the formation of cysts in the jaws possesses 
 unique features regarding their pathology and implicates the odontogenic 
 tooth - forming apparatus . in most classification schemes odontogenic 

cysts are divided in two major categories , namely the developmental 
 cysts and the cysts that are initiated in areas of acute or chronic 
 inflammation , with odontogenic keratocysts ( okc ) and periapical or 
 radicular cysts ( pacs ) as the most common types , respectively . the 
 world health organization ( who ) reclassified okc in its 2005 edition of 
 histological classification of odontogenic tumours and according to this 
 reclassification , the okc is now considered as a kcot .
it is defined as 
  a benign uni- or multicystic , intraosseous tumour of odontogenic 
 origin , with a characteristic lining of parakeratinized stratified 
 squamous epithelium that bears the potential for aggressive , 
 infiltrative behaviour  , a term that better reflects its neoplastic 
 nature .
several factors form the basis of this decision : a. the 
 clinical behaviour of the lesion , since kcot is locally destructive and 
 highly recurrent , b. the histopathologic characteristics , considering 
 that the basal layer of the kcot budding into connective tissue , in 
 addition to the mitotic figures that are frequently found in the 
 suprabasal layers [ 1,3 - 5 ] , and finally c. genetic alterations , are the 
 most important parameters .
they occur most commonly in the mandible , 
 especially in the posterior body and ramus regions
. it may be 
 solitary or multiple and the latter is usually characterizing the 
 inherited nevoid basal cell carcinoma syndrome  nbccs  .
kcots have a 
 high recurrence rate , ranging between 25% and 60% while when associated 
 with nevoid basal cell carcinoma syndrome  nbccs  or gorlin - goltz 
 syndrome , the recurrence rate is about 82% .
so far , there is only 
 limited evidence regarding the pathogenesis of the developmental cystic neoplasms 
 of the jaws and for this reason they are considering of unknown 
 aetiology .
the endoplasmic reticulum ( er ) is an 
 organelle with a major role in the synthesis of lipid and proteins and 
 leads many cellular processes such as organogenesis , transcriptional 
 activity , stress responses , and apoptosis [ 10 - 14 ] .
er is responsible for 
 the proper folding of the newly synthesized proteins that is facilitated 
 with the assistance of various er chaperones
. unfolded or 
 malfolded proteins are disposed by mechanisms implicating er - associated 
 protein degradation ( erad ) . when the amount of unfolded protein exceeds 
 the folding capacity of the er , human cells activate a homeostatic 
 defence mechanism designated as the upr that follows er stress [ 11 - 16 ] . among the various consequences of upr
is 
 also the upregulation of bip / grp78 and of other chaperones that is 
 considered diagnostic for the induction of er stress in a given tissue 
 [ 16 - 18 ] .
bip / grp78 binds to the hydrophobic region of unfolded proteins 
 via a substrate - binding domain and facilitates folding through 
 conformational change evoked by the hydrolysis of atp by the atpase 
 domain .
high mannose type 
 oligosaccharide is attached en 
 bloc to most proteins 
 synthesized in the er , and then trimmed sequentially [ 21 - 23 ] . when two 
 glucose residues are trimmed by glucosidase i or ii and the protein 
 contains only one glucose residue , calnexin and calreticulin bind and 
 fold the client protein .
when the last glucose residue is 
 trimmed by glucosidase ii , the client is released from calnexin and 
 calreticulin , and binds to udp - glucose - glycoprotein glucosyltransferase 
 .
if the protein is folded properly , it is released from the 
 enzyme and transported to the golgi apparatus .
if it is not folded 
 appropriately , udp - glucose - glycoprotein glucosyltransferase attaches one 
 glucose residue and returns it to calnexin and calreticulin .
calnexin and calreticulin 
 share a similar molecular structure and function , although they are 
 transmembrane and luminal proteins , respectively . considering the neoplastic nature of kcots , 
 in combination with their poorly defined aetiology we explored if er 
 stress is involved in disease development . specifically ,
we evaluated 
 the expression of the chaperones , bip / grp78 and calnexin in a panel of 
 kcots as compared to pacs and fbs . both of these markers is considered 
 to accurately reflect the induction of er stress which has been 
 associated with neoplastic development .
