the largest family of cancer testis antigens ( ctas ) and one of the most important is the family of melanoma antigen encoding genes ( mage ) .
members of the mage family are involved in resistance to apoptosis and cell cycle progression and have been associated with some features of neoplastic phenotype such as immortality , invasion , and immune evasion metastatic ability [ 13 ] .
one of its members , mage a3 , named melanoma antigen encoding gene a3 because it was first reported to encode a tumor - specific antigen on melanoma cell line , has been studied and used in trials of immunotherapy in various histological types of cancers because it is recognized by t - lymphocytes [ 5 , 6 ] . in tumor cells
abnormal expression of mage a3 gene has been observed in melanomas , lung carcinomas , head and neck cancers , leukemia , squamous cell carcinomas , breast cancers , multiple myelomas , astrocytomas , ovarian tumors , hepatocellular carcinomas , colorectal carcinomas , and thyroid carcinomas , and its abnormal expression is related to aggressive disease in most tumors , a fact that made many authors hypothesize on a possible role of mage a3 in malignant transformation and progression [ 4 , 79 ] .
although associated with features of aggressiveness , mage a3 might also play an important and beneficial role in tumors , acting as a stimulant of immune responses .
in fact , this protein has been tested on several in vivo models , aiming to determine its efficacy as an immune response inducer , and these preliminary results point out to a positive role of mage a3 in inducing tumor attack [ 10 , 11 ] .
although these tests have been conducted in different types of malignancy , studies of mage a3 protein in thyroid cancers are scarce and do not relate this protein to immune responses . the present study aimed to investigate a possible role of mage a3 in thyroid malignancy , analyzing its utility as a diagnostic and prognostic marker .
we also aimed to analyze whether mage a3 expression would be associated with the presence of infiltrated immune cells .
we studied 314 patients who underwent thyroid resection for thyroid cancer or thyroid nodules at ac camargo cancer center , so paulo , brazil .
regarding histological types , 154 out of the 195 malignant tissues were papillary thyroid carcinomas ( ptc ) and 41 follicular thyroid carcinomas ( ftc ) .
the most frequent histotype among papillary carcinomas was the classic variant ( cptc ) ( 101 cases ) , followed by the follicular variant ( fvptc ) ( 49 cases ) .
we also obtained benign tissue from patients , including 51 follicular adenomas ( fa ) and 51 goiter ( g ) , and 17 normal thyroid tissues obtained from the contralateral lobe of fa ( nt ) ( figure 1 ) .
aggressiveness at diagnosis was ascertained using american joint committee on cancer ( ajcc ) tnm system for differentiated thyroid carcinomas .
patients were managed according to lats and ata guidelines [ 12 , 13 ] and followed for a period of 12 to 175 months ( median = 35 ; mean = 41.1 ; sd = 26 months ) .
this study protocol was approved by the research ethics committees of the institutions involved ( 1259/09-c ) .
all the available clinical , surgical , and pathology reports , as well as follow - up data , were recorded .
all tumors were carefully and independently reviewed by two experienced pathologists ( jv and fas ) for diagnostic confirmation , and cases presenting conflicting results or areas of poor differentiation were excluded .
paraffin blocks of formalin - fixed tissues were collected and , in each case , the most representative area of the tumor , normal surrounding tissue , tumor areas of invasiveness , and metastatic tissue were selected and microdissected whenever available .
tissue microarrays ( tmas ) were built using the semiautomated tmarrayer ( beecher instruments , silver springs , md , usa ) .
five micrometer sections of tma were placed on electrically charged slides , deparaffinized , and rehydrated in decreasing concentrations of alcohol .
all tissue sections were subjected to heat - induced antigen retrieval using 10% citrate buffer ( 10  mm , ph 6.0 ) in a steamer ( 90c for 30 minutes ) .
tissues sections were then incubated overnight at 6c , with anti - mage a3 rabbit polyclonal antibody ( ls - b884 - 50ug , lifespan biosciences , seattle , wa , usa ) , diluted at 1  :  10 ; cd4 monoclonal 1f6 ( novocastra , newcastle upon tyne , uk ) diluted at 1  :  100 ; cd8 monoclonal sp16 ( thermo scientific , rockford , il , usa ) diluted at 1  :  100 .
