Identification of Metastasis-Associated Receptor Tyrosine Kinases
in Non–Small Cell Lung Cancer
Carsten Mu ¨ller-Tidow,
Joachim Schwa ¨ble,
Christian H. Brandts,
1Wolfgang E. Berdel,
1and Hubert Serve
2Bjo ¨rn Steffen,
3Paul M. Schneider,
1Department of Medicine, Hematology/Oncology;
Mu ¨nster; and
2Department of Medicine, Gastroenterology, University of Mu ¨nster,
3Department of Visceral and Vascular Surgery, University of Cologne, Cologne, Germany
Development of distant metastasis after tumor resection is
the leading cause of death in early-stage non–small cell lung
cancer (NSCLC). Receptor tyrosine kinases (RTK) are
involved in tumorigenesis but only few RTKs have been
systematically studied in NSCLC. Here, we provide quantita-
tive real-time reverse transcription-PCR expression data of
all RTKs (n = 56) in primary tumors of 70 patients with
early-stage (I-IIIA) NSCLC. Overall, 33 RTKs were expressed
in at least 25% of the patients. Several RTKs were
significantly expressed higher in tumors that ultimately
metastasized. The hazard risk for metastasis development
in stage I/II disease was increased at least 3-fold for tumors
with high expression levels of insulin receptor, neurotrophic
tyrosine receptor kinase 1, epidermal growth factor receptor,
ERBB2, ERBB3, platelet-derived growth factor receptor B B B,
fibroblast growth factor receptor 1, or leukocyte tyrosine
kinase. Relative risks were reduced 3-fold by expression of
EPHB6 or DKFZ1. Three members of the epidermal growth
factor receptor family were associated with a high risk of
metastasis, emphasizing the validity of our data. High ERBB3
expression was significantly associated with decreased
survival. Taken together, our genome-wide RTK expression
map uncovered the previously unknown value of several
RTKs as potential markers for prognosis and metastasis
prediction in early-stage NSCLC. The identified RTKs
represent promising novel candidates for further functional
analyses. (Cancer Res 2005; 65(5): 1778-82)
Non–small cell lung cancer (NSCLC) ranks among the most
frequent cancers in the world. Its mortality is only slightly lower
than its incidence indicating that most patients die from NSCLC
despite current therapy approaches. Only patients whose tumors
can be completely resected have a significant chance of cure.
Tumor resection is restricted to patients with stage I and II and
sometimes stage III disease. However, even patients with very
small tumors often develop distant metastasis. It is unclear
whether the metastatic potential of individual tumors develops
over time or whether the basic genetic program of the primary
tumor predetermines the metastatic capability (1). Whereas both
concepts seem reasonable, recent data from our lab and others’
indicate that a metastatic program is inherent to tumors that do
metastasize early (2, 3).
Several approaches have been used to dissect the mechanisms
that determine the metastatic potential (2, 4–7). We describe a
novel approach to identify metastasis-related genes and their
potential use for diagnostic purposes. Based on the knowledge
that a few receptor tyrosine kinases [RTK; e.g., epidermal growth
factor receptor (EGFR) and ERBB2] are known to play an
important role in solid tumor metastasis (8–10), we reasoned that
other RTKs might also be important. Besides EGFR and ERBB2,
only few RTKs have been studied for their involvement in
metastasis. Recently, we established a genome-wide RTK profiling
approach by real-time quantitative reverse transcription-PCR (RT-
PCR; ref. 11). Here, we provide data on the role of RTKs in the
metastasis of early-stage NSCLC tumors. In addition to the known
metastasis-associated genes EGFR and ERBB2, several RTKs
previously not known to be associated with the metastatic
process were identified as strong predictors for the development
of metastasis in early-stage NSCLC.
Materials and Methods
Non–Small Cell Lung Cancer Patient Samples. Primary tumor
specimens were obtained at the time of initial surgery for early-stage
NSCLC (12). Histologic and survival data were published previously (2, 7).
Patients with stage I/II disease had their tumors resected and did not
receive additional treatment, whereas stage IIIA patients were irradiated
following surgery. A long follow-up period of at least 36 months ensured
the correct classification of metastasizing and non-metastasizing
RNA Isolation and cDNA Preparation. The tumor samples were
pathohistologically analyzed for the percentage of tumor cells. Only
tumor biopsies with at least 70% cancer cells were used. RNA was
isolated using TRIzol reagent (Invitrogen, San Diego, CA). A total of 1 Ag
RNA from each sample was reverse-transcribed using oligo-d(T) primer
and Moloney murine leukemia virus reverse transcriptase (Clontech, Palo
Primer and Probe Design. Sequence information was obtained from
Genbank and previously published data (13). In brief, primers and probes
were designed to span exon-exon junctions and to be outside of the
conserved kinase domain using Primer Express software (Applied
Biosystems, Foster City, CA). The resulting primer and probe sequences
were verified by alignment. The GAPDH probe was labeled with 5V -VIC and
3V -TAMRA. All other probes were labeled 5V -FAM and 3V -TAMRA (Euro-
GenTec, Seraing, Belgium). Reliability of PCR amplification and detection
was verified on serial dilutions of standard cDNA before analysis of patient
Note: C. Mu ¨ller-Tidow and S. Diederichs contributed equally to this work.
