non - small - cell lung cancer ( nsclc ) is the most common form of the disease , accounting for approximately 85% of all cases . in patients with nsclc , some genetic and regulatory abnormalities have been considered responsible for the tumor survival advantage , limiting the survival benefit provided by the standard therapeutic options available . therefore , drugs targeting abnormal pathways could lead to more effective treatments for this often difficult disease . since the identification of some alterations in the expression of both epidermal growth factor / epidermal growth factor receptor ( egf / egfr ) in lung cancer pathogenesis , several therapeutic targeting agents have been employed for the treatment of lung tumors overexpressing these molecules [ 5 , 6 ] . at least three different types of agent for egf / egfr inhibition are currently in use : tyrosine kinase inhibitors ( tkis ) , monoclonal antibodies ( mabs ) , and molecular cancer vaccines . among them , nimotuzumab , a humanized therapeutic monoclonal antibody that neutralize the egfr , and cimavax - egf , a molecular vaccine that induces anti - egf antibodies neutralizing endogenous egf , have demonstrated promising results in patients with nsclc , alone or combined with established modalities [ 57 ] . nevertheless , methods to identify which patients are most likely to benefit from these targeted drugs are needed . in this way , the application of adequate immunohistochemical methods could permit a better evaluation of these molecules leading patients to a more appropriate therapeutic strategy . to date , one of the most explored pretreatment biomarkers is the status of the target molecules .   for these reasons , in this work we evaluated the tissue reactivity of ior egf / r3 mab , the murine counterpart of nimotuzumab , in lung carcinomas as well as , the ability of this mab to bind the extracellular domain of egfr inhibiting cell proliferation and inducing apoptosis in a nsclc cell line . additionally , we assessed the tissue expression of egf ligand using two different mabs : cb - egf1 and cb - egf2 . a number of 71 routinely processed , formalin - fixed , and paraffin - embedded archival samples with diagnosis of lung cancer were obtained from the pathology department of the national institute of oncology and radiobiology . five microns serial sections from each block were obtained in a lizt 1512 micrometer and mounted on plus slides ( dako s2024 , carpinteria , usa ) . all sections were attached to the slide by heating in a 60c oven for 1  h. afterward the slides were kept at room temperature and they were used within 30 days . the slides were dewaxed in xylene and rehydrated in decreasing ethanol series as usually and endogenous peroxidase activity was blocked with 0.03% hydrogen peroxide in methanol for 30 minutes at room temperature . afterward all sections were washed in distilled water for 10 minutes and then were rinsed with tbs ( tris / saline buffer solution ) for 5 minutes .   in order to verify the quality of the formalin - fixed and paraffin - embedded tissues a monoclonal antibody that detects an epitope common to many the slides were pretreated with 10  mm sodium citrate buffer at ph 6.0 for 10 minutes in a microwave oven at 600  w. the rest of sections were placed in a humid chamber and pretreated with 0.4% pepsin in 0.1n hydrochloric acid solution at 37c for 30 minutes . after pretreatments , the slides were washed gently in tap water and then with distilled water and tbs , as were described above . the samples were incubated with the primary mouse ior egf / r3 ( anti - egfr ) , cb - egf1 , cb - egf2 ( anti - egf ) , and mn-116 mabs for 1  h at room temperature . negative controls were performed by substituting primary antibody for tbs and sections of colonic adenocarcinoma of known positively for these antigens were taken as positive control . after two rinses in tbs the slides were incubated with a rabbit anti - mouse biotinylated secondary antibody ( dako e0354 , carpinteria , usa ) and abcomplex / hrp ( dako e0355 , carpinteria , usa ) both for 30 minutes at room temperature dilution 1  :  100 . between incubations , afterward , enzymatic activity was visualized with dab substrate chromogenic solution ( dako k3465 , carpinteria , usa ) and the tissues were counterstained with mayer 's hematoxylin ( dako s2020 , carpinteria , usa ) . briefly , all markers were evaluated for percentage of positive cells ( 0100% ) and the intensity of reaction ( 03 + ) . the results in agreement with two observers ( cher and rb ) were considered as final . afterward , the h - score was calculated for each specimen by