Background

The growth of solid tumors including esophageal cancer depends on angiogenesis for the supply of oxygen and nutrients for their continuous growth. Angiogenesis is regulated by cellular signaling mediated by vascular endothelial growth factor (VEGF) and its receptors [1, 2]. VEGF triggers its signaling via VEGFR1, VEGFR2 and VEGFR3 receptors, which are the members of receptor tyrosine kinase family. It has been reported that VEGFR1 or FLT had greater affinity for VEGF as compared to VEGFR2, but had lower tyrosine kinase activity [3,4,5]. VEGFR1 is one of the important receptors of VEGF angiogenesis signaling pathway and its expression is upregulated by hypoxia via HIF-1-dependent mechanism [6, 7]. VEGFR2 or KDR had higher affinity for VEGF-A and VEGF-E and lower affinity for VEGF-C and VEGF-D [8, 9]. von Willebrand factor is secreted by endothelial cells when VEGF binds with VEGFR2 and was reported to be one of the negative prognostic factors for many solid tumors [3, 10]. It has been documented that VEGF-VEGFR2 signaling cascade facilitates tumor growth, invasion and therapeutic resistance [11]. VEGFR1 and VEGFR2 have been described as major therapeutic targets for sorafenib [2]. VEGFR3 or FLT-4 has an affinity for VEGF-C and VEGF-D and its expression influenced the differentiation of lymphatic endothelial cells, tubulogenesis, proliferation, migration and survival of lymphatic endothelial cells [3, 8].

Biomarkers like single-nucleotide polymorphisms (SNPs) account for much of the genetic variations including disease susceptibility, prognosis and response to therapy. The angiogenic pathway, and hence the susceptibility and severity of cancer, may be affected by polymorphisms alone or in combination with environmental factors [12]. It has been reported that SNPs in VEGFRs may affect the production and functioning of protein, thus resulting in dysregulation of angiogenic pathway [13]. Several SNPs have been identified in the VEGFR2, some of which have the ability to alter gene expression, amount of circulating VEGFR2 levels and the efficiency with which VEGF binds to the receptor [14]. Genetic location of the VEGFR1, VEGFR2 and VEGFR3 polymorphisms is given in Fig. 1.

Fig. 1
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Genetic location of the screened polymorphisms

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Association of VEGFR1, VEGFR2 and VEGFR3 polymorphisms with risk of some of gastrointestinal tract (GIT) cancers has been reported in different populations. The G allele of VEGFR2-604A/G polymorphism was associated with increased risk of pancreatic cancer in Romanian population [15]. Combined TT + TC genotype of VEGFR2-604 T/C polymorphism was associated with improved overall survival in Danish colorectal cancer patients [16]. The CC genotype of VEGFR2 1192 T/C polymorphism was associated with improved survival in Danish colorectal cancer patients [16]. In Han Chinese population, TC genotype of VEGFR2 1192 T/C polymorphism was associated with low overall survival in hepatocellular cancer patients [17]. The T allele of VEGFR2 1416A/T polymorphism was found to be associated with increased risk of hepatocellular carcinoma in Portuguese population [18]. In Chinese gastric cancer patients, AA genotype of VEGFR2 1719 A/T polymorphism was associated with poor prognosis [19]. So far, there is no published study that has investigated the role of VEGFR1-710C/T and VEGFR3 rs72816988 polymorphisms in any of the GIT cancers.

Esophageal cancer is the eleventh most common cancer and is the seventh leading cause of mortality worldwide [20]. The highest regional standardized incidence and mortality of esophageal cancer was found in Eastern Asia, followed by Eastern Africa, Southern Africa and South-Central Asia [21]. According to Globocan 2020, nearly 79.7% of new esophageal cancer cases were found in Asia [22]. China had the largest number of new esophageal cancer cases, accounting for 67.3% of in Asia and 53.70% of cases worldwide, and India has the second highest number of new esophageal cancer cases in both Asia and the world, with 63,180 new cases [22]. In Punjab, esophageal cancer is the fourth leading cause of death in women and the second leading cause of mortality in men [23]. Histologically, esophageal cancer has two main subtypes, esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC), and they both differ in their incidence and risk factors profiling. Potential risk factors that one may have for being diagnosed with EAC are obesity, gastroesophageal reflux disease (GERD), male sex, white race and cigarette smoking (or a history of smoking) [24]. For ESCC, the potential risk factors are black race, smoking, alcohol drinking, diet rich in tea, coffee, tobacco chewing, and “chewers of areca nut” which is commonly consumed in regions such as Southeast Asia and India [25].