Requests for reprints: Carsten Mu ¨ller-Tidow, Department of Medicine A,
Hematology/Oncology, University of Mu ¨nster, Domagkstr. 3, 48149 Mu ¨nster,
Germany. Phone: 49-251-835-6229; Fax: 49-251-835-2673; E-mail: muellerc@uni-
I2005 American Association for Cancer Research.
Cancer Res 2005; 65: (5). March 1, 2005
Semiautomated Analysis of Gene Expression by Real-time
Quantitative Reverse Transcription-PCR. A semiautomated setup
(Tecan Genesis RP150 automated pipetting system) was established for
reliable and rapid RT-PCR analysis of 384 wells in parallel. The PCR
reaction mixture contained 600 nmol/L of each primer and 200 nmol/L
probe in a final volume of 22.5 AL. PCR conditions were 50jC/10 s, 95jC/
10 min, and 40 cycles of 95jC/15 s and 60jC/min in a real-time PCR
machine (ABI PRISM 7900 Sequence Detector, TaqMan). Expression levels
of RTKs or the housekeeping gene GAPDH were quantitated using a
fluorescence-based detection method described previously (11, 14, 15).
Relative gene expression levels were calculated using standard curves
generated by serial dilutions of a cDNA mixture by the SDS 2.0 software.
Expression levels for each gene and each sample were divided by the
GAPDH expression level. Several genes were independently analyzed twice
and P < 0.001.
Statistical Analysis. Statistical analyses were carried out using SPSS
11.0. Expression differences were tested for significance using Mann-
Whitney U and Kruskal-Wallis analysis. Cross-table analyses and
hazard risks were calculated using Fisher’s exact test. For survival
analyses, Cox regression analyses and Kaplan-Meier tests were done
and significance was calculated using the log-rank test. All tests were
executed two sided and the error level was set at 5% (P < 0.05). Owing
to this study designed to identify novel relevant RTKs in the met-
astatic process, Bonferroni or Sidak posthoc analyses were not done to
avoid type II errors despite the increased risk of type I errors (16).
Therefore, statistical values should be considered of exploratory
Analysis of Receptor Tyrosine Kinase Expression by Quan-
titative Real-time Reverse Transcription-PCR. From the human
genome project, a total of 56 RTKs have been identified. We have
recently analyzed the expression patterns of all RTKs in a wide
variety of human cancers (11). These analyses established vast
differences in the expression of RTKs in different types of human
In the current study, we analyzed the association of RTKs with
clinical features in patients with early-stage NSCLC. According to
quantitative real-time RT-PCR results, nine RTKs were expressed in
more than 75% of all NSCLC tumors, 18 RTKs were expressed in
more than 50% of the patients, and 33 RTKs were expressed in at
least 25% of the tumors (Fig. 1A). RTK expression patterns of
individual tumors showed a high degree of variability (Fig. 1B).
The different histologic NSCLC subtypes differed in RTK
expression levels: adenocarcinomas expressed higher levels
of RON (P = 0.02), c-KIT (P = 0.04), ERBB2 (P = 0.001), and ERBB3
Figure 1. RTK expression in NSCLC. A, RTK expression was analyzed by quantitative real-time RT-PCR. The bar diagram indicates the percentage of NSCLC
samples (n = 70) with detectable mRNA expression for each RTK. B, mRNA expression levels of all RTKs in 70 NSCLC samples grouped according to their
histologic subtype. Blue, samples without detectable expression. Expression levels of RT-PCR positive samples are indicated in log scale.
