Abstract
Background
This study was designed to detect the protein expression of epidermal growth factor receptor (EGFR) among serum, lymph node, and tumor tissues, and to discuss their relationship and clinical significance. We investigated whether EGFR levels in serum and lymph nodes could be used as an effective method for non-small cell lung cancer (NSCLC) to diagnose and assess clinical stage.
Methods
In 56 patients with NSCLC and 10 individuals with nonmalignant thoracic disease, we measured EGFR levels in serum using an enzyme immunoassay, and EGFR mRNA levels in lymph node and NSCLC tissues by quantitative real-time-polymerase chain reaction. We examined the correlation between them and with the clinical parameters.
Results
Serum EGFR levels substantially decreased after surgical treatment (P < 0.001). Serum EGFR levels were correlated with smoking, surgery, and pathological type after surgery (all P < 0.05). EGFR mRNA levels in lymph node and tumor tissues were correlated more closely with lymph node metastasis (P = 0.015. EGFR mRNA in tissues was higher than that of benign pulmonary diseases (P = 0.020). There was an obvious positive correlation among EGFR levels of serum and lymph node tissues (r = 0.764; P < 0.001), serum and tumor tissues (r = 0.616; P < 0.001), and lymph node and tumor tissues (r = 0.904; P < 0.001) in NSCLCs.
Conclusion
The data suggest that detecting EGFR levels in serum and lymph node tissues could be a simple and effective method to diagnose and assess the clinical stage in patients with NSCLC.
Keywords: EGFR, enzyme-linked immunosorbent assay, lung neoplasms, polymerase chain reaction
Introduction
Lung cancer is the most common cancer worldwide, representing approximately 12% of all new cancers.1,2 Approximately 80% of cases of this disease are non-small cell lung cancer (NSCLC). Most patients that have already been clinically diagnosed are in advanced stage. Overall, the prognosis for lung cancer remains dismal, as five-year survival rates are, at best, approximately 15%, despite the advances in treatment options available. Thus, early diagnosis and early treatment are particularly important to improve the survival rate.3,4 Because of the high incidence and poor prognosis, mainly a result of delays to diagnosis, it is difficult to obtain tumor tissue and painful for patients.5 There is, therefore, a clear need for a simple and effective method of diagnosis at the initial stage of NSCLC.
Epidermal growth factor receptor (EGFR) is a 170kDa transmembrane glycoprotein with an intracellular domain that exhibits tyrosine kinase activity. It is a member of the erbB gene family.6 EGFR is commonly expressed in normal cells. The ligands of EGFR include EGF and transforming growth factor alpha, which, upon binding of EGFR, stimulate cell growth. EGFR transmits intracellular signals resulting in the regulation of cell growth by dimerization, conformational changes, and internalization of EGFR molecules.7–15 EGFR mRNA and/or protein overexpression has been observed in many types of human malignancies, including breast, colorectal, gastric, and bladder cancer, as well as NSCLC.16,17 Approximately 80% of NSCLC patients have EGFR overexpression.18 Furthermore, tumors with a high expression of EGFR are characterised by metastasis in the early stage of NSCLC, easily relapse, and have poor survival prospects.19 Many reports show that EGFR expression in NSCLC is related to metastasis and proliferative activity.7,16,20–22 However, a study by Gomez-Roca et al.23found that EGFR status, analyzed by immunohistochemistry, showed discordance between the primary tumor and metastasis in about one third of the cases with NSCLC.
We investigated the expression of EGFR among serum, lymph node, and tumor tissues in NSCLC patients and their correlation in order to find a simple and effective method for the early diagnosis and clinical staging of NSCLC.
Materials and methods
Sample collection and storage
Data was collected from patients who underwent lung resection with curative intent in the period between January 2007 and October 2009. The final diagnosis was made pathologically, using surgically resected specimens. Fifty-six NSCLC patients constituted the trial group and 10 patients with benign thoracic diseases formed the control group. The mean age of patients was 60 years (range: 30–74 years). There were 26 adenocarcinomas (ADC), 24 squamous cell carcinomas (SCC), and six others. Thirty-eight patients had lymph node metastasis. The mean age of the contrast group was 52.61 years (range: 34–67 years); two had pulmonary hamartoma, four had pulmonary inflammatory pseudotumors, and four had pulmonary tuberculosis. (Table 1)
Table 1.
