esophageal squamous cell carcinoma ( escc ) is the sixth and fourth most common cancer in the world and in the developing countries , respectively , and also the second most frequent cause of cancer mortality in iran ( 1 , 2 ) . according to the latest medical reports , the highest rate of escc is in asia , in the so - called  esophageal cancer belt  which is extended from iran to china ( 3 ) . despite the advances in the diagnosis and treatment of escc ,
the rate of escc mortality has not yet improved ( 4 , 5 ) , reflecting the late detection of the disease and poor understanding of the cellular and molecular mechanisms for initiation and development of escc ( 6 , 7 ) .
tumor progression and development process is mediated by deregulation of different signaling pathways as well as mutual interactions between cancerous and normal cells ( 8) .
different studies have introduced the role of specific molecules such as cd44 family in tumor progression .
cd44 family is consisted of cell adhesion glycoproteins on the cell surface ( 9 , 10 ) .
cd44 is a single chain glycoprotein consisting of 4 functional domains including a preserved n - terminal extracellular domain , a non - preserved membrane proximal region , a preserved trans - membrane and a cytoplasmic tail domain .
the distal extracellular domain is primarily responsible for the binding of hyaluronic acid ( ha ) , while the proximal extracellular domain is the variable region , which can be different in various cd44 isoforms due to alternative splicing of cd44 mrna ( 11 ) .
the cytoplasmic tail of cd44 molecules exhibits a protein motif , which interacts with cytoskeletal proteins and other intracellular signaling molecules ( 12 ) .
the human cd44 gene is located on the short arm of chromosome 11 at a position described as 11p13 ( 13 ) , consisting of 20 exons .
although exons 1 to 5 and 16 to 20 are constant and splice together to make cd44s , the 10 variable exons ( also called v1-v10 ) will be spliced and enclosed in nursing insertion site between exons number five and sixteen .
previous studies have shown that in some cancers cd44 isoforms , cd44s and cd44v3 could play a major role in cancer invasion and metastasis ( 15 - 18 ) .
the expression of cd44v3 is related to advanced pathological stage and poor prognosis in colorectal cancer and plays an essential role in its invasion and metastasis ( 19 ) . in this study
, we retrospectively evaluated the level of cd44 mrna expression ( its standard form and v3 variant ) and its correlation with different clinicopathological features of patients with escc .
fresh tumoral and distant tumor - free esophageal tissues were obtained from fifty patients with escc through surgery at the imam reza and the omid hospitals of mashhad university of medical sciences , iran .
none of patients received any therapeutic interference such as chemo - therapy or radiation , prior to sampling .
after dissection , the samples were directly treated in rnalater solution ( qiagen , hilden , germany ) and transferred to 20 c till rna extraction .
the ethics committee of mashhad university of medical sciences approved the study and enrolled patients formally declared their consent to be recruited in the study .
histopathological characteristics of the neoplasmic samples , such as size , location , and grade of differentiation were recorded .
all tumoral tissues were also confirmed histologically to ensure that they contain at least 70% tumor cells ( 20 ) .
rna was extracted from the normal and tumoral samples , using the rneasy mini kit ( qiagene , hilden , germany ) .
synthesis of cdna was performed with oligo dt using first - strand synthesis kit ( fermentas , lithuania ) according to the manufacture s procedure .
expressional analysis of cd44s and cd44v3 was performed in triplicate reactions through a comparative threshold cycle / sybr in experienced methodology ( genet bio , korea ) in a real - time thermal cycler ( stratagenemx3000p , la jolla , ca ) using the primer sequences described in table 1 .
comparative real - time pcr was carried out using sybr green pcr master mix containing 6-carboxyl - x - rhodamine ( rox ) as a reference dye .
the sub - sequent thermal cycling program was applied : 10 min at 95 c as initial denaturation step , followed by 40 cycles of 15 sec at 95 c , 10 sec at 60 c and 1 min at 72c .
data was normalized for glyceraldehyd 3-phosphat dehyrogenase ( gapdh ) expression as an internal control by means of comparative threshold cycle method .
pcr reactions were accomplished in a 20 l total volume with 400 ng of cdna , 10 l sybr green pcr master mix ( genet bio , korea ) , ( nuclease - free water , and 0.6 l forward and reverse primers ( 10 pmol ) . over two - fold increase in mrna expression
was considered as overexpression , whereas minus two - fold decrease was considered as underexpression .
primer sequences used for comparative real - time pcr the statistical analysis was performed using spss 16.0 statistical package ( spss , il , us ) .
