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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 28  |  Issue : 2  |  Page : 140-144

Expression of cyclin D1 in oral squamous cell carcinoma and its correlation with histological differentiation: An immunohistochemical study


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 Submission26-Apr-2016
Date of Acceptance14-Nov-2016
Date of Web Publication02-Dec-2016

Correspondence Address:
Pallavi Kesarwani
Flat No. 2A Savitri Appartment, Harihar Singh Road, Morabadi, Ranchi - 834 008, Jharkhand
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-1363.195099

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   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 2021 Apr 13];28:140-4. Available from: https://www.jiaomr.in/text.asp?2016/28/2/140/195099


   Introduction Top


The cell cycle forms the basis of continuity of life and underlies the complexity of growth, renewal, and repair in all organisms. [1] 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). [2] These genetic alterations provide mechanism for the development of cancer, are putative targets of therapy, and/or may serve as diagnostic and prognostic markers. [3]

Cellular proliferation follows an orderly progression through the different phases of cell cycle, [4],[5] 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. [5],[6],[7]

Many studies have reported high levels of cyclin D1 expression in oral squamous cell carcinoma (OSCC), [8],[9],[10],[11],[12] 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 Top


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. [13] 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).

Labeling index

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. [14]

Statistical analysis

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 Top


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)

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   Discussion Top


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. [4] Deregulation/overexpression of cyclin D1 may, therefore, lead to shortening of G1 phase, increased cell proliferation, and reduced dependency on growth factors, [15] which might result in an accumulation of nonrepaired DNA mutations leading to disturbance in the normal cell cycle control and tumor formation. [4]

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. [16] 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. [17]

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. [17] and Angadi et al. [7] who have reported 63 and 70.7% positivity, respectively. Few studies conducted by Chetty et al. [18] and Fracchiolla et al. [6] 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. [10] and Shiang et al., [19] 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, [20] and the results were considered to be positive or negative correlating with the staining in the control epithelium. Hunter et al. [21] 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. [22] and Wu et al. [23] where they reported a positive correlation with the degree of differentiation. However, a similar study performed on lung carcinoma by Mate et al. [24] and OSCC by Lam et al. [17] 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. [25] 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. [26] The high expression of cyclin D1 with poor cellular differentiation is related to intense proliferative activity and invasiveness of the lesion. [17] In contrast, Wu et al. suggested that the absence of cyclin D1 can be related to the deregulation of other proteins in the cycle. [27]

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. [24] and Lam et al. [17]

Cyclin D1 expression has been correlated with various proliferation markers. Vicente et al. [28] found significant correlation between cyclin D1 and Ki-67. Koontongkaew et al. [29] 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. [30] 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, [17] while few have exclusively reported cytoplasmic staining and considered the same as positive. Falco et al. [31] 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 Top


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.

Acknowledgment

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

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
Poon RY. Cell cycle control. Encyclopedia Cancer 1997;1:246-55.  Back to cited text no. 1
    
2.
Michalides RJ, Veelen NM, Kristel PM, Hart AA, Loftus BM, Hilgers FJ, et al. Overexpression of Cyclin D1 indicates a poor prognosis in squamous cell carcinoma of the head and neck. Arch Otolaryngol Head Neck Surg 1997;123:497-502.  Back to cited text no. 2
    
3.
Shinozaki H, Ozawa S, Ando N, Suruta H, Terada M. Cyclin D1 amplification as a new predictive classification for squamous cell carcinoma of the esophagus, adding gene information. Clin Cancer Res 1996;2:1155-61.  Back to cited text no. 3
    
4.
Todd R, Hinds PW, Munger K, Rustgi AK, Opitz OS, Suliman Y, et al. Cell cycle dysregulation in oral cancer. Crit Rev Oral Biol Med 2002;13:51-61.  Back to cited text no. 4
    
5.
Pietenpol JA, Stewart ZA. Cell cycle checkpoint signaling: Cell cycle arrest versus apoptosis. Toxicology 2002;181-182:475-81.  Back to cited text no. 5
    
6.
Fracchiolla NS, Pruneri G, Pignataro L, Carboni N, Capaccio P, Boletini A, et al. Molecular and immunohistochemical analysis of the bcl-1/Cyclin D1 gene in laryngeal squamous cell carcinomas. Cancer 1997;79:1114-21.  Back to cited text no. 6
    
7.
Angadi PV, Krishnapillai R. Cyclin D1 expression in oral squamous cell carcinoma and verrucous carcinoma: Correlation with histological differentiation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:30-5.  Back to cited text no. 7
    
8.
Miyamoto R, Uzawa N, Nagaoka S, Hirata Y, Amagasa T. Prognostic significance of cyclin D1 amplification and overexpression in oral squamous cell carcinoma. Oral Oncol 2003;39:610-8.  Back to cited text no. 8
    
9.
Lin RJ, Lubpairee T, Liu KY, Anderson DW, Durham S, Poh CF. Cyclin D1 overexpression is associated with poor prognosis in oropharyngeal cancer. J Otolaryngol Head Neck Surg 2013;42:1-7.  Back to cited text no. 9
    
