Assessment of Tumor Angiogenesis by Expression of CD 105 in Ameloblastoma, Odontogenic Keratocyst and Central Giant Cell Lesion.

BACKGROUND: Angiogenesis is critical for tumor growth and reflects the aggressive behavior of invasive odontogenic lesions [like Ameloblastoma (AM), Odontogenic Keratocyst (OKC) and Central giant cell lesion (CGCL)]. Mean vascular density (MVD) shows the angiogenic potential and CD105 is an ideal endothelial biomarker due to its specificity to new blood vessels for MVD detection. The aim of the study was to compare the MVD (angiogenic potential) among AM, OKC and CGCL in comparison to Pyogenic Granuloma (PG) using CD105 biomarker. METHODS: Sixty-four primary cases of odontogenic invasive tumors (AM, OKC and CGCL) and PG, diagnosed clinically and histologically were included in the study, with 16 samples in each group. Tissue samples of peripheral AM, Peripheral GCL of jaws, malignant AM, and specimen with insufficient tissue were excluded. Tissue sections were embedded, processed and stained using Hematoxylin and Eosin (H and E). Immunohistochemistry was performed using antibodies against CD105, with positive brown cytoplasmic staining in the endothelial cells of neo-vasculature. Distinct countable, positively stained endothelial cell or clusters were evaluated under light microscope for identification of MVD. ANOVA and t-test were applied for statistical analysis of data. RESULTS: Highest MVD was displayed in CGCL (32.99±0.77) and the minimum was observed in OKC (7.21± 0.75) respectively. CGCL showed significantly higher MVD to AM, OKC and PG lesions (p <0.05). AM (8.07± 0.36) and Odontogenic Keratocyst (7.21± 0.75) showed comparable MVD, which was lower than PG (14.7± 0.96) and CGCL vascular density (p < 0.01) respectively. CONCLUSION: CGCL was most aggressive, with highest MVD among the investigated odontogenic lesions (OKC, AM and PG). The proliferative aggressive behavior of Odontogenic Keratocyst is comparable to AM due to comparable mean vascular density.
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Angiogenesis concept in odontogenic keratocyst: A comparative study.

CONTEXT: Recent reports have indicated that angiogenesis possibly affects the biologic behavior of the lesions. AIM: Given the different clinical behaviors of odontogenic keratocyst (OKC), the present study was undertaken to evaluate the concept of angiogenesis in pathogenesis and clinical behavior of OKC. SETTING AND DESIGN: This experimental study was carried out on 22 and 24 samples of OKCs and dentigerous cysts (DCs), respectively. METHODS: Immunohistochemical staining was approached using CD34 and vascular endothelial growth factor (VEGF) antibodies. The expression of VEGF was first reported by determining the counts of stained cells, including epithelial cells, fibroblasts, and endothelial cells, followed by the percentage of stained cells in each sample based on a 0-2 scoring system. The counts of CD34+ cells were reported in each group in the form of means ± standard deviations. In addition, the patterns of blood vessels in the samples prepared from the walls of both cysts were evaluated. STATISTICAL ANALYSIS USED: Mann-Whitney U-test, Chi-squared test, and t-test were used for analysis of data, and statistical significance was defined at p < 0.05. RESULTS: The expression percentage and scores of VEGF and the mean expression rate of CD34 were significantly higher in OKCs than DCs (p = 0.045, 0.000, and p = 0.58). Finally, there was a strong correlation between the expressions of the two markers in the samples (Correlation coefficient = 0.766). CONCLUSION: The present results indicate the angiogenesis may play an important role in the pathogenesis and the unique clinical behavior of OKC.

Interaction of stromal and microvascular components in keratocystic odontogenic tumors.