Metastasis-Associated RTKs in Non–Small Cell Lung Cancer
Cancer Res 2005; 65: (5). March 1, 2005
(P < 0.001), whereas large cell carcinomas expressed the highest
levels of fibroblast growth factor receptor 1 (P = 0.03). EPHB6
levels were high in squamous cell carcinomas (P = 0.009) and
expression of neurotrophic tyrosine receptor kinase 2 was notably
absent in adenocarcinomas (P = 0.01). In our study, patients with
stage I or II disease expressed significantly higher levels of c-KIT
(P = 0.04) and fibroblast growth factor receptor 1 (P = 0.047) in
comparison to stage IIIA patients. Expression levels of three
RTKs, MER (P = 0.01), EPHB2 (P = 0.04), and EPHB6 (P = 0.09,
not significant), differed with regard to the smoking history of
Receptor Tyrosine Kinases Predict Metastasis and Survival
in Early-stage Non–Small Cell Lung Cancer. The association
between RTK expression and metastasis or survival was analyzed
in all tumors (n = 62) and separately in stage I/II NSCLC
tumors (n = 44). For these analyses, patient samples were split
into two groups depending on the expression level of each RTK.
To avoid a bias in data analysis, the median of the analyzed
samples was used as a cutoff value for each RTK. Samples
expressing levels higher than the median were regarded as
‘‘high’’ whereas samples with expression levels below the median
were regarded as ‘‘low.’’ For RTKs with less than half of the
samples showing significant expression levels, samples with
detectable expression were regarded as ‘‘high.’’ We used this
dichotomized data to analyze the risk of patients with high level
expression of a certain RTK to metastasize. The hazard ratio
was calculated by cross-table analysis and is shown for stage I/II
patients (Fig. 2).
Two RTKs were associated with reduced risk of metastasis
(hazard ratio less than 0.3) when expressed in the primary tumor:
DKFZ1 or EPHB6 (Fig. 2). DKFZ1 expression was detected in 12
of 44 (27%) stage I/II NSCLC patients, whereas EPHB6 was
expressed in 18 of 44 patients (41%). Stage I/II patients with
tumors expressing either DKFZ1 or EPHB6 had an excellent
prognosis with only 2 of 23 (8.7%) patients developing distant
metastasis compared with 8 of 21 (38.1%) in patients with tumors
expressing neither RTK (Fig. 3A). Rates in metastasis development
were reflected in Kaplan-Meier plots indicating far better survival
for patients with tumors expressing either one or both RTKs
(DKFZ1 and EPHB6). This result was consistent if NSCLC tumors
from all stages were included (n = 62, P = 0.02) or if only tumors
from stage I/II were analyzed (n = 44, P = 0.02). The mean
survival differed significantly between 71 months (expression of
either DKFZ1 or EPHB6) versus 52 months (no expression of
either RTK; n = 62). In a multivariate Cox regression analysis
including age, sex, smoking status, tumor grade, and histology, the
expression of either DKFZ1 or EPHB6 emerged as an independent
prognostic factor for survival (P = 0.02 for n = 62; P = 0.018 for
n = 44 stage I/II only; Fig. 3B).
High expression of eight RTKs was associated with an at least
thrice increased risk of distant metastasis (Fig. 2). High expression
levels of the insulin receptor indicated a more than 7-fold increased
risk to develop metastasis (95% CI 1.3-40.2, P = 0.03) for patients
with stage I/II disease. The neurotrophic tyrosine receptor kinase 1/
nerve cell growth factor receptor 1 predicted a 5.6-fold increased
risk of metastasis (CI 1.2-26.1, P = 0.03) when overexpressed (Fig. 2).
Patients not expressing either of these RTKs did not suffer distant
metastasis (0 of 17 = 0.0%), whereas patients expressing either RTK
frequently developed metastases (10 of 27 = 37.0%; Fig. 4A).
In addition, the overall survival of patients expressing neither of
these two RTKs was significantly better in Kaplan-Meier and Cox
regression analyses (P = 0.033 and P = 0.014, respectively)
independent of tumor stage (Fig. 4B).
In accordance with published data (17), EGFR and ERBB2 were
among the genes predictive of metastasis (Fig. 2). In addition, the
EGFR family member ERBB3 was closely associated with
metastasis. High expression of either EGFR (33.3% metastasis
versus 13.0% metastasis in the low expressor group) or ERBB2
(33.3% versus 13.0%) or ERBB3 (33.3% versus 10.0%) indicated an
increased fraction of metastasizing tumors whereas ERBB4 did not
display this effect (Fig. 4A). In Kaplan-Meier survival analysis, stage
I/II patients with high expression of ERBB3 showed significantly
shorter survival (P = 0.044; Fig. 4B).
In this study, we provide a detailed map of RTK expression in
NSCLC. Our systematic approach corroborates the importance of
RTKs already known to be involved in NSCLC but also indicates
that other thus far neglected RTKs might have an important role
there. One focus of our study was to reveal their potential
involvement in metastasis. In recent years, we and others have used
Figure 2. Relative hazard to develop distant metastasis based on RTK
expression. Tumors were subdivided into a high or low expressing group
for each RTK. The relative risk to develop distant metastasis-associated with
high expression of the respective RTK was calculated for stage I and II patients
(n = 44). The dashed lines indicate the threshold for either 3-fold reduced or
3-fold increased risk of metastasis.
