PLEOMORFNOG ADENOMA , ADENOMA BAZALNIH ĆELIJA I POLIMORFNOG ADENOKARCINOMA NISKOG GRADUSA IMMUNOHISTOCHEMICAL AND KARYOMETRIC SIMILARITIES AND DIFFERENCES OF SALIVARY GLAND TUMORS BETWEEN PLEOMORPHIC ADENOMA , BASAL CELL ADENOMA AND POLYMORPHOUS LOW GRADE ADENOCARCINOMA

Uvod: Tumori pljuvačnih žlezda su veoma retke neoplazme. S obzirom na njihovu patohistološku sliku, ovi tumori predstavljaju veoma veliki dijagnostički izazov. Cilj: istaživanja je diferencijacija ova tri tipa tumora primenom imunohistohemijske i morfometrijske analize, kao i određivanje visine Ki67 proliferativnog indeksa. Materijal i metode: Istraživanje je obuhvatilo 44 tumora, 20 pleomorfnih adenoma, 12 adenoma bazalnih ćelija i 12 polimorfnih adenokarcinoma niskog gradusa. Analizirana je ekspresija Ki67, p53 i HER-2 antigena, kao markera proliferacije. U sklopu diferencijalne dijagnostike, analizirana je ekspresija CEA, EMA, GFAP, p63, vimentina, CK14, α-SMA, S-100 protein i WT1 antigena. Morfometrijska analiza vršena je u softverskom paketu „ImageJ” verzija 1.43u. Rezultati: Neoplastične ćelije u pleomorfnom adenomu su pokazale jaku ekspresiju GFAP, p63, WT1, vimentin i S100. U grupi od dvanaest polimorfnih adenokarcinoma niskog gradusa prisutna je difuzna ekspresija CK14, S100, vimentin i EMA su bili apsolutno eksprimirani, dok je αSMA bio negativan. Adenom bazalnih ćelija pokazuje pozitivnost na S-100, CEA, p63 i vimentin. Analizom vrednosti proliferativnog Ki67 indeksa ustanovljena je statistički značajna razlika u grupi pleomorfnog adenoma, što se dovodi u vezu sa čestim recidiviranjem. Morfometrijskom analizom se uočavaju veće vrednosti u grupi polimorfnog adenokarcinoma niskog gradusa, ali su statistički značajne razlike nađene samo za Feretov dijametar i integrisanu optičku gustinu u odnosu na pleomorfni adenom (p<0,05). U grupi adenoma bazalnih ćelija tumorske ćelije su pokazale statistički veće vrednosti za integrisanu optičku gustinu u odnosu na pleomorfni adenom (p<0,001). Zaključak: Za diferencijalnu dijagnozu tumora pljuvačnih žlezda, pored osnovne mikromorfološke, neophodna je i imunohistohemijska i morfometrijska analiza.

Introduction: Salivary gland tumors are extremely rare neoplasms.Given their pathohistological image, these tumors represent a great diagnostic challenge.The aim of our study was to differentiate these three tumor types by applying the immunohistochemical and morphometric analysis.Material and Methods: The entire study was conducted at the Center for Pathology and Pathological Anatomy in Niš.The study included 44 tumors, 20 pleomorphic adenomas, 12 basal cells adenomas and 12 polymorphous low-grade adenocarcinomas.The expression of Ki67, p53 and HER-2 antigens, as proliferation markers, was analyzed.The differential diagnostics also included the analysis of the expression of CEA, EMA, GFAP, p63, vimentin, CK14, α-SMA, S-100 protein and WT1 antigen.The morphometric analysis was done in the program pack "ImageJ" version 1.43u.The statistical analysis of data was done in the program pack SPSS 15.0.Results: Neoplastic cells in pleomorphic adenoma showed a strong expression of GFAP (20/20), p63 (20/20), WT1 (20/20), vimentin (18/20)  and S100 (16/20).Diffuse expression of CK14 (12/12) was present in the group of 12 polymorphous low-grade adenocarcinomas.S-100, vimentin and EMA were absolutely expressed, whereas α-SMA was negative.Basal cell adenoma showed negativity to S-100, CEA, p63 and vimentin.The analysis of the proliferative Ki67 index values pointed to a statistically significant difference in the pleomorphic adenoma group, which was associated with a frequent recurrence of this benign tumor.The analysis of morphometric characteristics showed higher values in the polymorphous low-grade adenocarcinoma group, but statistically significant differences were found only for the Feret diameter and the integrated optical density (p < 0.05).As for the basal cell adenoma group, tumor cells showed statistically higher values for the integrated optical density (p < 0.001).Conclusion: Apart from the basic micromorphological analysis, the differential analysis of salivary gland tumors also requires the immunohistochemical analysis as well as the monitoring of morphometric characteristics of these tumors' nuclei.

