Association of Polymorphic Variants of VEGF and KDR Genes with Development and Metastasing of Non-Small Cell Lung Cancer

Vascular endothelial growth factor (VEGF) is one of the most important and specific factors affecting angiogenesis in tumor development. VEGFR2 is a receptor encoded by the KDR gene. VEGF and VEGFR2 transmit a signal to intracellular tyrosine kinase cascades. Polymorphic variants of the VEGF and KDR genes significantly influence the expression levels of the endothelial growth factor and its receptor, which leads to a change in the activation of angiogenesis in oncopathological processes. In this study, the relationship between the polymorphic variants rs2010963, rs699947 and rs3025039 of the VEGF gene and rs1870377 and rs2071559 of the KDR gene was analyzed with the development of a specific histological type of non-small cell lung cancer and its clinical and morphological characteristics. It was established that the development of squamous cell carcinoma is associated with −634CC genotype of the VEGF gene and the genotypes containing −2578A allele of the VEGF gene reduce the likelihood of this cancer type development. The development of adenocarcinoma is associated with +936CC VEGF/1719TT KDR and +936CT VEGF/1719TT KDR combinations. In women with non-small cell lung cancer, −634GC genotype of the VEGF gene is associated with a greater degree of the primary lesion spread. Genotype −2578СС of the VEGF gene is associated with a higher degree of the primary tumor spread in the general group of patients and with regional metastases in women. Haplotypes −634G/−2578C/+936C are risky for the occurrence of metastases in regional lymph nodes in women.


Introduction
Lung cancer is one of the most common malignant neoplasms and the leading causes of death from cancer in men.Non-small cell lung cancer (NSCLC) constitutes 85% of all lung cancers and is a heterogeneous disease.Its most common histological types are adenocarcinoma (ADC) and squamous cell carcinoma (SCC).The key process in the NSCLC development is angiogenesis.One of the most important and specific factors affecting angiogenesis in tumor development is vascular endothelial growth factor VEGF [1] [2].The main receptor through which VEGF transmits a signal to intracellular tyrosine kinase cascades is the VEGFR2 receptor encoded by the KDR gene [3] [4].High VEGF expression is associated with tumor growth and metastatic process, while the inhibited VEGF expression results in suppressed tumor growth [5].The VEGF gene also triggers the activation of the protease cascade involved in the degradation of extracellular matrix, suppressing apoptosis, stimulates the endothelial cells survival, increases vascular permeability, inhibits the dendritic cells differentiation as well as activates tissue factors and monocytes migration [6].The VEGF gene is located on the short arm of chromosome 6 (6p21.3)and consists of eight exons and seven introns [7], the KDR gene encoding the VEGFR2 receptor is located in the 4q11 -q12 region of chromosome 4 and contains 26 exons, 25 introns [8].Polymorphic variants of the VEGF and KDR genes significantly influence the expression levels of the endothelial growth factor and its receptor, which leads to a change in the activation of angiogenesis in oncopathological processes.Therefore, the studies of molecular genetic disorders of these genes that will allow predicting the development and progress of this disease are of great importance.
In this research, we aim to study the relationship of the polymorphic variants rs2010963, rs699947, and rs3025039 of the VEGF gene and rs1870377 and rs2071559 of the KDR gene with the probability of development of an NSCLC specific histological type and clinical and morphological manifestations of this disease.

Object of Research
Peripheral blood samples of 234 patients with NSCLC, who were on treatment at Minsk City Clinical Oncology Clinic from 2004 to 2017, were used as biological material in our study.The diagnosis of lung cancer has been based on clinical picture of the disease, history data, bronchoscopy, X-ray examination and computed tomography, cytomorphology of sputum and tumor tissue biopsies.The study group consisted of patients with NSCLC stage 1 -4 according to the TNM А. N.

Methods of Research
Isolation of total DNA from peripheral blood was carried out using the method described by Mathew [9]

Statistical Processing of Results
The statistical analysis was carried out using the GraphPad InStat Version 3.05 application software and the SNPStats online program [10].For the validity check, when comparing genotype frequencies in groups, the standard Pearson χ 2 criterion or the Fisher's exact test for small sampling were used.The relationship between genotypes, development and course of the disease was assessed according to the odds ratio (OR (95% CI)) using codominant and dominant SNPStats models.

