mutations in the tyrosine kinase domain of the epidermal growth factor receptor ( egfr ) gene have been identified in non - small cell lung carcinoma ( nsclc ) , and they were correlated with clinical response to egfr  tyrosine kinase inhibitor ( tki ) treatment . patients with egfr mutations have a higher response rate to egfr - tkis ( 60%-80% ) than those with egfr  wild type or unknown mutation status ( 10%-20% ) . in addition , egfr - tki has been shown to be superior to carboplatin - paclitaxel as initial treatment . dna - based molecular methods have been used for selection of patients who would benefit from tki therapy ; however , they are tedious , expensive , and not routine in clinical laboratories . recently generated mutation - specific rabbit monoclonal antibodies against the two most common nsclc - associated egfr mutations ( e746-a750 deletion [ del ] in exon 19 and l858r point mutation [ l858r ] in exon 21 ) for detection of mutant egfr protein by immunohistochemistry ( ihc ) . ihc using these two mutation - specific antibodies has been suggested as a reliable screening method for egfr - tki therapy [ 4 - 8 ] . however , the immunostaining patterns reported in previous publications were contradictory ; some heterogeneous , while others were described as homogeneous [ 9 - 11 ] . biopsy samples , however , may be the only tumor materials available for confirming egfr mutation status , particularly in patients whose lung cancer is at an advanced stage and unresectable . in addition , tumor cells in the samples are often low in quantity or of insufficient quality for molecular assays , leading to a second biopsy procedure for obtaining additional tissues . it is therefore important to determine whether biopsy samples are suitable for detection of mutant egfr protein . there has been significant interest in total egfr ( tegfr ) protein expression , in particular , before the development of mutation - specific antibodies , however , its clinical significance is controversial . some studies reported a better outcome after egfr - tki treatment for tumors overexpressing the tegfr protein , whereas other studies did not [ 12 - 14 ] . to date , the literature contains limited data regarding the association between tegfr and mutant egfr proteins . in the current study , immunohistochemical analysis of both biopsies and resected tumor tissues from lung adenocarcinoma with known egfr mutation status by direct dna sequencing was performed using mutation - specific antibodies against egfr with e746-a750 deletion in exon 19 and l858r mutation in exon 21 . expression of tegfr was also investigated in order to determine possible correlations with mutant egfr proteins detected by mutation - specific antibodies . a total of 154 patients who underwent biopsy ( n=78 ) or surgical resection ( n=76 ) for pulmonary adenocarcinoma at the catholic university st . paraffin - embedded tissues were procured from biopsied or resected specimens in a blinded fashion to the clinical information and egfr mutation status , and ihc was performed using egfr mutation  specific antibodies . the study protocol was approved by the institutional review board ( irb ) of st . tissue corresponding to the precisely identified tumor areas on the hematoxylin and eosin ( h&e ) slides was scraped for subsequent dna extraction using a qiamp kit ( qiagen , valencia , ca ) . exons 18 to 21 of the tk domain in the egfr gene were amplified by polymerase chain reaction using specific primers , and dna sequencing was performed using the abi 3710 genetic analyzer ( applied biosystems , foster city , ca ) . a single block of paraffin - embedded tissue from biopsy specimens was cut in serial 4-m sections . for construction of tissue microarrays ( tmas ) from surgically resected specimens , the most representative tumor areas were identified on a selected h&e slide and marked by a pathologist ( j.y . ) . three replicate core samples , each measuring 2.0 mm in diameter , were obtained using a precise instrument , and arrayed on a recipient paraffin block . each case was tested using the following primary antibodies : del - specific monocloncal antibody ( pre - diluted , clone sp111 , ventana medical systems inc . , tucson , az ) , l858r - specific monoclonal antibody ( pre - diluted , clone sp125 , ventana medical systems inc . ) , and tegfr antibody ( 1:100 , clone sp9 , spring bioscience , pleasanton , ca ) . briefly , 4-m sections were deparaffinized in xylene , and rehydrated through a graded series of ethanol . slides were labeled with antibody and protocol - specific bar codes , and loaded into a benchmark xt automated slide stainer ( ventana medical systems inc . ) . the immunoreactions were detected using an ultraview universal dab detection kit ( ventana medical systems inc . ) and 3,3-diaminobenzidine , followed by counterstaining with hematoxylin and bluing reagent . immunohistochemical assessments were performed by an experienced pathologist ( j.y . ) without information on molecular - based egfr mutation status . immunoreactivity was scored based on membranous and/or cytoplasmic staining , as follows : 0 , no staining or faint staining in < 10% of tumor cells ; 1 + , weak staining in  10% of tumor cells ; 2 + , moderate staining in  10% of tumor cells ; 3 + , strong staining in  10% of tumor cells . for tma samples , the staining scores obtained in three cores were averaged and the result was taken as a representative score for each case . ibm spss ver . 