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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 16  |  Issue : 2  |  Page : 99-103

Immunohistochemical expression of minichromosome maintenance-3 protein in pleomorphic adenoma and mucoepidermoid carcinoma


1 Department of Oral Pathology, Faculty of Dentistry, Sinai University, Cairo, Egypt
2 Department of Oral Pathology, Faculty of Dentistry, Tanta University, Tanta, Egypt
3 Department of Oral Pathology, Faculty of Dentistry, Kafr El Sheikh University, Kafr Elsheikh, Egypt

Date of Submission16-Dec-2018
Date of Acceptance02-Apr-2019
Date of Web Publication23-Sep-2019

Correspondence Address:
Mohamed M Arafa
Department of Oral Pathology, Faculty of Dentistry, Sinai University, Cairo
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/tdj.tdj_43_18

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  Abstract 


Introduction
Salivary gland tumors represent a diverse set of tumors. Pleomorphic adenoma (PA) and mucoepidermoid carcinoma (MEC) are the most common benign and malignant salivary gland tumors respectively. Proliferation markers have been used to determine the behavior and prognosis of benign and malignant tumors. Minichromosome maintenance (MCM) proteins are family of six major members (MCM-2–7), which have similar biochemical functions and are essential for the process of DNA replication. MCM-3 is a member of this family, which present during proliferation of normal cells and neoplastic cells while absent in quiescent ones.
Aim
To assess the expression of MCM-3 in PA and MEC, and to correlate the expression of MCM-3 with the collected clinical data of PA and MEC.
Materials and methods
This retrospective study was performed using 40 paraffin-embedded tissue blocks of PA and MEC (20 cases each). Clinical data were collected. The specimens were stained by conventional hematoxylin and eosin staining to confirm the diagnosis and then immunohistochemical staining was performed by using antibody against MCM-3 to evaluate the expression.
Results
MCM-3 expression was significantly correlated to grades of MEC and significantly different between MEC and PA. Furthermore, MCM-3 expression was positively correlated to female cases of MEC.
Conclusion
MCM-3 may be used as a sensitive proliferative marker for PA and MEC.

Keywords: minichromosome maintenance-3, mucoepidermoid carcinoma, pleomorphic adenoma


How to cite this article:
Arafa MM, Raghib AM, Wahba OM. Immunohistochemical expression of minichromosome maintenance-3 protein in pleomorphic adenoma and mucoepidermoid carcinoma. Tanta Dent J 2019;16:99-103

How to cite this URL:
Arafa MM, Raghib AM, Wahba OM. Immunohistochemical expression of minichromosome maintenance-3 protein in pleomorphic adenoma and mucoepidermoid carcinoma. Tanta Dent J [serial online] 2019 [cited 2019 Nov 17];16:99-103. Available from: http://www.tmj.eg.net/text.asp?2019/16/2/99/267566




  Introduction Top


Salivary glands give rise to different pathologic conditions, ranging from cystic, inflammatory, tumor-like and neoplastic lesions [1]. Salivary gland tumors (SGTs) represent a diverse set of tumors with a broad range of biologic behaviors, ranging from completely benign tumors to low-grade malignancies to high-grade malignancies. In addition to this wide range in clinical behavior, SGTs can show extensive morphologic overlap. In many cases, a definitive diagnosis can be impossible without examination of the entire tumor. Despite the use of immunohistochemistry, challenges of morphologic overlap continue into the modern era of pathology [2].

Pleomorphic adenoma (PA) can be defined as a benign mixed tumor composed of epithelial and myoepithelial cells arranged with various morphological patterns, demarcated from surrounding tissues by fibrous capsule. It is one of the SGTs affecting both major and minor salivary glands [3]. PA represents about 60% of all SGTs. Among the major SGTs, PA is associated with parotid gland (53–77%), submandibular gland (44–68%), and minor salivary glands (4–6%). The annual incidence is ∼ 2–3.5 cases per 100 000 populations. PA occurs in individuals of all ages. PA occurs more often in females than in males (2: 1 ratio) [4].

