to detect bim deletion polymorphism , we performed two types of polymerase chain reaction ( pcr ) analysis , according to the method of ng et al . in brief , we used a single primer set that contains the deletion area in intron 2 and two separate primer sets designed for wild - type and deletion alleles . the dna was subjected to pcr amplification using primers designed to detect deletion site ( 2903 bp ) in intron 2 of the bcl2l11 gene . the resulting pcr products from the deletion ( 1285 bp ) and wild - type ( 4188 bp ) alleles were analyzed on agarose gels . in addition , the pcr products for the deletion ( 177 bp ) and wild - type ( 174 bp ) alleles were analyzed on agarose gel . we analyzed 20 cell lines , including the kcl-22 cell ( which was reported to have the bim deletion ) , and 30 dna samples from healthy japanese volunteers . we studied 70 patients with egfr mutation - positive nsclc who were treated with egfr - tki during the period from january 2008 to january 2013 . bim deletion polymorphism was analyzed by pcr in 58 samples of peripheral blood ( cell - free dna in 34 , leukocyte dna in 35 ) and on 24 formalin - fixed paraffin - embedded ( ffpe ) slides of surgical specimens ( 20 specimens of lung tissue and four of brain tissue ) ; both blood and tissue specimens were available for 12 patients . to confirm the validity of pcr analysis of two types of samples , we compared the results for bim deletion polymorphism identified in lung tissue on ffpe slides with those from peripheral blood ( cell - free dna or leukocyte dna ) from the same patients ( n = 12 ) . dna was extracted from ffpe slides using the qiaamp ffpe tissue kit ( qiagen kk , tokyo , japan ) . dna extraction blood samples were diluted in lysis solution to lyse the red cells and the white cell fraction was pelleted and washed once in phosphate - buffered saline . dna was extracted from the white cell pellets using the qiaamp dna mini kit ( qiagen kk , tokyo , japan ) . we retrospectively analyzed the clinical characteristics , response rate ( rr ) , disease control rate ( dcr ) , and toxicity of egfr - tki in patients with and without bim deletion polymorphism . toxicity was assessed according to national cancer institute common terminology criteria for adverse events , version 3 . we estimated pfs and overall survival ( os ) in patients with and without bim deletion polymorphism . the pfs of patients treated with egfr - tki was assessed from the date egfr - tki therapy was started to the earliest sign of disease progression as determined by findings from computed tomography or magnetic resonance imaging , according to the response evaluation criteria in solid tumors . os was defined as the period from the date of diagnosis until death from any cause . statistical analyses were conducted using spss software for windows , version 12.0 ( spss , tokyo , japan ) . differences in clinical characteristics , rr , dcr , and adverse events between patients with and without bim deletion polymorphism were compared using fisher s exact test . survival curves were drawn by the kaplan - meier method , and statistical analysis was performed using the log - rank test . we used univariate analysis and multivariate cox regression analysis to identify factors associated with shorter pfs . the investigated prognostic factors were age , sex ( male versus female ) , performance status ( 2 versus 1 versus 0 ) , brain metastasis ( yes versus no ) , bone metastasis ( yes versus no ) , pulmonary metastasis ( yes versus no ) , liver metastasis ( yes versus no ) , lymph node metastasis ( yes versus no ) , egfr mutation ( major mutations [ l858r and exon 19 deletion ] versus minor mutations [ other mutations ] ) , egfr - tki response ( partial response versus stable disease ) , smoking history ( pack - years ) , and bim deletion ( yes versus no ) . this single - center study was conducted at toho university omori medical center ( tokyo , japan ) and was approved by its human genome / gene analysis research ethical committee ( authorization number ; 24 - 1 ) . to detect bim deletion polymorphism , we performed two types of polymerase chain reaction ( pcr ) analysis , according to the method of ng et al . in brief , we used a single primer set that contains the deletion area in intron 2 and two separate primer sets designed for wild - type and deletion alleles . the dna was subjected to pcr amplification using primers designed to detect deletion site ( 2903 bp ) in intron 2 of the bcl2l11 gene . the resulting pcr products from the deletion ( 1285 bp ) and wild - type ( 4188 bp ) alleles were analyzed on agarose gels . in addition , the pcr products for the deletion ( 177 bp ) and wild - type ( 174 bp ) alleles were analyzed on agarose gel . we analyzed 20 cell lines , including the kcl-22 cell ( which was reported to have the bim deletion ) , and 30 dna samples from healthy japanese volunteers . we studied 70 patients with egfr mutation - positive nsclc who were treated with egfr - tki during the period from january 2008 to january 2013 . bim deletion polymorphism was analyzed by pcr in 58 samples of peripheral blood ( cell - free dna in 34 , leukocyte dna in 35 ) and on 24 formalin - fixed paraffin - embedded ( ffpe ) slides of surgical specimens ( 20 specimens of lung tissue and four of brain tissue ) ; both blood and tissue specimens were available for 12 patients . to confirm the validity of pcr analysis of two types of samples , we compared the results for bim deletion polymorphism identified in lung tissue on ffpe slides with those from peripheral blood ( cell - free dna or leukocyte dna ) from the same patients ( n = 12 ) . dna was extracted from ffpe slides using the qiaamp ffpe tissue kit ( qiagen kk , tokyo , japan ) . dna extraction blood samples were diluted in lysis solution to lyse the red cells and the white cell fraction was pelleted and washed once in phosphate - buffered saline . dna was extracted from the white cell pellets using the qiaamp dna mini kit ( qiagen kk , tokyo , japan ) . we retrospectively analyzed the clinical characteristics , response rate ( rr ) , disease control rate ( dcr ) , and toxicity of egfr - tki in patients with and without bim deletion polymorphism . toxicity was assessed according to national cancer institute common terminology criteria for adverse events , version 3 . we estimated pfs and overall survival ( os ) in patients with and without bim deletion polymorphism . the pfs of patients treated with egfr - tki was assessed from the date egfr - tki therapy was started to the earliest sign of disease progression as determined by findings from computed tomography or magnetic resonance imaging , according to the response evaluation criteria in solid tumors . os was defined as the period from the date of diagnosis until death from any cause . statistical analyses were conducted using spss software for windows , version 12.0 ( spss , tokyo , japan ) . differences in clinical characteristics , rr , dcr , and adverse events between patients with and without bim deletion polymorphism were compared using fisher s exact test . survival curves were drawn by the kaplan - meier method , and statistical analysis was performed using the log - rank test . we used univariate analysis and multivariate cox regression analysis to identify factors associated with shorter pfs . the investigated prognostic factors were age , sex ( male versus female ) , performance status ( 2 versus 1 versus 0 ) , brain metastasis ( yes versus no ) , bone metastasis ( yes versus no ) , pulmonary metastasis ( yes versus no ) , liver metastasis ( yes versus no ) , lymph node metastasis ( yes versus no ) , egfr mutation ( major mutations [ l858r and exon 19 deletion ] versus minor mutations [ other mutations ] ) , egfr - tki response ( partial response versus stable disease ) , smoking history ( pack - years ) , and bim deletion ( yes versus no ) . this single - center study was conducted at toho university omori medical center ( tokyo , japan ) and was approved by its human genome / gene analysis research ethical committee ( authorization number ; 24 - 1 ) . using the two types of pcr analysis , we analyzed 20 cell lines and 30 dna samples from healthy japanese volunteers . among the 20 cell lines , only kcl-22 showed bim deletion . as for dna samples , bim deletion polymorphism was present in six of the 30 ( 20% ) healthy volunteers . we confirmed the validity between blood samples ( leukocyte dna in 12 and cell - free dna in four ) and ffpe slides of surgical specimens ( lung tissue in 12 ) : bim deletion was detected in three of 12 patients ( 25% ) . we analyzed bim deletion polymorphism in 70 patients with egfr mutation - positive nsclc who were treated with egfr - tki . bim deletion polymorphism was present in 13 of the 70 patients ( 18.6% ) ; homozygous deletion was noted in one and heterozygous deletion in 12 . for the one case of homozygous deletion , pcr analysis using the primer set for the wild - type allele showed no amplification ( table 1 ) . presence of bim deletion in patients with egfr mutation - positive nsclc ( n = 70 ) there were no significant differences in the clinical characteristics , rr , dcr , or incidence of adverse events between patients with ( n = 13 ) and without ( n = 57 ) bim deletion polymorphism ( tables 2 and 3 ) . patient characteristics ( n = 70 ) comparison of clinical response and adverse events after egfr - tki therapy we estimated pfs and os in patients with and without bim deletion polymorphism . the patients with bim deletion polymorphism had significantly shorter pfs than did those without bim deletion polymorphism ( median , 227 versus 533 days ; p < 0.001 ; fig . 