Investigation of Vascular Endothelial Growth Factor Polymorphisms on Risk,Metastasis,Laterality,and Prognosis of Colorectal Cancer in Turkish Subjects

Abstract

Objectives:

Tumor angiogenesis is known to support the spread and invasion of tumor cells, allow distant organ metastasis and to result in poorer prognoses and increased mortality. Since vascular endothelial growth factor-A (VEGF-A) is the major regulator of angiogenesis, in the present study the associations of the VEGF-A +405G>C and −460C>T polymorphisms with risk, primary tumor location, prognosis and metastasis of colorectal cancer (CRC) were investigated in Turkish subjects.

Material and Methods:

A total of 153 subjects consist of 74 controls and 79 CRC diagnosed patients were included in the study. VEGF-A +405G>C and −460C>T polymorphisms were analyzed using the Agena MassARRAY platform.

Results:

The VEGF +405GC+CC genotypes were found to be significantly associated with left colon cancer (unadjusted OR = 5.208 95% CI: 1.064-25.496, p = 0.04). The VEGF −460TT and CT+TT genotypes were associated with reduced liver metastasis risk (OR = 0.080 95% CI: 0.009-0.689 p = 0.02 and OR = 0.191 95% CI: 0.039-0.925, p = 0.04, respectively). Patients with the VEGF +405GG genotype showed longer progression-free survival in response to bevacizumab treatment (Log rank = 6.92, p = 0.03).

Conclusion:

According to our results, the VEGF +405G>C and −460C>T polymorphisms were found to be associated with CRC prognosis, sidedness and metastases. Our findings need to be replicated in further studies.

Introduction

Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer-related death (Torre et al., 2015). By 2030, the global burden of CRC is expected to increase by 60% with 2.2 million new cases and 1.1 million deaths (Ferlay et al., 2013). One of the main causes of the high mortality in CRC is metastasis (Mlecnik et al., 2016). While 90% of CRC patients with early diagnosis can survive >5 years, unfortunately this rate remains around 10% for metastatic patients (Levin et al., 2008). Therefore, metastasis is considered as a keystone in tumor development because it leads tumor cells to spread.

Angiogenesis is a multistep biological process that leads to the formation of new capillary blood vessels from existing vascular systems and is controlled by local or systemic chemical signals (Hanahan and Folkman, 1996; Risau, 1997; Carmeliet, 2000). Angiogenesis, which is necessary for the development and maintenance of homeostasis in a healthy individual, is also an important and fundamental process in tumor development. The formed new vessels as a result of tumor angiogenesis provide oxygen and nutrients to growing tumors, supporting the spread and invasion of the tumor cells to the nearby normal tissue and allow distant organ metastasis (Mousa, 2000; Rajabi and Mousa, 2017; Li et al., 2018).

The vascular endothelial growth factor (VEGF) signaling pathway plays a key role in angiogenesis (Li et al., 2018). VEGF family consists of five secretory proteins (VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PlGF) and three tyrosine kinase receptors (VEGF receptor [VEGFR]-1, VEGFR-2, and VEGFR-3) (Ferrara et al., 2003; Macarulla et al., 2020). VEGF-A ligand is known to be the most important member of the VEGF system. Circulating VEGF-A is secreted by many cells, including malignant cells, and binds to two VEGF receptors (VEGFR-1 and VEGFR-2) that results in promoting the survival, proliferation, migration, and differentiation of endothelial cells (Dvorak, 2002; Hicklin and Ellis, 2005).

VEGF-A levels in tissues have been associated with cancer development, poor prognosis, poor survival, and therapy sensitivity in many in vitro and in vivo studies (Fu et al., 2014; Bendardaf et al., 2017; Dinami et al., 2020; Lacin and Yalcin, 2020; Van Cutsem et al., 2020; Mashima et al., 2021). Several variations on VEGF gene have potential to alter VEGF signaling and may directly affect VEGF levels in tissue and plasma (Koukourakis et al., 2004; Sa-Nguanraksa et al., 2013; O-Charoenrat, 2014; Innocenti et al., 2018).

