Angiogenesis is an essential process in the growth of the primary tumor and the development of metastasis . VEGF plays a key role in tumor angiogenesis and it has been identified in many malignancies, including head and neck squamous cell carcinoma [2, 7, 12, 13]. However, a number of different factors can regulate VEGF expression including hypoxia, cytokines, oncogenes and tumor suppressor genes . PTEN, a tumor suppressor gene, is a PI3K antagonist. PI3K has a role in many cellular events such as cell proliferation and survival, protein synthesis and tumor growth. PI3K and Akt have a role in tumor growth and angiogenesis by regulating VEGF and hypoxia-inducible factor-1 expression . Our results showed that there was no correlation between PTEN expression and VEGF expression which has not been investigated in the previous studies of the squamous cell carcinoma of the larynx. Also there was not a significant relationship between PTEN expression and tumor grade, tumor size and cartilage invasion in the laryngeal squamous cell carcinoma. This study demonstrates that VEGF expression is closely related to the poor clinicopathologic features including tumor grade, tumor size and lymph node metastasis of laryngeal squamous cell carcinoma.
After the first demonstration of the prognostic value of tumor angiogenesis in 1988 in malignant melanoma, VEGF expression has been studied in various tumor. The relationship of VEGF expression with parameters such as tumor differentiation, lymph node metastasis, and invasion depth has been reported in various tumors [12, 13, 15, 16]. The findings of studies assessing the relationship between VEGF expression and tumor differentiation, stage, and lymph node metastasis differ in head and neck carcinomas [13, 17, 18]. In a study reporting that there was no relationship between VEGF expression and tumor grade and localization in head and neck epidermoid carcinomas, it was reported that there was a correlation between increased VEGF expression and shortened life span . In a series of head and neck epidermoid carcinomas with only nine cases of laryngeal carcinoma, there was no relationship between VEGF expression and histological grade and lymph node metastasis but VEGF expression was correlated with life span, especially in tumors of the larynx and the oral cavity . Tae et al.  reported that there was no correlation between VEGF expression and tumor differentiation, stage, and localization in carcinomas of the head and neck region. They also stated that VEGF expression was at higher levels in benign lesions than in dysplasias and carcinomas, and suggested that VEGF may be effective in regulating the mucosa functions in normal physiological conditions. Unlike our study and other studies, VEGF expression was at higher levels in the group without lymph node metastasis in this study . The finding in our study that there was a significant relationship between VEGF expression and tumor differentiation, size, and lymph node metastasis supports the association of increased VEGF expression with invasive and aggressive forms of laryngeal tumors. The reasons for the different findings in our study may be the differences in assessment of VEGF expression and the fact that the other studies included tumors with different localizations in the head and neck region.
The relationship between MVD and prognostic parameters in head and neck epidermoid carcinoma could not be completely explained . In spite of studies finding a relationship between high MVD and tumor progression, tumor stage and grade, studies suggesting that there was no such relationship have also been reported [7, 8, 12, 20–22]. Tse et al.  conducted a study on 186 cases with head and neck epidermoid carcinomas including 34 cases with laryngeal carcinoma. They reported that there was no relationship between MVD and tumor stage, grade, localization, metastasis or prognosis. It was reported in this study that VEGF was strongly expressed but there was no relationship between VEGF expression and MVD in head and neck epidermoid carcinomas. MVD assessment was performed by two different methods by selecting hot-spot and random sites based on the fact that epidermoid carcinoma was poor for stroma and had little vascular proliferation and hot-spot sites could not be selected properly. It was reported in two assessments that there was no relationship between MVD and VEGF expression and clinicopathologic parameters. Kyzas et al.  reported that there was no relationship between MVD and tumor grade, localization and lymph node metastasis in 69 cases with head and neck epidermoid carcinoma, and although they found a tendency of increase in MVD with the increased VEGF expression, this increase was not significant. In this study it was reported that they performed with CD31 and CD34 and that the correlation between VEGF expression and MVD was weaker when CD34 was used . The increased MVD in less differentiated tumors in our study suggested that the increase in MVD may be related to loss of differentiation. There may be several reasons why we did not find a relationship between VEGF expression and MVD. VEGF expression is only one of the angiogenic factors. Many molecules such as angiogenin, interleukin 8 and 10, platelet-derived endothelial growth factor, fibroblast growth factor, and angiopoetins have a role in tumor angiogenesis . Besides forming new vessel buds, VEGF is also used as an autocrine growth factor for tumor cells. The presence of VEGF receptors on tumor cells in head and neck epidermoid carcinomas has been demonstrated . Besides, it is known that other cellular elements such as macrophages and fibroblasts express VEGF at various levels . This point of view may explain the lack of a significant correlation between VEGF expression and MVD. Another reason for the discrepancy between VEGF expression and MVD and between MVD and the other parameters may be the lack of a direct method to demonstrate angiogenesis . The conventional antibodies used to show vessels are not specific to active angiogenic vessels . It has been reported that using antibodies that show endothelial cell proliferation to assess tumor vascularization would be beneficial . The lack of a significant relationship between VEGF expression and MVD shows that tumor angiogenesis is a complex process .
In addition to extracellular signals such as growth factors, the activation of oncogenes or the mutations of tumor suppressor genes such as PTEN and p53 trigger tumor angiogenesis. PTEN mutations were reported in ovarian, brain, breast, thyroid, endometrium and stomach tumors [25–28]. The relationship between PTEN expression and tumor differentiation, lymph node metastasis, and invasion depth has been reported in tumors of the brain, prostate, breast, and stomach [26, 28, 29]. While the loss of PTEN expression has been found to be correlated with the loss of differentiation in tumors of the oral cavity, it has also been reported that there was no significant relationship between PTEN expression and the tumor stage, differentiation, lymph node metastasis in laryngeal carcinoma . Squarize et al.  found a negative relationship between PTEN expression and the grade in oral squamous cell carcinomas and reported that PTEN was an independent prognostic factor. In a study reporting that there was a decrease in PTEN expression in laryngeal tumors, there was no relationship between PTEN expression and tumor localization, tumor size, differentiation and stage . We could not find a correlation between PTEN expression and tumor size, differentiation, lymph node metastasis and invasion depth. The relationship between PTEN expression and lymph node metastasis was the opposite of the expected; therefore, further studies are required on this topic.
The relationship between PTEN and angiogenesis has been explained by PTEN being a PI3K antagonist and therefore inhibiting angiogenesis . It has been reported that the decrease in PTEN expression was correlated with VEGF expression and MVD increase in gastric and colon tumors [16, 33]. Chung et al.  reported in their study with invasive ductal carcinoma that there was no relationship between PTEN expression and VEGF expression and MVD. Similarly we were not found significantly correlation between PTEN and VEGF expression and MVD. Further studies are needed to clearly explain the relationship between PTEN expression and angiogenesis.
In conclusion, VEGF expression was found to be correlated with grade, tumor size and lymph node metastasis in laryngeal tumors. However there was no correlation between PTEN expression and poor prognostic clinicopathologic factors. Increased VEGF expression may be useful to choose patients who are candidates for anti-VEGF treatment . The lack of significant association between PTEN expression, VEGF expression and MVD indicates that to reveal the complex phenomenon of tumor angiogenesis in the laryngeal squamous cell carcinoma further investigation is needed.