initiation and early growth of primary epithelial cancers , and acquisition of 
 invasiveness is thought to herald the advanced stages of this multistep process that 
 eventually leads to metastatic dissemination , with life - threatening 
 consequences .
the genetic 
 control and biochemical mechanisms underlying the acquisition of the invasive phenotype 
 are mediated by the activation of an epithelial mesenchymal transition ( emt ) 
 program .
emt is a biologic 
 process that allows an epithelial cell to acquire the properties of a mesenchymal cell , 
 thereby promoting invasion into the surrounding stroma and intravasation of tumor 
 cells .
a number of distinct 
 molecular markers are engaged in order to initiate emt and enable its completion .
one 
 such marker is the s100a4 , a flament - associated calcium - binding protein , which is 
 associated with the cells of mesenchymal origin , and deemed to play a significant role 
 in emt .
s100a4 expression levels strongly correlate with motility , which is a central element of 
 the metastatic cascade . in oral 
 squamous cell carcinoma ( oscc ) , metastasis results in 90% of cancer associated 
 mortality . in this work
, we studied the role of s100a4 in oral cancer as a biomarker of 
 metastasis by assessing its expression in primary tumors from all the clinical stages of 
 oral cancer and determined the association of s100a4 expression with aggressive 
 clinico - pathological parameters including the pattern of invasion , distant metastasis 
 and recurrence .
assessment of s100a4 in oral cancer may be related to the phenotypic 
 alteration and thereby predict its metastatic potential .
the material for present study included a total of 57 formalin fixed paraffin 
 embedded tissue sections retrieved from the departmental archives . out of these , 47 
 were patients with carcinoma of the bucco - alveolar mucosal complex and of the foor of 
 the mouth .
fhe remaining 10 were healthy mucosal tissues , which were obtained at the 
 time of extraction of impacted teeth .
only surgically treated cases of oral squamous 
 cell carcinoma ( oscc ) with a minimum follow - up period of 5 years were included in the 
 study . institutional ethics committee approval ( iec 36/2012 )
was obtained from 
 kasturba hospital , manipal , prior to carrying out the study .
the patient demographics 
 and other details of the cases included in the study are presented in table 1 .
a chart analysis was carried out to 
 study the expression of s100a4 in all these cases by immunohistochemistry ( ihc ) . 

nt- no tobacco immunohistochemical staining was carried out using indirect streptavidin biotin 
 immunoperoxidase technique .
sections of 4 m thickness taken on amino propyl 
 triethoxysilane ( apes ) coated slides were incubated overnight at 48c in a slide 
 warmer to ensure complete adhesion of sections .
following tissue sections 
 deparaffinization and hydration through descending grades of alcohol , sections were 
 placed in phosphate buffered saline ( pbs ) for 30 minutes .
the slides were immersed in 
 a microwave resistant plastic staining jar containing antigen retrieval solution ( 10 
 mm sodium citrate buffer , ph 6.0 ) and maintained at 700 w power for 10 minutes . 

subsequently , the solution was allowed to cool at room temperature for at least 20 
 minutes .
the slides were incubated in 3% hydrogen peroxide for 5 minutes at room 
 temperature , following which slides were washed with pbs . to decrease the background 
 staining due to nonspecific binding of antibodies , the slides were incubated with 5% 
 bovine serum albumin ( bsa ) for 10 minutes at room temperature and washed with pbs for 
 5 minutes .
the researchers applied 100 l of the primary antibody ( rabbit polyclonal 
 anti - s100a4 antibody , 1:250 ) over the section completely , which was incubated for 60 
 minutes at 37c in a humidified chamber .
the slides were rinsed with pbs and placed 
 in the pbs bath for 5 minutes . after draining and wiping of the slides
, sections were 
 incubated in 100 l of biotinylated secondary antibody , anti - rabbit igg ( whole 
 molecule - peroxidase antibody produced in goat ) for 30 minutes at 37c .
the slides 
 were washed with pbs and placed in pbs bath for 5 minutes . for visualization , 
 sections were incubated with freshly prepared substrate solution 
 ( sigmafast 3 , 3'-diaminobenzidine tablets ) for 5 - 10 minutes . 