paraffin - embedded tissue specimens of kcots 
 ( 24 cases ) , pacs ( 9 cases ) and fibromas ( 5 cases ) were randomly selected 
 from the archives of the department of oral pathology , of the national 
 and kapodistrian university of athens , dental school spanning the years 
 2006 - 2011 and were analyzed by immunohistochemistry .
we have analyzed 
 the expression of chaperones bip / grp78 and calnexin in a panel of 24 
 kcots and 9 pacs .
in addition we have also 
 included in our analysis 5 fbs as controls since they represent lesions 
 of the connective tissue devoid of pathological findings in the 
 epithelium .
details of ethics approval no ethical issues are related to this study 
 since only paraffin - embedded archival material has been used and no data 
 related to the patients clinical records have been disclosed . therefore 
 the study did not require review by the institutional review board of 
 the university of athens .
immunohistochemistry was carried out in 
 formalin fixed , paraffin embedded tissue specimens . the antibodies used 
 were monoclonal rabbit anti - bip ( c50b12 ) , by cell signaling technology ; 
 1:100 and monoclonal mouse
anti - calnexin ( sc-46669 ) , by santa cruiz 
 biotechnology , santa cruz , ca , usa ; 1:75 .
immunostaining was performed 
 by using the superpicture polymer ( dab ) kit ( novocastra ) , following the 
 manufacturer s instructions . before evaluation
specimens 
 were evaluated blindly from two authors of the study ( i.c . , pathologist 
 and s.m .
the positive 
 immunohistochemical staining , was graded semiquantitatively by using a 
 5-tier scoring system and classified according to the intensity of the 
 labelling as : negative ( - ) , marginal ( + /-
) , mild ( + ) , moderate ( + + ) and 
 intense ( + + + ) .
in all specimens analyzed and for both 
 antigens , immunopositivity was relatively homogenous among cells and 
 varied only in terms of intensity .
as shown in 
 table 1 , bip / grp78 
 immunopositivity was detected in 18 out of 24 ( 75% ) kcots .
) in one sample , mild ( + ) in 10 , moderate ( + + ) in 6 and 
 very intense ( + + + ) in one specimen . with the exception of 3 specimens 
 exhibiting moderate or very intense immunopositivity and at which 
 bip / grp78 expression was primarily localized in the upper layers of the 
 epithelium ( figure 1a ) , in all other cases bip / grp78 immunopositivity 
 spanned full thickness of the epithelium ( figure 1b ) . as opposed to 
 kcots , pacs exhibited bip / grp78 immunopositivity in only 1 out of 9 
 ( 13% ) cases ( figure 2a ) while all five fbs were negative for bip / grp78 
 expression ( figure 2b ) suggesting that the overexpression of bip / grp78 
 in kcots was statistically significant ( p < 0.001 , x - test ) .
immunohistochemical 
 expression of bip / grp78 in keratocystic odontogenic tumours 
 ( x10 ) .
( a ) brown colour ( dab ) indicates staining in the upper 
 layers of the epithelium or ( b ) in the full thickness with 
 stronger intensity in the upper layers .
weak counterstaining 
 with haematoxylin was performed in all samples following 
 immunostaining ( original magnification x20 ) .
the 
 microphotograph shows negative staining for bip / grp78 in the 
 epithelium of periapical cyst ( a ) and negative staining for 
 calnexin in the epithelium of a fibroma ( b ) .
weak 
 counterstaining with haematoxylin was performed in all samples 
 following immunostaining ( original magnification x20 ) .
our results that are summarized 
 in table 1 show that calnexin expression is significantly higher ( p < 
 0.001 , x - test ) in kcots since it was expressed in 11 out of 
 24 kcots ( 46% ) but only one out of 9 ( 13% ) pacs and none of the 5 fbs 
 analyzed .
