various investigations have focused on it due to its biological activities , high tendency to recur and different growth mechanisms in comparison with other cystic lesions .
many studies have been conducted on cell - cycle associated proteins such as antiapoptotic markers ( tp53 , bcl-2 ) and cell proliferation markers ( ki67 , proliferating cell nuclear antigen , epidermal growth factor receptor [ egfr ] ) .
these studies have shown different biological and proliferative activities for the lining epithelium of kcot .
bcl-2 , which is located in the external membrane of mitochondria , endoplasmic reticulum and nuclear membrane , play an important role in apoptosis .
uncontrolled expression of bcl-2 has been found before histopathologic changes in the early stage of neoplasm .
epidermal growth factor receptor is the most important growth factor ligand on the cell surface .
overexpression of egfr - related genes is seen in many neoplasms , which causes the over sensitivity of cells to the normal level of growth factor .
therefore , it causes specific phenotypes of cells , which can affect the cellular reaction to the treatment .
kichi et al . have reported that bcl-2 is seen only in kcot and not in dentigerous cyst .
sandra et al . have reported strong expression of bcl-2 in ameloblastoma and suggested that antiapoptotic proteins are expressed more than apoptotic proteins in ameloblastoma . in a study done by shear , expression of egfr marker
the strongest reaction was related to kcot , and the weakest was in the radicular cyst .
indeed , because of specific activity of kcot in comparison with other odontogenic cysts , it is possible to evaluate the expression of egfr and bcl-2 proteins in kcot compared with dentigerous cyst and ameloblastoma .
expression of these proteins provides us with the knowledge about the biological activity of kcot . in this study ,
the expression of egfr and bcl-2 proteins in kcot was evaluated and compared with dentigerous cyst and ameloblastoma .
in a cross - sectional study , paraffin - embedded tissues of 16 kcot , 16 dentigerous cysts , and 16 ameloblastoma samples were obtained from the archives in department of oral pathology , dental school , isfahan university of medical sciences . in order to detect the specific antigens of egfr and bcl-2 ,
briefly , the main procedures were : serial sectioning ( in 3 - 4 um sections ) , deparafinization , rehydration , and antigen retrieval .
all specimens were then placed in the phosphate - buffered saline ( pbs ) and treated for 5 min in protein block solution to prevent any false staining .
the specimens were then incubated for 30 min with primary antibody of egfr ( ncl - egfr-384 ) clone egfr25 , bcl-2 ( ncl - bcl-2 - 486 ) clone 3.1 ( novocastra / germany ) .
furthermore , the sections were exposed to novolink polymer ( re7112 ) or a secondary antibody for 30 min and washed with pbs .
they were then incubated for 5 min with diaminobenzidine for visualization . after washing the slides
finally , the slides were mounted after being dried . to quantify the percentage of positive cells within the lesions of egfr and bcl-2 markers ,
the sections were observed by two oral pathologists separately by olympus light microscope ( cx21fs , tokyo , japan ) at 400 magnification .
the score of egfr staining and bcl-2 in each specimen / layer was calculated by the following formulas : 
 score of egfr staining in each layer = d  iscore of egfr staining in each specimen = td  tidistribution ( d ) : mean percentage of stained cells by 1000 cells in each layerintensity of staining in each layer ( i ) : negative ( 0 ) , weak ( + 1 ) , moderate ( + 2 ) , severe ( + 3)pattern of staining in each specimen ( p ) : membranous , cytoplasmic , membranous  cytoplasmictotal distribution of each ( td ) : mean distribution of the total layertotal intensity ( ti ) : mean intensity of total layer . 
 
score of egfr staining in each layer = d  i score of egfr staining in each specimen = td  ti distribution ( d ) : mean percentage of stained cells by 1000 cells in each layer intensity of staining in each layer ( i ) : negative ( 0 ) , weak ( + 1 ) , moderate ( + 2 ) , severe ( + 3 ) pattern of staining in each specimen ( p ) : membranous , cytoplasmic , membranous  cytoplasmic total distribution of each ( td ) : mean distribution of the total layer total intensity ( ti ) : mean intensity of total layer . according to the distribution ,
the intensity of bcl-2 staining was classified as below : d / td intensity < % 1:0/10 - 25%:+1/25 - 50%:+2/>50%:+3 .
whitney and kruskul  wallis tests using spss software ( spss inc . , chicago , il , usa ) .
the results of immunohistochemical staining of egfr marker showed that none of the kcot specimens expressed egfr with the score of 0 .
dentigerous cysts also indicated the score of 1.2 [ figures 1 and 2 , table 1 ] .
there was no difference between the intensity of egfr expression in ameloblastoma and dentigerous cyst specimens . on the other hand ,
91.8% of cells in ameloblastoma and 74.8% of cells in dentigerous cyst expressed this protein .
the pattern of expression in 94% of ameloblastoma specimens was membranous , and it was membranous - cytoplasmic in 100% of dentigerous cysts specimens .
there was a significant difference between egfr expression in peripheral and internal layers of ameloblastoma .
