|Year : 2016 | Volume
| Issue : 2 | Page : 140-144
Expression of cyclin D1 in oral squamous cell carcinoma and its correlation with histological differentiation: An immunohistochemical study
Anand Choudhary1, Pallavi Kesarwani2, Pravin Gaikwad3, Santhosh S Hiremath4, Renuka Gupta5, Sri Krishna Koppula1
1 Department of Oral Medicine and Radiology, Hazaribag College of Dental Sciences, Hazaribag, Jharkand, India
2 Department of Oral Pathology and Microbiology, Hazaribag College of Dental Sciences, Hazaribag, Jharkand, India
3 Department of Oral Pathology and Microbiology, Sukhmani Dental College, Dera Bassi, Punjab, India
4 Department of Oral Pathology and Microbiology, Vishnu Dental College, Bhimavaram, Andhra Pradesh, India
5 Department of Oral Pathology and Microbiology, Purvanchal Institute of Dental Sciences, Gorakhpur, Uttar Pradesh, India
|Date of Submission||26-Apr-2016|
|Date of Acceptance||14-Nov-2016|
|Date of Web Publication||02-Dec-2016|
Flat No. 2A Savitri Appartment, Harihar Singh Road, Morabadi, Ranchi - 834 008, Jharkhand
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Cyclin D1 oncogene is a key regulator of cell cycle progression associated with various human malignancies. Aim: To establish the prognostic significance of cyclin D1 in oral squamous cell carcinoma (OSCC) and to correlate its association with clinicopathological parameters. Materials and Methods: A retrospective study was carried out on 50 previously histopathologically diagnosed, formalin-fixed, paraffin embedded (FFPE) tissue samples of OSCC. Immunohistochemical analysis was performed in 50 cases of different grades of OSCC using a commercially available monoclonal anti-cyclin D1 antibody. Statistical Analysis Used: All the compiled data was statistically analyzed using Chi-square and Mann-Whitney test. P values of <0.05 were considered to be significant. Results: Overexpression of cyclin D1 was seen in 68% cases of OSCC with a significant correlation with younger age group; no correlation was reported with respect to gender and site. The intergroup comparison of the cyclin D1 expression between well with poorly differentiated OSCC exhibited a significant correlation. Conclusions: The relative contribution of cyclin D1 expression in OSCC was significant according to the present study and further needs to be determined in large cohorts.
Keywords: CCND1 protein, cell division cycle, cyclin D1
|How to cite this article:|
Choudhary A, Kesarwani P, Gaikwad P, Hiremath SS, Gupta R, Koppula S. Expression of cyclin D1 in oral squamous cell carcinoma and its correlation with histological differentiation: An immunohistochemical study. J Indian Acad Oral Med Radiol 2016;28:140-4
|How to cite this URL:|
Choudhary A, Kesarwani P, Gaikwad P, Hiremath SS, Gupta R, Koppula S. Expression of cyclin D1 in oral squamous cell carcinoma and its correlation with histological differentiation: An immunohistochemical study. J Indian Acad Oral Med Radiol [serial online] 2016 [cited 2022 Dec 7];28:140-4. Available from: http://www.jiaomr.in/text.asp?2016/28/2/140/195099
| Introduction|| |
The cell cycle forms the basis of continuity of life and underlies the complexity of growth, renewal, and repair in all organisms.  Cancer is a disease characterized by deregulation of cell cycle control. Alterations in the signaling pathway that ultimately lead to DNA replication and mitosis have been identified in various tumor types. These alterations include oncogenes that mediate cell cycle control (e.g., cyclins, cyclin-dependent kinases [CDK] and their inhibitors [CDI]), or genes that mediate stress or damage response (p53).  These genetic alterations provide mechanism for the development of cancer, are putative targets of therapy, and/or may serve as diagnostic and prognostic markers. 
Cellular proliferation follows an orderly progression through the different phases of cell cycle, , and at every next transition during cell cycle progression, signaling pathways screen the successful completion of upstream events prior to proceeding to the next phase. Cyclin D1 is a key regulatory protein at G1/S checkpoint of the cell cycle. The G1/S checkpoint is frequently altered in many epithelial tumors. ,,
Many studies have reported high levels of cyclin D1 expression in oral squamous cell carcinoma (OSCC), ,,,, though the association of cyclin D1 on clinicopathological parameters and prognosis in OSCC is inconclusive. Hence, the present study was undertaken to correlate immunopositivity of malignant cells with different grades of OSCC.
| Materials and Methods|| |
A total of 50 histopathologically diagnosed, formalin-fixed, paraffin embedded tissue samples of OSCC were collected from the archives of the Department of Oral Pathology and Microbiology, Institute of Dental Sciences, Bareilly. All the 50 blocks were categorized into different grades of OSCC based on Borders criteria into well-differentiated OSCC (WDSCC) - 16 cases, moderately-differentiated OSCC (MDSCC) - 19 cases, poorly-differentiated OSCC (PDSCC) - 15 cases. Normal oral mucosa was considered as positive controls and OSCC tissue with exclusion of primary antibody was taken as negative control.
