Is podoplanin expression associated with the proliferative activity of ameloblastomas?

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1 ORIGINAL ARTICLE Is podoplanin expression associated with the proliferative activity of ameloblastomas? KC Tjioe 1, DT Oliveira 1, CT Soares 2, JRP Lauris 3, JH Damante 1 (2012) 18, doi: /j x Ó 2012 John Wiley & Sons A/S All rights reserved 1 Department of Stomatology, Bauru School of Dentistry, University of Sa o Paulo, Bauru, Sa o Paulo; 2 Department of Pathology, Lauro de Souza Lima Institute, Bauru, Sa o Paulo; 3 Department of Community Dentistry, Bauru School of Dentistry, University of Sa o Paulo, Bauru, Sa o Paulo, Brazil OBJECTIVES: The aim of this study was to investigate the relationship between podoplanin expression and proliferative activity of ameloblastomas and remnants of the odontogenic epithelium from dental follicles (DF) of unerupted teeth. SUBJECTS AND METHODS: Thirty-three paraffinembedded ameloblastomas and thirty-two DF obtained of unerupted teeth were analyzed by immunohistochemistry using anti-human podoplanin and anti-ki-67 antibodies. Podoplanin expression in odontogenic epithelial cells was evaluated using a scoring method, and the Ki-67 labeling index was determined by the percentage of positive odontogenic cells. RESULTS: All ameloblastomas displayed podoplanin expression in ameloblast-like cells of the epithelial islands. Membranous expression of podoplanin in ameloblastomas was stronger than in the remnants of odontogenic epithelium (P = 0.001). Statistically significant difference was observed between the cytoplasmic and membranous expression of podoplanin in the remnants of odontogenic epithelium (P = 0.001). The index of epithelial odontogenic proliferative activity, verified by Ki-67 expression, was higher in ameloblastomas vs remnants of odontogenic epithelium (P < 0.001). No statistically significant correlation was identified between podoplanin and the cellular odontogenic proliferative activity in ameloblastomas and DF (P > 0.05). CONCLUSIONS: These results provide evidence that there is no connection between podoplanin immunostaining and odontogenic cellular proliferative activity and suggest a role for membranous podoplanin expression in the local invasion of ameloblastomas. (2012) 18, Correspondence: Kellen Cristine Tjioe, DDS, MSc, PhD Student, Department of Stomatology, Area of Pathology, Bauru School of Dentistry, Alameda Octávio Pinheiro Brisolla, 9-75 CEP , Bauru, São Paulo, Brazil. Tel: , Fax: , kellentjioe@yahoo.com.br Received 4 December 2011; revised 8 February 2012; accepted 20 February 2012 Keywords: ameloblastoma; podoplanin; dental follicle; odontogenic tumor; Ki-67 Introduction Podoplanin is a transmembrane glycoprotein that has been largely used as an identifying marker of the lymphatic endothelium (Schacht et al, 2003). Published findings indicate that this protein has a relatively broad spectrum of reactivity including its expression in a wide variety of benign and malignant oral tumors (Schacht et al, 2005; Yuan et al, 2006; Gonza lez-alva et al, 2009, 2011; Ito et al, 2009; Okamoto et al, 2009; Vormittag et al, 2009; Kreppel et al, 2010; Zustin et al, 2010; Friedrich and Zustin, 2011). In oral squamous cell carcinoma, the strong expression of podoplanin by malignant cells was associated with lymph node metastasis and poor clinical outcome (Schacht et al, 2005; Yuan et al, 2006; Wicki and Christofori, 2007; Ito et al, 2009; Vormittag et al, 2009; Kreppel et al, 2010). The expression of podoplanin in normal and tumorous odontogenic cells is a recent topic of study. During the murine odontogenesis, pre-ameloblasts exhibit a strong immunoreaction for podoplanin; however, once the pre-ameloblasts differentiate into secreting ameloblasts, podoplanin expression decreases drastically (Sawa et al, 2008; Imaizumi et al, 2010). In odontogenic tumors, such as ameloblastomas (AMs) (Gonza lez-alva et al, 2009; Zustin et al, 2010), keratocystic odontogenic tumors (Okamoto et al, 2009; Zustin et al, 2010), adenomatoid odontogenic tumors (Zustin et al, 2010), odontomas (Gonza lez-alva et al, 2011), and calcifying epithelial odontogenic tumors (Friedrich and Zustin, 2011), the expression of podoplanin is predominantly observed in the epithelial odontogenic cells in the peripheral areas of the tumor. In contrast, non-secreting ameloblasts from the odontoma and less active cells, such as the upper epithelial lining layers of keratocystic odontogenic tumors and ghost cells from calcifying epithelial odontogenic tumors, are