98.4% of the peripheral layer cells and 85.3% of the internal layer cells expressed egfr protein .
the intensity of egfr expression was also significantly different in peripheral and internal layers ( p = 0.01 ) . in the evaluation of different layers of dentigerous cysts ,
the scores of egfr expression in basal cells versus supra - basal and superficial layers were 1.85 and 0.47 , respectively .
( p < 0.01 ) the results of immunohistochemical evaluation of bcl-2 marker revealed that the intensity of bcl-2 expression was not significantly different in ameloblastoma and kcot .
the intensity of bcl-2 expression in ameloblastoma showed statistically significant differences compared with dentigerous cyst [ figures 3 and 4 , table 2 ] .
expression of bcl-2 protein with high intensity in all of the ameloblastoma specimens ( as a neoplastic lesion ) and kcot ( as a cystic lesion with neoplastic features ) indicated the abnormal control of cell - cycle in these two lesions .
this result can be due to the increase of cell vitality and invasive growth pattern of the lesion , which is similar to those reported by lo muzio et al .
strong expression of bcl-2 in the peripheral layers and minimal expression in the central layers of ameloblastoma indicated the high potential of cell vitality in peripheral layers .
it can be argued that bcl-2 has an important role in the survival of main cells in the peripheral layers of ameloblastoma .
higher expression of bcl-2 in the basal layer of kcot is related to the physiological potency of the basal layer as a cell source for epithelium . according to the present results , no expression of egfr ( as the most important protein in neoplastic proliferation )
no expression of this protein and high level of bcl-2 protein expression show that aggressive features of kcot are related to apoptotic proteins .
however , the aggressive potential of kcot is not as severe as that of a neoplasm such as ameloblastoma .
indeed , the significant expression of antiapoptotic proteins with lack of proliferative proteins can cause a neoplastic condition with less aggressive features .
high and severe expression of egfr and bcl-2 in ameloblastoma shows the neoplastic nature of this lesion .
, egfr expression causes an increase in the proliferation and aggressive activity of this lesion .
therefore , its treatment needs aggressive surgical techniques . in aggressive ameloblastoma cases , control of proliferation and cell vitality
the goal of investigations is to find less aggressive methods such as a monoclonal antibody of anti - egfr protein to prevent functional damage of the chewing system and deformity caused by surgical procedures .
anti - egfr drugs and radiotherapy have synergistic effects in controlling the neoplasm and decreasing the recurrence . in physiological conditions
all cases of dentigerous cyst indicated a membranous - cytoplasmic expression of egfr with lower value and distribution than ameloblastoma .
kichi et al . have reported the high distribution of tdt - mediated dutp - biotin nick end labeling positive cells in the superficial layers and lack of these cells in the basal and middle layers of dentigerous cyst . however , these findings show apoptosis in the superficial layer of dentigerous cyst . furthermore , in ameloblastoma ,
more severe expression and higher distribution of egfr protein in the peripheral layer of ameloblastoma have the same pattern as bcl-2 expression compared with the central layer .
li et al . have reported that the proliferative nature of cells decreases from the peripheral layer to the central layer of tumoral mass .
biological activities , high tendency to recur and growth mechanisms of kcot are different in comparison with other cystic lesions that are related to apoptotic proteins .
however , the aggressive potential of kcot is not as severe as that of the neoplasms such as ameloblastoma .
 background : keratocystic odontogenic tumor ( kcot ) is a developmental odontogenic cyst on which various investigations have been focused due to its biological activities , high tendency to recur and different growth mechanisms in comparison with other cystic lesions . 
 previous studies have shown different biological and proliferative activities for the lining epithelium of kcot . 
 the aim of this study was immunohistochemical evaluation of bcl-2 and epidermal growth factor receptor ( egfr ) expression in kcot compared with dentigerous cyst and ameloblastoma.materials and methods : formalin - fixed and paraffin - embedded tissue sections of 16 cases of kcot , 16 cases of dentigerous cyst and 16 cases of ameloblastoma were immunohistochemically analyzed to determine bcl-2 and egfr proteins expression . 
 biotin - stereotavidin method was used . 
 it was observed by two oral pathologists separately , and the data were analyzed by mann  
 whitney and kruskul  wallis . 
 p < 0.05 was considered as significant.results:regardless of staining intensity , all cases of ameloblastoma and kcot except dentigerous cases were positively stained for bcl-2 . 
 expression of bcl-2 was higher in the peripheral layer of ameloblastoma and basal layer of kcot . 
 furthermore , all cases of ameloblastoma and dentigerous cysts except kcot samples were positively stained for egfr . 
 expression of egfr was higher in the peripheral layer of ameloblastoma and basal layer of dentigerous cysts.conclusion:according to the expression of  bcl-2 in ameloblastoma and kcot , and no expression of egfr in kcot , it can be concluded that the biological activity and growth mechanisms of kcot are different compared with other cystic lesions . 
 however , the aggressive potential of kcot is not as severe as that of a neoplasm such as ameloblastoma . 
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION