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Odontoestomatología

versión impresa ISSN 0797-0374versión On-line ISSN 1688-9339

Odontoestomatología vol.22 no.35 Montevideo  2020  Epub 01-Jun-2020

http://dx.doi.org/10.22592/ode2020n35a7 

Research

Immunohistochemical profile of odontogenic myxoma, with emphasis on microvascular density and tumor aggressiveness markers

Zaira Gómez-Herrera1 
http://orcid.org/0000-0002-1925-0291

Celeste Sánchez-Romero2 
http://orcid.org/0000-0001-5365-2692

Gabriela Vigil-Bastitta3 
http://orcid.org/0000-0002-0617-1279

Vanesa Pereira-Prado4 
http://orcid.org/0000-0001-7747-6718

Estefania Sicco5 
http://orcid.org/0000-0003-1137-6866

Omar Tremillo-Maldonado6 
http://orcid.org/0000-0002-2798-1596

Ronell Bologna-Molina7 
http://orcid.org/0000-0001-9755-4779

1 Departamento de Atención a la Salud, Universidad Autónoma Metropolitana (UAM), México

2 Área de Patología Molecular Estomatológica, Facultad de Odontología, Universidad de la República, Uruguay, csr_90@hotmail.com

3 Área de Patología Molecular Estomatológica, Facultad de Odontología, Universidad de la República, Uruguay

4 Área de Patología Molecular Estomatológica, Facultad de Odontología, Universidad de la República, Uruguay

5 Área de Patología Molecular Estomatológica, Facultad de Odontología, Universidad de la República, Uruguay

6 Universidad Autónoma Metropolitana de México, unidad Xochimilco, México

7 Patología Molecular Estomatológica, Facultad de Odontología, Universidad de la República, Uruguay

Abstract:

In order to elucidate better the biological behavior of the odontogenic myxoma (OM), immunohistochemistry was performed in 31 samples, using markers related to mechanisms of tumor progression (adhesion, angiogenesis, apoptosis, inflammation and cell proliferation). Odontogenic epithelium was detected in four samples with CK19 and CD138, the latter had a low-expression in extracellular matrix (ECM) and a high-expression in tumor cells. The mean microvascular density (MVD) assessed with CD34 and VEGF-A, was 7.51 and 5.35 blood vessels respectively. A high-expression of Orosomucoid-1 and Mast Cell Tryptase was observed in tumor cells and ECM, while Calretinin was completely negative. The previously mentioned immunohistochemical profile, as well as the low expression of Ki-67, Bcl-2 and p53 and the relatively low MVD, suggests that the proliferative, anti-apoptotic and angiogenic activities do not represent the main growing mechanisms of OM, which could be associated to other events, such as immunomodulation and ECM degradation.

Keywords: odontogenic myxoma; immunohistochemistry; tumoral markers; angiogenesis.

Introduction

Odontogenic tumors (OT) include a group of lesions that primarily affect the gnathic bones, ranging from hamartomas to benign neoplasms and malignant tumors 1-2. Odontogenic myxomas (OM) are benign mesenchymal OT (with or without odontogenic epithelium); however, they exhibit an aggressive behavior: significant growth potential as well as a high recurrence rate (25%) 3.

Globally, OM represent between 2.2 and 17% of OT and affect mainly the posterior mandible, and occasionally the maxilla 4. Rare cases of peripheral OM have been reported, and they are less aggressive than central OM 5. OM occurs more frequently between the second and fourth decades of life (mean age of 28.6 years), and its clinical presentation is a slow growing volume increase, which is usually asymptomatic 6, so it may be large sized when diagnosed. Radiographically, its appearance may be unilocular (small lesions), or more commonly multilocular (classically described as “tennis racket” pattern) 3.

Microscopically, OM typically have fusiform to stellate cells dispersed in an abundant myxoid matrix composed mainly of glycosaminoglycans, which may or not have islands of odontogenic epithelium; the presence of collagen fibers is variable 3-4,6.

To date, there are few papers describing the molecular components of OM, so this study aims to determine the immunohistochemical profile of various tumor markers and to analyze microvascular density (MVD) in order to discuss its possible implications for the biological behavior of OM.

Methodology

Sample selection

Thirty-one cases of mandibular OM obtained from the Molecular Pathology Area of the School of Dentistry, Universidad de la República (Uruguay) were included. The samples were obtained by incisional biopsy, fixed in 10% formaldehyde and then included in paraffin blocks. This study was approved by the Ethics Committee of the School of Dentistry of Universidad de la República, under protocol number 091900-000113-14. Informed consent was obtained from all the study participants when the biopsy was performed.

Immunohistochemistry

For immunohistochemistry assays, 3µm sections of paraffin embedded OM tissues were made and placed on slides; they were then deparaffinized in xylol and subsequently hydrated with decreasing concentrations of alcohol. Antigen retrieval was performed with a citrate solution (pH 6.2) in a pressure cooker in microwave oven on full power for one minute. Endogenous peroxidases were blocked with hydrogen peroxide at 0.9% for 5 minutes. Primary antibodies were incubated for one hour (Table 1).

Table 1: Data of antibodies used in this study 

Later the sections were incubated with the secondary biotinylated anti-mouse/anti-rabbit antibody and with the streptavidin/peroxidase complex (LSA-B + Dako Corporation, Carpinteria CA, USA) for 30 minutes each. The reaction was visualized with 3.3’-diaminobenzidine-H2O2 substrate (Dako Corporation, Carpinteria, CA, USA). Finally, the sections were contrasted with Mayer’s hematoxylin.

The following positive controls were used: oral mucosa for CK19 and CD138; breast cancer for calretinin; oral carcinoma for orosomucoid-1 and p53; intestine for mast cell tryptase, CD34 and VEGF-A; and tonsil for Bcl-2 and Ki-67.

Microscopic examination

Reactions were considered positive when brown labeling was observed in cells (tumor cells, endothelial cells or islands of odontogenic epithelium), or in extracellular matrix (ECM). Labeling patterns (nuclear, cytoplasmic, membranous, or ECM) varied for each type of antibody.

Proteins assessed in islands of odontogenic epithelium: CK19 and CD138

Using an optical microscope, each section was fully visualized under a 10x objective to identify areas with positive results and then at 40x to confirm it was odontogenic epithelium. The result was expressed as positive or negative.

Proteins assessed in tumor cells and ECM: CD138, calretinin, orosomucoid-1, mast cell tryptase, Bcl-2, Ki-67, p53 and VEGF-A

The complete sections were visualized under the 40x objective to determine the immunoexpression percentage throughout the tumor tissue on the slide. Percentages were classified into four groups: 0%, negative expression; 1% to 10%, low expression; 11% to 50%, moderate expression and greater than 50%, high expression 7.

Proteins assessed in blood vessels: VEGF-A and CD34

The method described by Weidner et al. was used to determine MVD. Tissues were initially visualized using a 10x objective to identify three areas with the highest concentration of positive vessels (hot spots), where vessel counting was performed manually using a 40x objective. Finally, the average number of vessels in the five fields of each sample was calculated 8.

Results

Odontogenic epithelium positive for CK19 and CD138 was detected in four cases. Additionally, CD138 showed high immunopositivity in tumor cells from most samples (74.2%) and low expression in ECM in 38.7% of cases (Figures 1A, B).

Calretinin was negative in all OM samples. In contrast, high immunopositivity of orosomucoid-1 was observed in tumor cells as well as ECM in 100% of cases, while mast cell tryptase was detected in the form of mast cell granules, focusing on ECM and with high expression in tumor cells: 96.7% of cases (n=30) (Figures 2 A, D) (Figures 2 C, E, F).

Low labelling for Bcl-2 (cytoplasmic), Ki-67 (nuclear) and p53 (nuclear) was observed in tumor cells (n=18, 58.1%; n=28, 90.3% and n=12, 38.7% respectively).

