Expression of Matrix Metalloproteinases 7 and 9, Desmin, Alpha-Smooth Muscle Actin and Caldesmon, in Odontogenic Keratocyst Associated with NBCCS, Recurrent and Sporadic Keratocysts

Abstract

Nevoid basal cell carcinoma syndrome (NBCCS) associated odontogenic keratocysts (OKCs) show more aggressive behavior and it has a higher frequency of relapse than non-syndromic OKCs. Stromal myofibroblasts (MFs), characterized by α-smooth muscle actin (αSMA), desmin and caldesmon expression, and metalloproteinases (MMPs) have an essential role in the remodeling of the extracellular matrix (ECM). The aim of the study is to analyze the immunohistochemical expression of MMP-7, MMP-9, αSMA and other new markers in the study of OKCs MFs such as desmin and caldesmon in NBCCS-associated OKCs compared to recurrent and sporadic keratocysts. Fourty 40 patients (23 M and 17 F) underwent surgery to remove the OKCs. The histological sections in paraffin were incubated with markers antibodies and a semi-quantitative score was used to evaluate the immunoreactivity. Densitometric analysis showed a very significantly increased expression of αSMA, caldesmon, MMP-7 and MMP-9 in NBCCS-OKCs compared to non-syndromic OKCs (p < 0.001). However, desmin showed a not significant increased expression in non-syndromic OKC compared to NBCCS-OKCs specimens in which desmin was slightly or not at all expressed. NBCSS-OKCs showed a greater distribution of MFs compared to the other OKCs subtypes. Further studies will be needed to evaluate whether the different expressions of these markers can be correlated to a different clinical behavior.

Keywords: MMP-7; MMP-9; NBCCS; OKCs; alpha-SMA; caldesmon; desmin; immunohistochemistry; myofibroblasts.

Figure 1

Interaction between epithelial cells and myofibroblasts during connective wall invasion in NBCCS-OKC. (a), αSMA staining depicts myofibroblasts surrounding invading epithelial droplets (LSAB-HRP, ×10; nuclear counterstaining with hematoxylin); (b), further magnification of a (LSAB-HRP, ×40); invading droplets have been further examined on serial sections by digital pathology showing high expression of MMP-7; (c), digital pathology, ×40, MMP-7 in violet and nuclear staining in red pseudocolors); (d), digital pathology ×40, MMP-9 in violet and nuclear staining in red pseudocolors), whereas MMP-9 showed a low level of expression [arrows indicated area of epithelial nuclei].

Figure 2

NBCCS-OKC, magnifications ×10. (a), αSMA markedly expressed in plump fibroblasts both in the epithelium and in the connective tissue, ×10; (b), MMP-7 moderately expressed in the MFs localized in the sub-epithelium and in the epithelial layer, ×10; (c), MMP-9 markedly detected in the endothelial cells and moderately in the sub-epithelial MFs, ×10; (d), Recurrent OKC, αSMA expression around the vessels, ×10; (e), Solitary OKC, MMP-7 expression in the inflammatory area and in the basal and intermediate connective wall, ×10; (f), Solitary OKC, MMP-9 slightly expressed in the basal-intermediate connective wall and moderately in the phlogosis areas, ×10.

Figure 3

NBCCS-OKC (same case in Figure 2a–c), magnifications ×40. (a), αSMA expression, ×40; (b), MMP-7 expression, ×40; (c), MMP-9 expression, ×40.

Figure 4

(a), NBCCS-OKC, moderate desmin expression around the vessels, ×10; (b), Solitary OKC, caldesmon scattered expression, ×10; (c), Sporadic OKC, focal desmin expression, ×10; (d), NBCCS-OKC, caldesmon in plump fibroblasts in the epithelium and throughout the connective wall, ×10.

Figure 5

Densitometric analysis. A bar chart represents the comparison of the densitometric count (pixel2) of αSMA, caldesmon, MMP-7, MMP-9, and desmin immunostaining in the evaluated fields for NBCCS-OKCs tissue compared to solitary, sporadic and recurrent OKCs tissue, expressed by positive percentage, dark brown pixels of the analyzed fields. Data are presented as mean ± standard deviation (SD).