Results
The association study between polymorphic variants of VEGF and KDR genes and the risk of ADC and SCC development showed ( In the population under study, the relationship of the studied polymorphic variants of the KDR gene and the development of a specific histological NSCLC Since the signal transmission from VEGF into the cell is carried out through its receptor VEGFR2 encoded by the KDR gene, combinations of polymorphic variants of the VEGF and KDR genes were analyzed.Table 3 shows only significant combinations of these genes that affect the likelihood of the disease development both in the general group of patients with NSCLC and when divided into groups with ADC and SCC.It was revealed that the patients-carriers of +936CT/−906CC combination had a reduced risk of NSCLC development. When considering the effects of VEGF and KDR genotype combinations on the probability of a specific histological type of tumor development, it was found that +936CC/1719TT and +936CT/1719TT combinations are associated with the risk of development in specifically with ADC, but in the carriers of the homozygous combination +936CC/1719TT this association is of higher value (p = 0.02).The combination −634GC/1719TT reduces the risk of SCC development.
Analysis of the frequency distribution of polymorphic variants of genes under study in the groups of men and women with NSCLC is presented in Table 4.
It was established (Table 4) that −634C genotype of the VEGF gene is risky in terms of NSCLC development in men: in the group of patients, this genotype is occurring by 2.3 times more often as compared to the control group (OR = 2.26; 95% CI 1.03 -4.94; p = 0.05).The frequency distribution of this genotype in women with NSCLC does not differ from the corresponding control.The association study of polymorphic VEGF gene variants with the development of ADC or SCC in men and women showed that the genotypes containing −2578A allele of the VEGF gene contribute to the reduced SCC risk development in men (OR = 0.57, 95%CI: 0.35 -0.91; p = 0.02) and the genotypes containing +936T allele of the VEGF gene trigger the SCC development in men (OR = 1.62; 95%CI 1.02 -2.55; p = 0.04).
In women, there were no significant differences in the frequency occurrence of polymorphic VEGF gene variants between the control group and the patients with ADC and SCC (Table 5).
The assessed dependence of the degree of incidence of the primary NSCLC tumor on the polymorphic VEGF and KDR gene variants showed that in the carriers of −2578СС genotype of the VEGF gene the greater degree of incidence of the primary tumor (T2 -T4) is occurring more often (OR = 2.35; 95% CI: 1.21 -4.58; p = 0.02) than the smaller one (T1) (Figure 1).
Analysis of the relationship between the studied polymorphisms of VEGF and KDR genes with the primary tumor spread in men and women with NSCLC showed that −634GC genotype of the VEGF gene is more frequently occurring in women with a greater degree of the primary lesion spread (>3 cm) (OR = 4.80, 95%CI: 1.14 -20.09; p = 0.04) (Figure 2).There is also a tendency for the  increased frequency of occurrence of −634GG genotype of the VEGF gene in a group of patients with a tumor size less than 3 cm.In male patients, no associations of the polymorphic variants of the VEGF gene in −634C > G position with a tumor size were observed.
In the population under study, there were no statistically significant differences by the frequency of occurrence in men and women with NSCLC of polymorphic −2578С > A and +936C > T variants of the VEGF gene associated with a primary tumor size.Association study of polymorphic KDR gene variants with a degree of the primary lesion spread did not show statistically significant differences in men and women with NSCLC.
When analyzing the frequency distribution of polymorphic VEGF gene variants depending on the gender of patients with NSCLC, it was shown that −2578CC genotype of the VEGF gene affects the risk of regional metastasis in women with NSCLC (OR = 10.00,95%CI: 1.44 -69.26; p = 0.02) (Table 6).In men, the association was not identified.
No association of polymorphic KDR gene variants was identified in men and women with NSCLC with the presence of regional metastases.
Association analysis of VEGF gene haplotypes with regional metastases was carried out both in men and women with NSCLC.The statistical significance was reached only in the group of women as shown (Table 7): −634C/−2578C/+936C and −634G/−2578C/+936C haplotypes are risky for the metastasis occurrence in regional lymph nodes.