Clinically, the tumor is usually solitary and presents as a slowly growing painless firm and single nodular mass. Isolated nodules are generally outgrowths of the main nodule rather than a multinodular presentation. It is usually mobile unless found in the palate and can cause atrophy of the mandibular ramus when located in the parotid gland. Although it is classified as a benign tumor, PA has the capacity to grow to large proportions [5].

Histologically, it is highly variable in appearance. The main components are the capsule, epithelial and myoepithelial cells, and mesenchymal or stromal elements. The capsule varies in thickness and presence [6]. The epithelial component shows a wide variety of cell types including cuboidal, basaloid, polygonal squamous, spindle cell, plasmacytoid. The epithelium usually forms sheets or duct-like structures. There is a wide range of epithelial cellularity [6]. Myoepithelial cells may form a fine reticular pattern or sheets of spindle-shaped cells. A very distinctive appearance is seen when the myoepithelial cells are plasmacytoid or hyaline (Labvision, Fremont, California, USA) [7]. The mesenchymal element is mucoid, myxoid, cartilaginous or hyalinized and sometimes this tissue forms the bulk of the tumor [8].

Mucoepidermoid carcinoma (MEC) is the most common malignant epithelial neoplasm of the salivary glands in both adults and children [9]. MEC constituting less than 15% of all SGTs, 30% of all malignant salivary gland neoplasms that originate in both the major and minor salivary glands, 22% of malignant tumors in the major salivary gland and 41% of malignant tumors in the minor salivary gland [10]. Approximately 60% of MECs occur in the major salivary glands and 35% in the minor salivary glands. The parotid glands are the predominant site, representing 48% of cases, with 11% in the submandibular glands, and 1% in the sublingual glands. The palate and buccal mucosa are the most frequent sites in the minor salivary glands, followed by the antrum, tongue, gingiva, floor of the mouth, and nasal cavity [10]. MEC arising from the minor salivary glands of the palate having a perineural intracranial extension has been reported [11].

Microscopically, MEC has a mixture of mucous cells, intermediate cells, and epidermoid cells. Mucous cells are neoplastic cells that contain mucin. They vary in shape, having pale eosinophilic and foamy cytoplasm with distinct cell boundaries. Intermediate cells vary in appearance from small basaloid cells to slightly larger ovoid cells with scant pale and eosinophilic cytoplasm. Epidermoid cells have round to ovoid or polygonal shape with prominent nuclei, abundant eosinophilic cytoplasm, intercellular bridges [10],[12].

Minichromosome maintenance (MCM)-3 is a member of MCM protein family with a critical role in initiation of DNA replication. It is present during cellular proliferation of normal cells, premalignant and neoplastic cells but absent in cells that are in G0 phase [13]. Majority of human tumors overexpress MCM-3, it implicates that MCM-3 might facilitate the tumorigenesis by playing a role in the malignant transformation of cells and as a result, MCM-3 antibody is potentially valuable as a tumor marker. MCM-3 seems most directly associated to the growth ability of normal cells. It also seems to fit into the definition of a tumor marker because it can detect only cancer cells but not normal cells under the prolonged nutritional deprivation [14].


  Materials and Methods Top


This retrospective study included 40 paraffin-embedded tissue blocks of PA and MEC (20 cases each). Clinical data including (age, sex, site and size) were collected by reviewing the pathology records. Two adjacent tissue sections of paraffin-embedded blocks of PA and MEC were cut at 4 μm thickness, prepared for staining by hematoxylin and eosin to confirm the diagnosis and immunohistochemically stained to detect MCM-3 expression. For immuneohistochemical study, Primary antibody against MCM-3 (Labvision) and Streptavidin Biotin Complex Universal Kit (Labvision) were used. The dilution of MCM-3 was 1: 50 according to the manufacturer's instructions. For Evaluation and Quantification of immunostaining, sections for MCM-3 were analyzed and scored using image analysis software. Image analysis was done on a computer using the public domain and free software Image J (Image processing and analysis in Java, http://rsb.info.nih.gov/ij/). Assessment of MCM-3 expression using immunohistochemical (profiler) according to Varghese et al.[15] was done. The data obtained were statistically analyzed using SPSS system version 22 (IBM, Armonk, New York, USA). P values up to 0.05 were considered statistically significant in all analysis.


