Expression of VEGF₁₆₅b, VEGFR1, VEGFR2 and CD34 in benign and malignant tumors of parotid glands

,


Introduction
Malignant salivary gland tumors are highly invasive and frequently result in distant metastasis.The ability of cancer cells to invade stroma and then to metastasize to both the regional lymph nodes and distant tissue is a complex, multistage process.Tumor growth is regulated by the intensity of angiogenesis.Moreover, newly formed vessels have an impact on the ability of tumor cells to form metastases. Specific angiogenic molecules produced by tumor cells stimulate the surrounding capillary endothelial cells resulting in angiogenesis into tumor tissues.2][3][4][5][6] VEGF is a selective mitogen for vascular endothelial cells, which promotes angiogenesis and induces vascular permeability and therefore its inhibition could be integrated into treatment strategies. 3Several studies have evaluated whether VEGF immunohistochemical expression may be a prognostic factor for salivary gland carcinomas, but the results are conflicting or inconclusive.In our opinion, the possible source of these conflicting results may be the antagonistic antiangiogenic and proangiogenic properties of VEGF.
VEGF belongs to the platelet-derived growth factor (PDGF) superfamily and consists of glycoproteins with different structures and functions.It functions as a general tumor proangiogenic factor, but there are also different isoforms of VEGF with antiangiogenic properties.Two families of VEGF proteins are formed by an alternative splice-acceptor-site to give to 2 distinctive C-terminal sequences differing in their angiogenic properties.VEGF exerts its biological function by binding to its receptors: VEGFR1, VEGFR2 and VEGFR3.These 2 isoforms bind to VEGFR2 with the same affinity, but the binding of VEGF 165 b results in an insufficient activation of VEGFR2 and an impaired angiogenic response.The imbalance of VEGF 165 (proangiogenic) and VEGF 165 b (antiangiogenic) isoforms can underpin pathological angiogenesis.][9][10][11][12][13] The imbalance of VEGF isoforms in the pathogenesis of salivary gland tumors has not yet been examined.
Another very important issue is the distribution of these isoforms and their receptors in intratumoral and peritumoral tissues.Their different distribution in tumor and stromal cells may have a crucial role for tumor invasiveness. 7Although angiogenesis is difficult to measure directly in human tumors, there is increasing evidence that MVD may be an indirect marker of angiogenesis.One of the most common antibodies used for microvessel staining is CD34, which has been used in immunohistochemical studies to evaluate the intra-and peritumoral changes induced by VEGF. 14,15Despite the progress made in recent years in understanding the role of VEGF in salivary gland tumors, more data is needed to better elucidate the relationship between the expression of VEGF isoforms and tumor prognosis.To address this need, we determined the prognostic potential of these markers.
The purpose of this study was to evaluate and compare the immunohistochemical expression of VEGF 165 b, VEGFR1, VEGFR2, and CD34 in benign and malignant parotid gland tumors and in surgical margins from the same patients taken as a control group, and to explore the possible correlations between expression of these peptides and the clinicopathological features of tumors.

Study group
The study was performed on archived paraffin-embedded tissue samples derived from 70 patients with benign and malignant parotid gland tumors (25 with malignant tumor, 23 with pleomorphic adenoma, and 22 with Warthin's tumor), which were collected in the Department of Oncologic Pathology, Poznan University of Medical Sciences and in the Department of Pathomorphology, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, after surgical treatment of the primary tumor.Exclusion criteria included the presence of lymphoma, previous parotid gland surgery, and other parotid gland pathologies.Clinical examinations included ultrasound and, in selected cases, computed tomography (CT) or magnetic resonance imaging (MRI) to confirm the diagnosis.All tumors were described by location and size.Additionally, malignant tumors were evaluated for stage (7 th edition of the American Joint Committee on Cancer TNM staging), level of differentiation, regional lymph node metastasis status, and grade. 16Tumor diagnosis was performed by pre-and postoperative histopathological examinations.The tumor diagnosis was performed independently by 2 pathologists.Disease-free tumor resection margins, located at least 2 cm from the tumor, were used as controls.
The protocol for this study was approved by the Bioethics Committee of Poznan University of Medical Sciences, Poland (No. 744/11).All patients provided signed written informed consent.