Correlation between epidermal growth factor receptor (EGFR) levels in serum and clinical parameters before surgery
| Clinical parameter | n | EGFR levels (pg/mL) | t | P |
|---|---|---|---|---|
| Age | ||||
| Mean: 60 years | ||||
| <60 | 22 | 4424 ± 1389 | ||
| ≥ 60 | 34 | 4361 ± 1172 | 0.182 | 0.857 |
| Gender | ||||
| Male | 46 | 4359 ± 1342 | ||
| Female | 10 | 4509 ± 722 | −0.339 | 0.736 |
| Pathological subtype | ||||
| Adenocarcinoma | 26 | 4725 ± 812 | ||
| Squamous cell carcinoma | 24 | 4352 ± 1612 | ||
| Other | 6 | 4326 ± 1058 | 0.385 (F) | 0.681 |
| Differentiation | ||||
| Well differentiated | 9 | 4130 ± 1224 | ||
| Moderately differentiated | 28 | 4265 ± 1321 | ||
| Poorly differentiated | 19 | 4326 ± 1100 | 0.583 (F) | 0.566 |
| Size of tumor | ||||
| ≤3 cm | 14 | 4305 ± 1365 | ||
| >3 cm | 42 | 4628 ± 803 | −0.833 | 0.408 |
| Lymph node metastasis | ||||
| N0 | 18 | 4598 ± 1311 | ||
| N1 | 38 | 3939 ± 1000 | 1.883 | 0.065 |
| Stage | ||||
| I | 13 | 4163 ± 998 | ||
| II | 17 | 4189 ± 1661 | ||
| III | 23 | 4346 ± 1725 | ||
| IV | 3 | 4510 ± 1439 | 0.332 (F) | 0.713 |
| Smoking index | ||||
| ≤400 | 28 | 4031 ± 1256 | ||
| >400 | 28 | 4741 ± 1158 | −2.2 | 0.032 |
EGFR, epidermal growth factor receptor.
Fresh tumor and lymph node tissues were collected from NSCLC patients in the trial group, and focus and lymph node tissues were collected from the contrast group. Tissues and all serum samples (volume 4 mL, six hours prior to surgery, 24 hours after surgery) were stored at −80°C until assayed. All patients granted approval for the collected tissue and serum samples to be used.
Reagent and method
The goat anti-human enzyme-linked immune sorbent assay (ELISA) kit (Adlitteram Diagnostic Laboratories, USA), TRIzol reagent (Invitrogen, Carlsbad, CA, USA), RNA extract kit (Qiagen, Tokyo, Japan), reverse transcription kit, and quantitative real-time polymerase chain reaction (qRT-PCR) kit (Takara, Japan), were used. The EGFR Primer was designed using Primer Premier5 software and synthesised by Sangon. EGFR forward primers: 5′-GGACTCTGGATCCCAGAAGGTG-3′, reverse primers: 5′-GCTGGCCATCACGTAGGCTT-3′; GAPDHF forward primers: 5′-CAACAGCCTCAAGATCATCAGC-3′, reverse primers: 5′-TTCTAGACGGCAGGTCAGGTC-3′.
The expression of epidermal growth factor receptor (EGFR) in serum by enzyme-linked immune sorbent assay (ELISA)
The levels of EGFR in serum were measured using enzyme immunoassay kits according to the manufacturer's instructions. The absorbency was read at 450 nm for the standard curve solution and the specimens were analyzed by a microplate reader.
The detection of EGFR in tumor tissues and lymph node tissues by real-time-polymerase chain reaction (RT-PCR)
The total RNA was isolated from tissues by the TRIzol reagent. Total RNA quantification was carried out by the UV spectrophotometer. cDNA was synthesized using 500 ng of total RNA, 0.5ul of random six primers, and 0.5ul of PrimeScript reverse transcription enzyme mix1, following the manufacturer's recommended experimental conditions. EGFR gene expression was assessed by qRT-PCR on the Geneamp PCR System 9600 Detection System. The product was checked by 1% tris-borate (TBE) agarose gel electrophoresis. The PCR mixture contained 2.5 μL of template cDNA, 0.5 uL of primers, and 18ul of SYBR Premix Ex Taq and water to 25 μL. The amplification protocol consisted of 10 seconds at 95°C, followed by 40 cycles at 95°C for 15 seconds, at 57°C for 120 seconds and 72°C for 130 seconds. Duplicate assays were run for each sample and each plate included a standard curve and a negative control. The relative transcript quantification was calculated by the geNorm algorithm for Microsoft Excel and expressed in terms of arbitrary units.
