Immunohistochemical Expression of DAPK-1 in Oral Leukoplakia And Oral Squamous Cell Carcinoma: A Preliminary Study

Abstract

Introduction: The silencing of death-associated protein kinase 1 (DAPK-1) is an effective way of inactivating a tumor-suppressing mechanism. The aim of this study was to investigate the immunohistochemical expression of DAPK-1 in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC).

Methods: The immunohistochemical (IHC) detection of DAPK-1 was carried out in cases of OLs and OSCCs. DAPK-1 molecules' tissue distribution in OLs/OSCCs tissues was evaluated using semiquantitative immunohistochemistry in representative paraffin-embedded tissue samples (57 in total) from 2004-2019, retrieved from the archives of the Department of Oral Medicine/Pathology, School of Dentistry, Aristotle University of Thessaloniki, Greece and the St Lukas Hospital of Thessaloniki, Greece. The inclusion criterion was the presence of sufficient precancerous or cancerous biological material (estimated as more than 70% per tissue specimen) in the paraffin cubes. The exclusion criterion was the opposite, i.e. the lack of sufficient material due to previous sections. Statistics for IHC were evaluated by a non-parametric Mann-Whitney U Test. A two-sided p-value < 0.05 was considered statistically significant.

Results: DAPK-1 IHC expression was increased in OLs without dysplasia and with OLs with mild dysplasia compared to moderate/severe dysplasia (p=0.019, Mann-Whitney U Test) and OSCCs (p=0.003, Mann-Whitney U Test). Conclusions: DAPK-1 seemed to function as an oncosuppressor molecular biomarker, as its expression was decreased in areas of cellular dysplasia in OLs and in areas of OSCCs composed of less differentiated cells. The clinical application of this biomarker is that the positively stained, potentially malignant lesions are less likely to transition into malignancy, and cancerous lesions are more likely to behave non-aggressively. On the other hand, the lack of staining could signify the loss of this oncosuppressing ability, and it could be a potential prognostic biomarker for OSCC's aggressive biologic behavior if considered with other clinical parameters and a prognostic factor of malignant transformation of potentially malignant lesions. Since this is a preliminary study, more studies with larger sample sizes are required to support these conclusions.

Keywords: cancer biomarkers; cancer stem cells; dapk-1; immunohistochemistry ihc; oral leukoplakia; oral squamous cell carcinoma.

Figure 1. A: Overexpression of DAPK-1 in normal epithelium: cytoplasmic staining with only dispersed cells with nuclear staining (X40). B: Characteristic intense staining (Grade III) of all layers of epithelium, except the basal one in a case of OL without dysplasia (x20). C: Characteristic intense cytoplasmic expression of DAPK-1 in epithelial cells of OL without dysplasia (x40).

Figure 2. Α: Intense cytoplasmic expression of DAPK-1 in OL with mild dysplasia only in epithelial layers without dysplasia (most of spinous and granular) and absence in the lower 1/3 of ep-ithelium. Furthermore, it was observed a distinct staining in the interface between basal cells and basal membrane (x20) Β: Intense cytoplasmic staining of epithelial cells in a OL with mild dysplasia. Focal area of unstained cells among the intensively stained ones of the spinous layer (x40).

Figure 3. A: Positive staining of DAPK-1 is established in dispersed areas of the spinous layer and the full thickness of the granular one in a tissue section of moderate OL (X10), B: Positively stained epithelial cells in the granular layer showing nuclear as well as cytoplasmic expression of DAPK-1. Positive expression of DAPK-1 in the intercellular space of the spinous layer and few positively stained disperse cells in the upper part of it (X40). C: The expression of DAPK-1 in OL with severe dysplasia is limited to the granular layer (x10).

Figure 4. A: Dispersed epithelial islands of distinct positivity. Absence of staining in the epithelium in all layers but not the upper part of spinous and the granular layers where there is no dysplasia above neoplastic epithelial islands in a moderately differentiated case of OSCC (X20), B: Epithelial islands in a moderate differentiated OSCC case in which the more “differentiated” neoplastic cells in the center of the neoplastic islands show intense positive staining. Staining is either cytoplasmic or rarely nuclear (x20). C: Keratin pearl, in a case of well-differentiated OSCC, surrounded by DAPK-1 highly stained neoplastic epithelial cells as well as in the center of the neoplastic epithelial island. Staining is mainly cytoplasmic and, in some cases, membranous (x40).