Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Functional Promoter -308G>A Variant in Tumor Necrosis Factor α Gene Is Associated with Risk and Progression of Gastric Cancer in a Chinese Population

  • Yan Hong ,

    Contributed equally to this work with: Yan Hong, Zhijun Ge

    Affiliation Department of Surgery, Yixing People's Hospital, Yixing, China

  • Zhijun Ge ,

    Contributed equally to this work with: Yan Hong, Zhijun Ge

    Affiliation Department of Surgery, Yixing People's Hospital, Yixing, China

  • Changrui Jing,

    Affiliation Department of Plastic Surgery, Wuxi No.2 People's Hospital, Wuxi, China

  • Jun Shi,

    Affiliation Department of Surgery, Yixing People's Hospital, Yixing, China

  • Xiao Dong,

    Affiliation Department of Surgery, Yixing Cancer Hospital, Yixing, China

  • Fengying Zhou,

    Affiliation Department of Breast Surgery, Wuxi Maternal and Child Health Hospital, Wuxi, China

  • Meilin Wang,

    Affiliation Department of Molecular and Genetic Toxicology, School of Public Health, Nanjing Medical University, Nanjing, China

  • Zhengdong Zhang,

    Affiliation Department of Molecular and Genetic Toxicology, School of Public Health, Nanjing Medical University, Nanjing, China

  • Weida Gong

    gongweida2010@gmail.com

    Affiliations Department of Surgery, Yixing People's Hospital, Yixing, China, Department of Surgery, Yixing Cancer Hospital, Yixing, China, Cancer Center, Nanjing Medical University, Nanjing, China

Functional Promoter -308G>A Variant in Tumor Necrosis Factor α Gene Is Associated with Risk and Progression of Gastric Cancer in a Chinese Population

  • Yan Hong, 
  • Zhijun Ge, 
  • Changrui Jing, 
  • Jun Shi, 
  • Xiao Dong, 
  • Fengying Zhou, 
  • Meilin Wang, 
  • Zhengdong Zhang, 
  • Weida Gong
PLOS
x

Abstract

Background

Tumor necrosis factor-α (TNF-α) plays a crucial role in the development and progression of gastric cancer. A functional polymorphism, -308 G>A (rs1800629), which is located in the promoter of TNFA gene, has been suggested to alter the production of TNF-α and influence cancer risk. In the present study, we sought to investigate whether this polymorphism has effects on the risk and progression of gastric cancer in a Chinese population.

Methods

We genotyped the TNFA -308 G>A polymorphism using the TaqMan method in a two-stage case-control study comprising a total of 1686 gastric cancer patients and 1895 cancer-free subjects. The logistic regression was used to assess the genetic associations with occurrence and progression of gastric cancer.

Results

We found a significant association between the variant genotypes and increased risk of gastric cancer [P = 0.034, odds ratio (OR) = 1.39, 95% confidence interval (CI) = 1.01–1.67, GA/AA vs. GG]. Similar results were observed in the follow-up replication study. When combined the data from the two studies, we found a more significant association (P = 0.001, OR = 1.34, 95%CI = 1.13–1.59), especially for older subjects (>65 years). Furthermore, the patients carrying the variant genotypes had a significantly greater prevalence of T4 stage of disease (P = 0.001, OR = 2.19, 95%CI = 1.39–3.47) and distant metastasis (P = 0.013, OR = 1.61, 95%CI = 1.10–2.35).

Conclusions

Our results suggest that the functional promoter -308 G>A polymorphism in TNFA influence the susceptibility and progression of gastric cancer in the Chinese population.

Introduction

Gastric cancer is the fourth most common cancer and the second leading cause of cancer-related death worldwide, and particularly prevalent in certain countries, including China [1], [2]. Although the exact etiology of gastric cancer remains to be identified, accumulating epidemiological studies have shown that diet, smoking, alcohol and especially Helicobacter pylori (H. pylori) infection, are well-known causes of gastric cancer [3], [4]. However, a high prevalence of these risk factors do not always result in a high incidence of gastric cancer, which suggests that other susceptible factors such as genetic variations and environmental differences may additionally contribute to gastric carcinogenesis. Recently, emerging evidence has suggested that genetic polymorphisms in candidate genes were associated with susceptibility to gastric cancer [5], [6], [7].

