|Ahead of print publication
Comparative evaluation of immunohistochemical expression of MCM2 and Ki67 in oral epithelial dysplasia and oral squamous cell carcinoma
Sushmita Swain1, Roquaiya Nishat2, Sujatha Ramachandran3, Malvika Raghuvanshi4, Shyam Sundar Behura4, Harish Kumar4
1 The Tooth Dental Clinic, Kalinga Institute of Dental Sciences KIIT Deemed to be University, Bhubaneswar, Odisha, India
2 Department of Dentistry, Nalanda Medical College Hospital, Patna, Bihar, India
3 Dr. Raju Dental Care Ramanagar, Karnataka, India
4 Department of Oral Pathology and Microbiology, Kalinga Institute of Dental Sciences KIIT Deemed to be University, Bhubaneswar, Odisha, India
|Date of Submission||02-Jan-2020|
|Date of Decision||27-May-2020|
|Date of Acceptance||18-Jun-2020|
|Date of Web Publication||20-Aug-2021|
Department of Dentistry, Nalanda Medical College Hospital, Agamkuan Flyover, Sadikpur, Patna - 800 007, Bihar
Source of Support: None, Conflict of Interest: None
Aim and Objectives: The aim and objective of the study were to evaluate the immunohistochemical expression of proliferative markers, Ki67, and MCM2 in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC), to compare the relationship of their staining patterns, and to look for correlation between them, if any.
Materials and Methods: Thirty archival paraffin-embedded tissue blocks of previously diagnosed cases of OED, OSCC each, and 10 normal oral mucosa were used in the study. Immunohistochemical staining for MCM2 and Ki67 markers was done and the slides were individually evaluated for MCM2 and Ki67 expression, with immunopositivity determined on the basis of dark brown staining of the nucleus. The number of positively stained nuclei was counted in 10 representative areas and the data were charted and statistically analyzed.
Results: The overall mean expression of both the proteins increased progressively from normal mucosa to OED to OSCC. In normal mucosa, all positively stained nuclei were seen in the basal compartment of the epithelium, while in dysplastic cases, expression was seen toward the surface of squamous epithelium. In OSCC, the frequency of expression of MCM2 and Ki-67 proteins showed an inverse correlation with the degree of tumor differentiation. In well-differentiated cases, the positivity of either marker was restricted to the outermost layer of the tumor cells. In moderately differentiated cases, an expression of Ki-67 was more diffuse in inner layers, whereas the MCM2 antigen was found to be more intense and diffuse in both the inner and outer layers. Whereas in poorly differentiated SCC, positive expression was seen in most of the tumor cells, the mean expression of MCM2 was found to be higher than that of Ki67 in all cases.
Conclusion: MCM2, as a proliferation marker, is superior to Ki67 as it indicates the capacity of proliferation and the ability of DNA replication of a cell.
Keywords: Ki67, MCM2, oral epithelial dysplasia, oral squamous cell carcinoma, proliferation markers
|How to cite this URL:|
Swain S, Nishat R, Ramachandran S, Raghuvanshi M, Behura SS, Kumar H. Comparative evaluation of immunohistochemical expression of MCM2 and Ki67 in oral epithelial dysplasia and oral squamous cell carcinoma. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 7]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=324159
| > Introduction|| |
Oral epithelial dysplasia (OED) has been defined by the WHO as “altered epithelium with an increased likelihood for progression to squamous cell carcinoma.” The diagnosis and grading of OED is based on a combination of architectural and cytological changes. This reflects the loss of normal maturation and stratification pattern of surface epithelium, and plays a key role in assessing the probability for malignant transformation. OED is graded as mild, moderate, and severe based on whether dysplastic features are restricted to the lower third, middle third, or the upper third of the epithelium, respectively., Grading has a wide intra- and interobserver variability. The risk of progression into malignancy is associated with histological grade, but still, it is impossible to predict accurately which lesions will progress to oral malignancy and which will not. The rates of malignant transformation for mild, moderate, and severe dysplasias are 3%, 4%, and 43%, respectively. Squamous cell carcinoma is the most common malignancy occurring in the oral cavity.
All the diagnostic clinical adjuncts as well as histopathological evaluation by routine staining help in detecting whether dysplasia is present or not and if carcinoma has already set in, but there is no definitive diagnostic procedure to detect the malignant potential of dysplasias. Early detection of cellular changes by molecular markers enables a better understanding of the degree of malignant change of altered cells.