Tissue microarray paraffin blocks
Tissue microarray paraffin blocks (TMA) paraffin blocks were prepared for the evaluation of selected protein expression in the same location in the primary tissue sections.The primary paraffin embedded tissue sections (donor blocks) were re-embedded into paraffin-wax tissue blocks.Two tissue cylinders (2 mm in diameter) from each case were selected from the "donor blocks", punched from marked regions, and subsequently placed into the recipient TMA blocks using 3DHISTECH TMA Master v. 1.14 (3DHISTECH Ltd, Budapest, Hungary).

Immunohistochemistry
The paraffin blocks were cut into 4 µm tissue sections using a rotary microtome (Accu-Cut ® SMRTM200, Sakura, Japan).To establish immunohistochemical procedures, a series of positive control reactions were performed by determining the presence of antigens (The Human Protein Atlas). 17The negative control reactions were performed on additional tissue sections during proper immunohistochemical staining, by substituting the primary antibody for a solution of 1% bovine serum albumin (BSA) in phosphate-buffered saline.Immunohistochemical staining was performed according to protocols described in detail elsewhere. 2Immunohistochemical staining of selected tissue sections was performed using the monoclonal antibodies listed in Table 1.Epitopes were unmasked by Epitope Retrieval Solution high-pH (Dako Denmark A/S, Glostrup, Denmark) and then the slides were incubated overnight (16 h) with a primary antibody at 4˚C.The antibody complex was detected with EnVisionFlex Anti-Mouse/Rabbit HRP Labeled Polymer (Dako Denmark A/S, Glostrup, Denmark).Antigens were localized according to the presence of a brown reaction product, using DAB as a chromogen.Finally, the sections were counterstained with hematoxylin, dehydrated in increasing grades of ethyl alcohol (80%, 90%, 96%, and 99.8%) and mounted with Shandon Consul Mount (Thermo Fisher Scientific, Waltham, USA).
The results were analyzed using a light microscope (ECLIPSE E800; Nikon Instruments Europe, Amsterdam, the Netherlands), and the level of expression was estimated using morphometric principles.To assess the level of protein expression, we used the modified Remmele-Stegner scale (Index Remmele-Stegner IRS -immunoreactive score), according to the intensity of expression and the number of cells/tissue area positively expressed in our previous publications. 2,18Expression of VEGF 165 b, VEGFR1 and VEGFR2 was localized in the cytoplasm and graded as follows: (−) negative; (+) weak positive staining; (++) moderate positive staining; and (+++) strong positive staining.Expression of CD34 was assessed as a positive (>5 blood vessels in the field of view) or negative (<5 blood vessels in the field of view) staining.

Statistical analysis
The calculations were carried out with the use of Microsoft Excel 2010 and STATISTICA v. 10 software (StatSoft Inc., Tulsa, USA).The distributions of continuous variables obtained at each step of data processing were evaluated for normality using the Shapiro-Wilk test.The age of each group is expressed as a median with an interquartile range (IQR).Categorical variables resulting from immunohistochemistry are presented in contingency tables and they were tested depending on the number of cases using the χ 2 test or two-tailed Fisher's exact test.For paired nominal data, the on-line McNemar's test with continuity correction was used. 19Probability value <0.05 was considered statistically significant.

Results
There was a statistically significant difference in the expression of VEGFR1 in malignant tumors between females and males (Table 2).A statistically significant difference in the expression of VEGFR1 and a score of T classification in malignant tumors was found (Table 2).A statistically significant correlation between the expression of VEGF 165 b and VEGFR2 in pleomorphic adenomas was found (Table 3).There were no statistically significant differences in the expression of the selected proteins localized in the tumor and the surgical margin taken from the same patient (Table 4).