) in 5 , 
 mild ( + ) in 5 and moderate ( + + ) in one specimen while the single pac 
 that was positive for calnexin expression exhibited marginal 
 immunopositivity .
expression levels and localization of bip / grp78 and calnexin in kcots , pacs and fbs kcots = keratocystic odontogenic tumors ; pacs = periapical or radicular cysts ; fbs = fibromas ; ft = full thickness of the epithelium ; ul = upper layers ; ft / ul = full thickness with stronger intensity in the upper layers . in all but 3 cases that exhibited mild 
 immunopositivity and was localized in the upper layers of the epithelium 
 ( figure 3a ) , calnexin expression was spanned the full thickness of the 
 epithelium ( figure 3b ) .
( a ) brown colour ( dab ) indicates staining in the upper 
 layers of the epithelium or ( b ) in the full thickness .
weak 
 counterstaining with haematoxylin was performed in all samples 
 following immunostaining ( original magnification x20 . considering that calnexin and bip / grp78 are 
 both indicators of er stress we asked if their expression is correlated 
 in the same specimens .
indeed , out of the 38 specimens subjected to our 
 analyses , 29 were either positive or negative for both antigens while 
 only 9 exhibited expression for either bip / grp78 or calnexin .
this 
 observation confirms that bip / grp78 and calnexin are co - expressed ( p < 
 0.001 , x - test ) .
in order to better understand the pathogenic 
 mechanisms that underline the development of kcots we have hypothesized 
 that er stress and the resulting upr may be associated with their onset . 
 in order to test this hypothesis a bank of kcots specimens
were analyzed 
 by immunohistochemistry for the expression of bip / grp78 and calnexin , 
 two widely used chaperones that are considered diagnostic for cells 
 undergoing er stress [ 19 - 21,23 ] .
the expression of bip / grp78 and 
 calnexin in the kcots were compared to that in pacs that are cysts with 
 distinct pathologic features and pathogenetic mechanism from that of 
 kcots as well as in fbs at which the cell type affected does not involve 
 the epithelium but rather the stroma .
our results show a strong 
 overexpression of the er stress markers bip / grp78 and calnexin in the 
 kcots but not in the pacs and the fbs .
furthermore , these two chaperones 
 were frequently co - expressed in the same specimens , an observation that 
 implies that the overexpression of bip / grp78 and calnexin was not 
 coincidental but rather indicative for er stress induction and execution 
 of the upr .
it is conceivable that 
 this feature of the kcots , namely the misexpression of keratin , is 
 causatively associated with er stress . for reasons
not yet well 
 understood commitment to this keratinocyte differentiation program , may 
 induce er stress and activate the upr . whether this is due to 
 the ectopic overexpression of a specific protein such as keratin(s ) or 
 it is related to the perturbation of tissue homeostasis due to aberrant 
 differentiation
however , since neither pacs nor fbs 
 display evidence of er stress it is likely that the latter is linked to 
 kcots pathogenesis . consistently with this notion , recent findings 
 showed that in the skin , at which keratinization represents a normal and 
 physiological process , er stress - related chaperones bip / grp78 and hrd1 
 was elevated in cells of normal human epidermis that contain 
 keratinocytes undergoing differentiation .
these findings suggest 
 that keratinization , either in its physiological context such as in the 
 skin or in abnormal context such as in the kcots , are associated with er 
 stress induction .
furthermore , in the same study it has been 
 demonstrated that pharmacological interference with the execution of the 
 upr affected the differentiation of keratinocytes in 
 vitro , providing further clues regarding the causative link between 
 er stress and keratinocyte differentiation .
our results are 
 consistent with these findings and suggest that er stress is induced in 
 the odontogenic epithelium when cells are committed to the keratinocyte 
 differentiation program , during kcot development .
this is also supported 
 by the observation that especially bip / grp78 and to some extent calnexin 
 immunopositivity was frequently more intense in the upper layers of the 
 epithelium at which keratinization is more prominent .
studies comparing 
 the involvement of er stress to parakeratinized normal tissues , 
 hyperplastic and precancerous / dysplastic lesions of the oral epithelium 
 would bestow insightful understanding if bip / grp78 or calnexin may be 
 involved in parakeratinization .
indeed , a variety of diseases associated 
 with aberrant keratinization , such as hereditary keratoses , including 
 darier s disease , keratosis linearis with ichthyosis congenita and 
 keratoderma syndrome , erythrokeratoderma variabilis , and ichthyosis 
 follicularis with atrichia and photophobia syndrome , have been linked to 
 upr .