the advanced biotin - free polymer detection system was used ( dako , carpenteria , ca , usa ) . dab ( 3.3-diaminobenzidine - tetrahydrochloride ; sigma , st louis , ma , usa ) was applied as chromogen for five minutes , at room temperature .
slides were quantified by at least two of the authors ( mbm and/or mam and ecm ) and then submitted to other two independent experienced pathologists ( jv and fas ) , both blinded to tumor features for the final score .
an individual evaluation of each marker was completed for each spot tissue , estimating the number of positive cells per tma spot , considering an approximate area of 0.79  mm .
cells were considered positive for mage a3 when a cut - brown staining was observed in the cytoplasm as demonstrated in figure 2 .
visual evaluation was made for each tissue spot , estimating the percentage of positive tumor cells and the staining intensity .
the percentage of positive cells was graded as follows : 0 = no positive cell ; 1 = up to 25% positive cells ; 2 = 25 to 50% positive cells ; 3 = 50 to 75% positive cells ; 4 = more than 75% positive cells .
intensity was graded as follows : 0 = negative ; 1 = faint staining ; 2 = moderate staining ; 3 = strong staining . a final score was calculated adding both percentage of positive cells and immunostaining intensity , which ranged from 0 to 7 . for statistical purposes ,
cases scored from 0 to 2 were grouped as negative and cases scored from 3 to 7 were considered positive .
in addition to visual evaluation , we also analyzed the immunohistochemical expression of mage a3 using the acis - iii automated cellular imaging system ( chromavision medical systems , inc , dako ) .
briefly , each tissue spot was digitized to the system software and a numerical value proportional to the intensity and extension of brown staining was attributed by the computer analysis .
we considered the given numbers using the following formula : score = ( intensity  brown area)/(brown area + blue area ) .
we considered staining  median as negative , whereas all staining > median was considered positive in the survival analysis , as previously described [ 1416 ] . regarding tumor - infiltrating lymphocytes analyses
were considered positive for immunohistochemical markers when a clear - cut brown staining was observed in the typical corresponding cellular localization .
the cases were grouped into categories for statistical analysis : negative ( no positive cell ) and positive ( presence of positive cells in each spot ) .
the statistical analysis was carried out using the sas system for windows ( statistical analysis system ) , version 9.1.3 , service pack 3 , institute inc . , 2002 - 2003 , cary , nc , usa .
a multivariate logistic regression model was applied using tumor type as a dependent variable ; protein expression ; and clinical risk factors , including gender and age as explicative variables .
recurrence - free survival was calculated using kaplan - meier survival curves with log - rank comparison .
nonparametric analysis was performed using either chi - square or fisher 's exact test , as indicated .
the mann - whitney test was used to compare continuous or arranged measures between two groups ; kruskal - wallis test was used to compare three or more groups .
the predictive accuracy of mage a3 expression to predict malignancy was evaluated using receiver operating characteristic ( roc ) curve analysis based on predicted probabilities from logistic regression models .
as expected , dtc patients were predominantly females ( 81.52% ) with a mean age at diagnosis of 45.63  15.34 years ( range : 15 to 88 years ) .
dtc group did not differ from the individuals with benign thyroid diseases regarding gender or age at diagnosis .
unfortunately , we did not obtain sufficient metastatic tissues to be included in the statistical analyses ; thus , these tissues were excluded from further analysis .
the positivity of mage a3 protein was identified in the cytoplasm with a brown color .
intensity and positivity differences were observed among tissue types , both in the quantitative and the semiquantitative analyses , as demonstrated in figure 3 and table 1 . according to semiquantitative ( visual ) analysis ,
mage a3 was positive in 185 ( 94.87% ) out of the 195 cases of dtc and 74 ( 62.18% ) out of the nonmalignant thyroid samples analyzed ( p < 0.0001 ) .
ftcs presented a mean score of 3 for the percentage of positive cells and of 2 for intensity , cptcs presented a mean score of 3 for the percentage of positive cells and of 2 for intensity , fvptcs presented a mean score of 3 for the percentage of positive cells and of 2 for intensity , tcptcs presented a mean score of 3 for the percentage of positive cells and of 3 for intensity , fas presented a mean score of 2 for the percentage of positive cells and of 2 for intensity , gs presented a mean score of 1 for the percentage of positive cells and of 1 for intensity , and nts fas presented a mean score of 0 for the percentage of positive cells and of 0 for intensity .