Since overexpression of VEGF is known to result in tumor growth, metastasis, and poor prognosis (Frezzetti et al., 2017; Li et al., 2017; Cheng et al., 2018; Sopo et al., 2019), it would not be surprising that VEGF gene variations may have an influence on development, prognosis, and even treatment of cancer through VEGF expression.

We believe that it is very important to understand genetic mechanisms and the effects of their variations that may be effective in tumor formation, metastasis, drug response, and prognosis for successful CRC treatment. Following the idea, in the present study, we investigated the relationship of VEGF-A +405G>C and −460C>T polymorphisms on risk, primary tumor location, prognosis, and metastasis of CRC in Turkish subjects. Additionally, the relationships between the VEGF gene variations and prognosis of patients receiving anti-VEGF therapy were also investigated.

Materials and Methods

Subjects

The present investigation was approved by the Marmara University Local Ethics Committee with the protocol number 09.2018.174. Cases were selected randomly from histologically confirmed CRC patients without former inflammatory bowel disease or any of the known hereditary cancer history. Controls were selected randomly from volunteers without CRC and other malignancy or autoimmune disorder history. Both case and control subjects signed a well-written consent complying with the ethical criteria of the Helsinki Declaration. Five milliliters of EDTA blood samples was recruited from each patient at Group Florence Nightingale Sisli Hospital, Medical Oncology Department, and control subjects were recruited in the same hospital at other departments. Following the sampling, the blood samples were cold transferred to the Marmara University Molecular Metabolism Research Laboratory for genomic DNA isolation.

The 5-Fluorouracil or Capecitabine-based therapy regimens were applied in combination with oxaliplatin, irinotecan, cetuximab, or bevacizumab for treatment of CRC patients. Overall and progression-free survival times of patients were defined as the date from the beginning of the treatment until death from any cause, and survivors were censored at the date of last contact.

Genotyping

Genomic DNA was isolated from peripheral blood using the Kurabo, Quick Gene DNA Whole Blood Kit. VEGF-A rs2010963 (+405G>C or −634G>C) and rs8333061 (−460C>T or −1498 C>T) polymorphisms were genotyped in the study. Genotyping was performed with Agena MassARRAY platform. The platform combines iPLEX and Mass ARRAY technology (Agena Bioscience, San Diego, CA), which is based on Matrix-assisted laser desorption/ionization-time of flight Mass Spectrometry assay. Assay Design Suite software version 2.0 was utilized to design forward, reverse, and single base extension primers. PCR was conducted with 10 ng/μL DNA samples, deoxyribonucleotide triphosphates, forward and reverse primers, reaction buffers, and DNA polymerase (Agena iPLEX Gold Genotyping Kit).

PCR conditions were performed according to the protocol reported by Gabriel et al. (2009). Following the final PCR, for desalting the iPLEX Extension reaction, PCR products were treated with resin and then transferred to a 384-well Spectro-CHIP using Mass ARRAY Nanodispenser. Spectro-CHIPs were transferred to MALDI-TOF mass spectrometry analyzer and the data analyzed by Typer Analyzer v 4.0 software. Assay plate included both positive and negative controls.

Statistical analyses

Statistical analyses were performed using IBM SPSS statistics version 26. Data are expressed as numbers and percentage for discrete variables as mean ± standard error (min-max) for continuous variables. Nonparametric Mann-Whitney U test was used to compare continuous variables. χ² test was performed to analyze the accordance of genotype distributions with Hardy-Weinberg equilibrium and compared as a function of sex between case and controls. The comparisons of the genotype distributions between dichotomized groups (case/control, liver metastatic/nonmetastatic, left/right colon) were performed with χ² tests. Fisher's exact test was considered if the number of the subjects were <5. Binary logistic regression analysis was used to estimate odds ratio (OR) and 95% confidence intervals (CI).

Binary logistic regression analysis was performed as both unadjusted (case/control, right/left colon, liver metastatic/nonmetastatic groups) and adjusted in sex, age, and smoking (case/control group). Log-Rank test was used for comparisons of survival times. Survival times (month) were represented as mean ± standard error estimate (95% CI). p-values below 0.05 were considered as statistically significant.

Results

A total of 153 subjects, consisting of 74 controls (31 female, 43 male) and 79 CRC diagnosed patients (33 female, 46 male), were included in the study. Clinicopathological characteristics of the study group are summarized in Table 1.

Table 1.

Clinicopathological Characteristics of the Study Group

	Case, n (%)	Control, n (%)	p
Sex	33 (41.8)	31 (41.9)	0.98
Female	46 (58.2)	43 (58.1)
Male
Age	59.78 ± 1.30 (28-82)	53.75 ± 1.9 (22-85)	0.05
BMI	26.26 ± 0.5 (18.25-40.57)	27.28 ± 0.5 (19.05-41.62)	0.09
PFS	11.87 ± 8.5 (3-44)
OS	22.63 ± 17.48 (2-91)
Primary tumor location
Right	18 (22.8)
Left	61 (77.2)
Metastasis
No	15 (19.0)
Liver	61 (80.3)
Peritoneal	3 (4.7)
KRAS mutant
Yes	25 (31.6)
No	54 (68.4)
Dead	31 (42.5)
Irınotecan	23 (31.9)
Bevacizumab	23 (31.9)
Oxaliplatin	52 (72.2)
Cetuximab	16 (22.2)
Capecitabine	22 (27.8)
5-FU	57 (72.2)

Categorical variables were expressed as n (%). Age, weight, BMI, PFS, OS values are expressed as mean ± standard error (Min-Max).

BMI, body mass index; 5-FU, 5-Fluorouracil; OS, overall survival; PFS, progression-free survival.

Comparative genotype frequencies of VEGF +405G>C and −460C>T polymorphisms between cases and controls are given in Table 2. While the genotype distributions of the −460C>T polymorphism were in accordance with the Hardy-Weinberg equilibrium in cases and controls, +405G>C polymorphism deviated in controls.

Table 2.

Distribution of Genotype and Allele Frequencies of VEGF +405G>C and −460C>T Polymorphisms in Cases and Controls

	Case					Control					p ^a
		Allele frequency					Allele frequency				p ^a
+405G>C	n: 40 (%)	Wt	Mut	χ ²	p ^b	n: 53 (%)	Wt	Mut	χ ²	p ^b
GG	11 (27.5)	0.538	0.463	0.125	0.72	22 (41.5)	0.575	0.425	6.255	0.01	0.136
GC	21 (52.5)					17 (32.1)
CC	8 (20.0)					14 (26.4)
GG+GC/CC	32 (80.0)					39 (73.6)					0.623
GC+CC/GG	29 (72.5)					31 (58.5)					0.193
−460C>T	n: 74 (%)	Wt	Mut	χ ²	p ^b	n: 62 (%)	Wt	Mut	χ ²	p ^b
CC	27 (36.5)	0.635	0.365	2.045	0.15	15 (24.2)	0.540	0.460	2.513	0.11	0.219
CT	40 (54.1)					37 (59.7)
TT	7 (9.5)					10 (16.1)
CC+CT/TT	67 (90.5)					52 (83.9)					0.301
CT+TT/CC	47 (63.5)					47 (75.8)					0.139

Categorical variables were expressed as n (%).

p^a shows chi-square analysis results for comparisons of genotype distributions between case and control groups.

p^b and χ² show accordance with Hardy-Weinberg equilibrium.

p < 0.05 was considered statistically significant.

Mut, Mutant allele; VEGF, vascular endothelial growth factor; Wt, Wild-type allele.

The frequencies of VEGF +405G>C and −460C>T genotypes in cases and controls were not statistically significant (p = 0.136 and p = 0.219, respectively). The relationships between VEGF +405G>C and −460C>T polymorphisms and CRC risk are given in Supplementary Table S1. VEGF +405G>C and −460C>T polymorphisms were not found to be associated with CRC risk.

The genotype frequencies of VEGF +405G>C and −460C>T polymorphisms based on primary tumor localization are given in Table 3. In basic comparisons between right and left colon cancer patients, VEGF +405G>C genotype distributions were not found to be statistically significant. However, since GC+CC genotype frequencies were higher in the left colon than right colon (p = 0.08), association of GC+CC genotypes and left colon cancer was additionally examined with binary logistic regression analysis (Table 4). VEGF +405GC+CC genotypes were found to be significantly associated with left colon cancer (unadjusted OR = 5.208 95% CI: 1.064-25.496, p = 0.04).

