tumor samples were collected from 505 consecutive lung adenocarcinoma patients who underwent curative surgical resection at seoul national university bundang hospital between may 2003 and december 2012 . clinicopathological information and follow - up data were obtained by reviewing the medical and the pathological records of the enrolled patients . smoking history was defined as smokers who have smoked 100 cigarettes , and never - smokers who have never smoked or smoked less than 100 cigarettes in their life time . h.c ) independently reviewed the hematoxylin and eosin stained slides and classified the diagnosis according to the international association for the study of lung cancer / american thoracic society / european respiratory society ( iaslc / ats / ers ) criteria and the 2015 world health organization ( who ) classification system . histological grade was based on the predominant histological subtypes as follows : grade 1 , lepidic ; grade 2 , acinar and papillary ; and grade 3 , solid and micropapillary . tumors were staged according to the american joint committee on cancer , seventh staging system . progression - free survival ( pfs ) was measured from the date of surgery until disease progression or death . overall survival ( os ) was measured from the date of surgery to the time of death or last follow - up visit . the most representative areas were obtained for each tumor sample and arranged for tissue microarray ( tma ) . tissue cores with a diameter of 2 mm were embedded within tma blocks , which were sectioned into series of 4-m - thick slices and then stained with hematoxylin and eosin ; immunohistochemical labeling was then performed . immunohistochemistry for aqp1 , e - cadherin , and vimentin were performed according to the antibody manufacturer s instructions . the following antibodies were used : aqp1 ( b-11 , 1:100 , santa cruz biotechnology , santa cruz , ca , usa ) , e - cadherin ( spm471 , 1:150 , thermo fisher scientific , carlsbad , ca , usa ) , and vimentin ( 1:100 , 4a4 , zeta corporation , arcadia , ca , usa ) . aqp1 overexpression was defined when  25% showed membranous staining with loss of polarization , as previously reported . immunohistochemical stains of e - cadherin and vimentin were scored using a semi - quantitative evaluation for each case . the intensity of staining was scored on a four - point scale as follows : 0 , no staining ; 1 , weak staining ; 2 , moderate staining ; and 3 , strong staining . the final score was calculated by multiplying the intensity score to the fraction score , generating a total range of 0300 . scores of 0100 and 101300 were considered as negative and positive , respectively , as previously reported . translocation of anaplastic large cell lymphoma kinase ( alk ) was evaluated in 440 cases by fluorescence in situ hybridization analysis using a probe to alk ( vysis lsi alk dual color , break - apart rearrangement probe , abbott molecular , des plaines , il , usa ) ; translocation was observed in 28 out of 440 cases ( 6.4% ) . epidermal growth factor receptor ( egfr ) mutations of exon 18 to 21 and kras mutations of codon 12 and 13 were evaluated in 484 and 413 cases using polymerase chain reaction and direct dna sequencing , as previously described . egfr and kras mutations were identified in 49.0% ( 237/484 ) and 6.1% ( 25/413 ) of cases , respectively . the association between immunohistochemistry results and clinicopathological variables was assessed by chi - square test , fisher exact test , or spearman s rank correlation test . kaplan - meier analysis with log - rank test and multivariate cox regression analysis were performed for survival analysis . tumor samples were collected from 505 consecutive lung adenocarcinoma patients who underwent curative surgical resection at seoul national university bundang hospital between may 2003 and december 2012 . clinicopathological information and follow - up data were obtained by reviewing the medical and the pathological records of the enrolled patients . smoking history was defined as smokers who have smoked 100 cigarettes , and never - smokers who have never smoked or smoked less than 100 cigarettes in their life time . h.c ) independently reviewed the hematoxylin and eosin stained slides and classified the diagnosis according to the international association for the study of lung cancer / american thoracic society / european respiratory society ( iaslc / ats / ers ) criteria and the 2015 world health organization ( who ) classification system . histological grade was based on the predominant histological subtypes as follows : grade 1 , lepidic ; grade 2 , acinar and papillary ; and grade 3 , solid and micropapillary . tumors were staged according to the american joint committee on cancer , seventh staging system . progression - free survival ( pfs ) was measured from the date of surgery until disease progression or death . overall survival ( os ) was measured from the date of surgery to the time of death or last follow - up visit . the most representative areas were obtained for each tumor sample and arranged for tissue microarray ( tma ) . tissue cores with a diameter of 2 mm were embedded within tma blocks , which were sectioned into series of 4-m - thick slices and then stained with hematoxylin and eosin ; immunohistochemical labeling was then performed . immunohistochemistry for aqp1 , e - cadherin , and vimentin were performed according to the antibody manufacturer s instructions . the following antibodies were used : aqp1 ( b-11 , 1:100 , santa cruz biotechnology , santa cruz , ca , usa ) , e - cadherin ( spm471 , 1:150 , thermo fisher scientific , carlsbad , ca , usa ) , and vimentin ( 1:100 , 4a4 , zeta corporation , arcadia , ca , usa ) . aqp1 overexpression was defined when  25% showed membranous staining with loss of polarization , as previously reported . immunohistochemical stains of e - cadherin and vimentin were scored using a semi - quantitative evaluation for each case . the intensity of staining was scored on a four - point scale as follows : 0 , no staining ; 1 , weak staining ; 2 , moderate staining ; and 3 , strong staining . the final score was calculated by multiplying the intensity score to the fraction score , generating a total range of 0300 . scores of 0100 and 101300 were considered as negative and positive , respectively , as previously reported . translocation of anaplastic large cell lymphoma kinase ( alk ) was evaluated in 440 cases by fluorescence in situ hybridization analysis using a probe to alk ( vysis lsi alk dual color , break - apart rearrangement probe , abbott molecular , des plaines , il , usa ) ; translocation was observed in 28 out of 440 cases ( 6.4% ) . epidermal growth factor receptor ( egfr ) mutations of exon 18 to 21 and kras mutations of codon 12 and 13 were evaluated in 484 and 413 cases using polymerase chain reaction and direct dna sequencing , as previously described . egfr and kras mutations were identified in 49.0% ( 237/484 ) and 6.1% ( 25/413 ) of cases , respectively . all statistical analyses were performed using spss ver . 18.0 ( spss inc . , chicago , il , usa ) . the association between immunohistochemistry results and clinicopathological variables was assessed by chi - square test , fisher exact test , or spearman s rank correlation test . kaplan - meier analysis with log - rank test and multivariate cox regression analysis were performed for survival analysis . the tumor specimens in this study were from 505 lung adenocarcinoma patients , consisting of 247 male ( 48.9% ) and 258 female ( 51.1% ) patients . the mean age of patients was 62.9 years ( range , 21 to 83 years ) , with 302 non - smokers ( 59.8% ) and 203 smokers ( 40.2% ) . with respect to tumor pathology , 274 samples ( 54.3% ) were pathologic stage i , 93 ( 18.4% ) were stage ii , 109 ( 21.6% ) were stage iii , and 29 ( 5.7% ) were stage iv . according to the new iaslc / ats / ers adenocarcinoma classification and the 2015 who classification , 298 ( 59.0% ) were acinar , 83 ( 16.4% ) were papillary , 73 ( 14.5% ) were solid , 33 ( 6.5% ) were lepidic , 8 ( 1.6% ) were micropapillary , and 10 ( 2.0% ) were invasive mucinous subtypes . venous invasion , lymphatic invasion , and perineural invasion were observed in 25.7% ( 130/505 ) , 50.1% ( 253/505 ) , and 7.1% ( 36/505 ) of samples , respectively . aqp1 expression was observed in the vascular endothelial cells and the apicolateral surfaces of hyperplastic type ii pneumocytes around tumors . aqp1 was also detected in myoepithelial cells of bronchial glands and red blood cells ( data not shown ) . there was a significant association of aqp1 overexpression with venous invasion ( p=.035 ) and lymphatic invasion ( p=.039 ) . the recurrence rate of patients with aqp1 overexpression was significantly higher than that of patients without aqp1 overexpression ( p=.029 ) . aqp1 overexpression was not associated with higher histological grade ( p=.097 ) , pleural invasion ( p=.131 ) , and other clinicopathological variables or molecular characteristics , such as egfr and kras mutation and alk rearrangement . in total , immunohistochemical analyses of e - cadherin and vimentin were performed for 479 and 471 cases , respectively . loss of e - cadherin expression was observed in 201 of 479 cases ( 42.0% ) , and expression of vimentin was observed in 192 of 471 cases ( 40.8% ) . we compared the association of aqp1 overexpression to expression of e - cadherin or vimentin ( table 2 ) and found that aqp1 overexpression was correlated with loss of e - cadherin expression ( p=.011 ) and expression of vimentin ( p<.001 ) . at the time of analysis , the median pfs was 31.0 months ( range , 1 to 84 months ) and the median os was 39 months ( range , 1 to 84 months ) . during this time , 194 patients ( 38.4% ) suffered tumor recurrence and 90 patients ( 17.8% ) died from cancer . survival analysis using kaplan - meier and cox proportional hazards analyses were performed to evaluate the prognostic impact of aqp1 overexpression . as shown in fig . 2 , kaplan - meier revealed that pfs of patients with aqp1 overexpression was significantly shorter than that of the patients without aqp1 overexpression group ( p=.018 ) . univariate analysis indicated that larger tumor size ( p<.001 ) , higher histological grade ( p=.032 ) , pleural invasion ( p<.001 ) , venous invasion ( p<.001 ) , lymphatic invasion ( p<.001 ) , perineural invasion ( p=.043 ) , ptnm stage ( p<.001 ) , and aqp1 overexpression ( 46.1 months vs. 56.2 months , p=.020 ) were associated with shorter pfs ( fig . multivariate cox regression analysis demonstrated aqp1 overexpression to be an independent factor indicating poor prognosis with regard to pfs in patients with lung adenocarcinoma ( hazard ratio [ hr ] , 1.429 ; 95% confidence interval [ ci ] , 1.033 to 1.977 ; p=.031 ) . larger tumor size ( hr , 1.797 ; 95% ci , 1.336 to 2.418 ; p<.001 ) , pleural invasion ( hr , 1.372 ; 95% ci , 1.007 to 1.871 ; p=.045 ) , lymphatic invasion ( hr , 1.547 ; 95% ci , 1.113 to 2.151 ; p=.009 ) , and ptnm stage ( hr , 2.179 ; 95% ci , 1.586 to 2.995 ; p<.001 ) were also independent prognostic factors associated with shorter pfs in lung adenocarcinoma . for os , larger tumor size ( p<.001 ) , higher histological grade ( p=.021 ) , pleural invasion ( p<.001 ) , venous invasion ( p<.001 ) , lymphatic invasion ( p<.001 ) , perineural invasion ( p=.001 ) , vimentin expression ( p=.045 ) , and ptnm stage ( p<.001 ) reached statistical significance by univariate analysis . aqp1 overexpression was not associated with os ( 63.2 months vs. 70.1 months , p=.237 ) . in multivariate analysis , larger tumor size ( hr , 1.775 ; 95% ci , 1.137 to 2.771 ; p=.012 ) , venous invasion ( hr , 2.129 ; 95% ci , 1.352 to 3.354 ; p=.001 ) , and ptnm stage ( hr , 4.789 ; 95% ci , 3.026 to 7.578 ; p<.001 ) were statistically significant . the tumor specimens in this study were from 505 lung adenocarcinoma patients , consisting of 247 male ( 48.9% ) and 258 female ( 51.1% ) patients . the mean age of patients was 62.9 years ( range , 21 to 83 years ) , with 302 non - smokers ( 59.8% ) and 203 smokers ( 40.2% ) . with respect to tumor pathology , 274 samples ( 54.3% ) were pathologic stage i , 93 ( 18.4% ) were stage ii , 109 ( 21.6% ) were stage iii , and 29 ( 5.7% ) were stage iv . according to the new iaslc / ats / ers adenocarcinoma classification and the 2015 who classification , 298 ( 59.0% ) were acinar , 83 ( 16.4% ) were papillary , 73 ( 14.5% ) were solid , 33 ( 6.5% ) were lepidic , 8 ( 1.6% ) were micropapillary , and 10 ( 2.0% ) were invasive mucinous subtypes . venous invasion , lymphatic invasion , and perineural invasion were observed in 25.7% ( 130/505 ) , 50.1% ( 253/505 ) , and 7.1% ( 36/505 ) of samples , respectively . aqp1 expression was observed in the vascular endothelial cells and the apicolateral surfaces of hyperplastic type ii pneumocytes around tumors . aqp1 was also detected in myoepithelial cells of bronchial glands and red blood cells ( data not shown ) . there was a significant association of aqp1 overexpression with venous invasion ( p=.035 ) and lymphatic invasion ( p=.039 ) . the recurrence rate of patients with aqp1 overexpression was significantly higher than that of patients without aqp1 overexpression ( p=.029 ) . aqp1 overexpression was not associated with higher histological grade ( p=.097 ) , pleural invasion ( p=.131 ) , and other clinicopathological variables or molecular characteristics , such as egfr and kras mutation and alk rearrangement . in total , immunohistochemical analyses of e - cadherin and vimentin were performed for 479 and 471 cases , respectively . loss of e - cadherin expression was observed in 201 of 479 cases ( 42.0% ) , and expression of vimentin was observed in 192 of 471 cases ( 40.8% ) . we compared the association of aqp1 overexpression to expression of e - cadherin or vimentin ( table 2 ) and found that aqp1 overexpression was correlated with loss of e - cadherin expression ( p=.011 ) and expression of vimentin ( p<.001 ) . at the time of analysis , the median pfs was 31.0 months ( range , 1 to 84 months ) and the median os was 39 months ( range , 1 to 84 months ) . during this time , 194 patients ( 38.4% ) suffered tumor recurrence and 90 patients ( 17.8% ) died from cancer . survival analysis using kaplan - meier and cox proportional hazards analyses were performed to evaluate the prognostic impact of aqp1 overexpression . as shown in fig . 2 , kaplan - meier revealed that pfs of patients with aqp1 overexpression was significantly shorter than that of the patients without aqp1 overexpression group ( p=.018 ) . univariate analysis indicated that larger tumor size ( p<.001 ) , higher histological grade ( p=.032 ) , pleural invasion ( p<.001 ) , venous invasion ( p<.001 ) , lymphatic invasion ( p<.001 ) , perineural invasion ( p=.043 ) , ptnm stage ( p<.001 ) , and aqp1 overexpression ( 46.1 months vs. 56.2 months , p=.020 ) were associated with shorter pfs ( fig . multivariate cox regression analysis demonstrated aqp1 overexpression to be an independent factor indicating poor prognosis with regard to pfs in patients with lung adenocarcinoma ( hazard ratio [ hr ] , 1.429 ; 95% confidence interval [ ci ] , 1.033 to 1.977 ; p=.031 ) . larger tumor size ( hr , 1.797 ; 95% ci , 1.336 to 2.418 ; p<.001 ) , pleural invasion ( hr , 1.372 ; 95% ci , 1.007 to 1.871 ; p=.045 ) , lymphatic invasion ( hr , 1.547 ; 95% ci , 1.113 to 2.151 ; p=.009 ) , and ptnm stage ( hr , 2.179 ; 95% ci , 1.586 to 2.995 ; p<.001 ) were also independent prognostic factors associated with shorter pfs in lung adenocarcinoma . for os , larger tumor size ( p<.001 ) , higher histological grade ( p=.021 ) , pleural invasion ( p<.001 ) , venous invasion ( p<.001 ) , lymphatic invasion ( p<.001 ) , perineural invasion ( p=.001 ) , vimentin expression ( p=.045 ) , and ptnm stage ( p<.001 ) reached statistical significance by univariate analysis . aqp1 overexpression was not associated with os ( 63.2 months vs. 70.1 months , p=.237 ) . in multivariate analysis , larger tumor size ( hr , 1.775 ; 95% ci , 1.137 to 2.771 ; p=.012 ) , venous invasion ( hr , 2.129 ; 95% ci , 1.352 to 3.354 ; p=.001 ) , and ptnm stage ( hr , 4.789 ; 95% ci , 3.026 to 7.578 ; p<.001 ) were statistically significant . in the present study , we assessed the expression of aqp1 in 505 lung adenocarcinomas and evaluated the relationship between aqp1 overexpression and various clinicopathological factors and molecular characteristics , as well as the expression of emt - related markers . our study showed that aqp1 overexpression significantly correlated with several aggressive pathological factors and can be used as an independent prognostic factor for pfs in lung adenocarcinoma . recently , the functional roles of aqp1 protein expression have been studied in various cancers . previous studies have demonstrated that aqp1 is upregulated in several cancer tissues in vitro and in vivo , and aqp1 overexpression is associated with poor prognosis . hoque et al .   reported that upregulated aqp1 in lung cancer may play a role in cancer cell proliferation by resisting apoptosis . machida et al .   showed that aqp1 overexpression with a loss of polarization is associated with invasive growth and poor prognosis in lung adenocarcinomas . consistent with previous observations , our study showed that aqp1 overexpression tended to be more frequently observed in the high grade histological subtypes of adenocarcinomas although it was not statistically significant . we also analyzed the association of aqp1 overexpression with emt - related markers ( e - cadherin and vimentin ) . the loss of e - cadherin and increased expression of vimentin , both hallmarks of a mesenchymal phenotype , were frequently observed in tumors with aqp1 overexpression . we hypothesized that aqp1 may participate in tumor progression through emt ( loss of e - cadherin and vimentin expression ) in lung adenocarcinoma . emt is implicated in tumor progression , invasion , metastasis , and poor prognosis in lung cancer . recently aqp1 has been suggested to function as a linker molecule to promote emt , or to stabilize the cytoskeletal complex . similar results were reported in various cancers including colorectal cancer , breast cancer , and brain tumors , which is in line with our results . the exact biological and functional mechanism of aqp1 to promote emt needed to be clarifiied by further studies . of note , some studies demonstrated that tumor tissue showed intratumoral heterogeneity of aqp1 expression in brain tumor . thus , it is necessary to clarify the intratumoral heterogenous distribution of aqp1 overexpression in cancer and its clinical significance . in conclusion , we demonstrated that overexpression of aqp1 was significantly associated with venous invasion , lymphatic invasion , higher pathological stage , and cancer recurrence in lung adenocarcinomas . aqp1 was also deemed an independent marker of poor prognosis with regard to pfs . in particular , increased expression of aqp1 protein was associated with the expression of vimentin and loss of e - cadherin expression , suggesting that aqp1 overexpression may be involved in tumor cell invasion through facilitating emt .