Vascular endothelial growth factor ‑634G/C polymorphism is associated with increased breast cancer risk and aggressiveness
- Authors:
- Published online on: July 31, 2013 https://doi.org/10.3892/mmr.2013.1607
- Pages: 1242-1250
Abstract
Introduction
Breast cancer is the most prevalent cancer and the leading cause of cancer mortality in females worldwide, as well as in Thailand. Angiogenesis is the formation of new blood vessels and has been involved in the initiation and aggressiveness of breast cancer (1–3). The most important key modulator in this complex process is vascular endothelial growth factor (VEGF). VEGF plays a role in breast cancer (4) and the VEGF pathway is targeted in the treatment of breast cancer (5).
Human VEGF is localized on chromosome 6p21.3 and organized as eight exons, separated by seven introns (6,7). Several polymorphisms in the promoter and 5′ untranslated region of (UTR) of VEGF have been identified (8,9). Awata et al previously identified seven polymorphisms in the promoter region as well as 5′ and 3′UTR of VEGF in a Japanese population. Serum VEGF levels have also been found to be significantly higher in healthy subjects with the −634C/C genotype (10). However, in vitro experiments using lipopolysaccharide-stimulated peripheral blood mononuclear cells demonstrated that −634 G/G correlates with elevated VEGF production (9). In non-small cell lung cancer, a low VEGF expression in cancer tissue was significantly associated with the presence of the −2578C/C, −634G/G and −1154A/A and GA alleles in the VEGF promoter (11). The association between VEGF polymorphisms and breast cancer has been previously investigated (12). Based on these observations, we hypothesized that polymorphisms in the VEGF promoter and 5′UTR contribute to varied levels of VEGF expression, subsequently leading to susceptibility to aggressive breast cancer. To investigate this hypothesis, the association between VEGF polymorphisms and breast cancer susceptibility and aggressiveness was investigated, as well as mRNA expression in breast cancer tissue.
Materials and methods
Study population
The study population was recruited from the Division of Head, Neck and Breast Surgery (Siriraj Hospital, Bangkok, Thailand) between 2000 and 2003. Patients with histopathologically confirmed breast carcinoma were included in this prospective study and newly diagnosed breast cancer patients were included in the case group. Patients with histories of other types of cancer were excluded. Healthy individuals and patients who attended the hospital due to benign conditions with no previous diagnosis of cancer were included in the control group and frequency matched to the breast carcinoma cases with regard to age (±5 years). Informed consent was obtained from all subjects. This study was approved by the ethics committee of the Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Genotyping of VEGF polymorphisms
VEGF −634G/C and −1154G/A were genotyped by the allele refractory mutation system-PCR. PCR was performed as summarized in Table I. VEGF −1498C/T and −2578C/A were genotyped by the PCR-restriction fragment length polymorphisms. Representative PCR products were sequenced to validate the assay.
Assessment of VEGF mRNA expression levels
The correlation between VEGF polymorphisms and expression levels was determined in breast tissue. VEGF mRNA expression was assayed by semi-quantitative RT-PCR as described previously (13). A primer pair that amplified β-actin was employed to check RNA integrity and used as an internal control. Primer sequences, PCR conditions and the number of PCR cycles are presented in Table II. To account for variability between gels, an RT-PCR product from the MDA-MB-231 cell line was electrophoresed as a control on each gel. PCR product intensity was analyzed using GeneTools® software (Syngene, Cambridge, UK). mRNA levels were calculated as the ratio of tissue sample to corresponding β-actin and then corrected as a ratio to the MDA-MB-231 sample on the same scan. Each RNA sample was assayed in duplicate and in two separate settings.