OBJECTIVE: Little is known about the interaction of stromal components in odontogenic tumors. Thus, the aim of this study was to investigate mast cells (MCs), myofibroblasts, macrophages, and their possible association with angiogenesis and lymphangiogenesis in keratocystic odontogenic tumors (KCOTs). MATERIAL AND METHODS: Thirty cases of KCOTs were included and analyzed by immunohistochemistry for mast cell tryptase, α-SMA, CD34, CD163, and D240. For comparative purpose, 15 radicular cysts (CRs) and 7 pericoronal follicles (PFs) were included. RESULTS: There was an increase in MCs for RCs and this difference was significant when they were compared to KCOTS and PFs. A significant increase in the density of MFs was observed for KCOTs when compared to RCs and PFs (P = 0.00). No significant difference in CD163-positive macrophages (P = 0.084) and CD34-positive vessels (P = 0.244) densities was observed between KCOTs, RCs, and PFs, although KCOTs showed a higher density of all proteins. Significant difference in lymphatic vessel density was observed for KCOTs when compared to RCs and PFs (P = 0.00). Positive correlation was observed between mast cell tryptase and CD34 in KCOTs (P = 0.025). CONCLUSIONS: A significant interaction between the MC population and CD34-positive vessels in KCOTs supported the hypothesis that MCs and blood vessels contribute to the stromal scaffold of KCOT.

Histomorphometric comparative study of blood vessels and their pattern in follicular cyst, odontogenic keratocyst, and ameloblastoma.

OBJECTIVE: The present study aimed at assessment and histomorphometric analysis of intratumoral and peritumoral (cystic) blood vessels in odontogenic lesions and their pattern on their clinical behavior by immunohistochemistry and morphometry. METHODS: In a descriptive and analytical cross-sectional study, 45 paraffin blocks of ameloblastoma, odontogenic keratocyst, and follicular cyst were selected and stained immunohistochemically for CD34. In each slide, images of 3 microscopic fields with the highest microvessel density in intratumoral and peritumoral (cystic) areas were captured at 40× magnification with attached camera system. Inner vascular diameter (IVD) and outer vascular diameter (OVD), cross-sectional area (CSA), and the wall thickness (WT) of the vessels were measured with Motic Plus 2 software. The vascular pattern in odontogenic lesions was analyzed. RESULTS: Outer vascular diameter, IVD, and CSA of the vessels in peritumoral (cystic) areas were greater in ameloblastoma than keratocyst (P = 0.001) and follicular cyst (P < 0.001). However, WT of the blood vessels did not show any significant statistical difference among the 3 odontogenic lesions (P = 0.05). The differences in OVD, IVD (P = 0.8), CSA (P = 0.6), and WT (P = 0.4) of the blood vessels in intratumoral (cystic) areas were not statistically significant. The blood vessel pattern was circumferential in ameloblastoma, and it was directional in keratocyst and follicular cyst. CONCLUSIONS: Morphometric specifications of blood vessels (IVD, OVD, CSA) and their pattern in peritumoral (cystic) areas may influence the aggressive clinical behavior of ameloblastoma in comparison with keratocyst and follicular cyst.

Immunohistochemical analysis of bone resorption regulators (RANKL and OPG), angiogenic index, and myofibroblasts in syndrome and non-syndrome odontogenic keratocysts.

OBJECTIVE: The aim of this study was to immunohistochemically analyse bone resorption regulators (receptor activator of nuclear factor kappa B ligand [RANKL] and osteoprotegerin [OPG]), angiogenic index, and myofibroblasts in Gorlin syndrome-related odontogenic keratocysts (SOKCs) and non-syndrome odontogenic keratocysts (NSOKCs). STUDY DESIGN: Twenty-two SOKCs, 22 primary NSOKCs, and eight recurrent NSOKCs were evaluated by immunohistochemistry using anti-RANKL and anti-OPG antibodies. The angiogenic index was determined by microvessel count (MVC) using anti-CD34 antibody. Anti-α-smooth muscle actin (α-SMA) antibody was used for the identification of myofibroblasts. RESULTS: Analysis of the expression of RANKL and OPG in the epithelial lining and fibrous capsule did not reveal significant differences between groups (P>0.05). In the epithelial lining, the RANKL/OPG ratio was RANKL0.05). In the fibrous capsule, the ratio was RANKL=OPG in most primary (81.8%) and recurrent NSOKCs (75.0%) and in most SOKCs (45.5%) (P>0.05). No significant differences in the angiogenic index or number of myofibroblasts were observed between primary NSOKCs, recurrent NSOKCs, and SOKCs (P>0.05). CONCLUSIONS: The present results suggest that differences in the biological behaviour of SOKCs and NSOKCs may not be related to the expression of RANKL and OPG, to the RANKL/OPG ratio, to the angiogenic index, or to the number of myofibroblasts in these lesions.

