with the aggravation of environmental pollution , lung cancer is almost the most malignant tumor in the world with its high morbidity and mortality.1 the most common histologic subtype is non - small cell lung cancer ( nsclc ) , which accounts for 80% of all lung cancers.2 although much progress has been made in the treatment of lung cancer , early diagnosis is difficult and the majority of patients has progressed to an advanced stage when diagnosed .
the median survival rate for these patients is only 811 months.3 in 2004 , a landmark discovery had been made in that somatic mutations in the epidermal growth factor receptor ( egfr ) were associated with sensitivity to egfr tyrosine kinase ( tk ) inhibitors ( tkis ) ( egfr - tki).4,5 in subsequent large - scale randomized clinical trials , the relationship between egfr mutation status and efficacy of the egfr - tki drug was clearly explained.68 based on these findings , egfr mutation status in the tk domain can determine the treatment of advanced nsclc .
mutations associated with enhanced sensitivity to egfr - tkis are found in exons 1821 of the tk domain of egfr ; in particular , del e746-a750 in exon 19 and the l858r point mutation in exon 21 account for nearly 90% of all the mutations in egfr in lung cancer.7,9,10 nowadays , the detection of egfr mutation status in nsclc patients has become an expert consensus.11 different methodologies have been developed for molecular testing , such as the amplified refractory mutation system ( arms ) , high - resolution melting , dna direct sequencing , and next - generation sequencing ( ngs ) .
dna direct sequencing is considered the  gold standard  for the assessment of egfr mutation status in nsclc ; however , it is time consuming and laborious .
the arms method is widely used in the clinical testing ; however , the commercial assay kit for egfr is very expensive , and the experiment needs to be done under good experimental conditions with sophisticated real - time polymerase chain reaction ( pcr ) instruments.12 immunohistochemistry ( ihc ) is a well - established method that is widely applied in conventional pathological diagnosis .
egfr mutation - specific rabbit monoclonal antibodies against e746-a750 deletion and l858r ( cell signaling technology , inc . , danvers , ma , usa ) have been applied in ihc application .
this provides a simple and rapid screening method for assessing egfr mutation status.1315 a nanofluorescent material , fluorescent semiconductor nanocrystal quantum dots ( qds ) , have been widely used in labeling some molecules , such as streptavidin and antibodies , through carbodiimide chemistry , optionally using edac ( 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ) .
qd ( 605 nm)-labeled streptavidin emits bright red fluorescence with 605 nm as the maximum emission wavelength while being stimulated by an excitation light source < 580 nm , which is different from green background autofluorescence .
those labeled materials can be successfully applied to biological imaging and ihc detection of gene mutations , such as with human epidermal growth factor receptor 2 ( her 2 ) amplification in breast cancer.16,17 moreover , multiple markers can be visualized on one cell for in vitro multiplexed imaging ; for example , clinically significant tumor biomarkers including her2 , egfr , progesterone receptor , estrogen receptor , and mammalian target of rapamycin can be detected quantitatively and simultaneously in breast cancer cells using multicolor qds.18 qd - based immunofluorescence histochemistry ( qds - ihc ) is an established method ; it has been validated in many published papers.17,19,20 the staining signal detected by qds - ihc is much stronger with a lower background when compared with ihc.20 based on the application of egfr mutation - specific antibodies , this study was designed to develop a qd - based immunofluorescent approach for egfr mutation detection , which is a simple , quick , and highly sensitive molecular method for diagnosing egfr mutation status in nsclc samples .
fifty - five cases of formalin - fixed , paraffin - embedded ( ffpe ) nsclc specimens and ten cases of pleural effusion cell blocks from patients with lung adenocarcinoma were collected , which were provided by the central hospital of enshi autonomous prefecture and the hubei cancer hospital from january 2013august 2014 .
the cohort consisted of 13 squamous cell carcinomas , 50 adenocarcinomas , and two adenosquamous carcinomas .