From the representative tumor tissue block, two sections of approximately 3-4 μm thickness were cut on Poly L-Lysine coated slides and stained respectively by hematoxylin and eosin stain for histopathological grading by Broder's criteria.  Another section was utilized for immunohistochemical staining with cyclin D1 (DAKO EnVision TM FLEX). Sections were dewaxed, cleared in xylene, and rehydrated in descending grades of alcohol; antigen retrieval was carried out in a pressure cooker using citrate buffer (10Mm, pH 6.0) for 2-5 min. The slides were slowly allowed to cool down to room temperature. To block endogenous peroxidase activity, the sections were quenched by incubation in 3% hydrogen peroxide solution for 15 min. Consequently, the sections were incubated for 1 h with primary monoclonal anticyclin D1 antibody, followed by 45 min in secondary antibody and 30 min in streptavidin-biotin peroxidase conjugate. Later, the slides were incubated for 10 min with 3, 3-diaminobenzidine chromogen for antigen visualization and counterstained by Harris hematoxylin. All the steps were carried out at room temperature, and after each step the sections were washed with tris-buffer saline (pH 7.6).
Cyclin D1 protein expression was evaluated on the basis of the presence or absence of nuclear staining. Five representative fields that were most evenly stained were randomly selected in each case. All counts were performed on a research microscope using a gridded eyepiece at (400×) magnification. Two individual examiners performed the counts in order to remove the interobserver variability; in the few cases in which the assessment led to a large difference in results, consensus was reached after re-examination.
Total number of tumor cells in each slide should reach a minimum of 500, i.e., the sum of the denominators. The mean percentage of positive tumor cells was obtained by the simple formula shown below:
Mean percentage of IHC positive Cells (nA/B ) = Total no. of IHC positive cells × 100/Total no. of tumor cells
The average of the percentage thus obtained by the two observers for each slide was then used for scoring (labelling index), which were then graded on a scale of 0 to 3. An arbitrary score was assigned as follows: (0) for <10% positivity; (+1) for 10-30% positivity; (+2) for 30-50% positivity; (+3) for >50% positivity. 0 indicated negative staining, 1 indicated mild staining, 2 indicated moderate staining, and 3 indicated intense staining, based on a criteria used by Castle et al. 
All the compiled data were statistically analyzed using the Statistical Package for Social Sciences Software (SPSS) version 11.0, IBM Corporation, New York, U.S., using Chi-square and Mann-whitney test, to assess for statistical significance between the different parameters. P values of <0.05 were considered significant.
| Results|| |
Cyclin D1 immunoreactivity was reported in 68% of OSCC cases. Immunostaining was predominantly strict to nucleus but some tumor cells also showed simultaneous cytoplasmic staining. Hence, a heterogeneous pattern of staining was seen due to variation in protein levels during cell cycle progression. Absence of immunoreactivity was seen in positive control and as well in normal oral mucosal tissues. While analyzing cyclin D1 expression with clinicopathological features, it was found that the expression of cyclin D1 was significantly correlated with younger age group (<60) at P = 0.025, however, no statistical significance was reported in relation to gender (P = 0.938) and site (P = 0.386). According to histological differentiation, the present study suggested an inverse correlation of cyclin D1 expression with the degree of differentiation [Figure 1]; however, the results failed to reach statistical significance at P = 0.077. On analyzing labeling index score, the intergroup comparison of the cyclin D1 expression between WDSCC with MDSCC (P = 0.086) and MDSCC with PDSCC (P = 0.091) showed no significant correlation, while that of WDSCC with PDSCC exhibited a highly significant correlation at P = 0.002.
|Figure 1: The photomicrograph showing positive cyclin D1 expression in various grades of oral squamous cell carcinoma (a) in well-differentiated oral squamous cell carcinomas; (b) moderately differentiated oral squamous cell carcinomas; (c and d) poorly differentiated oral squamous cell carcinomas (×100)|
Click here to view
| Discussion|| |
Dysregulation of the cell cycle machinery is a fundamental hallmark of cancer progression. Cyclin D1 is expressed during the G1 phase of the cell cycle. Its fundamental role is to integrate extracellular (growth factor) signals with the cell cycle regulatory machinery.  Deregulation/overexpression of cyclin D1 may, therefore, lead to shortening of G1 phase, increased cell proliferation, and reduced dependency on growth factors,  which might result in an accumulation of nonrepaired DNA mutations leading to disturbance in the normal cell cycle control and tumor formation. 
