breast cancer is the most prevalent malignancy in women and affects about 1 in 8 women around the world ( 1 ) . therefore , investigation of early biomarkers and molecular aspects is valuable for improvement of breast cancer therapy and outcome . cancer - testis antigens ( cta ) are proteins with physiological expression restricted to adult testicular germ cells . they are down - regulated in somatic adult tissues but may be aberrantly re - expressed in various malignancies . the first cta was discovered by taking advantage of a newly developed dna - cloning method to identify targets of t - cell recognition . cytotoxic t lymphocytes ( ctl ) recognizing autologous tumor cells were obtained from a patient bearing melanoma with an unusually favorable clinical course . using the melanoma cell line mz2- mel and autologous ctl clones cytolytic to this line , mage-1 , subsequently was renamed as mage - a1 , and was identified as the target antigen . this was the first molecularly characterized tumor antigen eliciting autologous ctl responses in a cancer patient . further analysis of the mage - a family revealed 12 closely related genes clustered at xq28 . a growing number of tumor - associated antigens ( taa ) , with similar characteristics , identified by cellular or serological screening techniques , have been reported since . although some of them may be expressed in placenta as well , they are collectively referred to as cta . cta presently include 44 distinct gene families , some comprising multiple members , such as mage - a and gage1 , as well as splice variants , such as xage1a and xage1b , for a total of 89 transcripts . cta can be classified into those that are encoded on the x chromosome ( x - cta ) and those that are not ( non - x ctas ) ( 2 ) . to date , almost 100 genes and gene families encoding ctas have been identified . ctas mapping to chromosome x are referred to as x -ctas and are distinguished from non - x ctas located on other chromosomes ( 2 - 4 ) . x - ctas expression in breast cancer tissues is associated with a poor outcome and is more prevalent in higher grade and advanced stage tumors ( 5 - 9 ) . due to testis blood barrier and the immune privileged status of germinal cells ( 10 ) , expression of ctas in tissues other than testis can trigger an immune response . these antigens are also expected to become new candidates for cancer - specific immunotherapy , but little information is available on the comprehensive expression of ctas in a large number of samples of gastrointestinal and breast carcinomas ( 11 ) . expression of ctas of the mage family has been also reported in human breast carcinomas although only to a limited degree ( 12 ) . breast cancers , especially triple - negative cancers , show higher expression of ct genes , which is the prerequisite for any immunotherapeutic approach . ct genes have also gained attention for immunoprevention in high - risk patients ( 13 ) . the purpose of the present study is to assess immunohistochemical expression of cta mage-1 in tissue samples of invasive breast cancer and its correlation with known prognostic factors . a total of 113 patients with invasive breast cancer ( 112 ductal and one lobular ) were included . all patients were surgically treated at omid hospital in mashhad university of medical sciences , iran between 2011 and 2013 . data related to tumor size , grade , stage , estrogen receptor ( er ) and progesterone receptor ( pr ) status , human epithelial growth factor receptor 2 ( her-2/neu ) , and axillary lymph node status are summarized in table 1 . immunohistochemical staining of mage-1 was performed on the invasive breast cancer . for the detection of mage-1 protein , we used undiluted ncl - mage-1 monoclonal antibody ( mab ) , staining is described in detail elsewhere ( 14 ) . briefly , tissue slides from paraffin embedded breast cancer tumor samples were places on silane ( 3 aminopropyltriethoxysilane , a 3648 , sigma , st . , slides were heated in an 800-w microwave oven at maximum power for 30 minutes , held in 10 mmol / l edta buffer ( ph 6.0 ) for 5 minutes and then rinsed with a tris buffer solution ( pbc , ph 7.2 ) . to suppress endogenous peroxidase activity , after additional rinsing with pbc , they were incubated for 20 minutes with a 1:10 dilution of normal rabbit serum ( dakox0902 , dako a / s ) in a wet chamber at room temperature for 20 minutes to prevent non - specific binding of immunoglobulin . slides were then treated with undiluted mabs at room temperature for 90 minutes . the envision ( dako ) system was used as a secondary detection tool and diaminobenzidine tetrahydrochloride served as a chromogen . sections of normal human testis with intact spermiogenesis were used as positive controls for mage-1 mabs . this method takes into account percentages of positive cells ( scored on a 0 - 3 scale ) and the intensity of their staining ( scored on a 0 - 3 scale ) . the percentage of positive cells is then multiplied by the intensity of staining , and the final score ranges from 