CD34 and VEGF-A were positive in endothelial cells (blood vessels). Additionally, VEGF-A showed a predominantly high expression in OM tumor cells (n=24, 77.5%) (Table 2). MVD was 7.51 and 5.35 for CD34 and VEGF-A respectively (Figures 1C and 2D).

Most proteins that were detected in tumor cells and ECM showed a high immunoexpression, except for Bcl-2. Table 3 shows the pattern, level and immunoexpression distribution of each protein in OM cases.

Fig. 1: Immunohistochemical markers in OM. Small islands of odontogenic epithelium were positive for CK19 (A) and CD138 (B), which was also expressed in tumor cells and ECM. Only endothelial cells in blood vessels were positive for CD34 (C). (Original magnification, A-C: 400x). 

Fig. 2: Immunohistochemical profile of OM. High immunopositivity for orosomucoid-1 in tumor cells, endothelial cells and ECM (A). VEGF-A expression in tumor cells and endothelial cells (B). Bcl-2 expression in some tumor cells (C). Mast cell tryptase showed intense positivity with granular appearance in tumor cells, mast cells and ECM (D). Few nuclei positive for Ki-67 (E) and p53 (F). (Original magnification, A, B, C, E, F: 400x; D: 600x). 

Table 2: Distribution of marker expression in OM components 

Abbreviations. TC: tumor cells, ECM: extracellular matrix, OE: odontogenic epithelium, BV: blood vessels.

Table 3: Number and percentage of cases according to immunoexpression level 

Abbreviations. TC: tumor cells, M: mast cells. ECM: extracellular matrix. * VEGF-A expression in endothelial cells was quantified as microvascular density (see results section).

Discussion

It has been suggested that OM has an odontogenic origin based on the occasional presence of small islands of odontogenic epithelium, its appearance in the mandible and maxilla, and its histomorphological similarity to the mesenchymal tissue of the tooth germ 9.

In this study, 13.3% of the sample had CD138 and CK19 positive odontogenic epithelial islands. This is consistent with two previous studies where epithelial islands were detected using CK19 in 4.8% and 14% of cases 9-10). Additionally, our results ratify the usefulness of CD138 as an alternative marker of odontogenic epithelium in OM.

A high expression of CD138 was observed in tumor cells in most cases (73.30%), while in ECM, immunoexpression was mainly low (46.70% of cases). Contrary to our results, in the study conducted by Etemad-Moghadam et al. (2017), CD138 was negative in all the samples 11. The physiological role of CD138, jointly with the ECM, is to participate in the induction and regulation of proliferation by interacting with families of heparin-binding growth factors 12. Additionally, it interacts with other ECM components 13, some of which (type I collagen, fibronectin and tenascin) are part of the ECM of the OM 14. This suggests that, in addition to the structural maintenance function of an epithelial adhesion protein, upon release into the ECM, CD138 could participate in various signaling pathways by interacting with growth factors and other molecules present in the ECM.

Although calretinin expression has been described in ameloblastomas, and has been associated with enamel production in tooth germ, in this study it was negative in OM, as in previous studies 15-16. This absence of calretinin can be explained by the mesenchymal origin of OM, unlike ameloblastomas and the enamel organ 17-18.

A previous study determined MVD in OM by CD34 expression, with similar results to ours 7. Ameloblastoma has higher MVD compared to other odontogenic tumors and cysts, suggesting an association with a more aggressive behavior 19-20. In our study, MVD in OM was five times lower than in the ameloblastomas of the study conducted by Seifi et al. (2001), suggesting that angiogenesis could partially contribute to tumor growth in OM 19.

In OM, VEGF-A (one of the majors signaling proteins for angiogenesis) was expressed in endothelial cells and tumor cells. Recent researches suggest that VEGF-A immunoexpression in the epithelium of odontogenic cysts and epithelial OT, such as ameloblastoma, affects epithelial proliferation through an autocrine signaling, whereas angiogenic activity is mediated by a paracrine mechanism 7,21. However, in the only study describing VEGF-A expression in MO, it has been associated primarily with the angiogenic mechanisms of this tumor 7.

