Relationship Between EGFR Gene Mutations And Different Phenotypes Of Non-small Cell Lung Cancer

ObjectionLung cancer is one of the most threat malignant carcinoma with the characteristic of high incidence mortality rate. According to the clinical characteristics and pathological morphology, it can be divided into small cell lung cancer and non-small cell lung cancer (non-small-cell carcinoma, NSCLC), in which NSCLC accounts for about 75%-80%. The disappointing treatment outcome of NSCLC attribute to the advanced stage when patients are diagnosed. The WHO divided NSCLC into squamous cell carcinoma, adenocarcinoma and mixed type.Therefore,the adenocarcinoma can be divided into various subtypes according to the morphological characteristics as well.2015 WHO histological classification recommend using P63, CK7, TTF-1 and NapsinA to differentiate squamous cell carcinoma from adenocarcinoma. However, because of the complex structure of lung tissue, there are still some difficults to diagnose all the subtype of NSCLC. As a result, single chemotherapy scheme according to histologic subtype of NSCLC proved to be disappoint outcome. With the development of molecular biology, we found that about 30%of the NSCLC have EGFR gene mutations(especially adenocarcinoma),and EGFR tyrosine kinase inhibitors is an approved efficient treatment for it. But why are some of the squamous cancer expressed EGFR gene mutations,And which type of adenocarcinoma has higher risk of EGFR gene mutation is unknown. By using immunohistochemical method combined with histopathological observation to present a new classified method of NSCLC, and detecting the EGFR gene mutation by sequencing technology in those patients, we evaluated the relationship between EGFR gene mutation and NSCLC originated from different tissue.Methods106 NSCLC tissue specimens and their related clinical pathology materials were collected.First,we classified the tissue using the histopathological and genetic classification criteria of WHO of lung cancer in 2015. The expressions and localization of TTF-1/CK7,P63/NapsinA were detected by immunohistochemical staining.Then analyze the relationship between the four markers above and the clinic pathological parameters,to establish a novel tissue phenotype classification. The EGFR gene mutations were detected by sequencing technology. Then analyze the relationship between EGFR gene mutations and the clinic pathological parameters.The data were analyzed by statistical package SAS 9.4,and p≤0.05 for the difference was statistically significant.Results1. Localization and expression of P63,CK7,TTF-1 and NapsinAPositive signal of CK7 and NapsinA expression were located in the cytoplasm as indicated by a red staining, Positive signal of P63 and TTF-1 expression were located in the nucleus as indicated by a yellow-brown staining. P63 was mainly expressed in the large bronchial epithelial cells, rather than alveolar. CK7 is mainly expressed in respiratory bronchial and alveolar epithelial cells. TTF-1 is mainly expressed in the terminal bronchial epithelial cells and alveolar epithelial cells.NapsinA expression was positive in alveolar epithelial cells, which was not expressed in bronchial epithelial cells. The expression of P40 and P63 was almost identical in the respiratory epithelium of the large bronchial epithelial cells and the terminal epithelial cells. CK5&6 was not expressed.2. Expressions of NapsinA, P63, CK7 and TTF-1 in different subgroups of NSCLCThe positive expression rate of P63 in squamous cells carcinoma, lepidic predominant adenocarcinoma,acinar adenocarcinoma and solid adenocarcinoma were 100%,47.37%,55.56%,80%respectively, denoted a significant difference (p≤0.05).The positive expression rate of CK7 in squamous cells carcinoma, lepidic predominant adenocarcinoma,acinar adenocarcinoma and solid adenocarcinoma were 46.94%,100%,73.08%,100%respectively, denoted a significant difference (p<0.05).The positive expression rate of TTF-1 in squamous cells carcinoma, lepidic predominant adenocarcinoma,acinar adenocarcinoma and solid adenocarcinoma were 4.08%,89.47%,88.89%,70.00%respectively, denoted a significant difference (p≤0.05).The positive expression rate of NapsinA in squamous cells carcinoma, lepidic predominant adenocarcinoma,acinar adenocarcinoma and solid adenocarcinoma were 14.29%,78.95%,92.59%,60%respectively, denoted a significant difference (p≤0.05). In addition, we found 2 cases of NSCLC, which diagnosed as adenocarcinoma but the immunohistochemical staining showed CK7 expression only. According to the histopathological observation we inferred