a comparison of scores in different subtypes of thyroid lesions revealed that the visual analysis of ihc was able to discriminate some lesions , as demonstrated in table 1 .
mage a3 was expressed more in malignant than in benign lesions ( figure 3 ) .
based on the medians of expression given by acis iii , it was possible to perform a diagnostic roc curve for thyroid nodules .
the roc curve for a cutoff point of 71.93 may have identified a reasonable point of specificity and sensitivity .
in fact , mage a3 expression distinguished malignant from benign lesions ( p < 0.0001 ) with 66.70% sensitivity , 77.70% specificity , positive predictive value ( ppv ) of 85.92% , negative predictive value ( npv ) of 53.36% , and accuracy of 70.32% ( figure 3 ) .
this was also done with the follicular - patterned lesions , which constitute a major pathology challenge for diagnosis : cft , fa , and fvptc .
the comparison of ftc with fa suggested 78.550 as the cutoff point ( ftc group greater than or equal to 78.550 and fa less than that ) , showing a sensitivity of 71.40% , specificity of 78.80% , ppv of 73.14% , npv of 77.32% , and accuracy of 75.49% .
however , the roc curve for fvptc versus fa did not identify a good cutoff point . for a median expression of 76.660 ( fvptc group
76.660 or greater and smaller than that of fa ) the sensitivity was 53.30% , the specificity was 75% , ppv was 64.86% , npv was 64.98% , and accuracy was 64.93% ( figure 3 ) . concerning the quantitative analysis ,
the median expression of mage a3 was higher ( 75.077  30.419 ) in patients aged > 45 years than in patients aged < 45 years ( 85.646  32.625 ; p = 0.0272 ) and in females ( 84.763  30.375 ) than in males ( 65.840  32.044 ; p = 0.0016 ) .
regarding the characteristics of aggressiveness and invasion , tumor size , and multifocality , mage a3 was higher in tumors in which there was extrathyroidal invasion ( 83.421  30.880 ) than in cases without invasion ( 71.765  32.311 ) ( p = 0.0206 ) .
as expected , mage a3 was more expressed in patients with stage ii ( 91.072  22.983 ) when compared with stage i - tmn ( 76.060  32.875 ) ( p = 0.0107 ) as demonstrated in table 2 . however , there was correlation neither with tumor size and multifocality nor with disease - free interval .
we observed that mage a3 positive tumors were more likely to present cd8 + til 50% than cd8 til 46% ( p = 0.0346 ) .
when we compared mage a3 and cd8 positivity with clinical pathological features , mage a3 + cd8 + tumors were associated with less aggressive features : 31% of tumors presenting mage a3 + cd8 + phenotype did not present extrathyroidal invasion , whereas only 15% of mage a3 + cd8 were not invasive ( p = 0.0427 ) .
in addition , 35% of mage a3 + cd8 + tumors were < 2  cm , while 13% of mage a3 + cd8 were > 2  cm ( p = 0.0034 ) ( table 3 ) .
we did not find any correlation of mage a3 + cd8 + with outcome , although there was a trend of these tumors to evolve free of disease ( p = 0.0634 ) .
our results did not evidence any association between mage a3 expression and infiltration of cd4 + til .
many members of the mage family are normally expressed in the testis and placenta ; however , members of the a subfamily are found mainly in neoplastic tissues , leading some authors to identify the mage a family as a candidate of thyroid carcinogenesis mediator [ 17 , 18 ] .
although there are reports on the expression of members of mage family in thyroid cancers , reports concerning mage a3 are still insufficient [ 1821 ] . in this work , we demonstrated that mage a3 may help not only to identify malignancy in thyroid nodules but also to sort out follicular - patterned lesions ( i.e. , ftcs and fas ) .
the authors showed that the expressions of mage a1 and mage1/2 detected in fine - needle aspiration biopsies ( fnabs ) by reverse transcriptase - polymerase chain reaction ( rt - pcr ) distinguished fvptc from papillary hyperplasia in nodular goiters , facilitating the diagnosis of thyroid nodules and providing additional information to delineate ptc from papillary hyperplasia in fnab .
other functional studies reinforced these differences between mage expressions in thyroid lesions , suggesting that mage a3/6 and fgfr2-iiib participate in the development of thyroid cancer .
although not exactly similar , other authors have also reported associations between mage a3 and clinical pathological features of thyroid tumors .
demonstrated that mage a3 positivity was more frequent in ptcs smaller than 1  cm ( microcarcinomas ) , suggesting that the loss of this protein expression could be related to tumor progression . using an antibody that recognized both mage a6/a3 in 375 cases of thyroid tissues and 53 of metastatic disease , cheng et al . demonstrated that mage proteins are overexpressed in primary and metastatic thyroid tumors when compared with their surrounding normal tissues
furthermore , cytoplasmic mage score was related to tumor size and the number of lymph node metastases .
