Expression analysis of cyclin D, Ki-67, MCM3 and MCM2 in oral squamous cell carcinoma

The expression analysis of cyclin D1, Ki-67, MCM3 and MCM2 in oral squamous cell carcinoma to identify biomarkers is of interest. 45 formalin-fixed paraffin embedded tissue blocks collected from archives of Department of Oral and Maxillofacial Pathology and Oral Microbiology, Government Dental College and Hospital, Jamnagar, India were subjected to a retrospective cross-sectional immuno-histo-chemistry examination. 30 blocks of OSCC with histological diagnosis have 15 tissue blocks of well-differentiated oral carcinoma and 15 tissue blocks of moderately-differentiated oral carcinoma. 15 specimens of normal oral mucosa (NM) were also examined for comparison. In each of the categories, the immuno-histo-chemistry expression of cyclin D1, MCM 3, MCM 2, and ki67 was studied. Data shows that cyclin D1, Ki-67, MCM3 and MCM2 effectively indicate cellular proliferation for consideration as potential biomarkers of oral squamous cell carcinoma.


Background:
Prognosis and diagnosis of cancer is critical [1,2].A survivability rate of as high as 80% is reported for oral squamous cell carcinoma (OSCC) cases detected at the initial phase (T1N0); however, at later stages (T3-T4), this percentage drops to roughly 20%-30% [3,4].Research has confirmed that the development of genetic mutations and epigenetic anomalies in the functioning of genes associated with cell proliferation is the initial step towards oral carcinogenesis [5][6][7][8][9].Therefore, evaluating the behaviour of the tumour, the prognosis, and the survival rate of patients require an understanding of cell proliferation [10][11].To identify and measure the cell proliferation in oral cancer, a variety of proliferation biomarkers were established.In fact, research done in OSCC has shown the highest correlation between cyclins and cancer development [12,13].The G1 phase, which is the sole stage in which outside influences like growth regulators can affect the cell cycle, seems to be crucial for cyclin D1 among the various cyclins [14,15].There have been reports of cyclin D1 up-regulation and amplification in oral, head and neck, nasopharyngeal and laryngeal cancers [16,17].Similarly, a small number of studies showed that mini-chromosome maintenance (MCM) molecules can be utilised as proliferation biomarkers to assess tumour behaviour due to their activity in the initial phase of G1 [18,19].The proliferative scale and the prognostic aspect of OSCC patient longevity may both be estimated using MCM2 protein [20,21].
One key factor in tumour progression, the proliferative potential of neoplastic cells, is thought to be a significant prognostic predictor [14-16].Like another polypeptides that make up the MCM intricate, the MCM3 protein is found at reduced intracellular concentrations in differentiated and quiescent cells.Traditional indicators of cell division, that include the protein Ki-67, are frequently employed in the evaluation of a range of malignancies in humans, including neoplasms of the breast and prostate, sarcomas and lymphomas [22][23].MCM3 protein expression persists for longer compared to Ki-67 and it offers more accurate and sensitive data for assessing the proliferative characteristics of cell populations [19][20][21].In addition, it has been noted that the level of expression of the Ki-67 protein provides unclear knowledge concerning malignant neoplasia because it includes the entire percentage of cells in the cell cycle, irrespective of whether or not these cells will ultimately differentiate in the absence of any correlation to a malignant form [22][23].Furthermore, in apoptotic cells or in situations where DNA synthesis is inhibited, Ki-67 activity may also happen [24][25][26].It has been shown that certain human tumours, including salivary gland tumours, thyroid papillary carcinoma and melanoma engage MCM proteins as indicators of cellular proliferation [12,18,20].MCM2 protein positivity is thought to be a more accurate indicator of tumour prognosis in OSCC compared to .Therefore, it is of interest to document the expression of cyclin D, Ki-67, MCM3 and MCM2 in oral squamous cell carcinoma to glean biomarkers.

Methods and Materials:
Design of the study and selection of patient: Forty five archival collected formalin-fixed paraffin embedded tissue blocks were subjected to a retrospective cross-sectional immunohistochemistry examination.Thirty blocks of OSCC with histological diagnosis were included in the study that included fifteen tissue blocks of well-differentiated oral carcinoma and fifteen tissue blocks of moderately-differentiated oral carcinoma.Fifteen specimens of normal oral mucosa (NM) were examined (Table 1).In each of the two categories, the immuno histo-chemistry expression of cyclin D1, MCM 3, MCM 2, and ki67 was examined.

