Ⅰ. INTRODUCTION
Vimentin, an intermediate filament protein, is generally expressed in mesenchymal cells1). However, malignant epithelial cells can also express vimentin through a process known as epithelial-to-mesenchymal transition (EMT), which promotes the invasion and metastasis of carcinoma cells2). Intriguingly, vimentin expression has been observed in the epithelial component of benign odontogenic tumors, including ameloblastoma3-5), adenomatoid odontogenic tumor (AOT)5-7), ameloblastic fibroma8), and ameloblastic fibro- odontoma9). It has been speculated that EMT, secretory function, or cellular differentiation similar to that seen in odontogenesis may be associated with the expression of the mesenchymal marker in benign neoplastic epithelial cells in odontogenic tumors3-6,9).
WNT pathway-altered odontogenic tumors (WNT-OTs) are a recently proposed, genetically distinct group of odontogenic tumors characterized by mutations in WNT pathway genes, such as CTNNB1 (encoding β-catenin) and APC10-12). Calcifying odontogenic cyst (COC), dentinogenic ghost cell tumor (DGCT), and adenoid ameloblastoma (AA), which can be classified as benign WNT-OTs in a molecular context, also share histologic features, including ghost cells, dentinoid, clear cells, and whorled cellular condensations called morules10,11). The immunohistochemical features of benign WNT-OTs have been investigated in several recent studies, mostly focusing on nuclear accumulation of β -catenin as a hallmark of WNT pathway activation and expression of keratins as diagnostic markers10,11,13). However, their immunoreactivity for vimentin remains unclear14,15).
This study aimed to analyze the immunohistochemical expression of vimentin in benign odontogenic tumors, including WNT-OTs, with a focus on its localization within the epithelial component of each tumor type.
Ⅱ. MATERIALS and METHODS
Tissue samples
A total of 28 benign odontogenic tumors consisting of 11 WNT-OTs, 12 ameloblastomas, and 5 AOTs were included in this study. The 11 WNT-OTs consisted of 7 COCs, 2 DGCTs, and 2AAs. The 12 ameloblastomas consisted of 7 conventional ameloblastomas (CAs) and 5 unicystic ameloblastomas (UAs). The 5 AOTs were all follicular type. Non-decalcified, formalin-fixed, paraffin-embedded tissues were retrieved from the pathology files of Seoul National University Dental Hospital between 2004 and 2019. All diagnoses were confirmed based on the essential diagnostic criteria described in the latest WHO classification16). This study was granted exemption by the Institutional Review Board of Seoul National University Dental Hospital (No. ERI22030).
Immunohistochemistry
Immunohistochemical staining was performed using a Leica BOND-MAX autostainer (Leica Biosystems) and an in vitro diagnostic antibody against vimentin (clone V9; ready-to-use; PA0640, Leica Biosystems). Endothelial cells were used as an internal positive control. For immunohistochemical evaluation, neoplastic epithelial cells showing cytoplasmic staining of any intensity were considered positive. Based on the percentage of positive neoplastic epithelial cells, slides were classified into negative (<1%), focally positive (1%-49%), and diffusely positive (≥ 50%).
Ⅲ. RESULTS
The immunohistochemical results of this study are summarized in Table 1.
Vimentin immunoreactivity in WNT-OT
All WNT-OTs studied (11/11; 100%) were focally positive for vimentin. Morules were generally negative for vimentin, whereas a few layers of cells surrounding the morular structures were positively stained (Fig. 1A and B). Clear cells adjacent to or within dentinoid were consistently positive for vimentin (Fig. 1C and D). Some ghost cells were positive for vimentin with varying degrees of intensity, while others were completely negative (Fig. 1E and F).
Vimentin immunoreactivity in ameloblastoma
Two thirds of ameloblastomas studied (8/12; 66.7%)–5/7 (71.4%) CAs and 3/5 (60.0%) UAs–were focally positive for vimentin. In CA, vimentin was more frequently expressed in peripheral cells than in central cells, regardless of histologic subtype (Fig. 2A and B). Similarly, in UA, the basal layer showed higher vimentin expression than the upper layers (Fig. 2C).
Vimentin immunoreactivity in AOT
All AOTs studied (5/5; 100%) were focally positive for vimentin. Rosette- or duct-like structures, the main histologic features of AOT, were negatively stained. However, anastomosing strands located at the periphery of nodules showed consistent immunoreactivity for vimentin (Fig. 3A and B). Some nodular cells around small foci of calcification were also positive for vimentin (Fig. 3C).
Ⅳ. DISCUSSION
In this study, vimentin immunoreactivity was identified in the epithelial component of several types of benign odontogenic tumors. In ameloblastoma, it has previously been shown that vimentin is expressed in peripheral columnar cells only or in both peripheral and central cells3,5). Similarly in this study, vimentin immunoreactivity was observed more frequently in peripheral cells in CA and in the basal layer in UA than in central cells in CA and in the upper layers in UA, respectively. This intratumoral heterogeneity may be attributable to cellular differentiation during tumorigenesis. Vimentin expression in ameloblastoma has been explained by EMT involvement3-5), which is supported by previous findings that E-cadherin transcriptional repressors, including Twist and Snail, are expressed in tumor cells in ameloblastoma 17). In AOT, vimentin was expressed in peripheral anastomosing strands and cells around foci of calcifications in this study, as has been reported previously5-7). These findings may be explained by tumor cells of varying differentiation (nodular cells vs. peripheral cells) or secretory activity of some tumor cells, such as cells around calcifications, in AOT6,7).
This study presents novel findings on vimentin immunoreactivity in WNT-OTs. Although morules have been recognized as a distinctive histologic feature of AA, it has recently been demonstrated that other WNT-OTs, including COC and DGCT, also often have the same structures10,11,13,18). In addition to WNT-OTs, some tumor types, including endometrial and ovarian tumors and adamantinomatous craniopharyngioma, histologically show morular structures, which are immunoreactive for several markers, including β-catenin (nuclear), SATB2, CDX2, and CD1019-21). In this study, cells forming morules were negative for vimentin in contrast to adjacent cells with vimentin expression, further providing the immunohistochemical characterization of these unique histologic structures.
Clear cells are observed in both solid (DGCT and AA) and, less frequently, cystic (COC) WNT-OTs, mainly in close proximity to dentin-like material called dentinoid10,11,18). It has been demonstrated that clear cells in WNT-OTs show nuclear translocation of β-catenin, indicating activation of the WNT pathway in these cells10,11). Considering that nuclear β-catenin is observed during tooth development and that canonical WNT pathway activation is associated with dentin formation22), nuclear accumulation of β-catenin in clear cells may be related to the formation of dentinoid located in their vicinity. In a similar context, the consistent expression of vimentin in clear cells demonstrated in this study might be attributed to their secretory function or inductive effect.
Ghost cells have been recognized as a useful diagnostic clue for WNT-OTs16). Several hypotheses for the formation of ghost cells have been proposed, including aberrant keratinization, accumulation of enamel matrix proteins, and coagulative necrosis23). Interestingly, vimentin expression was identified in a subset of ghost cells in this study, which may contradict the aberrant keratinization hypothesis since vimentin is a type of intermediate filament protein distinct from cytokeratin1). Considering previous reports that cytokeratin is also expressed to varying degrees in ghost cells24), further studies are needed to clearly explain the mechanism underlying the co-expression of different types of intermediate filaments.
In conclusion, vimentin expression in epithelial cells is a common feature of benign odontogenic tumors including WNT-OTs. The intratumoral localization of vimentin in WNT-OTs provides insights into the nature of their histologic components.