this was followed by washing the slides in running tap water and counter staining 
 with mayer 's haematoxylin ( mhs1- haematoxylin solution , mayer 's 1 gil certified 
 hematoxylin * ph 2.4 ) for 5 minutes , after which the slides were washed in running tap 
 water for 1 minute .
briefy , the sections were dehydrated through ascending grades of 
 alcohol ( 70% , 95% and 100% ) for 2 minutes each , cleared in xylene for 1 minute , 
 mounted in a resinous media and coverslipped .
the neoplastic cell with cytoplasmic staining was viewed at 200 magnification , using 
 a light microscope .
the distribution of immunolabelling in neoplastic epithelial 
 cells was determined from a minimum of five representative high - power fields at the 
 invasive front .
a positive cell demonstrated a diffuse brown signal in the cytoplasm , 
 independent of its intensity . to eliminate any inter - observer bias ,
the degree of staining for s100a4 was 
 evaluated using a semi - quantitative scale29 .
tissues were graded as absent ( - ) when 
 there was no staining , low ( 1 + ) when less than 25% of the tumor cells were positive , 
 moderate ( 2 + ) when 25 to 75% of the tumor cells stained positively and diffuse ( 3 + ) 
 when more than 75% of the tumor cells were positive for s100a4 .
s100a4 expression was 
 then correlated with clinicopathological parameters including primary tumor site , 
 stage of tumor , degree of differentiation , lymph node metastasis and mode ( pattern ) 
 of invasion .
kendall 's 
 tau - b statistics was applied to assess the measure of agreement between two 
 observers .
chi - square test was used to study the association between the s100a4 
 staining and clinicopathological parameters .
the material for present study included a total of 57 formalin fixed paraffin 
 embedded tissue sections retrieved from the departmental archives . out of these , 47 
 were patients with carcinoma of the bucco - alveolar mucosal complex and of the foor of 
 the mouth .
fhe remaining 10 were healthy mucosal tissues , which were obtained at the 
 time of extraction of impacted teeth .
only surgically treated cases of oral squamous 
 cell carcinoma ( oscc ) with a minimum follow - up period of 5 years were included in the 
 study . institutional ethics committee approval ( iec 36/2012 )
was obtained from 
 kasturba hospital , manipal , prior to carrying out the study .
the patient demographics 
 and other details of the cases included in the study are presented in table 1 .
a chart analysis was carried out to 
 study the expression of s100a4 in all these cases by immunohistochemistry ( ihc ) . 

the antibodies and reagents were obtained from sigma  
 aldrich co. ( st . louis , mo , usa ) .
sections of 4 m thickness taken on amino propyl 
 triethoxysilane ( apes ) coated slides were incubated overnight at 48c in a slide 
 warmer to ensure complete adhesion of sections .
following tissue sections 
 deparaffinization and hydration through descending grades of alcohol , sections were 
 placed in phosphate buffered saline ( pbs ) for 30 minutes .
the slides were immersed in 
 a microwave resistant plastic staining jar containing antigen retrieval solution ( 10 
 mm sodium citrate buffer , ph 6.0 ) and maintained at 700 w power for 10 minutes . 

subsequently , the solution was allowed to cool at room temperature for at least 20 
 minutes .
the slides were incubated in 3% hydrogen peroxide for 5 minutes at room 
 temperature , following which slides were washed with pbs . to decrease the background 
 staining due to nonspecific binding of antibodies , the slides were incubated with 5% 
 bovine serum albumin ( bsa ) for 10 minutes at room temperature and washed with pbs for 
 5 minutes .
the researchers applied 100 l of the primary antibody ( rabbit polyclonal 
 anti - s100a4 antibody , 1:250 ) over the section completely , which was incubated for 60 
 minutes at 37c in a humidified chamber .
the slides were rinsed with pbs and placed 
 in the pbs bath for 5 minutes . after draining and wiping of the slides
, sections were 
 incubated in 100 l of biotinylated secondary antibody , anti - rabbit igg ( whole 
 molecule - peroxidase antibody produced in goat ) for 30 minutes at 37c .
the slides 
 were washed with pbs and placed in pbs bath for 5 minutes . for visualization , 
 sections were incubated with freshly prepared substrate solution 
 ( sigmafast 3 , 3'-diaminobenzidine tablets ) for 5 - 10 minutes . 

