spontaneous tumors of the rat submandibular gland are very rare , 1   ,   2   and most of them 
                 are diagnosed as adenomas or adenocarcinomas originating from acinar cells and 
                 ductal epithelia .
3   to our knowledge , there 
                 are few reports on detailed analyses of tumors in the submandibular glands of rats .   in the present case , the results of immunohistochemical and ultrastructural 
                 observations indicated that the rat submandibular gland tumor was probably derived 
                 from basal cells located in the duct .
the animal was a 10-week - old female galas rat 
                 ( brlhan : wist@jcl , purchased from clea japan , inc . , tokyo , japan ) that was assigned 
                 to the control group in a 4-week repeated - dose oral toxicity study and subjected to 
                 the final sacrifice according to the experimental protocol . during the treatment 
                 period
, there was no abnormality in clinical signs or body weight of the animal . at 
                 necropsy ,
a white nodule ( about 5  3 mm in size ) was found in the right 
                 submandibular gland . in other organs
the animal was treated in accordance with the rules for approval of animal 
                 experiments in sugi institute of biological science co. , ltd .  
the submandibular gland was routinely fixed in 10% phosphate - buffered formalin , and 
                 embedded in paraffin and sectioned .
immunohistochemical staining was performed using the following 
                 primary antibodies : cytokeratin ( ae1/ae3 ; 1:20 , dako , denmark ) , p63 ( 1:50 , dako , 
                 denmark ) , alpha - smooth muscle actin ( sma ) ( 1:50 , dako , denmark ) , vimentin ( 1:50 , 
                 dako , denmark ) , desmin ( 1:100 , dako , denmark ) , s-100 ( 1:400 , lvc , usa ) and 
                 proliferating cell nuclear antigen ( pcna ; 1:100 , dako , denmark ) .
the deparaffinized 
                 sections were incubated in the antibodies at room temperature for 1 hr and then in 
                 the envison+system - hrp labeled polymer ( dako , denmark ) at room temperature for 30 
                 min , and the staining was visualized with diaminobenzidine . for transmission 
                 electron microscopic examinations , the tissue kept in 10%
phosphate - buffered 
                 formalin was postfixed in 1% osmium , routinely processed to epoxy resin - embedded 
                 ultrathin sections and stained with uranyl acetate and lead citrate .  
histopathologically , oval to spindle - shaped and pale basophilic tumor cells 
                 proliferated closely and formed variably sized foci or grew diffusely in the 
                 submandibular gland ( figs .
the nodule separated by thin 
                 interstitial connective tissue and lobular structures were partially formed ( fig .
the border between the nodule and 
                 surrounding normal tissue was mainly clear , and the nodule partially spread into or 
                 invaded the surrounding normal tissue .
the nuclei of the tumor cells 
                 showed various sizes and many mitotic figures and contained poor chromatin and one 
                 to a few nucleoli ( fig .
ductal and acinar 
                 structures , cribriform patterns and keratinization were not found in the tumor .
immunohistochemically , the tumor cells showed a diffusely positive reaction for p63 
                 in the nuclei ( fig .
2 ) and for cytokeratin 
                 ( ae1/ae3 ) in the cytoplasm . there was a negative reaction for sma , vimentin , desmin 
                 and s-100 .
ultrastructurally , the tumor cells contained many tonofilaments in their cytoplasm 
                 and a few desmosomes at the intercellular portion ( fig .
the anti - p63 protein is a selective marker for both basal and myoepithelial 
                     cells , 4   ,   5   and the anti-sma protein is a selective marker for myoepithelial 
                 cells .
in addition , the reliable identification method of myoepithelial cells is 
                 thought to be an immunohistochemical method using anti-sma protein , together with 
                 anti - vimentin protein or anti - s-100 protein .
6 
                  ,   7   in the present case , the nuclei 
                 of the tumor cells were positive for anti - p63 protein , whereas no tumor cells showed 
                 a positive reaction for anti-sma protein . furthermore , the tumor cells were 
                 positive for cytokeratin ( ae1/ae3 ) and negative for vimentin , s-100 and desmin . 
                 based on the results of immunohistochemical examinations , the tumor cells were 
                 considered to be derived from basal cells .  
ultrastructurally , myoepithelial cells generally possess myofilaments with dense 
                     bodies . 8   in the present case ,
the tumor 
                 was not considered to originate from myoepithelial cells because there were no 
                 myofilaments in the cytoplasm .
since the tumor cells had many mitotic figures , a positive reaction to anti - pcna 
                 protein , and partially necrotic foci and infiltrated into the surrounding normal 
                 tissue , the tumor was regarded as a malignant tumor .  
based on these results , the tumor was diagnosed as a spontaneous basal cell 
                 carcinoma . in the normal submandibular gland ,
basal cells are located in the 
                 excretory duct 3 ; however , tonofilaments , the 
                 existence of which is one of the characteristic features of squamous cells , were 
                 observed in the cytoplasm of the tumor cells .
squamous cells are normally located in 
                 the end portion of the excretory duct in the submandibular gland . 3   ,   9 
                 therefore , the tumor is considered to have originated from the basal cells in the 
                 excretory duct near the opening to the oral cavity .
adenocarcinoma because this tumor did not clearly show the ductal structure .   in humans , tumors of the submandibular gland originating from basal cells
basal cell adenocarcinomas. the histologic 
                 appearance of the basal cell adenoma is dominated by relatively uniform , 
                 monomorphous proliferation of basaloid cells . while each tumor generally has a 
                 uniform histomorphologic architecture , variations among tumors allow categorization 
                 into solid , trabecular , tubular and membranous types .
basal cell adenocarcinoma is 
                 very similar to basal cell adenoma cytologically and histomorphologically and 
                 manifests infiltrative growth and incidental metastasis .
9   in rats , however , there are no reports on the classification 
                 of basal cell adenomas or adenocarcinomas in the submandibular gland .
 at necropsy , a white nodule ( about 5  3 mm in size ) was observed in the right 
                     submandibular gland of a 10-week - old female galas rat . 
 histopathologically , oval 
                     to spindle - shaped and pale basophilic tumor cells proliferated closely , and 
                     formed variably sized foci . 
 the nodule partially spread into or invaded the 
                     surrounding normal tissue , and necrotic foci were recognized in the tumor . 
                     
 immunohistochemically , the nuclei of the tumor cells showed a diffusely positive 
                     reaction for p63 , and the cytoplasm showed a diffusely positive reaction for 
                     cytokeratin and negative reaction for sma , vimentin , desmin and s-100 . 
 many 
                     tumor cells were positive for pcna . 
 ultrastructurally , the tumor cells contained 
                     many tonofilaments in the cytoplasm and a few desmosomes at the intercellular 
                     portion . 
 based on these findings , the tumor was diagnosed as a basal cell 
                     carcinoma originating from the duct in the rat submandibular gland . 