for each case , the hematoxylin and eosin sections were reviewed by at least two pathologists ( ygq and hlc ) .
this study was approved by the institutional ethics committee of the central hospital of enshi autonomous prefecture .
nsclc tissue sections ( 4 m thick ) were deparaffinized in xylene and rehydrated in a graded ethanol series .
qds - ihc was performed according to the manufacturer s instructions ( wuhan jiayuan quantum dots co. , ltd . ,
antigen retrieval was performed in ethylene diamine tetraacetic acid ( edta ) ( 10 mm ; ph 9.0 ) at 100c for 3 minutes , followed by cooling at room temperature for 30 minutes . for antibody bindings
, sections were first incubated in 2% bovine serum albumin ( bsa ) buffer ( sigma - aldrich co. , st louis , mo , usa ) at 37c for 30 minutes , and then three primary antibodies ( total egfr monoclonal antibody [ d38b1 ] , egfr del e746-a750 mutation - specific monoclonal antibody [ 6b6 ] , and l858r mutation - specific monoclonal antibody [ 43b2 ] ; cell signaling technology , inc . ) were diluted separately at 1:100 and manually applied to the sections .
after that , the slides were then washed three times with tris - buffered saline ( tbs ) with tween ( tbs - t ) ( 0.5% tween , 0.1 m tris - base , 0.9% nacl , and ph 7.6 ) for 5 minutes each time , and incubated in biotinylated goat antirabbit immunoglobulin g ( 1:300 dilution ; jackson immunoresearch inc . ,
finally , qd ( 605 nm)-labeled streptavidin ( 1:400 dilution in 2% bsa ; wuhan jiayuan quantum dots co. , ltd . ) was added to the sections and incubated at 37c for 30 minutes . after rinsing for three times ,
sections were sealed with 90% glycerine ( sigma - aldrich co. ) . during the process , negative control samples were performed in parallel , but the primary antibody was replaced with tbs buffer .
after washing the sections with tbs , diluted primary antibodies ( 1:100 ) were applied to cover the specimen .
after three washes in tbs for 3 minutes each , the slides were incubated for 30 minutes at room temperature with labeled polymer - horseradish peroxidase antirabbit secondary antibody ( envision kit ; dako denmark a / s , glostrup , denmark ) .
the qd staining signals were detected using olympus bx51 fluorescence microscopy equipped with an olympus micro dp72 camera ( olympus corporation , tokyo , japan ) .
the egfr - positive signal detected by qds - ihc was red , target - specific , bright , and photostable . the egfr - positive signal detected by traditional ihc was brown  yellow or brown .
immunostaining was evaluated by two different pathologists ( ygq and xdz ) using criteria based on published cutoffs .
the intensity of cytoplasmic and/or membrane staining , as well as the percentage of positive cells , was recorded .
staining intensity was scored from 0 to 3 + , as follows:21,22 0 if tumor cells had a complete absence of staining or faint staining intensity in < 10% ; 1 + if 10% of the tumor cells had faint staining ; 2 + if the tumor cells had moderate staining ; and 3 + if tumor cells had strong staining ( figure 1 ) .
accordingly , we classified scores of 0 and 1 + as negative and scores of 2 + and 3 + as positive . to assess the sensitivity and specificity of qds - ihc , we compared these results with those of real - time quantitative pcr .
extraction of genomic dna from ffpe nsclc tissue sections was performed using cobas sample preparation kits ( hoffman - la roche ltd . ,
the dna quality and purity were assessed using varioskan flash ( thermo fisher scientific , waltham , ma , usa).23 arms is a highly sensitive method ; it is a real - time pcr - based test . the amoydx egfr mutation test kit ( amoy diagnostics co. , ltd . , xiamen , people s republic of china ) has been widely used in the clinical laboratory .