Discussion
Although it is clear that there is a relationship between the expression of VEGF and the development of salivary gland tumors, the exact nature of these relationships remains to be fully explained.In the present study, we analyzed the tissue expression of VEGF 165 b, VEGFR1, VEGFR2, and CD34 in pleomorphic adenomas, Warthin's tumors and malignant tumors of the parotid glands to determine their potential value as prognostic and differentiating markers for these tumors.We also assessed expression of VEGF 165 b, VEGFR1, VEGFR2, and CD34 to check for correlations in tumorous and non-tumorous tissues, finding no difference between expression of these molecules in these different tissues.VEGF receptors correlated with selected   Taken together, these findings suggest that the balance of proangiogenic and antiangiogenic VEGF isoforms seems to have a limited influence on the development of parotid gland tumors, and therefore further research is required.
VEGF 165 b cannot be treated as a prognostic and differentiating factor.The correlation between VEGF 165 b and VEGFR2 in mixed tumors suggests the existence of an additional antiangiogenic pathway in poorly vascularized mixed tumors.There were some reports of correlations between the clinicopathological factors of salivary gland tumors and VEGF.According to Lim et al., increased VEGF expression correlated with lymph node metastasis, clinical stage, perineural invasion, vascular invasion, recurrence, and survival. 20Similar results were obtained by Lequerica-Fernández et al., who found a relationship between VEGF expression and neck node disease, clinical stage, survival and local control of the tumor. 6In our study, antiangiogenic VEGF 165 b did not correlate with better prognosis and a less severe clinical stage in malignant tumors.VEGF 165 b expression in intratumoral and peritumoral tissues was insufficient for the inhibition of tumor growth and for its invasiveness, or it was limited by VEGF 165 or other undetermined angiogenic factors.
The regulation of VEGF alternative splicing is unknown.VEGF gene expression is transcriptionally regulated by a diversity of factors including hypoxia, growth factors, * median (IQR); # C. ductale (n = 4); adenocarcinoma (n = 4); C. planoepitheliale kera (n = 4); C. adenoides cysticum (n = 3); C. mucoepidermale (n = 3); C. myoepithelial (n = 2); acinic cell carcinoma (n = 1); basal cell carcinoma (n = 1); C. glandulae salivare (n = 1); C. neuroendocrine (n = 1); C. ex pleomorphum (n = 1); TNM: T -size or direct extent of the primary tumor, N -degree of spread to regional lymph nodes, M -presence of distant metastasis; a two-tailed Fisher's exact test; b χ 2 for contingency tables.such as insulin-like growth factor 1 (IGF-1), transforming growth factor β1 (TGF-β1), transforming growth factor α (TGF-α), oncogenes and tumor suppressor genes. 10Ishibashi et al. postulated that hypoxia may be one of the factors inducing angiogenesis in salivary gland carcinomas by producing VEGF. 1 Distribution of these angiogenic factors in tumorous and non-tumorous tissues varies depending on the type of tissue and the local microenvironment.In our opinion, for a comprehensive assessment of the VEGF 165 b and VEGF 165 relationship and their distribution in tumor tissues, the influence of other possible splicing factors should be considered.
Among the molecules which were determined in our study, VEGFR1 expression correlated with the T score in malignant tumors.According to Younes et al., AEE788, a dual inhibitor of EGF and VEGF receptor tyrosine kinases, increased tumor and cell apoptosis, and decreased microvessel density, which correlated with a decrease in the incidence of vascular metastasis of salivary adenoid cystic carcinoma.This data showed that both receptors, EGFR and VEGFR, can be molecular targets for therapy of salivary ACC. 3 In the study by de Faria et al., VEGF and its receptor expression levels discriminated benign and malignant tumors of salivary glands but they cannot be treated as a predictor of metastasis from non-metastasizing tumors. 4In our study, VEGFR1 was weakly expressed but correlated with the T score in malignant tumors.This result may suggest its important influence in tumor growth and invasiveness.VEGFR1 seems to be a more sensitive marker of tumor growth than VEGFR2.Although VEGFR2 was more strongly expressed in our study than VEGFR1, it did not correlate with clinical data for malignant tumors.In our opinion, these positive correlations reflected the increased levels of proangiogenic VEGF isoforms in malignant tumors.Expression of VEGFR2 resulted from both VEGF 165 and VEGF 165 b activity.
VEGFR1 expression in malignant tumors varied for different genders.This correlation was observed only in malignant tumors.In a previous study by Lim et al., the male gender was connected with shorter survival in salivary gland carcinomas. 20Many studies have concentrated on the differences in survival in colon cancers according .3341 1 0 .6517 .6462 0 1 T -size or direct extent of the primary tumor, N -degree of spread to regional lymph nodes, M -presence of distant metastasis; a two-tailed Fisher's exact test; b χ 2 for contingency tables.