Discussion
At present, much attention is paid to the study of molecular-biologic markers characterizing the development and progression of malignant neoplasms.The studied VEGF and KDR gene polymorphisms play an important role in the oncological disease pathogenesis.Some studies show that −634C > G, −2578C > A, and +936C > T polymorphisms of the VEGF gene and -906T > C of the KDR gene are associated with the increased corresponding protein concentration in blood and correlate with an increased risk of the oncological diseases' development and progression [11] [12] [13].
Association of −634CC genotype of the VEGF gene with the probability of SCC development in the general group of patients and in men can be explained by the fact that −634C > G polymorphism of the VEGF gene is located in the 5'-untranslated region containing the binding elements of the MZF1 transcription factor [14].These sequences can be involved in regulation of the studied gene transcription, which is directly related to the expression of the VEGF gene.The genotype −634CC is associated with a higher serum concentration of VEGF as compared to CG and GG genotypes [15] [16] and with a higher vascular density in NSCLC cases [17].VEGF expression as compared to the A allele [18] [19].
Polymorphism +936C > T of the VEGF gene is localized in the 3'-untranslated gene region and affects the circulating VEGF concentrations in plasma [20] directly triggering angiogenesis [21], which can explain the risky significance in our population of +936T allele of the VEGF gene in men in the SCC development.
No such relationship was found in women with NSCLC.Squamous cell carcinoma is predominantly found in men and is hardly diagnosed in women, as known [22].Therefore, in our study the associations found for SCC correspond to the data characterizing a group of male patients.Polymorphic variants of the KDR gene do not separately influence the NSCLC development and its most common histological subtypes, but their combinations +936CC VEGF/1719TT KDR and +936CT VEGF/1719TT KDR significantly contribute to the risk of the ADC development.It is known that 1719A > T polymorphism of the KDR gene leads to the nonsynonymous substitution of amino acids, which affects the binding efficiency of VEGF to its VEGFR2 receptor [23].
In addition to the angiogenesis induction in tumors, VEGF promotes the spread of the tumor process.In particular, it increases vascular permeability in tumors, induces serine proteases, inhibits apoptosis of epithelial cells, which further promotes tumor spreading and metastazing [11] [12] [24].Among the clinical and morphological parameters characterizing these processes, the present study takes into account a degree of primary tumor (T) spread, the presence of metastases in regional lymph nodes (N) and distant organs (M).
It is known that −634C allele of the VEGF gene is associated with a higher serum concentration of VEGF [14].Among oncological patients with the GG genotype, the decreased expression of the VEGF gene is observed, and consequently, the primary lesion prevalence is lower than that in the GC and CC genotype carriers [15] [16], which confirms our results obtained with regard to the association of the heterozygous −634GC genotype of the VEGF gene with a higher degree of the primary lesion prevalence in women.
The relationship between −2578CC genotype of the VEGF gene with a higher degree of primary tumor spread in the general group of patients with NSCLC and the presence of regional metastases in women is consistent with the data of many studies showing that −2578C allele of the VEGF gene correlates with a higher level of VEGF expression than the A allele in cases of breast, large intestine, stomach and also lung cancer [16] [17] [25].The increased VEGF expression causes the increased vascular permeability and this leads to the increased interstitial and intratumoral pressure, promoting the penetration of tumor cells into the vascular bed and the metastases' formation [24].This can explain that in our study regional metastases are less common occurring in the homozygous carriers (−2578AA): a lower VEGF expression level in the presence of −2578A allele appears to protect −2578AA genotype carriers from the regional metastases' formation.
Other polymorphic variants of VEGF and KDR genes were not associated with a degree of the primary lesion spread and the presence of metastases in NSCLC cases.
VEGF triggers neoplastic angiogenesis resulting in the increased microcirculatory tumor vessels.Newly formed vessels start the supply of the tumor tissue with oxygen and nutrients, the tumor grows and continues to produce a large amount of VEGF.Polymorphism −2578C > A of the VEGF gene provides for the enhanced expression of a growth factor; +936C > T polymorphism contributes to the increased VEGF concentration in the blood plasma and −634C > G polymorphism is associated with the increased VEGF expression in patients with cancer diseases [11] [19] [21].Our study showed that the C allele of −2578C > A polymorphism influences largely the development of regional metastases in women with NSCLC.Analysis of haplotypes showed that this effect is enhanced when combined with the C allele of polymorphism +936C > T, which increases the VEGF concentration in the blood plasma.This in turn contributes to the enhanced VEGFR2 receptor expression through endothelial cells of tumor vessels, which stimulates cell growth and endothelial cell proliferation [23].This leads to the increased intratumoral pressure, the penetration of tumor cells into the vascular bed and the emergence of metastases.