Table 3.

Distribution of VEGF +405G>C and −460C>T Genotype Frequencies Based on Primary Tumor Localization and Presence of Liver Metastasis

	Primary tumor location		p	Liver metastasis		p
	Left colon (%)	Right colon (%)	p	No (%)	Yes (%)	p
VEGF +405G>C
GG	6 (19.4)	5 (55.6)	0.10	0 (0.0)	10 (31.3)	0.20
GC	18 (58.1)	3 (33.3)		5 (83.3)	15 (46.9)
CC	7 (22.6)	1 (11.1)		1 (16.7)	7 (21.9)
GG+GC/CC	24 (77.4)	8 (88.9)	0.66	5 (83.3)	27 (78.1)	0.77
GC+CC/GG	25 (80.6)	4 (44.4)	0.08	6 (100.0)	22 (68.8)	0.17
VEGF −460C>T
CC	17 (30.4)	10 (55.6)	0.15	2 (13.3)	25 (44.6)	0.04
CT	33 (58.9)	7 (38.9)		10 (66.7)	28 (50.0)
TT	6 (10.7)	1 (5.6)		3 (20.0)	3 (5.4)
CC+CT/TT	50 (89.3)	17 (94.4)	0.49	12 (80.0)	53(94.6)	0.10
CT+TT/CC	39 (69.6)	8 (44.4)	0.09	13 (86.7)	31 (55.4)	0.04

Categorical variables were expressed as n (%). p < 0.05 was considered statistically significant.

Statistically significant values were stressed as bold.

Table 4.

The Associations of VEGF +405GC+CC and −460CT+TT Genotypes and Left Colon Cancer Risk

	OR (95% CI)	p
VEGF +405G>C
GG	Reference
GC+CC	5.208 (1.064-25.496)	0.04
VEGF −460C>T
CC	Reference
CT+TT	2.868 (0.964-8.532)	0.06

Statistically significant values were stressed as bold.

CI, confidence interval; OR, odds ratio.

Similarly, VEGF −460C>T genotype distributions were not significantly different between right and left colon patients. However, in binary logistic regression analysis 460CT+TT genotypes were partially associated with left colon cancer, although not as much as +405G>C polymorphism (unadjusted OR = 2.868, 95% CI: 0.964-8.532, p = 0.06) (Table 4).

The distributions of VEGF +405G>C and −460C>T genotype frequencies according to the presence of liver metastasis are given in Table 3. Patients with a variant allele carrying VEGF −460CT+TT genotypes was found to be higher in nonmetastatic compared with liver metastatic patients (p = 0.04). Compared with the CC genotype, TT and CT+TT genotypes were associated with reduced liver metastasis risk (Table 5) (OR = 0.080 95% CI: 0.009-0.689 p = 0.02 and OR = 0.191 95% CI: 0.039-0.925, p = 0.04, respectively).

Table 5.

The Association of VEGF −460C>T Polymorphism and Liver Metastasis

VEGF −460C>T	OR (95% CI)	p
CC	Reference
CT	0.224 (0.045-1.122)	0.07
TT	0.080 (0.009-0.689)	0.02
CT+TT	0.191 (0.039-0.925)	0.04

Statistically significant values were stressed as bold.

VEGF +405G>C genotype distributions were not significantly different between liver and nonmetastatic patients (p = 0.20).

The associations between VEGF +405G>C and −460C>T polymorphisms and progression-free and overall survival of CRC patients are summarized in Supplementary Table S2. No statistically significant relationship was found between VEGF +405G>C and −460C>T polymorphisms and survival of CRC patients. However, although small sample size, we subcategorized our study group based on anti-VEGF agent bevacizumab use as BEV⁺ (bevacizumab prescribed) and BEV⁻ (as no bevacizumab prescription) and further investigated associations of patients' survival and VEGF +405G>C and −460C>T polymorphisms between these groups.

In general, survival times did not differ significantly between BEV⁺ and BEV⁻ groups (Log rank = 0.221 p = 0.638 for progression-free survival; Log rank = 1.288 p = 0.256 for overall survival, data not given in the table).