Introduction
Salivary gland tumors are extremely rare neoplasms.Given their pathological image, these tumors represent a great diagnostic challenge.The incidence of these tumors ranges from 3-6% of all tumors of the neck and head region 1,2 .The parotid gland is the most common site of salivary tumor glands, with the incidence of 80-85%, and almost 75% of these tumors are benign neoplasms.The second most frequent site is the submandibular gland with 10%, but one half of tumors of this localization is malignant neoplasms.The sublingual gland is affected in 1% of cases, however, about 80% of tumors are malignant.Minor salivary glands are the sites with the highest incidence of malignant tumors 3,4 .Epidemiological data show a different incidence in different ethnic groups and parts of the world, which further complicates the global incidence of these tumors 5,6 .The mean age of patients is 46-47 years, but the peak of some tumors is in the sixth and seventh decade of life 2 .
Pleomorphic adenoma, also referred to as mixed tumor (tumor mixtus), is the most common salivary gland neoplasm.It occurs at all ages, but the peak of incidence is in the fifth decade of life.It is equally represented among the sexes, with a slight domination in women.In 80% of cases, it is localized in the lower pole of the parotid gland.If localized in the deep cortex, it has a presentation of parapharyngeal tumor; they are usually of slow growth and painless.Minor tumors are presented as smooth, solid, mobile globules, whereas large tumors can damage the skin and mucosa which covers them.Multifocal and recurrent tumors may be attached to the surrounding tissue.They may sporadically appear with other tumors, especially with Warthin's tumor.The tumor size varies from 2 to 5 cm, however, they may reach enormous diameters as well.Pain and paresthesia may occur in cases of tumor incarceration.When it comes to the palate, they occur usually on the transition between the soft and hard palate, when they can be attached, due to the proximity of periosteum 7,8 .
Patohistološki, tumor je izgrađen od bazaloidnih ćelija, nejasnih međućelijskih granica, svetle citoplazme sa ovalnim do okruglim jedrima.Ćelije formiraju solidne, trabekularne ili tubularne formacije.satellite node.However, it is always in contact with the tumor.The outer surface of the tumor is lobular, it is homogenous at the intersection, and mostly white in color.When it comes to tumors with abundant cartilaginous or mixochondroid stroma, they have a pearly gloss.
Pathohistologically, the tumor is comprised of the epithelial, myoepithelial and mesenchymal component which can have mucoid, myxoid or chondroid appearance.The epithelial component forms nests or ductlike structures, and the cells themselves can be cuboidal, spindle-shaped, plasmocytoid, squamous or light cytoplasm cells.The epithelial component can sometimes be predominant in tumors, which are in such cases labelled as cellular pleomorphic adenomas, but it has no prognostic significance.Ducts are comprised of luminal, cuboidal cells and they can also have the abluminal layer of myoepithelial cells.Luminal cells sometimes have light cytoplasm and hyperchromic nuclei, which can be a great problem in the differential diagnosis of adenoid cystic or epithelial-myoepithelial carcinoma.The mesenchymal component of the tumor is mucoid, myxoid, cartilaginous or hyaline, and it can be the dominant component as well.Cells with mucoid substance are actually myoepithelial cells.Areas of bone metaplasia can also be seen here.Perennial tumors show a pronounced hyalinization, such that the epithelial component is present in traces and with signs of degeneration.Such tumors represent a great risk for malignant transformation [8][9][10] .
