[Abstract] Objective To investigate the non-small cell lung cancer (NSCLC) epithelial  mesenchymal transition (EMT) and epidermal growth factor receptor (EGFR) the relationship between the mutation and the clinical and pathological features. Methods  cadherin immunohistochemistry assay NSCLC carcinoma treated with E and wave protein expression. Using the chi-square test and logistic regression analysis to investigate the impact of clinical and pathological features and EGFR genotype in NSCLC EMT. Results 62 NSCLC specimens, epithelial phenotype 35.48% (22/62). EGFR mutations in NSCLC epithelial phenotype was significantly higher than that of wild-type (77.78% vs 18.18%; P <0.0001); women were significantly higher than men (54.55% vs 25%; P = 0.02); adenocarcinoma than other pathological type ( 39.47% vs 29.17%; P = 0.4087); slightly higher than smokers of non-smokers (42.42% vs. 27.59%; P = 0.2231); aged <60 years and age ≥ 60 age group difference was not statistically significant (43.33% vs 28.12%; P = 0.211), early lung cancer with advanced lung cancer group, the difference was not statistically significant (38.24% vs. 32.12%; P = .6178). Conclusion EGFR mutant females, non-smokers, adenocarcinoma tend epithelial phenotype consistent with the clinical features of the distribution of the EGFR tyrosine kinase inhibitor efficacy crowd.

Key words epithelial mesenchymal transition lung cancer with epidermal growth factor receptor tyrosine kinase inhibitor

    Lung cancer is the leading cause of cancer deaths in the world. Although clinicians continue to improve the diagnosis and treatment, the response of the lung cancer surgery and chemoradiotherapy is still Nanjinrenyi. Molecular targeted therapy because of its high selectivity, low toxicity and acclaimed. How to choose the appropriate patient population, and how to predict the efficacy of tumor molecular targeted therapy has also become the focus of increasing attention.

    Epithelial cells  mesenchymal transition (epithelial  mesenchymal transition, EMT) of epithelial cells in the process of interaction with the surrounding interstitial get some phenomenon of stromal cell-specific traits, E  cadherin (E  cadherin ), catenin (catenin) characterization of the epithelial type indicator protein loss and wave protein (vimentin) and fibronectin (fibronectin) characterization of interstitial-type indicator protein obtained as the main feature, and with tumor cells in situ invasion and distant metastasis is closely related to [1]. Recent studies have shown that epithelial type of lung cancer for EGFR (Epidermal growth factor receptor, EGFR) tyrosine kinase inhibitors (tyrosine kinase inhibitor, TKI) therapy interstitial changes sensitive prompts lung cancer the EMT the state is expected to become targeted therapy used in clinical predictors of [2,3]. The study on the relationship between the EMT status of non-small cell lung cancer (Non  small cell lung cancer, NSCLC) with EGFR genotype and the clinical and pathological features to explore, E  cadherin, vimentin, fibronectin EMT markers such as treatment predictor of the final clinical application to do a preliminary study.

    1 Materials and Methods

    1.1 Clinical data

    62 cases of NSCLC samples collected Shanghai Pulmonary Hospital from September 2003 to June 2004 underwent surgical resection and confirmed by pathology. Including 40 males and 22 females. A median age of 62 years (39 to 83 years old); pathological types: adenocarcinoma, 38 cases, 24 cases of squamous cell carcinoma, scales gland mixed cancer in two cases; revised lung cancer in 1997 UICC TNM pathologic staging: I of the 29 cases, five cases of Phase II, Phase III, 25 cases and three cases of stage IV. Examples before surgery were not receiving chemotherapy and radiotherapy.

    1.2 NSCLC, EGFR mutation analysis

    The Shanghai Hua Shunsheng material Co., a small amount of tissue cell genomic DNA quickly frozen tissue was extracted genomic DNA extraction and purification kit. Paez et al [4] reported 18, 19 of the EGFR gene using nested PCR exon amplification. Amplified fragment containing exon and its associated intron. PCR conditions: denaturation 95 ° C 15min; 35 times at 95 ° C 20s, 60 ° C for 30s and 72 ℃ 1min cycle; extension at 72 ° C for for 10 min. The PCR product sent Biological Technology Co., Ltd. Shanghai GeneCore sequencing EGFR gene 18, 19 and exon 21 mutations in the region.

    1.3 immunohistochemistry

    Immunohistochemistry (SP method) detecting NSCLC tissue of E  cadherin and vimentin expression. E  cadherin and vimentin antibodies are mouse anti-human monoclonal antibodies were purchased from Shanghai Long Island working concentration of 1:100. Paraffin sections after dewaxing, hydration, antigen retrieval processing join an anti-4 ℃ overnight. Secondary antibody at 37 ° C for 30 min; DAB color; hematoxylin; hydrochloride alcohol differentiate; dehydration and transparent; neutral resin sealing. Known positive sections positive control, PBS instead of primary antibody as negative control.