19.0 ( ibm co. , armonk , ny ) was used for analysis . all variables were estimated using pearson chi - square or fisher exact tests . a p - value of < 0.05 was considered statistically significant . immunohistochemical staining intensity ( score 0 - 3 ) and predictive probability of the logistic regression model based on mutation - specific antibodies and tegfr expression were used for construction of the receiver operating characteristic ( roc ) curves and for calculation of the area under the curves ( aucs ) . a total of 154 patients who underwent biopsy ( n=78 ) or surgical resection ( n=76 ) for pulmonary adenocarcinoma at the catholic university st . paraffin - embedded tissues were procured from biopsied or resected specimens in a blinded fashion to the clinical information and egfr mutation status , and ihc was performed using egfr mutation  specific antibodies . the study protocol was approved by the institutional review board ( irb ) of st . three 10-m thick sections were cut from the paraffin blocks . tissue corresponding to the precisely identified tumor areas on the hematoxylin and eosin ( h&e ) slides was scraped for subsequent dna extraction using a qiamp kit ( qiagen , valencia , ca ) . exons 18 to 21 of the tk domain in the egfr gene were amplified by polymerase chain reaction using specific primers , and dna sequencing was performed using the abi 3710 genetic analyzer ( applied biosystems , foster city , ca ) . a single block of paraffin - embedded tissue from biopsy specimens was cut in serial 4-m sections . for construction of tissue microarrays ( tmas ) from surgically resected specimens , the most representative tumor areas were identified on a selected h&e slide and marked by a pathologist ( j.y . ) . three replicate core samples , each measuring 2.0 mm in diameter , were obtained using a precise instrument , and arrayed on a recipient paraffin block . each case was tested using the following primary antibodies : del - specific monocloncal antibody ( pre - diluted , clone sp111 , ventana medical systems inc . , tucson , az ) , l858r - specific monoclonal antibody ( pre - diluted , clone sp125 , ventana medical systems inc . ) , and tegfr antibody ( 1:100 , clone sp9 , spring bioscience , pleasanton , ca ) . briefly , 4-m sections were deparaffinized in xylene , and rehydrated through a graded series of ethanol . slides were labeled with antibody and protocol - specific bar codes , and loaded into a benchmark xt automated slide stainer ( ventana medical systems inc . ) . the immunoreactions were detected using an ultraview universal dab detection kit ( ventana medical systems inc . ) and 3,3-diaminobenzidine , followed by counterstaining with hematoxylin and bluing reagent . immunohistochemical assessments were performed by an experienced pathologist ( j.y . ) without information on molecular - based egfr mutation status . immunoreactivity was scored based on membranous and/or cytoplasmic staining , as follows : 0 , no staining or faint staining in < 10% of tumor cells ; 1 + , weak staining in  10% of tumor cells ; 2 + , moderate staining in  10% of tumor cells ; 3 + , strong staining in  10% of tumor cells . for tma samples , the staining scores obtained in three cores were averaged and the result was taken as a representative score for each case . all variables were estimated using pearson chi - square or fisher exact tests . a p - value of < 0.05 was considered statistically significant . immunohistochemical staining intensity ( score 0 - 3 ) and predictive probability of the logistic regression model based on mutation - specific antibodies and tegfr expression were used for construction of the receiver operating characteristic ( roc ) curves and for calculation of the area under the curves ( aucs ) . the patient demographics and egfr mutation status predefined by direct dna sequencing are summarized in table 1 . there were 80 men and 74 women with a median age of 71 years ( range , 37 to 89 years ) . ninety - one patients ( 59.1% ) were never - smokers , 12 ( 7.8% ) were former smokers , and 51 ( 33.1% ) were current smokers . the t status distribution was as follows : 18.8% t1 , 34.4% t2 , 38.3% t3 , and 8.4% t4 . none of the patients had received neoadjuvant chemotherapy or radiotherapy prior to the biopsy or tumor resection . one patient ( 1.6% ) had egfr mutation in exon 18 , 25 ( 40.3% ) in exon 19 , three ( 4.8% ) in exon 20 , and 33 ( 53.2% ) in exon 21 ( table 2 ) . the mutation detected in exon 18 was a double missense mutation . in exon 19 , all were in - frame deletions : 14 ( 56% ) with e746-a750 deletion and the remaining 11 ( 44% ) with alternative in - frame deletions . in exon 20 , one was of the duplication type and the others of missense type . in exon 21 , e746-a750 deletion and l858r mutations accounted for 72.6% ( 45/62 ) of all egfr mutations . 