multiplication of the intensity of reaction and the grade of positive cells , resulting in a score ranging from 0 to 300 . subsequently , these scores were grouped as follow : 0 ( score 0 ) ; 1 ( scores < 150 ) ; 2 ( scores 150 ) . some morphological parameters such as histopathological classification , grade of differentiation , degree of cellular pleomorphism , and mitotic and necrosis indexes were evaluated for an expert pathologist ( cher ) in each tumor tissue using hematoxylin and eosin ( h / e ) staining . the degree of cell pleomorphism was evaluated considering some cytomorphologic characteristics such as cell and nuclear size , cellular shape , chromatin pattern , nucleoli , and amount of cytoplasm , and was scored as follows : 0 ( no evident cell pleomorphism ) , 1 ( low ) , 2 ( moderate ) , and 3 ( high ) cell pleomorphism . mitotic activity index ( mai ) was recorded by the evaluation of 10 high - power fields with 400x magnification ( 10x ocular , 40x objective ) in the most cellular area of the tumor ( containing the subjectively highest number of mitoses ) . finally , the mai was calculated by dividing cells out of total cells counted and expressed as previously described for cell pleomorphism . for the evaluation of the degree of tumor necrosis ( necrosis index ) on each section a low - power field with 100x magnification ( 10x ocular , 10x objective ) it was scored subjectively as follows : 0 ( no necrosis ) , 1 ( less than 50% of necrosis areas per field ) , and 2 ( more than 50% of necrosis areas per field ) . a human non - small - cell lung cancer nci - h125 ( atcc crl-5801 ) cell line was kindly provided by the research direction ( center of molecular immunology ) . cells were grown in dulbecco 's modified eagle 's media ( 12800 - 017 , gibco invitrogen co. , usa ) supplemented with 5% heat - inactivated fetal bovine serum ( 100082 , gibco invitrogen co. , usa ) . cells were maintained at 37c in a humidified atmosphere of air containing 5% co2 and were harvested using a trypsin - edta solution ( t-3924 , sigma - aldrich co. st . cells were seeded in a 6-well cell culture dishes at a density of 100.000 cells / well , in culture medium containing 5% fbs , and incubated at 37c and 5% co2 . twenty four hours later , the medium was changed to 1% fbs for serum starvation , and cells were exposed to different concentrations ( 12.5 , 25 , 50 , 100 , and 200  g / ml ) of the murine anti - egfr antibody ior egf / r3 for 96  h. an irrelevant mab was used as negative control ( t1  h mab raised against cd6 ) . also , additional doses of each antibody were added when freshly medium was replaced according to the following experimental design : treatment 1 ( no additional dose ) , treatment 2 ( one additional dose after 48  h ) , and treatment 3 ( additional doses every day ) . the cells were harvested by trypsinization , washed and resuspended in 0.5  ml facsflow ( becton - dickinson , usa ) . afterward , cells were incubated for 30 minutes with 0.5  ml of cold absolute ethanol ( hs002 - 97003 , spectrum chemical mfg co. , usa ) at 4c . for cell proliferation analysis , fixed cells were stained with propidium iodide / rnase solution for at least 30 minutes at room temperature . the percentage of cells in sub - g0-g1 , g0-g1 , s , and g2-m phases was analyzed using a facscan flow cytometer ( becton - dickinson , san jose , ca , usa ) equipped with a doublet discrimination module ( ddm ) , cellquest software ( version 2.0 ) and modfit lt ( verity software house , version 2.0 ) , respectively . to determine the apoptotic population , the sub - g0-g1 peak was also measured . each experiment was repeated at least twice on different days using different sub - cultures of nci - h125 cell line . the correlation between the reactivity of cb - egf1 and cb - egf2 mabs was assessed by spearman ranks correlation coefficients . the percent of cells in sub - g0-g1 , g0-g1 , s , and g2-m phases of the cell cycle according to the experimental design was compared using two - way anova followed by bonferroni post hoc test . for all tests , p < the percent of cell growth inhibition was calculated using the average of cell in s phase , according to the following formula : % of cell growth inhibition = ( control sample  treatment sample)/control sample 100 . the median patient age at presentation was 55.65 years ( ranged from 23 to 86 years ) . median overall survival of the population was 25.52 months ( ranged from 0.93 to 57.17 ) for sclc , 30.16 months ( ranged from 0.70 to 40.57 ) for patients with stage i , ii , or iiia nsclc , and 18.86 months for patients with stage iiib or iv nsclc . the stage was