we selected excised salivary gland tumors , excluding biopsy or consultation cases , from the surgical pathology files of the asan medical center from 1990 to 2012 . for retrieval of masc candidates , 196 salivary gland tumors ( mec , n=137 ; acicc , n=43 ; anos , n=11 ; cac , n=2 ; other unusual salivary gland neoplasm , n=3 )
the selection of study candidates was built on the histological features described by recent literature1,3 - 5 and sufficient amount of tissue available to construct a tissue microarray ( tma ) .
the criteria for selection among the above tumors included the presence of glandular formation or secretory activity .
the final specimens comprised 30 cases initially diagnosed as acicc ( n=16 ) , anos ( n=6 ) , mec ( n=3 ) , cac ( n=2 ) , salivary duct carcinoma ( sdc ; n=1 ) , squamous cell carcinoma ( n=1 ) , and oncocytic carcinoma ( n=1 ) . additionally , 6 conventional acicc cases , morphologically showing unequivocal serous acinar differentiation , and 2 sdc cases were selected for reference .
for all 38 cases , tmas were generated using a manual tissue arrayer ( pathology devices , westminster , md , usa ) .
two 1.0- or 1.5-mm cores were taken from donor blocks and arrayed into recipient blocks .
clinical data were obtained through review of medical records and pathologic staging was based on the cancer staging manual of american joint committee.8 disease - free survival was assessed from the time of histological diagnosis to recurrence or death , based on review of the patients ' medical records and information from the national health insurance .
a t - test was employed to characterize the relationships between quantitative variables and the fisher 's exact test was used to characterize the relationship between categorical variables .
disease - free survival periods with 95% confidence intervals ( ci ) were estimated using the kaplan - meier method , with statistical significance of differences between groups estimated by log - rank test . a p - value of < 0.05 was defined as statistically significant .
immunohistochemical stainings were performed using the ventana autostainer and ultra view dab detection kit ( ventana , tucson , az , usa ) according to manufacturer 's instructions .
fish was performed using a break - apart probe for the etv6 gene ( abbott molecular , des plaines , il , usa ) according to the manufacturer 's recommendations .
fish slides were examined with an olympus bx51 fluorescence microscope ( olympus , tokyo , japan ) using a 100 objective . through fitc and texas red band pass filters ,
a single green ( or red ) signal without a corresponding red ( or green ) signal in addition to a fused signal was considered negative ( non - rearranged ) in the present study .
red and green signals that were less than 2 signal diameters apart were considered as a single fused signal .
the average percentage of split signal in 6 referential aciccs , showing unequivocal serous acinar differentiation ( conventional acicc ) was 4.565 and standard deviation ( sd ) 3.042 .
thus , we considered cases , if more than 15% ( mean+3 sd , rounded up ) of examined nuclei showed a split signal , as positive for translocation which is the similar cut - off value used in previous literature.1 the slides were independently interpreted by 2 observers . if 10 - 20% of the analyzed cells showed a split signal , more cells were enumerated and the first and second cell count readings by the 2 observers were added together and a percentage was calculated .
valid fish results were obtained in 31 out of 38 cases . the laboratory used breast secretory carcinoma as a positive control .
from tma blocks , 4-m sectioned slides were obtained and prepared for immunohistochemistry and fish .
immunohistochemical stainings were performed using the ventana autostainer and ultra view dab detection kit ( ventana , tucson , az , usa ) according to manufacturer 's instructions .
fish was performed using a break - apart probe for the etv6 gene ( abbott molecular , des plaines , il , usa ) according to the manufacturer 's recommendations .
fish slides were examined with an olympus bx51 fluorescence microscope ( olympus , tokyo , japan ) using a 100 objective . through fitc and texas red band pass filters ,
a single green ( or red ) signal without a corresponding red ( or green ) signal in addition to a fused signal was considered negative ( non - rearranged ) in the present study .
red and green signals that were less than 2 signal diameters apart were considered as a single fused signal .