It has been suggested that chronic inflammation in gastric mucosa induced by the H. pylori infection is a critical step in the development of gastric cancer [8], [9]. In that case, genetic variations in inflammation-related cytokine genes may be potential susceptibility factors for this disease. One of the important cytokines associated with H. pylori infection is the tumor necrosis factor (TNF) –α which is encoded by the TNFA gene [10]. TNF-α is a pleiotropic cytokine mainly produced by activated monocytes and macrophages and plays an important role in the inflammatory response [11]. It has been suggested that over expression of TNF-α showed a significant severity-dose-response as risk markers from pre-neoplastic lesions to gastric cancer [12].

The TNFA gene is located within the human leukocyte antigen class III region on chromosome 6 (6p21) and is highly polymorphic. Polymorphisms in the TNFA gene have been shown to greatly influence its expression level [13]. Therefore, many studies attempted to investigate whether polymorphisms in the promoter of TNFA could be used as putative determinant factor of susceptibility for various diseases, including gastric cancer. Among the many polymorphisms in TNFA promoter, the TNFA-308 G>A polymorphism were wildly studied and there is functional study suggesting that the -308A allele is associated with increased TNF-α production [14]. As for gastric cancer, a number of studies were conducted to investigate the associations between the TNFA-308 G>A polymorphism and gastric cancer in various populations, however, the results form these studies remain inclusive [15], [16], [17], [18], [19]. To date, two published meta-analyses on this issue have suggested that the -308 G>A polymorphism was associated with gastric cancer in Western populations, but not in Asian populations [20], [21]. Herein, in the present study, we conducted a relative large two-stage case-control study that included a total of 1686 gastric cancer patients and 1895 cancer-free subjects to further evaluate the influence of the TNFA -308 G>A polymorphism on gastric cancer risk and progression in a Chinese population.

Materials and Methods

Ethics statement

The study was approved by the Institutional Review Board of the Nanjing Medical University, Nanjing, China. At recruitment, written informed consent was obtained from all participants involved in this study.

Study population

This is an ongoing molecular epidemiologic study of gastric cancer conducted in the First Affiliated Hospital of Nanjing Medical University, Nanjing, China. The design of the study and the inclusion criteria of the subjects were previously described elsewhere [22]. In brief, two independent hospital-based case-control studies were included in the present study. Overall, the test set included 750 gastric cases and 835 age and sex-matched controls recruited at the second affiliated hospital of Nanjing Medical University, Nanjing and Cancer Hospital of Nantong City, Nantong, China from March, 2006 to January, 2010, and the validation set included 936 cases and 1,060 controls enrolled from Yixing People's Hospital, Yixing, China from January, 1999 to December, 2006. All subjects were ethnic Han Chinese coming from different families and had no blood relationship. All the patients were newly diagnosed with histopathologically confirmed, incident gastric cancer and were consecutively recruited without restriction of age and sex. Histological sections of all cases were reviewed by two pathologists independently. Those patients that had previous cancer, metastasized cancer from other or unknown origin, or previous radiotherapy or chemotherapy were excluded. The controls were recruited from healthy subjects who were seeking physical examination in the outpatient departments at the corresponding hospital and were frequency-matched to the cases by age (±5 years) and sex. The cancer-free controls were genetically unrelated to the cases and had no individual history of cancer. Each patient donated 5 ml venous blood after providing a written informed consent. The response rate for case and control subjects was both above 85%.

DNA extraction and genotyping

The whole genomic DNA was isolated and purified from leucocytes of peripheral blood by proteinase K digestion and phenol/chloroform extraction. The TNFA -308 G>A polymorphism was genotyped using the TaqMan-MGB method (Applied Biosystems, Foster City, CA, USA), which uses two allele-specific TaqMan MGB probes and a PCR primer pair to detect the specific SNP target. The sequence of the primers and probes are available on request. The reaction mixture of 10 µL contained 20 ng genomic DNA, 3.5 µL of 2× TaqMan Genotyping Master Mix, 0.25 µL of the primers and probes mix and 6.25 µL of double distilled water. The amplification was performed under the following conditions: 50°C for 2 min, 95°C for 10 min followed by 45 cycles of 95°C for 15 sec, and 60°C for 1 min. Following the manufacturer's instructions, amplifications were conducted in the 384-well ABI 7900HT Real Time PCR System (Applied Biosystems, Foster City, CA, USA) and the allelic discrimination were performed using the SDS 2.4 software (Applied Biosystems, Foster City, CA, USA). The genotyping rates of these SNPs were all above 98%. To ensure the accuracy of genotyping, two negative experimental control (water) and two positive experimental controls with known genotype were included in each reaction plate. In addition, about 5% of the samples were randomly selected for repeated genotyping for confirmation; and the results were 100% concordant.

Statistical analysis

Before further analysis, the allele frequencies of TNFA -308G>A polymorphism in the controls of test set, validation set and combined set were assessed against departure from Hardy-Weinberg equilibrium (HWE) using a goodness-of-fit χ2-test. Differences in the distributions of age, sex and frequencies of genotypes of the TNFA -308G>A polymorphism between the cases and controls were evaluated by Pearson's χ2 test. The associations between the -308G>A genotypes and risk of gastric cancer as well as the clinical characteristics of the patients were measured by computing odds ratios (ORs) and 95% confidence intervals (CIs) from unconditional logistic regression analysis with the adjustment for age and sex. All the statistical analyses were performed with the software SAS 9.1.3 (SAS Institute, Cary, NC, USA) and a two-side P value of less than 0.05 was considered as statistically significant.

Results

Characteristics of the study population

The frequency distributions of demographic characteristics and clinical features of the two sets of study group are shown in Table 1. The cases and controls were matched by age and sex (P = 0.383 and 0.424 in the test set and P = 0.164 and 0.061 in the validation set, respectively). Although more older subjects (>65years) and female subjects were presented in the test set than in the validation set, no significant difference in age and sex between the cases and controls was observed when we combined the two populations (Table 1). In the test set, there were 295 cardia gastric cancer (CGC) patients and 455 non-cardia gastric cancer (NCGC) patients; 406 patients diagnosed as diffuse type of gastric cancer and 299 as intestinal type. Clinical TNM stage is categorized according to depth of invasion, lymph node metastasis and distant metastasis. The percent of TNM stage of patients in the test set from I to IV were 26.8%, 22.0%, 35.5%, and 15.7%, respectively. Of the 936 patients in the validation set, 358 were CGC and 578 were NCGC; 539 patients diagnosed as diffuse type of gastric cancer and 397 as intestinal type. Approximately 27.7%, 19.8%, 42.2% and 10.3% of the patients were found to have TNM stage I, II, III and IV diseases, respectively.

Association between the TNFA -308 G>A polymorphism and gastric cancer risk

The genotyping results of the TNFA -308 G>A polymorphism among the patients and controls in the two study set are presented in the Table 2. The observed genotype frequencies in the controls of test set, validation set and the combined set were all conformed to the HWE (P = 0.481, 0.448 and 0.946, respectively). We then used Pearson's χ2 test and logistic regression analysis to assess the association between TNFA -308 G>A polymorphism and gastric cancer risk. In the test set, we found that there were more variant genotypes (GA/AA) in the case group than that in the control group (P = 0.034). Compared with the individuals with GG genotype, we found those subjects with GA/AA genotypes had an increased risk of gastric cancer (adjusted OR = 1.39, 95%CI = 1.01–1.67). Similar associations were observed in the replication study (GA vs. GG: OR = 1.29, 95%CI = 1.02–1.65 and GA/AA vs. GG: OR = 1.31, 95%CI = 1.03–1.65). When we combined data from the two studies, a more significant association was observed (GA vs. GG: OR = 1.33, 95%CI = 1.12–1.59 and GA/AA vs. GG: OR = 1.34, 95CI% = 1.13–1.59).

thumbnail
Table 2. Genotype and allele frequencies of TNF-α -308 G>A polymorphism among the cases and controls and their associations with risk of gastric cancer.

https://doi.org/10.1371/journal.pone.0050856.t002

To evaluate if the TNFA -308 G>A polymorphism plays different role in subgroups of age, sex, tumor site and histological types, we then performed stratified analysis based on these variables. As shown in Table 3, the stratified analysis showed that the increased risk associated with the TNFA -308GG/GA genotypes was more evident among older subjects (>65years) (P<0.001, OR = 1.70, 95%CI = 1.28–2.25). Besides, no significant difference was observed in the stratification of sex, tumor site and histological types.

thumbnail
Table 3. Stratification analyses between TNFA -308 G>A polymorphism and gastric cancer risk in the combined group.