1 shows representative immunostaining with del - specific antibody , l858r - specific antibody , and tegfr antibody . immunoreactivity showed relatively homogeneous distribution between the cores of each tumor , and less than 9% had inconsistent findings in staining intensity with no effect on interpretation . the aucs for ihc with del - specific antibody and l858r - specific antibody were 0.843 with an optimal cutoff point of 0.5 ( p=0.001 ) and 0.873 with an optimal cutoff point of 0.5 ( p=0.001 ) , respectively ( data not shown ) . according to the optimal cutoff point of predictive probability by the logistic regression model , however , it was also positive in three patients with no e746-a750 deletion : one each with l745-a750 deletion in exon 19 , l858r mutation , and egfr wild type l858r - specific antibody correctly identified 24 of 31 patients with the corresponding gene mutation . two egfr wild type patients and one patient with t790 m mutation in exon 20 were also positive for l858r - specific antibody , whereas seven patients with l858r mutation were negative for l858r - specific antibody . the overall sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of del - specific antibody were 71.4% , 97.9% , 76.9% , and 97.2% , respectively ( table 5 ) . l858r - specific antibody showed 77.4% sensitivity , 97.6% specificity , 88.9% ppv , and 94.5% npv . no significant differences in mutant egfr detection rate ( 23/78 [ 29.5% ] vs. 22/76 [ 28.9% ] ; p > 0.05 ) , or in sensitivity , specificity , ppv , and npv were observed between biopsy samples and resected samples . the sensitivity , specificity , ppv , and npv of tegfr antibody were 29% , 77.2% , 46.2% , and 61.7% , respectively ( data not shown ) . no correlation was observed between tegfr expression and egfr mutation ( p=0.284 ) , del - specific antibody ihc ( p=0.64 ) , or l858r - specific antibody ihc ( p=0.125 ) ( table 6 ) . in the current study , we compared the expression status of two egfr mutation  specific antibodies between biopsies and resection samples from pulmonary adenocarcinoma , which had been previously genotyped by direct dna sequencing . we also investigated tegfr protein for evaluation of its possible correlation with mutant egfr proteins detected by mutation - specific antibodies . the major findings of our study are as follows : ( 1 ) ihc with mutation - specific antibodies correctly identified egfr mutation status in 89% ( 137/154 ) , showing negative immunostaining in 94.5% ( 103/109 ) of patients with egfr wild type or mutations other than e746-a750 deletion and l858r mutation , and positive reaction in 75.6% ( 34/45 ) of patients with corresponding mutations ; ( 2 ) no significant differences in the egfr mutation frequency were observed between biopsies and resection specimens ; ( 3 ) tegfr expression showed no association with immunoreactivity for both mutation - specific antibodies . specific antibodies against egfr with e746-a750 deletion and l858r for identification of mutant egfr proteins by ihc , and reported a sensitivity of 92% and specificity of 99% as compared with direct dna sequencing . although accumulating data have suggested ihc using the two mutation - specific monoclonal antibodies as a simple , rapid , and costeffective screening tool for discriminating lung cancer patients responsive to egfr - based therapies [ 4 - 10 ] , the reported sensitivities and specificities were quite variable . in several investigations on nsclc , the specificities of both antibodies were relatively high ( 77%-100% ) , whereas the sensitivities of del - specific and l858r - specific antibodies ranged from 40% to 94% and 24% to 100% , respectively [ 5,6,9,10,15 - 17 ] . all used the same two antibodies ( clone 43b2 specific for e746-a750 deletion and clone 6b6 specific for l858r mutation , cell signaling technology , danvers , ma ) , so that the wide range of sensitivities and specificities is most likely not due to the antibody used . in addition , we used egfr mutation - specific antibodies purchased from a different manufacturer ( clones sp111 and sp125 , respectively , ventana medical systems inc . ) . del - specific ihc showed a sensitivity of 71.4% and specificity of 97.1% , while l858r - specific ihc showed a sensitivity of 77.4% and specificity of 97.6% , compatible with previous reports . similar results were obtained by the most recent investigation using the same antibodies as in the current study . the wide range of sensitivities and specificities may be attributed to scoring systems , which play a critical role in ihc - based analyses . several scoring schemes for interpretation of mutation - specific ihc have been adopted in published papers . immunoreactivity was classified on the basis of staining