In the present study, molecular markers, namely Ki67 and MCM2, have been used as immunohistochemical markers in OED and oral squamous cell carcinomas (OSCCs) to evaluate and determine their prognostic efficacy. Ki-67, a well-known proliferative marker, is associated with cell proliferation. Ki-67 protein is present during all active phases of the cell cycle (G1, S, G2, and mitosis) but is absent from resting cells (Go)., MCM2, belonging to the family of minichromosome maintenance 2–7 proteins, is engaged in recognition and control of DNA replication. MCM2 is expressed in all phases of cell cycle making it a much more sensitive marker of proliferation since it detects cells that are “licensed to proliferate” and capable of initiating DNA replication.
Exploring the functional expression of biomarkers like Ki67 and MCM2 offers the opportunity to move from a tumor model of temporal determinism to one of the biological determinism, as carcinogenesis is defined by the molecular characteristics of tumor and the host. Hence, the aim of the study was to evaluate and compare the expression pattern of MCM2 and Ki-67 in OED and OSCC in the oral cavity and to look for correlation if any between MCM2 and Ki67 expression.
| > Materials And Methods|| |
The present retrospective study was carried out on 30 cases of OED (Group A), 30 OSCC (Group B), and 10 normal oral mucosa (Group C) retrieved from the archives. OED cases were further subdivided into 10 mild, 10 moderate, and 10 severe cases. Similarly, OSCC cases were also divided into 10 well-differentiated, 10 moderately differentiated, and 10 poorly differentiated cases by a single observer to avoid any bias.
Serial sections of 4-μ thickness were obtained from the archival tissue specimens, as this thickness allows optimal staining. The sections of groups A, B, and C were first subjected to routine hematoxylin and eosin examination. Further, OSCC cases were graded according to the Broder's classification and OED cases in consonance with the 2005 WHO classification system., Later, other sections of all the three groups were subjected for immunohistochemical analysis using Ki-67 and MCM2 antibody.
Immunohistochemical analysis was performed using PolyExcel HRP/DAB detection system (PathnSitu Biotechnologies Pvt. Ltd.). The sections were deparaffinized and rehydrated. Antigen retrieval was done in a pressure cooker containing buffer solution prepared by diluting 1 part of × 50 Tris-EDTA epitope retrieval buffer (pH-9) in 49 parts of distilled water. The tissue sections were then covered with 3% hydrogen peroxide, incubated for 5–10 min, and gently washed with immunowash buffer to block any endogenous peroxidase activity. Primary antibody incubation was done using monoclonal Ki-67 and MCM2 antibodies for 1 h at room temperature in a humid chamber. Target binder and HRP polymer were then added for 12 min each, followed by the application of DAB chromogen for 5 min. The slides were then counterstained using hematoxylin for 2–3 min, following which they were washed, dehydrated, and mounted using DPX mounting media.
The slides were observed under magnification of ×400 using a light microscope. Representative areas in OED were selected according to the most dysplastic areas and in OSCC from the invasive tumor front. The slides were individually evaluated for Ki-67 and MCM2 and immunopositivity was determined on the basis of dark brown staining of the nucleus. An eyepiece graticule was used and the epithelial–connective tissue interface was taken as the base of the square. All epithelial cells inside the area of the graticule were counted. The number of positively stained nuclei was counted in 10 representative areas (1000 cells in each area) using a manual cell counter, data were charted, and the mean was calculated. Kruskal–Wallis and Mann–Whitney U-tests were used for the statistical analysis using SPSS 22.0 software (IBM, Armonk, New York, USA). P < 0.001 was considered to be highly significant.
| > Results|| |
Nuclear staining was noted in all the cases, and statistical analysis of our study showed that the overall expression rate of both the proteins increased progressively from normal oral mucosa to OED to OSCC.
In normal mucosa, all the nuclei were present in the basal and suprabasal compartment of squamous epithelium. In dysplastic squamous epithelium, there was the persistence of MCM2 and Ki-67 expression in compartments in which they are normally absent or sparse, especially toward the surface of squamous epithelium [Figure 1] and [Figure 2].
|Figure 1: Photomicrograph showing Ki-67 and MCM2 expression in normal oral mucosa|
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|Figure 2: Photomicrograph showing Ki-67 and MCM2 expression in severe dysplasia|
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A decreased MCM2 expression was seen in the dysplastic squamous epithelium toward the surface, and in case of invasive SCC, MCM2 downregulation was seen toward the center of cell nests – that is, in areas of differentiation. Moreover, a correlation was found between the expression of MCM2 and the histological grade of OSCC, with higher MCM2 expression in poorly differentiated tumors.
