pulmonary adenocarcinoma is a major histological subtype of lung cancer , and has a constant tendency to increase in japan , irrespectively of sex . its incidence rates were 43% and 67% of all histological types of japanese lung cancer cases during 19992003 in males and females , respectively.1 over the last decade , in contrast to squamous cell carcinoma , treatment for adenocarcinoma has developed dramatically . this histology has benefited from a new cytotoxic antitumor drug , pemetrexed,2 and a molecularly targeted antibody bevacizumab.3 furthermore , adenocarcinoma has been divided into two subsets according to genetic information : adenocarcinoma harboring a driver mutation , either positive egfr mutation4 or alk rearrangement;5 and adenocarcinoma without these driver mutations . nowadays , treatment strategies differ markedly between these two subsets . for the former subset , specific tyrosine - kinase inhibitors are indispensable . on the other hand , for the latter subset platinum - based combination regimens with or without bevacizumab are recommended as the first - line treatment . however , almost all patients experience progression during or after first - line chemotherapy . some of them require salvage chemotherapy . some survey studies have revealed a trend of patients with advanced non - small - cell lung cancer who have received first- and later - line chemotherapy.615 however , there is no study that has focused on patients with egfr wild - type adenocarcinoma and followed their course of chemotherapy . our retrospective study for adenocarcinoma with wild - type egfr aimed to investigate 1 ) what the rate of patients who had received first- , second , or third - line chemotherapy was and 2 ) who benefited from chemotherapy . the inclusion criteria were : 1 ) histologically or cytologically diagnosed with pulmonary adenocarcinoma ; 2 ) stage iiib or iv , defined by the seventh tnm ( tumor , node , metastasis ) classification of lung cancer by the union for international cancer control16 ( staging by sixth edition of the uicc classification was reclassified according to seventh edition ) ; ( 3 ) diagnosed between june 2007 and march 2015 at our institution ; ( 4 ) egfr wild - type status examined by lsi medience corporation ( tokyo , japan ) using the peptide nucleic acid - locked polymerase chain - reaction clamp method.17 the exclusion criteria were : 1 ) patients who were diagnosed at our hospital , but thereafter transferred to other hospitals for aggressive treatment ; 2 ) diagnosis and introduction of any aggressive treatment were performed at another hospital , but thereafter transferred to our hospital for later - line treatment ; 3 ) adenocarcinoma combined with other histological types ; 4 ) immunohistochemically positive alk gene rearrangement ; 5 ) egfr mutations were not examined . in japan , fluorescence in situ hybridization and immunohistochemistry for alk gene rearrangement were approved in april 2012 and june 2014 , respectively . 1 ) in order to investigate predictive factors influencing introduction of systemic chemotherapy , we compared patients who had received chemotherapy ( chemotherapy group ) with those who had not received chemotherapy ( nonchemotherapy group ) . 2 ) to find prognostic factors influencing survival after each line of chemotherapy , we extracted three cohorts of patients who had started first- , second- , or third - line regimens between june 2007 and november 2015 . we excluded chemotherapies that started after december 2015 . during the study period in japan , erlotinib , pemetrexed , bevacizumab , nab - paclitaxel , and nivolumab were approved for non - small - cell lung cancer in october 2007 , may 2009 , november 2009 , february 2013 , and december 2015 , respectively . patients background data , laboratory data , criteria and definitions of overall response rate ( rr ) , overall survival ( os ) , and progression - free survival ( pfs ) in this study followed those of our previous studies.18,19 in this study , we added creatinine clearance estimated by cockcroft gault formula as an explanatory variable at diagnosis and at the start of each chemotherapy . we added 0.2 mg / dl to serum creatinine concentrations measured by the enzymatic method , in order to adjust the difference between creatinine values measured by the jaffe method and the enzymatic method.20 in principle , laboratory data comparing between the chemotherapy and nonchemotherapy groups were obtained on the last day before the examination leading to the confirmed diagnosis of malignancy . however , alkaline phosphatase ( alp ) values were not measured before the examination in three and one cases in the chemotherapy and nonchemotherapy groups , respectively . instead , we used the data on the nearest day after the examination in those four cases . height could not be measured in three patients in the nonchemotherapy group , because they could not stand by themselves . the osaka police hospital ethics committee approved this study ( number 501 ) and waived the requirement for informed consent , because our data were retrospective and deidentified . continuous , discrete , and categorical variables are expressed as mean  standard deviation , median ( range ) , and frequency , respectively . normality of distribution and homogeneity of variances were assessed by shapiro  wilk and f - tests , respectively . comparing relative frequencies , discrete variables , and normally distributed continuous variables between two groups , we used c or fisher s exact test , mann  whitney u test , and student s t - test , respectively . thereafter , all variables with a p - value < 0.2 were included in the subsequent univariate logistic regression analysis . the laboratory data were divided by the optimal cutoff values and transformed into dichotomous variables . for receiver - operating characteristic curves and areas under the curve , cutoff points were decided as the value that gave maximal joint sensitivity and specificity . logistic regression analysis and cox proportional - hazard analysis were used to find factors influencing outcome variables , introduction of chemotherapy , and survival after chemotherapy , respectively . all variables with a p - value < 0.1 in univariate analysis were included in the subsequent multivariate analysis . when a moderate - to - strong correlation ( pearson s correlation coefficient , r0.4 ) was observed between two laboratory data and a risk of multicollinearity was a concern , we arbitrarily excluded either of the coefficients from candidate explanatory variables in the multivariate analysis . backward - stepwise selection based on p - value was used to select variables that were entered into the multivariate model . these results were expressed as odds ratios ( ors ) or hazard ratios ( hrs ) and 95% confidence intervals ( cis ) . all statistical analyses were performed with ezr ( saitama medical center , jichi medical university , saitama , japan ) , which is a graphical user interface for r ( r foundation for statistical computing , vienna , austria).21 the inclusion criteria were : 1 ) histologically or cytologically diagnosed with pulmonary adenocarcinoma ; 2 ) stage iiib or iv , defined by the seventh tnm ( tumor , node , metastasis ) classification of lung cancer by the union for international cancer control16 ( staging by sixth edition of the uicc classification was reclassified according to seventh edition ) ; ( 3 ) diagnosed between june 2007 and march 2015 at our institution ; ( 4 ) egfr wild - type status examined by lsi medience corporation ( tokyo , japan ) using the peptide nucleic acid - locked polymerase chain - reaction clamp method.17 the exclusion criteria were : 1 ) patients who were diagnosed at our hospital , but thereafter transferred to other hospitals for aggressive treatment ; 2 ) diagnosis and introduction of any aggressive treatment were performed at another hospital , but thereafter transferred to our hospital for later - line treatment ; 3 ) adenocarcinoma combined with other histological types ; 4 ) immunohistochemically positive alk gene rearrangement ; 5 ) egfr mutations were not examined . in japan , fluorescence in situ hybridization and immunohistochemistry for alk gene rearrangement were approved in april 2012 and june 2014 , respectively . 1 ) in order to investigate predictive factors influencing introduction of systemic chemotherapy , we compared patients who had received chemotherapy ( chemotherapy group ) with those who had not received chemotherapy ( nonchemotherapy group ) . 