.   observed that there was a strong staining of this protein in large tumors and in metastasis , thus , providing additional evidence that this protein may be involved in the formation of metastasis and tumor progression . using an antibody that only recognized mage a
we observed an increased expression of mage a3 in the presence of extrathyroidal invasion , and more advanced stages of disease pointed out to the same direction , leading us to believe that this protein is related to tumor progression and aggressiveness .
furthermore , we demonstrated a possible clinical utility of mage a3 expression in the identification and characterization of malignancies of follicular lesions .
although the real physiologic role of mage 3 gene has yet to be identified , the cancer specificity of this gene expression may be important because of its possible application in tumor - specific immunotherapy .
in fact , mage a3 has been studied as a possible immune target in other types of tumors and may be interesting for differentiated thyroid cancers refractory to radioiodine therapy .
furthermore , we demonstrated that although mage a3 has been related to some features of tumor aggressiveness , when tumors were mage a3 + and able to recruit cd8 + expression , aggressiveness not only disappeared but also was associated with better prognostic features such as stage i , smaller tumors ( < 2  cm ) , younger age ( 45 years ) , and absence of extrathyroidal invasion .
there was a trend of mage a3 + cd8 + tumors to evolve free of disease .
these results reinforce the hypothesis of a role of mage a3 as a recruiter / inducer of cd8 + tumor infiltrating lymphocytes , a set of immune cells that our group had already associated with better outcome and less aggressive disease [ 15 , 16 ] .
as already mentioned , the members of the mage family have been recently considered as tumor antigens , therefore , able to induce and recruit several types of immune cells to attack tumors . in a recent study ,
grard et al . demonstrated that mage a3 may be an efficient inducer of long - lasting antitumor responses .
in fact , several groups have demonstrated that vaccines using mage a3 as immune response inducer might work for different types of advanced cancers , such as lung cancers , melanomas , renal cancers , and esophageal and even head and neck cancers , all with a considerable efficacy [ 2427 ] .
there are no reports on the usage of mage a3 as an immune vaccine in thyroid cancer until the present moment , but undoubtedly , our study opens this possibility for a promising future scenario , especially considering patients with metastatic or refractory disease . in conclusion , we demonstrated that although mage a3 does not seem to solve diagnostic problems , such as the diagnosis of follicular - patterned lesions , it may play an important role in malignant thyroid nodules , potentially discriminating cases of worse outcome and presenting an interesting perspective for new researches on differentiated thyroid cancer immunotherapy .
 
 background . we aimed to investigate a possible role of mage a3 and its associations with infiltrated immune cells in thyroid malignancy , analyzing their utility as a diagnostic and prognostic marker . 
 materials and methods . 
 we studied 195 malignant tissues : 154 ptcs and 41 ftcs ; 102 benign tissues : 51 follicular adenomas and 51 goiter and 17 normal thyroid tissues . 
 mage a3 and immune cell markers ( cd4 and cd8 ) were evaluated using immunohistochemistry and compared with clinical pathological features . 
 results . 
 the semiquantitative analysis and acis iii analysis showed similar results . 
 mage a3 was expressed in more malignant than in benign lesions ( p < 0.0001 ) , also helping to discriminate follicular - patterned lesions . 
 it was also higher in tumors in which there was extrathyroidal invasion ( p = 0.0206 ) and in patients with stage ii disease ( p = 0.0107 ) . 
 mage a3 + tumors were more likely to present cd8 + til ( p = 0.0346 ) , and these tumors were associated with less aggressive features , that is , extrathyroidal invasion and small size . 
 there was a trend of mage a3 + cd8 + tumors to evolve free of disease . 
 conclusion . 
 we demonstrated that mage a3 and cd8 + til infiltration may play an important role in malignant thyroid nodules , presenting an interesting perspective for new researches on dtc immunotherapy . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion