lung carcinoma is the mostly frequently diagnosed cancer in the world and is also the most common cause of cancer deaths in males and females all over the world . female lung cancer , with mortality higher than that of the combined mortality from breast and colon / rectum cancer , accounted for 26% of the estimated cancer deaths in 2012 . a recently study , in which never smokers with lung cancer were frequently women , demonstrated an increased prevalence of egfr mutations in never - smoking lung cancer patients with familial cancer history . hsieh et al . also reported a much higher mutation of loci related to nsclc in females than in males , as detected by sequencing . this trend of rapidly increasing incidence of lung carcinoma in females has recently drawn much attention . lung cancer is broadly classified as small cell lung cancer originating from neuroendocrine cells versus non - small cell lung cancer ( nsclc ) originating from bronchial epithelial cell precursors . nsclc can be divided into 3 types : squamous cell carcinoma , adenocarcinoma , and large cell carcinoma . among all types of lung carcinoma , non - small cell lung cancer ( nsclc ) accounts for approximately 85% of cases . the 5-year survival rate is still about 17% , despite improvements in cancer therapies over the past few decades . although many indicators of lung carcinoma have been found , there has been limited work in developing indicators from the perspective of potentially unique or vulnerable subpopulations , such as females . as the understanding of lung carcinoma evolves , researchers found that a kinase , epidermal growth factor receptor ( egfr ) , is overexpressed in 4080% of nsclc patients , and is therefore an important target of interest for therapy in this disease . egfr is the expression product of the proto - oncogene c - erbb-1 and possesses tyrosine kinase ( tk ) activity . in general , reported that the expression rate of egfr was 50% in female nsclc cases , which revealed the indicator potential of egfr with the increasing incidence of female nsclc . recent studies have shown that the egfr receptor is overexpressed in nsclc and several other types of cancers . clinical research has demonstrated that egfr inhibitors have a relatively good therapeutic effect on female lung carcinoma , particularly adenocarcinoma . according to a report , use of egfr mutations - specific tyrosine kinase inhibitors ( tki ) , such as erlotinib , gefitinib , or afatinib , is the most effective treatment strategy of nsclc that is targeted to egfr . gefitinib and erlotinib significantly increased overall survival ( os ) and progression - free survival ( pfs ) compared with placebo or best support care ( bsc ) . in this study , we investigated the expression of egfr in chinese female nsclc patients , trying to explore the relationship between egfr expression and the clinical and pathological characteristics in prognosis of female nsclc patients . our results showed that high expression of egfr was observed in female nsclc and its expression was closely related to pathology of the tumor type , tnm staging , and lnm . our study shows that egfr is an important indicator of disease progression and prognosis in female nsclc , and that it can be used to investigate the target therapy in female nsclc . we enrolled 62 female patients diagnosed with pathologic stages i to iv nsclc from january 2000 to june 2002 at the archives of the changzheng hospital . of the 62 specimens from patients aged 3275 years ( mean , 56 years ) , none had received chemotherapy , radiotherapy , or any other anticancer therapies ; 46 had adenocarcinoma , 15 had bronchioloalveolar carcinoma ( bac ) , and 16 had squamous cell carcinoma ( scc ) . poorly - differentiated , moderately - differentiated , and well - differentiated carcinoma was observed in 22 , 25 , and 15 cases , respectively . lymph node metastasis was observed in 37 cases and non - lymph node metastasis was observed in 25 cases . stage i , stage ii , stage iii , and stage iv were observed in 7 , 25 , 28 , and 2 cases , respectively . the survival period was expressed in months until death or the last recorded follow - up ; this period was determined through telephone or mail inquiries until oct 31 , 2005 . thirty - four patients survived less than or equal to 3 years and 28 patients survived more than 3 years . ten control samples were randomly selected from the nonmalignant lung tissue of female patients during the study period . this investigation was approved by the research ethics committee and the written informed consent was obtained from all individuals . according to personal medical records and individual interviews from research group and control group , a uniform epidemiology questionnaire was designed to obtain patients information , including sex , age , smoking , and alcohol consumption . a biotin - free immunohistochemical staining technique was used for detecting egfr expression according to the manufacturer s instructions ( dako ) . serial sections were placed on l - lysine coated slides , deparaffinized in xylene , and rehydrated through an ethanol gradient . antigen retrieval was achieved by slide immersion in pepsin solution for 15 min at 37c . endogenous peroxidase activity was blocked by incubating the sections in 3% hydrogen peroxide for 15 min , and nonspecific binding was blocked with 10% goat serum for 15 min . the sections were then incubated with anti - egfr monoclonal antibody ( 1:1000 ) at 4c overnight in a humid chamber . subsequently , the sections were incubated with envision reagent for 30 min at room temperature . the sections were visualized with the application of diaminobenzidine substrate chromogen solution and hematoxylin counterstain . the positive cell percentage was scored as follows : 05% ( score 1 ) , 5%25% ( score 2 ) , 5175% ( score 3 ) , and > the staining intensity was scored as follows : no staining ( score 1 ) , brown - yellow ( score 2 ) , and chocolate brown ( score 3 ) . the si score was the sum of the positive cell percentage and staining intensity scores . an si score of 0 to 2 indicated negative expression , and a score of 3 to 7 indicated positive expression . we looked for associations between egfr expression and various patient characteristics , including age , smoking history , pathology of the tumor type , tnm staging , and lnm . for statistical analysis , the chi - square test was conducted to analyze associations between egfr expression and the different variables . the cumulative survival rate was determined by the kaplan - meier product - limit method . the prognosis of female patients with nsclc was examined by using a multivariate cox proportional hazard regression model . statistical analysis was carried out using a commercially available computer program ( spss version 10.0 ; spss ; chicago , il ) . we enrolled 62 female patients diagnosed with pathologic stages i to iv nsclc from january 2000 to june 2002 at the archives of the changzheng hospital . of the 62 specimens from patients aged 3275 years ( mean , 56 years ) , none had received chemotherapy , radiotherapy , or any other anticancer therapies ; 46 had adenocarcinoma , 15 had bronchioloalveolar carcinoma ( bac ) , and 16 had squamous cell carcinoma ( scc ) . poorly - differentiated , moderately - differentiated , and well - differentiated carcinoma was observed in 22 , 25 , and 15 cases , respectively . lymph node metastasis was observed in 37 cases and non - lymph node metastasis was observed in 25 cases . stage i , stage ii , stage iii , and stage iv were observed in 7 , 25 , 28 , and 2 cases , respectively . the survival period was expressed in months until death or the last recorded follow - up ; this period was determined through telephone or mail inquiries until oct 31 , 2005 . thirty - four patients survived less than or equal to 3 years and 28 patients survived more than 3 years . ten control samples were randomly selected from the nonmalignant lung tissue of female patients during the study period . this investigation was approved by the research ethics committee and the written informed consent was obtained from all individuals . according to personal medical records and individual interviews from research group and control group , a uniform epidemiology questionnaire was designed to obtain patients information , including sex , age , smoking , and alcohol consumption . a biotin - free immunohistochemical staining technique was used for detecting egfr expression according to the manufacturer s instructions ( dako ) . serial sections were placed on l - lysine coated slides , deparaffinized in xylene , and rehydrated through an ethanol gradient . antigen retrieval was achieved by slide immersion in pepsin solution for 15 min at 37c . endogenous peroxidase activity was blocked by incubating the sections in 3% hydrogen peroxide for 15 min , and nonspecific binding was blocked with 10% goat serum for 15 min . the sections were then incubated with anti - egfr monoclonal antibody ( 1:1000 ) at 4c overnight in a humid chamber . subsequently , the sections were incubated with envision reagent for 30 min at room temperature . the sections were visualized with the application of diaminobenzidine substrate chromogen solution and hematoxylin counterstain . the positive cell percentage was scored as follows : 05% ( score 1 ) , 5%25% ( score 2 ) , 5175% ( score 3 ) , and > the staining intensity was scored as follows : no staining ( score 1 ) , brown - yellow ( score 2 ) , and chocolate brown ( score 3 ) . the si score was the sum of the positive cell percentage and staining intensity scores . an si score of 0 to 2 indicated negative expression , and a score of 3 to 7 indicated positive expression . we looked for associations between egfr expression and various patient characteristics , including age , smoking history , pathology of the tumor type , tnm staging , and lnm . for statistical analysis , the chi - square test was conducted to analyze associations between egfr expression and the different variables . the cumulative survival rate was determined by the kaplan - meier product - limit method . the prognosis of female patients with nsclc was examined by using a multivariate cox proportional hazard regression model . statistical analysis was carried out using a commercially available computer program ( spss version 10.0 ; spss ; chicago , il ) . positive staining of egfr was observed in the cytoplasm and cytomembrane of tumor cells , which was characterized by brownish - yellow particles . staining efficacy varied among different tissues , as indicated by the pathological images ( figure 1 ) . egfr expression was positive in 70.97% ( 44/62 ) of female nsclc cases , while it was significantly negative in the control group with nonmalignant lung tissues ( p=0.000014 ) . the expression of egfr in female nsclc showed dramatic differences in different pathology of the tumor type , with or without lnm , and various tnm staging ( p<0.05 ) , indicating the various expression types of egfr with different pathological characteristics . however , no remarkable correlations between egfr expression and age , smoking history , tumor size , tumor site , tumor differentiation degree , or tumor distal metastases were found ( p>0.05 ) . of the 44 cases with positive egfr expression , survival time was less than or equal to 3 years in 28 cases and greater than 3 years in 16 cases . in the 18 cases with negative egfr expression , the survival time was less than or equal to 3 years in 6 cases and greater than 3 years in 12 cases . the 3-year survival rate of the group with positive egfr expression was significantly lower than that of the group with negative egfr expression ( p 0.0295 ) . the kaplan - meier survival curve based on the relationship between the different levels of expression of egfr and survival time is shown in figure 2 . a cox proportional risk regression model was constructed according to patient s age , pathology of the tumor type , tumor size , tumor site , lnm , surgical method , egfr expression , and survival time . the analysis of the cox proportional risk model indicated that the postoperative survival time was significantly associated with pathology of the tumor type and lnm ( p<0.05 ) . adenocarcinoma with lnm was an independent poor prognostic factor ( i.e. , positive cox proportional risk regression model ) ( table 3 ) ; however , patient age , tumor size , tumor site , surgical method , and egfr expression were not independent prognostic factors . positive staining of egfr was observed in the cytoplasm and cytomembrane of tumor cells , which was characterized by brownish - yellow particles . staining efficacy varied among different tissues , as indicated by the pathological images ( figure 1 ) . egfr expression was positive in 70.97% ( 44/62 ) of female nsclc cases , while it was significantly negative in the control group with nonmalignant lung tissues ( p=0.000014 ) . the expression of egfr in female nsclc showed dramatic differences in different pathology of the tumor type , with or without lnm , and various tnm staging ( p<0.05 ) , indicating the various expression types of egfr with different pathological characteristics . however , no remarkable correlations between egfr expression and age , smoking history , tumor size , tumor site , tumor differentiation degree , or tumor distal metastases were found ( p>0.05 ) . of the 44 cases with positive egfr expression , survival time was less than or equal to 3 years in 28 cases and greater than 3 years in 16 cases . in the 18 cases with negative egfr expression , the survival time was less than or equal to 3 years in 6 cases and greater than 3 years in 12 cases . the 3-year survival rate of the group with positive egfr expression was significantly lower than that of the group with negative egfr expression ( p 0.0295 ) . the kaplan - meier survival curve based on the relationship between the different levels of expression of egfr and survival time is shown in figure 2 . a cox proportional risk regression model was constructed according to patient s age , pathology of the tumor type , tumor size , tumor site , lnm , surgical method , egfr expression , and survival time . the analysis of the cox proportional risk model indicated that the postoperative survival time was significantly associated with pathology of the tumor type and lnm ( p<0.05 ) . adenocarcinoma with lnm was an independent poor prognostic factor ( i.e. , positive cox proportional risk regression model ) ( table 3 ) ; however , patient age , tumor size , tumor site , surgical method , and egfr expression were not independent prognostic factors . the expression of egfr in tumor tissues is closely related to pathology of the tumor types , differentiation degree , lnm , and tumor invasion ; moreover , a correlation has been observed between egfr expression and patient prognosis . in this study , we recruited chinese female nsclc patients as subjects to explore the expression of egfr in female nsclc . the expression rate of egfr was 70.97% in chinese female nsclc cases , which was higher than that reported in previous studies . with more detail classification of pathology in our research , we found that the expression of egfr in female adenocarcinoma was higher than that in scc and even higher than that in female bac ; the expression of egfr was positively correlated with lnm and tnm staging , and it was not associated with patient age , smoking history , tumor size , tumor site , differentiation degree , and distal metastases . some studies confirmed that female adenocarcinoma and bac were also associated with egfr gene mutations ; therefore , the high level of expression of egfr in female nsclc might be related to female egfr gene mutation [ 1618 ] , which corresponded with our immunohistochemical results . some studies have indicated that the expression of egfr is associated with a reduction in disease - free survival time and total survival time , poor prognosis , easy recurrence , late staging , and high metastasis rate , but other studies have reported contradictory findings . our study showed that the 3-year survival rate of female nsclc patients with positive egfr expression was clearly lower than that of patients with negative egfr expression . the cox proportional risk model analysis demonstrated that the postoperative survival time of the patients was significantly associated with pathology of the tumor type and lnm ( p<0.05 ) , and that adenocarcinoma with lnm was the independent poor prognostic factor . currently , in the treatment of tumors , target therapy of lung cancer has drawn much attention . egfr is an important target for tumor inhibition , which affects the signal transduction system of tumor cells and suppresses tumor proliferation , invasion , metastases , and angiogenesis . clinical research has validated the therapeutic effects of the egfr - tk inhibitor in female patients with adenocarcinoma . therefore , the study of egfr expression in chinese female nsclc can predict the pathogenesis , development , and prognosis of the disease in chinese female nsclc patients and can also be a good choice for the target therapy of female nsclc . the expression of egfr is high ( 70.97% ) in female nsclc and the positive egfr expression exhibited a reduction in 3-year survival rate compared with those of the negative egfr expression . our results also showed that egfr expression was closely related to pathology of the tumor type , tumor - node metastasis staging , and lymph node metastasis , which could be used as a promising indicator of nsclc in chinese female patients . a more comprehensive study that includes male patients and different races is needed to determine if our results have universal applicability .