this study included 40 cases of lung adenocarcinoma treated with surgery alone or surgery and postoperative adjuvant therapy at our institute between 2000 and 2011 . selected patients had ais , mia , or small adenocarcinoma ( sad ) and no other malignant tumors within five years of their diagnosis of lung adenocarcinoma . patients were defined as ever - smokers if they had smoked > 20 packs of cigarettes or 12 oz of tobacco in their lifetime , or smoked > 1 cigarette / day or > 1 cigar / wk for 1 year.9 the clinical records reviewed included data on age , gender , smoking history , tumor size , pathologic stage , operative mode , and mortality outcomes . the tumor sections were examined , with special attention to the following details : classification of the pulmonary adenocarcinoma according to the new iaslc / ats / ers classification system , presence or absence of lepidic growth , extent of invasion , predominant invasive pattern including acinar , papillary , solid , or micropapillary growth , presence or absence of solid or micropapillary growth using a 5%-cutoff , presence or absence of lymph node metastasis , lymphatic invasion , and tumor necrosis . in accordance with the updated iaslc / ats / ers classification system , ais cases were selected according to the following criteria : localized sad ( 2 cm ) with growth of neoplastic cells along pre - existing alveolar structures ; lack of stromal , vascular , or pleural invasion ; absence of papillary or micropapillary patterns ; and absence of intra - alveolar tumor cells . tumors were subclassified as mia in cases with a small solitary adenocarcinoma ( 2 cm ) with a predominantly lepidic pattern and 5 mm invasion in the greatest dimension of any one focus . the invasive component to be measured in mia was defined as follows : histological subtypes other than a lepidic pattern ( i.e. , acinar , papillary , micropapillary , or solid ) or tumor cells infiltrating myofibroblastic stroma . the invasive component was measured morphometrically and a 5-mm cutoff was used to distinguish mia from lepidic predominant adenocarcinoma ( lpa ) . for cases that contained multiple tumor foci , mia was excluded if the tumor invaded the lymphatics , blood vessels , pleura , or contained tumor necrosis . lpa and non - lepidic adenocarcinoma with > 5 mm and 2 cm invasion in diameter were classified as sad and were divided further into acinar , papillary , and micropapillary based on their predominant invasive pattern . forty cases of lung ais and adenocarcinoma measuring 2 cm in its widest dimension were analyzed for nkd1 expression after selecting one representative section of tumor from each case . for immunohistochemistry , 4 m - thick sections were deparaffinized using immunoautostainer ( leica bond - max , leica , newcastle upon tyne , uk ) hydrated through graded alcohols to water , and washed in distilled water . antigen retrieval was performed by immersing slides in citrate buffer ( ph 6.0 ) , heating for 20 minutes , and cooling for 10 minutes . after washing in distilled water , endogenous peroxidase activity slides were then washed in distilled water , placed in tris - buffered saline , incubated for 30 minutes with monoclonal antibody to nkd1 ( epitomics , burlingame , ca , usa ) at a 1:400 dilution , and then washed in buffer . slides were washed , incubated with polymers ( bond polymer refine kit , leica ) for 30 minutes , washed in buffer again , and counterstained with hematoxylin . the expression of nkd1 was classified into five groups according to percentage of positively stained cells : 0 , negative ; 1 , 1 - 25% ; 2 , 26 - 50% ; 3 , 51 - 75% ; 4 , 76% . the staining intensity was evaluated as follows : 0 , negative ; 1 , weak ; 2 , moderate ; and 3 , strong . cases with faintly stained cytoplasms were categorized as " weak , " whereas deeply stained were considered to be " strong , " and intermediate ones " moderate . " the expression of nkd1 in normal lung parenchyma was evaluated after randomly selecting ten cases with normal lung tissue with at least 2 cm distance from the tumor . in a normal lung , alveolar pneumocytes were weakly stained in the cytoplasm , whereas bronchial epithelium were weakly to moderately stained in the cytoplasm , especially in the apical portions . scores from normal lung tissue ranged from 3 to 6 ( mean , 4.3 ) as indicated in fig . there were two cases with a score of 3 , four cases with a score 4 , five cases with a score 5 , and one case with a score 6 . on the basis of these results , a score of 3 or less was considered to be " reduced expression , " 4 or 5 were " normal expression , " and scores of 6 or more were regarded as " overexpression . " the overall survival rate was evaluated using the kaplan - meier method , and statistical differences in survival times were determined using log - rank testing . this study included 40 cases of lung adenocarcinoma treated with surgery alone or surgery and postoperative adjuvant therapy at our institute between 2000 and 2011 . selected patients had ais , mia , or small adenocarcinoma ( sad ) and no other malignant tumors within five years of their diagnosis of lung adenocarcinoma . patients were defined as ever - smokers if they had smoked > 20 packs of cigarettes or 12 oz of tobacco in their lifetime , or smoked > 1 cigarette / day or > 1 cigar / wk for 1 year.9 the clinical records reviewed included data on age , gender , smoking history , tumor size , pathologic stage , operative mode , and mortality outcomes . the tumor sections were examined , with special attention to the following details : classification of the pulmonary adenocarcinoma according to the new iaslc / ats / ers classification system , presence or absence of lepidic growth , extent of invasion , predominant invasive pattern including acinar , papillary , solid , or micropapillary growth , presence or absence of solid or micropapillary growth using a 5%-cutoff , presence or absence of lymph node metastasis , lymphatic invasion , and tumor necrosis . in accordance with the updated iaslc / ats / ers classification system , ais cases were selected according to the following criteria : localized sad ( 2 cm ) with growth of neoplastic cells along pre - existing alveolar structures ; lack of stromal , vascular , or pleural invasion ; absence of papillary or micropapillary patterns ; and absence of intra - alveolar tumor cells . tumors were subclassified as mia in cases with a small solitary adenocarcinoma ( 2 cm ) with a predominantly lepidic pattern and 5 mm invasion in the greatest dimension of any one focus . the invasive component to be measured in mia was defined as follows : histological subtypes other than a lepidic pattern ( i.e. , acinar , papillary , micropapillary , or solid ) or tumor cells infiltrating myofibroblastic stroma . the invasive component was measured morphometrically and a 5-mm cutoff was used to distinguish mia from lepidic predominant adenocarcinoma ( lpa ) . for cases that contained multiple tumor foci , mia was excluded if the tumor invaded the lymphatics , blood vessels , pleura , or contained tumor necrosis . lpa and non - lepidic adenocarcinoma with > 5 mm and 2 cm invasion in diameter were classified as sad and were divided further into acinar , papillary , and micropapillary based on their predominant invasive pattern . forty cases of lung ais and adenocarcinoma measuring 2 cm in its widest dimension were analyzed for nkd1 expression after selecting one representative section of tumor from each case . for immunohistochemistry , 4 m - thick sections were deparaffinized using immunoautostainer ( leica bond - max , leica , newcastle upon tyne , uk ) hydrated through graded alcohols to water , and washed in distilled water . antigen retrieval was performed by immersing slides in citrate buffer ( ph 6.0 ) , heating for 20 minutes , and cooling for 10 minutes . after washing in distilled water , endogenous peroxidase activity slides were then washed in distilled water , placed in tris - buffered saline , incubated for 30 minutes with monoclonal antibody to nkd1 ( epitomics , burlingame , ca , usa ) at a 1:400 dilution , and then washed in buffer . slides were washed , incubated with polymers ( bond polymer refine kit , leica ) for 30 minutes , washed in buffer again , and counterstained with hematoxylin . the expression of nkd1 was classified into five groups according to percentage of positively stained cells : 0 , negative ; 1 , 1 - 25% ; 2 , 26 - 50% ; 3 , 51 - 75% ; 4 , 76% . the staining intensity