Protocol for evaluation and correlation of CD44 immunoexpression in tumor tissue and surgical margins of oral squamous cell carcinoma with three-year survival: A retrospective study

Background and rationale

The commonest type of cancer in head and neck region is oral squamous cell carcinoma (OSCC), due to its high rates of occurrence and mortality. More than 90% of cases of oral and oropharyngeal cancer are OSCC cases.¹ Although OSCC treatment techniques (surgery, radiation, chemotherapy) have improved, survival has not. The overall five-year survival rate is less than 50% globally.²

The squamous epithelia inside the mouth give rise to OSCC. According to the cancer stem cells hypothesis, a tumor’s core contains a subpopulation of multipotent cells that cause tumor differentiation, continuation, and its metastasis to other places. Cancer stem cells (CSCs) are thought to be resistant to conventional therapy and are capable of producing new tumor cells that genetically resembles the original tumor. The ability of CSCs to regenerate themselves causes recurrence of disease. We must find and eliminate these self-renewing cells in order to improve survival.

Identification of these cells is done by detecting expression of CSCs markers which are present either on their surfaces or within the cells. There are various cancer stem cell markers such as CD44, CD133, L1CAM and SOX2.

Cluster of differentiation 44 (CD44) is a surface glycoprotein present on tumor cells. It regulates cell proliferation, adhesion, migration and invasion of cancer stem cell. Various studies have reported that CD44 immunoexpression is an independent predictor for prognosis.^3–5

In order to improve prognosis, surgical removal of tumor should be done with adequate negative surgical margins. Immunoexpression of molecular markers in surgical margins indicates poor prognosis of disease and increased risk of local recurrence.⁶

According to a literature search, no study has been done on CD44 expression in surgical margins of OSCC; therefore, we aim to assess immunoexpression of CD44 in surgical margins of OSCC, in order to evaluate its relationship with histologic types and overall survival.

Trial design

Retrospective cohort study.

Study setting

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 and Research, Sawangi (Meghe) Wardha.

Eligibility criteria

The inclusion criteria of this study will comprise of 80 surgically treated patients who have been clinically and histopathologically diagnosed with OSCC. Follow-up data will be kept in record for disease-free survival for three years before the study starts. The cases that will be excluded from the studies who previously underwent surgery for recurrence, had a history of oral cancer, recurring or distant disease and underwent preoperative chemotherapy, or radiation therapy.

Intervention

Paraffin-embedded tissue blocks having suitable mass of tumor and acceptable amount of normal tissue will be cut into 4-μm thick tissue pieces. All tissue sections will be processed for CD44 immunohistochemistry. After that, light microscopic evaluation of CD44 expression will be done. A dark brown color of membranous staining of neoplastic cells will be evaluated for CD44 positivity which have to be observed in the tumor core, in the basal layer, because most of stem cells are present in the basal layer of oral mucosa and in the invasive front of tumor.

Outcomes

Primary outcome

The present study will measure the immunohistochemical expression of CD44 tumor tissue and surgical margins of OSCC, in order to evaluate the prognosis of OSCC.

Secondary outcome

Immunoexpression of CD44 may vary in different grades of OSCC and accordingly affect the overall and disease-free survival.

Sample size

Considering the prevalence of OSCC as 70.7% in the outpatient department of Oral Pathology and Microbiology, Sharad Panwar Dental College and Hospital, using the single proportion formula (Angadi et al., 2007), the sample size is calculated by applying the formula:

Where

Z²_{1-α/2 -} The significance level at five (5)%

i.e. 95% confidence interval = 1.96

p - Sample showing positive CD 44 expression focally in small group cells in the basal layer of epithelium = 70.7% = 0.707

$\mathrm{E}-\text{Error of margin}=10\%=0.10$

$\mathrm{n}={1.96}^2\times 0.707\times \left(1-0.707\right)/{0.10}^2$

n = 80

Article reference: 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 Mar;103(3):e30-5. doi: 10.1016/j.tripleo.2006.09.011. Epub 2007 Jan 2. PMID: 17197212.⁷

Immunostaining

Paraffin-embedded tissue blocks will be cut into 4 μm thick tissue pieces (fixed with formalin). They will be transferred to glass slides made of 3-amino propyl triethoxy silane (APES). The slides will be deparaffinized with xylene and rehydrated with reduced ethanol concentrations. The slides will be heated in TRIS-EDTA for five minutes in a pressure boiler to perform antigen retrieval. With 3% H₂O₂, endogenous peroxidase activity will be blocked for 10 minutes. The plates will be exposed to ultraviolet block reagent for five minutes. Slides will be incubated with the main CD44 antibody for one hour at room temperature. After that, the secondary antibody will be added for 30 minutes with horse radish peroxidase. 3′-diaminobenzidine tetrahydrochloride will be used as the substrate chromogen for staining, and Harris Hematoxylin will be used for counterstaining. Sections will first be dehydrated, then cleared, mounted, and finally observed under a light microscope.

Dissemination

The resulting research article will be published in an indexed journal.

Study status

The study has not started yet.

Ethical considerations

Ethical approval received from Datta Meghe Institute of Higher Education and Research, Sawangi, Wardha (IEC reference number DMIHER (DU)/IEC/2023/841).