1 ) . there was no significant difference in os ( median , 1176 versus 1363 days ; p = 0.27 ; fig . 2 ) . multivariate cox regression analysis showed that bim deletion polymorphism was the strongest independent indicator of shorter pfs ( hazard ratio [ hr ] , 3.99 ; 95% confidence interval [ ci ] , 1.8648.547 ; p < 0.001 ; table 4 ) . indicators of shorter pfs after egfr - tki treatment patients with bim deletion polymorphism had significantly shorter progression - free survival than did those without bim deletion polymorphism ( median , 227 versus 533 days ; p < 0.001 ) . there was no significant difference in overall survival between patients with and without bim deletion polymorphism ( median , 1176 versus 1363 days ; p = 0.27 ) . using the two types of pcr analysis , we analyzed 20 cell lines and 30 dna samples from healthy japanese volunteers . among the 20 cell lines , only kcl-22 showed bim deletion . as for dna samples , bim deletion polymorphism was present in six of the 30 ( 20% ) healthy volunteers . we confirmed the validity between blood samples ( leukocyte dna in 12 and cell - free dna in four ) and ffpe slides of surgical specimens ( lung tissue in 12 ) : bim deletion was detected in three of 12 patients ( 25% ) . we analyzed bim deletion polymorphism in 70 patients with egfr mutation - positive nsclc who were treated with egfr - tki . bim deletion polymorphism was present in 13 of the 70 patients ( 18.6% ) ; homozygous deletion was noted in one and heterozygous deletion in 12 . for the one case of homozygous deletion , pcr analysis using the primer set for the wild - type allele showed no amplification ( table 1 ) . presence of bim deletion in patients with egfr mutation - positive nsclc ( n = 70 ) there were no significant differences in the clinical characteristics , rr , dcr , or incidence of adverse events between patients with ( n = 13 ) and without ( n = 57 ) bim deletion polymorphism ( tables 2 and 3 ) . patient characteristics ( n = 70 ) comparison of clinical response and adverse events after egfr - tki therapy the patients with bim deletion polymorphism had significantly shorter pfs than did those without bim deletion polymorphism ( median , 227 versus 533 days ; p < 0.001 ; fig . 1 ) . there was no significant difference in os ( median , 1176 versus 1363 days ; p = 0.27 ; fig . 2 ) . multivariate cox regression analysis showed that bim deletion polymorphism was the strongest independent indicator of shorter pfs ( hazard ratio [ hr ] , 3.99 ; 95% confidence interval [ ci ] , 1.8648.547 ; p < 0.001 ; table 4 ) . indicators of shorter pfs after egfr - tki treatment patients with bim deletion polymorphism had significantly shorter progression - free survival than did those without bim deletion polymorphism ( median , 227 versus 533 days ; p < 0.001 ) . there was no significant difference in overall survival between patients with and without bim deletion polymorphism ( median , 1176 versus 1363 days ; p = 0.27 ) . bim deletion polymorphism is a germline alteration that affects egfr - tki  related apoptosis . in a study that screened 2597 healthy individuals , bim deletion polymorphism was present in 12.3% of east asians but absent in africans and europeans . in the present study , bim deletion polymorphism was present in 13 of 70 japanese patients ( 18.6% ) with egfr mutation - positive nsclc and in six of 30 healthy japanese volunteers ( 20% ) , a statistically insignificant difference . the overall frequency of bim deletion polymorphism in our study ( 19% , n = 100 ) was higher than that noted in a previous report . reported homozygous bim deletions in patients with mantle - cell lymphoma , and homozygous bim deletion was found in 0.5% of east asians . among the present 70 japanese patients with nsclc , one ( 1.4% ) had homozygous deletion and 12 had heterozygous deletion . future studies should investigate the characteristics of patients with homozygous bim deletion polymorphism to determine if this genotype results in worse clinical outcomes when compared with heterozygous bim deletion . there were no significant differences between clinical characteristics , response to egfr - tki , or incidences of adverse events due to egfr - tki among patients with and without bim deletion polymorphism . thus , it is difficult to distinguish between patients with and without bim deletion polymorphism on the basis of clinical characteristics alone . bim knockdown was reported to prevent foxo3 ( i.e. , fkhrl1 , a member of the forkhead transcription factor subfamily)-mediated overproduction of reactive oxygen species and apoptosis . bim deletion polymorphism might affect egfr - tki  related lung injury by preventing overproduction of reactive