Material and Method: Nine solid ameloblastomas , 12 unicystic ameloblastomas, 13 odontogenic keratocyst and 12 dentigerous cyst were assessed in this study. The slides were examined at x400 magnification. Finally the lesions were divided into two groups based on microscopic examination, “low expression” and “high expression”.

Results: There were no significant differences between the lesions, except that fascin expression was slightly higher in unicystic ameloblastomas in comparison to other groups in intensity and count of the immunostaining cells.

Conclusion: The results of this study suggest that local aggressiveness does not result in fascin expression. We suggest more studies with more samples, assessing expression of different proteins be done in the future.

Epithelial cell junctions are mostly dependent on cellcell or cell-matrix interactions which help to stabilize the epithelial cells and prevent their migration. In epithelial malignancies, invasion to basal lamina and metastasis occurs due to elimination of these junctions. Fascin is considered in the migration and invasion of carcinoma cells since this actin-cross-linking protein is known to be responsible for controlling the guiding system of cell migration.

Many studies have reported the upregulation of fascin expression in various tumors. Moreover it has been reported that high expression of fascin is associated with high motility of cells, poor differentiation, advanced grade and stage and metastasis ^11,12. Fascin helps to stabilize the actin bundles in protrusions and it therefore seems to be used by cancerous cells to assemble stable and durable invasive protrusions (called invadopodia) that help invasion into the matrix ^13,14.

Ameloblastoma is slow growing and locally aggressive odontogenic epithelial tumor with a low tendency to metastasize and a high recurrence rate ¹⁵.

Odontogenic keratocyst (OKC), formerly known as keratocystic odontogenic tumor (KCOT), is a common odontogenic cyst that originates from the dental lamina or from its residue; occurring at any age, and is more common in men than women with a 2:1 ratio ¹⁶). Because of the high proliferation rate, aggressiveness and recurrence rate of epithelial cells of this cyst, in 2005 WHO reclassified odontogenic keratocyst as benign intra-osseous neoplasia, called KCOT ¹⁷. However, this odontogenic lesion is now listed as odontogenic keratocyst in the 2017 classification of developmental odontogenic cysts ¹⁶.

Dentigerous cyst (DC) is the second most common odontogenic cyst (following radicular cyst) which is typically seen as a radiolucency surrounding the crown of an impacted tooth. DC is not as aggressive as ameloblastoma or OKC ¹⁸.

In this article, we assessed and compared fascin expression in 4 lesions which differ in aggressiveness: OKC, two types of ameloblastoma (solid -which is believed to be more aggressive- and Unicystic) and DC to find out whether the fascin expression is associated with aggressiveness of these lesions or not.

Thus, 46 paraffin blocks including 9 solid ameloblastomas (SA), 12 unicystic ameloblastomas (UA), 13 OKCs and 12 DCs were assessed in this study.

After obtaining sections with a thickness of 4μm, immunohistochemical staining was carried out with the monoclonal anti-fascin antibody (Clone 55K-2 Dako, Glostrup, Denmark) diluted 1:50. For each case, a section with the primary antibody omitted was used as negative control and the cytoplasm of endothelial cells was used as internal positive control.

In microscopic assessment, the slides were examined at x400 magnification (Figure 1A-F) by an oral and maxillofacial pathologist who was blinded to the clinical characteristics of the samples. Immunoexpression of fascin in tumor cells was evaluated in terms of extent and intensity. The extent of immunostaining was scored according to Terence K. Lee ¹⁹: 1-negative: less than 10% of cells, 2-weak: 11-50% of cells, 3-moderate: 51-80% of cells and 4-strong: more that 81% of cells. Moreover, the staining intensity of the samples was categorized according to the cytoplasmic staining of cells into: 1-faint, 2-moderate and 3-severe. Finally, to determine the expression level of fascin, a Combined Immunoreactivity Score (CIS) was calculated by adding the score of extent to the score of intensity for each case. The samples were further classified into 4 groups: Negative expression: staining in less than 10% of cells regardless their staining intensity, weak expression: CIS equal to 3, moderate expression: CIS equal to 4 or 5, strong expression: CIS equal to 6 or 7.

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Figure 1: A) High expression of fascin in basal and suprabasal layers in odontogenic keratocyst. Note immunoactivity of stomal fibroblasts and endothelial cells as positive internal control (IHC; x100). B) Low expression of fascin only in basal layer in odontogenic keratocyst (IHC; x100). C) High expression of fascin in basal and suprabasal stratum reticulum layers in unicyctic ameloblastoma (IHC; x400). D) Low expression of fascin in unicyctic ameloblastoma. Note immunoactivity of stromal fibroblasts and endothelial cells even in low epithelial lining reaction (IHC; x100). E) High expression of fascin in dentigerous cyst (IHC; x100). F) Low expression of fascin in dentigerous cyst (IHC; x100).

In statistical analyses, the first 2 groups were merged as the “low expression” group, and the groups 3 and 4 merged as the “high expression” group. Research data were analyzed using the Statistical Package for Social Sciences (SPSS), version 18. The Kruskal-Wallis and two by two Dunn Tests were used. P values less than 0.05 were considered significant.

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Table I: Scores of stained-cell counts, staining intensities and group classifications based on immunohistochemical assessments

In order to assess aggressiveness of lesions, some studies have examined the extra cellular matrix and connective tissue proteins such as fibronectin, laminin, tenascin, RANK, RANKL, and OPG in these lesions. Many of them found significant differences in the expression levels of these proteins among DC, ameloblastoma and OKC ^24-27.

All mentioned markers are exclusively expressed in connective tissue cells except laminin which is expressed in epithelial tissue. In this study we assessed fascin, as another epithelial cell marker. Fascin is also known as the protein of motility ^11,12. No wonder that expression of fascin can be associated with aggressive and invasive behaviors ^28,29 as seen in many epithelial cancers ¹³. Fascin is believed to be a biomarker that indicates aggressiveness and migration of tumors. However, our results contradict this hypothesis as we observed no significant upregulation of fascin in aggressive lesions. In addition, one meta-analysis study demonstrated that fascin may have potential as a novel biomarker for early identification of aggressive and metastatic tumors ¹². In our study, the only statistically significant difference among the four lesions was the increased fascin expression in unicystic ameloblastoma in comparison to the other three. There is a possibility that fascin might be more expressed in carcinomas than cyst walls or odontogenic tumors. Also since the lesions of this study were mostly intra-bony, there were no epithelial barriers to their progression. Moreover these lesions do not metastasize. Considering these, we suggest that fascin might be more involved in cell invasion and migration (as in carcinomas) than local aggressiveness. Lee et al. suggested that fascin over-expression might enhance oral squamous cell carcinoma aggressiveness, possibly by interacting with E-cadherin expression ¹⁹. There is no E-cadherin interaction in progression of odontogenic lesion versus progression of carcinomas. Therefore, according to Lee theory ^19, our suggestion based on more involvement of fascin in invasion and migration than local aggressiveness in benign odontogenic lesion is justifiable. Furthermore, Li et al. showed that fascin is an integral component of invadopodia -the finger-like protrusions using by cancer cells which help them invade into and degrade extracellular matrix- and it is important for the stability of actin in invadopodia ²⁸. Epithelial cells of benign odontogenic lesions like DC, OKC or ameloblastoma that we assessed in this study do not use invadopodia for progression, and this may be a reason for less involvement of fascin in local aggressiveness.

It is accepted that immunohistochemistry is a complex metric due to the use of different scoring systems to assess the extent of staining in different specimens but the scoring of fascin is a continuous measurement and in most publications researchers categorized specimens into high/positive or low/negative fascin based on different semi-quantitatively assessed cut-off points ¹². We also assessed the fascin expression in two groups of low and high expression as in most previous publications. However, this study has several limitations. First, we did not assess whether these lesions had perforated the cortical bone and invaded local soft tissue. Second, we just examined fascin expression and not other proteins or markers. We suggest more studies with more samples, and assessing expression of different proteins in the future.

CONFLICT of INTEREST
The authors declare no conflict of interest.

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