    Judgment: the cell membrane and (or) color results prompted positive cytoplasm yellow-brown stain particles. Random observation 10 high power field, according to the slice in the number of cancer-positive, no, and the percentage of positive cells expressing the results are divided into: negative (-) to yellow-brown staining or staining positive cells <10%; weakly positive ( +) positive cells ≥ 50% from 10% to 24% of the number of positive cells; moderately positive (+ +) from 25% to 49% of the number of positive cells; strongly positive (+ + +).

    1.4 statistical methods

    Calculated using the chi-square test analysis of the the EMT phenomenon and the age, gender, tumor stage, histological type, smoking status and EGFR genotype and other factors of the relationship; logistic regression analysis to further test the relationship between various factors and EMT state; Kappa values ​​of E  cadherin staining and vimentin staining characterization the EMT state of consistency test. All statistical analyzes were performed using SAS9.0, P <0.05 was considered statistically significant.

    2 Results

    2.1 NSCLC, EGFR mutation analysis

    62 cases of lung cancer, 18 patients with presence of EGFR gene mutation, the mutation rate was 29.03% (18/62). 12 cases of deletion mutations, six cases of point mutations in the EGFR gene point mutation or deletion mutations were heterozygous. All deletion mutation occurs in exon 19 codon EGFR TK region. All point mutations in codon 858 in exon 21, resulting in T  G conversion. But not detected exon 18 of the gene mutation, as shown in Figure 1.

    2.2 E  cadherin and vimentin expression in NSCLC with EGFR genotype

    E  cadherin is mainly targeted at the tumor cell membrane, a small amount of cytoplasmic staining in NSCLC tissues, the positive expression rate of 35.48% (22/62), as shown in Figure 2. EGFR gene mutations in tumor E  cadherin expression levels were significantly higher than the wild-type EGFR (77.78% vs 18.18%, P <0.0001).

    vimentin is expressed mainly in the membrane and cytoplasm of positive expression rate was 67.74% (42/62), as shown in Figure 3. More mutant EGFR wild-type vimentin expression levels were significantly higher (84.09% vs 27.78%). Two specimens of E  cadherin and vimentin express both positive.

    2.3 lung cancer the EMT status with EGFR genotype and clinical pathological features

    Yauch [3] reported membrane staining / cytoplasmic staining and Western blot results are in good agreement, using the results of the immunohistochemical characterization of the EMT state with high reliability. E  cadherin-positive, vimentin negative characterization of epithelial-type contrary characterization interstitial. Kappa values ​​analysis suggests that a higher consistency (Kappa = 0.9281 E  cadherin and vimentin staining to determine the results of the EMT status; 95% confidence interval, 0.8303 to 1.0259; P <0.0001), and therefore E  cadherin staining characterization of lung cancer EMT status.

    Statistical results show that: the epithelial phenotype of NSCLC accounted for 35.48% (22/62). Epithelial phenotype in EGFR mutant significantly higher than that of wild-type (77.78% vs 18.18%; P <0.0001); women were significantly higher than men (54.55% vs 25%; P = 0.02); pathological type of adenocarcinoma is higher than other disease. the difference was not statistically significant (39.47% vs 29.17%; P = 0.4087); non-smokers, slightly higher than the smokers, the difference was not statistically significant (42.42% vs. 27.59%; P = 0.2231); aged <60 years and age ≥ 60-year-old group difference was not statistically significant (43.33% vs 28.12%; P = 0.211), early lung cancer with advanced lung cancer group, the difference was not statistically significant (38.24% vs. 32.12%; (P = .6178), as shown in Table 1 Table 162 cases EMT markers of tumor of the lung cancer patients with EGFR gene *: squamous cell carcinoma, mixed carcinoma and adenocarcinoma scales gland; **: Kappa consistency analysis prompted E  cadherin and vimentin staining high consistency, we chose E  cadherin staining characterization of lung cancer EMT state P (chi-square test) calculated in accordance with the results of E  cadherin staining Logistic regression analysis showed that the epithelial phenotype and is closely related to EGFR mutations (odds ratio, 0.094; 95% confidence interval, 0.026 to 0.349; P = 0.0004). Logistic regression analysis of epithelial and mesenchymal type was no significant difference in age, clinical stage, and smoking status stratified not line with the epithelial type relationship of these factors.

