Expression And Relationship Of Aromatase And PCNA In Salivary Gland Mucoepidermoid Carcinoma

Objectives: Mucoepidermoid carcinoma is one of the mostcommon salivary gland malignancy and comprises 12 % of allsalivary gland tumours or 30% of all salivary glandmalignancies.It can be divided into well differentiated,moderately differentiated and poorly differentiated typeaccording to histopathological criterion.Currently,the primarytreatment for salivary gland MEC is surgical resection.Thesuccess of endocrine therapy for sex hornone-dependent tumorsprovides a new thought for treating salivary gland MEC.Studiesin recent years show that there are estrogen receptors in salivarygland MEC tissues and the disturbance of sex hormonemetabolizing may be correlated with the occurrence anddevelopment of salivary gland MEC.Cytochrome P450aromatase plays an important role in the process of conversionfrom androgen to estrogen. It can transform androgen to estrogen,and modulate estrogenic and/or androgenic actions.Proliferating cell nuclear antigen is usually regarded as anindex of determinating cell proliferating and its expression levelin the nuclear is correlated with with the activity of cellproliferation. The activity of tumor cell proliferation can beknown by examinationing PCNA of tumor cells.There is obviousrelationship between aromatase and PCNA in mammary glandcarcinoma tissues and the local estrogens catalysed by aromatasein tumor tissues played an important role in the tumor cellproliferating process.We adopted immunohistochemical method to investigate theexpression and immunolocation of aromatase and PCNA intissues of normal salivary gland and MEC in order to discuss thefunction of aromatase in salivary gland MEC and the relationshipbetween aromatase and proliferation of tumor cells,then toprovide theoretical evidences for treating salivary gland MEC byaromatase inhibitor.Methods: Formalin-fixed,paraffin-embedded blocks of 40salivary tissues with MEC and 17 normal salivary tissues wereselected and divided into experimental group (MEC) and controlgroup (normal), respectively. 4 sections with thickness of 4-5μmwere cut from each block, one for HE staining, one for negativecontrol, the others for immunohistochemical staining ofaromatase and PCNA, respectively. The immunohistochemicalstaining was operated as following: the paraffin sections weredeparaffinized by normal method; placed them in methanolcontaining 5% H₂O₂ for 25 min to block endogenous peroxidaseactivity; put them into microwave oven to repair antigens for 15min at 92-98℃, cooled at room temperature; the section weretreated with normal goats serum for 30min at 37℃; after beingwiped the serum off, the sections were incubated with theprimary antibodies for one night at 4℃; the sections were treatedwith biotinglated secondary antibodies for 25 min at 37℃; andwith peroxidaze-conjugated streptavidin for 20 min at 37℃;stained with DAB and conterstained with hematoxylin,dehydrated in graded rthamal, cleared in xylene and finallymounted. Except for the normal goat serum-blocking step, thesections were washed with PBS for 3×5 min after each treatment.The positive control was positive breast carcinoma tissuesexpressing aromatase and PCNA. As a negative control, thesections were treated with PBS instead of the primary antibodies.The positive expression of aromatase is that there weregranules of yellow to brown in cytoplasm and/or membrane andstaining is stronger than that of background . Negative expressionis that there was no positive cell.Positive expression of PCNA is that there were granules ofyellow to brown in nucei and the staining is stronger than that ofbackground.Criterion of grade:Five representative visual fieldswere selected under light microscope(400×) and positive cellpercentage, which is also called proliferating index (PI),wascounted according to numbers of possive cells/all observingcells.PI=0 means negative(-),1%～25 % was weak positive(+),26%～50% was moderate positive(++), >50% was strongpositive(+++).All data were statistically analyzed by SPSS11.5.Chi-squaretest was used in comparing expression rate and the Fisher exactprobabilities was used when necessary. The rank sum test wasused in comparing staining intensity.Results1 Expression of aromatase1.1 Aromatase was expressed in both MEC group and normalsalivary gland group.Aromatase was expressed mainly in mucouscell of MEC with positive rate 55%(22/40)and acinus cell ofnormal salivary gland with positive rate 17.65%(3/17). Thedifference of expression rate between MEC and normal groupwas statistically significant (P<0.05).1.2 Expression rate of aromatase in MEC tissues from differentsex was not statistically significant(P>0.05).1.3 Expression rate of aromatase in MEC tissues from differentsalivary gland was not statistically significant(P>0.05).1.4 Expression rate of aromatase in MEC tissues from differentdegree of differentiation was not statistically significant(P>0.05).2 Expression of PCNA2.1 All samples in MEC group are positive for PCNA and itspositive expression rate is 100%(40/40).There were 11 weakpositive,17 moderate positive and 12 strong positive samples inMEC group.The positive expression rate of normal salivary glandis 11.76%(2/17)and two positive samples are all weak positive.These differences of expression rate and expression intensitybetween MEC and normal group were statistically significant(P<0.05).2.2 Expression intensity of PCNA in MEC tissues from differentsex was not statistically significant(P>0.05).2.3 Expression intensity of PCNA in MEC tissues from differentsalivary gland was not statistically significant(P>0.05).2.4 Expression intensity of PCNA rises gradually with thedepression of differentiation degree of salivary gland MEC.3 Expression intensity of PCNA in aromatase positive MECtisues is stronger than that in aromatase negative MEC tissues.Conclusions1. Aromatase was expressed in both salivary gland MEC tissuesand normal salivary tissues.The expression rate of aromatase inMEC tissues is stronger than that in normal salivary tissues.2. Expression rate of aromatase in MEC tissues from differentsex, different salivary gland and different degree ofdifferentiation was not statistically significant.3. PCNA was expressed in both salivary gland MEC tissues andnormal salivary tissues. Expression rate and expression intensityof PCNA were higher in MEC tissues than those in mormalsalivary gland tissues.4. The difference of expression intensity of PCNA for differentdegree of differentiation was statistically significant and it risesgradually with the depression of differentiation degree of salivarygland MEC.The proliferating index of PCNA can be regarded asan important parameter of histological grading for salivary glandMEC.5. Expression intensity of PCNA from different sex and fromdifferent salivary gland were not statistically significant.6. The estrogens catalysed by aromatase over expressed in local...