenocarcinoma histology. The percentage of early stage
(I or II) NSCLC in the control group is only slightly higher
than the ALK(+) group (24.0% versus 10.6%) because of the
inclusion of the young cohort patients who underwent surgical resection with mostly early stage NSCLC. Regardless, the
differences of the histology and stage distribution between the
ALK(+) and ALK(−) groups are not statistically significant
(Table 1) EGFR mutation was detected in three of 50 ALK(−)
Copyright © 2014 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology ® • Volume 9, Number 5, May 2014
TABLE 1. Clinical Characteristics of the Study Patients
Age at diagnosis (median,
25th–75th)
Female (%)
Smoking
Never
Current
Former
Histology
Adenocarcinoma
Squamous
Large cell
Poorly differentiated
Others
Stage
I
II
III
IV
EGFR mutation
Death
Survival month (median,
25th—75th)
ALK (−)
ALK (+)
N = 73
N = 19
p
53.0 (43.4–67.5)
51.0 (40.5–63.6)
0.44
38 (52.1%)
8 (42.1%)
0.61
0.01
14 (19.2%)
27 (37.0)
32 (43.8%)
10 (52.6%)
2 (10.5%)
7 (36.8%)
54 (74.0%)
10 (13.7%)
4 (5.5%)
2 (2.7%)
3 (4.1%)
18 (94.7%)
0
1 (5.3%)
0
0
0.36
11 (15.5%)
6 (8.5%)
20 (28.1%)
34 (47.9%)
3 (6.0%)
24
12.6 (4.9–30.0)
1 (5.3%)
1 (5.3%)
6 (31.6%)
11 (57.9%)
0
5
32.0 (14.4–83.1)
0.69
1.0
0.16
tumors, but none of the ALK(+), which is consistent with previously established mutual exclusion of EGFR mutation and
ALK gene rearrangement.12,13
MET Receptor Protein Expression by IHC
The results of MET IHC were available in 77 patients
(84%), the missing data are mainly because of lack of tissue.
The pattern of immunostaining observed was both cytoplasmic and membranous. The staining is tumor specific and there
was no significant background staining of the normal lung tissue. Representative examples of various staining intensities are
shown in Figure 1. The percentage of MET IHC score 0, 1+,
2+ and 3+ were 5.5%, 27.8%, 50.0%, and 16.7% in ALK(+)
group, compared with 28.8%, 33.9%, 23.7%, and 13.6% in
ALK(−) group, respectively (Fig. 2). The frequency of MET
high expression (IHC score 2 or 3) was significantly higher in
ALK(+) group statistically (66.7% versus 37.3%, p = 0.03; 1.8fold) than in the ALK(−) group. Using the MET high expression
definition of 2+ or 3+ in ≥50% cells, the frequency of MET
high expression remains significantly higher in ALK(+) group
than ALK(−) controls (58.8% versus 26.7%, p = 0.02; 2.2-fold).
The median (25th–75th) H-score was also significantly higher
in ALK(+) group than ALK(−) controls (60 [0–150] versus 152
[74–211], p = 0.03).
We also observed significantly higher frequency of
MET receptor high expression in adenocarcinoma than for
other histology types (52.6% versus 20%, p = 0.02) in our
study. Finally, we performed subgroup analysis in adenocarcinoma alone, which demonstrated 70.6% of ALK(+) and 45.0%
High MET Expression in ALK(+) NSCLC
of ALK(−) tumors had MET high expression (p = 0.09). In the
subgroup of advanced NSCLC (stage 3 or 4, N = 59), MET
high receptor expression also remained significantly higher
in ALK(+) tumors compared with the controls (68.8% versus
37.2%, p = 0.04).
HGF Ligand Expression by IHC
The results of HGF IHC were available in 75 patients
(82%), the missing data are mainly because of lack of tissue. The pattern of immunostaining observed was mainly
cytoplasmic. Overall, the IHC staining seems more diffuse
than MET IHC, with a predominantly intratumoral staining pattern albeit with some minor stromal positivity also.
Representative examples of various staining intensities are
shown in Figure 3. The percentage of HGF IHC score 0, 1+,
2+, and 3+ were 0%, 66.7%, 27.8%, and 5.5% in ALK(+)
group, compared with 22.8%, 61.4%, 15.8%, and 0% in
ALK(−) group, respectively (p = 0.02; Fig. 4). Only 1 case
with IHC 3+ staining was identified in the entire ALK(+)
cohort, but none in the ALK(−) cohort. HGF high expression
(IHC score 2 or 3) was higher in ALK(+) group, although
the difference was not statistically significant (33.3% versus
15.8%, p = 0.17). The MET and HGF IHC expression levels
(score 0–3) were found to be significantly correlated (Pearson
correlation coefficient = 0.36, p = 0.001).