the average percentage of split signal in 6 referential aciccs , showing unequivocal serous acinar differentiation ( conventional acicc ) was 4.565 and standard deviation ( sd ) 3.042 .
thus , we considered cases , if more than 15% ( mean+3 sd , rounded up ) of examined nuclei showed a split signal , as positive for translocation which is the similar cut - off value used in previous literature.1 the slides were independently interpreted by 2 observers .
if 10 - 20% of the analyzed cells showed a split signal , more cells were enumerated and the first and second cell count readings by the 2 observers were added together and a percentage was calculated .
valid fish results were obtained in 31 out of 38 cases . the laboratory used breast secretory carcinoma as a positive control .
among the 31 technically successful cases , 13 cases showed etv6 translocation , reclassified as masc , and 10 cases showed intact etv6 , categorized as mimic masc ( fig .
the other 8 cases served as a reference , including 6 conventional aciccs and 2 sdcs , and showed intact etv6 . on average ,
57.65% of examined cells showed a split signal with etv6 translocation ( masc ) in 13 cases , while 9.55% of examined cells showed a split signal in 10 cases with intact etv6 ( mimic masc ) .
the percentage of cells with a split signal ranged between 20.75% and 78.57% in cases with etv6 translocation , and between 3.10% and 12.96% in cases with intact etv6 .
the clinicopathologic features of 23 masc candidates with etv6 translocation ( masc , n=13 ) or with intact etv6 ( mimic masc , n=10 ) , are summarized in table 2 . for referential comparison , conventional aciccs ( n=15 )
although no differences in gender distribution were found between masc and mimic masc cases , a noticeable male predilection was observed compared with conventional aciccs , which affected females more frequently than males ( f : m=12:3 ) .
the average age in the mascs , mimic mascs , and conventional aciccs was in the fifth decade . both mascs and
the parotid gland was the most frequently involved site in mascs , mimic mascs , and conventional aciccs and the tumor size in mascs ranged from 0.7 cm to 2.5 cm ( mean , 1.77 cm ) .
only 4 cases of masc showed extraparenchymal extension , and any lymph node involvement at the time of surgery was not observed .
all patients were initially treated by surgery , and 3 masc , 3 mimic masc , and 2 conventional acicc patients received postoperative radiotherapy and all are currently alive .
three masc patients developed local recurrence ( 3/13 , 23.1% ) with median recurrence time of 44 months after diagnosis ( range , 10 to 101 months ) and 2 mimic masc patients developed metastases to neck lymph nodes ( 2/10 , 20.0% ) at 62 months and 70 months after diagnosis , while an acicc patient developed local recurrence ( 1/15 , 6.7% ) at 39 months after diagnosis .
in summary , masc , mimic masc , and conventional acicc showed no statistically significant differences in gender , age , site , pathologic t stage , and disease - free survival .
histologically , the most frequent features in the 13 masc cases were well circumscribed , encapsulated , or multinodular masses , showing papillary - cystic , microcystic , solid , follicular or their combination growth pattern .
some cells were smaller than typical acinar cells appearing to have an increased nuclear to cytoplasmic ratio , and showed eosinophilic or finely granular cytoplasm and ovoid basophilic nuclei , known as intercalated duct - like cells .
other cells showed a more abundant or vacuolated cytoplasm and low - grade wrinkled nuclei .
numerous variable - sized spaces were frequently observed and the spaces were usually clear but sometimes contained pinkish- to grayish blue - colored amorphous materials , which were positive for periodic acid - schiff .
as stated in previous literature , the above - mentioned characteristics were predominant features of masc and corresponded to microcystic , papillary - cystic , or follicular type of acicc.1 - 5 these usual morphological masc features are illustrated in fig .
one case was macrocystic , measuring up to 2.5 cm , and partially filled with intraluminal papillary proliferation .
nevertheless , epithelial configuration of the cyst lining and papillary projection was similar to the mentioned masc .