https://doi.org/10.1371/journal.pone.0050856.t003

Association between the TNFA -308 G>A polymorphism and clinical characteristics of the patients with gastric cancer

Polymorphisms in candidate genes have been suggested to influence cancer progression. We then evaluated whether the TNFA -308 G>A polymorphism has effects on the clinical characteristics of the patients, including TNM stage, distant metastasis, lymph node metastasis and depth of tumor infiltration. As shown in Table 4, we found that the patients carrying the variant genotypes had a significantly greater prevalence of T4 stage of invaded depth (P = 0.001, OR = 2.19, 95%CI = 1.39–3.47) and distant metastasis (P = 0.013, OR = 1.61, 95%CI = 1.10–2.35), indicating the polymorphism might be associated with gastric cancer progression.

thumbnail
Table 4. The association between TNF-α-308 G>A polymorphism and the progression of gastric cancer.

https://doi.org/10.1371/journal.pone.0050856.t004

Discussion

The main findings of our study are the significant associations between the TNFA -308 G>A polymorphism and gastric cancer risk and progression. TNF-α is an important immune mediator in inflammatory response and has been suggested to be an endogenous tumor promoter in human carcinogenesis [23], [24], [25]. In gastric cancer, Senthilkumar et al. demonstrated that TNF-α mRNA and protein expressions were significantly increased in gastric adenocarcinoma patients, by using RT-PCR, western blotting and immunohistochemistry [12]. The influence of the -308G>A polymorphism on the expression level of TNFA has been previously investigated. Karen et al. showed that the -308G>A polymorphism could affect the affinity of factor binding and result in a factor binding to the -308A allele but not the -308G allele, which then lead to an increased expression of TNFA [14]. Considering that TNF-α is up-regulated in gastric cancer, it is possible that individuals carrying the -308A allele (ie. GA/AA), which is associated with increased level of TNF-α, have an increased risk for gastric cancer. Therefore, it is biologically plausible that the TNFA -308G>A confers individuals' susceptibility to gastric cancer.

To date, several molecular epidemiological studies have been conducted to investigate the association between the TNFA -308G>A polymorphism and gastric cancer risk. However, the results from these studies remain inconclusive. There are two published meta-analyses on the associations between the TNFA -308G>A and gastric cancer risk that have identified this polymorphism as a risk factor for gastric cancer in Caucasian populations but not in Asian populations [20], [21]. When reviewed the two meta-analyses, we found only 3 studies with a total of 513 cases and 714 controls and 5 studies with a total of 833 cases and 1375 controls in Chinese populations were included in the meta-analyses of Zhang et al. [20] and Gorouhi et al. [21], respectively (Table 5). The number of studies and the sample size of these studies may be too limited to generate a stable result. Of these studies, only Lu et al. found a significant association between TNFA -308GA genotype and increased risk of gastric cancer risk (OR = 1.81, 95%CI = 1.04–3.14). Our study further identify the TNFA -308G>A polymorphism as a risk factor for gastric cancer in Chinese population, which may be of value in evaluating the role of TNF- α in gastric carcinogenesis. As indicated by Power and Sample Size Calculation software, our study had a 96% power to detect an OR of 1.50 or greater and a minimal OR of 0.62 with an exposure frequency of 9% at the current sample size (1686 cases and 1895 controls), which suggests that the sample size of our study is efficient.

thumbnail
Table 5. Comparison of the sample size of the studies conducted in Chinese populations.

https://doi.org/10.1371/journal.pone.0050856.t005

In addition, we found that the TNFA -308G>A polymorphism was associated with increased risk of gastric cancer among subgroups of older subjects (age>65 years) rather than younger subjects. This observation is well supported by a large body of studies, which link weak immune system with ageing [26], [27]. The immune system of aged individuals undergoes alterations that may account for an increased susceptibility to malignancies [26]. It has been proposed that high expression of TNF- α might be involved in tumor growth and spread through influencing tissue remodeling and stromal development [28], [29]. In our study, we also found the TNFA -308G>A polymorphism was associated with clinical aggressiveness such as advanced tumor stage and distant metastasis. Similar observation has also been reported in other study [30]. If confirmed by additional studies, this polymorphism might help to accurately predict the clinical course of gastric cancer. However, these results should be interpreted with cautious because there is possibility that the associations with poor prognosis are due to a late stage at diagnosis.