that it could be originated from the salivary gland tissue in the airway. P63 is expressed in all squamous cell carcinomas, whereas CK5&6 is expressed only in highly differentiated squamous cell carcinoma. The expression of P40 and P63 was almost identical to that of squamous cell carcinoma.3. Relationship between the expression of P63,CK7,TTF-1 and NapsinA and the clinic pathological parameters of NSCLC.The expression of P63,TTF-1 and NapsinA in NSCLC were significantly related to gender (p≤0.05), P63 expressd mainly in male,because squamous cell carcinoma happen in most male smokers.TTF-1 and Napsin were mainly expressed in female, because TTF-1 expressed mainly in the terminal bronchiole epithelial cells and alveolar epithelial cells of adenocarcinoma, and NapsinA expressed mainly in alveolar lobular units and alveolar of adenocarcinoma expression. Therefore, it is more common in women. There was no statistical significance between the expression of CK7 and gender (p> 0.05). The expression of P63 and TTF-1 in NSCLC were both significantly related to smoking (p≤0.05). There was no statistical significance between P63,CK7,TTF-1,NapsinA and the grade of cellular differentiation(p>0.05). And there were no significant relationship between the expression of P63,CK7,TTF-1,NapsinA and other clinic pathological parameters of NSCLC.4. Relationship between different phenotypes of NSCLC and the clinic pathological parameters.Respiratory Bronchial epithelium cancer, bronchioloalveolar cancer and alveolus epithelium cancer were significantly related to gender, smoking, cellular differentiation and tumor size (p≤0.05). Respiratory Bronchial epithelium cancer is mainly happen in male smokers, and the tumor is larger. Alveolar epithelial cancer occurs frequently in non-smoking female, and its tend to be moderately differentiated,and there were no significant relationship with other clinic pathological parameters of NSCLC (p>0.05)5. Relationship between different phenotypes of NSCLC and EGFR gene mutations.EGFR gene mutation rate in respiratory bronchial epithelium cancer, bronchioloalveolar cancer and alveolus epithelium cancer were 27.12%,54.55%,60.00% respectively, denoted a significant difference (p≤0.05)6. Relationship between EGFR gene mutations and the clinic pathological parameters of NSCLC.EGFR gene mutations were related to smoking (p≤0.05),whereas there were no significant relationship between EGFR gene mutations and the other clinic pathological parameters of NSCLC (p>0.05)Conclusions1.By using immunohistochemical method combined with histopathological observation, we present a new classification which divided NSCLC into respiratory bronchial epithelium cancer, bronchioloalveolar cancer,alveolus epithelium cancer and secreting gland cancer. Respiratory bronchial epithelium cancer include squamous cells cancer (CK5&6±,P63+,CK7-,TTF-1-,NapsinA-), adenocarcinoma (CK5&6-,P63+,CK7+,TTF-1±-,NapsinA-) and squamous adenocarcinoma (CK5&6±,P63+,CK7+,TTF-1±,NapsinA-) 。 Bronchioloalveolar cancer usually showed solid adenocarcinoma,acinar adenocarcinoma and papillary thyroid carcinoma (CK5&6-,P63+,CK7+,TTF-1+,NapsinA+). Alveolus epithelium cancer usually showed lepidic predominant adenocarcinoma,acinar adenocarcinoma and solid adenocarcinoma (CK5&6-,P63-,CK7+,TTF-1+,NapsinA+). Secreting gland cancer showed CK7 expression only. P63 is expressed in all squamous cell carcinomas, whereas CK5&6 is expressed only in highly differentiated squamous cell carcinoma. The expression of P40 and P63 was almost identical to that of squamous cell carcinoma.2.Respiratory bronchial epithelium cancer, bronchioloalveolar cancer and alveolus epithelium cancer were significantly related to gender, smoking, the grade of cellular differentiation and tumor size, but there were no significant relationship with other clinic pathological parameters of NSCLC.3.NapsinA was significantly related to gender, P63 and TTF-1 were both significantly related to gender and smoking. Meanwhile, the expression of TTF-1 was related to the cellular differentiation. These evidences indicate that NapsinA, P63 and TTF-1 may play important roles in the progression of NSCLC.4.EGFR gene mutation rate in respiratory bronchial epithelium cancer, bronchioloalveolar cancer and alveolus epithelium cancer denoted a significant difference, which indicated that alveolus epithelium is more inclined to have EGFR gene mutations.5. EGFR gene mutations were related to smoking (p≤0.05),whereas there were no significant relationship between EGFR gene mutations and the other clinic pathological parameters of NSCLC (p> 0.05).