Statistical analysis
Distribution of VEGF allele frequencies and genotypes among the case and control groups was analyzed using the χ2 test. Odds ratios (ORs) and 95% confidence intervals (CIs), obtained from unconditional logistic regression, were used to measure the strength of the association between VEGF polymorphisms and susceptibility and aggressiveness of breast cancer. Individual haplotypes were determined using the PHASE program available at http://www.stat.washington.edu/stephens/phase.html (14). The end-point of overall survival (OS) analysis was breast cancer-associated mortality. The disease-free survival (DFS) analysis end-point was cancer recurrent/metastasis or breast cancer-associated mortality. DFS and OS time was calculated as the time from diagnosis to the end point of the study, censoring at the date of last contact or non-cancer mortality. The survival curves were determined using a Kaplan-Meier product-limit method. Statistical significance between the survival curves was assessed using the log-rank test. Multivariate analysis was performed to evaluate the effect of prognostic factors on OS, using the Cox proportional hazards model. P<0.05 was considered to indicate a statistically significant difference. mRNA levels were calculated as the ratio of tissue sample to corresponding β-actin and corrected as a ratio to the MDA-MB-231 cell line on the same scan. Each RNA sample was assayed in duplicate and in two separate settings.
Results
Correlation between VEGF genotypes and breast cancer susceptibility
Genotyping was performed on 483 breast cancer patients and 524 controls. −634C allele distribution was significantly higher in breast cancer patients compared with control subjects (33.23 vs. 23.71%; P<0.001). VEGF −634G/C and −634C/C genotype distributions were significantly higher in breast cancer patients (GC, 41.20 and CC, 12.63 vs. GC, 25.06 and CC, 11.18%; P<0.001). Allele and genotype frequency distributions of other loci were not found to be different. The mean age of the control group was 48.56±14.45 years (SEM). The mean age of the breast cancer patients was 50.8±11.326 years (SEM). The mean age of the breast cancer and control groups were statistically different [OR, −2.238; 95% CI, (−5.20)-(−3.957); P=0.011], thus, ORs and 95% CIs calculated by logistic regression were adjusted for age. Individuals with the −634G/C genotype had an increased risk of breast cancer when compared with the −634G/G genotype (OR, 2.544; 95% CI, 1.852–3.496; P<0.001). Homozygous CC had an increased risk when compared with −634G/G (OR, 1.600; 95% CI, 1.030–2.485; P=0.036; Table III). VEGF polymorphisms in other loci did not demonstrate any increased risk for breast cancer.
Correlation between VEGF genotypes and clinicopathological parameters
Table IV shows known clinicopathological parameters and demographic factors of the breast cancer patients. Numerous patients received surgery (mastectomy in 385 patients and wide excision in 91 patients). The patients with invasive carcinoma who underwent wide excision received radiotherapy. VEGF −634G/C genotype was associated with tumor size >20 mm (OR, 1.638; 95% CI, 1.103–2.434; P=0.015), perineural invasion (OR, 2.261; 95% CI, 1.217–4.202; P=0.010) and stage II–IV at diagnosis (OR, 1.915; 95% CI, 1.255–2.944; P=0.003). Separate analysis of invasive ductal carcinoma revealed a marked association with tumor size >20 mm (OR, 1.722; 95% CI, 1.097–2.703; P=0.018), perineural invasion (OR, 2.36; 95% CI, 1.227–4.539; P=0.010) and stage II–IV at diagnosis (OR, 2.078; 95% CI, 1.237–3.490; P=0.006). The VEGF −1498C/C genotype correlated with decreased risk of lymphovascular invasion (LVI; OR, 0.308; 95% CI, 0.102–0.927; P=0.036).
Haplotype analysis
The −2578C/−1498T/−1154G/−634G haplotype was the most common haplotype in the two groups (frequency, 0.3778 and 0.4739, respectively). Permutation testing revealed a significant difference between haplotype frequencies in the breast cancer and control groups (P=0.01). CTGG and CTGC haplotype copy number distributions were significantly different between the groups. Bearing 1 or 2 copies of the CTGG haplotype had a protective effect against breast cancer (OR, 0.55; 95% CI, 0.42–0.73; P<0.001). By contrast, bearing 1 or 2 copies of the CTGC haplotype was found to have an increased risk for breast cancer (OR, 1.81; 95% CI, 1.39–2.35; P<0.001). Patients with 1 or 2 copies of the CTGC haplotype significantly correlated with a tumor size >20 mm, (OR, 1.60; 95% CI, 1.09–2.35; P=0.0126), presence of perinural invasion (PNI; OR, 1.84; 95% CI, 0.97–3.52; P=0.046) and stage II–IV (OR, 1.73; 95% CI, 1.14–2.61; P=0.0062). Patients with 1 or 2 copies of ACAG haplotype exhibited a reduced risk for LVI and poorly differentiated cell types (OR, 0.74; 95% CI, 0.48–1.15; P=0.1656 and OR, 0.74; 95% CI, 0.46–1.17; P=0.1768, respectively).
