Identification of Metastasis-Associated Receptor Tyrosine Kinases in Non–Small Cell Lung Cancer

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.

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 patients.

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 μg RNA from each sample was reverse-transcribed using oligo-d(T) primer and Moloney murine leukemia virus reverse transcriptase (Clontech, Palo Alto, CA).

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 5′-VIC and 3′-TAMRA. All other probes were labeled 5′-FAM and 3′-TAMRA (Euro-GenTec, Seraing, Belgium). Reliability of PCR amplification and detection was verified on serial dilutions of standard cDNA before analysis of patient samples.

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 μL. PCR conditions were 50°C/10 s, 95°C/10 min, and 40 cycles of 95°C/15 s and 60°C/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 reproducibility was always excellent with a correlation coefficient r > 0.95 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 α error level was set at 5% (P < 0.05). Owing to this study designed to identify novel relevant RTKs in the metastatic 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 significance.

Analysis of Receptor Tyrosine Kinase Expression by Quantitative 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 tumors.

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 (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 the patients.

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 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 neuroblastoma 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.

Taken together, our findings indicate that besides the well-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.

Grant support: Research in our lab is supported by grants from the WilhelmSander-Foundation (2001.086.2), DFG (Heisenberg program Mu1328/3-1) and German Cancer Aid (Deutsche Krebshilfe 10-2155-Mü3).

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