Statistical analysis
Statistical analysis was performed by the SPSS 17.0 for Windows software. Quantitative data was analyzed by numerous tests, including an independent sample t-test, Paired t-test, analysis of variance, and line correlation analysis. A P-value of <0.05 was considered statistically significant.
Results
The expression of EGFR in serum
We found no correlation between EGFR expression and gender, age, pathological type, tumor differentiation level, pathological stage or tumor size (P > 0.05). The serum EGFR expression of patients with lymph node metastases was higher than that of patients without lymph node metastases, however there was no statistical significance between them (P > 0.05). Patients whose smoking index was more than 400 had higher EGFR expression levels than patients with an index less than 400 (P < 0.05). These results are detailed in Table 1.
A substantial decrease was detected in serum EGFR levels after surgery, compared to levels prior to surgery, revealing a statistical significance (2778 ± 857 pg/mL vs. 4386 ± 1249 pg/mL, P < 0.001) (Fig. 1). After surgery, EGFR expression levels showed statistical significance between different pathological types (adenocarcinoma group and squamous carcinoma, adenocarcinoma group and adenosquamous carcinoma group, P < 0.05, respectively) (Fig 2). There was no difference in serum EGFR levels of NSCLC patients in comparison with those in non-malignant disease controls (4385 ± 1250 pg/mL vs. 4169 ± 505, P > 0.05).
Figure 1.
Expression of epidermal growth factor receptor (EGFR) in serum before and after surgical treatment. EGFR expression in 56 patients: P < 0.001, by paired t-test.
Figure 2.
Serum epidermal growth factor receptor (EGFR) levels in different pathological subtypes before and after surgical treatment. EGFR expression in 56 patients: P < 0.05, by paired t-test.
The expression of EGFR in lymph node tissues
The expression of EGFR mRNA in lymph node tissues had no correlation with gender, age, pathological type, tumor differentiation level, smoking index, size or stage of tumors (P > 0. 05) (Table 2). EGFR levels in lymph node tissues were higher in NSCLC patients with lymph node metastasis than in patients without lymph node metastasis (P < 0.05). The expression of EGFR mRNA in lymph node tissues in NSCLC cases was higher than that of benign pulmonary diseases (5.99 ± 0.92 pg/mL vs. 5.27 ± 0.48 pg/mL, P < 0.05).
Table 2.
Correlation between epidermal growth factor receptor (EGFR) levels in lymph nodes and clinical parameters
| Clinical parameter | n | EGFR levels (pg/mL) | t | P |
|---|---|---|---|---|
| Age | ||||
| Mean: 60 years | ||||
| <60 | 22 | 6.06 ± 0.94 | ||
| ≥ 60 | 34 | 5.94 ± 0.93 | 0.962 | 0.659 |
| Gender | ||||
| Male | 46 | 5.95 ± 0.96 | ||
| Female | 10 | 6.15 ± 0.77 | −0.619 | 0.538 |
| Pathological subtype | ||||
| Adenocarcinoma | 26 | 5.59 ± 1.26 | ||
| Squamous cell carcinoma | 24 | 5.82 ± 1.07 | ||
| Other | 6 | 5.49 ± 1.12 | −0.613 (F) | 0.487 |
| Differentiation | ||||
| Well differentiated | 9 | 5.46 ± 0.87 | ||
| Moderately differentiated | 28 | 5.71 ± 1.01 | ||
| Poorly differentiated | 19 | 5.93 ± 1.34 | 1.279 (F) | 0.181 |
| Size of tumor | ||||
| ≤ 3 cm | 14 | 5.94 ± 1.01 | ||
| >3 cm | 42 | 6.09 ± 0.60 | −0.493 | 0.624 |
| Lymph node metastasis | ||||
| N0 | 18 | 5.56 ± 0.84 | ||
| N1-2 | 38 | 6.19 ± 0.90 | 2.522 | 0.015 |
| Stage | ||||
| I | 13 | 5.63 ± 1.01 | ||
| II | 17 | 5.65 ± 1.48 | ||
| III | 23 | 5.87 ± 1.23 | ||
| IV | 3 | 6.01 ± 0.89 | 0.613 (F) | 0.542 |
| Smoking index | ||||
| ≤400 | 28 | 5.86 ± 0.95 | ||
| >400 | 28 | 6.13 ± 0.89 | −1.104 | 0.275 |
EGFR, epidermal growth factor receptor.