the associations between cd44s / cd44v3 mrna expression levels and different clinicopathological aspects were analyzed by 2 squared test .
all p values were two - tailed and p- values < 0.05 were considered statistically significantly .
fresh tumoral and distant tumor - free esophageal tissues were obtained from fifty patients with escc through surgery at the imam reza and the omid hospitals of mashhad university of medical sciences , iran .
none of patients received any therapeutic interference such as chemo - therapy or radiation , prior to sampling .
after dissection , the samples were directly treated in rnalater solution ( qiagen , hilden , germany ) and transferred to 20 c till rna extraction .
the ethics committee of mashhad university of medical sciences approved the study and enrolled patients formally declared their consent to be recruited in the study .
histopathological characteristics of the neoplasmic samples , such as size , location , and grade of differentiation were recorded .
all tumoral tissues were also confirmed histologically to ensure that they contain at least 70% tumor cells ( 20 ) .
rna was extracted from the normal and tumoral samples , using the rneasy mini kit ( qiagene , hilden , germany ) .
synthesis of cdna was performed with oligo dt using first - strand synthesis kit ( fermentas , lithuania ) according to the manufacture s procedure .
expressional analysis of cd44s and cd44v3 was performed in triplicate reactions through a comparative threshold cycle / sybr in experienced methodology ( genet bio , korea ) in a real - time thermal cycler ( stratagenemx3000p , la jolla , ca ) using the primer sequences described in table 1 .
comparative real - time pcr was carried out using sybr green pcr master mix containing 6-carboxyl - x - rhodamine ( rox ) as a reference dye .
the sub - sequent thermal cycling program was applied : 10 min at 95 c as initial denaturation step , followed by 40 cycles of 15 sec at 95 c , 10 sec at 60 c and 1 min at 72c .
data was normalized for glyceraldehyd 3-phosphat dehyrogenase ( gapdh ) expression as an internal control by means of comparative threshold cycle method .
pcr reactions were accomplished in a 20 l total volume with 400 ng of cdna , 10 l sybr green pcr master mix ( genet bio , korea ) , ( nuclease - free water , and 0.6 l forward and reverse primers ( 10 pmol ) . over two - fold increase in mrna expression
was considered as overexpression , whereas minus two - fold decrease was considered as underexpression .
the statistical analysis was performed using spss 16.0 statistical package ( spss , il , us ) .
the associations between cd44s / cd44v3 mrna expression levels and different clinicopathological aspects were analyzed by 2 squared test .
all p values were two - tailed and p- values < 0.05 were considered statistically significantly .
fifty patients including 24 males ( 48% ) and 26 females ( 52% ) enrolled in this study .
the surgical samples were obtained before any other treatment ensuring that the histopathological characteristics of the samples were not affected with the therapeutic interference .
the patients mean age and standard deviation at the time of diagnosis was 61.7312.14 years ( ages ranged from 30 to 87 years ) .
samples were dissected from middle ( 25 of 50 ) , lower ( 22 of 50 ) or upper ( 3 of 50 ) regions of the esophagus .
most tumor samples ( 48 of 50 ) were in stages ii or iii of tumor growth , and only two samples were in stage i. based on the histopathological analyses , 8 tumor samples ( 16% ) were classified as poorly differentiated , whereas 10 ( 20% ) and 32 ( 64% ) specimen were well and moderately differentiated , respectively .
furthermore , 21 of the tumors ( 42% ) had metastasized to the lymph nodes .
correlations between cd44 isoforms s and v3 genes expression and clinicopathological characteristics of the esophageal squamous cell carcinoma patients we analyzed cd44s and cd44v3 mrna expressions in fifty tumor samples compared to their paired non - neoplastic esophageal epithelium by quantitative real time - pcr .
the y - axis displays fold change of gene expression , and the x - axis demonstrates the number of patients . a two - fold increase in gene expression in tumor samples
was considered to be overexpressed state , whereas a minus two - fold decrease was considered as underexpression .
the average was thought as neither change nor normal expression significant overexpression of cd44s and cd44v3 mrna was observed in 13 ( 26.0% , p=0.03 ) and 11 ( 22.0% , p=0.007 ) specimens , respectively .
the minimum and maximum levels of mrna expression fold change for cd44s were -3.96 and 13.80 fold , respectively ( meansd , 1.222.59 ) , while these levels for cd44v3 were -5.53 and 11.30 fold ( 0.852.89 ) .
