Expression of the Embryonic Cancer Stem Cells' Biomarkers SOX2 and OCT3/4 in Oral Leukoplakias and Squamous Cell Carcinomas: A Preliminary Study

Abstract

Introduction: Cancer stem cells (CSCs) are incriminated for initiating the process of carcinogenesis either de novo or through the transformation of oral potentially malignant disorders (OPMDs) to oral squamous cell carcinoma (OSCC). The aim of this study was to detect the expression of embryonic-type CSC markers OCT3/4 and SOX2 in OSCCs and oral leukoplakias (OLs), the most common of OPMDs.

Materials and methods: The study type is experimental, and the study design is characterized as semiquantitative research, which belongs to the branch of experimental research. The experiment was conducted in the Department of Oral Medicine/Pathology, School of Dentistry, Aristotle University of Thessaloniki, Greece. This study focuses on the semiquantitative immunohistochemical (IHC) pattern of expression of CSCs protein-biomarkers SOX2 and OCT3/4, in paraffin embedded samples of 21 OSCCs of different grades of differentiation and 30 cases of OLs with different grades of dysplasia, compared to five cases of normal oral mucosa in both terms of cells' stain positivity and intensity. Statistical analysis was performed through SPSS 2017 Pearson Chi-square and the significance level was set at 0.05 (p=0.05). The expression of the respective genes of SOX2 and OCT3/4 was studied through quantitative polymerase chain reaction (qPCR), in paraffin-embedded samples of 12 cases of OLs with mild/non dysplasia and 19 cases moderately/poorly differentiated OSCCs(n=19) and five normal mucosa using the Independent Paired T-test.

Results: The genes SOX2 and Oct3/4 were expressed in all examined cases although no statistically significant correlations among normal, OL and OSCC, were established. A nuclear/membrane staining of OCT3/4 was noticed only in three out of 21 OSCCs but in none of OLs or normal cases (without statistical significance). A characteristic nuclear staining of SOX2 was noticed in the majority of the samples, mostly in the basal and parabasal layers of the epithelium. SOX2 was significantly detected in the OSCCs group (strong positivity in 17/21) than in the OL group (30 cases, mostly mildly stained) (p-value=0.007), and the normal oral epithelium (mild stained, p=0.065). Furthermore, SOX2 was overexpressed in well differentiated OSCCs group (5/OSCCs, strongly stained) rather than in mildly dysplastic and non-dysplastic OLs samples (14/OLs, mildly stained) (p-value =0.035).

Conclusion: The characteristic expression of SOX2 but not of OCT3/4 in OLs' and OSCCs' lesions suggests the presence of neoplastic cells with certain CSC characteristics whose implication in the early stages of oral tumorigenesis could be further evaluated. The clinical use of SOX2, as prognostic factor, requires further experimental evaluation in larger number of samples.

Keywords: cancer stem cells; oct3/4; oral cancers; oral leukoplakia; sox2.

Figure 1. OCT3/4 IHC staining

A) A cancerous nest in a moderately differentiated OSCC comprised of cancer cells positive to OCT3/4 (blue arrows) (Χ40); B) Cancerous foci are noticed, with cancer cells positive to OCT3/4 (blue arrows) in poorly differentiated OSCC (Χ40); C) Adjacent to moderately differentiated OSCC, the dysplastic epithelium exhibits the typical membrane staining of the interface area between the basal layer and the basal membrane  zone (green arrows) (Χ40). Nuclear staining is also noticed in individual cells (blue arrows); D) Adjacent to moderately differentiated OSCC, the dysplastic epithelium exhibits the typical membrane staining of the interface area between the basal epithelial layer and the basal membrane (blue arrows) (Χ40). Membrane staining is also noticed in individual cells at the parabasal layer (yellow arrows). OSSC: Oral squamous cell carcinoma; IHC: Immunohistochemical

Figure 2. SOX2 IHC staining

A) The nuclear staining of SOX 2 is noticed, mostly in the lower of the epithelium, in a case of non-dysplastic OL, characteristically, at the interface area between the basal epithelial layer and the basal membrane (blue arrows). Scattered, positively stained, cells are noticed also in the prickle layer (yellow arrows) (X40); B) The nuclear staining of SOX2 is noticed in the lower twp-thirds of the epithelium (blue bracket) in a case of moderately dysplastic OL (X20). Typical nuclear staining at the basal cells is noticed, at the interface area between the basal epithelial layer and the basal membrane (blue arrows); C) A well-differentiated OSCC manifests cancerous foci with small cells positive to SOX2 (blue arrows) (Χ20). Lack of staining is also noticed in scattered peripheral cancer cells (green arrows); D)  A cancerous focus is noticed with SOX2 positive CSCs (green arrows), among muscle cells, in a moderately differentiated OSCC (Χ40); E) The nuclear staining of SOX 2 (blue arrows) is sporadically noticed in the lower third of the epithelium (blue bracket), in one case of normal oral mucosa (X40). OL: Oral leukoplakia OSSC: ; IHC: Immunohistochemical; CSC: Cancer stem cell