The expression of EGFR in tumor tissues
The expression of EGFR mRNA in tumor tissues had no correlation with gender, age, pathological type, tumor differentiation level, smoking index, size or stage of tumors (P > 0. 05) (Table 3). EGFR levels in tumor tissues were higher in NSCLC patients with lymph node metastasis than in patients without lymph node metastasis (P < 0.05). The expression of EGFR mRNA in tumor tissues in NSCLC cases was higher than that of benign pulmonary diseases (6.27 ± 0.96 pg/mL vs. 5.37 ± 0.48 pg/mL, P < 0.05).
Table 3.
Correlation between epidermal growth factor receptor (EGFR) levels in non-small cell lung cancer (NSCLC) tissues and clinical parameters
| Clinical parameter | n | EGFR levels (pg/mL) | t | P |
|---|---|---|---|---|
| Age | ||||
| Mean: 60 years | ||||
| <60 | 22 | 5.16 ± 1.09 | ||
| ≥ 60 | 34 | 5.83 ± 1.04 | 1.025 | 0.778 |
| Gender | ||||
| Male | 46 | 5.98 ± 0.96 | ||
| Female | 10 | 6.24 ± 0.88 | −0.419 | 0.763 |
| Pathological subtype | ||||
| Adenocarcinoma | 26 | 5.69 ± 1.66 | ||
| Squamous cell carcinoma | 24 | 5.84 ± 1.17 | ||
| Other | 6 | 5.45 ± 1.82 | −0.609 (F) | 0.492 |
| Differentiation | ||||
| Well differentiated | 9 | 5.19 ± 1.07 | ||
| Moderately differentiated | 28 | 5.81 ± 1.61 | ||
| Poorly differentiated | 19 | 5.95 ± 1.34 | 1.285 (F) | 0.174 |
| Size of tumor | ||||
| ≤3 cm | 14 | 5.32 ± 1.39 | ||
| >3 cm | 42 | 5.79 ± 1.97 | −0.519 | 0.707 |
| Lymph node metastasis | ||||
| N0 | 18 | 5.15 ± 0.86 | ||
| N1 | 38 | 6.21 ± 0.93 | 2.212 | 0.012 |
| Stage | ||||
| I | 13 | 5.48 ± 1.21 | ||
| II | 17 | 5.86 ± 1.48 | ||
| III | 23 | 5.96 ± 1.13 | ||
| IV | 3 | 6.26 ± 0.89 | 0.602 (F) | 0.457 |
| Smoking index | ||||
| ≤400 | 28 | 5.64 ± 1.06 | ||
| >400 | 28 | 6.15 ± 0.96 | −1.124 | 0.202 |
EGFR, epidermal growth factor receptor.
Correlation analysis of EGFR expression among serum, lymph node, and tumor tissues
The serum EGFR levels were correlated with its expression in lymph node tissues (P < 0.001, r = 0. 764) (Fig. 3). We found that the higher the EGFR expression in the lymph node tissues, the higher the expression was in the serum. The serum EGFR levels were also correlated with the tumor tissues (P < 0.001, r = 0. 616) (Fig. 4). The data suggests that EGFR mRNA levels in the serum are derived from NSCLC tumor tissue specimens. The EGFR levels in the tumor tissues were correlated more closely with their expression in the lymph node tissues (P < 0.001, r = 0.904) (Fig. 5). The higher the EGFR expression was in the lymph node tissues, the higher the EGFR expression was in the tumor tissues.
Figure 3.
Correlation of epidermal growth factor receptor (EGFR) levels between serum and lymph node tissues. EGFR levels in serum and lymph node tissues in 56 patients: P < 0.001, r = 0.764 by line correlation analysis.
Figure 4.
Correlation of epidermal growth factor receptor (EGFR) mRNA levels in serum and tumor tissues. EGFR levels in serum and in tumor ttissues in 56 patients: P < 0.001, r = 0.616 by line correlation analysis.
Figure 5.
Correlation of epidermal growth factor receptor (EGFR) mRNA levels in lymph node and tumor tissues. EGFR levels in lymph node and tumor tissues in 56 patients: P < 0.001, r = 0.904 by line correlation analysis.