    3 Discussion

    EGFR targeted drugs has shown good clinical efficacy, but not all patients from small molecule EGFR TKI (gefitinib and erlontinib) treatment benefit. The gefitinib and erlontinib for second-or third-line treatment for NSCLC, objective response rate between 9% to 19%, the other about 50% of cases occur progression. Numerous studies have been found effective in Asians, women, non-smokers, adenocarcinoma patients. However, trying to distinguish molecular predictors patient population for EGFR TKI therapy study results also unsatisfactory. Reasonable to assume initially EGFR targeted therapy effective EGFR expression in lung tissue paraneoplastic organizations, but the analysis of animal models and clinical patient specimens results show that high tumor EGFR expression is not a predictor of EGFR TKI therapy sensitive; same , EGFR of tyrosine kinases (tyrosine kinase, TK) functional domain mutation is closely related to the treatment of sensitivity conclusion widely recognized, but studies have shown that about 10% EGFR wild-type patients also benefit from EGFR TKI treatment to obtain more good effect [4  8]. Therefore, it is necessary to find new energy more effectively predict EGFR TKI sensitivity indicators.

    EMT is a common physiological process of formation and development of biological embryos, the EMT also exist in a variety of pathological processes. The latest research shows: the expression E  cadherin NSCLC sensitive to EGFR TKI therapy and express vimentin / fibronectin of NSCLC, the EGFR TKI insensitive [2,3]. When the EMT of lung cancer, after re-expression of E  cadherin tumor cells restore sensitivity to EGFR TKI therapy. Yauch such reports are not sensitive to erlotinib lung cancer often combined markers vimentin and (or) fibronectin expression [2,3], and thus speculate E  cadherin expression levels predict NSCLC patients as a new biological effect of erlotinib clinical school mark. Tumor decreased sensitivity after the EMT of EGFR TKI therapy, can be understood as The EMT may reduce the growth or proliferation of tumor cells on the needs of the EGFR signaling pathway. For example, when the phenotype of interstitial cells continued Akt activation, triggering a non-EGFR-dependent PI3K/Akt signaling pathway activation, resulting in the treatment of tumor cells to erlotinib and other sensitive [9]. So, the EMT status of the tumor is closely related to EGFR TKI treatment reactivity. In the present study, we examined the EMT status of the 62 cases of lung cancer, and then explores the tumor the EMT status with EGFR genotype, as well as the relationship of the clinical and pathological features.

    Tissue staining showed that 77.78% of the EGFR gene mutations in patients with E  cadherin expression prompted positive epithelial type, patients with EGFR mutations EGFR TKI clinical efficiency similar prompt E  cadherin expression levels may predict NSCLC patients with EGFR TKI clinical indicators of efficacy reliable. 18.18% in E  cadherin expression only in wild-type EGFR, vimentin expression was 84.09%, mainly interstitial. Lack of effective treatment of sensitive biological marker in patients with wild-type EGFR gene, EGFR TKI treatment and tolerability indicators exon 20 mutations K  RAS mutations in the Chinese population, the incidence is very low, and observed in this study to of E  cadherin expression rate reported in the literature of EGFR in patients with wild-type EGFR TKI clinical efficiency is similar, therefore, more than leather or interstitial to distinguish those who are most likely to benefit from targeted EGFR therapy patient population has important The clinical significance. Our findings seem to cause structural activation of mutant EGFR mesenchymal transition of tumor cell migration, invasion and distant metastasis inconsistent results [10]. Mechanism worthy of further study, because the occurrence of EMT requires the involvement of many signaling pathways in Src, MAPK, and PI  3K, these signaling pathways involved in the EGFR intracellular signal transduction and are closely related to EGFR TKI resistance As for the EGFR signal transduction pathway is a relationship with EMT is not yet clear. Speculated that epithelial type or interstitial affected by the impact of EGFR mutations, patients with mutant tumors often isolated epithelial type, while wild-type tumors appear interstitial type conversion. 62 NSCLC patients, female patients with epithelial phenotype significantly higher than men, the difference was statistically significant; the adenocarcinoma type as well as non-smokers also tend to show the epithelial phenotype, consistent with the clinical gefitinib effective population characteristics. Noteworthy is the epithelial phenotype differences between the earlier lung and late stage lung cancer was not found. Since EMT and tumor invasion and metastasis, in advanced lung cancer should be rendered more interstitial change. Another possibility can be explained early lung cancer and advanced lung epithelial phenotypic differences between results, i.e. distant metastasis of the tumor more performance between qualitative, and the primary tumor is still more rendering epithelial type, and we obtain of both primary tumor tissue.

    In summary, this study found that EGFR mutations, women, non-smokers, adenocarcinoma tend epithelial phenotype distribution with EGFR TKI efficacy crowd clinical features consistent; further analysis showed that the mutant and wild-type EGFR expression of E  cadherin rate and reported in the literature for the group of patients with EGFR TKI clinical efficiency similar. Suggesting that the epithelial type or interstitial type is likely to predict clinical efficacy of EGFR TKI as a factor.