ephrin receptors ( eph ) are the largest family of tyrosine kinase receptors ( rtks ) , divided into 2 distinct classes ( a and b ) based on sequence homology of their extracellular domain , which largely determines their ephrin ligands . in general , a - class ephrins ( ephrin a1a6 ) bind to the a - class eph receptors ( eph - a1a10 ) and b - class ephrins ( ephrin b1b3 ) bind to b - class receptors ( ephb1b6 ) , although cross - reactions have also been described . interaction between ephrin receptors and their ligands ( ephrins ) occurs at sites of cell - to - cell contact , since both molecules are membrane - bound , or between plasma membrane clusters ( microdomains ) that transform into clearly defined signaling centers upon eph / ephrin complex formation . the cellular response to eph stimulation by ligands includes a wide range of biological effects exerted through several signaling pathways in a bi - directional mode . this is considered a unique property of eph / ephrin interaction and consists of forward and reverse signaling components . eph / ephrin signaling has mainly been implicated in angiogenesis , cell shape formation , adhesion and motility   in normal developmental and in pathological processes . ubiquitous expression of eph and ephrins in the vast majority of normal human tissues has suggested a crucial role in the maintenance of organized and differentiated cellular function in adults , although specific expression patterns by organ have also been described . more recently , tumorigenesis has been related to transcriptional alterations of eph genes through somatic mutations and epigenetic silencing , especially in more advanced stages of tumor progression , suggesting a potential role for ephs in tumor cell migration and metastasis . recent studies have been focused mainly on eph - a2 expression and the role of eph - a2/ephrin - a1 interaction in several human malignancies , indicating a crucial role in tumor development and progression . eph - a2 was found to be widely expressed in highly proliferating human tissues and overexpressed in several types of malignancy , such as melanoma , prostate , breast , ovarian , esophageal , liver , lung and gastrointestinal cancer [ 10,1318 ] . according to a theoretical , but realistic , model proposed by wykosky and debinski , nonphosphorylated eph - a2 may stimulate oncogenic signaling pathways , leading to increased cell migration and invasion due to eph - a2 gene overexpression or lack of ephrina1-induced down - regulation . the expression levels of eph receptor in pathological processes seem to correlate positively with the degree of malignant transformation , whereas this has not been well documented for the ephrin ligands . the predictive value of eph - a2 expression pattern for tumor grade , stage , recurrence and/or survival has also been investigated in ovarian , gastric , lung and prostate cancers , suggesting a substantial correlation between eph - a2 overexpression and poor clinical prognosis in most cases [ 17,1922 ] . the diversity of eph - mediated functions in virtually every stage of tumorigenesis from development to metastasis and interference in cell - to - cell interactions and the tumor microenvironment have made this receptor family an attractive target for novel drug design in cancer therapeutics . thyroid cancer is generally considered a curable malignancy in its early stages , although management of suspect thyroid lesions has continuously evolved during the last decades and is updated on a regular basis , indicating the presence of diagnostic pitfalls and limitations of the current patient workup . several markers have been investigated in an effort to increase the diagnostic accuracy of fine - needle aspiration biopsy in the preoperative setting [ 2325 ] . at the same time , novel prognostic indices are being evaluated for their role in the optimal postoperative management of thyroid cancer [ 2629 ] . unlike the other above - mentioned solid organ tumors , the expression of ephs in human benign and malignant thyroid lesions has not yet been evaluated . in light of the above considerations , the present study aimed to examine the immunohistochemical expression of eph - a2 and -a4 in 131 patients with benign and malignant thyroid lesions and to assess their diagnostic utility . we also aimed to evaluate the association of eph - a2 and eph - a4 expression with important clinicopathological characteristics for the management of patients with malignant thyroid tumors , such as tnm stage , and capsular , lymphatic and vascular invasion . we used 131 formalin - fixed , paraffin - embedded thyroid specimens from an equal number of patients who had undergone surgery for benign or malignant thyroid disease . the who histological classification of thyroid tumors was used , according to which 59 malignant ( 47 papillary , 5 follicular , 5 medullary and 2 anaplastic thyroid carcinomas ) and 72 benign ( 