Survival analysis
The median follow-up time was 65 months (range, 1.25–136.7 months). Among 446 patients, there were 37 mortalities during the study period, 30 of which were breast cancer-related. Locoregional recurrence was observed in 29 patients. Distant metastasis occurred in 86 patients. The Kaplan-Meier survival curves of various VEGF genotypes are shown in Fig. 1. Patients with the −634CC genotype (5 mortalities of 57 patients) succumbed to breast cancer in the first 21 months following breast cancer diagnosis. Univariate analysis between clinicopathological parameters, VEGF genotypes and corresponding 5-year survival are provided in Table V. Age >50 years correlated with lower 5-year OS. Larger tumor size, LVI, PNI, estrogen receptor- and progesterone receptor-negative, regional lymph-node positive, distant metastasis and advanced staging were the major prognostic factors for OS and DFS. No statistically significant correlation was found between VEGF genotypes and 5-year survival in the univariate analysis. Factors with a P-value ≤0.2 were included in the Cox regression analysis and poor differentiation, presence of PNI, PR-negative, axillary nodal metastasis and having the −634C/C and −1154G/A genotypes were found to significantly correlate with increased hazard ratio (HR) for DFS. HRs were 3.050 (95% CI, 1.354–6.871; P=0.007) and 2.452 (95% CI, 1.384–4.343; P=0.002) for the −634C/C and −1154G/A genotypes, respectively.
VEGF mRNA expression in breast cancer tissue
VEGF mRNA expression was evaluated in 124 breast cancer tissues. Characteristics of breast cancer patients are provided in Table VI. Expression ranged between 0 and 3.27 with a median of 1.10. Patients with the VEGF −634C/C genotype had significantly higher VEGF mRNA in breast cancer tissue compared with those with the −634G/G or −634G/C genotype (Fig. 2). Patients with heterozygous −1154G/A, −1498C/T and −2578A/C exhibited lower VEGF mRNA when compared with homozygous −1154G/G, −1498T/T and −2578C/C. Due to the presence of outliers and a small number of patients with the homozygous −1154A/A, −1498C/C and −2578A/A genotypes, VEGF mRNA expression in these groups appeared to be high. Following exclusion of the outlier, VEGF mRNA expression was decreased in patients with homozygous −1154A/A, −1498C/C and −2578A/A (data not shown).
Correlation between VEGF mRNA levels and clinicopathological parameters
Breast cancer patients were classified into low and high expression groups using a median value of 1.10. Patient distribution in each group and clinicopathological parameters are provided in Table VII. Elevated VEGF expression correlated with a tumor size >20 mm (OR, 2.476; 95% CI, 1.047–5.858; P=0.039), axillary nodal metastasis (OR, 2.288; 95% CI, 1.110–4.713; P=0.025) and presence of LVI (OR, 2.406; 95% CI, 1.142–5.070; P=0.021).
Discussion
In the current study, it was observed that alteration of nucleotides from G to C at −634, resulted in an increased risk of breast cancer. However, previous studies have not reported this correlation in breast cancer (15–20). In the present study, the −634G/C genotype was significantly associated with more aggressive features. Due to a limited number of patients with the −634C/C genotype, the difference was not observed to be statistically significant. This was consistent with a previous study by Balasubramanian et al and Jin et al which reported that an alteration of G to C at this position was associated with a larger tumor size and high grade breast cancer (16,18). By contrast, Langsenlehner et al observed a significant correlation between the −634G/C and −634C/C genotypes and smaller tumor size (19). Survival analysis revealed a significant correlation between the −634C/C genotype and lower DFS. However, OS of the patients with different −634G/C genotypes was similar, which may be due to the relatively short term follow up of this study. By contrast, the survival analysis of 1,455 Chinese breast cancer patients revealed that patients with the −634G/G genotype had a lower OS compared with those with the −634C/C genotype, however, this polymorphism was not found to correlate with DFS (21). The variance in the demographic results of the cancer population may contribute to discrepancies observed. In the present study, 26.09% of the patients were diagnosed as stage III and IV and 63.77% of the patients received chemotherapy, while in a study by Lu et al, only 11.34% of the patients were diagnosed as stage III and IV and a large number of patients received chemotherapy (93.95%) (21).