the assay was carried out according to the manufacturer s protocol for the kit with the lightcycler 480 ii real - time pcr system ( hoffman - la roche ltd . ) . upon completion
, the results were analyzed according to the criteria defined by the manufacturer s instructions .
statistical analysis was performed using the statistical software spss version 17.0 ( ibm corporation , armonk , ny , usa ) .
cohen s kappa was used to determine intraobserver agreement and the agreement between qds - ihc , ihc , and arms .
a kappa value between 0.81 and 1.0 was defined as a nearly perfect agreement , between 0.41 and 0.80 as a moderate agreement , between 0.21 and 0.40 as a fair agreement , and between 0.00 and 0.20 as a slight agreement .
all tests were two - sided , and a p - value of < 0.05 was considered statistically significant .
fifty - five cases of formalin - fixed , paraffin - embedded ( ffpe ) nsclc specimens and ten cases of pleural effusion cell blocks from patients with lung adenocarcinoma were collected , which were provided by the central hospital of enshi autonomous prefecture and the hubei cancer hospital from january 2013august 2014 .
the cohort consisted of 13 squamous cell carcinomas , 50 adenocarcinomas , and two adenosquamous carcinomas .
for each case , the hematoxylin and eosin sections were reviewed by at least two pathologists ( ygq and hlc ) .
this study was approved by the institutional ethics committee of the central hospital of enshi autonomous prefecture .
nsclc tissue sections ( 4 m thick ) were deparaffinized in xylene and rehydrated in a graded ethanol series .
qds - ihc was performed according to the manufacturer s instructions ( wuhan jiayuan quantum dots co. , ltd . ,
antigen retrieval was performed in ethylene diamine tetraacetic acid ( edta ) ( 10 mm ; ph 9.0 ) at 100c for 3 minutes , followed by cooling at room temperature for 30 minutes . for antibody bindings , sections were first incubated in 2% bovine serum albumin ( bsa ) buffer ( sigma - aldrich co. , st louis , mo , usa ) at 37c for 30 minutes , and then three primary antibodies ( total egfr monoclonal antibody [ d38b1 ] , egfr del e746-a750 mutation - specific monoclonal antibody [ 6b6 ] , and l858r mutation - specific monoclonal antibody [ 43b2 ] ; cell signaling technology , inc . ) were diluted separately at 1:100 and manually applied to the sections .
specimens were incubated at 4c overnight with those three primary antibodies , respectively . after that
, the slides were then washed three times with tris - buffered saline ( tbs ) with tween ( tbs - t ) ( 0.5% tween , 0.1 m tris - base , 0.9% nacl , and ph 7.6 ) for 5 minutes each time , and incubated in biotinylated goat antirabbit immunoglobulin g ( 1:300 dilution ; jackson immunoresearch inc . ,
finally , qd ( 605 nm)-labeled streptavidin ( 1:400 dilution in 2% bsa ; wuhan jiayuan quantum dots co. , ltd . ) was added to the sections and incubated at 37c for 30 minutes .
after rinsing for three times , sections were sealed with 90% glycerine ( sigma - aldrich co. ) . during the process , negative control samples were performed in parallel , but the primary antibody was replaced with tbs buffer .
resected tumor specimens were stained simultaneously using these three antibodies according to the manufacturer s instructions .
after washing the sections with tbs , diluted primary antibodies ( 1:100 ) were applied to cover the specimen .
after three washes in tbs for 3 minutes each , the slides were incubated for 30 minutes at room temperature with labeled polymer - horseradish peroxidase antirabbit secondary antibody ( envision kit ; dako denmark a / s , glostrup , denmark ) . following three washes in tbs
the qd staining signals were detected using olympus bx51 fluorescence microscopy equipped with an olympus micro dp72 camera ( olympus corporation , tokyo , japan ) .
the egfr - positive signal detected by qds - ihc was red , target - specific , bright , and photostable . the egfr - positive signal detected by traditional ihc was brown  yellow or brown .
immunostaining was evaluated by two different pathologists ( ygq and xdz ) using criteria based on published cutoffs .