Immunohistochemistry:
The conventional IHC method was used to assess the expression of cyclin D1, MCM3 MCM2, and Ki-67 using the following antibodies (Table 2).The tonsils for cyclin D1, MCM3, MCM2, and ki-67 were included in the positive control sections, which received the same treatment as the test categories.

Immunohistochemical analysis:
Positive staining was demonstrated by the existence of a browncolored final product at the target antigen location.Nuclear staining varied in intensity across all cases.The staining intensity was examined to determine the degree of stain absorption.On each slide, ten randomly chosen fields were magnified at a magnification of ×40.The following scale was used to rate the staining intensity of each section [11][12].
No stain = 0 Mild stain= 1 Moderate stain = 2 Intense stain= 3 After scanning the complete section of the epithelium, the area of coloured epithelial cells was measured in order to ascertain the expression profile and the amounts of protein accumulation in the epithelial layers [17].
After the slides were viewed at ×40 magnification using an Olympus CX21 light microscope, illustrative photomicrographs were made of each slide in five different hotspot areas in order to calculate the labeling index (LI).After that, an image processing programme called ImageJ (http://imagej.nih.gov/ij/) was used to analyse the photomicrographs.The total number of tumour cells in each slide was computed until a minimum of 400 cells were attained, i.e addition of the denominators (x).The percentage of IHC positive tumour cells per hot spot (A) was also determined [8].
The following formula was used to compute LI [9].

Statistical Analysis
Statistical analysis was performed on the collected data using SPSS Version 17.0 (SPSS, Inc., Chicago, IL, USA).After doing a Mann-Whitney U test and Kruskal-Wallis ANOVA, P < 0.05 was deemed statistically significant  Studies have demonstrated a correlation between tumours with elevated levels of Ki-67 expression and a decreased probability of survival and recurrence.In OSCC, Ki-67 has also been demonstrated to be a strong predictive indicator of survival and recurrence [21][22][23][24].A previous study claim that because the MCM protein complex is involved in cellular proliferation, modifications that lead to an increase in this helicase's activity are associated with the development of cancer [13][14][15].Consequently, studies have demonstrated a negative prognosis for brain tumours, salivary gland tumours, thyroid carcinomas, melanoma, and twelve other types of malignancies when MCM3 expression is increased [15][16][17][18][19].
The mean LI of cyclin D1 and ki-67 was found to be lower in the current study than the mean LI of MCM3 and MCM2 in the study groups.This may be the result of the MCM2 and MCM3 proteins, which are produced in the nucleus of a cell from the initial G1 phase onward and which recognize both cycling as well as noncycling cells having proliferative capacity across the cell cycle [15][16][17][18][19].Therefore, compared to cyclin D1 and ki 67, MCM2 and MCM 3 can be more useful biomarkers for cell proliferation in OSCC.To validate the current findings, more research with a bigger sample size is required.

Conclusion:
Data shows that MCM3 and MCM 2 are potential biomarkers compared to cyclin D1 and Ki-67 in OSCC.

Table 7 : Comparison of values of labeling index between cyclin D1, MCM 2, Ki 67 and MCM3 in normal mucosa, oral squamous cell carcinoma
[21][22][23][24]activity can also occur in apoptotic cells or in conditions where DNA synthesis is suppressed[21][22][23][24].Research has demonstrated that MCM [15,16]lements may contribute to the development of oral cancer separately or in combination[15,16].As the tumour grows, the chances of survival decrease, hence it is imperative to detect it as soon as possible.For cases of oral squamous cell carcinoma (OSCC) found in the early stages, a survivability rate of up to eighty percent has been observed; however, at later stages, this percentage falls to about twenty percent to thirty percent [21-24].Studies have shown that the first step towards the development of oral carcinogenesis is the emergence of genetic mutations and epigenetic abnormalities in the regulation of genes linked to cell proliferation [12-16].Thus, an awareness of cell proliferation is necessary in order to assess the behaviour of the tumour, the prognosis, and the survival rate of patients [13-17].Many proliferation markers were developed in order to detect and quantify the cell proliferation in oral cancer.In fact, studies conducted in OSCC have revealed the strongest link between cyclins, MCMs and the onset of cancer [16-21].proliferative traits of cell populations than Ki-67 since it is expressed for a longer period of time [19-23].Furthermore, as noted by previous study, the expression level of the Ki-67 protein encompasses the total percentage of cells in the cell cycle, regardless of whether or not these cells will eventually differentiate into a malignant form, thus providing ambiguous information regarding malignant neoplasia [17-23].express Ki-67 is frequently associated with the clinical progression of tumours, particularly breast and lung cancers [22-26].