Immunohistochemically, ductal epithelial cells are positive to EMA, CEA GFAP, CK14, whereas the myoepithelial component is positive to αSMA, p63, vimentin, S100 and GFAP.A positive reaction with WT1 has been noticed in recent years.Namely, modified epithelial cells exhibit a pronounced cytoplasmic expression of proteins 7 .
Basal cell adenoma is a rare benign salivary gland neoplasm of basaloid phenotype.In general, it is found in 1-3% of cases of all salivary gland tumors, with the peak in the seventh decade of life.It is more frequent in women, with a ratio of 2:1.It is usually localized in large salivary glands, especially in the parotid gland.In terms of clinical presentation, it is a clearly circumscribed, mobile node of solid consistency 7,11,12 .
Pathohistologically, the tumor is composed of the basaloid cells of blurry intercellular boundaries, with light cytoplasm with oval to round nuclei.The cells form solid, trabecular or tubular formations.In the solid growth type, tumor nests are of different shape with peripheral radial cell arrangement (palisading), separated from each other by thick bundles of collagen fibers.The trabecular type is characterized by the basaloid cells which form narrow strips or trabeculae, separated by the vascular stroma.In the tubular type, ductal structures are dominant.The membranous type is characterized by wide strips of hyaline binder at the periphery of tumor nests, as well as intracytoplasmic inclusions.
Immunohistochemically, the cells at the periphery of tumor beaches express p63, vimentin, SMA and S100, whereas luminal cells are positive to CK14, CEA and S100.Having analyzed the expression of markers, we concluded that basal cell adenoma was histogenetically of intercalated channel cells origin 7 .
Cystic structures, squamous and oncocytic (in the tubular type) differentiation, as well as the cribriform growth, can be seen in each variant of adenoma 7,13,14 .
Polymorphous low-grade adenocarcinoma is the primary malignant epithelial tumor of the salivary glands, with a rather polymorphous presentation, monomorphic cells, the infiltrative growth and a low metastatic potential.The incidence of the tumor is 26% of all intraoral carcinomas.It is more common in elderly population (around 70% of patients is 50-70 years of age), with a predominance in women with a 2:1 ratio.In about 60% of cases, it is localized on the palate, then on the buccal mucosa, in the retromolar area, on the upper lip and the floor of the tongue.It is rarely found in large salivary glands.It is presented in the form of a painless mass which may be accompanied by bleeding, telangiectasia and ulceration 7,13 .
Macroscopically, the tumor has clear boundaries, but without a capsule.It is of firm consistency, dirty-yellow in color, and of a lobulated appearance.
Pathohistologically, the tumor is composed of the monomorphic cells, of small to medium size, with small, oval and hyperchromatic nuclei, but without clearly visible nucleoli.Mitosis and necrosis are rare.The term polymorphous in the name of this tumor stands for a solid, cribriform, tubular, trabecular, fascicular (streaming), linear (indian file) and cystic growth type.
The stroma can be hyalinized, mucoid or fibrous, which can further complicate the differential diagnosis.
The differential diagnosis includes pleomorphic adenoma and adenoid cystic carcinoma.

Aim
Given the polymorphism and overlapping of the micromorphological presentation of pleomorphic adenoma, basal cell adenoma and polymorphous low-grade adenocarcinoma, the aim of our study was to differentiate these three tumor types by applying the immunohistochemical and morphometric analysis.