intensity , sometimes multiplied by the percentage values , both of which are highly dependent on the researcher . some assessed immunostaining in membrane only , others in membrane and/or cytoplasm , or even based on cytoplasmic staining of tumor cells alone . furthermore , when using a four - grading scale ( score 0 - 3 ) ,  1 + was defined as positive by some investigators , and  2 + was considered positive by others [ 7 - 10,19 ] . recently , an optimized protocol of an intensity of 2 + or more in membrane and/or cytoplasm of > 10% tumor cells for positivity was shown to be the most appropriate way to interpret the egfr mutation  specific ihc . in our study , we performed roc analyses to determine the best cutoff for detection of egfr mutations , and found it to be a score of 1 + or more . consensus of universally accepted criteria in interpreting the results as positive or negative should be reached in order to produce objective and reproducible results before using mutation - specific ihc as a substitute for molecular methods . observed different staining intensity in at least one of three tma cores each from 15/33 positive tumors ( 45% ) . on the other hand , xiong et al .   used whole tissue sections and reported homogeneity in staining pattern . this prompted us to investigate whether the type of specimen ( biopsy vs. resection ) would make a difference in determining mutation status by ihc . in the current study , observed staining patterns were homogeneous in both biopsies and tma cores obtained from each patient . in addition , no significant differences in mutant egfr protein detection rates were found between the biopsy samples and tmas ( 29.5% vs. 28.9% , p > 0.05 ) . fan et al .   described a heterogeneous expression pattern in some of their cases with no details given , and speculated the causal relationship between heterogeneity and lower sensitivity of del - specific antibody in their biopsy samples . however , they also detected the egfr mutation phenotype in 32.1% of biopsies and 36.3% of resection samples ( p > 0.05 ) with high specificity ( 97.9%-100% ) , which is in concordance with our results , suggesting that ihc using two mutation - specific antibodies may be performed as reliably in biopsies as in resection samples . therefore , when biopsied specimens are the only materials available for egfr status in patients at advanced stage , and tumor cells in the samples are insufficient or inadequate for dna - based molecular methods , using ihc with mutation - specific antibodies may preclude another biopsy procedure to obtain ample tissues . the overall sensitivity of ihc with del - specific antibody for all exon 19 mutations was low ( 44% ) . this is because del - specific antibody is relatively specific for the frequent e746-a750 deletion and can not detect the alteration . in the current study , 11 of 25 patients with exon 19 mutations had alternative in - frame deletions other than e746-a750 deletion , and thus almost exclusively did not react with del - specific antibody . our observation of the lack of correlation between egfr mutation and tegfr ihc is not surprising . identification of tegfr protein by ihc did not always reflect the detection of egfr mutation , suggesting that mechanisms other than egfr mutation may also play a role in tegfr expression . as in the study herein , all previous publications , to the best of our knowledge , used tegfr antibodies detecting the external domain ( ed ) of egfr . reported a correlation of tegfr with mutation - specific antibodies , using another ed - specific antibody ( clone 2 - 18c9 , dako , carpinteria , ca ) for tegfr . . a histologically inhomogeneous series of lung cancer may be one possible explanation for this discrepancy . ed - specific antibodies bind to receptors containing ed and do not discriminate between active and inactive receptors , whereas intracellular domain ( id)specific antibody would detect active or even truncated forms of the receptor . one study demonstrated that egfr expression evaluated by ed - specific antibody did not predict patient survival , but egfr protein expression using an id - specific antibody was a significant predictor of response to gefitinib in nsclc patients . investigations on whether egfr id - specific antibody ihc is correlated with mutant egfr , and has an impact on diagnostic power in the ihc interpretation of the mutation - specific antibodies , may be of great help in more accurate assessment of egfr mutation status . ihc with egfr mutation  specific antibodies exhibited extremely high specificity ( 94.5% ) with good sensitivity ( 75.6% ) in both biopsies and resection specimens specific antibodies may preclude a second biopsy procedure to obtain additional tissues for identification of egfr mutations by molecular biology techniques in biopsies from advanced cancer , especially when tumor cells in the samples are limited . negative cases , however , require further molecular - based analyses for identification of other mutations with low frequency .