Genetic Polymorphism of VEGF-1154 (A/G) in Laryngeal Squamous Cell Carcinoma

Archives of Medical Research 39 (2008) 209e211

ORIGINAL ARTICLE

Genetic Polymorphism of VEGF-1154 (A/G) in Laryngeal Squamous Cell Carcinoma ¨ zlen Tubay Ba ¨ nal,a Murat U ¨ nal,b O Zeynep Nil U gdato glu,a Gu¨rbu¨z Polat,a and U gur Atika a

Department of Biochemistry and bDepartment of Otorhinolaryngology, Mersin University School of Medicine, Mersin, Turkey Received for publication August 17, 2007; accepted September 24, 2007 (ARCMED-D-07-00379).

Background. Angiogenesis has been shown to be increased in various human tumors including head and neck squamous cell carcinoma (SCC). Vascular endothelial growth factor (VEGF) is thought to be one of the most important angiogenic factors in tumorigenesis. In this study, we aimed to investigate whether polymorphism of VEGF1154 (A/G) genotypes are associated with the risk of laryngeal SCC. Methods. A prospective, randomized, case-control study in a tertiary university hospital was done. The study group consisted of 57 Caucasian patients with laryngeal SCC and 89 control subjects. Blood samples were obtained before surgery or from the patients under follow-up to 5 years after surgery. VEGF-1154 (A/G) genotypes were detected by realtime polymerase chain reaction with thermal cycler system. Results. According to the high-risk (GG) genotype, the difference between the patient and control groups was statistically significant (OR 0.43, 95% CI 5 0.19e0.95, p 5 0.037). Conclusions. GG genotype of the VEGF gene may increase the risk of laryngeal SCC in this population. VEGF gene polymorphism may be an important potential genetic and therapeutic marker of laryngeal SCC. Ó 2008 IMSS. Published by Elsevier Inc. Key Words: VEGF polymorphism, Laryngeal cancer, Genetic predisposition.

Introduction Squamous cell carcinoma (SCC) of the larynx is one of the most frequent malignancies occurring in the head and neck region. The intensity of angiogenesis has been shown to be increased in various human tumors including head and neck SCC (HNSCC). It is now generally accepted that tumor angiogenesis is important both for the growth of a primary neoplastic tumor and also for the development of metastasis (1). Among these angiogenic factors, vascular endothelial growth factor (VEGF) is thought to be one of the most important. VEGF induces the proliferation, differentiation, and migration of vascular endothelial cells; increases the permeability of the capillaries; and enhances the survival of endothelial cells by preventing their apoptosis (2e4). In several studies, increased microvessel density and overexpression of VEGF in HNSCC was also associated with Published previously online November 15, 2007. ¨ nal, Mersin Universitesi Tip Address reprint requests to: Dr. Murat U Fak. Hastanesi, KBB AD, Zeytinlibahce cad., 33079, Mersin, Turkey; E-mail: [email protected]; [email protected]

metastasis, recurrence, and poor prognosis. However, the association between angiogenesis and tumorigenesis of HNSCC is not well defined (5,6). Genetic host factors, including the VEGF genotype, must also contribute to the laryngeal cancer development mechanisms. Several single nucleotide polymorphisms have been described in the VEGF gene, some of which have been shown to be associated with differential expression of VEGF in vitro, and two of them (positions 2578 and 1154) are located in the VEGF promoter (7). The aim of this study is to investigate the possible relation between the VEGF-1154 (A/G) gene polymorphism and the laryngeal SCC. Materials and Methods The study group consisted of 57 Caucasian patients with laryngeal SCC. There were 54 men with a mean age of 55 years (range, 37e72 years) and three women with a mean age of 47 years (range, 32e55 years) and 89 control subjects, 53 men and 36 women; mean age 50 years (range, 28e73 years). This was a hospital-based case-control study

0188-4409/08 $esee front matter. Copyright Ó 2008 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2007.09.008

¨ nal et al./ Archives of Medical Research 39 (2008) 209e211 U

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conducted at the University of Mersin Hospital from 1999 to 2006. Patients and control subjects were from the same geographic region (southern Turkey) and of the same ethnic origin (Turkish-Caucasian). Patients and control subjects were unrelated. All participants gave informed consent and the institutional review board approved the study. All patients were primarily treated with surgical intervention (total or partial laryngectomy with or without neck dissection). Tumor localization was supraglottic in 24 cases, glottic in 14 cases, and transglottic in 19 cases. According to pathological TNM, 23 patients had stage IeII disease, and 34 had stage IIIeIV disease. We tried to match the control and study groups according to age and smoking status. Table 1 summarizes the patient and control group characteristics. Blood samples (5 mL) were obtained before surgery or during follow-up for up to 5 years after surgery (mean follow-up, 33 months; range, 6e60 months). DNA Extraction and Genotyping of VEGF Blood was collected in EDTA-containing tubes, and DNA was extracted from the leucocytes by high pure template preparation kit (Roche Diagnostics, GmbH, Mannheim, Germany). Primers for VEGF were as follows: VEGF1154 common primer: CCC CGC TAC CAG CCG ACT T, VEGF-1154 A (low expresser): GCC CGA GCC GCG TGT GGA A, and VEGF-1154 G (high expresser): GCC CGA GCC GCG TGT GGA G. All DNAs were amplified using these oligonucleotide primers. A PCR mixture was obtained with 5 mM dNTP, primers, control primers, 10X PCR tampon, distilled water, 25 mM MgCl2, 100% sucrose, 10 mM cresol red, and Taq polymerase enzyme. A polymerase chain reaction was performed by adding DNA samples on to the PCR mixture in a thermal cycler system (GeneAmp PCR 9700; Applied Biosystems, Foster City, CA). PCR procedure was done according to the literature (8). Samples were then loaded on 2% agarose gel for electrophoresis (90 V, 40 min). Agarose gel was evaluated under ultraviolet light and polymorphisms were classified as AA, AG, and GG (Figure 1). Table 1. Characteristics of the study groups Characteristics