the tumor showed focal proliferation of the epithelial lining and even cribriform appearance in some relatively small islands .
these features were also reminiscent of cac , however , epithelial lining cells were similar to masc rather than the cuboidal , clear , tall columnar , or oncocytic features of cac .
the third case showed a distinct morphologic change previously referred to as dedifferentiation or high - grade transformation of acicc.9 the tumor was composed of 2 distinct areas with an obvious transformation zone .
histological features of one area corresponded to microcystic and papillary - cystic masc and the other area showed poor zymogen granule containing - basophilic polymorphous cells , forming solid nests with comedonecrosis ( fig .
4 ) . a diversity of morphological features with intact etv6 was also obtained . among 10 mimic mascs ,
3 cases could be interpreted as cac . though the cytomorphologic features were not uniform , the tumor cells were slightly different from masc , showing cuboidal , tall columnar , clear to oncocytic features . additionally , the epithelial lining of cysts was more evenly thin in cac than masc . additionally , when papillary projections were present , they were mostly large papillae covered with columnar or tall cuboidal cells .
others showed nonspecific morphologies , corresponding to anos . infiltrating nests of clear cells , glandular growth in the prominent sclerosing background , or solid nests surrounded by very thin fibrovascular septa with reticular and abundant cytoplasm were other examples of mimic masc .
two cases of mecs lacking mucous and epidermoid cells were initially similar to masc , but careful examination can help in distinguishing mec from masc .
most staining patterns were strong and diffuse , except for a case showing dedifferentiation ( fig .
three out of 10 mimic mascs expressed s100 protein and no expression was observed in 6 conventional aciccs .
gross cystic disease fluid protein 15 ( gcdfp-15 ) immunostains were focally positive for 2 out of 13 mascs , 3 out of 10 mimic mascs , and none of the conventional aciccs .
dog1 was expressed in 3 conventional aciccs and 1 masc , with weak apical or apicoluminal membranous staining .
estrogen receptor ( er ) and progesterone receptor ( pr ) were all negative for masc , mimic masc and conventional aciccs ( table 3 ) .
among the 31 technically successful cases , 13 cases showed etv6 translocation , reclassified as masc , and 10 cases showed intact etv6 , categorized as mimic masc ( fig .
the other 8 cases served as a reference , including 6 conventional aciccs and 2 sdcs , and showed intact etv6 . on average ,
57.65% of examined cells showed a split signal with etv6 translocation ( masc ) in 13 cases , while 9.55% of examined cells showed a split signal in 10 cases with intact etv6 ( mimic masc ) .
the percentage of cells with a split signal ranged between 20.75% and 78.57% in cases with etv6 translocation , and between 3.10% and 12.96% in cases with intact etv6 .
the clinicopathologic features of 23 masc candidates with etv6 translocation ( masc , n=13 ) or with intact etv6 ( mimic masc , n=10 ) , are summarized in table 2 . for referential comparison , conventional aciccs ( n=15 )
although no differences in gender distribution were found between masc and mimic masc cases , a noticeable male predilection was observed compared with conventional aciccs , which affected females more frequently than males ( f : m=12:3 ) .
the average age in the mascs , mimic mascs , and conventional aciccs was in the fifth decade . both mascs and
the parotid gland was the most frequently involved site in mascs , mimic mascs , and conventional aciccs and the tumor size in mascs ranged from 0.7 cm to 2.5 cm ( mean , 1.77 cm ) .
only 4 cases of masc showed extraparenchymal extension , and any lymph node involvement at the time of surgery was not observed .
all patients were initially treated by surgery , and 3 masc , 3 mimic masc , and 2 conventional acicc patients received postoperative radiotherapy and all are currently alive .