In the present study, on the basis of age, it was found that the cyclin D1 expression in younger individuals (<60 years) is higher with statistically significant correlation. These results can be attributed to genetic defects and partly to difference in carcinogen exposure between the two age groups. Changing patterns in the incidence of oral cancer suggest that there may be mechanistic difference with regard to tumor progression between young and old individuals, and therefore, now the younger group of population are at a risk of developing aggressive disease.  No significant correlation was found in relation to gender, and site distribution may be attributed to the relatively small sample size or racial differences and might be due to variation in tumor biology at different sites in OSCC. 
Overexpression of cyclin D1 is a common genetic event in head and neck squamous cell carcinoma. In the present study, overexpression of cyclin D1 was reported in 68% cases of OSCC. This was in accordance with previous studies done by Lam et al.  and Angadi et al.  who have reported 63 and 70.7% positivity, respectively. Few studies conducted by Chetty et al.  and Fracchiolla et al.  showed a very low percentage of positivity of 29 and 39%, respectively. A very high cyclin D1 expression of 95 and 97% has been reported by Swaminathan et al.  and Shiang et al.,  respectively. Thus, there is wide variation in the reported positive IHC reactivity in the literature. In case of normal oral epithelium, the expression was restricted to the basal and parabasal layers of epithelium,  and the results were considered to be positive or negative correlating with the staining in the control epithelium. Hunter et al.  suggested that cyclin D1 is an activator of the cell proliferation, and because peripheral cells are the most proliferating ones, they express more cyclin D1. Loss of cyclin D1 in mitotic cell is suggested to be due to the inactivation of this protein by the end of S phase and because of its short half-life.
The present study suggested a positive correlation for cyclin D1 expression with lack of differentiation, which is in contrast with previously reported studies of Saawarn et al.  and Wu et al.  where they reported a positive correlation with the degree of differentiation. However, a similar study performed on lung carcinoma by Mate et al.  and OSCC by Lam et al.  stated that differentiation in addition to proliferation is a major target of cyclin D1 impingement upon mechanisms of development and neoplasia. This was supported by a report by Shapek et al., where a direct relationship between cyclin D1 overexpression and inhibition of myogenic differentiation in myoblasts was explained in an experimental model.  This raises a possibility that cyclin D1 overexpression may play a role in inhibition of tumor cell differentiation in some cell types, probably by binding with nonfunctional CDK.  The high expression of cyclin D1 with poor cellular differentiation is related to intense proliferative activity and invasiveness of the lesion.  In contrast, Wu et al. suggested that the absence of cyclin D1 can be related to the deregulation of other proteins in the cycle. 
Furthermore, in the present study, on intergroup comparison of labeling index score for cyclin D1 expression between WDSCC with MDSCC and MDSCC with PDSCC showed no significant correlation, whereas that of WDSCC with PDSCC exhibited a significant one. Hence, the present study suggests that the alteration of cyclin D1 protein is not only associated with higher cell proliferation but it also contributes importantly in the process of tumor differentiation, which is in accordance to previously published literature by Mate et al.  and Lam et al. 
Cyclin D1 expression has been correlated with various proliferation markers. Vicente et al.  found significant correlation between cyclin D1 and Ki-67. Koontongkaew et al.  suggested that inappropriate expression of p53, pRb, cyclin D1, or CDK4 is likely to contribute to the development of oral and pharyngeal cancers. Shintani et al.  reported elevated expression of cyclin D1 along with other cell cycle regulatory proteins such as cyclin E, CDK2, and loss of p12, p16, and p27 may contribute to the multistep process of carcinogenesis. Few studies have reported cyclin D1 immunoreactivity confined to the nucleus, some suggested cytoplasmic staining along with nucleus,  while few have exclusively reported cytoplasmic staining and considered the same as positive. Falco et al.  explained that the transit of cyclin D1 between nuclear and cytoplasmic compartments occurs via nuclear pores, thus helping in maintaining cell homeostasis. The present study also showed cytoplasmic staining of atypical cells along with nuclear staining.
| Conclusion|| |
The relative contribution of cyclin D1 expression in OSCC needs to be determined in large cohorts. The present study opens a window of opportunity for further studies and research in this field. An opportunity to select a group of patients for more intensive treatment and follow-up, which may contribute significantly to the patient's well-being, by establishing cyclin D1 as a better prognostic marker.
We would like to thank all the staff members of Oral Pathology and Oral Medicine Department for their support and guidance.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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