Annals of African Medicine

ORIGINAL ARTICLE
Year
: 2015  |  Volume : 14  |  Issue : 4  |  Page : 188--192

Relative expression of α-smooth muscle actin and matrix metalloproteinases-2 in ameloblastoma of a black African sub-population


Akinyele O Adisa1, Samuel E Udeabor2, Bukola F Adeyemi1, Kubesch Alica3, Patrick Booms3, Shahram Ghanaati3, Robert A Sader4,  
1 Department of Oral Pathology, College of Medicine, University of Ibadan, Ibadan, Nigeria
2 Department of Oral and Maxillofacial Surgery, College of Health Sciences, University of Port Harcourt, Rivers, Nigeria; Department for Oral Cranio Maxillofacial and Facial Plastic Surgery, Medical Center of the Goethe University Frankfurt, Frankfurt am Main; REPAIR Laboratory Institute of Pathology, University Medical Center, Johannes Gutenberg University, Mainz, Germany
3 Department for Oral Cranio Maxillofacial and Facial Plastic Surgery, Medical Center of the Goethe University Frankfurt, Frankfurt am Main, Germany; REPAIR Laboratory Institute of Pathology, University Medical Center, Johannes Gutenberg University, Mainz, Germany
4 Department for Oral Cranio Maxillofacial and Facial Plastic Surgery, Medical Center of the Goethe University Frankfurt, Frankfurt am Main, Germany

Correspondence Address:
Shahram Ghanaati
Department for Oral Cranio.Maxillofacial and Facial Plastic Surgery, Medical Center of the Goethe University Frankfurt, Frankfurt am Main, Germany

Abstract

Background: Ameloblastoma although a benign odontogenic tumor, is locally invasive. The abundant presence of myofibroblasts (marked by α-smooth muscle actin [α-SMA]) in the stroma and expression of matrix metalloproteinase-2 (MMP-2) in the neoplastic or stromal cells have been linked with the tumor's ability for both local and distant spread. We aim to estimate the relative expression of α-SMA and MMP-2 in ameloblastoma from a black African subgroup to gauge their relative potential for enhancing local invasiveness and hence, their prospects as possible chemotherapeutic targets. Materials and Methods: Twenty-five formalin-fixed paraffin-embedded blocks of ameloblastoma cases from Nigeria were prepared for antibody processing to α-SMA (Dako Monoclonal Mouse Anti-Human α-SMA antibody clone 1A4) and MMP-2 (Abcam Mouse Monoclonal Anti-MMP-2 antibody [CA-4001/CA719E3C] ab3158). The score for percentage positivity of the tumor cells and the score for staining intensities were then multiplied in order to generate an immunoreactive score. Results: α-smooth muscle actin was only expressed in the fibrous connective tissues adjacent to the tumor islands while MMP-2 was expressed in the ameloblasts, stellate reticulum, and the connective tissues in varying proportions. All the variants analyzed expressed α-SMA mildly or moderately, except for the follicular variant that either did not express α-SMA or expressed it mildly. The highest number of strong immunoreactivity to MMP-2 in the ameloblast region was found in the plexiform variant. Conclusion: Chemotherapeutic targeting of both molecules may, therefore, be a vital step in the control of local ameloblastoma invasiveness.



How to cite this article:
Adisa AO, Udeabor SE, Adeyemi BF, Alica K, Booms P, Ghanaati S, Sader RA. Relative expression of α-smooth muscle actin and matrix metalloproteinases-2 in ameloblastoma of a black African sub-population.Ann Afr Med 2015;14:188-192


How to cite this URL:
Adisa AO, Udeabor SE, Adeyemi BF, Alica K, Booms P, Ghanaati S, Sader RA. Relative expression of α-smooth muscle actin and matrix metalloproteinases-2 in ameloblastoma of a black African sub-population. Ann Afr Med [serial online] 2015 [cited 2022 Aug 9 ];14:188-192
Available from: https://www.annalsafrmed.org/text.asp?2015/14/4/188/152075


Full Text

 Introduction



Ameloblastoma is a benign, locally infiltrative odontogenic neoplasm with an unpredictable tendency for metastasis.[1] Some proteins have been described as supporting the local and distant spread of this neoplasm and a significant correlation was found to exist between the presence of myofibroblasts (marked by α-smooth muscle actin [α-SMA]) and matrix metalloproteinase-2 (MMP-2) expression in ameloblastoma.[2] The abundant presence of myofibroblasts in the stroma and expression of MMP-2 in the neoplastic or stromal cells were also significantly correlated with rupture of the cortical jawbone, which was considered an important prognostic marker of ameloblastoma aggressiveness. They then suggested that the abundant presence of myofibroblasts and expression of MMP-2 in solid ameloblastomas might be associated with a more aggressive infiltrative behavior.