2 674 negative for podoplanin (Zustin et al, 2010; Friedrich and Zustin, 2011; Gonza lez-alva et al, 2011). In benign odontogenic cysts, such as dentigerous cysts (Gonza lez-alva et al, 2009; Zustin et al, 2010) and odontogenic orthokeratinized cysts (Okamoto et al, 2009), the epithelial cells do not express podoplanin except when associated with inflammatory infiltrate (Okamoto et al, 2009). The reason why podoplanin expression is enhanced during the times of high cellular activity and inflammatory response remains unknown (Okamoto et al, 2009). Sawa et al suggested that this protein may be associated with ectodermal cell proliferation. Interestingly, both mitotic activity and podoplanin expression within the ameloblastoma are coincident (i.e., virtually restricted to the peripheral epithelial cells of the tumor cords and strands) (Sandra et al, 2001). Therefore, this study was designed to determine the potential role of podoplanin in benign tumor progression by attempting to identify an association between podoplanin and Ki-67 expression in odontogenic epithelial cells from ameloblastomas and dental follicles (DF). Subjects and methods All paraffin-embedded tissue specimens from both ameloblastomas and DF collected between 1980 and 2009 were retrieved from the Laboratory of Pathology in the Department of Stomatology of Bauru School of Dentistry, University of Sa o Paulo. The study population included 71 ameloblastomas and 182 DF. The inclusion criteria were as follows: (i) patients with microscopically confirmed diagnoses of solid multicystic ameloblastoma or unicystic ameloblastoma determined by the sum of the clinical, radiographic, and microscopic data; (ii) DF of patients with asymptomatic, unerupted, and non-inflamed teeth confirmed both clinically and microscopically; (iii) presence of at least 200 remnants of dental lamina and or enamel reduced epithelium cells; and (iv) the availability of the paraffin block with a sufficient amount of tissue for microscopic analysis. The samples that met the inclusion criteria consisted of 33 ameloblastomas and 32 DF. These samples were analyzed by immunohistochemistry for both podoplanin and Ki-67 expressions. The diagnosis of ameloblastoma was reviewed according to the World Health Organization histological classification of odontogenic tumors (Barnes et al, 2005). This study was approved by the Research Ethics Committee of the Bauru School of Dentistry, University of Sa o Paulo (process number ). Immunohistochemistry Formalin-fixed 4 lm sections of ameloblastomas and DF of unerupted teeth were obtained from the pathology archive and subjected to immunohistochemistry analysis using anti-podoplanin and anti-ki-67 antibodies to determine their expression in odontogenic cells. After antigen retrieval using 10 mm citrate buffer, ph 6.0, in a domestic pressure cooker (model Eterna 4½ L; Nigro, Araraquara, Brazil) for 4 min, endogenous peroxidase activity was blocked by incubating in 3% H 2 O 2 for 20 min. Each ameloblastoma or dental follicle section was incubated overnight at 4 C in a 1:200 dilution of the primary monoclonal anti-podoplanin antibody (D2-40 clone, code#3619-1; Dako North America, Inc., Carpinteria, CA, USA) or a 1:50 dilution of the anti-ki-67 antibody (MIB-1 clone, code#7240-1; Dako North America, Inc.). Primary antibody dilutions were made in a phosphate-buffered saline (PBS) bovine serum albumin (code#a2153; Sigma, Co., St Louis, MO, USA) solution to block non-specific reactions. After the primary antibody incubation, each section was incubated using the Advance HRP Link System (code #K4067; Dako North America, Inc.) for 30 min at 37 C. Both antibodies (podoplanin and Ki-67) were detected using 3.3 diaminobenzidine tetrahydrochloride (cod# D-5637; Sigma). Sections were counterstained with Mayer s hematoxylin before being dehydrated and cover slipped. Palatine tonsils were used as the positive control for both antibodies. The primary antibody was omitted during immunohistochemical staining for the negative control. Immunostaining evaluation For the evaluation of cytoplasmic and or membranous podoplanin expression by epithelial odontogenic