eight samples ( 16.0% ) showed overexpression of both genes , whereas 34 samples ( 68.0% ) showed normal or under expression of both cd44v3 and cd44s genes ( table 3 ) .
association between cd44s and cd44v3 gene expression there is a significant correlation between cd44s and cd44v3 mrna expression among escc specimens ( p=0.002 ) . by analyzing concomitant gene expression
, we found that the samples with high level of cd44v3 gene expression , also expressed high level of cd44s ( figure 2 ) .
regression plot representing correlation between cd44s / cd44v3 genes expression ( p=0.002 ) to estimate the impact of cd44s and cd44v3 mrna expression on clinicopathological features , we analyzed the correlation between cd44s and cd44v3 expression and various clinicopathological variables ( table 2 ) .
a significant correlation was detected between the expression of both genes and the depth of tumor invasion ( p=0.001 ) .
furthermore , expression of both genes was significantly correlated to the stage of tumor progression ( p=0.03 ) .
another valuable significant correlation was revealed between cd44s and cd44v3 genes expression and the grade of tumor differentiation ( p=0.033 ) .
in fact , 2 of 13 ( 15.38% ) tumor samples , which overexpressed cd44s and 3 of 11 ( 27.27% ) tumor samples , which overexpressed cd44v3 were well differentiated ( wd ) , indicating the early expression of these genes in the process of dedifferentiation .
in addition , expression of both genes was significantly associated with the lymph node metastasis ( p=0.04 ) .
five of 13 cd44s overexpressed tumors ( 38.46% ) and 6 of 11 cd44v3 overexpressed tumors ( 54.54% ) did not show any metastasis to the lymph nodes , while 2 of 13 cd44s overexpressed tumors ( 15.38% ) and 1 of 11 cd44v3 overexpressed tumors ( 9.09% ) had lymph node metastasis .
overexpression of both genes was significantly higher in males ( p=0.03 ) in comparison to females ( cd44s : meansd : 1.843.08 and 0.631.91 , respectively and cd44v3 : 1.152.76 and 0.553.04 , respectively ) .
cd44v3 mrna expression was inversely correlated to the depth of tumor invasion ( p=0.015 ) .
five of 11 tumors ( 45.45% ) , which overexpressed cd44v3 were t1 , and 6 of 11 ( 54.54% ) tumors that overexpressed cd44v3 were t2 .
there were no significant associations between cd44s and cd44v3 expressions alone and other clinicopathological variables such as age and tumor size ( p>0.05 ) .
we analyzed cd44s and cd44v3 mrna expressions in fifty tumor samples compared to their paired non - neoplastic esophageal epithelium by quantitative real time - pcr .
the y - axis displays fold change of gene expression , and the x - axis demonstrates the number of patients . a two - fold increase in gene expression in tumor samples
was considered to be overexpressed state , whereas a minus two - fold decrease was considered as underexpression .
the average was thought as neither change nor normal expression significant overexpression of cd44s and cd44v3 mrna was observed in 13 ( 26.0% , p=0.03 ) and 11 ( 22.0% , p=0.007 ) specimens , respectively .
the minimum and maximum levels of mrna expression fold change for cd44s were -3.96 and 13.80 fold , respectively ( meansd , 1.222.59 ) , while these levels for cd44v3 were -5.53 and 11.30 fold ( 0.852.89 ) .
eight samples ( 16.0% ) showed overexpression of both genes , whereas 34 samples ( 68.0% ) showed normal or under expression of both cd44v3 and cd44s genes ( table 3 ) .
there is a significant correlation between cd44s and cd44v3 mrna expression among escc specimens ( p=0.002 ) . by analyzing concomitant gene expression
, we found that the samples with high level of cd44v3 gene expression , also expressed high level of cd44s ( figure 2 ) .
to estimate the impact of cd44s and cd44v3 mrna expression on clinicopathological features , we analyzed the correlation between cd44s and cd44v3 expression and various clinicopathological variables ( table 2 ) .
a significant correlation was detected between the expression of both genes and the depth of tumor invasion ( p=0.001 ) .
furthermore , expression of both genes was significantly correlated to the stage of tumor progression ( p=0.03 ) .
another valuable significant correlation was revealed between cd44s and cd44v3 genes expression and the grade of tumor differentiation ( p=0.033 ) .
in fact , 2 of 13 ( 15.38% ) tumor samples , which overexpressed cd44s and 3 of 11 ( 27.27% ) tumor samples , which overexpressed cd44v3 were well differentiated ( wd ) , indicating the early expression of these genes in the process of dedifferentiation .