this was followed by washing the slides in running tap water and counter staining 
 with mayer 's haematoxylin ( mhs1- haematoxylin solution , mayer 's 1 gil certified 
 hematoxylin * ph 2.4 ) for 5 minutes , after which the slides were washed in running tap 
 water for 1 minute .
briefy , the sections were dehydrated through ascending grades of 
 alcohol ( 70% , 95% and 100% ) for 2 minutes each , cleared in xylene for 1 minute , 
 mounted in a resinous media and coverslipped .
the neoplastic cell with cytoplasmic staining was viewed at 200 magnification , using 
 a light microscope .
the distribution of immunolabelling in neoplastic epithelial 
 cells was determined from a minimum of five representative high - power fields at the 
 invasive front .
a positive cell demonstrated a diffuse brown signal in the cytoplasm , 
 independent of its intensity . to eliminate any inter - observer bias ,
the degree of staining for s100a4 was 
 evaluated using a semi - quantitative scale29 .
tissues were graded as absent ( - ) when 
 there was no staining , low ( 1 + ) when less than 25% of the tumor cells were positive , 
 moderate ( 2 + ) when 25 to 75% of the tumor cells stained positively and diffuse ( 3 + ) 
 when more than 75% of the tumor cells were positive for s100a4 .
s100a4 expression was 
 then correlated with clinicopathological parameters including primary tumor site , 
 stage of tumor , degree of differentiation , lymph node metastasis and mode ( pattern ) 
 of invasion .
kendall 's 
 tau - b statistics was applied to assess the measure of agreement between two 
 observers .
chi - square test was used to study the association between the s100a4 
 staining and clinicopathological parameters .
positive s100a4 expression in the cytoplasm of the tumor cells was observed in 30 out of 
 47 cases ( 63.82% ) of oscc .
kendall 's tau - b test between the two observers showed a very 
 high level of agreement for the expression of s100a4 ( 0.8 , p<0.001 ) . in the control 
 group , none of oral mucosal tissues were positive for s100a4 in the cytoplasm of the 
 epithelium .
out of 47 cases studied , 1/6 of stage i , 1/2 of stage ii , 13/17 of stage iii and 15/22 
 of stage iv cases showed positive s100a4 expression ( table 2 ) . a strong association was noted between the stage of the tumor and 
 the expression score of s100a4 ( p=0.007 ) .
s100a4 expression was positive in 12/14 
 ( 85.7% ) node positive stage iii cases , of which 7/12 were ( 3 + ) and 5/12 were ( 2 + ) for 
 s100a4 expression . among the node negative cases , 1/3 cases showed ( 3 + ) for s100a4 
 expression .
likewise , s100a4 expression was positive in 14/15 ( 93.3% ) stage iv cases , of 
 which 13/14 were ( 3 + ) and 1/14 was ( 2 + ) . among the 7 node
negative cases , 1/7 was ( 3 + ) 
 and the remaining 6/7 cases were negative for s100a4 expression . although the 
 cytoplasmic expression of s100a4 was strongly associated with lymph node involvement 
 ( p<0.001 ) , there was no association between s100a4 expression in lymph node positive 
 cases and primary size of the tumor ( p=0.15 ) .
association of s100a4 expression with clinical stage , lymph node involvement , 
 tumor size , metastasis , recurrence , grade of tumor and pattern of invasion wdscc - well differentiated squamous cell carcinoma ; mdscc - moderately 
 differentiated squamous cell carcinoma ; pdscc - poorly differentiated squamous 
 cell carcinoma with regard to distant metastasis ( m ) , 11/22 ( 50% ) cases in stage iv were positive for 
 s100a4 expression , out of which 10/11 were ( 3 + ) and 1/11 was ( 2 + ) for s100a4 expression . 