cells, 20 fields in each ameloblastoma or dental follicle (magnification 400 ) were digitally captured using an Axiocam camera (Axiocam MR3; Zeiss, Jena, Germany) attached to a light microscope (Axioskop 2 Plus; Zeiss) and recorded by Axiovision software (Axiovision 4.6; Zeiss). A semi-quantitative method was used to score the podoplanin expression in the epithelial odontogenic cells, as previously described (Soini et al, 2001). Scoring was based on (a) the intensity of the immunostaining in the epithelial odontogenic cells (0 = absent, 1 = weak, 2 = moderate, 3 = strong, and 4 = very strong) and (b) the percentage of positive odontogenic cells (0 = 0% positive cells, 1 = <25% positive cells, 2 = 25 50% positive cells, 3 = 50 75% positive cells, and 4 = >75% positive cells). The final immunostaining score was determined by the sum of (a) + (b). Final scores ranged from 0 to 8 (0 = absent, 1 4 = weak, and 5 8 = strong). Membranous and cytoplasmic podoplanin expressions in the epithelial cells of ameloblastomas and DF were analyzed separately. For Ki-67 staining, the labeling index (LI) (number of positive epithelial cells total cells 100) was determined. At least 500 outer layer epithelial cells in AMs and all epithelial remnant cells in DFs were counted. Fields of both ameloblastomas and DF were captured in the region in intimate contact with the connective tissue. Statistical analysis The Mann Whitney test was used to compare the independent groups, and the Wilcoxon test was used to compare the dependent groups. The correlation between podoplanin and Ki-67 expression was calculated using Spearman s correlation coefficient. The level of significance was set at 5% for all tests.

3 Results Clinical features The age of patients with ameloblastoma ranged from 9 to 68 years (mean of 34 years). A predominance of women (75.8%) was observed in relation to men (24.2%). The most common location of the tumor was the posterior mandible (15 cases) followed by concomitant posterior and anterior mandible (four cases), anterior mandible (four cases), anterior maxilla (three cases), and posterior maxilla (one case). The location of the ameloblastomas remained unknown in six patients. The distribution of tumors according to the histopathological type is listed in Table 1. Seventeen male and 15 female patients comprised the samples of DF from the unerupted teeth selected for this study. Their ages ranged from 9 to 32 years (mean of 17.6 years). The vast majority of DF, 84%, were from posterior teeth, 12.5% were from anterior teeth, and 9.4% did not have this information available. Podoplanin expression in odontogenic epithelial cells from ameloblastomas and dental follicles The expression of podoplanin in follicular ameloblastomas was predominantly observed in the peripheral columnar cells of tumoral islands, while the reticulum stellate-like cells exhibited virtually no immunostaining (Figure 1a). The outer cells of the plexiform AMs expressed podoplanin based on immunohistochemistry staining. In a few specimens, its expression reached the central cells. The expression of podoplanin was quite variable in the unicystic AMs. Its positivity was restricted to the basal and supra-basal layers of the cystic lining in some tumors (Figure 1b) and spread throughout the extension of the tumoral epithelium in others. Focal areas of granular cells were encountered in two AM specimens (Figure 1c), and the acanthomatous areas did not express podoplanin (Figure 1d). Membranous and cytoplasmic immunostaining of podoplanin were analyzed in the peripheral cells of cords, strands, and islands of ameloblastomas, and the results can be seen in Table 2. The difference between the membranous and cytoplasmic expression of podoplanin in ameloblastomas was not statistically significant (P = 0.130). The vast majority of central cells from the islands of ameloblastomas displayed absent or weak immunoreactivity for podoplanin (71.9%) (Figure 1a), while in 28.1% of tumors, it was strong. One case did not contain enough central cells for analysis. Table 1 Distribution of ameloblastomas according to the histopathologic type (Barnes et al, 2005) Type N % Follicular Plexiform Unicystic (luminal) Unicystic (mural) Mixed 1 3 Total The expression of podoplanin in the odontogenic epithelial cells of DF was observed predominantly in areas in contact with