the intensity of cytoplasmic and/or membrane staining , as well as the percentage of positive cells , was recorded .
staining intensity was scored from 0 to 3 + , as follows:21,22 0 if tumor cells had a complete absence of staining or faint staining intensity in < 10% ; 1 + if 10% of the tumor cells had faint staining ; 2 + if the tumor cells had moderate staining ; and 3 + if tumor cells had strong staining ( figure 1 ) .
accordingly , we classified scores of 0 and 1 + as negative and scores of 2 + and 3 + as positive . to assess the sensitivity and specificity of qds - ihc , we compared these results with those of real - time quantitative pcr .
extraction of genomic dna from ffpe nsclc tissue sections was performed using cobas sample preparation kits ( hoffman - la roche ltd . ,
the dna quality and purity were assessed using varioskan flash ( thermo fisher scientific , waltham , ma , usa).23
arms is a highly sensitive method ; it is a real - time pcr - based test . the amoydx egfr mutation test kit ( amoy diagnostics co. , ltd . , xiamen
, people s republic of china ) has been widely used in the clinical laboratory .
the assay was carried out according to the manufacturer s protocol for the kit with the lightcycler 480 ii real - time pcr system ( hoffman - la roche ltd . ) . upon completion
, the results were analyzed according to the criteria defined by the manufacturer s instructions .
statistical analysis was performed using the statistical software spss version 17.0 ( ibm corporation , armonk , ny , usa ) .
cohen s kappa was used to determine intraobserver agreement and the agreement between qds - ihc , ihc , and arms .
a kappa value between 0.81 and 1.0 was defined as a nearly perfect agreement , between 0.41 and 0.80 as a moderate agreement , between 0.21 and 0.40 as a fair agreement , and between 0.00 and 0.20 as a slight agreement .
all tests were two - sided , and a p - value of < 0.05 was considered statistically significant .
tissue sections from small biopsies , pleural effusion cell blocks , and surgery were successfully stained by egfr mutation - specific antibodies , and these antibodies showed distinct immunoreactivity ( red signals ) for the tumor cells , as presented in figure 1 .
a positive signal of the total egfr protein detected by qds - ihc and ihc was moderate to strong in all 65 samples , which was regarded as the positive control ( figure 2 ) .
the positive rates for the egfr mutation detected by qds - ihc and ihc were 40.0% ( 26/65 ) and 36.9% ( 24/65 ) , respectively . in the qds - ihc method ,
12 ( 46.2% ) patients showed e746-a750-specific staining and 14 ( 53.8% ) patients were l858r mutant - specific ( table 1 ) .
figure 2 shows the representative images of the same surgical case of cancer , which carried the l858r mutation and total egfr .
the cancer cells were strongly stained by the total egfr antibody ( figures 2a and b ) ; moreover , the cancer cells were positively stained for the anti - l858r antibody ( figures 2c and d ) and negative for the del e746-a750 deletion . simultaneously ,
egfr mutations were also detected by qds - ihc and ihc using mutation - specific antibodies at the pleural effusion cell blocks ( figures 3a  d ) and small biopsies ( figures 4a  d ) .
egfr mutations detected by qds - ihc and ihc exhibited significant difference between squamous cell carcinoma and adenocarcinoma ( p<0.05 ) . in all the 65 nsclc specimens , the mutation rates for egfr detected by adx - arms was 46.15% ( 30/65 ) , 52.00% ( 26/50 ) for adenocarcinoma , 15.38% ( 2/13 ) for squamous cell carcinoma , and 100% ( 2/2 ) for adenosquamous carcinoma . a significant difference in egfr mutations was observed between adenocarcinoma and squamous cell carcinoma ( p=0.018 ) .
adx - arms could detect eleven cases of exon 19 deletion , 14 cases of exon 21 l858r mutations , two cases of exon 20 point mutations ( s768i ) , and one case of exon 20 insertion .