found to be associated with the overall survival of nsclc patients ( p = 0.0405 ; log rank test ) ( figure 1 ) . the tissues showed a good morphology , preserving undamaged the molecular antigenic determinants by means of the immunoreactivity of the monoclonal antibody against cytokeratin . no correlation between the egfr expression and the overall survival of nsclc patients was observed ( p = 0.9560 ; log rank test ) ( figure 2 ) neither with tumor and patient characteristics ( data not shown ) . nevertheless , a statistically significant difference was obtained when the overexpression of egfr in stage i  iiia and iiib  iv was compared ( p = 0.0264 ; chi - square test ) . the pattern of staining of ior egf / r3 mab was finely granular and was mainly located in the plasmatic membrane of malignant cells , although their cytoplasm was also decorated ( figures 3(a ) and 3(b ) ) . the reactivity of ior egf / r3 mab was evidenced in 37/59 ( 62.71% ) of nsclc samples . according to the histopathological classification , 11/20 ( 55.00% ) squamous cell carcinoma , 13/23 ( 56.52% ) adenocarcinoma , 5/6 ( 83.33% ) large cell carcinoma , and 8/10 ( 80.00% ) of other minor types represented were recognized by the ior egf / r3 ( table 3 ) . we used two different anti - egf ligand monoclonal antibodies ( kindly provided by the center for genetic engineering and biotechnology , havana , cuba ) . the percent of positive cases according to the histopathological classification of the tumors is extensely described in table 4 . the pattern of staining of these mabs was finely granular and mainly located in cytoplasm , although a membrane staining was also observed . in addition , an extracellular pattern of staining was detected ( figures 3(c ) and 3(d ) ) . a slight increase in the intensity of reaction noteworthy , a significant correlation was detected when the reactivity of cb - egf1 and cb - egf2 mabs were compared ( p < 0.0001 , rs = 0.5429 ; spearman test ) . however , no correlation between the egf expression and the overall survival of patients was observed for both cb - egf1 and cb - egf2 mabs ( p = 0.9144 and p = 0.2706 ; log rank test , resp . ) . no differences were also evidenced when the reactivity of both cb - egf1 and cb - egf2 mabs were compared in stage i  iiia and iiib  iv ( p = 0.8154 and p = 0.2743 ; chi - square test , resp . ) . the dual expression of both egfr and egf was observed in 2/12 ( 16.67% ) and 34/59 ( 57.63% ) of sclc and nsclc , respectively . a significant correlation was detected when the expression of egfr was compared with the reactivity of cb - egf1 mab in nsclc samples but not in sclc ( p = 0.0006 , rs = 0.4319 and p = 0.3784 , rs = 0.2798 , respectively ; spearman test ) . similar results were obtained with cb - egf2 mab ( p = 0.0035 , rs = 0.3740 and p = 0.5918 , rs = 0.1725 ; spearman test ) . additionally , no correlation between the dual expression of both egfr and egf with tumor and patient characteristics was obtained ( data not shown ) neither with the overall survival of nsclc patients ( p = 0.7321 and p = 0.9235 ; log rank test ) . we evaluated the ability of ior egf / r3 mab to mediate specific biological functions such as cell growth inhibition and induction of apoptosis in a non - small - cell lung cancer cell line . growth curve profiles were evaluated in nci - h125 following the addition of different antibody concentrations of ior egf / r3 ( 12.5 , 25 , 50 , 100 and 200  g / ml ) in a period of 48 , 72 and 96  h. no differences in the inhibition of cell proliferation were detected after 48 and 72  h ( data not shown ) . the percent of inhibition ranged from 50.30 to 65.64% ( s phase ) as compared to the irrelevant control , obtaining the maximum of cell growth inhibition at 100  g / ml ( figure 4(a ) ) . no induction of apoptosis was observed with this experimental design ( sub g0-g1 peak , p = 0.3062 ; anova ) . to investigate whether the biological activity of ior egf / r3 is dose - dependent , additional doses of mabs were added when freshly medium was replaced . the addition of ior egf / r3 caused a significant inhibition on cell proliferation 48  h after mab was added to the culture ( treatment 2 ) ( s phase , p = 0.0015 ; anova ) , reaching the maximal inhibition at 96  h ( treatment 3 ) ( s phase , p = 0.0053 ; anova ) , as compared to the treatment 1 ( s phase , p = 0.0149 ; anova ) used as control . the exposure to ior egf / r3 mab caused a more marked inhibition on proliferation in a dose - dependent manner . curiously , after 96  h of incubation and adding additional doses of ior egf / r3 mab ( treatment 3 ) , an increase in the percentage of cells