0 ( no staining ) to 9 ( diffuse and strong staining ) . the final results were further classified as 0 ( no staining ) , 1 ( score 1 , 2 , 3 ) , 2 ( score 4 , 5 , 6 ) and 3 ( score 7 , 8 , 9 ) . for statistical analysis , mage-1 scores of 0 and 1 were considered negative , whereas scores 2 and 3 were considered positive . the association between immunohistochemical data and different clinicopathological parameters were evaluated by chi2 and t - test . the computer program spss 16 software ( spss for windows , 2007 ) was used . a total of 113 patients with invasive breast cancer ( 112 ductal and one lobular ) were included . all patients were surgically treated at omid hospital in mashhad university of medical sciences , iran between 2011 and 2013 . data related to tumor size , grade , stage , estrogen receptor ( er ) and progesterone receptor ( pr ) status , human epithelial growth factor receptor 2 ( her-2/neu ) , and axillary lymph node status are summarized in table 1 . immunohistochemical staining of mage-1 was performed on the invasive breast cancer . for the detection of mage-1 protein , we used undiluted ncl - mage-1 monoclonal antibody ( mab ) , staining is described in detail elsewhere ( 14 ) . briefly , tissue slides from paraffin embedded breast cancer tumor samples were places on silane ( 3 aminopropyltriethoxysilane , a 3648 , sigma , st . louis mo , usa ) . after de paraffinization , slides were heated in an 800-w microwave oven at maximum power for 30 minutes , held in 10 mmol / l edta buffer ( ph 6.0 ) for 5 minutes and then rinsed with a tris buffer solution ( pbc , ph 7.2 ) . to suppress endogenous peroxidase activity , after additional rinsing with pbc , they were incubated for 20 minutes with a 1:10 dilution of normal rabbit serum ( dakox0902 , dako a / s ) in a wet chamber at room temperature for 20 minutes to prevent non - specific binding of immunoglobulin . the envision ( dako ) system was used as a secondary detection tool and diaminobenzidine tetrahydrochloride served as a chromogen . sections of normal human testis with intact spermiogenesis were used as positive controls for mage-1 mabs . this method takes into account percentages of positive cells ( scored on a 0 - 3 scale ) and the intensity of their staining ( scored on a 0 - 3 scale ) . the percentage of positive cells is then multiplied by the intensity of staining , and the final score ranges from 0 ( no staining ) to 9 ( diffuse and strong staining ) . the final results were further classified as 0 ( no staining ) , 1 ( score 1 , 2 , 3 ) , 2 ( score 4 , 5 , 6 ) and 3 ( score 7 , 8 , 9 ) . for statistical analysis , mage-1 scores of 0 and 1 were considered negative , whereas scores 2 and 3 were considered positive . the association between immunohistochemical data and different clinicopathological parameters were evaluated by chi2 and t - test . a p value < 0.05 was considered significant . for all statistical analyses , the computer program spss 16 software ( spss for windows , 2007 ) was used . mage-1 expression ( score  2 + ) was detected in 34/111 ( 30.1% ) of patients ( figure 1 ) . a , strong nuclear and cytoplasmic staining of most of neoplastic cells ( h & e , 100  ) ; b , strong nuclear and cytoplasmic staining of most of neoplastic cells ( h & e , 100  ) . mage-1 expression ( score  2 + ) was detected in 36/111 ( 31.8% ) of patients ( figure 1 ) . expression of mage-1 was significantly associated with lymph node ( p = 0.003 ) breast cancers , but no association was found between mage-1 cytoplasmic expression and tumor size , age , her-2 status , tumor stage , grade , and er/ pr status . expression of mage-1 was significantly associated with tumor size ( p = 0.018 ) and lymph node ( p = 0.042 ) breast cancers . no association was found between mage-1 nuclear expression and age , her-2 status , tumor stage , grade , and er/ pr status . mage-1 expression ( score  2 + ) was detected in 34/111 ( 30.1% ) of patients ( figure 1 ) . a , strong nuclear and cytoplasmic staining of most of neoplastic cells ( h & e , 100  ) ; b , strong nuclear and cytoplasmic staining of most of neoplastic cells ( h & e , 100  ) . mage-1 expression ( score  2 + ) was detected in 36/111 ( 31.8% ) of patients ( figure 1 ) . table 3 presents associations between mage-1expression with clinicopathological variables . expression of mage-1 was significantly associated with lymph node ( p = 0.003 ) breast cancers , but no association was found between mage-1 cytoplasmic expression and tumor size , age , her-2 status , tumor stage , grade , and er/ pr status . expression of mage-1 was significantly associated with tumor size ( p = 0.018 ) and lymph node ( p = 0.042 ) breast cancers . no association was found between mage-1 nuclear expression and age , her-2 status , tumor stage , grade , and er/ pr status . breast cancer is among the leading causes of death in women worldwide ( 16 ) . the search for human tumor antigens as potential immunotherapeutic targets represents an appealing therapeutic concept since decades