both exon changes such as exon 19 deletion and exon 20 t790 m mutations , or exon 18 g719x and exon 20 s768i mutations could be detected simultaneously in the same case ( table 1 ) .
thirty - five wild - type egfrs were also noted , and there were two invalid results , mainly due to the fact that there was little tissue available ; as such , the concentration of extracted dna was very low ( 2.54 ng/l and 2.04 ng/l ) .
we then compared the mutation status between adx - arms and immunostaining - based egfr .
of the 26 patients with positive qds - ihc staining , all of the egfr mutations ( exon 19 deletion and exon 21 l858r mutation ) were detected by adx - arms ; however , adx - arms could detect four cases of exon 18 or exon 20 changes .
if the adx - arms results were true , the sensitivity of qds - ihc in detecting egfr mutations , as compared with adx - arms , was 86.7% ( 26/30 ) ; the specific for both antibodies was 100.0% ( 26/26 ) .
two cases with the exon 21 l858r point mutation , as identified by qds - ihc , were negatively stained by ihc using the egfr mutation - specific antibody .
ihc sensitivity was relatively low ( 80.0% ; 24/30 ) and the specificity was 92.31% ( 24/26 ) .
when detecting egfr mutations , qds - ihc and adx - arms demonstrated perfect consistency (   = 0.882 ; p<0.01 ) .
excellent agreement was observed between ihc and adx - arms when detecting egfr mutations (   = 0.826 ; p<0.01 ) .
tissue sections from small biopsies , pleural effusion cell blocks , and surgery were successfully stained by egfr mutation - specific antibodies , and these antibodies showed distinct immunoreactivity ( red signals ) for the tumor cells , as presented in figure 1 .
a positive signal of the total egfr protein detected by qds - ihc and ihc was moderate to strong in all 65 samples , which was regarded as the positive control ( figure 2 ) .
the positive rates for the egfr mutation detected by qds - ihc and ihc were 40.0% ( 26/65 ) and 36.9% ( 24/65 ) , respectively . in the qds - ihc method ,
12 ( 46.2% ) patients showed e746-a750-specific staining and 14 ( 53.8% ) patients were l858r mutant - specific ( table 1 ) .
figure 2 shows the representative images of the same surgical case of cancer , which carried the l858r mutation and total egfr .
the cancer cells were strongly stained by the total egfr antibody ( figures 2a and b ) ; moreover , the cancer cells were positively stained for the anti - l858r antibody ( figures 2c and d ) and negative for the del e746-a750 deletion . simultaneously ,
egfr mutations were also detected by qds - ihc and ihc using mutation - specific antibodies at the pleural effusion cell blocks ( figures 3a  d ) and small biopsies ( figures 4a  d ) .
egfr mutations detected by qds - ihc and ihc exhibited significant difference between squamous cell carcinoma and adenocarcinoma ( p<0.05 ) .
in all the 65 nsclc specimens , the mutation rates for egfr detected by adx - arms was 46.15% ( 30/65 ) , 52.00% ( 26/50 ) for adenocarcinoma , 15.38% ( 2/13 ) for squamous cell carcinoma , and 100% ( 2/2 ) for adenosquamous carcinoma .
a significant difference in egfr mutations was observed between adenocarcinoma and squamous cell carcinoma ( p=0.018 ) .
adx - arms could detect eleven cases of exon 19 deletion , 14 cases of exon 21 l858r mutations , two cases of exon 20 point mutations ( s768i ) , and one case of exon 20 insertion .
both exon changes such as exon 19 deletion and exon 20 t790 m mutations , or exon 18 g719x and exon 20 s768i mutations could be detected simultaneously in the same case ( table 1 ) .
thirty - five wild - type egfrs were also noted , and there were two invalid results , mainly due to the fact that there was little tissue available ; as such , the concentration of extracted dna was very low ( 2.54 ng/l and 2.04 ng/l ) .