VEGF/VEGFR pathway is the key regulator of angiogenesis and plays an important role in carcinogenesis [26]. From the early phases of carcinogenesis to the final stage of the disease, angiogenesis plays a significant role in esophageal cancer, and angiogenesis-related agents are being investigated as potential targets for new treatments for esophageal cancer [27]. Therefore, the present study aimed to evaluate the association of VEGFR1-710C/T, VEGFR2-604 T/C (rs2071559), VEGFR2 1192 G/A (rs2305948), VEGFR2 1719A/T (rs1870377) and VEGFR3 (rs72816988) polymorphisms with esophageal cancer risk in patients from Punjab, North-west India. Identification of association of SNPs can aid in predicting the clinical response to the various therapeutic drugs used in the treatment of esophageal cancer. To best of our knowledge, this is the first study evaluating the association of five VEGFR polymorphisms with esophageal cancer risk.

Material and methods

Study subjects

The present case–control study was carried out in accordance with the guidelines of the Helsinki Declaration and was approved by the ethics committee of Guru Nanak Dev University, Amritsar, Punjab, India. In this case–control study design, 310 esophageal cancer patients (137 males and 173 females) and 325 (144 males and 181 females) age and gender matched healthy controls from same ethnicity were investigated based on the predefined inclusion and exclusion criteria (Table 1). The sample size was calculated by using online software Cats Power Calculator (https://csg.sph.umich.edu/abecasis/cats/gas_power_calculator/index.html) using data of minor allele frequency of VEGFR1, VEGFR2 and VEGFR3 polymorphisms from dbSNP (1000 Genome Data). The threshold for significance was set at 0.05, and relative risk was set at 1.5. Five milliliter intravenous blood sample of each subject was collected in EDTA vials after obtaining the written informed consent from all the subjects. The patients were investigated at Sri Guru Ram Das Institute of Medical Sciences and Research, Amritsar, Punjab, India. The blood samples were transported to the Department of Human Genetics, Guru Nanak Dev University, Amritsar, in an ice box from the site of sample collection. Unique code was given to each sample and was stored at − 20℃ till further processing. Demographic characteristics of study participants are shown in Table 2.

Table 1 Inclusion and exclusion criteria for case and controls
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Table 2 Demographic characteristics of the study participants
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Genomic DNA extraction and genotyping of VEGFR polymorphisms

Genomic DNA was extracted from blood samples using standard phenol chloroform method with few modifications [28]. The procedure of DNA extraction is given in Supplementary file 1. Quantity and quality of DNA samples was analyzed on 1% agarose gel. VEGFR1-710C/T, VEGFR2-604 T/C (rs2071559), VEGFR2 1192 G/A (rs2305948), VEGFR2 1719A/T or 1416A/T (rs1870377) and VEGFR3 (rs72816988) polymorphisms were screened by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method (Figs. 2, 3, 4, 5, 6).

Fig. 2
figure 2

A Photograph of 2.4% agarose gel showing the digested products. B Sequencing electropherogram representing CC genotype. C CT genotype of VEGFR1-710C/T polymorphism

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Fig. 3
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A Photograph of 2.4% agarose gel showing the digested products. B Sequencing electropherogram representing AA genotype. C AT genotype and D TT genotype of VEGFR2 1719A/T polymorphism

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Fig. 4
figure 4

A Photograph of 2.4% agarose gel showing the digested products. B Sequencing electropherogram representing TT genotype. C TC genotype and D CC genotype of VEGFR2-604 T/C polymorphism

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Fig. 5
figure 5

A Photograph of 2.4% agarose gel showing the digested products. B Sequencing electropherogram representing GG genotype. C GA genotype and D AA genotype of VEGFR2 1192G/A polymorphism