Conclusion
Thus, the study showed that the development of squamous cell carcinoma is associated with −634CC genotype of the VEGF gene and the genotypes containing −2578A allele of the VEGF gene reduce the likelihood of this cancer type development.Regardless of patients' gender, the development of adenocarcinoma is associated with +936CC VEGF/1719TT KDR and +936CT VEGF/1719TT KDR combinations.In women with non-small cell lung cancer, regardless of the histological type of tumor, −634GC genotype of the VEGF gene is associated with a greater degree of the primary lesion spread.Genotype −2578СС of the VEGF gene is associated with a higher degree of the primary tumor spread in the general group of patients and with regional metastases in women.Haplotypes −634G/−2578C/+936C are risky for the occurrence of metastases in regional lymph nodes in women.

Figure 1 .
Figure 1.Genotypes' association of −2578C > A polymorphism of the VEGF gene in the patients with NSCLC showing the extent of the primary tumor spread.

Figure 2 .
Figure 2. The relationship of polymorphic VEGF gene variants and a degree of the primary lesion spread in men and women with NSCLC.

Figure 3 .
Figure 3. Association of −2578С > A polymorphism genotypes of the VEGF gene with regional metastases in patients with NSCLC.
Genotypes containing −2578A allele of the VEGF gene also show protective characteristics in relation to the SCC development in the general group of patients and in men.Single nucleotide substitution C > A of the VEGF gene in the position of −2578 bp is localized in the promoter region.The gene polymorphism in this region can weaken the VEGF expression affecting the tumor development.According to literary sources, −2578C allele correlates with a higher

Table 1 .
Shchayuk et al.Classification of Malignant Tumours (2009) who underwent surgery.The group of NSCLC included the most common histological types of NSCLC: 126 patients with squamous cell carcinoma and 108 with adenocarcinoma without primary multiple cancer (181 men and 53 women).Sociodemographic and clinical characteristics of NSCLC patients are shown in Table 1.The average patients' age Sociodemographic and clinical characteristics of NSCLC patients and control group.

Table 2 .
Distribution of the VEGF gene genotypes' frequency in the control group and in patients with ADC and SCC.
type was not established.The frequency of occurrence of polymorphic variants of this gene in patients with ADC and SCC was close to the frequency of these variants in the control groups.

Table 3 .
Significant combinations of VEGF and KDR gene genotypes in patients withNSCLC and in the control group.

Table 4 .
The frequency distribution of polymorphic VEGF gene variants in men and women with NSCLC and in the corresponding control.It was shown that the genotypes containing −2578A allele of the VEGF gene have protective characteristics in the NSCLC development in men (OR = 0.65, 95% CI 0.43 -0.99, p = 0.04).At the next stage of work, the influence of molecular genetic disorders in the VEGF and KDR genes was studied for the probability of a certain histological type of NSCLC development in men and women.The frequency distribution of genotypes by the polymorphic loci of the VEGF gene in the control group and in the groups of patients with ADC and SCC, depending on gender, is presented in

Table 5 .Table 5 .
Frequency distribution of polymorphic VEGF and KDR gene variants among men and women with ADC and SCC.

Table 6 .
Frequency distribution of VEGF gene genotypes in men and women with NSCLC depending on the presence of regional metastases.

Table 7 .
Association of VEGF gene haplotypes with regional metastases in women with