we then compared the mutation status between adx - arms and immunostaining - based egfr .
of the 26 patients with positive qds - ihc staining , all of the egfr mutations ( exon 19 deletion and exon 21 l858r mutation ) were detected by adx - arms ; however , adx - arms could detect four cases of exon 18 or exon 20 changes .
if the adx - arms results were true , the sensitivity of qds - ihc in detecting egfr mutations , as compared with adx - arms , was 86.7% ( 26/30 ) ; the specific for both antibodies was 100.0% ( 26/26 ) .
two cases with the exon 21 l858r point mutation , as identified by qds - ihc , were negatively stained by ihc using the egfr mutation - specific antibody .
ihc sensitivity was relatively low ( 80.0% ; 24/30 ) and the specificity was 92.31% ( 24/26 ) . when detecting egfr mutations , qds - ihc and adx - arms demonstrated perfect consistency (   = 0.882 ; p<0.01 ) .
excellent agreement was observed between ihc and adx - arms when detecting egfr mutations (   = 0.826 ; p<0.01 ) .
the superior optical and electronic properties of qds over conventional organic dyes , such as high brightness , high photostability , continuous absorption , narrow emission bandwidth , and the ability to simultaneously excite multiple fluorescent colors , make them attractive labels for the development of qds - ihc imaging for multiplexing cancer biomarker detection on ffpe tissues.24,25 in our study , we confirmed that qds - ihc is a simple and standardized method for detecting egfr mutations , and it has high sensitivity and specificity when compared with real - time pcr .
in addition , the development of specific antibodies against egfr mutation proteins might be useful for the diagnosis and treatment of lung cancer .
in general , patients with advanced nsclc ( stage iiib ) do not benefit from surgery alone and are best managed by initial chemotherapy , chemotherapy plus radiation therapy , or radiation therapy alone .
egfr mutation status plays a critical role in the therapeutic decision making for these patients . nowadays
, egfr - tkis have been recommended as a first - line therapy in nsclc patients with activating mutations of egfr , including gefitinib , erlotinib , afatinib , and so on .
several clinical studies have recently shown that egfr - tkis are superior to chemotherapy in nsclc patients with an egfr - activating mutation.26,27 therefore , it is highly important to evaluate the egfr mutation status in advanced nsclc patients , especially before any clinical therapy decision is undertaken .
exon 19 del e746-a750 and exon 21 l858r point mutations represent the majority of egfr mutations.27,28 analysis of egfr mutations has become an important tool for targeted therapy in lung cancer , and recently , many efforts have been made to find a more specific and sensitive method ( including arms and ngs ) to detect them.29,30 when compared with conventional dna direct sequencing , targeted ngs provides a more accurate and clinically useful molecular classification method for lung adenocarcinoma.30 however , these techniques are relatively expensive for routine use in clinical laboratories , and they depend on the quality of the samples . the goal of our study was to evaluate the accuracy and sensitivity of qds - ihc in detecting egfr mutations in nsclc when compared with traditional ihc and arms .
our results showed that egfr mutations in 40.0% ( 26/65 ) of nsclc patients were detected by qds - ihc , 12 ( 46.2% ) cases showed e746-a750-specific staining , and 14 ( 53.8% ) patients were l858r mutant - specific .
we observed nearly perfect consistency between the positive immunostaining results of qds - ihc and adx - arms when detecting egfr mutations status .
the sensitivity of qds - ihc when detecting egfr mutations , as compared with adx - arms , was 86.7% ( 26/30 ) ; both demonstrated antibody specificity of 100.0% ( 26/26 ) .
however , ihc sensitivity was relatively low ( 80.0% ; 24/30 ) and the specificity was 92.31% ( 24/26 ) . despite the small number of cases in our study , we also identified that qds - ihc combined with egfr mutation - specific antibodies to detect an egfr mutation has a high specificity and sensitivity in nsclc patients .