Discussion
EGFR is important in normal and neoplastic epithelial cell growth, which is involved in cellular proliferation and differentiation. Its overexpression may lead to apoptosis, adherence, aggressive metastasis, and induce mitogenesis of NSCLC.24
Several reports have shown that serum EGFR levels are associated with aggressive cancer development, such as in kidney and gastric cancer. However, analysis of serum EGFR levels in NSCLC patients and what these levels represent is controversial. A report by Sasaki et al.7 used an enzyme immunoassay to detect the serum EGFR levels of 106 lung cancer patients and 16 patients with non-malignant thoracic disease. It showed that lung cancer patients with lymph node metastasis had significantly higher EGFR levels than those in patients without lymph node metastasis (P = 0.0228). Thus, this report considered that serum EGFR levels were correlated closely with tumor metastasis. Comparing EGFR levels in serum in NSCLC patients with those in non-malignant disease controls did not reveal any statistical difference (P = 0.8083). Sasaki et al., found that EGFR plays an important role in cancer development, however, EGFR levels were not elevated in patients with lung cancer. There was no correlation between serum EGFR levels in the lung cancer patients and their clinical stage or pathological subtype. A report by Miura et al.25 measured serum EGFR mRNA levels using a qRT-PCR assay and detected EGFR protein levels using immunohistochemistry. Serum EGFR mRNA was more closely related to that in the cancer tissues (P = 0.002, r = 0.96). This data suggests that serum EGFR mRNA is derived from lung cancer tissue specimens. It shows that the number of tumors and clinical staging were significantly correlated with EGFR mRNA expression (both P < 0.05). It tended to be expressed more strongly in SCC than in ADC. However, there was no statistical significance between mRNA expression and pathological type of cancer. A report by Ahn et al.16 showed a correlation between EGFR expression and tumor cell proliferative activity, as measured with the Ki-67 proliferation index. However it showed no significant correlations between EGFR expression in NSCLC and other clinicopathological parameters, such as tumor size, nodal metastasis, angiogenesis, and prognosis. EGFR was expressed in 18 (28%) of 65 NSCLC samples. EGFR-positive was higher in squamous tumors (35%) than other NSCLCs (23%), although there was no statistical significance (P = 0.308).
We collected fresh frozen tumor and lymph node tissues to determine EGFR levels. Gallegos Ruiz et al. reported that research using fresh tumor tissue yielded more reliable results than using paraffin embedded tissue.26
Our data shows that smoking index and surgery had statistical differences on serum EGFR levels, especially levels taken before and after surgery, which showed significant differences (P < 0.001). The serum EGFR expression in NSCLC patients had no correlation with gender, age, pathological type, tumor differentiation level or tumor size (P > 0.05), which was also recorded in previous reports. However a report by Okabe et al.27 showed that smoking had a correlation with EGFR mutation in NSCLC patients, and that gefitinib had an obvious effect on non-smokers and very infrequent smokers. Our data revealed a correlation between smoking and serum EGFR expression in NSCLC patients, which, in turn, revealed that EGFR mutation and expression may have a correlation. It is worth further research. Miura et al.25 reported that EGFR mRNA levels after surgery tended to decrease than before treatment, however this decrease was not statistically significant (P = 0.065). In our study, EGFR expression levels after surgery also decreased compared to those prior to surgery (P < 0.001), which suggested that serum EGFR levels are derived from lung cancer tissue. Post surgery, there was statistical difference between EGFR expressions in serum and pathology type (P < 0.05). The expression of EGFR in the adenocarcinoma group was higher than in the squamous carcinoma and other pathology types. However, there was no difference between serum EGFR levels in NSCLC patients in comparison with those in non-malignant disease controls (P > 0.05). It may be that EGFR in tumor tissues does not reach the blood pool to the same extent as EGFR in metastases. EGFR levels in serum tended to elevate in NSCLC patients with lymph node metastasis compared to patients without lymph node metastasis, but this elevation was not statistically significant (P = 0.065), However, Sasaki et al. did find a statistical difference between them (P < 0.05).7 It may be that sample sizes in our study were too limited.