2 ) to find prognostic factors influencing survival after each line of chemotherapy , we extracted three cohorts of patients who had started first- , second- , or third - line regimens between june 2007 and november 2015 . we excluded chemotherapies that started after december 2015 . during the study period in japan , erlotinib , pemetrexed , bevacizumab , nab - paclitaxel , and nivolumab were approved for non - small - cell lung cancer in october 2007 , may 2009 , november 2009 , february 2013 , and december 2015 , respectively . patients background data , laboratory data , criteria and definitions of overall response rate ( rr ) , overall survival ( os ) , and progression - free survival ( pfs ) in this study followed those of our previous studies.18,19 in this study , we added creatinine clearance estimated by cockcroft gault formula as an explanatory variable at diagnosis and at the start of each chemotherapy . we added 0.2 mg / dl to serum creatinine concentrations measured by the enzymatic method , in order to adjust the difference between creatinine values measured by the jaffe method and the enzymatic method.20 in principle , laboratory data comparing between the chemotherapy and nonchemotherapy groups were obtained on the last day before the examination leading to the confirmed diagnosis of malignancy . however , alkaline phosphatase ( alp ) values were not measured before the examination in three and one cases in the chemotherapy and nonchemotherapy groups , respectively . instead , we used the data on the nearest day after the examination in those four cases . height could not be measured in three patients in the nonchemotherapy group , because they could not stand by themselves . the osaka police hospital ethics committee approved this study ( number 501 ) and waived the requirement for informed consent , because our data were retrospective and deidentified . continuous , discrete , and categorical variables are expressed as mean  standard deviation , median ( range ) , and frequency , respectively . normality of distribution and homogeneity of variances were assessed by shapiro  wilk and f - tests , respectively . comparing relative frequencies , discrete variables , and normally distributed continuous variables between two groups , we used c or fisher s exact test , mann  whitney u test , and student s t - test , respectively . thereafter , all variables with a p - value < 0.2 were included in the subsequent univariate logistic regression analysis . the laboratory data were divided by the optimal cutoff values and transformed into dichotomous variables . for receiver - operating characteristic curves and areas under the curve , cutoff points were decided as the value that gave maximal joint sensitivity and specificity . logistic regression analysis and cox proportional - hazard analysis were used to find factors influencing outcome variables , introduction of chemotherapy , and survival after chemotherapy , respectively . all variables with a p - value < 0.1 in univariate analysis were included in the subsequent multivariate analysis . when a moderate - to - strong correlation ( pearson s correlation coefficient , r0.4 ) was observed between two laboratory data and a risk of multicollinearity was a concern , we arbitrarily excluded either of the coefficients from candidate explanatory variables in the multivariate analysis . backward - stepwise selection based on p - value was used to select variables that were entered into the multivariate model . these results were expressed as odds ratios ( ors ) or hazard ratios ( hrs ) and 95% confidence intervals ( cis ) . all statistical analyses were performed with ezr ( saitama medical center , jichi medical university , saitama , japan ) , which is a graphical user interface for r ( r foundation for statistical computing , vienna , austria).21 among 194 patients who met the inclusion criteria , 148 patients were able to receive chemotherapy ( chemotherapy group ) , but 46 could not ( nonchemotherapy group ) . on the other hand , 62 patients were excluded from analysis , because their egfr - mutation status was unknown . among these 62 patients , 19 ( 31% ) received chemotherapy . alk - immunohistochemistry screening was performed in 22 patients ( 48% ) of the nonchemotherapy group and 59 ( 40% ) of the chemotherapy group . in the chemotherapy group , 89 and 48 patients proceeded to second- and third - line chemotherapy , respectively ( figure s1 ) . patients in the chemotherapy group were younger ( 679.5 vs 73.99.7 years , p<0.01 ) , included a lower proportion of stage iv ( p<0.01 ) , kept better eastern cooperative oncology group ( ecog ) performance status ( ps ) ( p<0.01 ) , and survived longer ( median 404 vs 87 days , p<0.01 ) ( table 1 ) . as for laboratory data , there were significant differences in absolute lymphocyte count ( 1,721570 vs 1,332562 cells/l , p<0.01 ) , hemoglobin ( 13.51.6 vs 11.91.8 g / dl , p<0.01 ) , creatinine clearance ( 59.418.8 vs 46.321.6 ml / min , p<0.01 ) , serum sodium concentration ( 1393.3 vs 137.83.9 , p=0.04 ) , alp ( 297.6158.9 vs 351.9184.7 , p=0.04 ) , and crp ( 2.13.4 vs 4.83.5 , p<0.01 ) ( table 1 ) . age 75 years , stage iv , ecog ps 2 , neutrophil count 8,356 