Material and Methods
The entire study was conducted in the Center for Pathology and Pathological Anatomy in Niš.The analyzed material was a tissue obtained by the surgical procedure or biopsy of salivary glands, from the Maxillofacial Surgery Clinic in Niš.The gender ratio varied depending on the tumor, but there was a slight predominance of women, especially in cases of malignant tumors.The age of the patients also varied, ranging from 12 to 75 years, especially in benign lesions.Malignity in children was not registered.Upon the applied intervention, the tissue was fixed in 10% formalin, as recommended by the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 16 .The study included 44 representative salivary gland tumors, 20 pleomorphic adenomas, 12 basal cells adenomas and 12 polymorphous lowgrade adenocarcinomas.Forty out of the total of 44 examined specimens were operational, whereas the remaining 4 were biopsy materials.A more detailed review of the analyzed tumors is presented in Table 1.The preliminary diagnostics included the microscopic analysis of the obtained preparations.Simultaneously, a representative tissue sample was determined, i.e. part of the tumor area with minimal necrosis and inflammatory infiltrate on which immunohistochemical staining had been done.Analizirana je ekspresija Ki67, p53 i HER-2 antigena, kao marker proliferacije.U sklopu diferencijalne dijagnostike analizirana je ekspresija karcino-embrionalnog antigena, epitelnog membranskog antigena, kiselog glijalnog fibrilarnog proteina, p63 antigena, vimentina, citokeratina 14, α-glatkomišićnog aktina, S-100 protein i Vilms tumor 1 antigena.Pravljene su digitalne mikrofotografije, kako osnovnih tako i preparata dobijenih imunohistohemijskim bojenjem.
Considering the polymorphism of salivary gland tumors, one of the parameters for the selection of tissue samples was the most polymorphous image on the pathohistological preparation.The healthy tissue of the salivary gland served as the control.
We analyzed the expression of Ki67, p53 and HER-2 antigens, as well as the expression of proliferation makers.The expression of the carcinoembryonic antigen, epithelial membrane antigen, glial fibrillary acidic protein, p63 antigen, vimentin, cytokeratin 14, α-smooth muscle actin, S-100 protein and Wilms' tumor 1 antigen was analyzed within the differential diagnosis.Digital photomicrographs of both basic and preparations obtained from immunohistochemical staining were made.
The staining of nuclei was considered positive for the following markers: Ki67, p53, p63 and WT1.Positive membrane staining was analyzed for HER-2, membrane and cytoplasmic staining for CEA and EMA, whereas nuclear and cytoplasmic staining was analyzed for S-100.Positive cytoplasmic staining could be seen in αSMA, GFAP, SK14 and vimentin.

software, Wayne
Rasband, National Institutes of Health, Bethesda, Maryland, USA).Photomicrographs, in colour, were obtained using a high-resolution digital camera (Nikon, DS-Fil, Tokyo, Japan), which was connected to the microscope (Nikon, ECLIPLE 50i, Tokyo, Japan).After that, the picture was transferred to the compatible computer and the analysis of nuclear parameters was carried out using the pack.The eight-bit picture was manually processed after the calibration, using a computer mouse.We analyzed 100 random tumor cell nuclei, at the magnification of x40, i.e. cells that did not overlap.We also analyzed six nuclear parameters: area, perimeter, circularity, roundness, Feret diam-eter and integrated optical density.

Statistical data analysis
The statistical analysis of data was performed in the program pack SPSS 15.0.The obtained results are presented in tables.
Continuous variables are presented by basic statistical parameters: mean value ( X ), standard deviation (SD), median (Me) as a measure of central tendency, range, i.e. minimal and maximal values.Quantitative characteristics of the examined variables were determined based on frequency (n) and the percentage share (%).
Depending on the size of the sample, the normality of the distribution of continuous variables was examined using the Kolmogrov-Smirnov or Shapiro-Wilk test.
