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despite the improvement in the diagnostic and therapeutic strategies , oral squamous cell carcinoma ( oscc ) remains a major public health problem ; it is the eighth most common cancer worldwide .
oral leukoplakia ( olk ) and oral submucous fibrosis ( osf ) are potentially malignant disorders with a high risk of transitioning to oscc .
the potential for malignant transformation of olk and osf lesions with evidence of epithelial dysplasia ranges from 5%-15% and 3%-19% , respectively .
therefore , attempts are being made to identify specific molecular biomarkers to predict which lesions have the potential to be malignant .
activation of the canonical wnt pathway occurs by binding extracellular ligands to the frizzled receptor and the low - density lipoprotein ( lrp5/6 ) co - receptors , which ultimately leads to stabilization of -catenin .
-catenin translocates to the nucleus , interacts with tcf / lef family , and activates transcription by replacing repressor complexes like the groucho repressor complex .
the human dickkopf family comprises four members ( dkk1 , 2 , 3 , and 4 ) , which function as inhibitors of the canonical wnt pathway .
dkk3 is a potential tumor suppressor gene ( tsg ) that is localized on 11p15 , a locus often deleted in cancerous cells .  
dkk3 interacts with the krm receptors intracellularly , which exerts its inhibitory effect on wnt signaling either by binding to btrcp , thus blocking -catenin from entering the nucleus ,   or by increasing the localization of -catenin in the cell membrane .
dkk3 has been shown to be downregulated in various cancer tissues , suggesting that this gene may be a tsg , while hypermethylation of its promoter correlates with the occurrence of cancers .
since the expression of dkk3 in potentially premalignant disorders has not been examined , in this study we aim to assess and compare immunohistochemical expression of dkk3 in olk and osf as well as correlation with clinicopathological features .
seventy - five formalin - fixed paraffin - embedded blocks with histologically proven nom , osf , olk , and oscc were collected retrospectively .
the samples consisted of 5 nom , 14 osf without dysplasia , 16 osf with dysplasia , 18 olk with mild dysplasia , 12 olk with moderate / severe dysplasia , and 10 oscc .
all patients in this study provided informed consent , and the study was approved by the school of medicine , central south university .
oscc was diagnosed according to who criteria of 2005 . according to pindborg s criteria ,
osf cases were further categorized as early stage ( n=5 ) , moderately advanced ( n=16 ) , or advanced ( n=9 ) .
nom samples were obtained from the buccal mucosa during the surgical removal of third molars .
the biopsy samples were fixed in 10% neutral buffered formalin , left 24 h at room temperature , routinely processed for histology and embedded in paraffin wax .
four - micrometer - thick sections were cut , dewaxed in xylene , and dehydrated in a graded alcohol .
after the slides were rinsed , endogenous peroxidase activity was blocked by treatment with 3% h2o2 for 15 min at room temperature .
the slides were pretreated with citrate buffer ( 10% citrate buffer stock in distilled water , ph 6.0 ) and microwaved for 20 min to retrieve the antigen .
non - reactive staining was blocked by 1.0% goat serum in tris - buffered saline ( ph 6.0 ) applied for 3 min .
primary rabbit polyclonal antibodies against human dkk3 ( ap1523a , abgent , san diego , ca , usa ; 1:50 ) were diluted in phosphate - buffered saline ( pbs , ph 7.4 ) and then incubated for 1 h at 37c in a humidified chamber .
after the slides were rinsed with pbs , they were incubated with a secondary antibody ( biotinylated polylink ; dako , hamburg , germany ) for 30 min at 37c in a humidified chamber and then rinsed with pbs .
dkk3 expression and epithelial thickness was measured in all tissue samples using imagej , java - based image processing software ( national institute of mental health , bethesda , md , usa ) .
the mean values for each group of measurements were compared using the student s t - test .
the chi - square test was used to determine the relationship between dkk3 expression and clinicopathological parameters .
dkk3 expression was compared between different olk and osf groups using the mann - whitney u test .
differences with a probability value of less than 0.05 were considered to be statistically significant .
seventy - five formalin - fixed paraffin - embedded blocks with histologically proven nom , osf , olk , and oscc were collected retrospectively .
the samples consisted of 5 nom , 14 osf without dysplasia , 16 osf with dysplasia , 18 olk with mild dysplasia , 12 olk with moderate / severe dysplasia , and 10 oscc .
all patients in this study provided informed consent , and the study was approved by the school of medicine , central south university .
oscc was diagnosed according to who criteria of 2005 . according to pindborg s criteria ,
osf cases were further categorized as early stage ( n=5 ) , moderately advanced ( n=16 ) , or advanced ( n=9 ) .
nom samples were obtained from the buccal mucosa during the surgical removal of third molars .
the biopsy samples were fixed in 10% neutral buffered formalin , left 24 h at room temperature , routinely processed for histology and embedded in paraffin wax .
four - micrometer - thick sections were cut , dewaxed in xylene , and dehydrated in a graded alcohol .
after the slides were rinsed , endogenous peroxidase activity was blocked by treatment with 3% h2o2 for 15 min at room temperature .
the slides were pretreated with citrate buffer ( 10% citrate buffer stock in distilled water , ph 6.0 ) and microwaved for 20 min to retrieve the antigen .
non - reactive staining was blocked by 1.0% goat serum in tris - buffered saline ( ph 6.0 ) applied for 3 min .
primary rabbit polyclonal antibodies against human dkk3 ( ap1523a , abgent , san diego , ca , usa ; 1:50 ) were diluted in phosphate - buffered saline ( pbs , ph 7.4 ) and then incubated for 1 h at 37c in a humidified chamber .
after the slides were rinsed with pbs , they were incubated with a secondary antibody ( biotinylated polylink ; dako , hamburg , germany ) for 30 min at 37c in a humidified chamber and then rinsed with pbs .
dkk3 expression and epithelial thickness was measured in all tissue samples using imagej , java - based image processing software ( national institute of mental health , bethesda , md , usa ) .
the mean values for each group of measurements were compared using the student s t - test .
chicago , il , usa ) . the chi - square test was used to determine the relationship between dkk3 expression and clinicopathological parameters .
dkk3 expression was compared between different olk and osf groups using the mann - whitney u test .
differences with a probability value of less than 0.05 were considered to be statistically significant .
dkk3 staining was observed in 60 of the samples and was lost in 15 . in nom , dkk3 was localized only in the cell membrane of suprabasal cell layers , whereas the basal cell layer was negatively stained except for some scattered spots of weak dkk3 expression .
it was localized in both the cell membrane and cytoplasm . however , the percentage of cytoplasmic expression in cases of olk with severe dysplasia was higher compared to olk with mild dysplasia . in contrast , the membranous expression of the suprabasal cells was higher in olk with mild dysplasia compared to olk with severe dysplasia ( figure 1 c - f ) .
dkk3 expression was detected in 15 of 30 tissue samples of patients with osf ( table 2 ) .
dkk3 expression was detected in 13 cases of osf without dysplasia and was lost in 1 , whereas dkk3 was detected in 2 cases of osf with dysplasia and was lost in 14 .
a significant difference was observed when comparing dkk3 staining among cases of osf with and without dysplasia ( figure 2 a - f ) .
dkk3 expression was observed in 4 out of 5 patients in the early stage , 9 out of 16 in the moderately advanced stage , and 2 out of 9 in the advanced stage .
there was a gradual decline in dkk3 expression while epithelial atrophy progressed , but this was not statistically significant .
no dkk3 expression was detected in the cell membrane of any osf case . in the early stage of osf
, dkk3 expression was localized in the cytoplasm of the basal and suprabasal layers , but it was only localized in the cytoplasm of the basal layer in the moderately advanced and advanced stages ( figure 2 a - f ) .
cytoplasmic dkk3 expression was detected in all oscc cases ( figure 3 a , b ) .
the relationship between dkk3 expression and the various clinicopathological characteristics of olk and osf was studied . only the dysplastic status of osf , which shows significant correlation with dkk3 expression , other shows no significance ( tables 1 and 2 ) . when dkk3 expression was compared between olk groups and osf groups ,
an important aim of this study was to assess and compare dkk3 expression in olk and osf patients .
the current study is the first to investigate dkk3 expression in both olk and osf . in nom
, dkk3 was expressed on the cell membrane at suprabasal layers and was absent at the basal layer . in olk ,
the percentage of dkk3-positive cytoplasmic expression in mild dysplasia was less compared with severe dysplasia .
further , the staining was localized in the cytoplasm of the basal layer in both mild and severe dysplasia .
our finding was in line with the discovery that dkk3 expression gradually increases from nom to olk and from olk to oscc .