61 hyperplastic nodules and 11 hashimoto thyroiditis ) thyroid cases were observed . among papillary thyroid carcinoma cases , thyroid malignancies were grouped according to the latest tnm classification ( american joint committee on cancer  ajcc cancer staging manual , seventh edition )   as : stage i , 40 cases ( 67.8% ) ; stage ii , 8 cases ( 13.6% ) ; stage iii , 8 cases ( 13.6% ) ; and stage iv , 3 cases ( 5% ) . a female predominance was also noted in our study population , which reached 5:1 in the subset of malignant thyroid tumors . the median size of malignant thyroid tumors was 8 mm ( range 0.980 mm ) . eph - a2 and eph - a4 immunostaining was performed on formalin - fixed , paraffin - embedded thyroid tissue sections using rabbit polyclonal anti - eph - a2 ( s-20 ) and anti - eph - a4 ( h-77 ) primary igg antibodies ( santa cruz biochemicals , santa cruz , ca , usa ) . briefly , 4 m - thick tissue sections were dewaxed in xylene and were brought to water through graded alcohols . antigen retrieval ( citrate buffer at ph 6.1 and microwave heating ) was performed according to the manufacturer s instructions . to remove the endogenous peroxidase activity , sections were treated with freshly prepared 0.3% hydrogen peroxide in methanol in the dark for 30 minutes ( min ) at room temperature . non - specific antibody binding was then blocked using sniper ( sniper , biocare medical , walnut , creek , ca , usa ) , a specific blocking reagent for mouse and rabbit primary antibodies , for 5 min . the sections were then incubated for 1 hour at room temperature , with the primary antibodies diluted 1:100 in phosphate - buffered saline ( pbs ) . after washing 3 times with pbs , sections were incubated at room temperature with biotinylated linking reagent ( biocare medical ) for 10 min , followed by incubation with peroxidase - conjugated streptavidin label ( biocare medical ) for 10 min . the resultant immune peroxidase activity was developed in 0.5% 3,3-diaminobenzidine hydrochloride ( dab ; sigma , st . louis , mo , usa ) in pbs containing 0.03% hydrogen peroxide for 3 min . sections were counterstained with harris hematoxylin and mounted in entellan ( merck , darmstadt , germany ) . appropriate negative controls were performed by omitting the primary antibody and/or substituting it with an irrelevant anti - serum . as a positive control , pancreatic cancer tissue sections with known increased eph - a2 and eph - a4 immunoreactivity were used . follicular cells proliferative capacity was assessed immunohistochemically , using a mouse anti - human ki-67 antigen ; igg1k antibody ( clone mib-1 , dakopatts , glostrup , denmark ) as previously described . specimens were considered positive for eph - a2 , eph - a4 and ki-67 when more than 5% of the follicular cells were stained . the percentage of positively stained follicular cells was obtained by counting at least 1000 cells in each case by 2 independent observers ( s.t . and p.a . both membraneous and cytoplasmic staining was taken into consideration in determining eph - a2 and eph - a4 immunoreactivity . ki-67 immunoreactivity was classified into 2 categories ( below and over mean value ) according to the percentage of positively stained follicular cells exceeding the mean percentage value , as previously reported . as there are no clinically relevant cut - off values available for eph - a2 and eph - a4 expression levels in thyroid lesions , a quantitative scoring system was applied based on previously published reports for other immunohistochemical markers on thyroid tissue lesions [ 3437 ] . the immunoreactivity of follicular cells for eph - a2 and eph - a4 was scored according to the percentage of positively stained cells as follows : 0 = 04% of follicular cells positive ( considered as negative staining ) , 1 = 530% of follicular cells positive , 2 = 3160% of follicular cells positive , 3 = 61100% of follicular cells positive , and their intensity scored as 0 = negative staining , 1 = weak staining , 2 = intermediate staining , and 3 = intense staining . immunoreactivity was also classified , using the term total immunohistochemical ( ihc ) score , as  negative or weak  for a total score 02 ,  moderate  for a total score 34 , and strong  for a total score 57 . in this way we ensured that each group had a sufficient and more homogeneous number of cases in order to be comparable with the other groups . the differences in eph - a2 and eph - a4 expression between the histopathological types of thyroid lesions , as well as the association with the clinicopathological parameters in the subgroup of malignant cases , were investigated . possible associations were analyzed with the pearson s chi - square test and linear correlations were investigated using spearman s rho coefficient and cramer s v test for categorical data . a kruskal - wallis analysis was made for ordinal variables , followed by a mann - whitney u test for significant