MET Gene Amplification by DDISH
MET DDISH assay was successfully performed in 63
cases (68%), including nine repeated cases by using our own
modified protocol. The lack of valid result in the remaining samples was primary because weak signals for MET
and or CEP7 and nonspecific pigmentation in background.
Representative examples of MET gene amplified and nonamplified cases are illustrated in Figure 5. We identified nine
cases with MET gene amplification and only one of them was
ALK(+). However, the percentage difference of MET gene
amplification between ALK(+) and control group did not reach
statistical significance (7.1% versus 18.4%, p = 0.43), given
the limited sample size. The number of cases with MET IHC
score 0, 1+, 2+, and 3+ here were two (25.0%), zero (0%), two
(25.0%), and four (50.0%) in nine cases (one case has missing MET IHC data) with MET gene amplification, and seven
(17.9%), 18 (46.2%), nine (23.1%), five (12.8%) in 54 cases
(15 cases have missing MET IHC data) without MET gene
amplification, respectively. A statistically significant correlation between MET expression by IHC and MET gene amplification by DDISH also was not identified (coefficient 0.21,
p = 0.16). We observed that a high proportion of tumor tissues
had MET aneusomy in both study cohorts (30.8% in ALK(+)
and 44.4% in ALK(−), p = 0.5), although the clinical impact
is uncertain.
MET Receptor Expression and Crizotinib
Response in ALK(+) Patients
Of the 19 ALK(+) patients, 10 were treated with crizotinib, which is a dual ALK/MET inhibitor. Two had documented complete response and six had partial response. Of
these 10 patients, eight had MET IHC results available and
Copyright © 2014 by the International Association for the Study of Lung Cancer
649
Journal of Thoracic Oncology ® • Volume 9, Number 5, May 2014
Feng et al
FIGURE 3. Representative examples of HGF IHC staining with score 0 to 3 using a human HGF affinity purified goat polyclonal
antibody (R&D). The pattern of immunostaining was mainly cytoplasmic.
100%
ALK(-)
P=0.17
ALK(+)
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
0
1+
2+
3+
0/1+
HGF IHC intensity
six of them demonstrated high MET expression (2+/3+; two
complete response and four partial response). Two MET
­IHC-negative patients had partial response to crizotinib. We
are not able to draw any statistical conclusion about the association of MET expression and crizotinib response in this
small sample size cohort. There is one patient in our entire
cohort whose tumor harbored both ALK rearrangement and
MET gene amplification, but unfortunately he died before he
had a chance to receive crizotinib therapy. His tumor exhibited
650
2+/3+
FIGURE 4. Distribution of HGF IHC signal intensity in ALK(−) and ALK(+) NSCLC.
moderate MET expression (score 2) and weak HGF expression (score 1) by IHC.
DISCUSSION
Our study is the first to report that MET expression
is significantly increased in ALK(+) NSCLC. Tsuta et al21
studied MET pathway alteration in 906 surgically resected
NSCLC cases, which were further divided by EGFR gene
mutation or ALK gene rearrangement. They found the
Copyright © 2014 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology ® • Volume 9, Number 5, May 2014
High MET Expression in ALK(+) NSCLC
FIGURE 5. Representative examples of MET gene amplification by dual probe (black MET probe and red Chromosome
7 probe), dual hapten bright field in situ hybridization (DDISH). MET gene amplification is defined as the ratio of MET/
Chromosome 7 ≥ 2.0.
c-MET, p-MET expression, and MET gene copy number
were similar in 17 ALK(+) NSCLC compared with the rest
of the cohort. A few methodology differences are noticeable between ours and the study by Tsuta et al21. MET IHC
staining intensity variation among different tumors and
intratumoral heterogeneity are rather often seen. A four-tier
(0–3) intensity scoring system is often used in most studies but a universally accepted level of cutoff for MET high
expression has not been established yet. In the study Tsuta
et al21, c-MET IHC positivity was defined as “those exhibiting staining in ≥10% of cells” and the staining intensity
was not part of the definition. This may account for substantial variability in data interpretation. Regardless, they
reported c-MET-positive in 26.4% ALK wild type, which is
comparable with our result in ALK(−) group (37.3% using
MET 2+ or 3+ in ≥10% cells, 26.7% using MET 2+ or 3+ in
≥50% cells), the study by Dziadziuszko et al19 (25% using
MET 2+ or 3+ in ≥50% cells) and our previous study.22
More importantly, the study by Tsuta et al21 was conducted
in surgically resected samples, whereas our cases are most
metastatic NSCLC. It is possible that MET expression levels have significant difference between resectable and metastatic ALK(+) NSCLC. So the different conclusion is likely
related to sample selection and possible different definition
of MET high expression.