cells/l , lymphocyte count < 1,414 cells/l , hemoglobin < 13.2g / dl , sodium < 139 meq / l , creatinine clearance < 50.4 ml / min , alp 293 iu / l , and crp 0.53 mg / dl  while multivariate analysis extracted the four variables ecog ps 2 ( or 0.08 , 95% ci : 0.030.21 ; p<0.01 ) , hemoglobin < 13.2 g / dl ( or 0.13 , 95% ci : 0.050.36 ; p<0.01 ) , creatinine clearance < 50.4 ml / min ( or 0.36 , 95% ci : 0.150.9 ; p=0.03 ) , and crp 0.53 mg / dl ( or 0.33 , 95% ci : 0.120.88 ; p=0.03 ) ( table 2 ) . table 3 shows pretreatment characteristics and laboratory data at the start of first- , second- , and third - line chemotherapy , respectively . carboplatin - based combination regimens were most frequently used in the first - line setting , while nonplatinum monotherapy regimens were used in the second- and third - line settings ( table s2 ) . rr , disease - control rate , and pfs gradually diminished every line ( table s3 ) . as factors predicting os from the start of first- , second- , and third - line chemotherapy , univariate cox - hazard analysis detected that the variables female , ecog ps 2 , neutrophil count , lymphocyte count , monocyte count , creatinine clearance , sodium concentration , and crp in first - line therapy ( table 4 ) ; age 75 years , male , ecog ps 2 , neutrophil count , lymphocyte count , monocyte count , hemoglobin , red cell distribution width ( rdw ) , platelet count , creatinine clearance , sodium concentration , ldh , alp , crp , and interval between first and second lines in second - line therapy ( table 5 ) ; and ecog ps 2 , neutrophil count , lymphocyte count , monocyte count , sodium concentration , ldh , alp , crp , and interval between first and third lines in third - line therapy ( table 6 ) . subsequent multivariate cox - hazard analysis extracted age 75 years ( hr 1.56 , 95% ci : 1.012.39 ; p=0.04 ) , ecog ps 2 ( hr 3.22 , 95% ci : 1.985.24 ; p<0.01 ) , lymphocyte count ( hr 0.65 , 95% ci : 0.470.89 ; p<0.01 ) , and crp ( hr 1.07 , 95% ci : 1.021.11 ; p<0.01 ) in first - line therapy ( table 4 ) ; female ( hr 0.43 , 95% ci : 0.240.77 ; p<0.01 ) , neutrophil count ( hr 1.26 , 95% ci : 1.131.4 ; p<0.01 ) , lymphocyte count ( hr 0.42 , 95% ci : 0.260.67 ; p<0.01 ) , creatinine clearance ( hr 0.81 , 95% ci : 0.680.95 ; p<0.01 ) , sodium concentration ( hr 0.24 , 95% ci : 0.090.6 ; p<0.01 ) , and interval between first- and second - line chemotherapy ( hr 0.88 , 95% ci : 0.80.95 ; p<0.01 ) in second - line therapy ( table 5 ) ; age 75 years ( hr 3.33 , 95% ci : 1.38.53 ; p=0.01 ) , body mass index ( hr 3.26 , 95% ci : 1.278.36 ; p=0.01 ) , neutrophil count ( hr 1.26 , 95% ci : 1.061.51 ; p=0.01 ) , lymphocyte count ( hr 0.42 , 95% ci : 0.190.92 ; p=0.03 ) , sodium concentration ( hr 0.06 , 95% ci : 0.010.27 ; p<0.01 ) , ldh ( hr 1.4 , 95% ci : 1.031.90 ; p=0.03 ) , and crp ( hr 1.45 , 95% ci : 1.211.73 ; p<0.01 ) in third - line therapy ( table 6 ) . this retrospective study is the first to clarify the trajectory of patients with advanced adenocarcinoma with egfr wild - type status . we found several factors leading to introduction of chemotherapy and longer survival after first- , second- , and third - line chemotherapy . our study revealed a flow of patients with adenocarcinoma with egfr wild - type status . 1 ) 76% of patients who had been diagnosed with adenocarcinoma with egfr wild - type status received chemotherapy . this proportion was higher than 67% , the proportion of patients with squamous cell carcinoma during the same study period in our hospital.19 however , we are afraid that this proportion might be overestimated . we did not examine egfr - mutation status intentionally in some patients with poor general condition and/or severe complications at diagnosis . therefore , our non - chemotherapy group excluded some patients who appeared unfit for both aggressive treatment and tyrosine - kinase inhibitors at diagnosis . 2 ) 61% and 34% of patients who received first - line chemotherapy proceeded to second- and third - line chemotherapy , respectively . when calculating these proportions , we excluded three and three patients who remained not to progress after first- and second - line chemotherapy , respectively . these proportions were consistent with 65% and 34% , those of patients with advanced pulmonary squamous cell carcinoma in our previous study.19 therefore , the receipt rate of salvage chemotherapy was not affected by histological type . oncologists should keep in mind that approximately a third of our patients may drop out of chemotherapy during or after the ongoing line . therefore , we should select the best regimen at the upcoming line for each patient , rather than the best sequence of later - line regimens . we detected some predictive markers for unlikely introduction of chemotherapy and survival benefit of each line of chemotherapy . 