When interpreting our results, some other limitations should also be concerned. First, since our study was hospital-based design, the possibility of selection bias of subjects that were associated with a particular genotype might exist. However, the genotype distributions in our controls were similar to distributions reported in other Chinese studies and conformed to HWE. Therefore, the selection bias in terms of genotype distributions would not be substantial. Second, H. pylori infection status is a well-known cause of gastric cancer and has been suggested to influence the expression of TNF- α [31]. Genetic variations in TNFA may interact with this factor to affect gastric caner risk. Unfortunately, lack of available information on H. pylori infection status in our study limited us to further explore these interactions. Thus, further studies with more detailed data on H. pylori infection are warranted.

In conclusion, our results suggest the -308G>A polymorphism in the promoter of TNFA has a significant influence on the occurrence and progression of gastric in the Chinese population. Although the associations appeared to be statistically significant in our population, these findings should be independently verified by other prospective studies with larger sample size simultaneous of well-matched controls and unbiased-matched homogeneous participants.

Author Contributions

Conceived and designed the experiments: WG ZZ ZG. Performed the experiments: YH FZ. Analyzed the data: YH FZ WG ZG JS. Contributed reagents/materials/analysis tools: YH XD CJ. Wrote the paper: YH FZ WG ZG MW.

References

  1. 1. Parkin DM, Bray F, Ferlay J, Pisani P (2005) Global cancer statistics, 2002. CA Cancer J Clin 55: 74–108.
  2. 2. Hohenberger P, Gretschel S (2003) Gastric cancer. Lancet 362: 305–315.
  3. 3. Uemura N, Okamoto S, Yamamoto S, Matsumura N, Yamaguchi S, et al. (2001) Helicobacter pylori infection and the development of gastric cancer. N Engl J Med 345: 784–789.
  4. 4. Crew KD, Neugut AI (2004) Epidemiology of upper gastrointestinal malignancies. Semin Oncol 31: 450–464.
  5. 5. Yoshida T, Ono H, Kuchiba A, Saeki N, Sakamoto H (2010) Genome-wide germline analyses on cancer susceptibility and GeMDBJ database: Gastric cancer as an example. Cancer Sci 101: 1582–1589.
  6. 6. Wang M, Zhang R, He J, Qiu L, Li J, et al. (2012) Potentially functional variants of PLCE1 identified by GWASs contribute to gastric adenocarcinoma susceptibility in an eastern Chinese population. PLoS One 7: e31932.
  7. 7. Shi Y, Hu Z, Wu C, Dai J, Li H, et al. (2011) A genome-wide association study identifies new susceptibility loci for non-cardia gastric cancer at 3q13.31 and 5p13.1. Nat Genet 43: 1215–1218.
  8. 8. You WC, Zhang L, Gail MH, Chang YS, Liu WD, et al. (2000) Gastric dysplasia and gastric cancer: Helicobacter pylori, serum vitamin C, and other risk factors. J Natl Cancer Inst 92: 1607–1612.
  9. 9. Peek RM Jr, Blaser MJ (2002) Helicobacter pylori and gastrointestinal tract adenocarcinomas. Nat Rev Cancer 2: 28–37.
  10. 10. Yea SS, Yang YI, Jang WH, Lee YJ, Bae HS, et al. (2001) Association between TNF-alpha promoter polymorphism and Helicobacter pylori cagA subtype infection. J Clin Pathol 54: 703–706.
  11. 11. Thalmaier U, Lehn N, Pfeffer K, Stolte M, Vieth M, et al. (2002) Role of tumor necrosis factor alpha in Helicobacter pylori gastritis in tumor necrosis factor receptor 1-deficient mice. Infect Immun 70: 3149–3155.
  12. 12. Senthilkumar C, Niranjali S, Jayanthi V, Ramesh T, Devaraj H (2011) Molecular and histological evaluation of tumor necrosis factor-alpha expression in Helicobacter pylori-mediated gastric carcinogenesis. J Cancer Res Clin Oncol 137: 577–583.
