Study of correlation between vascular endothelial growth factor receptor and some prognostic factors in papillary carcinoma of thyroid gland

1. Introduction

ccording to latest reports of the Ministry of Health of Iran, thyroid cancer is the seventh most common cancer in women. Age-specific incidence rates in men and women are 2.80 and 1.60, respectively.Also the incidence of thyroid cancer in women and men is 1.19% and 4.36% of all cancers, respectively. The most common age of thyroid cancer in women is 45-48 years and in men 80-84 years (1). In United States the incidence of thyroid cancer is lower and includes less than 1% of all cancers (2% in women and 0/5% in men). The annual mortality from thyroid cancer is 6 per million. Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer which includes 80% of all thyroid malignancies (female to male ratio 2/1) (2, 3). PTC appears as a solitary nodule then

through thyroid lymphatic vessels spreads to subcapsular and pricapsular area. It presents with a palpable mass in 80% of children and 29% of adult and can metastases to lung or bone (4). After diagnosis of cancer for determining of the prognosis and to choose the type of treatment, multiple clinical parameters such as TNM staging, pathological andmolecular factors are used. Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) are considered as factors in determining the prognosis. VEGF is the strongest known protein that leads to specific angiogenic and mitogenic change in endothelial cell and increases the vascular permeability. This factor induces significant angiogenic response in vivo (5-9). The new vessels endothelial cell growth is dependent on VEGF (10). Three endothelial receptors with high affinity to VEGF are known. These receptors are VEGFR-1/ Flt-1، VEGFR-2/ Flk-1/ KDR, VEGFR-3/ Flt-4 that transmit signals during vessel formation (11). VEGFR-1 and VEGFR-2 are cell surface tyrosine kinase receptors which localized endothelial cells during embryonic development. Association of VEGF gene expression patterns and its receptors show that these proteins play an important role in vessels development in fetal period (12, 13). VEGFR-Tks is a member of tyrosine kinase large family and play an important role in messaging pathway leads to proliferation, differentiation, migration and anabolism of cells. Recently, Bernatches et al. used oligomers against FLK-2 and FLt-2 and show that proliferation, differentiation and migration of endothelial cells and platelet-activating factor are dependent on Flk-2, whereas inhibition of Flt-1 has no effect on VEGF activates. These studies showed that targeting VEGFR-2 messaging process could be effective in prevention of angiogenesis in tumors (14). VEGF and its receptors have a role in angiogenesis in many solid tumor like cancers of breast, colon, urinary bladder, gastric, liver, and prostate. Since solid tumors growth are dependent on angiogenesis, several therapeutic strategies based on targeting the VEGF path-way as anti-cancer therapy is being formed. Main approaches are prevention of the secretion of VEGF, neutralization of VEGF in capillary blood circulation and prevention of VEGF binding to its receptors (15-20). The purpose of this study was to determine the VEGF receptor expression as a prognostic marker in the prognosis of papillary carcinoma of thyroid gland.

2. Materials and Methods

Data of 70 diagnosed cases of papillary carcinoma of thyroid gland during the period of 2009 to 2011 were retrieved from the files of Department of Pathology of Mostafa Khomeini hospital, Tehran, Iran.

The data were analyzed according to age, gender, tumor size and lymph node status.Tissue blocks were cut at 5 micron, stained by conventional Hematoxylin and Eosin method to identify tumor and lymph node metastasis.

Another slide with 3 micron thick sections was prepared. These slides were deparaffinized in xylene and rehydrated through graded concentrations of ethanol. After washing with 10% phosphate buffered saline (PBS), the slides were placed in H2O2- methanol solution (1/9) for 10 minutes, washing with 10% PBS. Then they were placed in EDTA at 120 ºc for 15 minutes. The slides were chilled at room temperature, and washed with 10% PBS.

At next step, the slides were immunohisto- chemically stained according to kit instruction (Novacastra, UK).

At the end of immunostaining, we used Mayers Hematoxylin for counterstaining, then rinising slides with distilled water, rehydrated them through graded concentrations of ethanol, mounted with entellane and coverslipped. The expression or absence of VEGFR was examined by light microscopy.

Statistical analysis was performed using SPSS statistical software, Mann-Whitney and Chi-Square. We used '' percent'' for descriptive purpose. A p-value ≤ 0.05 was considered statistically significant.

3. Results

This study was done on 92 cases of papillary carcinoma of thyroid gland. The mean age of patients was 39.32± 16.93 years, ranging from 13 to 80 years. Meanwhile, 40.2% (37 cases) were positive for VEGFR expression and 59.8% (55 cases) were negative.

"Figure 1" details these data. 14.1% (13) cases were male and 85.9% (79 cases) were female (Figure 2). In this study, 23.9% (22 cases) had lymph node metastasis and 76.1% (70 cases) were negative for lymph node metastasis (Figure 3).

Figure.1: The frequency of VEGFR expression in papillary carcinoma of thyroid gland

Figure.2: The frequency of gender of patients with papillary carcinoma of thyroid

Figure.3: The frequency of Lymph nodes involvement

Tumor size ranged from 0.5 to 14 cm (mean 3.6± 22 cm).

Using Chi-Square, no significant relationship between the expression of VEGFR and the patients age was found (p=0.633).

Table 1 details the numbers of VEGFR positive and negative cases in both sexes. From 37 cases

with VEGFR expression, 14 cases had lymph node metastasis and from 55 cases with no expression of VEGFR, 47 cases found with no lymph node metastasis.

According to Chi-Square, meaningful relationship was observed between the absence of VEGFR expression and un-ivolvement of lymph nodes(p=0.01). These data are shown in Table 2.

Using Mann-Whithney test, no significant relationship was found between VEGFR expression and tumor size (p=0.458) (Table3).

Table 1: Frequency of VEGFR

Table 2: The correlation between lymph node involvement and VEGR expression

Table 3: The correlation between tumor size and VEGR expression