ago . recent advances in molecular characterization of human tumor - associated antigens have paved the way toward active specific immunotherapy of cancer ( 17 ) . ctas are of particular interest , because they are expressed in a very limited number of healthy tissues typically including hla class i negative spermatogonia , while they are expressed in a wide range of malignancies ( 18 ) . , we analyzed expression of mage-1 antigen on archival paraffin - embedded samples of invasive breast cancer tissue of 113 patients and correlated their expression with other clinicopathological variables . to our knowledge , this is the first report specifically examining expression of mage-1 , at the protein level , in breast cancer . mage-1 cytoplasmic expression ( score  2 + ) was detectable in 30.1% and nuclear expression ( score  2 + ) was detectable in 31.8% of patients . data on mage - a and ny - eso-1 expression in literature are highly variable . the frequency of multispecific mage - a and ny - eso-1 positivity in published studies ranges between 17 and 74% and 2% - 40% , respectively ( 2 ) . stefan found a 18% positive cta7 ( mage - c1 ) , defined as immunoreactivity in more than 50% of tumor cells , in 124 women with invasive breast cancer ( 16 ) . in the study of recurrent ductal breast cancer , bandic found 74% of mage - a and 40% of ny - eso-1-positivity in samples ( 19 ) . these discrepancies observed between studies may be due to different antibodies used for ct detection or difference in scoring system . some authors define positive x - cta expression according to percentage of cells ( 19 - 21 ) , while others combine the extent and intensity of cta expression using semi - quantitative scoring systems ( 22 , 23 ) . studies exploring potential prognostic significance of cta expression in breast cancer have yielded contradictory findings . some authors found that expression of cta is associated with poorly differentiated histological phenotypes ( 20 , 22 ) . others found no association between their expression and various pathological parameters ( 19 ) or only an association between mage - a1 and ki-67 labeling index ( 23 ) . according to the present study ( table 3 ) , a positive nuclear expression mage-1 status correlated significantly with lymph nodes status ( p = 0.042 ) , and positive cytoplasmic expression mage-1 status correlated significantly with lymph nodes ( ln ) status ( p = 0.003 ) . positive nuclear expression mage-1 status correlated significantly with tumor size ( p = 0.018 ) . however , the expression of mage-1 was not associated with other typical adverse clinicopathological features , namely tumor stage , and pathological grade . in badovinac crnjevic study ( 2 ) mage - a10 expression was significantly associated with er - negative ( p = 0.002 ) , pr - negative ( p = 0.002 ) and her-2-negative ( p = 0.044 ) tumors . they showed that mage - a10 was frequently expressed in the triple negative ( tn ) subgroup of patients , where the majority ( 85.7% ) of tumors expressed cta ( 19 ) . curigliano showed a significantly higher expression of mage - a ( 26% ) in tn breast cancers compared with er - positive tumors ( 10% ) ( p = 0.07 ) ( 23 ) . although the exact biological function of ctas is still unknown , future studies will hopefully allow more insights into the activities of ctas in tumor cells on the molecular level . in conclusion , due to mage-1 expression ( score  2 + ) in about 30% of our patients , even more frequent than her2 positivity in breast cancer
backgroundaberrant expression of cancer - testis antigens ( cta ) in breast carcinoma tissue , and its natural expression in the testis , the tissue away from the immune system , makes them good candidates for cancer immunotherapy and vaccines designing.objectivesthe aim of this study was to assess the expression of a cta ( mage-1 ) in invasive breast cancer and its correlation with prognostic factors.methods paraffin blocks of breast cancer tissues from 113 patients operated in 2011 - 2013 were stained for mage-1expression by immunohistochemistry ( ihc ) . 
 the associations of mage-1 expression with known prognostic factors were assessed by statistical analysis using spss 16.resultsmage-1 expression was found in cancer cell cytoplasms of 30.1% of patients , with different degrees of intensity , ( 23.9% moderate and 6.2% strong ) . 
 nuclear staining turned positive in 31.8% , stratified from moderate in 26.5%to to strong in 5.3% . 
 there was a significant association between the number of lymph nodes involved and both nuclear ( p = 0.042 ) and cytoplasmic ( p = 0.003 ) mage-1 expression . 
 there was also a significant correlation between the nuclear expression of mage-1 and tumor size ( p = 0.018 ) . 
 cytoplasmic expression of mage-1 increased with increasing pathologic grade of tumors although the association was not statistically significant ( p = 0.119).conclusionscta mage-1 has significant association with some prognostic factors in breast cancer and may have the role of a prognostic factor .