undergoing apoptosis was obtained as compared to the irrelevant mab ( sub - g0-g1 peak , p = 0.0290 ; anova ) ( figures 4(c ) and 4(d ) ) . up to date , tnm staging system ( tumor , node , metastasis ) after surgery is considered the most important prognostic factor in nsclc [ 12 , 13 ] . tnm staging system also leads the clinicians to the selection of a more appropriate conventional treatment ( e.g. , surgery , radiation , chemotherapy ) for nsclc patients . in this study , we showed an increased overall survival of patients at stages i  iiia as compared with the group of patients at stages iiib  iv . it is known , the prognosis of patients at stages iiib  iv nsclc is poor due to the effectiveness of the conventional modalities of treatment . therefore , the application of newer treatment modalities in order to improve patient survival and overall quality of life is mandatory [ 12 , 13 ] . consequently , in the last years the egfr pathway inhibition has been accepted as an option for the first- , second- and third - line therapy of nsclc .   in nsclc , the overexpression of egfr has been reported to be ranging from 40% to 89% [ 15 , 16 ] . here , we obtained the overexpression of egfr in about 62% of nsclc using the ior egf / r3 mab . the ability of ior egf / r3 mab to recognize human egfr in frozen lung carcinoma tissues by immunohistochemistry was previously reported . moreover , the efficacy of the 99mtc - labeled ior egf / r3 mab for the detection of epithelial - derived tumors , their metastases and recurrences by radioimmunoscintigraphy was previously evaluated . in addition , no correlation between the expression of egfr and the overall survival of patients was observed . the overexpression of egfr has been associated with a more aggressive disease and reduced survival in a variety of tumors types , but in nsclc the evidences are less convincing . additionally , we showed that blocking egf binding to the receptor , in a nsclc - derived cell line ( nci - h125 ) by means of ior egf / r3 significantly decreased tumor cell proliferation and induced apoptosis . the ior egf / r3 mab recognizes an epitope located in the extracellular domain of the human egfr with high affinity . nevertheless , it is known that therapy with murine - derived mabs is limited by their tendency to develop human anti - mouse antibodies response ( hama ) . for this reason , a humanized therapeutic version of ior egf / r3 mab ( nimotuzumab ) was developed in our center . in clinical trials , nimotuzumab has exhibited promising results used as a sensitizer to radio- or chemotherapy in advanced nsclc patients positive for egfr expression [ 23 , 24 ] .   usually , the selection of patients to any anti - egfr therapy using a neutralizing mab is based on the tissue reactivity of a different anti - egfr mab for diagnostic purposes . on the contrary , nimotuzumab was obtained by transplanting the complementary determining regions ( cdrs ) of the ior egf / r3 mab to a human framework . nimotuzumab and egf / r3 showed a very similar immunohistochemical pattern of recognition of fetal , adult and some malignant tissues , including sclc and nsclc . in this way , the evaluation of egfr expression by ihc using the ior egf / r3 mab could provide more specific information in the selection of nsclc patients to nimotuzumab treatment . in a previous report , egfr was significantly more expressed in stage iiia when compared to earlier stages ( i - ii ) , suggesting that expression increases stepwise from precancerous lesions to more advanced stages of cancer . nevertheless , in this work , patients at stage i  iiia displayed higher level of egfr expression as compared with those at stages iiib  iv . interestingly , we obtained a significant correlation when the expression of egfr and egf was compared , although , no differences in the expression of egf between nsclc stages were evidenced . it is known , the upregulation of egfr expression by egf is considered a mechanism that promotes the development and progression of tumors . our results permit to suggest a major activation state of the egf / egfr system in earlier stage of the disease , due to egfr is mainly activated by the binding of its ligands . in line with this , some authors have suggested that growth factor / receptor loop is more important for lung tumor formation than for tumor progression [ 28 , 29 ] . notoriously , the dual expression of egfr and egf was observed in about 60% of nsclc patients . in this way , our data also support the potential use of combined passive and active immunotherapy in tumors overexpressing both molecules , not depending of the stage of the disease . on the other hand , the identification of deregulated expression of egf family ligands in lung cancer pathogenesis has permitted to consider their potential use as therapeutic targeting agents . here , we found the tissue overexpression of egf in at least the 70% of nsclc samples , although no correlation between the egf expression and the overall survival of patients was obtained . in a previous report , both increased levels of serum egf as well as increased reactivity to egf were found in nsclc patients . nevertheless , a correlation of higher level of serum egf and poorly overall survival of patients was evidenced . nevertheless , we obtained statistical significant differences when reactivity of cb - egf1 and cb - egf2 mabs were compared . in a previous report using competition assays , it was demonstrated that cb - egf1 and cb - egf2 bind to different antigenic determinants of egf . while cb - egf1 is a highly specific mab to human egf , the cb - egf2 mab also reacts with the murine egf . these results could support the major reactivity of cb - egf1 mab evidenced in our study . interestingly , an increasing in the overall survival of advanced nsclc patients treated with cimavax - egf has been reported [ 6 , 7 ] . in this way , the evaluation of egf expression on both serum and tumor section using the cb - egf1 mab could lead to a better selection of nsclc patients to cimavax - egf therapy . finally , the egfr is commonly overexpressed in nsclc , but it is rare in sclc [ 15 , 31 , 32 ] . in our study , we obtained the overexpression of egfr in about 33% of sclc . by the contrary , the immunostaining was observed mainly located in the plasmatic membrane of malignant cells . additionally , we detected the expression of egf in about 17% of tumors , although no correlation between the expression of egfr and egf was detected . previously , kaseda et al . published both no egf binding activity in 6 sclc using 125i - egf joint to no egfr amplification in those specimens tissues . nevertheless , the activity of the egfr pathway in sclc has been previously demonstrated . in spite of the fact that our data is very small ; the exploration of the potential use of both egfr and egf as target for sclc immunotherapy could be of interest . in this way , experiments in order to evaluate the functionability of the egf / egfr system in sclc are being planned in our laboratory . in summary , we reported the tissue reactivity of ior egf / r3 mab , the murine counterpart of nimotuzumab , in both sclc and nsclc . the anti - proliferative activity and the capacity to induce apoptosis of egf / r3 mab in a nsclc cell line after binding the extracellular domain of egfr were also demonstrated . in addition , we showed the immunohistochemical recognition of two different anti - egf ligand monoclonal antibodies as well as their correlation with the expression of egfr . the dual expression of both egf and egfr support the potential use of passive and active immunotherapy against these molecules alone or combined with established modalities . moreover , our data permit to consider the development of diagnostic kits using ior egf / r3 mab and cb - egf1 mabs in order to a better selection of patients to nimotuzumab and cimavax - egf treatments , respectively .
adequate methods to identify which lung cancer patients are most likely to benefit from the targeted drugs against both epidermal growth factor receptor / epidermal growth factor ( egfr / egf ) are needed . 
 for this reason , we evaluated both the tissue reactivity of ior egf / r3 monoclonal antibody ( mab ) in human lung carcinomas and its biological activity in nci - h125 cells . 
 additionally , we assessed the tissue expression of egf using two mabs , cb - egf1 and cb - egf2 . 
 the overexpression of egfr was detected in 33.33% and 62.71% of small - cell lung carcinoma ( sclc ) and non - small - cell lung carcinoma ( nsclc ) , respectively . 
 the ability of ior egf / r3 mab to bind the extracellular domain of egfr inhibiting cell proliferation and inducing apoptosis in nci - h125 cells was also demonstrated . 
 the egf expression was observed in about 17% and 70% of sclc and nsclc , respectively . 
 however , differences in the reactivity of cb - egf1 and cb - egf2 were evidenced . 
 a dual expression of egfr and egf was observed in 16.67% and 57.63% of sclc and nsclc patients , respectively . 
 but , a correlation between them was only obtained in nsclc . 
 our results permit to recommend the development of diagnostic kits using ior egf / r3 and/or cb - egf1 mabs in order to achieve a better selection of patients to egfr / egf - targeting treatment .