To evaluate the significance of the difference (p) of continuous variables between two independent groups of subjects, the Student's t-test of independent samples was used in case of the normal distribution of data, i.e. the Mann-Whitney U test in case of the distribution which deviates from the normal one.The standard value p < 0.05 was defined as the threshold of statistical significance.
ANOVA was used to test the significance of the difference between several independent groups, and based on the Levene's test for the homogeneity of variances, a sequential post-hock analysis was carried out, i.e. multiple comparisons using the Tukey's HSD (for homogenous variances) or the Tamhane's test in cases of non-homogenous variances.
To test the statistical significance of differences in absolute frequencies between samples, we used the χ² test, or the Fisher's exact probability test, if the absolute frequency of a sample was less than 5.
The results of immunohistochemical analysis of the expression of proliferation markers and intermediary filaments, as well as other markers analyzed in the study, are shown in Table 2.
We analyzed all four types of tumor growth in the basal cell adenoma group.Twelve cases from this tumor group were present.In general, the most common was the solid tumor growth.The expression in luminal and basal cells was also monitored.The absence of a positive reaction was noticed for GFAP.WTI, CEA, S100 showed focal positivity, vimentin and CK14 were expressed in basaloid cells in the tubular type, i.e. at the periphery of tumor nests in the solid growth type (Figure 6).
Diffuse expression of CK14 (12/12) was present in the group of 12 polymorphous low-grade adenocarcinomas.S100, vimentin and EMA were absolutely expressed, whereas αSMA was negative.Focal expression of WT1 was noticed, especially in the cribriform parts of the tumor (Figure 7).
The analysis of the proliferative Ki67 index values showed a statistically significant difference in the pleomorphic adenoma group (Tumor mixtus), which was associated with frequent recurrence of this benign tumor.

Results of the analysis of morphometric characteristics
The analysis of morphometric characteristics showed higher values in the polymorphous low-grade adenocarcinoma group, but statistically significant differences were found only for Feret diameter and integrated optical density (p < 0.05), as well as higher values for integrated optical density in the basal cell adenoma group (p < 0.001).

Discussion
A great diagnostic dilemma may arise in the differentiation of pleomorphic adenoma and polymorphous low-grade adenocarcinoma.It may be a problem especially when it comes to small, incisional biopsies.It that case, a false picture about the infiltrative growth may be created regarding minor salivary gland tumors where pleomorphic adenoma does not possess the capsule, but may have focal extensions in the neighboring glands and present itself as a malignant neoplasm.Both tumor types consist of relatively uniform cells which are characterized by the absence of atypia 17 .When it comes to polymorphous low-grade carcinoma, the perineural invasion is a very important parameter for malignity, and it cannot be determined based on a small biopsy sample.
In such cases, it is necessary to apply immunohistochemistry. Firstly, markers of myoepithelial differentiation and GFAP are applied.Back in 1982, Nakazato et al. pointed to the cross-over expression of GFAP, vimentin and cytokeratin in the differentiation of pleomorphic adenoma 18 .GFAP exhibited a very complex cytoplasmic expression in ductal epithelial and myoepithelial adenoma cells, whereas insignificant positivity was registered in the cells of polymorphous lowgrade adenoma 19 .Our results show a complete absence of the positive reaction of GFAP in this type of carcinoma, and absolute positivity in pleomorphic adenoma, which is an accordance with large studies dealing with the differential diagnosis of these two entities 20,21 .
Moreover, a very significant finding in the differentiation includes the expression of αSMA in pleomorphic adenoma which is absent in polymorphous low-grade adenocarcinoma 22 .In recent years, WT1 has been used for the determination of neoplastic cells.Leader and Langman emphasized the significance of this marker in their studies.WT1 showed absolute cytoplasmic positivity in abluminal cells with myoepithelial differentiation of pleomorphic adenoma 23 .Those cells exhibited the expression of p69.In polymorphous low-grade adenocarcinoma, the expression varied depending on the variant of cell arrangement.In accordance with the findings of published studies, our results emphasize the importance of immunohistochemical detection of myoepithelial cells in the diagnostics of polymorphous low-grade adenocarcinoma.