Table 2. Clinical characteristics of patients with malignant parotid gland cancers, pleomorphic adenoma and Warthin's tumor, and the association of selected parameters with the distribution of VEGF 165 b, VEGFR1, VEGFR2 assessed as negative (-) for no or weak intensity of expression (0 and 1) and positive (+) for moderate and strong intensity of expression (2 and 3), and CD34 expression assessed as negative (-) or positive (+) by immunohistochemistry (cont.) gland carcinomas were explored.EGF and VEGF belong to the same family of growth factors and their functions overlap very often.In our view, a larger-scale study would be needed to explain the relation between gender and angiogenic factors in parotid tumors.There were no statistically significant differences in the expression of molecules determined in the study and localized in the tumor and surgical margin taken from the same patient.Similar results were obtained by Tayama et al.In this study, VEGF was expressed on similar levels in both tumor and stromal cells.Tumor   to gender-dependent polymorphism of the VEGF gene. 21imilar associations between gender, age and family history -dependent VEGF gene polymorphism and the increased risk of osteosarcoma -were determined in studies by Tie et al. 22 Patel et al., in contrast, did not observe any demographic and gender predispositions for Warthin's tumor compared to other benign salivary gland tumors.However, in the growth of benign tumors, VEGF and its isoforms seem to play a limited role. 23Polymorphisms of VEGF and their receptors in salivary gland tumors have not been yet determined.Previously, only the influence of EGF, its receptor EGFR and their polymorphism in salivary In our study, VEGF 165 b correlated with VEGFR2 in mixed tumors.This is a highly unexpected result, because the stroma of pleomorphic adenoma is always poor in terms of vascularity.This is a contradiction to the general idea of highly vascular neoplasm architectures on which antitumor therapeutic strategies have been devised.On the other hand, in the poorly vascularized tissues of mixed tumors, the hypoxic conditions promote higher VEGF expression.Swelam et al. demonstrated VEGF and its receptors in normal duct epithelial and myoepithelial cells as well as in tumor cells in ductal structures and in myxochondroid stromata.Additionally, they determined at least 4 proangiogenic VEGF isoforms, among which VEGF 121 was the most enhanced.They also observed higher HIF-1α levels in mixed tumors. 5These previous results suggested that mixed tumor cells produce VEGF in several functional forms for their own proliferation or differentiation, and that VEGF expression is controlled by the hypoxic circumstances of poorly vascularized mixed tumors.Our results also revealed the existence of an antiangiogenic VEGF 165 b/ VEGFR2 pathway in mixed tumors.In our opinion, this pathway is strictly associated with the special histological structure or special conditions of a mixed tumor.However, we do not explain which mixed tumor cells might be a source of this antiangiogenic mechanism and whether it is primary or secondary to the proangiogenic VEGF 165 , and which factors can foster its production.
The development of parotid gland tumors is not associated with deterioration in the balance between proangiogenic and antiangiogenic VEGF isoforms.VEGF 165 b levels did not correspond with a less severe clinical stage of parotid gland tumors.Furthermore, this cannot be treated as a prognostic or differentiating factor of malignant and benign parotid gland tumors.Without a comprehensive assessment of the VEGF 165 b/VEGF 165 ratio in tumor and stromal tissues and the influence of their receptors, VEGFR1 and VEGFR2, its expression has limited importance.In our opinion, a more important issue is the ratio of proangiogenic/antiangiogenic VEGF isoforms in the tissue in question.VEGFR1 expression correlated with selected clinicopathological data of malignant tumors, indicating an additional field

3 -
clinicopathological data of malignant tumors, indicating their possible role as a prognostic marker of salivary tumors.

Table 1 .
Primary antibodies used in immunohistochemistry (IHC)

Table 2 .
Clinical characteristics of patients with malignant parotid gland cancers, pleomorphic adenoma and Warthin's tumor, and the association of selected parameters with the distribution of VEGF 165 b, VEGFR1, VEGFR2 assessed as negative (-) for no or weak intensity of expression (0 and 1) and positive (+) for moderate and strong intensity of expression (2 and 3), and CD34 expression assessed as negative (-) or positive (+) by immunohistochemistry

Table 4 .
Associations of VEGF 165 b, VEGFR1, VEGFR2, and CD34 expression assessed as weak (-) or strong (+) between the tumor and surgical margins taken as a control in the groups studied * McNemar's test with the continuity correction for paired data.

Table 3 .
Associations of VEGF 165 b, VEGFR1, VEGFR2, and CD34 expression in malignant and benign parotid tumors