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Fig. 6
figure 6

A Photograph of 2.4% agarose gel showing the digested products. B Sequencing electropherogram representing GG genotype and C GA genotype of VEGFR3 (rs72816988) polymorphism

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The targeted regions of VEGFR1, VEGFR2 and VEGFR3 were amplified using published primer sequence [29,30,31]. The reaction volume used for amplification was 15 µl, and it contained 75 ng of template DNA, 1.5 µl of 10X Taq Buffer A with 15 mM MgCl2, 6 pmol of each primer, 0.3 µl of dNTPs mixture and 1 U Taq DNA polymerase. Amplified PCR products were analyzed on 2% agarose gel. PCR products were further digested with specific restriction enzymes as per manufacturer’s instructions. For VEGFR1-710C/T polymorphism, NlaIII restriction enzyme (New England BioLabs) was used to digest the 665-bp PCR products. For VEGFR2-604 T/C polymorphism, BsmI restriction enzyme (New England BioLabs) was used to digest the 290-bp PCR products. For VEGFR2 1192G/A polymorphism, BstZ17I-HF restriction enzyme (New England BioLabs) was used to digest the 262-bp PCR products. For VEGFR2 1719A/T polymorphism, AluI restriction enzyme (New England BioLabs) was used to digest the 404-bp PCR products. For VEGFR3 (rs72816988) polymorphism, the amplified products of 218 bp were digested with AciI restriction enzyme (New England BioLabs). The digestion was done for overnight at 37 °C for VEGFR1-710C/T, VEGFR2 1192G/A, VEGFR2 1719A/T and VEGFR3 (rs72816988) polymorphisms, whereas for VEGFR2-604 T/C polymorphism digestion was done at 65 °C. Restriction digestion products were analyzed on 2.4% agarose gel. Genotype was assigned to each sample on the basis of fragments obtained after digestion. The amplification and genotype conditions are given in Table 3. Randomly 10% samples were repeated by Sanger sequencing to revalidate the results and 100% concordance was found (Figs. 2, 3, 4, 5, 6).

Table 3 Details of analyzed VEGFR polymorphisms and genotyping conditions
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Statistical analysis

The Hardy–Weinberg equilibrium (HWE) was evaluated to compare the observed and expected genotype frequencies among controls using Chi-square test. The differences in the genotype and allele frequencies of VEGFR1, VEGFR2 and VEGFR3 polymorphisms between the patients and controls were compared. Odds ratio (OR) and 95% confidence intervals were calculated by MedCalc software [32] to find the association of alleles and genotypes with esophageal cancer risk. SNPstats online software was used to study the different genetic models and haplotypes [33]. p-value less than 0.05 was considered as statistically significant for all the statistical analysis.

Results

A total of 310 esophageal cancer patients and 325 healthy controls were analyzed in this study. The genotype distribution for the studied VEGFR2 polymorphisms was in Hardy–Weinberg equilibrium in controls.

Association of VEGFR1-710C/T polymorphism

The frequency of the CC and CT genotype of VEGFR1-710C/T polymorphism was 98.39 vs 94.46% and 1.61 vs 5.54% in patients and controls, respectively (Table 4). TT genotype was not observed in any of the subjects. CT genotype (OR = 0.28; 95%CI, 0.10–0.76; p = 0.01) and T allele (OR = 0.28; 95%CI, 0.10–0.77; p = 0.01) was found to be significantly associated with decreased risk of esophageal cancer.

Table 4 Comparison of genotype and allele frequency of VEGFR polymorphisms between esophageal cancer patients and controls
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Association of VEGFR2 polymorphisms

The frequency of TC genotype of VEGFR2-604 T/C polymorphism was higher in controls (Table 4) and was associated with decreased risk of esophageal cancer (OR = 0.66; 95%CI, 0.44–0.97; p = 0.03). After stratification of the data according to gender, TC genotype of VEGFR2-604 T/C was found to be significantly associated with decreased risk of esophageal cancer in male group (OR = 0.50; 95%CI, 0.28–0.89; p = 0.02) (Table 5). Genetic model analysis of VEGFR2-604 T/C polymorphism showed a decreased risk of esophageal cancer under codominant (OR = 0.66; 95%CI, 0.44–0.97; p = 0.03) and dominant model (OR = 0.66; 95%CI, 0.46–0.96; p = 0.03) (Table 6). In male group, decreased esophageal cancer risk was observed under codominant (OR = 0.50; 95%CI, 0.28–0.89; p = 0.02) and dominant model (OR = 0.53; 95%CI, 0.31–0.92; p = 0.02) (Table 7).