The distribution of Ki-67 and MCM2 antigen-positive cells in the tumor nests of well-differentiated squamous cell carcinoma tissues was usually restricted to the outermost layer of the tumor cells [Figure 3]. In cases of moderately differentiated carcinomas, the expression of Ki-67 antigen in the tumor nests was found to be more diffuse in the inner layers, whereas MCM2 antigen was found to be more intense and diffuse in both inner and outer layers. In poorly differentiated squamous cell carcinoma, the expression was seen in most of the tumor cells [Figure 4]. In addition to this, the higher intensity of MCM2 also revealed the site of tumor invasive front where the cells are most aggressive in nature.
|Figure 3: Photomicrograph showing Ki-67 and MCM2 expression in well-differentiated squamous cell carcinoma|
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|Figure 4: Photomicrograph showing Ki-67 and MCM2 expression in poorly differentiated squamous cell carcinoma|
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Ki-67 with MCM2 expression in OED, OSCC, and normal mucosa showed a significant positive correlation. The mean expression of MCM2 was found to be customarily higher than that of Ki67 regardless of the histologic differentiation type in dysplasia or the grade of carcinoma.
The following tables and graphs depict the results obtained in our study. [Table 1] represents a comparison of mean MCM2 and Ki-67 expression in different grades of OED and OSCC by Kruskal–Wallis test. [Table 2] shows intergroup comparison of the mean MCM2 and Ki-67 expression using Mann–Whitney U-test, wherein the mean values of both the markers were found to be significantly more among OSCC group than OED which was significantly more than the control group. [Figure 5] shows the graphical representation of MCM 2 and Ki-67 expression in the different grades of OED, wherein the expression profile was found to be severe OED > moderate OED > mild OED. [Figure 6] represents the comparison of mean MCM2 and Ki-67 expression in well-, moderately, and poorly differentiated OSCC, wherein the maximum expression was observed in poorly differentiated OSCC, followed by moderately differentiated and well-differentiated OSCC. [Table 3] shows the correlation between the expression of MCM2 and Ki67 markers in OED, OSCC, and control using the Pearson's correlation test, and a significant positive correlation was found between the two markers.
|Table 1: Comparison of mean MCM2 and Ki67 expression in different grades of oral epithelial dysplasia and oral squamous cell carcinoma by Kruskal-Wallis test|
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|Table 2: Inter-group comparison of mean MCM2 and Ki67 expression using Mann-Whitney U-test|
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|Table 3: Correlation between expression of MCM2 and Ki67 markers in oral epithelial dysplasia, oral squamous cell carcinoma, and control using the Pearson's correlation test|
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|Figure 5: Graphical representation of MCM2 and Ki-67 expression in different grades of oral epithelial dysplasia|
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|Figure 6: Graphical representation of mean comparison of mean MCM2 and Ki67 expression in well-, moderately, and poorly differentiated oral squamous cell carcinoma|
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| > Discussion|| |
Cell proliferation is regarded as one of the most important biologic mechanisms in oncogenesis. Proliferative activity of cells can be measured by immunohistochemistry using antibodies against proliferation-associated antigens, which helps to assess the prognosis and gives a fair appraisal of the degree of biological aggression. Assessment of cell kinetic information by immunohistochemical methods using antibodies against MCM2 and Ki-67 antigen has the recompense of not only conserving cellular and tissue architecture but also being applicable to routine archival formalin-fixed, paraffin-embedded specimens. A thorough search of PubMed database suggests that no studies have been done in the Indian subcontinent comparing the two markers in OSCC and OED, and hence, this study was undertaken.
Our study showed that the mean expression of both the proteins increased progressively from normal mucosa to OED to OSCC. In normal mucosa cases, all the positively stained nuclei were present in the expected proliferative transit compartment, i.e., the basal compartment of squamous epithelium. On the contrary, in cases of dysplastic epithelium, both the proteins were expressed in the majority of cells, extending to the surface layers. In OSCC, the frequency of expression of MCM2 and Ki-67 showed an inverse correlation with the degree of tumor differentiation. Moreover, a significant positive correlation was detected between the two markers. The mean expression of MCM2 was found to be higher than Ki-67 irrespective of the stage of differentiation of carcinoma or grade of dysplasia. MCM2 provides a more reliable and useful means of rapidly evaluating the growth fraction of normal and neoplastic cell populations in phases other than G0 and mitotic phase since MCM2 is expressed in normal or neoplastic cells even in the early G1 phase, while Ki67 is undetectable during this phase.