it could be argued that keratinization and thus er stress 
 induction is irrelevant to the oncogenic stimulus that induces kcots 
 development .
however , to the extent that keratinization reflects 
 aberrant differentiation of the cells and considering that neoplastic 
 development is indeed a disease of aberrant differentiation , er stress 
 is causatively linked to the disease development .
the results of the present study may provide 
 clues regarding the pathogenesis of kcots .
which specific branches of 
 upr are activated during kcot development and whether its inhibition is 
 sufficient for the reversal of keratinization and regression of kcots 
 remains to be explored .
furthermore , it would be of particular interest 
 to investigate the involvement of epithelial to mesenchymal 
 transition - associated markers in the pathogenesis of kcot and their 
 potential link to er stress . in view of recent findings linking 
 mutations in ptch1 gene to kcots development , and considering that the 
 ptch1 gene s product is a secreted ligand
this is also 
 supported by the ability of ptch1 to act on post er smo and to modulate 
 its activity without affecting its overall expression levels .
the detection of er stress 
 and activation of the upr may have diagnostic value for kcot 
 characterization especially in cases at which diagnosis is not clear 
 especially in inflamed kcots   at which infiltration of the epithelium 
 by inflammatory cells masks their typical histopathologic 
 characteristics , raising issues of differential diagnosis .
more 
 importantly , considering that er stress influences keratinocyte 
 differentiation we may postulate that inhibition of upr may have 
 adjuvant therapeutic value for the management of kcots , particularly 
 those with aggressive behaviour and often recurrences , at which surgery 
 is the treatment of choice .
indeed , recently developed chemical 
 chaperones that can inhibit specific branches of the upr may be 
 beneficial for the therapy of kcots patients .
this is the first demonstration of the 
 involvement of endoplasmic reticulum stress in the pathogenesis of 
 keratocystic odontogenic tumours .
understanding the precise mechanism by 
 which endoplasmic reticulum stress is involved the development of 
 keratocystic odontogenic tumours may find application in the diagnosis 
 and management of the disease .
this study was supported by a research grant 
 from the empeirikion foundation ( granted to i.c . ) .
 abstractobjectives odontogenic keratocysts ( okcs ) are developmental 
 cysts that have been reclassified according world health organization 
 ( who ) , to keratocystic odontogenic tumours ( kcots ) , a term that better 
 reflects their neoplastic nature . the aim of present study is to 
 evaluate the induction of stress of the endoplasmic reticulum and 
 execution of the resulting unfolded protein response in keratinocystic 
 odontogenic tumours.material and methodswe analyzed by 
 immunohistochemistry the expression of the chaperones bip / grp78 and calnexin in 
 24 cases of kcots . as controls , we have used 9 cases of periapical or radicular 
 cysts ( pacs ) and 5 cases of fibromas ( fbs ) . 
 the pacs and the fbs were included 
 in the analysis , as pacs are the most common type of inflammatory odontogenic 
 cysts of and fbs , as lesions of the connective tissue with unaffected 
 epithelium.resultsanalysis revealed a strong association between 
 both bip / grp78 and calnexin expression and kcots : 18 out of 24 ( 75% ) kcots 
 expressed bip / grp78 as opposed to 1 out of 9 ( 13% ) pacs , and none of 5 fbs 
 evaluated ( p < 0.001 , x2-test ) . 
 calnexin was expressed in 11 out of 
 24 kcots ( 46% ) but only one out of 9 ( 13% ) pacs , and none of the 5 fbs analyzed 
 ( p < 0.001 , x2-test).conclusionsstudy results imply 
 that induction of endoplasmic reticulum stress maybe of diagnostic value in 
 keratocystic odontogenic tumours characterization . 
 in addition to recent 
 findings suggesting that endoplasmic reticulum stress plays a causative role in 
 keratinization of epithelia , pharmacological interference with the execution of 
 the unfolded protein response should be considered for the management of 
 keratocystic odontogenic tumours . 
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
ACKNOWLEDGMENTS AND DISCLOSURE STATEMENTS