  Results Top


In normal salivary gland, only ductal epithelium showed positive immunoreactivity to MCM-3 as shown in [Figure 1]a. While, acinar cells of both mucous and serous secreting cells revealed negative immunoreactivity to MCM-3. Seventeen out of 20 (85%) PA specimens showed variable positivity to MCM-3 with mean value of 1.15 ± 0.67. While, three (15%) specimens were negative. Low positive (+1) immunoreaction to MCM-3 was observed in 11 (55%) cases. While, the remaining six (30%) cases showed positive (+2) immunoreactivity. MCM-3 expression was detected in epithelial cells arranged in sheets, islands and duct-like structures as shown in [Figure 1]b and [Figure 1]c. Also, myoepithelial cells revealed positive immunoreaction. Whereas, negative immunoreactions were observed in different types of PA stroma as shown in [Figure 1]d. Regarding clinical data, no statistical correlation was found between expression of MCM-3 and parameters of age, sex, site and size.
Figure 1: Minichromosome maintenance (MCM)-3 expression. (a) A photomicrograph of normal salivary gland exhibits positive immunoreactivity of MCM-3 in the ductal epithelium (red arrows) (MCM-3, X200). (b) A photomicrograph of PA illustrates positive (+2) expression of MCM-3 detected in sheets of epithelial cells (red arrows) (MCM-3, X200). (c) A photomicrograph of PA exhibits positive nuclear immunoreaction of duct like structures (red arrows) (MCM3, X400). (d) A photomicrograph of PA reveals MCM-3 expression in myoepithelial cells (red arrows) and negative immunostaining of myxoid tissue (green arrows) (MCM-3, X400). (e) A photomicrograph of low-grade MEC illustrates positive (+2) expression of MCM-3 detected in both epidermoid cells (red arrows) and mucous cells (green arrows) (MCM-3, X200). (f) A photomicrograph of high-grade MEC reveals high positive (+3) expression of MCM-3 in epidermoid cells (red arrows) (MCM-3, X200).

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Eighteen out of 20 (90%) of MEC specimens showed variable positivity to MCM-3 with mean value 1.75 ± 0.85. While, two (10%) specimens were negative. MCM-3 expression was detected mainly in epidermoid cells and intermediate cells with strong intensity. While mucous cells revealed weak immunostaining as shown in [Figure 1]e. Low positive (+1) immunoreaction to MCM-3 was observed in four (20%) cases while, 11 (55%) cases showed positive (+2) immunoreactivity and three (15%) cases revealed high positive (+3) immunoreactivity. Low-grade MEC expressed nuclear immunoreaction with mean value 1.40 ± 0.70. Negative immunoreactivity to MCM-3 was detected in one (10%) case and four (40%) cases revealed low positive (+1) immunoreactivity. While, five (50%) cases revealed positive (+2) immunoreactivity. High-grade MEC expressed nuclear immunoreaction with mean value 2.10 ± 0.88. Negative immunoreactivity to MCM-3 was detected in one (10%) case while, six (60%) cases revealed positive (+2) immunoreactivity and three (30%) cases revealed high positive (+3) immunoreactivity as shown in [Figure 1]f. Regarding clinical data, there was a significant positive correlation between expression of MCM-3 and sex of MEC cases (P = 0.013) as MCM-3 expression increased in females. No statistical correlation was found between expression of MCM-3 regarding parameters of age, site and size. There was a significant difference in MCM-3 expression between the grades of MEC (P = 0.034) as shown in [Table 1]. Also, there was a significant difference in MCM-3 expression between MEC and PA cases as shown in [Table 2].
Table 1: Relation between minichromosome maintenance-3 expression in low-grade and high-grade mucoepidermoid carcinoma cases

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Table 2: Relation between minichromosome maintenance-3 expression in mucoepidermoid carcinoma and pleomorphic adenoma cases

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  Discussion Top


PA and MEC are the most common benign and malignant SGTs respectively. Hematoxylin and eosin stained tissue sections are used routinely for diagnosis of these tumors. These tumors are managed differently therefore, there is a need to use prognostic markers [16]. MCM-3 proteins might represent a useful proliferative marker in SGTs, as this marker identify not only cycling cells, but also noncycling cells with proliferative potential [17]. So, this study used MCM-3 to assess the expression in PA and MEC cases.