In our study, the expression of EGFR mRNA in lymph node and NSCLC tumor tissues in NSCLC patients is correlated with lymph node metastasis, and the EGFR expression in the lymph node metastasis group is higher than in patients without lymph node metastasis. It shows a statistical difference between them (both, P < 0.05). There were also statistical differences in EGFR levels of NSCLC lymph node and tumor tissues (both, P < 0.05) between NSCLC patients and those in non-malignant disease controls. However, it had no correlation with gender, age, pathological type, tumor differentiation level, smoking index, size or stage of tumors (all, P > 0.05). As EGFR mRNA in NSCLC lymph node and tumor tissues was significantly related to lymph nodal metastasis, it may indicate that EGFR is an appropriate biomarker for NSCLC metastasis.
Our correlation findings were: (i) serum EGFR levels are correlated with those of lymph node tissues (r = 0.764, P < 0.001); (ii) the expression of EGFR in serum was related to those in NSCLC tumor tissues (r = 0.616, P < 0.001); and (iii) EGFR levels in lymph node tissues were significantly related to levels in NSCLC tissues (r = 0.904, P < 0.001).
Conclusion
In conclusion, detecting EGFR levels in serum and lymph node tissues is an easy and feasible method, which may replace detecting EGFR levels in tumor tissues for NSCLC patients. It may represent a simple and effective method for diagnosis and clinical staging of NSCLC. However, because of our small sample size, caution should be exercised in the interpretation of our preliminary results. Further confirmation by larger studies is required.
Acknowledgments
This work was supported by a grant from Medical and Health Project of Health Department of Shandong Province (No. 2007HW137).
Disclosure
No authors report any conflict of interest.
References
- Hansen HH. Treatment of advanced non-small cell lung cancer. BMJ. 2002;325:452–453. doi: 10.1136/bmj.325.7362.452. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Youlden DR, Cramb SM, Baade PD. The international epidemiology of lung cancer: geographical distribution and secular trends. Thorac Oncol. 2008;3:819–831. doi: 10.1097/JTO.0b013e31818020eb. [DOI] [PubMed] [Google Scholar]
- Alberg AJ, Ford JG, Samet JM. Epidemiology of lung cancer: ACCP Evidence-based Clinical Practice Guidelines (2nd edition) Chest. 2007;132(3 Suppl):29S–55S. doi: 10.1378/chest.07-1347. [DOI] [PubMed] [Google Scholar]
- Jemal A, Siegel R, Ward E, et al. Cancer statistics 2008. CA Cancer J Clin. 2008;58:71–96. doi: 10.3322/CA.2007.0010. [DOI] [PubMed] [Google Scholar]
- De Palma G, Mozzoni P, Acampa O, et al. Expression levels of some antioxidant and epidermal growth factor receptor genes in patients with early-stage non-small cell lung cancer. J Nucleic Acids. 2010 doi: 10.4061/2010/147528. ; Article ID 147528. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Balsara BR, Pei J, Mitsuuchi Y, et al. Frequent activation of AKT in non-small cell lung carcinomas and preneoplastic bronchial lesions. Carcinogenesis. 2004;25:2053–2059. doi: 10.1093/carcin/bgh226. [DOI] [PubMed] [Google Scholar]
- Sasaki H, Yukiue H, Mizuno K, et al. Elevated serum epidermal growth factor receptor level is correlated with lymph node metastasis in lung cancer. Int J Clin Oncol. 2003;8:79–82. doi: 10.1007/s101470300014. [DOI] [PubMed] [Google Scholar]
- Berghmans T, Meert AP, Martin B, Ninane V, Sculier JP. Prognostic role of epidermal growth factor receptor in stage III nonsmall cell lung cancer. Eur Respir J. 2005;25:329–335. doi: 10.1183/09031936.05.00060804. [DOI] [PubMed] [Google Scholar]
- Hubbard SR, Miller WT. Receptor tyrosine kinases: mechanisms of activation and signaling. Curr Opin Cell Biol. 2007;19:117–123. doi: 10.1016/j.ceb.2007.02.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yarden Y, Sliwkowski MX. Untangling the ErbB signaling network. Nat Rev Mol Cell Biol. 2001;2:127–137. doi: 10.1038/35052073. [DOI] [PubMed] [Google Scholar]