the ectomesenchymal tissue. In the remnants of the odontogenic epithelium island, the peripheral epithelial cells were those predominantly immunostained for podoplanin (Figure 2a). Likewise, the layer composed by the compaction of the outer epithelium, stratum intermedium, and stellate reticulum was positive for podoplanin, while the reduced ameloblasts were negative (Figure 2b). The percentage of cells expressing cytoplasmic and membranous podoplanin in the remnants of odontogenic epithelium can be found in Table 3. The membranous expression of podoplanin was higher than the cytoplasmic expression of this protein in the remnants of the odontogenic epithelium within the DF (P = 0.001). Membranous expression of podoplanin was more frequent in ameloblastomas than in the DF (P = 0.001); however, cytoplasmic podoplanin staining was not significantly different between AMs and DFs (P = 0.833). Podoplanin expression and proliferative activity The proliferative activity, visualized by Ki-67 staining, was observed mainly in the peripheral layer of the epithelial islands, the cystic lining of unicystic ameloblastomas (Figure 3a), and in the DF s epithelial odontogenic remnants. The mean LI was 2.4% for ameloblastomas (range, %) and 0.4% for DF (range, %). Based on the median of LI, the AMs and DFs were divided into three groups: absent, 4%, and >4% staining of the odontogenic epithelial proliferating cells. About 21.2%, 48.5%, and 30.3% of ameloblastomas displayed absent, 4%, and >4% Ki-67-positive staining of the odontogenic epithelial cells, respectively. Conversely, 78.1% of DF did not exhibit proliferative activity, 15.6% displayed 4% (Figure 3b), and 6.3% had >4% proliferating cells. The mitotic activity was statistically higher in AMs than in DFs (P < 0.001). There was no statistically significant correlation between cytoplasmic (r = )0.15 and P = 0.396) and membranous (r = 0.00, P = 0.989) expression of podoplanin and Ki-67 in ameloblastomas (Table 4). Similarly, no statistically significant correlation was identified between the cytoplasmic (r = 0.15 and P = 0.421) and membranous (r = )0.09 and P = 0.629) expression of podoplanin and Ki-67 in the DF (Table 4). Discussion The expression of podoplanin has been observed in some odontogenic tissues and tumors (Sawa et al, 2008; Gonza lez-alva et al, 2009, 2011; Okamoto et al, 2009; Imaizumi et al, 2010; Zustin et al, 2010; Friedrich and Zustin, 2011); however, the exact role of this protein and the significance of its expression in odontogenic cells are still unclear. Here, all ameloblastomas displayed membranous and or cytoplasmic expression of podoplanin, and its 675

4 676 (a) (b) (c) (d) Figure 1 Immunohistochemical reactivity for podoplanin in (a) follicular ameloblastoma, (b) unicystic ameloblastoma, (c) granular cells in ameloblastoma, and (d) acanthomatous ameloblastoma. Podoplanin expression is positive in the outer cells of the odontogenic epithelial islands (a, c, d) and negative in (a) the reticulum stellate-like, (b) granular, and (c) acanthomatous cells of ameloblastomas Table 2 Membranous and cytoplasmic expression of podoplanin by the odontogenic epithelial peripheral cells in ameloblastomas Podoplanin Cytoplasmic Membranous N % N % Absent P = ns Weak Strong Total ns, no statistically significant. P value determined by Wilcoxon signed-rank test. expression was consistent with the cell type and tumor location. The epithelial tumoral cells in intimate contact with the stroma were stained strongly positive for podoplanin (Figures 1a,b,d), while the central cells of the islands were negative (Figure 1a). These results corroborated the previous reports of Gonza lez-alva et al (2009) and Zustin et al (2010). Interestingly, a similar pattern of podoplanin expression in the odontogenic epithelium was found in murine developing teeth where pre-ameloblasts strongly express podoplanin until they differentiate into mature ameloblasts, at which time the podoplanin immunoreactivity decreases (Sawa et al, 2008; Imaizumi et al, 2010). Moreover, in odontomas, the secreting ameloblast-like cells display a strong podoplanin immunoreaction, whereas in non-secreting ameloblast-like cells, the expression is absent (Gonza lez-alva et al, 2011). These findings appear to provide the evidence of a relationship between podoplanin and odontogenic cellular activity (i.e., the protein is expressed during intense proliferative activity in odontogenic cells and when these cells reach maturity or a stable state, there is a reduction or lack of podoplanin immunoreactivity). Our results reinforce this hypothesis because the odontogenic tumoral cells with low mitotic activity, such as the central stellate reticulum-like cells, keratinized acanthomatous cells, and granular cells (Figures 1a,c,d) did not express podoplanin. Inclusively, as proposed by Gonza lez-alva et al (2009), this pattern of distribution of podoplanin immunostaining, according to the cellular subtype in ameloblastomas, may be helpful to the classification of odontogenic tumors (Gonza lez-alva et al, 2009). (a) (b) Figure 2 Immunohistochemical reactivity for podoplanin in (a) the epithelial remnants of the dental lamina and (b) the reduced enamel epithelium of the dental follicles of unerupted teeth. (a) Strong membranous and cytoplasmic podoplanin expression by peripheral cells in the remnants of the dental lamina (b) Lack of podoplanin expression by reduced ameloblasts and strong immunostaining by basal layer of reduced enamel epithelium

5 Table 3 Membranous and cytoplasmic expression of podoplanin in epithelial cells of the remnants of the odontogenic epithelium from dental follicles Podoplanin Cytoplasmic Membranous N % N % Absent P = 0.001* Weak Strong Total P value determined by Wilcoxon signed-rank test. *Statistically significant. We also evaluated the expression of podoplanin in the odontogenic epithelial remnants of DF from unerupted teeth. The epithelial component of a dental follicle consists of the epithelial rests of the dental lamina and the reduced enamel epithelium (Damante and Fleury, 2001). These both represent quiescent and mature odontogenic epithelium that has performed its function and often remains dormant in the ectomesenchymal tissue indefinitely. Therefore, the epithelium of the dental follicle was used as a control group to compare to AM islands and cords. Similar to the odontogenic epithelium of ameloblastomas, membranous and or cytoplasmic podoplanin immunoreactivity was observed in the peripheral cells of dental lamina remnants (i.e., those adjacent to the surrounding connective tissue) (Figure 2a). Epithelial islands with dystrophic calcification lacked podoplanin expression. In the reduced enamel epithelium, the basal cells were positive for podoplanin while the reduced ameloblasts exhibited no staining (Figure 2b). Sawa et al and Imaizumi et al described the expression of podoplanin during bud, cap, and bell stages of murine odontogenesis. The current study described the sequence of this process. After the conclusion of the secretory activity, the ameloblasts reduce their size and the number of cellular organelles as a consequence of ending their secreting and proliferative activities, assuming a more quiescent state. Our results showed that at this moment, the so-called reduced ameloblasts do not express podoplanin. Indeed, other low active areas of the DF, such as the dystrophic calcification areas of the epithelial islands, were negative for podoplanin. These data support the concept that podoplanin may be necessary for the proliferative activity of the epithelial odontogenic cells. Moreover, our data reinforce the report by Zustin et al, (2010) that described the strong membranous podoplanin reactivity of several separated and deeper epithelial remnants of the dental lamina in tooth germs, while most centrally located islands and those showing squamous metaplasia were negative. Some authors suggested that podoplanin may be associated with cellular proliferative activity based on its expression during odontogenesis (Sawa et al, 2008; Imaizumi et al, 2010) once during this process, the protein expression is restricted to areas with high mitotic rates, such as the dental lamina, the terminal portion of Hertwig s sheath and pre-ameloblasts. These cited regions display high Ki-67 expression (Guven et al, 2007), which reinforces the hypotheses of Imaizumi et al (2010) and Sawa et al (2008). In ameloblastomas, the same occurs (i.e., proliferative activity is virtually restricted to the outer layer of epithelial islands) (Figure 3a) (Sandra et al, 2001). This pattern of staining was observed in our study either. However, despite podoplanin expression being found in areas of