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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Intra-epithelially entrapped blood vessels in ameloblastoma.

BACKGROUND: The ameloblastoma is a benign but locally aggressive odontogenic neoplasm with a high recurrence rate. While significant progress has been made in our understanding regarding the role of tumoral vasculature relative to the diverse behavioral characteristics of this tumor, no attention has been paid to a distinct subset of blood vessels entrapped within its epithelial compartment. As vascular niches are known to influence tumoral growth, clarification of these vessels is important. The objectives of this study were to investigate the morphologic characteristics of intra-epithelially entrapped blood vessels (IEBVs) in ameloblastoma and to speculate on their relevance. MATERIALS AND METHOD: Here, we evaluated the frequency, microvessel density (MVD), morphology, and distribution pattern of IEBVs in 77 ameloblastoma of different subtypes based on their immunoreactivity for endothelial markers (CD34, CD31, CD105), vascular tight junction protein (claudin-5), pericyte [α-smooth muscle actin (α-sma)], and vascular basement membrane (collagen IV). RESULTS: IEBVs were heterogeneously detected in ameloblastoma. Their mean MVD (CD34 = 15.46 ± 7.25; CD31 = 15.8 ± 5.04; CD105 = 0.82 ± 0.51) showed no significant correlation with different subtypes, and between primary and recurrent tumors (P > 0.05). These microvessels may occur as single/clusters of capillary sprouts, or formed compressed branching/non-branching slits entrapped within the epithelial compartment, and in direct apposition with polyhedral/granular neoplastic epithelial cells. They expressed proteins for endothelial tight junctions (claudin-5-positive) and pericytes (α-sma-positive) but had deficient basement membrane (collagen IV weak to absent). Aberrant expression for CD34, CD31, and CD105 in tumor epithelium was variably observed. CONCLUSIONS: Although rare in occurrence, identification of IEBVs in ameloblastoma could potentially represent a new paradigm for vascular assessment of this neoplasm.

Expression of CD34 and maspin in ameloblastoma from a West African subpopulation.

Ameloblastoma is a locally invasive odontogenic tumor with a high recurrence rate. Its local invasiveness is aided by angiogenesis, which can be correctly estimated by CD34. On the other hand, maspin decreases the local invasive and metastatic capability of cancer cells and functions as an angiogenesis inhibitor. We aim to assess the association between maspin expression and microvessel density in ameloblastoma. Twenty-five formalin-fixed paraffin-embedded (FFPE) blocks of ameloblastoma cases were prepared for antibody processing to CD34 and maspin. Positive immunohistochemical staining was marked by brown cytoplasmic/membrane coloration for CD34 and by nuclear/cytoplasmic coloration for maspin. At the ×40 magnification, we counted blood vessels in two areas of dimension; 300 × 400 µm (area A) and 150 × 200 µm (area B) adjacent to the tumor region to assess relative dispersion of the vessels bordering the tumor. The overall approximate microvessel density (MVD) for area A = 11 (minimum 2, maximum 21) and that for area B = 5 (minimum 1, maximum 10). The MVD in the area A of plexiform ameloblastoma was similar to that of the unicystic, while the hemangiomatous variant had the highest MVD for area A. Maspin positivity was present only in the cytoplasm of ameloblast, stellate reticulum, and the fibrous connective tissue in varying proportions. There was no evidence of the anti-angiogenesis effect of maspin in ameloblastoma from this study. The significance of cytoplasmic localization of maspin in the ameloblasts and stellate reticulum cells needs further investigation.

Vascularized mandibular anterior ameloblastoma - an entity still unresolved.

Vascularized ameloblastoma is a bewildering entity whose existence is questionable from its origin to nosology and its very characterization as a distinct variant of ameloblastoma. This uncertainty is largely because of a fewer number of documented cases and loss of long-term follow-up. The current paper describes two cases of ameloblastoma in the mandibular anterior region, which had features of so-called "hemangiomatous ameloblastoma" as it was originally described. Understanding its pathophysiology based on various views and clinical implications in terms of its biologic behavior are brought to light in this paper.

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.

Tumor angiogenesis: role in locally aggressive biological behavior of ameloblastoma and keratocystic odontogenic tumor.

BACKGROUND: The purpose of this study was to assess and compare angiogenesis in ameloblastoma, keratocystic odontogenic tumors, dentigerous cysts, and normal oral mucosa. METHODS: Angiogenesis was assessed in 28 ameloblastoma-36 keratocystic odontogenic tumors, 28 dentigerous cysts, and 19 normal oral mucosa by measuring the mean vascular density (MVD), total vascular area (TVA) and mean vascular area (MVA). Immunohistochemistry was carried out by using CD105. RESULTS: The nonsignificant difference of MVD was noted between ameloblastoma and keratocystic odontogenic tumors (p = .174). TVA and MVA were significantly higher in ameloblastoma than keratocystic odontogenic tumors, normal oral mucosa, and dentigerous cysts (p < .001). MVD, TVA, and MVA were significantly higher in keratocystic odontogenic tumors than normal oral mucosa and dentigerous cysts (p < .001). CONCLUSION: The results suggest that tumor angiogenesis may play an important role in locally invasive aggressive biologic behavior of ameloblastoma and keratocystic odontogenic tumor. The angiogenesis could be a potent target for developing antiangiogenic therapeutic strategies, particularly in recurrent cases of odontogenic tumors.