Myofibroblasts are found in the stroma of neoplasms and by expressing proteinases they can affect tumor infiltration and advancement. The presence of myofibroblasts has been reported in ameloblastoma as being closely apposed to the neoplastic cells.[2] MMPs are enzymes that can cleave extracellular matrix and basement membrane components. Within the MMP family, the MMP-2 has been most associated with ameloblastoma invasiveness.[3] The presence of this enzyme in ameloblastoma has been used to explain its local invasiveness and tumor advancement. MMP-2 also contributes to angiogenesis by degrading the proteins that keep the vessel walls solid. This proteolysis allows the endothelial cells to escape into the interstitial matrix as seen in sprouting angiogenesis. This activity of MMP encourages tumor growth. Thus, the inhibition of MMPs will prevent the formation of new capillaries and inadvertently prevent tumor advancement.[4]

We aim to estimate the relative expression of α-SMA and MMP-2 in ameloblastoma from a black African subgroup to gauge their relative potential for enhancing local invasiveness and hence their prospects as possible chemotherapeutic targets.

 Materials and Methods



Twenty-five formalin-fixed paraffin-embedded blocks of ameloblastoma cases from the Oral Pathology Department of the University College Hospital and University of Ibadan, Nigeria were sectioned and stained with hematoxylin and eosin for re-evaluation and inclusion. At the REPAIR laboratory, Institute of Pathology, School of Medicine, University of Mainz Germany, sections were prepared for antibody processing to α-SMA and MMP-2 for each specimen, using the specification of the manufacturers and established standardized protocol.[5] A material transfer agreement was signed between the institutions, and the tissue blocks were labeled with numbers instead of names to conceal patients' identities. The sections were deparaffinized, hydrated, and then rinsed in phosphate-buffered solution (PBS). They were immersed in heat-induced epitope retrieval citrate buffer of concentration 15 mMol and pH 6.0, diluted 1:10 with distilled water and incubated at 94°C for 10 min. They were then placed in fresh citrate, cooled in water for 20 min, and then rinsed in PBS for 6 min. Positive and negative controls were employed for each antibody.

EnVision FLEX peroxidase blocking reagent was added to each section for 5 min, and the sections were rinsed in PBS for 6 min. The specimen was incubated for 30 min with 1:100 dilutions of Dako Monoclonal Mouse Anti-Human α-SMA antibody clone 1A4 and Abcam Mouse Monoclonal anti-MMP-2 antibody (CA-4001/CA719E3C) ab3158, rinsed with PBS, followed by incubation with undiluted EnVision

FLEX/horseradish peroxidase for 20 min. A volume of 1 ml diaminobenzidine solution was added to cover the specimen, followed by incubation in a humidity chamber for 15 min. The sections were then immersed in aqueous Meyer's hematoxylin and rinsed in distilled water for 5 min. The tissue was dehydrated and subsequently rinsed with xylene. Distyrene plasticizer in xylene mounting fluid was then applied, and a cover slip placed.

The positive cells were quantified as a percentage of the total number of cells and assigned to one of five categories (0, <5%; 1, 5–25%; 2, 26–50%; 3, 51–75%; 4, >75%). The percentage of positivity of the tumor cells and the staining intensities were then multiplied in order to generate an immunoreactive score. The product of the proportion and intensity scores were calculated such that a final score of 0 indicated no expression, 0–4 indicated weak expression, 5–8 indicated moderate expression and 9–12 indicated strong expression. Each sample was examined and scored by two independent oral pathologists using a conventional diagnostic microscope (Eclipse 80i, Nikon, Tokyo, Japan) and further image analysis was done with the NIS-Elements AR software (version 4.10.03, Nikon, Tokyo, Japan).

The data were analyzed using version 20 of the SPSS (IBM corp.). Qualitative data were compared using Chi-square statistics. Quantitative data were summarized using mean, standard deviation, and confidence interval and compared using Student's t-test and/or one-way analysis of variance test. The level of significance was set at P < 0.05.