cellular proliferative activity, there was not a statistically significant correlation between podoplanin expression and the mitotic activity of the odontogenic epithelia in ameloblastomas and DF (Table 4). Nevertheless, membranous expression of podoplanin was significantly stronger in ameloblastomas than in DF. Furthermore, in DF, the epithelial membranous immunoreactivity for podoplanin was significantly stronger than its cytoplasmic expression (Table 3). The current study is the unique that separately analyzed membranous and cytoplasmic podoplanin expression; therefore, comparisons to other reports are difficult. However, Zustin et al (2010) showed a strong membranous and cytoplasmic anti-podoplanin reaction at the invasive edge of ameloblastomas. Gonza lez-alva et al (2009) described positivity for podoplanin in AM, which was often observed on the cell membrane, at cell cell boundaries, and in the cytoplasm of the peripheral columnar cells. Therefore, our findings are in accordance with Zustin et al (2010) and Gonza lez-alva et al (2009). Taken together, they show an enhanced podoplanin expression in areas of local invasion of ameloblastomas, suggesting a role of this protein in such process. The tumoral invasion relies on intense active remodeling of the cellular actin cytoskeleton. In both human 677 (a) (b) Figure 3 Immunohistochemical reactivity of Ki-67 in (a) follicular ameloblastoma and (b) dental follicle

6 678 Table 4 Correlation of cytoplasmic and membranous podoplanin expression with the proliferative activity of the odontogenic epithelial cells from ameloblastomas and dental follicles Podoplanin expression keratinocytes and in MCF7 breast cancer cells, the forced expression of podoplanin led to a dramatic change of cellular morphology with a significant decrease of cellular stress fibers and a concomitant formation of filopodia-like membrane protrusions, even in the presence of E-cadherin expression (Wicki et al, 2006; Wicki and Christofori, 2007). Moreover, podoplanin is expressed in podocytes and myofibroblasts (Wetterwald et al, 1996; Hata et al, 2008), both cell types with a high amount of cytoskeletal remodeling. Adding to these findings the increased membranous podoplanin expression in the peripheral cells of ameloblastomas, it is possible to speculate that this protein may be associated with the remodeling of the cytoskeleton of neoplastic cells. However, the podoplanin is unable to bind itself to the actin filaments. The ERM membrane proteins (ezrin, radixin, and moesin) are involved in the organization of the cortical cytoskeleton by linking filamentous actin to the apical membrane of cells (Neisch and Fehon, 2011). It was demonstrated that human podoplanin directly interacts with ezrin and moesin (Scholl et al, 1999),and that the overexpression of podoplanin in HaCaT keratinocytes leads to a marked increase in phosphorylation of ezrin (Martin- Villar et al, 2006; Wicki et al, 2006). These findings indicate a possible association between podoplanin and ezrin to mediate the cellular motility in the process of tumoral invasion (Gonza lez-alva et al, 2009). Based on previous studies, as well as the evidence provided here, it may be hypothesized that the membranous expression of podoplanin may be the evidence of local invasion of epithelial odontogenic cells in ameloblastomas. Indeed, the conflicting results about the role of podoplanin in odontogenic tumors reinforce the importance of elucidating how this molecule participates in the growth of these tumors. Acknowledgements We would like to thank the Fundac ão de Amparo à Pesquisa do Estado de Sa o Paulo (FAPESP) for supporting this study (grants # and # ). Conflicts of interest None to declare. Ameloblastomas Ki-67 Dental follicles r P r P Cytoplasmic ) ns ns Membranous ns ) ns ns, no statistically significant. r and P values calculated by Spearman s correlation coefficient. Author contributions Dr. Kellen Cristine Tjioe contributed to the conception and design of the project, acquisition, analysis, interpretation of data, and drafting and submission of the manuscript. Prof. Dr. Denise Tostes Oliveira, Prof. Dr. Jose Humberto Damante contributed to the conception and design of the project, analysis and interpretation of data, and drafting and revising critically the manuscript. 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