Cancer Res 2005; 65: (5). March 1, 2005
various techniques to identify genes that are associated with the
risk of distant metastasis in NSCLC revealing novel genes linked to
metastasis (2, 7, 12). Several groups have tried educated guess
approaches and have identified promising metastasis markers (e.g.,
refs. 5, 6, 18). The best known example is the EGFR which
constitutes an important target for directed therapies (19). Owing
to RTKs often being at the mechanistic basis of pathogenetic
processes in cancer, we decided to study the expression of all RTKs
in NSCLC metastasis. We chose quantitative real-time RT-PCR
because of its high specificity and unparalleled sensitivity.
First, expression levels of eight genes were associated with an
increased risk of metastasis. Among these, three EGFR family
members were found. Because EGFR and ERBB2 are well known
to be associated with metastasis (17), these findings provide
evidence for the reliability and validity of our approach. Our data
further indicate that ERBB3, a ‘‘kinase dead’’ RTK that forms
heterodimers with EGFR and ERBB2 (20, 21), is closely linked to
NSCLC metastasis. RTKs can exert oncogenic functions either due
to overexpression or activating mutations (e.g., ref. 22). In the
EGFR, a paradigm for an overexpressed oncogene due to, for
example, gene amplification (23), mutations were recently
identified and closely associated with patients’ response to
gefitinib (24, 25). None of the other screened RTKs contained
mutations (25) whereas mutations of ERBB2 in lung cancer have
recently been found (26). Despite the clinically relevant EGFR
mutations, overexpression of EGFR and possibly of other RTKs
remains a dominant mechanism within tumor pathogenesis due
to high EGFR expression found in almost all patients whereas
mutations were relatively rare.
In addition to EGFR family members, several RTKs were newly
identified to confer an increased risk of metastasis. The insulin
receptor and its related growth factors and receptors are well
known to be associated with tumor cell growth (27) but a
potential role in NSCLC metastasis has not been considered
before. The neurotrophic tyrosine receptor kinase 1/nerve cell
growth factor receptor 1 receptor is overexpressed in neuro-
blastoma but associates with a rather good prognosis in this
tumor entity (28). In human lung cancer cell lines, nerve growth
factor signaling stimulates clonal growth (29). We conclude that
overexpression of RTKs could not only contribute to oncogenic
transformation but might also enhance metastatic properties.
Figure 3. DKFZ1 and EPHB6 expression associates with reduced risk of
metastasis and increased survival. A, early-stage NSCLC patients (stage I/II,
n = 44) expressing neither DKFZ1 nor EPHB6 exhibited the highest risk of
distant metastasis (38.1%) whereas patients expressing both RTKs were
associated with a much lower incidence of metastasis (8.7%). B, in
Kaplan-Meier and Cox regression analyses (see text), expression of DKFZ1
or EPHB6 was significantly associated with a survival benefit for all
patients (n = 62) or specifically for stage I/II patients (n = 44).
Figure 4. Insulin receptor, neurotrophic tyrosine receptor kinase 1, and ERBB3
expression associates with increased risk of metastasis and decreased
survival. A, expression of insulin receptor, neurotrophic tyrosine receptor kinase
1, or one EGFR family member (EGFR, ERBB2, or ERBB3) in early-stage
NSCLC correlated with an increased proportion of patients developing distant
metastasis. B, in all analyzed NSCLC patients, expression of either insulin
receptor or neurotrophic tyrosine receptor kinase 1 contributed to significantly
impairedoverall survival in Kaplan-Meier and Cox regression analyses. For
ERBB3, a similar correlation was found for early-stage NSCLC patients.
Metastasis-Associated RTKs in Non–Small Cell Lung Cancer
Cancer Res 2005; 65: (5). March 1, 2005
Taken together, our findings indicate that besides the well- Download full-text
known EGFR family, several other RTKs could play a role in NSCLC
metastasis. The RTK expression differences can serve as markers
for prognosis and metastasis prediction. This RTK expression map
will guide future analyses to uncover the detailed functions of RTKs
in NSCLC pathogenesis and metastasis. Our data provide the basis
for additional basic studies in vitro and in vivo.
Received 9/17/2004; revised 11/29/2004; accepted 12/23/2004.
Grant support: Research in our lab is supported by grants from the Wilhelm
Sander-Foundation (2001.086.2), DFG (Heisenberg program Mu1328/3-1) and German
Cancer Aid (Deutsche Krebshilfe 10-2155-Mu ¨3).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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