Tumor-associated macrophages and angiogenesis: a statistical correlation that could reflect a critical relationship in ameloblastoma.

Neoplasm growth is determined not only by the tumor cells themselves, but also by the tumor microenvironment. Increased densities of macrophages and activation of angiogenesis have been identified as common events in the progression of several neoplasms. Ameloblastoma is one of the most frequent odontogenic tumors and an excellent model for the study of neoplasm progression due to the different clinical variants that it exhibits. Here, by immunohistochemical studies using antibodies against CD68 and CD34, we evaluated the density of macrophages and microvessels associated to 45 paraffin-embedded ameloblastomas. In solid/multicystic ameloblastoma (SMA), we observed significantly higher densities of both macrophages and microvessels than in unicystic (UA) and desmoplastic (DA) ameloblastomas. Likewise, higher densities of macrophages and microvessels were found in UA than in DA. Furthermore, a predominance of intratumoral and peritumoral macrophage infiltrates was seen in SMA, while in UA, both macrophages and microvessels were also detected in the wall of the cysts. In contrast, DA had scant macrophages and microvessels, mainly situated distant from tumoral cells. In addition, a high correlation between macrophage and microvessel densities was observed in the samples (r=0.9623). Our results suggest that these two tumor microenvironmental elements could have an important role during ameloblastoma progression.

Ultrasonographic evaluation of mandibular ameloblastoma: a preliminary observation.

OBJECTIVE: The purpose of this study was to demonstrate ultrasonographic characteristics of mandibular ameloblastoma and assess the value of ultrasonography in diagnosis of the tumor. STUDY DESIGN: Nineteen subjects with ameloblastomas in the mandibles were examined with ultrasonography. Locations, sizes, internal echoes, boundaries, and blood flow of the tumors were observed and documented. Ultrasonographic appearances of the tumors were compared with histopathological findings. Sensitivity and specificity of Doppler flow signals for prediction of active tumor proliferations were calculated. RESULTS: The main sonographic features of the tumor appeared as a complex cystic mass with solid contents. Most tumors (15/19, 79%) showed no or minimal flow signals on color Doppler flow imaging (CDFI), whereas the remaining 4 lesions demonstrated abundant flow signals. The sensitivity and specificity of the Doppler flow signals for prediction of active tumor proliferations were 100% and 94%, respectively. The ultrasonographic appearances could be classified into 4 types: multilocular (10/19, 53%), honeycomb (4/19, 21%), unilocular (3/19, 16%), and local severe destructive (2/19, 10%). CONCLUSION: Ultrasonography can be used as an effective supplementary diagnostic method for mandibular ameloblastomas. CDFI of tumor vascularity could be used to predict active tumor proliferations.

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.

[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.

Association between vascular endothelial growth factor (VEGF) expression and tumor angiogenesis in ameloblastomas.

BACKGROUND: Expression of vascular endothelial growth factor (VEGF), a major angiogenic factor, and microvessel density (MVD), assessed by the use of anti-CD34 antibody, were immunohistochemically examined in benign and malignant ameloblastomas, as well as tooth germs, to clarify the possible role of angiogenesis in epithelial odontogenic tumors. METHODS: Specimens of 5 tooth germs, 35 benign ameloblastomas and 5 malignant ameloblastomas were examined by immunohistochemistry using anti-VEGF and CD34 monoclonal antibodies. RESULTS: Immunoreactivity for VEGF was detected in both normal and neoplastic odontogenic epithelial cells, and weakly in microvessels near odontogenic epithelial cells, suggesting that this angiogenic factor acts on endothelial cells via a paracrine mechanism in odontogenic tissues. Both benign and malignant ameloblastomas showed elevated VEGF expression as compared to tooth germs. VEGF expression was low in keratinizing cells in acanthomatous ameloblastomas and granular cells in granular cell ameloblastomas, and acanthomatous ameloblastomas showed the lowest VEGF reactivity among the subtypes of ameloblastomas. MVD in both benign and malignant ameloblastomas was higher than that in tooth germs, indicating increased demands for blood in the neoplastic tissues. CD34-positive microvessels in follicular ameloblastomas were numerous and small, whereas those in plexiform ameloblastomas were scattered and dilated. MVD tended to depend on VEGF expression levels in both benign and malignant ameloblastomas. CONCLUSIONS: VEGF was considered to be an important mediator of angiogenesis in these epithelial odontogenic tumors, and up-regulation of VEGF might be associated with neoplastic or malignant changes of odontogenic epithelial cells.

Hemangiomatous ameloblastoma: Clinical, radiologic, and pathologic features.

A hemangiomatous ameloblastoma was present in the third molar region of the left mandible of a 26-year-old woman. The histology and radiologic features of this tumor differed from those of a conventional ameloblastoma. Its histologic features were consistent with those of a hemangiomatous ameloblastoma, and its standard radiologic features and computed tomography mimicked that of fibro-osseous lesions, whereas magnetic resonance imaging suggested a vascular lesion. The behavior and prognosis of the hemangiomatous ameloblastoma are uncertain because of the small number of documented cases and lack of long-term follow-up, but are thought to be similar to those of the conventional type. The relevant clinical, radiologic, and pathologic features of this case are presented.