findings regarding the location of the expression either in the cell membrane of the nom or in the cytoplasm of the olk and osf . the precise mechanism by which dkk3 expression shifts from cell membrane to the cytoplasm is not fully clear
. however , this does not appear to be through inhibition of the canonical wnt/-catenin signaling pathway .
the most striking finding in the present study was the loss of dkk3 expression in 50% of osf cases .
in contrast to olk cases , the percentage of positive cytoplasmic expression was higher in cases of osf without dysplasia than in cases of osf with dysplasia ( p=0.0001 ) .
it is suggested that dkk3 is involved in scc carcinogenesis in the head , neck , and oral regions .
showed that by performing a genome - wide loss of heterozygosity ( loh ) analysis .
an allele at the dkk3 locus is frequently deleted in head and neck scc suggesting that dkk3 functions as a tsg .
dkk3 knockdown in oscc - derived cells resulted in reduced cell migration and invasion and may have an oncogenic function that is independent of wnt signaling .
an intriguing question was raised : why was dkk3 expressed in all olk cases , but loss of protein expression was reported in 50% of osf cases ? to answer this question , we should first understand the cause of osf development , which is still not clear .
therefore , it is tempting to hypothesize that there may be underlying factors responsible for loss of dkk3 expression in osf with dysplasia . the possible explanation for aberrant dkk3 expression may be the chemicals in areca nuts : arecoline , arecaidine , guvacine , and guvacoline .
these compounds have been proposed to be highly cytotoxic and genotoxic to cultured human buccal epithelial cells .
teh et al . demonstrated that genomic instability is due to loh in several chromosomal loci containing known oncogenes and tsgs associated with head and neck carcinogenesis in osf tissues .
whether this reflects that dkk3 is instable or hypermethylation occurs in the promoter remains to be confirmed by molecular biology techniques .
surprisingly , a gradual loss / reduced expression of dkk3 in osf cases , from overexpression in the early stage to partial loss in the moderately advanced stage , and then to severe loss in the advanced stage was observed . in other words ,
dkk3 expression progressively decreased with increased severity of epithelial atrophy from the early to advanced stages of osf .
however , the results were not significant ( p=0.547 ) . in their investigation in colorectal cancer , zitt et al
. reported that dkk3 positive samples showed a higher mean microvessel count than did dkk3 negative samples . suggesting that dkk3 can be considered a putative pro - angiogenic protein in colorectal cancer . since osf occurs because of the stromal changes , which undergo progressive hyalinization , decrease in vascularity and cellularity .
we hypothesized that dkk3 expression in osf samples may decrease with the decrease in vascularity of the stromal layer .
recently , dkk3 is the clinical target of gene therapy in prostate , gastric scirrhous and renal fibrosis .
. demonstrated that dkk3 might serve as a diagnostic marker to identify the degree of atrophy and fibrosis in human patients with different types of chronic kidney diseases .
findings as well as our study findings , dkk3 may serve as a diagnostic marker to identify different stages of osf based on the severity of epithelial atrophy .
further , it may also help to assess the osf carcinogenesis based on the dysplastic status of osf , as most predictions about the onset of malignancy are currently based on the severity of dysplasia .
therefore , further well - designed study with large sample size and multiple markers are needed .
we aimed to assess and compare the expression of dickkopf homolog 3 ( dkk3 ) , a possible tumor suppressor gene ( tsg ) , in oral leukoplakia ( olk ) and oral submucous fibrosis ( osf ) using immunohistochemistry . 
 seventy - five cases of normal oral mucosa ( nom ) , olk , osf , and squamous cell carcinoma ( oscc ) were studied . 
 dkk3 was expressed in all cases of nom , olk and oscc . 
 there was steady increases in the percentage of the positive cells progressing toward oscc . 
 the expression was localized in the cytoplasm and cell membrane of cell affected by olk with mild dysplasia and olk with severe dysplasia . 
 no significant association was observed between dkk3 expression and dysplastic status of olk . 
 loss of dkk3 expression was observed in 15 of 30 cases in the osf group , which was significantly associated with histological grade of osf ( p<0.0001 ) . 
 the percentage of positive cells gradually declined with the increasing severity of epithelial atrophy . a significant difference ( p<0.01 ) 
 was observed when comparing dkk3 expression among different groups of olk and osf cases . 
 dkk3 may have diverse expressions in oral premalignant lesions . 
 loss of dkk3 expression in dysplastic / advanced stage of osf may imply a high risk of progression to oral cancer .