a strong association could be established between the s100a4 expression in tumor cells 
 and distant metastasis ( p=0.004 ) . likewise ,
11/47 ( 23.4% ) cases that recurred after 
 treatment were either in stage iii ( 2/11 ) or stage iv ( 9/11 ) .
all these cases showed 
 ( 3 + ) expression for s100a4 . among the stage iii cases that recurred ,
one was node 
 positive and the other was negative , whereas all the 9 stage iv cases that recurred were 
 node positive .
there was a strong positive association between the s100a4 expression and 
 rate of recurrence ( p=0.002 ) .
with regard to the pattern of invasion among the positive cases , 1/6 cases of stage i 
 showed a type 1 ( figure 1 ) pattern and 1/2 cases 
 in stage ii showed type 2 ( figure 2 ) pattern .
out 
 of the 13 stage iii cases that showed positive s100a4 expression , 2 were type 1 , 2 
 others were type 2 , 3 were type 3 ( figure 3 ) , 3 
 were type 4c ( figure 4 ) and 3 were type 4d ( figure 5 ) .
in the same manner , out of the 15 stage iv 
 cases that showed positive s100a4 expression , 2 were type 3 , 4 were type 4c and 9 were 
 type 4d .
there was a strong association ( p<0.001 ) between s100a4 expression and 
 pattern of invasion of oscc
. however , there was no association seen between the s100a4 
 expression and histological grade of the oscc ( p=0.27 ) . oral squamous cell carcinoma ( oscc ) showing type 1 well defined borderline pattern 
 of invasion ( s100a4 , 20x ) oral squamous cell carcinoma ( oscc ) showing type 2 cord like pattern of invasion 
 ( s100 a4 , 20x ) oral squamous cell carcinoma ( oscc ) showing type 3 pattern of invasion with groups 
 of cells with no distinct borderline ( s100a4 , 20x ) oral squamous cell carcinoma ( oscc ) showing type 4c diffuse cord like pattern of 
 invasion ( s100a4 , 20x ) oral squamous cell carcinoma ( oscc ) showing type 4d diffuse widespread pattern of 
 invasion ( s100a4 , 20x )
oral cancer is one of the major cancers worldwide , with a low five - year survival rate 
 that has not significantly changed over the past few decades .
metastasis , which is responsible for 90% of the cancer 
 associated mortality , is one of the poorly understood factors in cancer progression .
an 
 important step for the invasion and metastasis is the epithelial mesenchymal transition 
 ( emt ) , a complex process induced by modifications of multiple pathways leading to a 
 spectrum of epithelial cellular changes , including loss of polarity and cohesion , 
 increased motility , and the acquisition of mesenchymal phenotype .
emt is critical for 
 the invasion , progression , and metastasis in epithelial carcinogenesis .
metastasis promoting s100a4 is a typical representative of s100 family of ca2 + -binding 
 multifunctional proteins with dual extra- and intracellular function .
the increased expression of the s100a4 
 in invasive front of the tumor suggests the pivotal role played by s100a4 in invasion of 
 tumor cells into the surrounding matrix .
the acquisition of invasive phenotype with 
 altered pattern of adhesion is a result of e - cadherin down regulation by s100a4 , which 
 the tumor cells acquire .
further influences cell - matrix adhesions by interacting with liprin [ 1 and masking their 
 protein kinase c - mediated phosphorylation sites .
inhibition of the liprin [ 1-lar complex by s100a4 loosens cell 
 adhesion and allows cell invasion .
cell invasion is also facilitated by matrix 
 metalloproteinases ( mmps ) , as s100a4 causes metastasis not only by affecting the 
 motility of cells but also by affecting its invasive properties through influencing the 
 expression of mmps and their endogenous inhibitors .
a positive correlation of the s100a4 and mmp2 expression in 
 oesophageal squamous cell carcinoma has been reported .
s100a4 is located at the leading edge of migrating cells , where it induces the formation of fexible protrusions . 

the 
 s100a4-myosin ii - a interaction does not only increase cell motility , but also enhances 
 cell polarization and directed migration .
s100a4 also binds to the septins 2 , 6 and 7 , which play a central 
 role in cytokinesis , cell polarity determination , cytoskeletal reorganization , and 
 membrane dynamics10 , .
s100a4 binds to annexin ii on the endothelial surface , a complex that 
 activates the tissue plasminogen activator , which in turn converts the plasminogen to 
 plasmin , activating prommps .
ca - dependent binding of s100a4 to metap2 
 modulates the metap2 activity , which could promote endothelial growth and angiogenesis . 