Comparison of angiogenesis in keratocystic odontogenic tumours, dentigerous cysts and ameloblastomas.

OBJECTIVE: The aim of the present study was to evaluate and compare angiogenesis in keratocystic odontogenic tumours, dentigerous cysts (DCs) and ameloblasomas using monoclonal antibody against CD34. MATERIALS AND METHODS: Microvessel density was assessed in a total of 53 cases including 20 keratocystic odontogenic tumours, 13 DCs and 20 ameloblastomas (14 solid and six unicystic variants). Microvessel density was expressed as the mean number of microvessels per high-power-field. RESULTS: Statistically significant differences in mean microvessel density were observed between keratocystic odontogenic tumours, DCs and solid ameloblastomas (P < 0.001). Mean microvessel density was significantly higher in solid ameloblastomas compared with both keratocystic odontogenic tumours and DCs; and was also significantly higher in keratocystic odontogenic tumours than in DCs. CONCLUSION: Within the limitations of the present study, it can be suggested that angiogenesis may be one of the mechanisms possibly contributing to the different biological behaviours of keratocystic odontogenic tumours, DCs and solid ameloblastomas.

Expression of inducible nitric oxide synthase and vascular endothelial growth factor in ameloblastoma.

The purposes of this study were to determine the correlation between the expression of inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) in ameloblastoma and to examine the relationships of this expression to angiogenesis and the clinical and biological behaviors of the tumor. Immunohistochemical staining with streptavidin peroxidase was used to analyze iNOS and VEGF expression, and CD34 was used to evaluate microvascular density (MVD) in 35 ameloblastomas (24 primary tumors and 11 recurrences) and 5 malignant ameloblastomas. Ten odontogenic keratocysts (OKCs) served as controls. On relational analysis, positive and VEGF expression and MVD counts increased in this order: OKCs, primary ameloblastoma, recurrent ameloblastoma, and malignant ameloblastoma. Differences between the ameloblastomas and OKCs were significant (P < 0.05). Among ameloblastomas, MVD counts increased with increasing expression of iNOS and VEGF (P < 0.05), and iNOS expression and VEGF expression were positively correlated (r = 0.66, P < 0.05). Inducible nitric oxide synthase expression and VEGF expression may be closely related to the angiogenesis and invasive biological behavior of ameloblastomas.

Ultrasonography and Doppler ultrasonography in the evaluation of intraosseous lesions of the jaws.

OBJECTIVES: To evaluate the efficacy of ultrasonography and colour and power Doppler ultrasonography for diagnosis of intraosseous lesions of the jaws and to correlate the contents of the lesion with the histological findings. METHODS: This study included 20 patients referred to the oral surgery clinic for treatment. All patients had intraosseous jaw lesions in the maxilla or mandible. Ultrasonographic examinations were performed and, according to the ultrasonography findings, the jaw lesions were classified into three groups: cystic, semisolid and solid. Additionally, colour and power Doppler ultrasonography examinations were performed to evaluate blood flow in all patients. After the ultrasonography examination, the patients underwent surgical treatment. The correlation between ultrasonography and Doppler ultrasonography findings of the lesions and histological findings was investigated. RESULTS: 22 lesions were identified in 20 patients. Of the five lesions with histological findings of inflammatory granulation tissue, ultrasonography identified four of them that showed a solid appearance. Vascularization was detected in both internal and external areas of these lesions with colour and power Doppler ultrasonography. Of the 17 odontogenic cystic lesions, the ultrasonography examination showed a simple cystic appearance in 5 lesions, a complex cystic appearance in 3 lesions, a semisolid appearance in 6 lesions and a solid appearance in 1 lesion. Two lesions were inconclusive on ultrasonographic examination. CONCLUSIONS: Ultrasonography can provide accurate information on the content of intraosseous lesions of the jaws before any surgical procedure. Additionally, Doppler ultrasound can show vascularization of the lesion. However, there was no correlation between the ultrasound findings and the definitive histological diagnosis.