The clinical and therapeutic implications of higher MET
receptor expression in ALK(+) NSCLC patients are currently
unknown. Crizotinib was demonstrated as a bona fide MET
inhibitor in a previous case report, a NSCLC patient with de
novo MET amplification but no ALK rearrangement achieved
a rapid and durable respond to crizotinib.9 Because of crizotinib is a dual inhibitor of MET and ALK, one may postulate
that the status of MET expression may impact the efficacy
of crizotinib in ALK(+) NSCLC under therapy. Nonetheless,
our study consists of only a small cohort of ALK(+) patients
who were treated with crizotinib and we are not able to
test the hypothesis if MET high expression is associated
with tumor response and clinical outcomes from crizotinib
therapy. Overall, our results here represent an interesting
hypothesis-generating observation that warrants further
­
studies in larger cohorts. The oncogenic role and molecular mechanism of high MET expression in ALK(+) NSCLC
is particularly worthy of further investigation. Our results
raise a question whether there might be cross-talk signaling
between the oncogenic rearranged-ALK and MET activation
through deregulated expression of the receptor, although the
observed high MET expression could alternatively represent
only a bystander effect in this unique molecular subgroup of
ALK(+) NSCLC. The underlying mechanism for high MET
expression in ALK(+) NSCLC could be MET gene amplification (unlikely in our cohort), transcriptional upregulation,
alternative splicing or mutation. Unfortunately, there was not
sufficient sample and tissue materials to further characterize
the MET gene expression level, alterations, and mutations
in the MET high expression tumors, especially in ALK(+)
NSCLC. Regardless, the potential combined therapeutic
effect of MET inhibitor plus ALK inhibitor (especially the
second-generation ones such as LDK-378, which does not
possess MET inhibitory activity) would be of great interest
to further explore.
High MET expression has recently been identified as
potential biomarker for patient selection for MET inhibitory
agents, which has been shown in the phase 2 trial of MET
inhibitor tivantinib (ARQ197) in advanced HCC,18 the phase
3 tivantinib in combination with erlotinib in advanced NSCLC
(MARQUEE) trial8 and the phase 2 trial of onartuzumab
(MetMAb) combined with erlotinib in advanced NSCLC.18
The inclusion criterion of high MET expression in patient
selection in some ongoing phase 3 trials reflects our current
emerging understanding of MET expression level in relation
to therapeutic outcomes and their prediction.
MET gene amplification has previously been evaluated in preclinical and clinical studies. Multiple studies have
reported primary MET gene amplification to be in the wide
range of 2 to 21% in NSCLC,2,19,23,24 although its impact on
clinical outcomes such as survival is still controversial.25,26
Correlation between MET expression and MET gene amplification has been reported in some previous studies,16,19,21
although some controversies remain.27,28 We found that the
correlation between MET receptor expression and gene
amplification was quite poor in our study. The variation of
tumor sample characteristics, testing methods, and the definition of MET gene amplification, may explain some of the
outcome differences among reported studies. In our study, we
found that MET gene amplification is uncommon (one of 19
patients) in ALK(+) NSCLC, but more frequent in ALK(−)
Copyright © 2014 by the International Association for the Study of Lung Cancer
651
Journal of Thoracic Oncology ® • Volume 9, Number 5, May 2014
Feng et al
NSCLC. Our results indicate MET gene amplification may
not be the predominant underlying genomic mechanism that
activates MET signaling pathway in ALK(+) NSCLC. Instead,
other alternative mechanisms such as MET gene upregulation, alternative splicing or mutation would be worth further
exploration, especially in ALK(+) NSCLC. Unfortunately, we
do not have sufficent sample and tissue available to test these
hypotheses.
MET gene amplification was tested traditionally
by quantitative real-time polymerase chain reaction or
FISH.2,23,24 The DDISH was initially developed for HER2
gene amplification in breast cancer and study showed nearperfect agreement between the PathVysion and the DDISH
assays.29 Dziadziuszko et al19 also accessed MET gene copy
number by silver in situ hybridization (same as DDISH) in
surgically resected NSCLC. The success rate of DDISH in
the 189 samples was 74%. We are currently evaluating the
concordance of MET DDISH and FISH in solid tumors.