Table 5 Analysis of VEGFR polymorphisms and esophageal cancer risk in male and female subjects
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Table 6 Relationship between genetic models of VEGFR2 polymorphisms and the risk of esophageal cancer
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Table 7 Genetic model analysis of VEGFR2 polymorphisms with esophageal cancer risk in male and female subjects
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In female group, GA genotype of VEGFR2 1192G/A polymorphism was found to be significantly associated with decreased esophageal cancer risk (OR = 0.54; 95%CI, 0.31–0.95; p = 0.03). Genetic model analysis of VEGFR2 1192G/A polymorphism revealed a significantly decreased esophageal cancer risk in codominant (OR = 0.54; 95%CI, 0.31–0.95; p = 0.03), dominant (OR = 0.56; 95%CI, 0.32–0.96; p = 0.04), and overdominant model (OR = 0.54; 95%CI, 0.31–0.95; p = 0.03) in female group. We further compare the genotype distribution of VEGFR2 1192G/A polymorphism between male patients and female patients and observed that GA genotype was significantly associated with increased risk of esophageal cancer in male patients as compared to female patients (Table 8). There was no significant difference in the genotype and allele frequencies of VEGFR2 1719A/T polymorphism between patients and controls (Table 4).

Table 8 Association of VEGFR polymorphisms with esophageal cancer risk in male and female patients
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Association of VEGFR3 (rs72816988) polymorphism

The frequency of the GG and GA genotype of VEGFR3 (rs72816988) polymorphism was 95.16 vs 94.46% and 4.84 vs 5.54% in patients and controls, respectively. AA genotype was not observed in any of the subjects. There was no significant difference in genotype and allele frequencies between patients and controls (Table 4).

Haplotype analysis

To evaluate the combined effect of VEGFR2 polymorphisms in the susceptibility to esophageal cancer, haplotype analysis was performed. In total subjects, haplotype C-604 A1719A1192 was significantly associated with the decreased esophageal cancer risk (OR = 0.44; 95%CI, 0.23–0.84; p = 0.01), whereas haplotype C-604 A1719G1192 was marginally associated with the decreased cancer risk (OR = 0.74; 95%CI, 0.54–1.01; p = 0.06). Haplotype C-604 A1719G1192 was significantly associated with the decreased esophageal cancer risk in male group (OR = 0.48; 95%CI, 0.28–0.80; p = 0.006) (Table 9).

Table 9 VEGFR2 polymorphism haplotypes and esophageal cancer risk
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Discussion

In the present study, we have investigated the association of VEGFR1-710C/T, VEGFR2-604 T/C, VEGFR2 1192 G/A, VEGFR2 1719A/T and VEGFR3 (rs72816988) polymorphisms with esophageal cancer risk. Researchers have investigated these polymorphisms in different cancers and results are variable (Supplementary Tables 1–4). So far, the role of these polymorphisms has not been explored in esophageal cancer. In the present study, T allele of VEGFR1-710C/T polymorphism was significantly associated with decreased risk of esophageal cancer. Till date, there is no published study on VEGFR1-710C/T polymorphism in gastrointestinal tract cancer. Association of T allele with reduced breast cancer risk has been reported in Spanish population [29], whereas no association of VEGFR1-710C/T polymorphism with breast cancer risk has been reported in patients from Punjab North-west India [34].

The promoter polymorphism VEGFR2-604 T/C changes the binding site for transcription factor E2F in KDR promoter region, which can downregulate KDR expression [14]. In the present study, TC genotype of VEGFR2-604 T/C polymorphism was significantly associated with reduced risk of esophageal cancer. The TC + CC combined genotype of VEGFR2-604 T/C polymorphism was associated with decreased esophageal cancer risk in dominant model. Contrary to our results, combined TC + CC genotype was significantly associated with increased risk of colorectal cancer in Korean population [35]. The C allele of VEGFR2-604 T/C polymorphism was associated with increased risk of pancreatic cancer in Romanian population [15]. However, no correlation of VEGFR2-604 T/C polymorphism has been observed with gastric cancer [19] and hepatocellular carcinoma in Chinese population [17]. In Danish population, TT + TC genotype of VEGFR2-604 T/C was associated with improved overall survival in colorectal cancer patients [16].

VEGFR2 1192G/A polymorphism located in third NH2 terminal Ig-like domains in the extracellular region is crucial for ligand binding [14]. In the present study, no association was found between VEGFR2 1192G/A polymorphism and esophageal cancer in total subjects. However, combined GA + AA genotype was significantly associated with decreased esophageal cancer risk in female group. Association of combined GA + AA genotype of VEGFR2 1192G/A polymorphism with increased colorectal cancer risk has been documented in Korean population [35]. In Danish population, GG genotype of VEGFR2 1192G/A polymorphism was associated with improved survival in patients having colorectal cancer [16]. GA genotype was associated with lower overall survival in hepatocellular cancer Han Chinese patients [17]. No correlation was observed between VEGFR2 1192G/A polymorphism and gastric cancer in Chinese population [19].

In the present study, VEGFR2 1719A/T polymorphism was not associated with esophageal cancer risk. VEGFR2 1719A/T polymorphism was not associated with recurrence and overall survival in esophageal adenocarcinoma patients who underwent surgery [36]. Similarly, no association of VEGFR2 1719A/T polymorphism has been reported in hepatocellular carcinoma in Han Chinese [17] and colorectal cancer in Danish patients [16]. The T allele of VEGFR2 1719A/T polymorphism was associated with increased hepatocellular cancer risk in Portuguese patients [18], whereas AA genotype was associated with poor prognosis in Chinese gastric cancer patients [19].

No significant association was observed between VEGFR3 (rs72816988) polymorphism and esophageal cancer risk in the present study. Till date, there is no published study on VEGFR3 (rs72816988) polymorphism in GIT cancer. Relationship between VEGFR3 (rs72816988) polymorphism with the clinical outcomes of renal cell carcinoma patients treated with sorafenib [37] and sunitinib [31] has been studied, and no association was found in both of these studies.

In the present study, C-604 A1719A1192 haplotype of VEGFR2 was significantly associated with decreased esophageal cancer risk in total subjects, whereas C-604 A1719G1192 haplotype was associated with decreased esophageal cancer risk in male group. Association of C-604G1192 and C-604A1192 haplotypes with an increased risk to colorectal cancer has been reported in Korean patients [35]. The response of VEGFR polymorphisms with different therapies in GIT cancers has been studied in different populations and reported association with disease survival (Supplementary Tables 5–7).

Strength of the study

So far, the present case–control study is the first study which have analyzed the association of VEGFR1-710C/T, VEGFR2-604 T/C, VEGFR2 1192 G/A, VEGFR2 1719A/T and VEGFR3 (rs72816988) polymorphisms with esophageal cancer risk. It provides the baseline data for genetic polymorphisms of angiogenic pathway.

Limitations of the study

The present study only focuses on the population of Punjab, North-west India, with a limited sample size; however, the frequency of genetic polymorphisms often varies between different ethnic groups.

Conclusion and future directions

In the present study, we found that VEGFR1-710C/T, VEGFR2-604 T/C and VEGFR2 1192G/A polymorphisms were associated with decreased risk of esophageal cancer in the patients from Punjab, North-west India. In future, further studies with larger sample size on different ethnic groups are required to better understand the role of VEGFR polymorphisms in the development and progression of esophageal cancer. Understanding the relationship between VEGFR polymorphisms and esophageal cancer risk can aid in identifying individuals at higher risk and facilitate early detection and intervention which is crucial for better prognosis. In future, studies examining the relationship between VEGFR polymorphisms and the response of esophageal cancer patients to chemotherapeutic drugs are required. This will help to understand how these polymorphisms affect treatment response and aid in the provision of personalized medicine, which aims to maximize therapeutic outcomes and minimizing adverse effects.