in addition , expression of both genes was significantly associated with the lymph node metastasis ( p=0.04 ) .
five of 13 cd44s overexpressed tumors ( 38.46% ) and 6 of 11 cd44v3 overexpressed tumors ( 54.54% ) did not show any metastasis to the lymph nodes , while 2 of 13 cd44s overexpressed tumors ( 15.38% ) and 1 of 11 cd44v3 overexpressed tumors ( 9.09% ) had lymph node metastasis .
overexpression of both genes was significantly higher in males ( p=0.03 ) in comparison to females ( cd44s : meansd : 1.843.08 and 0.631.91 , respectively and cd44v3 : 1.152.76 and 0.553.04 , respectively ) .
cd44v3 mrna expression was inversely correlated to the depth of tumor invasion ( p=0.015 ) .
five of 11 tumors ( 45.45% ) , which overexpressed cd44v3 were t1 , and 6 of 11 ( 54.54% ) tumors that overexpressed cd44v3 were t2 .
there were no significant associations between cd44s and cd44v3 expressions alone and other clinicopathological variables such as age and tumor size ( p>0.05 ) .
escc is an extremely invasive tumor of the gastrointestinal system , and novel prognostic , diagnostic and therapeutic modalities are immediately required .
these requirements can lead to explore key markers involved in the development and progression of the escc . in this study
, we analyzed mrna expression of the stem cell markers cd44s and cd44v3 in tumoral and adjacent non - tumoral tissues of fifty patients with escc .
previous studies have shown that the specific molecular structure of each isoform can influence the cell behavior uniquely .
the isoforms vary according their position of n / o - linked glycosylation modification , which exist in the extracellular domain . changing in these positions
influences the affinity of isoforms with various ligands , leading to different modulations of intracellular signaling pathway ( 21 ) .
several studies have shown the overexpression of cd44 and its variants in variety of malignancies .
it has been shown that the expression of this gene and its isoforms is increased significantly in different malignant tissues including breast cancer ( 22 ) , leukemic hematopoiesis ( 23 ) , head and neck squamous cell carcinoma ( 17 ) , oral cancer ( 24 ) and barrett s esophagus ( 25 ) .
takahashi et al have shown that there is a significant correlation between cd44 and grade of tumor cell differentiation in prostate cancer ( 26 ) . in this study , we also observed overexpression of cd44s ( 26% ) and cd44v3 ( 22% ) in escc patients .
many studies have shown the correlation between cd44 variants gene expression and depth of tumor cells invasion and metastasis in human cancers ( 27 ) .
although wang et al have illustrated that the expression of cd44v3 has a direct correlation with tumor invasion in head and neck cancer ( 17 ) , nonetheless , suzuki et al have described that decreased expression of cd44v3 and v6 is associated with depth of tumor invasion in lung cancer ( 28 ) .
this may be due to specific physiological state of esophagus and micro environment of tumor cells .
we showed that the simultaneous increased expression of cd44s and cd44v3 genes was associated with clinical features of escc patients .
further detailed studies on a large patient population are required to elucidate the exact role of cd44s and its variants expression to introduce a specific marker for prognosis of esophageal carcinoma .
 objective(s):cd44 is a member of the cell adhesion molecules family . 
 naturally , cd44s , along with cd44v3 influence the cell motility , migration , and adhesion , while in tumor cells they lead to tumor invasion , progression , and metastasis . 
 the purpose of this research is to evaluate the cd44s and cd44v3 expression in esophageal squamous cell carcinoma ( escc ) and to reveal their correlations with clinicopathological features of patients.materials and methods : fresh tumoral and distant tumor - free esophageal tissues were obtained from 50 patients with escc . using quantitative real - time pcr , the expression levels of cd44s and cd44v3 were quantified and compared in both groups of cells . 
 the patients had not received any therapeutic interference , such as chemotherapy or radiation , prior to sampling.results:significant overexpression of cd44s and cd44v3 mrna was observed in 13 ( 26.0% , p=0.03 ) and 11 ( 22.0% , p=0.007 ) tumor specimens , respectively . 
 the expression of the genes were significantly correlated not only with each other ( p=0.0001 ) , but also with differentiation grade of tumor ( p=0.033 ) , stage of tumor progression ( p=0.003 ) , and depth of tumor invasion ( p=0.00 ) . 
 in addition , low level of cd44v3 mrna expression was attended to be associated with tumor invasion.conclusion:there is no correlation between cd44s expression with clinicopathological features of patients ; however , simultaneous expression of these genes has an important effect on tumorigenesis . 