Expression of transforming growth factor-beta 1 (TGF-beta 1) in odontogenic cysts.

AIM: To evaluate the positivity to transforming growth factor-beta 1 (TGF-beta 1) in different types of odontogenic cysts. METHODOLOGY: A total of 30 radicular cysts (RCs), 27 follicular cysts (FCs) and 28 odontogenic keratocysts (OKCs) were evaluated for immunohistochemical analysis of TGF-beta 1. TGF-beta 1 was evaluated in blood vessels, stromal cells (fibroblasts) and pluristratified squamous epithelium. TGF-beta 1 expression was determined by evaluating the number of positive elements. TGF-beta 1 expression was determined by evaluating 1000 cells in the pluristratified squamous epithelium (500 in the basal and parabasal layers, and 500 in the superficial layer) and 500 cells (the fibroblasts in the stroma) for each specimen, and counting the number of positive cells. The number of positive vessels was evaluated in 10 high power fields (HPF). The Chi-square test was used to evaluate differences between the two groups (RC + FC and OKC). A P-value <0.05 was considered to indicate statistical significance. RESULTS: A higher and statistically significant positivity was found in the basal-suprabasal epithelial layers (P=0.0011), superficial epithelium (P=0.053) and stromal cells (P=0.0002) of orthokeratotic and parakeratotic OKC as compared with RC and FC. CONCLUSIONS: These differences suggest that control of the cell cycle may be abnormal in orthokeratotic OKCs. These OKCs may have an intrinsic growth potential not present in other cyst types.

[Expression of ICAM-1 and VCAM-1 in human ameloblastoma and odontogenic keratocyst].

OBJECTIVE: To study the expression of ICAM-1 and VCAM-1 in human ameloblastoma (AB) and odontogenic keratocyst (OKC) and investigate the relation of ICAM-1 and VCAM-1 with the pathologic characteristic of AB and OKC. METHODS: The specimens of 38 cases of AB, 10 cases of OKC, 7 cases of normal oral mucosa (NOM) were examined by streptovidin-biotin method. Their expression was analyzed combining the pathologic characteristics. RESULTS: Comparing AB with OKC and NOM, there was a significant difference in ICAM-1 and VCAM-1 (P<0.05). The positive expression of ICAM-1 in AB was 65.2%, much higher than that in NOM(14.3%). There was no difference between the expression of ICAM-1 in OKC(60.0%) and in NOM. The positive vessels number stained by VCAM-1 in AB was significantly higher than that in OKC and NOM. The expression of ICAM-1 and VCAM-1 had no correlation to age, sex, histological types, lesion locations(P>0.05). CONCLUSION: ICAM-1 and VCAM-1 play an important role in genesis, development of AB and OKC and are related to cell differentiation and proliferation.

A comparative stereologic and ultrastructural study of blood vessels in odontogenic keratocysts and dentigerous cysts.

Blood vessels were investigated both stereologically and ultrastructurally in keratocyst and dentigerous cyst. The volume and surface densities of blood vessels in 15 keratocysts and dentigerous cysts were analyzed stereologically. No significant differences were found between them using these parameters, suggesting that their overall vascularity may be similar. However, the ultrastructural study showed marked differences between blood vessels in these two types of cysts. It was observed that fenestrated capillaries were found only in keratocysts. In addition, degeneration of endothelial lining associated with thrombosis was also a prominent feature of this cyst. While ruptured endothelium, narrow lumen and Weibel-Palade bodies were characteristic of vessels in dentigerous cyst. The presence of fenestrated capillaries in keratocyst and not in dentigerous cyst might indicate a rapid transfer of fluid to meet the demand of the relatively active proliferating epithelium, which may be promoted by growth factors released from platelets in those thrombosed vessels.