Basal cell adenoma was classified by the World Health Organization in 1991.By that time, it had been referred to as nonpleomorphic adenoma or monomorphic adenoma.Based on the histological presentation, growth type and neoplastic cell arrangement, it was divided into several variants: solid, which is the most common, tubular, trabecular and membranous 15 .The tumor usually exhibits many growth type variants, with one of them being dominant, based on which further morphological subclassification of basal cell adenoma is done.They all have the fibrous stroma, but without myxochondroid areas which can be seen in pleomorphic adenoma.Cystic degeneration, squamous metaplasia, keratinization and the cribriform growth have increased the possibility of diagnostic errors for this tumor.In general, tumor nests consist of dark, blue cells at the periphery, which often have a palisade arrangement.Somewhat larger cells of lighter cytoplasm occupy the central parts.Myoepithelial differentiation exists in all variants and it is not obvious on standard H&E preparations, however, the immunohistochemical analysis has proven it 24 .The differential diagnosis of basal cell adenoma includes polymorphous low-grade adenocarcinoma and pleomorphic adenoma, as well as some of myoepithelioma variants.The immunohistochemical analysis of our results, in accordance with data from the literature, showed that cells at the periphery of tumor nests express p63, vimentin, SMA and S100, whereas cells in the interior or luminal cells, if we talk about the tubular variant, were positive to SK14, CEA and S100.
Having analyzed the expression of markers, we concluded that basal cell adenoma was histogenetically of intercalated channel cells origin 25 .
The evaluation of the mitotic index, primarily the Ki67 proliferative index, proved to be a very important parameter in the differential diagnosis, predicting biological behavior and aggressiveness in many tumors.Our study included 42 salivary gland tumors, both benign and malignant.We analyzed the proliferative index and, in correlation with other clinical-pathological characteristics, we obtained significant results in the differential diagnosis of these neoplasms.
For the purposes of our study, we used 5% as a threshold for the Ki67 proliferative index, but the existing literature offers studies which were conducted using higher values for tumor aggressiveness, i.e. >10% 26,27 .In our study, recurrent cellular pleomorphic adenoma of the soft palate showed the highest value.
Similar to the results of our study, Shida et al. obtained slightly higher values of the Ki67 proliferative index in the basal cell adenoma group 28 .Our results match the results of Horii et al., which indicate the highest proliferative activity of pleomorphic adenoma in the group of benign tumors 29 .By comparing the expressions of p53 and Ki67, Saghravanian et at.state that they are of great importance for the differentiation of polymorphous low-grade adenocarcinoma and adenoid cystic carcinoma.In their study, 24% of tumor cell nuclei showed the expression of Ki67, contrary to only 3.88% in the polymorphous low-grade adenocarcinoma group.Based on great variability of the obtained results in our study, as well as results from other researchers, we believe that a low value of the proliferative index is not necessarily always strictly related to a low-grade tumor.The results of our study show that the value of the Ki67 proliferative index varies depending on the case and the histological type of the tumor itself.
The examination of the expression of p53 and Ki67 is a widespread method in the determination of the evolution and prognosis of malignant tumors of all localizations.p63 is the most commonly identified mutated gene in malignant neoplasms, especially present in breast, stomach, liver and prostate cancer.The expression of p53 is associated with the negative prognosis of a disease, and it is usually present in aggressive malignant Weber je sa svojim saradnicima analizirao ekspresiju p53 u grupi benignih tumora.Pokazali su da ekspresija proteina postoji jedino u grupi pleomorfnog adenoma i mioepitelioma.Svoje rezultate su tumačili kao povećanu sklonost tumora ka malignoj alteraciji 30 .Korišćenjem softverskog paketa "ImageJ" analizirano je šest jedarnih parametara: površina, perimetar, cirkularnost, zaobljenost, Feretov dijametar i integrisana optička gustina.