In the OM samples tested, high orosomucoid-1 immunoexpression was observed in tumor cells and ECM. This confirms the findings of the two studies that reported orosomucoid-1 overexpression in OM by immunohistochemistry and proteomics 7-22.

Macroscopically, OM appears as a highly viscous mucous mass, which, according to our results, could be partly due to the presence of orosomucoid-1 (which is a mucoprotein) in the ECM. It is widely accepted that the structural viscosity of the OM enables it to infiltrate bone and invade 7. In turn, García Muñoz et al. (2012) proposed that overexpression of orosomucoid-1 in OM could play a major role in tumor cell growth and invasion potential by inhibiting antitumor immune response 22.

Orosomucoid-1 has been shown to participate in VEGF-A regulation and induction 23-24. Thus, overexpression of these proteins in OM in both, tumor and endothelial cells, could indicate an interaction between orosomucoid-1 and VEGF-A in this tumor, suggesting a collaborative proangiogenic role 7.

In this study, most cases expressed high immunopositivity for mast cell tryptase (96.70%) in tumor cells, mast cells, and the ECM. A previous study including seven cases of OM reported no expression of mast cell tryptase; however, the sample size may not be representative 25. Another study conducted in a larger sample (62 cases) found mast cell tryptase in 72.6% of OM and it was mainly located in mast cells, which have large amounts of active tryptase stored, than, when released, can degrade fibronectin (one of the components of the OM’s ECM). This suggests that, to some extent, ECM degradation could be mediated by the release of mast cell tryptase 9-26. Additionally, there is an association between the presence of mast cells and mast cell tryptase with bone resorption in odontogenic cysts 27, and although the incidence of mast cells in OM varies according to different studies, it has been described that these cells are frequently distributed adjacent to residual bone trabeculae 28. These findings suggest that ECM degradation and bone remodeling processes via tryptase contribute significantly to the invasion potential of OM (27, -28).

The imbalance between apoptosis mechanisms and cell proliferation accounts for significant tumorigenic molecular alterations in various neoplasms.

In this study we evaluated p53, Bcl-2 and Ki-67, which are proteins related to apoptotic, anti-apoptotic and cell proliferation processes respectively. The apoptosis induced by p53 has been shown to be blocked by Bcl-2 29. Both markers, p53 and Bcl-2, showed a low immunoexpression in most OM, which is consistent with previous reports 9,30. Likewise, Ki-67 showed a low proliferation rate in our cases, which is consistent with the work of various authors 9,30. These results could indicate that, due to the low level of anti-apoptotic and proliferative activity in this tumor, these would not be the main mechanisms associated with the growth potential and aggressiveness of OM.

Conclusions

CK19 and CD138 labelling is useful for detecting odontogenic epithelium in OM. The high expression of orosomucoid-1 in the ECM may indicate it is a structural component that contributes to its viscosity and, therefore, facilitates invasion. Orosomucoid-1 also showed a VEGF-A-like pattern in tumor and endothelial cells, suggesting proangiogenic collaborative activity, possibly involved in tumor development. Anti-apoptotic and proliferative activity do not seem to be crucial mechanisms in the aggressive behavior of OM. However, ECM degradation and bone resorption mediated by mast cell tryptase could be significant in the high invasive potential of OM.

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Authorship contribution: 1. Conception and design of study 2. Acquisition of data 3. Data analysis 4. Discussion of results 5. Drafting of the manuscript 6. Approval of the final version of the manuscript ZGH, CSR and RBM have contributed in 1,2,3,4,5,6. GVB and VPP have contributed in 3,4,5,6. ES has contributed in 4,5,6. OTM has contributed in 1,2,3.

Editor’s opinion: This article has been accepted by the Odontoestomatología’s editor Dra. Vanessa Pereira-Prado

Received: July 25, 2019; Accepted: December 09, 2019

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