this process of angiogenesis favors the metastasis of the tumor to the regional lymph 
 nodes .
elevated s100a4 expression has shown to be the marker of metastasis and poor prognosis 
 in patients with pancreatic ductal adenocarcinoma , prostate adenocarcinoma , colorectal cancer4 , and breast cancer . a higher level of s100a4 expression has shown to correlate with 
 auxillary lymph node metastasis and overall patient survival .
evidence suggesting the role for s100a4 in hnscc , especially in oscc , is scarce . 
 elevated s100a4 as a prognostic factor in metastatic cases of oscc was demonstrated by 
 moriyama - kita , et al .
( 2004).our 
 findings showing increased expression of s100a4 in cases with regional lymphnode 
 involvement and metastasis further emphasizes the role of s100a4 in oral cancer patients 
 with poor prognosis .
the final step in the metastatic cascade is the migration of the cells to a distant site 
 and secondary colonization . in our study
this finding has been elucidated 
 earlier in papillary thyroid carcinoma and pancreatic cancer , in which poor outcome of patients was related to increased s100a4 
 expression coupled with decreased e cadherin expression .
the increased expression of s100a4 in our study showed a significant correlation with 
 the overall clinical stage of tumor , a finding that has been noted also in colorectal 
 carcinoma and pancreatic 
 cancer .
however , no 
 association could be drawn between the elevated s100a4 level and the primary tumor size 
 or histological grading in our analysis .
( 2014 ) in lung 
 cancer cells , in which s100a4 was frequently overexpressed , irrespective of histological 
 subtype . in our study a strong association between s100a4 expression and pattern of invasion 
 could be elicited , with an increased expression noted in type 4c and type 4d pattern of 
 invasion .
similar observations have been made in malignant melanomas where increased 
 expression of s100a4 in the nodular variant was noted , linking this association to the 
 pattern of invasion .
significantly , our observation of high s100a4 expression with tumor recurrence is a 
 novel finding .
s100a4 has been identified as a candidate marker for maintaining the 
 stemness in head and neck cancer initiating cells .
s100a4 controls the notch and 
 pten / pi3k / akt signaling pathways , which regulate the self - renewal and tumorigenicity of 
 stem cells or cancer stem cells . 

s100a4 signaling pathways thus play a major role in the maintenance of cancer initiating 
 cell population and contribute for the tumor recurrence .
in summary , the data from our study adds weight to the growing body of evidence that 
 s100a4 can be used to identify the subgroup of patients at high risk of metastasis and 
 recurrence in oscc cases , and that targeting s100a4 signaling might be a potential 
 therapeutic target for increasing the patient survival .
 s100a4 , a biomarker of epithelial mesenchymal transition ( emt ) , plays an important 
 role in invasion and metastasis by promoting cancer cell motility . in oral squamous 
 cell carcinoma ( oscc ) , metastasis results in 90% of cancer associated mortality.objectiveto investigate the role of s100a4 expression as an important component of the 
 epithelial mesenchymal transition ( emt ) program in oral squamous cell carcinoma 
 ( oscc).material and methodss100a4 protein expression was assessed semi - quantitatively by immunohistochemistry 
 in 47 histologically confirmed cases of oral squamous cell carcinoma ( oscc ) and 10 
 normal oral mucosal biopsies . 
 the association between the s100a4 overexpression 
 and the aggressive features of oscc were analyzed by x2 test.resultsmoderate to strong cytoplasmic expression of s100a4 was observed in 30 out of 47 
 specimens of oscc ( 64% ) . 
 overexpression of s100a4 was significantly associated 
 with the clinical stage , lymph node involvement , metastases , pattern of invasion 
 and recurrence ( p<0.05).conclusions100a4 expression represents an important biomarker of prognostic significance 
 that may be used to identify a subset of patients at high risk of invasion and 
 metast 
INTRODUCTION
MATERIAL AND METHODS
Patient details
Immunohistochemistry
Staining interpretation
Statistics
RESULTS
DISCUSSION
CONCLUSION