The associations of VEGF +405G>C genotypes and progression-free survival of CRC patients in BEV⁺ and BEV⁻ groups are summarized in Table 6. VEGF +405 GG genotype resulted with longer progression-free survival in BEV⁺ patients compared with other genotypes (Log rank = 6.92, p = 0.03). Similarly, patients with GC+CC genotypes had shorter progression survival times compared with patients with GG genotype (Log rank = 4.61, p = 0.03). No statistically significant relationship was found between +405G>C polymorphism and progression-free survival in BEV⁻ group.

Table 6.

Effects of VEGF +405G>C and −460C>T Polymorphisms on Progression-Free Survival of Bevacizumab-Prescribed and Unprescribed Colorectal Cancer Patients

	BEV⁺	Log rank	p	BEV⁻	Log rank	p
VEGF +405G>C
GG	43.50 ± 0.35 (42.81-44.19)	6.92	0.03	11.86 ± 1.31 (9.30-14.42)	0.34	0.84
GC	10.69 ± 1.90 (6.96-14.42)			18.00 ± 3.31(11.51-24.49)
CC	6.50 ± 1.50 (3.56-9.44)			18.00 ± 0.00 (18.00-18.00)
GG+GC/CC	22.36 ± 6.27 (10.07-34.6)	3.64	0.06	18.19 ± 2.53 (13.23-23.16)	0.33	0.57
GC+CC/GG	9.74 ± 1.59 (6.63-12.85)	4.61	0.03	18.85 ± 2.40 (14.15-23.55)	0.11	0.74

Values were represented as mean ± standard error estimate (95% CI).

Statistically significant values were stressed as bold.

BEV⁺, bevacizumab-prescribed CRC patients. BEV⁻, CRC patients with no bevacizumab prescription; CRC, colorectal cancer.

VEGF −460C>T polymorphism was not found to be associated with progression-free survival in both BEV⁺ and BEV⁻ groups (shown in Supplementary Table S3). No association was detected in both VEGF +405G>C and −460C>T polymorphisms and overall survival times of the patients between BEV⁺ and BEV⁻ groups (shown in Supplementary Table S4).

Discussion

The importance of tumor angiogenesis in growth and survival of solid tumors is well known (Hasina and Lingen, 2001; Gupta and Qin, 2003). In addition, angiogenesis contributes to the development of cancer by facilitating the metastatic spread of tumor cells (Kumar et al., 2004). Therefore, angiogenesis is a keystone in a successful metastasis. For this reason, we investigated the effects of VEGF polymorphisms in CRC metastasis in the present study. VEGF −460TT and CT+TT genotypes were associated with reduced liver metastasis risk (OR = 0.080 95% CI: 0.009-0.689 p = 0.02 and OR = 0.191 95% CI: 0.039-0.925, p = 0.04, respectively). VEGF +405G>C polymorphism was not found to be associated with metastasis in our study group.

Similar to our results, do Espírito Santo et al. (2017) evaluated the association between VEGF +405G>C and −460C>T polymorphisms and liver metastasis and reported −460C>T polymorphism was associated to be reduced liver metastasis (OR = 0.32; p = 0.048). The researchers did not report the same association for +405G>C polymorphism (do Espírito Santo et al., 2017). Chae et al. (2008) on the other hand, subcategorized their study groups based on the presence of distant organ metastasis, and they detected +405GC genotype to be nearly associated with reduced metastasis risk (p = 0.05), but not record the same association with CC genotype. Koutras et al. (2012) reported that VEGF +405G>C and −460C>T polymorphisms were not associated with liver metastasis.

Although the presence of conflicting studies, results from our study and do Espírito Santo et al. may indicate that VEGF −460C>T variation displays metastasis-preventing attitude in CRC. Since it is known that VEGF −460TT genotype results with decreased VEGF mRNA expressions compared with TC and CC genotypes in CRC (Yamamori et al., 2004), the VEGF −460C>T single nucleotide polymorphism might be protective against liver metastasis in CRC. However, further in vivo and in vitro studies are needed to understand by which mechanisms VEGF −460C>T polymorphism protects against metastasis.