In the present study, no correlation was found between −1154G/A polymorphisms and breast cancer risk, consistent with previous studies in Caucasian populations (18,20,22). No correlation between breast cancer aggressiveness and −1154G/A polymorphisms was observed, consistent with a study by Jin et al(18). Breast cancer susceptibility or aggressiveness was not associated with −1498C/T polymorphisms, consistent with previous large case-control studies in Asian and Caucasian populations (15,16). There was no association between −2578A/C polymorphisms and breast cancer susceptibility/aggressiveness as previously observed in Caucasian populations by Jin et al and Langsenlehner et al(18,19). By contrast, two additional studies in Caucasian populations revealed conflicting results. Schneider et al reported an association between the −2578A/A genotype and breast cancer risk while Jacobs et al reported that −2578C was associated with an increased risk of invasive breast cancer (17,20). However, the latter study stated the importance of LD of −2578A/C and −1154G/A. Thus, the association of the two polymorphisms and breast cancer risk may not be individually demonstrated.
Haplotype analysis revealed that the −2578C/−1498T/ −1154G/−634G haplotypes had a protective effect against breast cancer. Patients with the −2578C/−1498T/−1154G/−634C haplotype had an increased risk of breast cancer and were associated with a tumor size >20 mm, stage II–IV and PNI. In a Swedish population, haplotypes −2578C/−634C were significantly associated with a large tumor size and higher grade. Having 2 copy numbers of haplotypes −2578A/−634G was associated with lower tumor grade (18). Jacobs et al reported that the −2578A/−1154A/−634G haplotypes correlated with a reduced risk of breast cancer in an American population (17). These observations and the observations of the present study indicate that haplotypes bearing the−634C allele contribute to increased breast cancer risk and aggressiveness. Non-replication of genetic association results is common in genetic epidemiology. In addition, the polymorphisms on the promoter region and 5′UTR were in LD. Interpretation of haplotype analysis revealed that the alleles included in the haplotypes may be associated with other functional polymorphisms that were not assessed. Comparison of haplotype analysis may be complicated due to variation in alleles in the haplotype, different software used to generate the haplotype and determining haplotype frequency.
The VEGF −634CC genotype correlated with elevated levels of mRNA expression. Elevated VEGF mRNA expression correlated with tumor size >20 mm, lymph node involvement and presence of LVI. These observations are consistent with a previous study employing the RT-PCR technique. Gomez-Esquer et al demonstrated a correlation between VEGF mRNA expression higher than the 25th percentile and more aggressive features in 103 breast cancer patients (23).
Incorporation of bevacizumab, a humanized monoclonal antibody that targets VEGF in chemotherapy, is a rapidly evolving area in the treatment of breast cancer. An association study of VEGF polymorphisms in 180 advanced breast cancer patients treated with paclitaxel alone or with bevacizumab and 183 untreated patients, revealed that the VEGF −2578A/A genotype was associated with an improved median OS in the combination arm when compared with AC combined with the CC genotype. The VEGF −1154A/A genotype also demonstrated an improved median OS when compared with GG combined with the GA genotype in the combination arm (24). These observations indicate that selection methods to identify the patients suitable for anti-VEGF therapy must be established. −634G/C polymorphisms may identify populations at risk and predict the outcome of breast cancer. It is possible that genotyping of VEGF −634 polymorphisms in breast cancer patients may be used to select appropriate patients for anti-angiogenesis treatment.
Acknowledgements
This study was supported by the Faculty of Medicine Siriraj Hospital, Mahidol University.
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