associations only . we used 131 formalin - fixed , paraffin - embedded thyroid specimens from an equal number of patients who had undergone surgery for benign or malignant thyroid disease . the who histological classification of thyroid tumors was used , according to which 59 malignant ( 47 papillary , 5 follicular , 5 medullary and 2 anaplastic thyroid carcinomas ) and 72 benign ( 61 hyperplastic nodules and 11 hashimoto thyroiditis ) thyroid cases were observed . among papillary thyroid carcinoma cases , thyroid malignancies were grouped according to the latest tnm classification ( american joint committee on cancer  ajcc cancer staging manual , seventh edition )   as : stage i , 40 cases ( 67.8% ) ; stage ii , 8 cases ( 13.6% ) ; stage iii , 8 cases ( 13.6% ) ; and stage iv , 3 cases ( 5% ) . a female predominance was also noted in our study population , which reached 5:1 in the subset of malignant thyroid tumors . the median size of malignant thyroid tumors was 8 mm ( range 0.980 mm ) . eph - a2 and eph - a4 immunostaining was performed on formalin - fixed , paraffin - embedded thyroid tissue sections using rabbit polyclonal anti - eph - a2 ( s-20 ) and anti - eph - a4 ( h-77 ) primary igg antibodies ( santa cruz biochemicals , santa cruz , ca , usa ) . briefly , 4 m - thick tissue sections were dewaxed in xylene and were brought to water through graded alcohols . antigen retrieval ( citrate buffer at ph 6.1 and microwave heating ) was performed according to the manufacturer s instructions . to remove the endogenous peroxidase activity , sections were treated with freshly prepared 0.3% hydrogen peroxide in methanol in the dark for 30 minutes ( min ) at room temperature . non - specific antibody binding was then blocked using sniper ( sniper , biocare medical , walnut , creek , ca , usa ) , a specific blocking reagent for mouse and rabbit primary antibodies , for 5 min . the sections were then incubated for 1 hour at room temperature , with the primary antibodies diluted 1:100 in phosphate - buffered saline ( pbs ) . after washing 3 times with pbs , sections were incubated at room temperature with biotinylated linking reagent ( biocare medical ) for 10 min , followed by incubation with peroxidase - conjugated streptavidin label ( biocare medical ) for 10 min . the resultant immune peroxidase activity was developed in 0.5% 3,3-diaminobenzidine hydrochloride ( dab ; sigma , st . louis , mo , usa ) in pbs containing 0.03% hydrogen peroxide for 3 min . sections were counterstained with harris hematoxylin and mounted in entellan ( merck , darmstadt , germany ) . appropriate negative controls were performed by omitting the primary antibody and/or substituting it with an irrelevant anti - serum . as a positive control , pancreatic cancer tissue sections with known increased eph - a2 and eph - a4 immunoreactivity were used . follicular cells proliferative capacity was assessed immunohistochemically , using a mouse anti - human ki-67 antigen ; igg1k antibody ( clone mib-1 , dakopatts , glostrup , denmark ) as previously described . specimens were considered positive for eph - a2 , eph - a4 and ki-67 when more than 5% of the follicular cells were stained . the percentage of positively stained follicular cells was obtained by counting at least 1000 cells in each case by 2 independent observers ( s.t . and p.a . ) blinded to the clinical data , with complete observer agreement . both membraneous and cytoplasmic staining was taken into consideration in determining eph - a2 and eph - a4 immunoreactivity . ki-67 immunoreactivity was classified into 2 categories ( below and over mean value ) according to the percentage of positively stained follicular cells exceeding the mean percentage value , as previously reported . as there are no clinically relevant cut - off values available for eph - a2 and eph - a4 expression levels in thyroid lesions , a quantitative scoring system was applied based on previously published reports for other immunohistochemical markers on thyroid tissue lesions [ 3437 ] . the immunoreactivity of follicular cells for eph - a2 and eph - a4 was scored according to the percentage of positively stained cells as follows : 0 = 04% of follicular cells positive ( considered as negative staining ) , 1 = 530% of follicular cells positive , 2 = 3160% of follicular cells positive , 3 = 61100% of follicular cells positive , and their intensity scored as 0 = negative staining , 1 = weak staining , 2 = intermediate staining , and 3 = intense staining . immunoreactivity was also classified , using the term total immunohistochemical ( ihc ) score , as  negative or weak  for a total score 02 ,  moderate  for a total score 34 , and strong  for a total score 57 . in this way we ensured that each group had a sufficient and more homogeneous number of cases in order to be comparable with the other groups the differences in eph - a2 and eph - a4 expression between the histopathological types