Protocol for assessment and correlation of Brandwein-Gensler criteria with P53 immunoexpression in oral squamous cell carcinoma: A retrospective study

Background and rationale

The commonest type of malignancy in the head and neck region is oral squamous cell carcinoma (OSCC). It is also one of the top 10 malignancies in the world.¹ The median survival for OSCC, which is less than 50% in five years, has improved very little despite recent improvements in treatment methods.² The main reason for death in people with OSCC is loco-regional recurrence. Many factors, including the size of tumor, its grade, the negative resection margin, lymph node metastases, perineural invasion, etc., have an effect on local recurrence and survival. Brandwein provided a risk model that includes scores for the following three factors: worst invasion pattern (WPI), lymphocytic host response (LHR) and perineural invasion (PNI). This risk model accounts for overall survival and local recurrence.³ Low-, moderate-, and high-risk categories are determined by adding these scores.³

When neoplastic cells enter tissues at the tumor/host contact, this is referred to as having the worst pattern of invasion.⁴ The WPI is notably related to poor prognosis, metastases of the lymph nodes, and loco-regional recurrence rates.⁵ In the risk model, lymphocyte density at the tumour interface serves as a histological measure of the LHR. Strong, intermediate, or weak are the three categories.⁶ PNI is the development of cancerous cells inside one of the three layers of a nerve structure or the existence of tumour foci outside of the nerve that cover more than one-third of the nerve’s circumference.⁷ PNI has been labelled as an OSCC parameter with a high level of risk.⁷ PNI is additionally referred to as a predictor of survival and local recurrence.⁵

In the Brandwein-Gensler risk model, WPI with type 4 and 5 scores shows scattered neoplastic cells, which are masked by marked chronic inflammatory cells and are difficult to identify. p53 is considered to be one of the most significant prognostic markers.² p53 is a protein present in the nucleus of epithelial cells produced by a tumor suppressor gene known as TP53. It regulates cell division and apoptosis.⁸ p53 overexpression in OSCC is thought to be a sign of poor prognosis.² In this study, we determine if there is a correlation between the Brandwein-Gensler criteria and p53 immunoexpression in OSCC.

Objectives

Study design and setting

This is a retrospective cohort study, which will be carried out in the Department of Oral and Maxillofacial Pathology and Microbiology, Sharad Pawar Dental College and Hospital, Sawangi, Wardha, Maharashtra. Ethical approval was provided by the Institutional Ethical Committee, Datta Meghe Institute of Higher Education and Research, Sawangi, Wardha.

IEC reference number: DMIHER (DU)/IEC/2023/840. Date: 31/03/2023.

Eligibility criteria

Inclusion criteria

Exclusion criteria

Intervention

In this retrospective study, a total of 80 previously unused, randomly selected samples of histopathologically diagnosed OSCC from 2019 to 2022 will be retrieved from the archives of the Department of Oral and Maxillofacial Pathology and Microbiology, Sharad Pawar Dental College and Hospital in the form of paraffin blocks. Blocks will be cut and tissue sections will be taken on slides. Slides will be deparaffinized and rehydrated in distilled water. Mayers Hematoxylin staining will be carried out for one minute. The slides will be washed 4–5 times with tap water or until the stain has been removed. Counterstain for one minute in alcohol-eosin without rinsing. Dehydrate by going through three 95% EtOH changes and two 100% EtOH changes, each lasting one minute. Clear in three Xylene changes, lasting one minute each. Then mounting will be done. After processing and staining with H & E stain, slides will be evaluated under a microscope (OLYMPUS- CX21iFS1) to assess Brandwein-Gensler criteria (WPI, LHR and PNI). After evaluation of Brandwein-Gensler criteria, the cases will be studied for immunohistochemical expression of p53. This will be carried out by AJCC’s criteria and Worst pattern of invasion, perineural invasion and lymphocytic hot response will be evaluated. Brown nuclear staining in the cell will be examined for p53 immunoexpression and will be evaluated as per the following criteria: No staining (0), light staining (1+), medium staining (2+), and heavy staining (3+).² A focal positive is defined as less than 10% of tumor cells, which are intermediate to weakly stained, whereas a positive is defined as more than 10% of cancer cells, which are moderate to strongly stained.²

Data measurement

The risk scoring system is adapted from Ref. 3. The effectiveness of the Brandwein Gensler Risk Model, which combines three significant pathological variables, in forecasting the course of surgically treated OSCC, will be examined in this study. Worst invasion pattern, perineural invasion, and lymphocytic host response are these pathogenic variables.

In worst pattern of invasion:

In lymphocytic host response:

In perineural invasion:

Evaluation of risk for oral squamous cell carcinoma⁵:

Immunostaining

The DO-7 clone of the DAKO anti-human p53 protein and the DAKO EnVision method will be used for p53 immunohistochemistry. Blocks of paraffin-embedded tissue (fixed with formalin) will be divided into 4 mm thick tissue slices using a microtome. The tissue slices will be placed on “3-amino propyl triethoxy silane” (APES) glass slides. Slides should be deparaffinized in two 5-minute cycles of xylene or a xylene replacement. Slides should be transferred to 100% alcohol, changed twice for three minutes each, and then once through 95% alcohol for three minutes. For ten minutes, incubate slices in a 3% H₂O₂ solution in methanol to inhibit endogenous peroxidase activity. To perform antigen retrieval, the slides will be cooked in TRIS-EDTA for five minutes in a pressure cooker.

Endogenous peroxidase activity will be inhibited for ten minutes with 3% hydrogen peroxide. The UV block reagent will be applied to the slides for a duration of five minutes. Slides were incubated for one hour at room temperature (DO-1) with mouse monoclonal anti-p53 primary antibody (PAb 240), which is BSA-free (PathnSitu). Horse radish peroxidase will be utilized for 30 minutes after the secondary antibody. Three′-diaminobenzidine tetrahydrochloride will be the substrate chromogen used for staining, and Harris Hematoxylin will be used for counterstaining. The sections will be dehydrated, cleaned, mounted, and then examined under an Olympus CX21iFS1 microscope.

Both quantitatively and subjectively, nuclear staining for p53 is assessed. p53 immunoexpression will be evaluated as per following criteria: Absence of staining (0), light staining (1+), medium staining (2+), and heavy staining (3+) present.² A focal positive is defined as less than 10% of tumor cells which are intermediate to weakly stained, whereas a positive is defined as more than 10% of cancer cells which are moderate to strongly stained.

Dissemination

Once complete the study results will be published in an indexed journal.

Study status

The study will be starting in January 2024.