1 ) patients with poor ps , low hemoglobin concentration , low creatinine clearance , and higher crp value were unlikely to receive chemotherapy . irrespective of histology , ps was a common predictive factor.18,19 low hemoglobin was also common with squamous cell carcinoma,19 while this study additionally detected low creatinine clearance and crp as influential factors . 2 ) in our previous study of squamous cell carcinoma,19 ps was a common predictive factor at any time of the chemotherapeutic course . on the other hand , in this study , absolute lymphocyte count was a common predictive factor from first- to third - line chemotherapy . ratios of neutrophils2227 or monocytes to lymphocytes have been reported to be predictive markers for os in advanced lung cancers . we did not assess ratios of these leukocyte differentiations as predictive markers in this study . as described earlier , we did not examine egfr - mutation status in all patients with adenocarcinoma . we did not evaluate variables related to nutrition status , such as serum albumin concentration , because we did not measure these data routinely . our analyses focused exclusively on medical and physical conditions . whether or not to start chemotherapy might depend not only on medical conditions but also on many other issues . approximately 76% of patients with adenocarcinoma with egfr wild - type status were treated with first - line chemotherapy . of those patients , 61% and patients with poor ps , low hemoglobin concentration , low creatinine clearance , and higher crp value tended not to receive chemotherapy . absolute lymphocyte count was a common predictive factor from first- to third - line chemotherapy . receiver - operating characteristic curve analysis for optimal cutoff values of laboratory data at diagnosis for the introduction of chemotherapy abbreviations : alp , alkaline phosphatase ; auc , area under the curve ; ccr , creatinine clearance ; ci , confidence interval . abbreviation : tki , tyrosine - kinase inhibitor . treatment response and discontinuation from initiation of first- , second- , or third - line chemotherapy . abbreviation : ci , confidence interval .
backgroundpulmonary adenocarcinoma , recently benefited by new cytotoxic and molecularly targeted drugs , has been classified by driver mutations , such as egfr mutations . 
 the aim of this study was to research the proportions of patients treated with first- to third - line chemotherapy and to find influential factors for the introduction of chemotherapy and survival benefit from chemotherapy.materials and methodsdata were collected retrospectively on patients who met the following criteria : adenocarcinoma , diagnosed between june 2007 and march 2015 at our hospital , stage iiib or iv , and egfr wild type . a nonchemotherapy group of patients who did not receive chemotherapy was compared with a chemotherapy group of patients who received it . 
 the patients who had received first- to third - line chemotherapy between june 2007 and november 2015 at our hospital were also analyzed.resultsduring the study period , 46 patients did not receive chemotherapy , while 148 , 89 , and 48 received first- , second- and third - line chemotherapy , respectively . as predictive factors for unlikely chemotherapy , 
 multivariate logistic analysis detected eastern cooperative oncology group ( ecog ) performance status ( ps ) 2 , hemoglobin < 
 13.2 g / dl , creatinine clearance ( ccr ) < 50.4 ml / min , and crp 0.53 mg / dl . as factors predicting shorter survival after chemotherapy , multivariate cox proportional - hazard analyses detected age 75 years , ecog ps 2 , lower lymphocyte counts , and higher crp for the first line ; female , higher neutrophil counts , lower lymphocyte counts , reduced ccr , hyponatremia , and shorter interval between first- and second - line chemotherapy for the second line ; and age 75 years , body mass index < 18.5 kg / m2 , higher neutrophil counts , lower lymphocyte counts , hyponatremia , higher lactate dehydrogenase , and higher crp for the third line.conclusionapproximately 76% of patients were treated with first - line chemotherapy . 
 of those patients , 61% and 34% proceeded to second- and third - line chemotherapy , respectively . for patients with poor ps , anemia , reduced ccr , and higher crp , it is difficult to introduce chemotherapy .