DDISH has been recently used in MET gene amplification in
NSCLC and other malignancies.16,17,19 Compared with traditional FISH, DDISH has several potential advantages. First,
assessment can be made using conventional light microscopy with preserved cell morphology based on an automated
platform. Additionally, the DDISH slides are more stable
and can be preserved for review for several years. Compared
with quantitative real-time polymerase chain reaction, which
was often used in detecting MET gene amplification, DDISH
is a fully automated process, may be more easily applied in
routine clinical practice and preserves morphologic context
while assessing genotype.
Our study has certain limitations. We included some
young patients with surgical resected NSCLC in the ALK(−)
control group which introduced some imbalance between the
two groups, including age, histology, and stage distribution.
However, these differences were not statistically significant
between the two groups. Dziadziuszko et al19 studied MET
protein expression by IHC in surgically resected NSCLC and
found no difference among different histology or pathological
stages. Our study has a relative small sample size and confirmation in a larger study is warranted.
In summary, our study evaluated the MET signaling pathway, using assays to investigate MET and HGF
expression levels and MET gene amplification in a unique
population of ALK(+) NSCLC that is known to be sensitive to dual ALK and MET inhibitor crizotinib. Compared
with ALK(−) NSCLC, we found that MET receptor expression is significantly higher in the ALK(+) group. MET
gene amplification is a rare event in this particular patient
population, suggesting that it is unlikely the predominant
mechanism of inducing high MET expression and pathway
activation in ALK(+) NSCLC. Validation of our findings in
an independent study with larger sample size is of interest
and warranted.
ACKNOWLEDGMENTS
This study is supported by Cleveland Clinic Research
Program Committees Pilot Research Award 2012 to 2013 (Y.F.
and P.C.M.) and a Department of Defense Promising Lung
652
Cancer Clinician Research Award (W81XWH-10-1-0690;
P.C.M.). The authors appreciate Denise Hatala at the
Cleveland Clinic Lerner Research Institute Imaging Core for
her technical contribution to MET/HGF IHC work.
REFERENCES
1.Feng Y, Thiagarajan PS, Ma PC. MET signaling: novel targeted inhibition and its clinical development in lung cancer. J Thorac Oncol
2012;7:459–467.
2.Beau-Faller M, Ruppert AM, Voegeli AC, et al. MET gene copy number
in non-small cell lung cancer: molecular analysis in a targeted tyrosine
kinase inhibitor naïve cohort. J Thorac Oncol 2008;3:331–339.
3.Olivero M, Rizzo M, Madeddu R, et al. Overexpression and activation
of hepatocyte growth factor/scatter factor in human non-small-cell lung
carcinomas. Br J Cancer 1996;74:1862–1868.
4. Engelman JA, Zejnullahu K, Mitsudomi T, et al. MET amplification leads
to gefitinib resistance in lung cancer by activating ERBB3 signaling.
Science 2007;316:1039–1043.
5. Cappuzzo F, Jänne PA, Skokan M, et al. MET increased gene copy number and primary resistance to gefitinib therapy in non-small-cell lung cancer patients. Ann Oncol 2009;20:298–304.
6.Bean J, Brennan C, Shih JY, et al. MET amplification occurs with
or without T790M mutations in EGFR mutant lung tumors with
acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci U S A
2007;104:20932–20937.
7.Sequist LV, Waltman BA, Dias-Santagata D, et al. Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors.
Sci Transl Med 2011;3:75ra26.
8.Scagliotti G, Novello S, Ramlau R, et al. MARQUEE: A randomized,
double-blind, placebo-controlled, phase 3 trial of tivantinib (ARQ 197)
plus erlotinib versus placebo plus erlotinib in previously treated patients
with locally advanced or metastatic, non-squamous, non-small-cell lung
cancer (NSCLC). European Cancer Congress 2013. Abstract E17–1821.
9.Ou SH, Kwak EL, Siwak-Tapp C, et al. Activity of crizotinib
(PF02341066), a dual mesenchymal-epithelial transition (MET) and
anaplastic lymphoma kinase (ALK) inhibitor, in a non-small cell
lung cancer patient with de novo MET amplification. J Thorac Oncol
2011;6:942–946.
10.Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature
2007;448:561–566.
11.Kwak EL, Bang YJ, Camidge DR, et al. Anaplastic lymphoma
kinase inhibition in non-small-cell lung cancer. N Engl J Med
2010;363:1693–1703.
