Vascular Endothelial Growth Factor (VEGF) Expression in Normal, Dysplastic and Neoplastic Squamous Epithelium of Oral Mucosa

Squamous cell carcinoma (SCC) is by far the most common malignancy of head and neck region with an estimated 5-year survival rate of 40% upon diagnosis. Multifactorial etiology has made this sinister pathology a poor candidate for therapy, with an added burden of poor quality of life despite treatment [1,2]. The characterization of molecular targets involved in initiation, progression and metastasis of SCC can play a vital role in devising the future therapeutics based on these oncotargets [3]. Vascular endothelial growth factor (VEGF) is a protein, which directly affects vascular endothelial cells, stimulates the proliferation of endothelial cells, and helps in chemotaxis of macrophages and granulocytes. Since malignant cells are rapidly dividing cells and nutrients are required in higher quantities, neovascularization, possibly via the overexpression of VEGF is needed. Previous studies have shown that VEGF is the main factor in neovascularization leading to tumor growth and metastasis [4-7]. Studies have shown that, as compared to normal squamous epithelium, the dysplastic and neoplastic mucosa have high expression of VEGF [8,9]; while other studies have found little or no correlation between VEGF expression and vascularity in oral cancers [10,11,12]. In order to examine the association between VEGF overexpression and malignant SCC, we compared the VEGF expression among normal, dysplastic and neoplastic mucosa [11,12].

This study was conducted in Department of Oral Pathology, Isfahan University of Medical Sciences dental school. Sixty samples of oral squamous epithelium from sixty individuals were obtained from the archives of the pathology laboratory, 20 in each group (normal, dysplastic and neoplastic).Only samples that had sufficient tissue and little inflammation were considered for this study. The normal mucosae were obtained from the patients who had undergone a dental implant with no mucosal pathology. Our study protocol was approved by Isfahan research and ethical committee. After standard laboratory protocol for sample handling, the tissues were immunohistochemically stained with the biotin-streptavidin (B-SA) method to detect the specific VEGF antigen [10]. Briefly, the main procedure included serial sectioning (in 3-4 μm sections), deparafinization, rehydration, and antigen retrieval. All specimens were placed in phosphate- buffered saline[PBS], treated with protein block (RS 1102) for 5 minutes to prevent any false staining, and then the specimens were incubated for 30 minutes with primary antibody of VEGF clone VG1 (DAKO, Denmark). Further, the sections were exposed to Novolink polymer (RE7112) or secondary antibody for 30 minutes and washed in PBS. Then they were incubated with DAB (Diamino-benzidin) for 5 minutes for visualization, and after washing, the slides were counter-stained with hematoxylin and finally after drying, the slides were mounted. The sections were observed by two pathologist using 100 and 400 magnifications of light microscope. Cells which were positive for VEGF were scored. Five non- overlapping fields were selected randomly and based on endothelial stain intensity each specimen was assigned a specific score. Where the epithelial cytoplasm stained stronger than endothelial cell, the field score would be 3 where it was in good harmony with endothelial cell it would be 2 and if it was weaker than endothelium it would receive a score of 1. The score of 0 was recorded if it was not stained. [10] Finally the mean intensity of 5 fields in one specimen recorded. The data were statistically analyzed by Mann Whitney and Kruskalwallis tests (SPSS, ver.10.0).

The study population consisted of 60 paraffin embedded tissues of 20 normal mucosa, 20 dysplastic mucosa and 20 SCC. Immuno-histochemical examination found positive reaction for VEGF in all samples of SCC, dysplastic, and normal mucosa. The mean expression of VEGF in SCC was 24.65, 16.35 in dysplastic mucosa, and 15.5 in normal mucosa. VEFG expression was significantly higher in the SCC group as compared to the dysplastic group (p=0.02) or normal group (P=0.001). On the other hand, there was no difference in the VEGF expression between the dysplatic mucosa and normal mucosa groups (p=0.11)

Oral SCC is the most common oral cancer. SCC arises from dysplastic epithelial cells and invades the underlying connective tissue in forms of cords and islands [13].Angiogenesis is a natural essential phenomenon in growth of tumors [4]. The effect of angiogenesis via VEGF on tumor metastasis and prognosis has been well-studied; VEGF directly affects the vascular endothelial cells and promote their proliferation and migration [14]. Over expression of VEGF and extensive angiogenesis has been reported in some precancerous oral lesions [6, 14]. About 10-20% of pre-cancerous lesions lead to oral SCC, therefore, recognizing the related molecular mechanism may help to highlight biological progression [15, 16]. In this study normal mucosa, dysplastic mucosa and SCC lesions of oral cavity were evaluated for angiogenic growth factors. To achieve this purpose, VEGF which is one of the important growth factor in angiogenesis, was examined. The results of this study show that the mean expression of VEGF in SCC is significantly higher than dysplastic or normal mucosa (24.65). Concomitantly, in previous studies, over expression of VEGF in malignant lesions were suggested [9, 12, 17]. Our study validates their findings. Although the mean VEGF expression in dysplastic mucosa (16.35) was higher than normal mucosa (15.5), the difference was not statically significant (P=0.108). A previous study found a significantly higher VEGF expression in dysplastic mucosa as compared to normal mucosa. The authors of that study further noted that VEGF expression may decrease during the tumorigenesis [6]. Thus, their findings are in line with our results that VEGF has a significant role in increased angiogenesis in SCC lesions in comparison with dysplastic and normal mucosa. In contrast to our and others findings, some studies have found no association between VEGF expression and malignancy. Sauter [18] did not find VEGF overexpression in malignant lesions; while calile et al [19] stated that VEGF merely has a physiologic role in tumors and no significant role in dysplastic and malignant changes. Moreover, it has been suggested that VEGF overexpression plays significant role in primary transformation of tumors at an earlier stage but later on genetics has greater influence on progression and metastasis [19].

In summary, we have shown that SCC has significantly higher expression of VEGF than dysplastic and normal epithelium. This finding confirms the previously reported role of VEGF in neovascularization in malignant cells. Whether VEGF expression is a physiological need of malignant tissues for increased blood flow or it plays some role in malignant transformation needs to be studied.

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