three masc patients developed local recurrence ( 3/13 , 23.1% ) with median recurrence time of 44 months after diagnosis ( range , 10 to 101 months ) and 2 mimic masc patients developed metastases to neck lymph nodes ( 2/10 , 20.0% ) at 62 months and 70 months after diagnosis , while an acicc patient developed local recurrence ( 1/15 , 6.7% ) at 39 months after diagnosis .
in summary , masc , mimic masc , and conventional acicc showed no statistically significant differences in gender , age , site , pathologic t stage , and disease - free survival .
histologically , the most frequent features in the 13 masc cases were well circumscribed , encapsulated , or multinodular masses , showing papillary - cystic , microcystic , solid , follicular or their combination growth pattern .
some cells were smaller than typical acinar cells appearing to have an increased nuclear to cytoplasmic ratio , and showed eosinophilic or finely granular cytoplasm and ovoid basophilic nuclei , known as intercalated duct - like cells .
other cells showed a more abundant or vacuolated cytoplasm and low - grade wrinkled nuclei .
numerous variable - sized spaces were frequently observed and the spaces were usually clear but sometimes contained pinkish- to grayish blue - colored amorphous materials , which were positive for periodic acid - schiff .
as stated in previous literature , the above - mentioned characteristics were predominant features of masc and corresponded to microcystic , papillary - cystic , or follicular type of acicc.1 - 5 these usual morphological masc features are illustrated in fig .
one case was macrocystic , measuring up to 2.5 cm , and partially filled with intraluminal papillary proliferation .
nevertheless , epithelial configuration of the cyst lining and papillary projection was similar to the mentioned masc .
the tumor showed focal proliferation of the epithelial lining and even cribriform appearance in some relatively small islands .
these features were also reminiscent of cac , however , epithelial lining cells were similar to masc rather than the cuboidal , clear , tall columnar , or oncocytic features of cac .
the third case showed a distinct morphologic change previously referred to as dedifferentiation or high - grade transformation of acicc.9 the tumor was composed of 2 distinct areas with an obvious transformation zone .
histological features of one area corresponded to microcystic and papillary - cystic masc and the other area showed poor zymogen granule containing - basophilic polymorphous cells , forming solid nests with comedonecrosis ( fig .
4 ) . a diversity of morphological features with intact etv6 was also obtained . among 10 mimic mascs ,
3 cases could be interpreted as cac . though the cytomorphologic features were not uniform , the tumor cells were slightly different from masc , showing cuboidal , tall columnar , clear to oncocytic features . additionally , the epithelial lining of cysts was more evenly thin in cac than masc . additionally , when papillary projections were present , they were mostly large papillae covered with columnar or tall cuboidal cells .
infiltrating nests of clear cells , glandular growth in the prominent sclerosing background , or solid nests surrounded by very thin fibrovascular septa with reticular and abundant cytoplasm were other examples of mimic masc .
. two cases of mecs lacking mucous and epidermoid cells were initially similar to masc , but careful examination can help in distinguishing mec from masc .
most staining patterns were strong and diffuse , except for a case showing dedifferentiation ( fig .
three out of 10 mimic mascs expressed s100 protein and no expression was observed in 6 conventional aciccs .
gross cystic disease fluid protein 15 ( gcdfp-15 ) immunostains were focally positive for 2 out of 13 mascs , 3 out of 10 mimic mascs , and none of the conventional aciccs .
dog1 was expressed in 3 conventional aciccs and 1 masc , with weak apical or apicoluminal membranous staining .
estrogen receptor ( er ) and progesterone receptor ( pr ) were all negative for masc , mimic masc and conventional aciccs ( table 3 ) .
in this study we investigated the new salivary gland tumor , known as masc , to clarify the clinical , histological , and immunohistochemical characteristics , and to determine any pathognomonic features helpful in diagnosing mascs .
the first masc report focused on features reminiscent of breast secretory carcinoma.1 in recent years , there have been several studies extending the clinical , histological , and immunohistochemical properties of mascs.3,5 however , masc clinicopathologic features appear to be indistinctive and the molecular demonstration of etv6 rearrangement is the only diagnostic method .
regardless of the originality of this entity , a salivary gland tumor with etv6 translocation has yet to be documented in korea .
the present study compared masc candidate groups with an intact etv6 and etv6 translocation . to evaluate mascs with an emphasis on easy identification of specific characteristics , consideration of the ideal cut - off value to assess the etv6 fish results was necessary . in this study ,
the negative control cut - off value ( mean+3 sd , 13.688 ) showed a 99.7% ci for intact etv6 .
the cut - off value used ( 15% ) was slightly higher than the negative control cut - off value .
moreover , the interpretation of a single colored signal without a corresponding colored signal in addition to a fused signal as negative could decrease the percentage of positive cells for etv6 translocation .
the highest negative value for etv6 translocation was 12.96% and the lowest positive value was 20.75% .
this result shows the importance of determining an accurate cut - off value in a study .
overall , papillary - cystic , microcystic , or their mixture types were predominantly observed .
aciccs , according to the current world health organization classification , are comprised of heterogeneous tumors .
many cell types , including acinar , intercalated duct type , vacuolated , clear , and nonspecific glandular cells , and variable growth patterns such as solid , microcystic , papillary - cystic , and follicular , are described as constituents of acicc .
anos is also a heterogeneous tumor group , and their diagnosis is based on the lack of features that characterize other salivary gland adenocarcinomas .
according to prior studies and our results , a considerable number of anos and zymogen granule - poor aciccs belong to the masc group .
the concept that masc is a distinctive entity of salivary gland tumors requires more consideration , however , no zymogen granule - rich acicc or sdc has shown an etv6 rearrangement .
our study revealed previously undescribed histology in etv6-rearranged tumors , such as a solid pattern resembling dedifferentiated acicc and a large cyst with sparse papillary proliferation .
chiosea et al.4 reported that the results of etv6 fish in 4 cases of acicc with high - grade malformation were all negative for etv6 translocation .
apart for a few exceptions , the histological outline of masc appears evident , and mimic masc cases without an etv6 translocation could be differentiated from masc by careful histological examination .
dog1 , known as a novel marker of salivary acinar and intercalated duct differentiation , was not helpful.10 gcdfp-15 , er , and pr were considered a possibility due to their mammary - like character , but were not expressed in masc .
the s100 protein was highly sensitive , but showed low specificity to differentiate masc from mimic masc .
, masc could be a molecularly well - defined salivary gland neoplasm , encompassing some portions of acicc and anos , but its histological spectrum and clinical implication need further investigation .
 backgrounda new tumor entity of the salivary glands , mammary analogue secretory carcinoma ( masc ) with etv6-ntrk3 translocation , has recently been proposed . 
 masc was originally diagnosed as adenocarcinoma , not otherwise specified ( anos ) , or acinic cell carcinoma ( acicc ) by the current world health organization classification . 
 we aimed to identify masc cases by molecular tests , and to characterize their clinical , histological , and immunohistochemical features.methodsthirty cases of masc candidates were selected after review of 196 salivary gland tumors , and subjected to break - apart etv6 fluorescence in situ hybridization ( fish ) , and immunohistochemical study for s100 protein , gross cystic disease fluid protein 15 , dog1 , estrogen receptor , and progesterone receptor.resultsvalid fish results were obtained in 23 cases , and 13 positive cases were retrieved . 
 mascs were histologically varied , and the most frequent features observed in 10 cases were low - grade papillary / cystic / glandular patterns , intraluminal secretory materials , ovoid / wrinkled nuclei , and relatively abundant granular eosinophilic cytoplasms , corresponding to papillary - cystic or follicular types of acicc . 
 all cases showed diffuse immunopositivity for s100 protein . 
 three cases developed recurrences , but all patients remained alive.conclusionsmasc could be a molecularly well - defined salivary gland neoplasm , encompassing some portions of acicc and anos , but its histological spectrum and clinical implication require further investigation . 