was evaluated as follows : 0 , negative ; 1 , weak ; 2 , moderate ; and 3 , strong . cases with faintly stained cytoplasms were categorized as " weak , " whereas deeply stained were considered to be " strong , " and intermediate ones " moderate . " the expression of nkd1 in normal lung parenchyma was evaluated after randomly selecting ten cases with normal lung tissue with at least 2 cm distance from the tumor . in a normal lung , alveolar pneumocytes were weakly stained in the cytoplasm , whereas bronchial epithelium were weakly to moderately stained in the cytoplasm , especially in the apical portions . scores from normal lung tissue ranged from 3 to 6 ( mean , 4.3 ) as indicated in fig . 1 . there were two cases with a score of 3 , four cases with a score 4 , five cases with a score 5 , and one case with a score 6 . on the basis of these results , a score of 3 or less was considered to be " reduced expression , " 4 or 5 were " normal expression , " and scores of 6 or more were regarded as " overexpression . " the overall survival rate was evaluated using the kaplan - meier method , and statistical differences in survival times were determined using log - rank testing . a p - value of < 0.05 was considered statistically significant . our patient population was composed of 17 men and 23 women aged 35 - 79 years ( mean , 56.9 years ) . smoking history was available in all 40 patients , with 11 ( 27.5% ) classified as ever - smokers and 29 ( 72.5% ) as never - smokers . the subtypes of lung tumor were as follows : 5 ( 12.5% ) ais , 8 ( 20.0% ) mia , and 27 ( 67.5% ) sad . these various clinicopathologic features were compared according to nkd1 expression ( tables 2 , 3 ) . among the 40 samples , 2a , b ) , normal expression in 15 ( 37.5% ) cases ( fig . 2c , d ) , and overexpression in 17 ( 42.5% ) cases ( fig . 2e , f ) . overexpression of nkd1 was closely related to the predominant papillary pattern , showing increased expression in all cases of lung adenocarcinoma with papillary pattern ( p=0.026 ) . among five samples of ais staged as tis , four cases showed normal expression ( 80% ) and one case showed overexpression ( 20% ) with no statistically significant differences ( p=0.189 ) . nkd1-reduced expression was observed in 2 out of 8 mia cases ( 25% ) and in 6 out of 27 sad cases ( 22.2% ) , while no cases of ais had nkd1-reduced expression ( p=0.189 ) . the rate of normal or increased nkd1 expression in adenocarcinoma with lepidic predominant growth ( 11/13 , 84.7% ) was higher than that without lepidic predominant growth ( 21/27 , 77.8% ) , but this was not a statistically significant difference ( p=0.349 ) . the rate of reduced nkd1 expression in adenocarcinoma with solid predominant growth ( 1/4 , 25% ) was higher than that without solid predominant growth ( 7/36 , 19.4% ) , with no statistical significance ( p=0.821 ) . follow - up data were available for 40 patients and the mean follow - up period was 33.7 months ranging from 1 to 156 months . thirty - six patients were alive and free of disease at the mean follow - up period of 35.6 months . one patient died 14 days after surgery and was excluded from the analysis . according to our univariate analysis , factors that statistically influenced survival included presence of lymph node metastasis ( p=0.001 ) and presence of micropapillary pattern ( p=0.001 ) as indicated in table 4 . regarding histologic subtypes , all patients with ais and mia were alive and free of disease , while 3 out of 27 sad patients had poor prognosis and died from their disease ( ais vs sad , p=0.453 ; mia vs sad , p=0.296 ) ( fig . expected survival time decreased as expression of nkd1 became overexpressed , which was the opposite effect to what had been expected , however there was no statistical significance ( reduced vs normal , p=0.763 ; reduced vs overexpressed , p=0.599 ; normal vs overexpressed , p=0.550 ) . our patient population was composed of 17 men and 23 women aged 35 - 79 years ( mean , 56.9 years ) . smoking history was available in all 40 patients , with 11 ( 27.5% ) classified as ever - smokers and 29 ( 72.5% ) as never - smokers . the subtypes of lung tumor were as follows : 5 ( 12.5% ) ais , 8 ( 20.0% ) mia , and 27 ( 67.5% ) sad . these various clinicopathologic features were compared according to nkd1 expression ( tables 2 , 3 ) . among the 40 samples , 2a , b ) , normal expression in 15 ( 37.5% ) cases ( fig . 2c , d ) , and overexpression in 17 ( 42.5% ) cases ( fig . 2e , f ) . overexpression of nkd1 was closely related to the predominant papillary pattern , showing increased expression in all cases of lung adenocarcinoma with papillary pattern ( p=0.026 ) . among five samples of ais staged as tis , four cases showed normal expression ( 80% ) and one case showed overexpression ( 20% ) with no statistically significant differences ( p=0.189 ) . nkd1-reduced expression was observed in 2 out of 8 mia cases ( 25% ) and in 6 out of 27 sad cases ( 22.2% ) , while no cases of ais had nkd1-reduced expression ( p=0.189 ) . the rate of normal or increased nkd1 expression in adenocarcinoma with lepidic predominant growth ( 11/13 , 84.7% ) was higher than that without lepidic predominant growth ( 21/27 , 77.8% ) , but this was not a statistically significant difference ( p=0.349 ) . the rate of reduced nkd1 expression in adenocarcinoma with solid predominant growth ( 1/4 , 25% ) was higher than that without solid predominant growth ( 7/36 , 19.4% ) , with no statistical significance ( p=0.821 ) . follow - up data were available for 40 patients and the mean follow - up period was 33.7 months ranging from 1 to 156 months . thirty - six patients were alive and free of disease at the mean follow - up period of 35.6 months . one patient died 14 days after surgery and was excluded from the analysis . according to our univariate analysis , factors that statistically influenced survival included presence of lymph node metastasis ( p=0.001 ) and presence of micropapillary pattern ( p=0.001 ) as indicated in table 4 . regarding histologic subtypes , all patients with ais and mia were alive and free of disease , while 3 out of 27 sad patients had poor prognosis and died from their disease ( ais vs sad , p=0.453 ; mia vs sad , p=0.296 ) ( fig . expected survival time decreased as expression of nkd1 became overexpressed , which was the opposite effect to what had been expected , however there was no statistical significance ( reduced vs normal , p=0.763 ; reduced vs overexpressed , p=0.599 ; normal vs overexpressed , p=0.550 ) . noguchi et al.10 analyzed the histology of small lung adenocarcinoma and classified it according to the presence or absence of bac components and other histologic characteristics . this seminal study brought to attention the importance of bac and its association with excellent prognosis , including 100% 5-year survival . subsequent studies11 - 16 elucidated various histologic features of prognostic significance in lung adenocarcinomas with bac components , including size of scar , percentage of lepidic growth , percentage of papillary growth , vascular invasion , and size or pattern of invasion . based on these studies , new concepts were introduced , such as including the subgroups of ais and mia , according to the new iaslc / ats / ers classification system . in accordance with earlier studies , our results suggested better tumor behavior in mia ( 5 mm ) compared to sad , which supports distinguishing these classes of tumors from stage t1a cancers . in this study , in contrast , among 27 patients with sad , 2 ( 7.4% ) had lymph node metastasis , and 3 ( 11.1% ) died of disease . the overall survival curve ( fig . 3 ) also identified a relationship among ais , mia , and sad that was not statistically significant . there are several limitations to this study including its small sample size and short follow - up period , which likely contributed to our statistically insignificant results . as seen in fig . 3 , patients of ais and mia had shorter follow - up than those with sad . this limited data likely reflects a recent increase in the early detection of small lung adenocarcinoma , owing to advances in imaging technique . we reviewed all surgically resected lung adenocarcinomas less than 2 cm in size since 2000 , as well as cases of ais and mia were observed in our institute only after 2007 . there is still a need for future studies to validate these findings based on standardized pathologic criteria . although there was not a large enough sample size to conduct a powerful statistical analysis of mia cases , our univariate analysis demonstrated , as expected , that lymph node metastasis and micropapillary patterns were indeed unfavorable prognostic markers ( table 4 ) . the aforementioned variables were mostly observed in cases of sad , supporting the idea that among t1a lung adenocarcinomas , mia may be distinguished from sad with obviously different outcomes . increasing reports of nkd1 have demonstrated up - regulation of nkd1 mrna levels in lung cancer , as well as colorectal adenomas and hepatoblastomas.8,17,18 conversely , nkd1 protein expression was down - regulated in some gastric cancers and nsclc . zhang et al.8 reported nkd1 protein in nsclc . in their study , 100 cases of nsclc , including 33 cases of squamous cell carcinoma , and 76 cases of adenocarcinoma , underwent immunohistochemical staining with nkd1 polyclonal antibody ; their results demonstrated reduced nkd1 protein levels with elevated nkd1 mrna associated with poor differentiation , high ptnm stage , lymph node metastasis , and poor prognosis . the authors hypothesized that a post - translational modification of nkd1 or protein degradation may have been involved in the discordance between reduced nkd1 expression and up - regulated nkd1 mrna . these findings also suggested that nkd1 depletion could up - regulate dvl-1 and -catenin protein , enhancing the invasive ability of lung cancer cells . a statistically significant association was found between reduced nkd1 expression and nodal metastasis ( p=0.003 ) , which was observed in previous studies , and further implies an unfavorable prognosis in lung adenocarcinoma with reduced nkd1 expression . this discordant result might have been due to a limited experimental group in this study , which included ptis- and pt1a - staged lung adenocarcinoma . moreover , a monoclonal nkd1 antibody was applied in this study , whereas polyclonal nkd1 antibodies were used in previous studies . nkd1 was expressed differently in the papillary subtype compared to non - papillary subtypes , with several possible implications . hypotheses include that nkd1 protein in the papillary subtype may be less fragile than in the non - papillary subtype , making it less vulnerable to degradation or post - translational modification compared to the non - papillary subtype . overexpression of nkd1 in the papillary subtype suggests that nkd1 may have a specific role in the pathogenesis of distinct histologic subtypes of lung adenocarcinoma , and may specifically drive different pathways , which remain unidentified . pulmonary adenocarcinoma is histologically heterogeneous and currently , there have been several reports of different molecular or immunohistochemical expressions of distinct histologic subtypes in lung adenocarcinoma.19,20 to the best of our knowledge , this is the first study to investigate the relationship between nkd1 expression and clinicopathological features , with special attention to the histologic pattern in small lung adenocarcinoma . though there were several reports on the therapeutic implications of ais , mia , and sad,21 - 23 the appropriateness of limited resection remains unclear and should be subject to further clinical trials . ongoing clinical trials should define selection criteria for limited resection in patients with small lung adenocarcinoma , which will encompass not only histologic parameters but also immunohistochemical and molecular aspects of the cancer . in conclusion however , our study was limited by statistically insignificant results likely due to our small sample size and short - term follow - up . future studies should validate our results , as well as try to verify a favorable prognosis with mia and to identify selection criteria for limited surgical resection . based on previous reports investigating the invasive ability of nkd1 in nsclc , different expressions of nkd1 protein in small lung adenocarcinoma suggest its potential role in invasion assessment . though having failed to support those speculations , this study demonstrated that down - regulated nkd1 expression was closely associated with nodal metastasis and altered expression was associated with the papillary subtype , suggesting that nkd1 is indeed a likely unfavorable marker .