Torres-Rendon et al. conducted a study to identify the expression pattern of MCM2 and Ki-67 in normal mucosa, OED, and their subsequent OSCC. They observed MCM2 and Ki-67 expression to increase progressively from normal mucosa to OED to OSCC. They also found increased expression of MCM2 than Ki67 and reported MCM2 expression to be higher in OED cases with malignant progression, thereby concluding MCM2 to be a novel growth and prognostic biomarker. All of these findings were in concordance with our study. Bhattacharya et al. evaluated the expression of Ki67, p53, and Bcl-2 in oral leukoplakia and OSCC patients and found a significant increase of Ki-67 expression in OSCC cases than leukoplakia cases along with significant high p53 and Ki67 expression in moderately and poorly differentiated OSCC, thereby summarizing that these markers could be used as predictive markers for poor prognosis in such cases. Our study showed similar results.
Valverde et al. evaluated and compared the immunoexpression of MCM3 and Ki-67 in OSCC and reported greater MCM3 expression than Ki67 in tumors, while tumor-free resection margins and nonneoplastic oral mucosa exhibited greater Ki-67 expression than MCM3, thus arriving at the conclusion that Ki-67 immunoexpression was influenced by inflammation. Their findings suggested both the markers to be reliable to assess cellular proliferation, but since MCM3 expression was not influenced by external factors, it was considered to be a superior marker. Our study also showed MCM2, a member of the same minichromosome maintenance protein family, to be a better marker, and hence, was in consonance with this study. Lopes et al. also studied the expression of Ki-67 and MCM3 in OSCC and reported similar greater expression of MCM3. They also found a statistically significant result on evaluating Ki-67 immunostaining and overall survival, as well as an association between tumor size and Ki-67 expression, whereas concerning MCM3, no statistically significant results were found.
Shalash et al. examined the cellular distribution of MCM2 in OSCC and reported variable cellular localization similar to our study, i.e., well-differentiated cases showing positivity along the periphery of epithelial cell nests and at invasive fronts while central cores of cell nests lacked positivity. Moderately differentiated cases revealed expression along the periphery of epithelial cell nests, extending into the middle third, while the central cores showed a negative reaction. On the contrary, most of the poorly differentiated cases showed a widespread positivity involving all the malignant squamous epithelial cells. Scott et al. immunohistochemically examined 66 oral tissue samples and reported greater frequency of Mcm-2 expression in surface layers of moderate/severe dysplasia and SCC compared to benign keratosis/mild dysplasia, a finding similar to our study.
Takkem et al. investigated Ki-67 expression in OED, OSCC, and normal oral mucosa. They reported variable expression patterns similar to our study. However, they reported statistically significant difference between the expression in OED and normal oral mucosa, OSCC and normal oral mucosa, but not between OED and OSCC. This finding was not in concordance with our study. Verma et al. studied the expression of Ki-67 at the invasive tumor front of OSCC and reported higher expression in poorly differentiated OSCC signifying actively proliferating malignant cells at invasive tumor front, a finding similar to our study. Birajdar et al. studied the expression of Ki-67 in normal oral epithelium, leukoplakic oral epithelium, and OSCC and reported Ki-67 labeling index restricted to basal and parabasal layers of normal oral epithelium in basal, suprabasal, and spinous layers in OED. Ki-67 positive cells in OSCC were located in the periphery of tumor nests than the center. Similar expression pattern was noted in our study.
Bettendorf et al. studied the prognostic relevance of Ki-67 in 329 cases of OSCC and reported a highly significant inverse correlation between Ki-67 labeling index, stroma/tumor proportion, and the degree of keratinization, whereas no correlation could be established between Ki-67 labeling index and all the other histological and clinical parameters. They concluded that Ki-67 staining alone had no prognostic relevance in oral cancer. Gonzales-Moles et al. analyzed the prognostic value of Ki-67 in oral cancer and its relationship with Ki-67 expression in precancerous epithelium and reported Ki-67 expression to be higher in well-differentiated versus poorly differentiated carcinomas and that Ki-67 expression had no impact on survival. They concluded that Ki-67 lacked prognostic value, probably because it is a marker of the total fraction of proliferating cells, corresponding not only to cells in constant proliferation but also to proliferating cells destined for terminal differentiation.
| > Conclusion|| |
We thereby conclude that MCM2, as a proliferation marker, is superior to Ki-67 as it indicates a licensed capacity of proliferation and DNA replication of a cell. Further studies with larger sample size including demographics and lymph node sections are necessary to assess their value in prediction of lymph node metastasis with an association to age, sex, and habit history, thereby helping in the prognosis of the disease.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3]