12. Gainor JF, Varghese AM, Ou SH, et al. ALK rearrangements are mutually
exclusive with mutations in EGFR or KRAS: an analysis of 1,683 patients
with non-small cell lung cancer. Clin Cancer Res 2013;19:4273–4281.
13.Wong DW, Leung EL, So KK, et al.; University of Hong Kong Lung
Cancer Study Group. The EML4-ALK fusion gene is involved in various
histologic types of lung cancers from nonsmokers with wild-type EGFR
and KRAS. Cancer 2009;115:1723–1733.
14.Cui JJ, Tran-Dubé M, Shen H, et al. Structure based drug design of
crizotinib (PF-02341066), a potent and selective dual inhibitor of
­mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic
lymphoma kinase (ALK). J Med Chem 2011;54:6342–6363.
15. Minca EC, Portier BP, Wang Z, et al. ALK status testing in non-small cell
lung carcinoma: correlation between ultrasensitive IHC and FISH. J Mol
Diagn 2013;15:341–346.
16. Lee HE, Kim MA, Lee HS, et al. MET in gastric carcinomas: comparison
between protein expression and gene copy number and impact on clinical
outcome. Br J Cancer 2012;107:325–333.
17.Yan B, Lim M, Zhou L, et al. Identification of MET genomic amplification, protein expression and alternative splice isoforms in neuroblastomas. J Clin Pathol 2013;66:985–991.
18. Santoro A, Rimassa L, Borbath I, et al. Tivantinib for second-line treatment
of advanced hepatocellular carcinoma: a randomised, ­placebo-controlled
phase 2 study. Lancet Oncol 2013;14:55–63.
19.Dziadziuszko R, Wynes MW, Singh S, et al. Correlation between MET
gene copy number by silver in situ hybridization and protein expression
Copyright © 2014 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology ® • Volume 9, Number 5, May 2014
by immunohistochemistry in non-small cell lung cancer. J Thorac Oncol
2012;7:340–347.
20.Shaw AT, Yeap BY, Mino-Kenudson M, et al. Clinical features and outcome of patients with non-small-cell lung cancer who harbor ­EML4-ALK.
J Clin Oncol 2009;27:4247–4253.
21.Tsuta K, Kozu Y, Mimae T, et al. c-MET/phospho-MET protein expression and MET gene copy number in non-small cell lung carcinomas.
J Thorac Oncol 2012;7:331–339.
22. Ma PC, Tretiakova MS, MacKinnon AC, et al. Expression and mutational
analysis of MET in human solid cancers. Genes Chromosomes Cancer
2008;47:1025–1037.
23.Onitsuka T, Uramoto H, Ono K, et al. Comprehensive molecular analyses of lung adenocarcinoma with regard to the epidermal growth factor
receptor, K-ras, MET, and hepatocyte growth factor status. J Thorac
Oncol 2010;5:591–596.
24.Onozato R, Kosaka T, Kuwano H, Sekido Y, Yatabe Y, Mitsudomi T.
Activation of MET by gene amplification or by splice mutations deleting
the juxtamembrane domain in primary resected lung cancers. J Thorac
Oncol 2009;4:5–11.
High MET Expression in ALK(+) NSCLC
25.Cappuzzo F, Marchetti A, Skokan M, et al. Increased MET gene copy
number negatively affects survival of surgically resected non-small-cell
lung cancer patients. J Clin Oncol 2009;27:1667–1674.
26. Kanteti R, Yala S, Ferguson MK, Salgia R. MET, HGF, EGFR, and PXN
gene copy number in lung cancer using DNA extracts from FFPE archival samples and prognostic significance. J Environ Pathol Toxicol Oncol
2009;28:89–98.
27.Kondo S, Ojima H, Tsuda H, et al. Clinical impact of c-Met expression
and its gene amplification in hepatocellular carcinoma. Int J Clin Oncol
2013;18:207–213.
28.Gunia S, Erbersdobler A, Hakenberg OW, Koch S, May M. C-MET is
expressed in the majority of penile squamous cell carcinomas and correlates with polysomy-7 but is not associated with MET oncogene amplification, pertinent histopathologic parameters, or with cancer-specific
survival. Pathol Res Pract 2013;209:215–220.
29.Kosa C, Kardos L, Kovacs J, Szollosi Z. Comparison of dual-color
­dual-hapten brightfield in situ hybridization (DDISH) and fluorescence
in situ hybridization in breast cancer HER2 assessment. Pathol Res Pract
2013;209:147–150.
Copyright © 2014 by the International Association for the Study of Lung Cancer
653