- Raymond E, Faivre S, Armand JP, et al. Epidermal growth factor receptor tyrosine kinase as a target for anticancer therapy. Drugs. 2000;60(Suppl):15–23. doi: 10.2165/00003495-200060001-00002. [DOI] [PubMed] [Google Scholar]
- Yarden Y. The EGFR family and its ligands in human cancer: signalling mechanisms and therapeutic opportunities. Eur J Cancer. 2001;37(Suppl):S3–8. doi: 10.1016/s0959-8049(01)00230-1. [DOI] [PubMed] [Google Scholar]
- Mischel PS, Cloughesy TF. Targeted molecular therapy of GBM. Brain Pathol. 2003;13:52–61. doi: 10.1111/j.1750-3639.2003.tb00006.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100:57–70. doi: 10.1016/s0092-8674(00)81683-9. [DOI] [PubMed] [Google Scholar]
- Brand TM, Iida M, Li C, Wheeler DL. The nuclear epidermal growth factor receptor signaling network and its role in cancer. Discov Med. 2011;12(66):419–432. [PMC free article] [PubMed] [Google Scholar]
- Ahn JH, Kim SW, Hong SM, et al. Epidermal growth factor receptor (EGFR) expression in operable non-small cell lung carcinoma. J Korean Med Sci. 2004;19:529–535. doi: 10.3346/jkms.2004.19.4.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jin Y, Li JP, Tang LY, et al. Protein expression and significance of VEGF, EGFR and MMP-9 in non-small cell lung carcinomas. Asian Pac J Cancer Prev. 2011;12:1473–1476. [PubMed] [Google Scholar]
- Milas I, Komaki R, Hachiya T, et al. Epidermal growth factor receptor, cyclooxygenase-2, and BAX expression in the primary non-small cell lung cancer and brain metastases. Clin Cancer Res. 2003;9:1070–1076. [PubMed] [Google Scholar]
- Herbst RS, Bunn PA., Jr Targeting the epidermal growth factor receptor in non-small cell lung cancer. Clin Cancer Res. 2003;9(16 Pt 1):5813–5824. [PubMed] [Google Scholar]
- Rao C, Hu Q, Ma J, et al. Comparison of the epidermal growth factor receptor protein expression between primary non-small cell lung cancer and paired lymph node metastases: implications for targeted nuclide radiotherapy. J Exp Clin Cancer Res. 2010;29:7. doi: 10.1186/1756-9966-29-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Badalian G, Barbai T, Rásó E, Derecskei K, Szendrôi M, Tímár J. Phenotype of bone metastases of non-small cell lung cancer: epidermal growth factor receptor expression and K-RAS mutational status. Pathol Oncol Res. 13:99–104. doi: 10.1007/BF02893484. [DOI] [PubMed] [Google Scholar]
- Koo JS, Kim SH. EGFR and HER-2 status of non-small cell lung cancer brain metastasis and corresponding primary tumor. Neoplasma. 2011;58(1):27–34. doi: 10.4149/neo_2011_01_27. [DOI] [PubMed] [Google Scholar]
- Gomez-Roca C, Raynaud CM, Penault-Llorca F, et al. Differential expression of biomarkers in primary non-small cell lung cancer and metastatic sites. J Thorac Oncol. 2009;4:1212–1220. doi: 10.1097/JTO.0b013e3181b44321. [DOI] [PubMed] [Google Scholar]
- Tagliaferri P, Tassone P, Blotta S, et al. Antitumor therapeutic strategies based on the targeting of epidermal growth factor-induced survival pathways. Curr Drug Targets. 2005;6:289–300. doi: 10.2174/1389450053765897. [DOI] [PubMed] [Google Scholar]
- Miura N, Nakamura H, Sato R, et al. Clinical usefulness of serum telomerase reverse transcriptase (hTERT) mRNA and epidermal growth factor receptor (EGFR) mRNA as a novel tumor marker for lung cancer. Cancer Sci. 2006;97:1366–1373. doi: 10.1111/j.1349-7006.2006.00342.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gallegos Ruiz MI, Floor K, Rijmen F. Grünberg K, Rodriguez JA, Giaccone G. EGFR and K-ras mutation analysis in non-small cell lung cancer: comparison of paraffin embedded versus frozen specimens. Cell Oncol. 2007;29:257–264. doi: 10.1155/2007/568205. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Okabe T, Okamoto I, Tamura K, et al. Differential constitutive activation of the epidermal growth factor receptor in non-small cell lung cancer cells bearing EGFR gene mutation and amplification. Cancer Res. 2007;67:2046–2053. doi: 10.1158/0008-5472.CAN-06-3339. [DOI] [PubMed] [Google Scholar]