 Results



The gender distribution of patients was approximately equal, and 18 (72.0%) were in the third decade of life. α-SMA was only expressed in the fibrous connective tissues adjacent to the tumor islands. About 75% of the samples that did not express α-SMA were from males while all the cases with strong expression were from females. All the variants analyzed expressed α-SMA mildly or moderately, except for the follicular variant that either did not express α-SMA or expressed it mildly [Table 1].{Table 1}

Matrix metalloproteinase-2 was expressed in the ameloblasts, stellate reticulum, and the connective tissue in varying proportions. Strong expression of MMP-2 was noted in the ameloblast and stellate reticulum in 16.0% and 8.0% of cases, respectively [Table 2]. The ameloblast in the cystic variant had the highest frequency of zero MMP-2 expression, closely followed by the fibrous connective tissue of the plexiform variant [Table 3]. The highest number of strong immunoreactivity in the ameloblast region was found in the plexiform variant, but there was no strong expression of MMP-2 in the adjacent fibrous connective tissue of any histologic variant [Table 3]. [Figure 1] and {Figure 2] show the various levels of expression for α-SMA and MMP-2.{Table 2}{Table 3}{Figure 1}{Figure 2}

 Discussion



Myofibroblasts have been found to be the most prominent stromal cell type in the tumor microenvironment [6] and their role in the support of tumor growth and progression through the secretion of growth factors, extracellular matrix proteins, and the stimulation of angiogenesis has been documented.[7],[8],[9] This has, therefore, become the basis for a possible potential target for therapeutic intervention. α-SMA is one of the immunohistochemical markers that are used to define the presence of these myofibroblasts in the tumor immediate environment.[10] In this study, α-SMA was expressed only in the fibrous connective tissue adjacent to the islands of odontogenic epithelium, this is in agreement with the report by Fregnani et al.[2] and that by Vered et al.[11] further linked the level of α-SMA expression with aggressiveness of ameloblastoma and found that solid ameloblastoma expressed levels comparable to that seen in squamous cell carcinoma.

It was therefore suggested that anti-myofibroblast agents be used in odontogenic tumors with high α-SMA expression to reduce their size before surgical intervention. In our study, most of the moderate to strong α-SMA expression was seen in the plexiform and unicystic variants. In a review of 3677 cases of ameloblastoma, approximately one-third were found to be plexiform [12] and this gives a representation of cases that may benefit from possible chemotherapy.

Even though a study by Pechkovsky et al.[13] showed no differential α-SMA expression according to gender, our study showed that 75% of the tissue samples that did not express α-SMA were from males while all the cases with strong expression were from females. Currently, we have no explanation for this variation.

Several studies have demonstrated that MMP-2 plays an important role in tumor invasion and aggressive behavior in ameloblastoma.[14],[15] We found that MMP-2 was expressed in the ameloblast, stellate reticulum, and the adjacent fibrous connective tissue stroma, and this is in agreement with a report by Sah et al.[1] Zhong et al.[16] reported strong MMP-2 expression in the central and peripheral cells of ameloblastoma islands in 65% of studied cases. We found strong expression of MMP-2 in the ameloblast and stellate reticulum in 16.0% and 8.0% of cases, respectively. Except for population disparities, we have no other explanation for this wide margin of difference in percentage expression of MMP-2.

The plexiform variant in this study had moderate to strong α-SMA expression and also strong immunoreactivity for MMP-2 in the ameloblast region compared to other variants, this may suggest a more aggressive biology than the other variants. Histological observation shows that plexiform ameloblastoma does have the most invasive interlacing pattern compared to all the other variants. Considering the fibrous connective tissue microenvironment in this study, there was no strong expression of MMP-2 in the adjacent fibrous connective tissue of any histologic variant but all variants expressed α-SMA mildly or moderately.

 Conclusion



We, therefore, suggest that to control the tumor microenvironment of ameloblastoma with chemotherapeutics, targeting myofibroblasts is appropriate while for local tumor invasiveness, anti-MMP-2 agents should be considered. Targeting both molecules simultaneously may, therefore, be more effective for tumor control.

 Acknowledgments



The authors wish to thank Mrs. Verena Hoffmann for her excellent technical assistance in the course of this study.

Source of Support:

Nil

Conflict of Interest:

None declared.

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