10.
Swaminathan U, Joshua E, Umadevi K, Ranganathan K. Expression of p53 and Cyclin D1 in oral squamous cell carcinoma and normal mucosa: An immunohistochemical study. J Oral Maxillofac Pathol 2012;16:172-7.  Back to cited text no. 10
  Medknow Journal  
11.
Das SN, Khare P, Singh MK, Sharma SC. Correlation of cyclin D1 expression with aggressive DNA pattern in patients with tobacco-related intraoral squamous cell carcinoma. Indian J Med Res 2011;133:381-6.  Back to cited text no. 11
[PUBMED]  Medknow Journal  
12.
Shenoy AM. Cyclin D1 over expression as a prognostic factor in patients with tobacco-related intraoral squamous cell carcinoma. Indian J Med Res 2011;133:364-5.  Back to cited text no. 12
[PUBMED]  Medknow Journal  
13.
Anneroth G, Batsakis J, Luna M. Review of literature and a recommended system of malignancy grading in oral squamous cell carcinoma. Scand J Dent Res 1987;95:229-49.  Back to cited text no. 13
    
14.
Castle JT, Cardinali M, Kratochvil FJ, Abbondanzo SL, Kessler HP, Aucair PL, et al. P53 and Cyclin D1 staining patterns of malignant and premalignant oral lesions in age dependent populations. Oral Surg Oral Med Oral Radiol Endod 1999;88:326-32.  Back to cited text no. 14
    
15.
Donnellen R, Chetty R. Cyclin D1 and human neoplasia. J Clin Pathol 1998;51:1-7.  Back to cited text no. 15
    
16.
Macfarlane GJ, Zheng T, Marshall JR, Boffetta P, Niu S, Brasure J, et al. Alcohol, tobacco, diet and the risk of oral cancer: A pooled analysis of three case-control studies. Oral Oncol Eur J Cancer 1995;31B: 181-7.  Back to cited text no. 16
    
17.
Lam KY, Irene OL, Yuen AP, Kwong DL, William W. Cyclin D1 expression in oral squamous cell carcinomas: Clinicopathological relevance and correlation with p53 expression. J Oral Pathol Med 2000;29:167-72.  Back to cited text no. 17
    
18.
Chetty R, Chetty S. Cyclin D1 and retinoblastoma protein expression in esophageal squamous cell carcinoma. J Clin Pathol Mol Pathol 1997;50:257-60.  Back to cited text no. 18
    
19.
Shiang FH, Sou DC, Wen YC, Chen IH, Chun TL, Hung MW, et al. Cyclin D1 overexpression and poor clinical outcomes in Taiwanese oral cavity squamous cell carcinoma. World J Surg Oncol 2012;10:1-7.  Back to cited text no. 19
    
20.
Rousseau A, Lim MS, Lin Z, Jordan RC. Frequent cyclin D1 gene amplification and protein overexpression in oral epithelial dysplasias. Oral Oncol 2001;37:268-75.  Back to cited text no. 20
    
21.
Hunter T, Pines J. Cyclins and cancer II: Cyclin D and CDK inhibitors come of age. Cell 1994;79:573-82.  Back to cited text no. 21
    
22.
Saawarn S, Astekar M, Saawarn N, Dhakar N, Sagari SG. Cyclin D1 Expression and Its Correlation with Histopathological Differentiation in Oral Squamous Cell Carcinoma. Scientific World Journal 2012;2012:978327.  Back to cited text no. 22
    
23.
Wu M, Putti TC, Bhuiya TA. Comparative study in the expression of p53, EGFR, TGF- alpha, and cyclin D1 in verrucous carcinoma, verrucous hyperplasia and squamous cell carcinoma of head and neck region. Appl Immunohistochem Mol Morphol 2002;10:351-6.  Back to cited text no. 23
    
24.
Mate JL, Ariza A, Aracil C, Lopez D, Isama TM, Piteira JP. Cyclin D1 overexpression in non-small cell lung carcinoma; correlation with ki67 labelling index and poor cytoplasmic differentiation. J Pathol 1996;80:395-9.  Back to cited text no. 24
    
25.
Shapek SX, Rhee J, Spicer DB, Lassar AB. Inhibition of myogenic differentiation in proliferating myoblasts by cyclin D1- dependent kinase. Science 1995;267:1022-4.  Back to cited text no. 25
    
26.
Bruce E. Clurman, James M. Roberts. Cell Cycle and Cancer. J Natl Cancer Institute 1995;87:1499-501.  Back to cited text no. 26
    
27.
Neves AD, Mesquita RA, Novelli MD, Todda E, De Sousa SO. Comparison between Immunohistochemical Expression of Cyclin D1 and p21 and Histological Malignancy Graduation of Oral Squamous Cell Carcinomas. Br Dent J 2004;15:93-8.  Back to cited text no. 27
    
28.
Vicente JC, Zapatero AH, Frerno MF, Lopez AJ. Expression of Cyclin D1 and Ki-67 in squamous cell carcinoma of the oral cavity: Clinicopathological and prognostic significance. Oral Oncol 2002;38:301-8.  Back to cited text no. 28
    
29.
Koontongkaew S, Chareonkitkajorn L, Chanvitan A, Leklakriangrak, Amornphimolthan P. Alterations of p53, pRb, cyclin D1 and cdk4 in human oral and pharyngeal squamous cell carcinomas. Oral Oncol 2000;36:334-9.  Back to cited text no. 29
    
30.
Shintani S, Mihara M, Nakahara Y, Kiyota A, Yegama Y, Matsumura T, et al. Expression of cell cycle control proteins in normal epithelium, premalignant and malignant lesions of oral cavity. Oral Oncol 2002;38:235-43.  Back to cited text no. 30
    
31.
Falco MD, Fedele V, Luca LD, Penta R, Cottone G, Cavallotti I, et al. Evaluation of cyclin D1 expression and its subcellular distribution in mouse tissues. J Anat 2004;205:405-12.  Back to cited text no. 31
    


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