Patients (n 5 57)

Control subjects (n 5 89)

Mean age Sex Male Female Smoking habit Smoking Nonsmoking Tumor location Glottic Supraglottic Subglottic Transglottic

55

50

54 3

53 36

57 e

42 47

14 24 e 19

Figure 1. A sample view of the PCR results. First row represents the internal control band and the second row shows the genotypes of patients (1e5); patients 1e3: AG, patients 4e5: GG.

Statistical Analysis The association between VEGF polymorphisms and laryngeal SCC was modeled through multivariate logistic regression analysis. Odds ratios and confidence intervals were used to analyze the occurrence of VEGF-1154 (A/G) genotype in patients with laryngeal SCC compared with the control groups. Significance testing was carried out by combining chi-square tests and then comparing the two independent proportions. In this study, the maximum type I error was set at .05. Results The AA genotype was not seen in both patient and control groups. The AG genotype was 56% in patient group and 37.08% in control group, respectively. The GG genotype was detected in a rate of 44% in patient group, and 62.92% in control group, respectively. According to the high-risk (GG) genotype, the difference between the patient and control groups was statistically significant (OR 0.43, 95% CI 5 0.19e0.95, p 5 0.037). Table 2 summarizes the genotype and allelic frequencies of the study and control groups. Discussion The VEGF genotype divides into low expression (homozygote AA), intermediate (heterozygote AG) and high expression (homozygote GG) phenotypes by Mendelian inheritance. We hypothesized that patients with high Table 2. Genotype and allelic frequencies of the study and control groups n Genotypes AA AG GG Allele frequencies Allele A Allele G

Control

Patient

0 (0%) 33 (37%) 56 (63%)

0 (0%) 32 (56%) 25 (44%)

33 (18.5%) 145 (81.5%)

26 (29%) 64 (71%)

VEGF Polymorphism in Laryngeal Cancer

expression of VEGF genotype have more aggressive and metastatic tumor behavior than low expression or heterozygote genotypes. To our knowledge, this is the first study investigating this relationship in the Western literature, and according to the high-risk (GG) genotype, the difference between the patient and control groups was statistically significant. Although it is possible that patients with the high-risk genotype have more aggressive disease, this should be supported with a prospective study that examines the association between genotype and survival. VEGF gene is located on chromosome 6p12 and five genes encoding VEGF-like proteins (AeE) have been identified, each of which generates different isoforms by alternative splicing mechanisms (9). The main mechanism for VEGF upregulation is hypoxia, which occurs frequently in rapidly growing tumors. VEGF proteins themselves are the most potent inducers of receptor upregulation. Receptor binding of VEGF is a central event in the angiogenic cascade, resulting finally in increased microvessel density (MVD). MVD represents the clinical correlate for neoangiogenesis, and increased MVD in tumors has been associated with adverse clinical outcome. However, a clear correlation between VEGF, VEGF receptors, and clinical parameters has not yet been established in HNSCC. Riedel et al. investigated the circulating level of VEGF in sera from patients with HNSCC as well as from healthy normal controls, VEGF serum concentration being significantly higher in HNSCC (10). Teknos et al. found that serum VEGF levels were significantly elevated in patients with advanced laryngeal carcinoma vs. healthy controls, and elevated pre-treatment serum VEGF levels tended to indicate a more aggressive disease state and a poorer overall survival in advanced laryngeal carcinoma (11). Yaylaci et al. demonstrated a clear correlation between tumor vascularity and VEGF expression in the samples of laryngeal SCC (12). On the other hand, there has been no study investigating the relationship between the genetic polymorphism of VEGF and laryngeal SCC. Previously, Ku et al. investigated the VEGF gene-460C/T polymorphism as a biomarker for oral cancer and found that the frequency of the ‘TT’ homozygote in the oral cancer group was much higher than in the control group (13). As with Ku et al., we could not analyze the possible link between the disease’s aggressiveness and age, tumor location, neck status, and tumor stage due to small sample size. Also, Yapijakis et al. recently demonstrated that low VEGF production allele of the þ936C/T polymorphism is strongly associated with increased risk for oral cancer (14). In conclusion, many investigators believe that survival can be improved if there are effective methods of identification of the patient who is at high risk for the development of malignancy. Epidemiology of genetic features may help in the detection and prevention of laryngeal SCC. This approach may lead the clinicians to identify high-risk individuals (15,16). We conclude that GG genotype of the VEGF gene may increase the risk of laryngeal SCC in this population. VEGF

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gene polymorphism may be an important potential genetic and therapeutic marker of laryngeal SCC. The major limitations of our study are the modest sample size and matching problem in controls and patients according to gender and age. Thus, further studies are necessary to delineate the potential role of VEGF polymorphism in the susceptibility to laryngeal SCC in a larger population.

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