furthermore , when compared with adx - arms and traditional ihc , qds - ihc for egfr mutation detection can be performed on very small biopsy specimens and pleural effusion cell blocks .
this is especially the case in instances when the number of tumor cells of these precious biopsy samples could not reach dna or rna extraction requirements . according to our knowledge ,
this was the first report on the detection of egfr mutations using qds - ihc in nsclc patients .
our study also showed that these methods could precisely detect egfr exon 19 deletion or exon 21 l858r mutation status ; adx - arms had the best sensitivity , which could also detect exon 18 point mutation and exon 20 insertion .
nevertheless , adx - arms was more expensive and required strict conditions for routine use in clinical laboratories , which is difficult to carry out and popularize in a basic hospital setting .
lung adenocarcinoma showed higher egfr mutations than squamous cell carcinoma ; this mutation was identified by three methods , and it was found that adenocarcinoma exhibits different biological behaviors and requires a different therapeutic strategy .
in the present study , we found that qds exhibit excellent photostability , a broad excitation spectrum , and a long fluorescence lifetime.20,31 qds - ihc could accurately detect egfr mutation protein localization in nsclc . taken together
, the qds - ihc technique achieves levels of sensitivity and specificity that are sufficient for detecting egfr mutation signals in ffpe surgery , biopsy , and cell block specimens , while minimizing costs and optimizing therapeutic options . combining this method with adx - arms
 backgroundepidermal growth factor receptor ( egfr ) mutation status plays an important role in therapeutic decision making for non - small cell lung cancer ( nsclc ) patients . 
 since egfr mutation - specific antibodies ( e746-a750del and l858r ) have been developed , egfr mutation detection by immunohistochemistry ( ihc ) is a suitable screening test . on this basis , we want to establish a new screening test , quantum dots immunofluorescence histochemistry ( qds - ihc ) , to assess egfr gene mutation in nsclc tissues , and we compared it to traditional ihc and amplification refractory mutation system ( arms).materials and methodsegfr gene mutations were detected by qds - ihc , ihc , and adx - arms in 65 cases of nsclc composed of 55 formalin - fixed , paraffin - embedded specimens and ten pleural effusion cell blocks , including 13 squamous cell carcinomas , two adenosquamous carcinomas , and 50 adenocarcinomas.resultspositive rates of egfr gene mutations detected by qds - ihc , ihc , and adx - arms were 40.0% , 36.9% , and 46.2% , respectively , in 65 cases of nsclc patients . 
 the sensitivity of qds - ihc when detecting egfr mutations , as compared to adx - arms , was 86.7% ( 26/30 ) ; the specificity for both antibodies was 100.0% ( 26/26 ) . 
 ihc sensitivity was 80.0% ( 24/30 ) and the specificity was 92.31% ( 24/26 ) . 
 when detecting egfr mutations , qds - ihc and adx - arms had perfect consistency (   = 0.882 ; p<0.01 ) . 
 excellent agreement was observed between ihc and adx - arms when detecting egfr mutations (   = 0.826 ; p<0.01).conclusionqds - ihc is a simple and standardized method to detect egfr mutations with its high sensitivity and specificity , as compared with real - time polymerase chain reaction . 
 in addition , the development of specific antibodies against egfr mutation proteins might be useful for the diagnosis and treatment of lung cancer . 
Introduction
Materials and methods
Patients and samples
QD immunofluorescence histochemistry staining
Immunohistochemistry to detect EGFR mutation
QDs-IHC and IHC scoring
DNA extraction from NSCLC FFPE tissues and cell blocks
Detection of EGFR mutations in exon 1821 by ARMS
Statistical analysis
Results
EGFR mutation detected by QDs-IHC and IHC using mutation-specific antibodies in NSCLC
EGFR mutation detected by ADx-ARMS
Comparison of EGFR mutation detection by QDs-IHC, IHC, and ADx-ARMS
Discussion
Conclusion