CRCs may have different histological, pathological, molecular, or genetic features depending on the location of primary tumor (Benedix et al., 2010; Lee et al., 2015; Plastiras et al., 2019). Although tumors in the right colon are rare compared with left, right colon cancers have worse prognosis and higher mortality (Benedix et al., 2010; Stintzing et al., 2017). Therefore, we believe primary tumor location in CRC has the potential to be one of the evaluation criteria of cancer development and progression. It is known that variations on gene regions that encode some members of VEGF signaling pathway, including VEGF-A, affect CRC depending on primary tumor location (Riera et al., 2018; Grassadonia et al., 2019). Since VEGF expression levels are known to vary depending on the location in colon (Bendardaf et al., 2008; Szajewski et al., 2014), we thought that genetic variations of VEGF might be associated with occurrence of tumors in a specific location.

Only a limited number of studies have focused on the relationship of angiogenesis-related VEGF polymorphisms and location of primary tumor. It is difficult to get clear results due to different classification criteria of study groups in these limited studies. Slattery et al. (2014) subclassified their study group based on primary tumor location as rectum and colon, and reported VEGF +405GC+CC genotypes to be associated with rectal cancer (p = 0.007). Similarly, Jang et al. considered the primary tumor locations as colon and rectum, but unlike Slattery et al. (2014) and they did not detect a significant relationship between VEGF +405G>C polymorphism and primary tumor localization (Jang et al., 2013b). In another study, study group was subclassified as colon, rectum and rectosigmoid, and no correlation was found between both +405G>C and −460C>T polymorphisms and the primary tumor location (Koutras et al., 2012).

In our study, we subcategorized our study group as right (cecum, ascending colon, hepatic flexure, and transverse colon) or left (splenic flexure, descending colon, sigmoid colon, and rectum) colon patients. VEGF +405GC+CC genotypes were found to be associated with left colon cancer (OR = 5.208 95% CI: 1.064-25.496, p = 0.04). Since rectum is involved in the left colon, our results partially support the study conducted by Slattery et al. (2014). But still, further studies with larger sample size are needed to validate our results.

In the present study, we also investigated the association of VEGF +405G>C and −460C>T polymorphisms with CRC risk in Turkish subjects. Although VEGF +405GC genotype was found to be nearly associated with increased CRC risk in both unadjusted (OR = 2.471 95% CI: 0.941-6.490, p = 0.066) and the adjusted models (OR = 2.643 95% CI: 0.916-7.624, p = 0.072), results were not statistically significant. Similar (Dassoulas et al., 2009; Antonacopoulou et al., 2012; Jang et al., 2013a) and opposite results (Chae et al., 2008; Zhao et al., 2012; Guo et al., 2014) were reported by researchers in different populations. No significant relationship was found between VEGF −460C>T polymorphism and CRC risk in Turkish subjects. In a different study conducted with CRC patients and healthy controls in the Turkish population, VEGF −460C>T genotype distributions also were not found significantly different in cases and controls (Jannuzzi et al., 2015). Dassoulas et al. (2009) reported no association between VEGF −460C>T polymorphism and CRC risk; whereas other studies reported the opposite (Maltese et al., 2009; Zhao et al., 2012).

Bevacizumab, a recombinant humanized IgG1 monoclonal antibody, is the first antiangiogenic agent approved for metastatic CRC treatment (Hurwitz et al., 2004). Favorable outcomes in progression-free survival, overall survival, and response rate have been achieved in metastatic CRC patients treated with chemotherapy+bevacizumab compared with chemotherapy alone (Botrel et al., 2016; Ruan et al., 2018).

Although clinical use is increasing, the efficacy or drug resistance of bevacizumab varies widely among patients. The different bevacizumab responses mainly occur due to genetic variations, which effects function of the proteins that involve directly or indirectly in angiogenesis (Novillo et al., 2020). These variations are also associated with the etiology and clinical outcomes of metastatic CRC (Loupakis et al., 2013; Ulivi et al., 2015).

The efficiency of bevacizumab treatment at the population level is limited due to multiple resistance mechanisms. Since the molecular genetic mechanisms underlying the different bevacizumab responses have not yet been clarified enough, attempts to individualize treatment for bevacizumab remain insufficient. Thus, we finally evaluated the effects of VEGF +405G>C and −460C>T polymorphisms on progression-free and overall survival times of patients regarding bevacizumab use.