oxygen species . further studies are needed to clarify the relationship between egfr - tki  related pneumonitis and bim . bim deletion polymorphism , a germline alteration , is thought to be associated with intrinsic resistance to egfr - tki and would likely result in primary resistance and no response to treatment . however , the present clinical outcomes were probably due to acquired resistance : when compared with patients without bim polymorphism , those with bim deletion polymorphism had similar rrs and dcrs but shorter pfs . the reasons for these findings remain to be investigated . it has been hypothesized that egfr - tki  induced apoptosis does not completely depend on the bim pathway and that tumor response to egfr - tki in patients with bim deletion might depend on other proapoptotic regulators , which could have less - prolonged clinical activity than those of the bim pathway . a second hypothesis is that bim deletion polymorphism itself results in relative resistance to egfr - tki . kuroda et al . showed that cancer cells were sensitive to small changes in bim protein concentrations and that bim protein concentration exerted a dose - dependent effect on apoptosis and the degree of tki resistance . in a report by faber et al . , pfs was shorter ( 4.7 versus 13.7 mo , p = 0.007 ) among patients with low bim rna expression , which appeared to correlate with high bim protein expression on immunohistochemistry . the rr after egfr - tki was worse among patients with low bim rna expression ( 44% ) than among those with high bim rna expression ( 77% ) , although the difference was not statistically significant . carriers  that have varied bim expression and clinical responses that are modulated by genetic or epigenetic interactions , a possibility that warrants further study . although cells with bim deletion polymorphism show decreased induction of exon-4containing transcripts after tki exposure , the response after prolonged tki exposure should be investigated . ng et al . reported that patients with bim deletion polymorphism had significantly shorter pfs than did patients without bim deletion polymorphism after egfr - tki treatment ( 6.6 versus 11.9 mo , p = 0.0027 ) , but they did not report rr or os . our present study in a japanese population yielded similar results : bim deletion polymorphism was an independent indicator of shorter pfs . however , there was no significant difference in os among patients with and without bim deletion polymorphism . multivariate cox regression analysis showed that indicators of shorter os were egfr - tki  related pneumonitis ( hr , 3.52 ; 95% ci , 1.1903.860 ; p = 0.023 ) , brain metastasis ( hr , 2.14 ; 95% ci , 1.0994.165 ; p = 0.025 ) , and smoking history ( hr , 1.001 ; 95% ci , 1.0001.001 ; p = 0.026 ) . egfr - tki  related pneumonitis developed only in patients without bim deletion polymorphism ( n = 8 , 14% ) but has a detrimental effect on chemotherapy given after pneumonitis . thus , egfr - tki  related pneumonitis might have reduced os among the present patients without bim deletion , which possibly explains the lack of a significant difference in os between patients with and without bim deletion polymorphism in the present study . bh3-mimetic drugs and histone deacetylase inhibitors may be able to surmount bim - associated resistance to egfr - tki . our findings suggest that although there was no significant difference in rr or os among patients with and without bim deletion polymorphism , the addition of these drugs might prolong pfs . however , this study was a retrospective study at a single center . a prospective multicenter study should be conducted to investigate the clinical significance of bim deletion polymorphism on egfr - tki therapy . in addition , egfr - tki  related pneumonitis should be considered in any randomized prospective study of the clinical benefit of bh3-mimetic drugs or histone deacetylase inhibitors for patients with bim deletion polymorphism . in conclusion , bim deletion polymorphism , a germline alteration , was successfully detected by pcr analysis of samples of peripheral blood and ffpe slides of surgical specimens , thus providing a minimally invasive and convenient detection method . bim deletion polymorphism was the strongest indicator of shorter pfs among patients with egfr mutation - positive nsclc treated with egfr - tki . our results indicate that new treatment strategies should be established for patients with bim deletion polymorphism . we thank atsushi kakimoto , hiroki todoroki , and satoshi natsume of srl ( tokyo , japan ) and hiroyuki mano and manabu soda of jichi medical university ( tochigi , japan ) for their help . we are also grateful to r. j. turner of toho university and david kipler for their review of the language of this article .