of thyroid lesions , as well as the association with the clinicopathological parameters in the subgroup of malignant cases , were investigated . possible associations were analyzed with the pearson s chi - square test and linear correlations were investigated using spearman s rho coefficient and cramer s v test for categorical data . a kruskal - wallis analysis was made for ordinal variables , followed by a mann - whitney u test for significant associations only . eph - a2 positivity ( ihc score > 0 ) was noted in 75 out of 131 ( 57.3% ) thyroid tissue specimens . positive staining was observed in 74.6% ( 44/59 ) of malignant thyroid tumors , 39.3% ( 24/61 ) of hyperplastic nodules and 63.6% ( 7/11 ) of hashimoto thyroiditis cases . in figure 1 , representative eph - a2 immunostaining for papillary carcinoma , hyperplastic nodules and hashimoto thyroiditis cases are depicted ( figure 1a  c , respectively ) . cases with malignant thyroid lesions showed significantly increased incidence of eph - a2 positivity compared to those with benign thyroid lesions ( cramer s v=0.343 , p<0.001 ) . eph - a2 positivity was definitely distinctive between cases with papillary carcinoma and hyperplastic nodules ( z=3.877 , p<0.001 , figure 2a ) . no significant differences between malignant and hashimoto thyroiditis or benign hyperplasia and hashimoto thyroiditis cases were noted ( p=0.457 and p=0.137 , respectively ) . significant intergroup differences were also not observed for all possible comparisons of malignant histopathological types ( data not shown ) . eph - a2 positively stained cases of thyroid lesions , either malignant or benign , exhibited overall higher proliferative capacity , as shown by ki-67 immunoreactivity ( p=0.04 ) , although no statistically significant difference was observed in favor of malignant lesions ( data not shown ) . the mean total score of eph - a2 positive malignant cases was significantly higher than that of benign cases ( cramer s v=0.361 , p=0.001 ) . significantly higher ihc score for malignant compared to hyperplastic thyroid lesions was also noted ( z=3.899 , p<0.001 , figure 2b ) . papillary carcinomas showed significantly higher eph - a2 expression than hyperplastic thyroid nodules ( z=4.270 , p<0.001 ) , whereas this was not found for other types of malignant thyroid lesions ( i.e. , follicular , medullary or anaplastic cancer ) . the mean total ihc score of malignant tumors was 3.252.06 , with 40.7% of them exhibiting moderate ( total score 3 or 4 ) and 33.9% strong ( total score 57 ) eph - a2 expression . on the other hand , the mean total score of benign thyroid lesions was 1.82.2 , with a significantly lower proportion of strong eph - a2 expression ( 12.5% , p<0.05 , table 2 ) . eph - a2 expression was not differentiated between conventional type cases and follicular variant cases of papillary carcinoma ( data not shown ) . eph - a4 positivity ( ihc score > 0 ) was noted in 83 ( 63.4% ) of 131 thyroid tissue specimens . positive staining was observed in 62.7% ( 37/59 ) of malignant lesions , 63.9% ( 39/61 ) of hyperplastic nodules and 63.6% ( 7/11 ) of hashimoto thyroiditis cases . in figure 1 , representative eph - a4 immunostaining for papillary carcinoma , hyperplastic nodules and hashimoto thyroiditis cases are depicted ( figures 1d , e and f , respectively ) . no significant differences were noted between cases with malignant and benign thyroid lesions in respect to eph - a4 positivity ( figure 2c ) . the mean total ihc score of eph - a4-positive malignant cases was 3.472.87 , with a remarkable proportion of 47.5% showing strong eph - a4 expression ( total score 57 ) . benign thyroid lesions yielded a mean ihc score of 2.962.46 , with strong eph - a4 expression in 33.3% of cases ; however , significant differences were not observed ( figure 2d ) . non - parametric comparison of the mean ranks by histopathological type of thyroid cancer revealed significantly higher ihc score for cases with papillary carcinoma compared to those with hyperplastic nodules ( z=2.724 , p=0.006 ) , although this effect was not detected for other histopathological types of malignant lesions . in table 3 , the proliferative capacity of all positively stained thyroid lesions for eph - a4 was similar to eph - a2-positive samples ( ie , increased ki-67 immunoreactivity ) ( p=0.01 ) without significant difference between malignant and benign lesions ( data not shown ) . eph - a4 expression was not differentiated between conventional subtype cases and follicular variant cases of papillary carcinoma ( data not shown ) . we also assessed whether eph - a2 and eph - a4 expression exerts a diagnostic effect in the subgroup of patients with malignant thyroid lesions . for both ephs , positive staining and ihc score were not correlated with capsular , lymphatic and vascular invasion . the associations of the clinicopathological characteristics of malignant thyroid lesions ( ie , tumor size [ t ] and nodal status [ n ] ) with eph - a2 and eph - a4 expression were also investigated . overall , 40 t1 ( 67.8% ) , 13 t2 ( 22% ) , 3 t3 ( 5.1% ) and 3 t4 ( 5.1% ) malignant thyroid cases were analyzed . t1 cases expressed eph - a2 in 72.5% ( 29/40 ) , with a mean total ihc score of 3.222.08 , t2 cases in 69.2% ( 9/13 ) with a mean total ihc score 2.922.43 , t3 and t4 cases in 100% ( 3/3 in both groups ) with a mean total ihc score 4.331.15 and 4.0 , respectively . eph - a4 expression was detected in 65% ( 26/40 ) of t1 cases with a mean total score 3.562.82 , 46.2% ( 6/13 ) of t2 tumors with a mean total score 2.462.96 , 66.7% ( 2/3 ) of t3 tumors with a mean total score 4.333.79 and 100.0% ( 3/3 ) of t4 tumors with a mean total score 5.661.53 . although t3 and t4 malignant cases showed increased total score for both eph - a2 and eph - a4 , significant correlations could not be extracted . similarly , nodal status and the tnm stage were not significantly correlated to eph - a2 or eph - a4 expression . moderate to strong eph - a2 and eph - a4 expression was associated with high ki-67 protein statement , but this association was not statistically significant . the associations between eph - a2 and eph - a4 immunoreactivity and the clinicopathological characteristics of the thyroid samples of our study are presented in table 4 . ephrin receptors and the bidirectional signaling to their ephrin ligands have been shown to actively participate in solid tumor development and/or progression . substantial evidence has documented the presence of eph / ephrin complex formation and/or overexpression in response to oncogenic mutations   that directly affect tumor immunology , cell motility and adhesion , vessel invasion and metastatic potential . notably , a - class eph receptors ( epha ) have been involved in tumor angiogenesis and vascularization , mainly through positive and negative stimulation [ 4042 ] . in this aspect , several studies have suggested that eph - a2 was overexpressed in a variety of malignant tumors , being associated with clinicopathological parameters crucial for patients management and prognosis , whereas there is no comprehensive research on eph - a4 member [ 17,1922,31 ] . in this regard , the present study aimed to evaluate the clinical significance of eph - a2 and eph - a4 expression in human benign and malignant thyroid lesions . the present study shows that eph - a2 but not eph - a4 expression was enhanced in cases with malignant compared to those with benign thyroid lesions , and especially in papillary carcinomas compared to hyperplastic nodules . expression of mutant inactive eph - a2 variants resulted in tumor mass reduction , whereas eph - a2 upregulation was correlated with tumor stage and progression in several cancers . modification of cytoskeletal architecture and loss of expression of adhesion molecules such as e - cadherin have been considered as possible effects of eph - a2 overexpression , which may in turn lead to cancer cell migration and metastasis . more specifically , the ratio of eph receptor to ligand density seems to play a major role in vivo by modulating integrin function and increasing tissue invasiveness . however , the role of this potent bidirectional receptor in tumorigenesis and cell migration has been considered far more complex due to ligand - dependency . a dynamic temporal interaction between eph - a2 , its ligands ( mainly ephrin a1 ) , and tumor microenvironment might reasonably account for the crucial role of eph - a2 in several types of cancers . in line with overexpression patterns observed in other solid organ malignancies , eph - a2 receptor seems to play a role in thyroid tumorigenesis , probably associated with the increased proliferative activity of malignant thyroid lesions . expression of eph - a2 gene , located on chromosome 1p36 , was limited to normal tissues with high proliferation rates   ( e.g. , skin and small intestine ) , although a systematic analysis of eph - a2 expression in normal tissues has not been done to date . moreover , we found enhanced proliferative capacity of follicular cells ( reflected by ki-67 protein statement ) expressing eph - a2 or eph - a4 . although no distinction could be made between malignant and benign thyroid lesions on the basis of this finding , significant correlations between increased eph receptor expression and upregulated proliferative status of thyroid cells might underlie the presence of a common pathogenetic deviation from normal development in all types of thyroid pathology . substantial studies have documented an oncogenic effect of eph - a2 even in the absence of related ligands , which may elicit malignant transformation and metastatic potential in normal cells . according to ruoslahti et al , eph - a2 overexpression appears to alter extracellular matrix adhesions rather than affecting cell proliferation , due to mislocalization and inability of the