  13. 13. Raabe T, Bukrinsky M, Currie RA (1998) Relative contribution of transcription and translation to the induction of tumor necrosis factor-alpha by lipopolysaccharide. J Biol Chem 273: 974–980.
  14. 14. Kroeger KM, Carville KS, Abraham LJ (1997) The -308 tumor necrosis factor-alpha promoter polymorphism effects transcription. Mol Immunol 34: 391–399.
  15. 15. Li C, Xia B, Yang Y, Li J, Xia HH (2005) TNF gene polymorphisms and Helicobacter Pylori infection in gastric carcinogenesis in Chinese population. Am J Gastroenterol 100: 290–294.
  16. 16. Lu W, Pan K, Zhang L, Lin D, Miao X, et al. (2005) Genetic polymorphisms of interleukin (IL)-1B, IL-1RN, IL-8, IL-10 and tumor necrosis factor {alpha} and risk of gastric cancer in a Chinese population. Carcinogenesis 26: 631–636.
  17. 17. Wu MS, Huang SP, Chang YT, Shun CT, Chang MC, et al. (2002) Tumor necrosis factor-alpha and interleukin-10 promoter polymorphisms in Epstein-Barr virus-associated gastric carcinoma. J Infect Dis 185: 106–109.
  18. 18. Fei BY, Xia B, Deng CS, Xia XQ, Xie M, et al. (2004) Association of tumor necrosis factor genetic polymorphism with chronic atrophic gastritis and gastric adenocarcinoma in Chinese Han population. World J Gastroenterol 10: 1256–1261.
  19. 19. Guo W, Wang N, Li Y, Zhang JH (2005) Polymorphisms in tumor necrosis factor genes and susceptibility to esophageal squamous cell carcinoma and gastric cardiac adenocarcinoma in a population of high incidence region of North China. Chin Med J (Engl) 118: 1870–1878.
  20. 20. Zhang J, Dou C, Song Y, Ji C, Gu S, et al. (2008) Polymorphisms of tumor necrosis factor-alpha are associated with increased susceptibility to gastric cancer: a meta-analysis. J Hum Genet 53: 479–489.
  21. 21. Gorouhi F, Islami F, Bahrami H, Kamangar F (2008) Tumour-necrosis factor-A polymorphisms and gastric cancer risk: a meta-analysis. Br J Cancer 98: 1443–1451.
  22. 22. Zhou F, Zhu H, Luo D, Wang M, Dong X, et al. (2012) A Functional Polymorphism in Pre-miR-146a Is Associated with Susceptibility to Gastric Cancer in a Chinese Population. DNA Cell Biol
  23. 23. Grimm M, Lazariotou M, Kircher S, Hofelmayr A, Germer CT, et al. (2010) Tumor necrosis factor-alpha is associated with positive lymph node status in patients with recurrence of colorectal cancer - indications for anti-TNF-alpha agents in cancer treatment. Anal Cell Pathol (Amst) 33: 151–163.
  24. 24. Komori A, Yatsunami J, Suganuma M, Okabe S, Abe S, et al. (1993) Tumor necrosis factor acts as a tumor promoter in BALB/3T3 cell transformation. Cancer Res 53: 1982–1985.
  25. 25. Fujiki H, Suganuma M (2011) Tumor promoters–microcystin-LR, nodularin and TNF-alpha and human cancer development. Anticancer Agents Med Chem 11: 4–18.
  26. 26. Ginaldi L, De Martinis M, D'Ostilio A, Marini L, Loreto MF, et al. (1999) The immune system in the elderly: II. Specific cellular immunity. Immunol Res 20: 109–115.
  27. 27. Pawelec G, Barnett Y, Forsey R, Frasca D, Globerson A, et al. (2002) T cells and aging, January 2002 update. Front Biosci 7: d1056–1183.
  28. 28. Balkwill F (2002) Tumor necrosis factor or tumor promoting factor? Cytokine Growth Factor Rev 13: 135–141.
  29. 29. Fujiki H, Suganuma M, Komori A, Yatsunami J, Okabe S, et al. (1994) A new tumor promotion pathway and its inhibitors. Cancer Detect Prev 18: 1–7.
  30. 30. Correa GT, Bandeira GA, Cavalcanti BG, de Carvalho Fraga CA, dos Santos EP, et al. (2011) Association of -308 TNF-alpha promoter polymorphism with clinical aggressiveness in patients with head and neck squamous cell carcinoma. Oral Oncol 47: 888–894.
  31. 31. Harris PR, Mobley HL, Perez-Perez GI, Blaser MJ, Smith PD (1996) Helicobacter pylori urease is a potent stimulus of mononuclear phagocyte activation and inflammatory cytokine production. Gastroenterology 111: 419–425.