Oral squamous cell carcinoma (OSCC) is a malignant epithelial tumor with poor prognosis. OSCC is one of the top causes of death in India and Southeast Asia. ¹ Oral squamous cell carcinoma caused due to chewing tobacco, such as panmasala, gutkha, gudakhu, khaini, and other products composed of tobacco. ^{2 , 3} Patients with middle-aged to older ages typically affected by OSCC. ⁴ OSCC have a high rate of proliferation of malignant epithelial cells. The prognosis of OSCC depends on recommended surgical margin.

In the surgical margin, the epithelial cells may be altered by field cancerization. There is an increase in the proliferation of epithelial cells in OSCC which is control by cell cycle regulators like Cyclin D1. ⁵

Cyclin control the orderly phases of the cells through the G1, S, G2, and M phases of the cell cycle. ^{6 , 7} It plays a role in cell cycle induction, transcriptional control, and DNA damage-induced death. ² (CCND1) Cyclin D1, 11q13 location of chromosomes, carries the cell cycle from the G1 to the S phase by encoding a vital cell cycle regulating protein. ^{8 ‐ 10} The control of the cell cycle is facilitated by Cyclin D1, proto-oncogenes and an enzyme known as Cylin-dependent kinase 4 (CDK4). It also interacts with other transcriptional factor (TFs) and controls the activity of transcriptional cofactors. ⁴ Lack of regulation of the cell cycle mechanism is an essential phase in the carcinogenic change. Cyclin D1 is linked to a poor prognosis for patients, including aggressive malignancies, recurrence, protracted remission, etc. ³

This study will be conducted in the Department of Oral Pathology and Microbiology, Sharad Pawar Dental College in collaboration with Central Research Laboratory, Datta Meghe Institute of Higher Education & Reasearch, Sawangi (Meghe) Wardha.

Inclusion criteria •

Paraffin embedded tissue sections of well differentiated squamous cell carcinoma (WDSCC), moderately differentiated squamous cell carcinoma (MDSCC), poorly differentiated squamous cell carcinoma (PDSCC) histopathologically, their surgical margins and normal oral mucosa (NOM) will be retrieved from archival of department.

Exclusion criteria •

Patients who have a history of pre-malignant lesion, recurrent or distant metastasis and pre-operative chemotherapy, radiation therapy and the patients previously having undergone surgery for recurrence (except biopsy).

The present study will find the immunohistochemical expression of cyclin D1 tumor tissue and surgical margins of OSCC, in order to evaluate the prognosis of OSCC.

There will be a variation in the immunoexpression of Cyclin D1 among different grades of oral squamous cell carcinoma, its surgical margin and normal oral mucosa.

Considering the prevalence of OSCC as 70.7% in the outpatient department of Oral Pathology and Microbiology, using a single proportion formula, the sample size is calculated via the expression below. ¹¹ n ≥ Z 2 1 − α / 2   ∗ p ∗ 1 − p / d 2

Where,

Z ² _{1-α/2 -} The level of significance at 5 %

i.e. 95 % confidence interval =1.96

p - Sample demonstrating +ve Cycline D1 expressions in tiny groups of cells in the basal layer of the

epithelium = 70.7% = 0.707

E - Errors of margins = 10 % = 0.10

n = 1.96 ²x 0.707 x (1-0.707) /0.10 ²

n = 80

Formula reference - Angadi PV, Krishnapillai R. Cyclin D1 expression in OSCC and verrucous carcinoma: correlation with histological differentiation. OS, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology. 2007 Mar 1;103(3):e30-5

A paraffin block having suitable mass of tumor and acceptable amount of normal tissue is selected. On Poly-L-Lysinecoated slides, sections of having thickness of three micrometers are cut & placed. For De-paraffinization, sections are placed in the xylene solution. Sections are rehydrated by subjecting them to descending concentrations of alcohol (70-100%). In order to wash sections, tap water is used. Washing time for sections in distilled water is 60 seconds. After washing all the sections are transferred to Coplin’s jar containing the retrieval buffer solution. The solution which is used for Antigen retrieval is composed of 30 ml of retrieval solution in 1500 mili-liters of distilled water for fifteen to twenty minutes in the pressure cooker. Cooling is done at room temperature.

Sections are dipped once in distilled water. Sections are washed with Tris buffer solution for at least 5 minutes at room temperature. This step is repeated thrice. For peroxidase blocking a mixture of “3,5 Hydrogen Peroxide and Methanol” is used for thirty minutes. Tris buffer solution is used for washing the sections 3 times for 5 minutes each. Primary antibody cyclin D1 is applied at room temperature for 1 hour. Envision technique is performed by utilizing a labelled polymer for 30 minutes at room temperature. For washing the sections tris buffer solution is used thrice for five minutes.

The application of “DAB (3,3’-diaminobenzidine)” substrate is done for fifty to twenty minutes. The working DAB solution is comprised of following –one millilitre of DAB buffer & twenty five microliters of “DAB concentrate”. This time, washing of sections is done by Tris buffer, for 15-20 minutes. Sections are cleaned in distilled water. For counter staining Mayer’s hematoxylin is used, which is done for five minutes. Again, the washing of sections is done under tap water and then dried. Once the sections are dried; mounte in DPX. After that a microscopic examination is done. Calculations will be made to determine the median percentage per slide (labelling index) and the proportion of IHC positive tumour cells per hot spot. Labelling indices of 1-35%, 36-70%, and 71-100%, respectively, each received a score of 1, 2, or 3. According to their microscopic appearance, Cyclin D1 immunostaining intensity will be assigning each a score of 0, 1, 2, or 3 will indicate whether it is weak, intermediate, or strong. Cyclin D1 expression was classified as weak (scoring 1-4), moderate (score 5-8), or strong based on a final expression score that was obtained by multiplying the labelling index score by the intensity score (score 9–12). We will tabulate and do the necessary statistical analysis on all relevant clinical, histopathological, and immunohistochemical information that we will gather.

Will be published in an indexed journal.

Not started yet.

Ethical approval received from Datta Meghe Institute of Higher Education and Research, Sawangi, Wardha.

IEC reference number- DMIHER (DU)/IEC/2023/842.

Informed written consent was obtained from the patient before surgery and before the before the write-up of the protocol as per the data requirement of this study.