receptors to bind to their ligands . moreover , excessive eph - a2 production was shown to reduce focal - adhesion kinase ( fak ) phosphorylation , thus inhibiting integrin - mediated cell adhesion and enhancing cell motility and invasion . however , possible clinical implications of eph - a2 expression on local progression of malignant thyroid lesions were not generated in the present work . thyroid capsule disruption , a significant pathological determinant influencing the choice of postoperative treatment of malignant tumors , as well as lymphatic or blood vessel invasion , which negatively influence prognosis , were not significantly correlated to eph - a2 and eph - a4 expression . t3 and t4 malignant lesions showed a higher ihc score  moderate to high  for eph - a2 and eph - a4 , compared to t1 and t2 tumors ; however , significant correlations could not be made , possibly due to the small number of locally advanced thyroid cancers . similarly , the 8 cases with nodal involvement could not be reliably related to eph - a2 and eph - a4 expression . with regard to the tnm stage of thyroid cancers of our study , a high proportion of early - stage tumors ( i.e. , t1 and t2 ) positively expressed eph - a2 ( 72.5% and 69.2% , respectively ) with a moderate ihc score . this finding is consistent with available data for eph - a2 expression in colorectal cancer and has been attributed to high microvessel density in eph - a2-positive tumor areas , thus emphasizing the role of eph receptors through angiogenesis in earlier stages of tumorigenesis . thyroid cancer is 3 times more prevalent in women than in men ; however , the role of sex hormones in its pathogenesis has not yet been elucidated . in this context , several studies have documented a possible implication of estrogen receptor ( er)- and/or - in thyroid carcinoma growth and progression . notably , dual targeting of eph - a2 and estrogen receptor ( er ) was shown to restore tamoxifen sensitivity in er / epha2-positive breast cancer . eph - a2 overexpression was also associated with aggressive phenotypic features and inversely with er and progesterone receptor ( pr ) expression in endometrioid endometrial carcinoma patients , making eph - a2 a potential therapeutic target , especially in hormone receptor - negative endometrial carcinoma . further studies could be conducted to evaluate the expression profile of both eph - a2 and er in thyroid carcinoma patients and especially in cases where a strong association between er and thyroid neoplastic transformation has been documented , such as in the case of papillary carcinoma arising in premenopausal women . our results suggest that upregulation of eph - a2 but not eph - a4 expression may be associated with the malignant transformation of thyroid neoplasia . on the other hand , correlations of eph - a2 and eph - a4 with clinicopathological parameters , which may reinforce their role in tumor invasion and metastasis through modification of adhesive properties of malignant cells , were not found . however , these findings are provisional and need to be further confirmed in cohort studies including a higher proportion of patients with advanced nodal and metastatic disease . subset analysis of histological subtypes of papillary and follicular carcinoma , with the latter the most difficult for pathologists to diagnose , is also recommended .
summarybackgroundephrin receptors ( ephs ) are frequently overexpressed in a wide variety of human malignant tumors , being associated with tumor growth , invasion , metastasis and angiogenesis . 
 the aim of the present study was to evaluate the clinical significance of eph - a2 and eph - a4 expression in human benign and malignant thyroid lesions.material/methodseph-a2 and eph - a4 protein expression was assessed immunohistochemically on paraffin - embedded thyroid tissues from 131 patients with benign and malignant lesions.resultseph-a2 was significantly overexpressed in malignant compared to benign thyroid lesions ( p<0.001 ) . 
 papillary carcinoma cases presented significantly increased eph - a2 expression compared to those with hyperplasia nodules ( p<0.001 ) . 
 eph - a4 expression was not differentiated between cases with malignant or benign thyroid lesions . 
 papillary carcinoma cases presented significantly increased eph - a4 expression compared to those with hyperplasia nodules ( p=0.006 ) . in the subgroup of malignant thyroid lesions , eph - a2 and eph - a4 expression was not associated with tnm stage , capsular , lymphatic or vascular invasion.conclusionsthe present data suggest that eph - a2 , but not eph - a4 , overexpression may be associated with the malignant transformation of thyroid neoplasia . 
 further studies conducted on cohorts including a higher proportion of patients with advanced nodal and metastatic disease are recommended to draw definite conclusions on the clinical significance of eph proteins in thyroid neoplasia .