According to our results, progression-free survival times of CRC patients with VEGF +405GG genotype was found to be longer only in the BEV⁺ group compared with the GC+CC genotypes (Log rank = 4.61, p = 0.03, respectively). Our results may indicate that bevacizumab treatment results in longer progression-free survival in CRC patients with VEGF +405GG genotype. Studies focused on relationship between VEGF +405G>C polymorphism and CRC survival show heterogeneity in terms of both content and results. Dassoulas et al. (2009) reported that a 6-year survival of CRC patients with +405CC genotype was lower compared with other genotypes. Hansen et al. (2011) found the +405GC genotype to be associated in borderline with progression-free survival in mCRC patients treated with first-line capecitabine and oxaliplatin. Do Espírito Santo et al. (2017) found +405CC genotype to be associated with 5-year survival of CRC patients in multivariate analysis, but the researchers did not detect any significant relationship in univariate analysis.

In many studies conducted with CRC patients, where BEV was prescribed in combination with fluorouracil, irinotecan, or oxaliplatin-based chemotherapies, no significant relationship was found between VEGF +405G>C polymorphism and progression-free and/or overall survival (Loupakis et al., 2011a, 2011b; Pander et al., 2011; Koutras et al., 2012; Papachristos et al., 2019). Many studies indicate that VEGF +405GG genotype causes decreased VEGF protein levels and angiogenicity in tumor cells (Awata et al., 2002; Koukourakis et al., 2004). It is also known that those patients with low or no VEGF expression show better survival compared with patients with high VEGF expression (Lee et al., 2000; Ferroni et al., 2005; Kuramochi et al., 2006; Altomare et al., 2007). Therefore, it may be predicted that VEGF +405GG genotype affects CRC prognosis through VEGF expression levels. However, since VEGF +405G>C polymorphism was not found to be related with VEGF protein levels in CRC patients under FOLFOXIRI+BVZ treatment (Loupakis et al., 2011a), further studies in larger study groups are needed to fully elucidate the role of VEGF +405GG polymorphism in CRC prognosis.

The heterogeneous results may occur due to differences in size, content, analysis methods, and observation times of the studies, or due to differences in ethnicity, genetic backgrounds, drug regimens, or disease characteristics of the patients in study groups. For this reason, it is difficult to define the association of VEGF +405G>C and −460C>T polymorphisms with survival clearly. However, if each study group is defined in detail and the factors that have potential effects on survival are standardized, perhaps a common denominator might be found and the effects of these polymorphisms on survival processes might be clarified.

Conclusion

In the present study, we investigated the associations of VEGF +405G>C and −460C>T polymorphisms with CRC risk, primary tumor location, metastasis, and prognosis as a response to bevacizumab therapy. While VEGF +405G>C polymorphism was associated with left colon cancer and progression-free survival as a response to bevacizumab treatment, VEGF −460C>T polymorphism was found to be related to liver metastasis in CRC patients. It is obvious that our study has limitations such as small sample size, heterogeneous treatment regimens, and these limitations may be limiting our results. However, we suggest that our findings would contribute to the understanding of the genetic background of CRC and treatment process by providing data on personalized therapy regimens. Thus, our conclusions should be verified in further studies with larger populations.

Footnotes

Authors' Contributions

M.C. contributed to the experimental molecular genetic analysis, study design, and literature. E.N., U.I.K., N.D.S., and C.C. as the clinicians selected and provided the patient to participate in this study, and blood samples and clinical data of the patients. B.S. contributed to the whole study management, development of the final protocol of the experiments, interpretation of the results, and organization of the article. All authors were involved in preparing the article.

Ethics Approval and Consent to Participate

This study was approved by the Marmara University Local Ethics Committee with the protocol number 09.2018.174. Informed consent was obtained from all individual participants included in the study.

Availability of Data and Materials

The datasets used and/or analyzed during this study are available from the corresponding author on reasonable request.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by the Marmara University Scientific Research Projects Coordination Unit. Grant No: FEN-C-DRP-110718-0407.

Supplementary Material

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