Weber et al. analyzed the expression of p53 in the benign tumor group.They showed that the expression of protein existed only in the pleomorphic adenoma and myoepithelioma groups.They interpreted their results as an increased tendency of tumors to malignant alteration 30 .
Six nuclear parameters were analyzed using the software pack "ImageJ": area, perimeter, circularity, roundness, Feret diameter and integrated optical density.
The differences found for the nuclear parameters which refer to the size of nuclei (area and perimeter), as well as the shape of nuclei (circularity and roundness), were slightly higher in the polymorphous lowgrade adenocarcinoma group.The values of Feret diameter and integrated optical density significantly depend on the nature and type of the tumor (p < 0.05).
The available literature offers a very small number of studies dealing with the morphometric characteristics of tumors.Using light microscopy, we can notice some of the characteristics of nuclei.It can be clearly seen whether it is the case of hyperchromatic nuclei, large or small, whether atrophy is present, what chromatin is like, whether the edges are even, or the area serrated.The differential diagnosis of salivary gland tumors is often quite difficult, and the morphometry enables the quantification of a pathohistological finding, and therefore reduces the possibility for errors in setting the final diagnosis.The morphometric analysis of salivary gland tumors has been applied so far, but the number of studies which have compared the results of these analyses in different tumor types is rather small.In previous studies, the authors mostly examined nuclear area, circularity, perimeter and integrated optical density using different software packs 31,32 .Layfield published the results for these parameters in his study, and showed their diagnostic significance in the differentiation of benign and malignant mixed tumors of the parotid gland 33 .Prvulović et al. published a study on the differentiation of ductal and lobular breast carcinoma on cytological material.They also emphasized the applicability of this method, not only in the differentiation, but also in grading the ca-melitusom tip I, i pokazali kako su jedarni parametri statistički veći u ovoj grupi bolesnika 35 .Obad-Kovačević je u svoje istraživanje uključila morfometrijske karakteristike benignih i malignih tumora parotidne žlezde, proučavajući karakteristike cele ćelije.Došli su do zakljčka da je odnos površine jedara i citoplazme znatno veći, u korist jedara, kod malignih tumora 36 .
Vrednosti morfometrijskih parametara, integrisana optička gustina i Feretov dijametar statistički su veće u grupi polimorfnog adenokarcinoma niskog gradusa.U grupi adenoma bazalnih ćelija vrednosti za integrisanu optičku gustinu statistički su veće u odnosu na grupu pleomorfnih adenoma.rcinoma itself 34 .Oz et al. published a study on the morphometric characteristics of oral mucosa cells in patients with diabetes mellitus type I, and showed that nuclear parameters were higher in this group of patients 35 .Obad-Kovačević et al. included morphometric characteristics of benign and malignant tumors of the parotid gland in their research by studying the characteristics of the entire cell.They concluded that the ratio of the area of nuclei and cytoplasm was considerably higher in malignant tumors, in favor of nuclei 36 .

Conclusion
Pleomorphic adenoma was positive to S-100, GFAP, CK14, αSMA, CEA, EMA and WT1, whereas basal cell adenomas showed positivity to S-100, CEA, p63 and vimentin.The expression of HER2 in the benign tumor group was completely absent.Polymorphous low-grade adenocarcinoma showed a positive immunohistochemical reaction to CK14, p63, EMA, S100 and vimentin.
Pleomorphic adenoma showed the highest proliferative activity.The obtained results were in accordance with the frequency of tumor recurrence, as well as the degree of malignant alteration.
The values of morphometric parameters -integrated optical density and Feret diameter -were statistically higher in the polymorphous low-grade adenocarcinoma group.The basal cell adenoma group showed statistically higher values for integrated optical density compared to the pleomorphic adenoma group. .

Table 1 .
Characteristics of patients

Table 4 .
Morphometric characteristics of tumor cell nuclei