In this study, MCM-3 expression was negative in acinar cells of normal salivary gland which was in consistent with Abdalla et al. [18]. Also in agreement with Vargas et al.[19] who reported that MCM-2 was rarely expressed in normal salivary gland tissue. This finding might be explained by the fact that the acinar cells of normal salivary gland are in fully differentiated state. Whereas, the ductal epithelium of normal salivary gland showed positive immunoreactivity to MCM-3. This finding was in a line with Ashkavandi et al. [20]. This might be due to the proliferative potentiality of the ductal epithelium.

In this study, no significant correlation was found between MCM-3 expression and clinical data including age, sex, site and size of PA cases. This was in agreement with Vargas et al.[19] who found no significant correlation between MCM-2 expression and clinical data including age, sex, site and tumor size of PA cases. This was explained by Carreon-Burciaga et al.[21] who stated that clinical features do not directly affect tumor cell proliferation. In this study, no significant correlation was detected between MCM-3 expression in MEC and the clinical parameters including age, site and size, these findings were in line with Vargas et al.[19] who reported that no significant correlation between MCM-2 expression in MEC and clinical data.

Interestingly, this study revealed a positive significant correlation of MCM-3 expression with female cases of MEC comparing to male cases. This finding was in agreement with Lee et al.[22], Liu et al.[23] and Dudderidge et al.[24] It may be explained by the hormonal changes of females in this study. On contrary, Vargas et al.[19] reported that no significant correlation between MCM-2 expression in MEC and sex. Regarding to MCM-3 expression in histologic sections of PA, 85% of cases revealed immunoreactivity to MCM-3 with prominent expression in myoepithelial and epithelial cells. This finding was in line with Ashkavandi et al.[20].

In this study, MCM-3 immunostaining preferentially yielded a nuclear pattern, although some cases showed positive nuclear and cytoplasmic immunoreactivity, a finding which was observed by Kodani et al.[25], Chatrath et al.[26], Torres-Rendon et al.[27] and Vargas et al.[19] and considering this observation as a nonspecific reaction. However, Labib et al.[28] explained this by the fact that in S phase of the cell cycle, nearly the whole amount of MCM proteins dissociate from the chromatin, leaving only a small fraction bound to regions of un-replicated DNA. Subsequently, during G2/M phase, MCM proteins are absent on chromatin and are detectable predominantly in cytoplasm where they later undergo enzymatic degradation.

Using computer image analysis, this study revealed that the MCM-3 expression increased with increasing histological grade of MEC, where low-grade cases showed lower expression, while the high-grade cases showed higher expression of MCM-3. This finding was in accordance with Abdalla et al.[18] who reported that MCM-3 expressed more in high-grade MEC than low-grade MEC but, without statistical significant. Also, Ghazy et al.[29] and Vargas et al.[19] revealed an increase of MCM-2 expression from low-grade MEC to high-grade MEC but, without statistical significant. This finding may be explained by that, in cancer, differentiated neoplastic cells tend to grow and spread at a slower rate than undifferentiated or poorly differentiated cells, which lack the structure and function of normal cells and grow uncontrollably. Therefore, the withdrawal of cells from the cell cycle into differentiated state is coupled with down-regulation of MCM-3 expression.

Furthermore, this study revealed a significant difference of MCM-3 expression between MEC and PA. In a line with, Ashkavandi et al.[20] who found a significant difference of MCM-3 expression between MEC and PA. Whereas, Vargas et al.[19] found an increase of MCM-2 expression between MEC and PA without statistically significant.


  Conclusion Top


MCM-3 is a sensitive proliferative marker for SGTs.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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