Japanese Journal of Clinical Oncology Advance Access originally published online on June 9, 2009
Japanese Journal of Clinical Oncology 2009 39(8):497-501; doi:10.1093/jjco/hyp056
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© The Author (2009). Published by Oxford University Press. All rights reserved
No Association of the NFKB1 Insertion/Deletion Promoter Polymorphism with Survival in Patients with Gastric Cancer
1 Department of Oncology/Hematology, Kyungpook National University Hospital, Kyungpook National University School of Medicine
2 Department of Radiology, Kyungpook National University Hospital, Kyungpook National University School of Medicine
3 Department of the Advanced Medical Technology Center for Diagnosis and Prediction, Kyungpook National University Hospital, Kyungpook National University School of Medicine
4 Department of Internal Medicine, Kyungpook National University Hospital, Kyungpook National University School of Medicine
5 Department of Surgery, Kyungpook National University Hospital, Kyungpook National University School of Medicine, Daegu, Korea
For reprints and all correspondence: Wansik Yu, Department of Surgery, Kyungpook National University Hospital, Kyungpook National University School of Medicine, 50 Samduck 2-Ga, Jung-Gu, Daegu 700-712, Korea. E-mail: wyu{at}mail.knu.ac.kr
Received February 27, 2009; accepted May 4, 2009
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Abstract |
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 TOP Abstract INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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Objective: The present study analyzed the functional insertion/deletion polymorphism in the promoter region of NFKB1 gene and their impact on the prognosis for patients with gastric adenocarcinoma.
Methods: Four hundred and seventy two consecutive patients with curatively resected gastric adenocarcinoma were enrolled in the present study. The genomic DNA was extracted from paraffin-embedded tissue and the –94 insertion/deletion ATTG polymorphism of NFKB1 determined using a polymerase chain reaction–restriction fragment length polymorphism assay.
Results: The NFKB1 promoter gene polymorphism was successfully amplified in 97.8% of the cases. There were no sexual differences in relation to the genotype and allele. No correlation was observed between the frequency of the genotype or allele and the T, N or M stage. The multivariate survival analysis showed no association between the NFKB1 –94 insertion/deletion promoter polymorphism and the disease-free survival or overall survival of the patients with gastric cancer.
Conclusions: The functional NFKB1 promoter polymorphism was not found to be a prognostic marker for Korean patients with surgically resected gastric adenocarcinoma.
Key Words: gastric cancer • NFKB1 • polymorphism • prognosis
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INTRODUCTION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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Although gastric cancer is increasingly being diagnosed at an early stage, resulting in 10-year survival rates of between 80% and 95% in the Far East, gastric adenocarcinoma is still the second leading cause of cancer-related death worldwide (1,2). The role of genetic polymorphisms, which is an important determinant of endogenous causes of cancer, in relation to the risk or prognosis of gastric cancer has attracted increasing interest, probably because of advances in DNA analysis technologies and human genome knowledge. However, most previous studies have only addressed the effect of genetic variants of metabolic enzymes and inflammation mediators (3).
Nuclear factor-
B (NF-B) is a major transcription regulator of immune response, apoptosis and cell growth control genes, and there are five members of the NF-B family in mammals: p50/p105, p65/RelA, c-Rel, RelB and p52/p100. Although many dimeric forms of NF-B have been detected, the major form of NF-B is a heterodimer of the p50 and p65/RelA subunits, encoded by the NFKB1 and RelA genes, respectively (4). A common insertion/deletion (–94 insertion/deletion ATTG) located between two putative key promoter regulatory elements in the NFKB1 gene was identified, which seems to be the first potential functional NFKB1 genetic variation. The presence of a 4 bp deletion resulted in the loss of binding to nuclear proteins, leading to reduced promoter activity (5).
A research has shown that the deletion (ATTG1 allele) was associated with an increased risk for an inflammatory intestinal disorder-ulcerative colitis, but subsequently other study failed to replicate this association (5–7). It has also been reported that the NFKB1 insertion/deletion promoter polymorphism is associated with the susceptibility of oral squamous cell carcinoma, nasopharyngeal cancer and bladder tumor (8–10). Moreover, Riemann et al. (11) reported the relationship between this polymorphism and survival of colorectal and renal cell carcinoma.
However, no study has yet been published that has investigated the single-nucleotide polymorphism (SNP) of the NFKB1 promoter and their relationship to the clinical outcomes of gastric cancer. Therefore, the present study analyzed the functional insertion/deletion polymorphism in the promoter region of NFKB1 gene and their impact on the prognosis for patients with gastric adenocarcinoma.
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PATIENTS AND METHODS |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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Study Population
All the tissues investigated in this study were obtained from consecutive Korean patients (n = 472) who had undergone a surgical gastrectomy between January 2000 and December 2001 at Kyungpook National University Hospital (Daegu, Korea). Written informed consent for gene expression analyses including SNPs was received from all the patients before surgery, and the study was approved by the Institutional Research Board at Kyungpook National University Hospital. The diagnosis and staging of gastric carcinoma were assessed according to the WHO classifications (12) and TMN classifications set out by the American Joint Committee on Cancer (AJCC) (13).
Genotyping of NFKB1 Polymorphism
The genomic DNA was extracted from paraffin-embedded tumor-bearing tissue using a Wizard genomic DNA purification kit (Promega, Madison, WI, USA). The –94 insertion/deletion ATTG polymorphism of NFKB1 was then determined using a polymerase chain reaction (PCR)–restriction fragment length polymorphism assay. The primers generating amplicons of 305 bp (for deletion allele) or 309 bp (for insertion allele) were sense: 5'-CCGCACCAAAAACCAGTAGAG-3' and antisense: 5'-AGGGAAGCCCCCAGGAAG-3'. The PCRs were performed in a 20 µl reaction volume containing 100 ng genomic DNA, 25 pmol/l each primer, 0.2 mmol/l deoxynucleotide triphosphates, 10 mmol/l Tris–HCl (pH 8.3), 50 mmol/l KCl, 1.5 mmol/l MgCl2 and 1 U of Taq polymerase (Takara Shuzo Co., Otsu, Shiga, Japan). The PCR cycle conditions consisted of an initial denaturation step at 94°C for 5 min, followed by 35 cycles of 30 s at 94°C, 30 s at 62°C, 30 s at 72°C and a final elongation at 72°C for 10 min. The digested PCR products were resolved on a 3% agarose gel and stained with ethidium bromide for visualization under UV light. For quality control, the genotyping analysis was done blind as regards the subjects. The selected PCR-amplified DNA samples (n = 2, for each genotype) were also examined by DNA sequencing to confirm the genotyping results.
Statistical Analysis
The genotypes for each SNP were analyzed as a three-group categorical variable (referent model), and those were also grouped according to the dominant and recessive model. Categorical data were compared using 
2 test and continuous variables using two-sided variance. Overall survival (OS) was measured from the day of surgery until the date of death or last follow-up. Disease-free survival (DFS) was calculated from the day of surgery until recurrence or death from any cause. The survival estimates were calculated using the Kaplan–Meier method. The difference in OS or DFS according to the polymorphism of NFKB1 promoter was compared using log-rank tests. Cox's proportional hazard regression model was used for the multivariate survival analyses, and the analyses were always adjusted for age (<60 versus 
60 years), sex (male versus female), stage (0 to IV) and adjuvant chemotherapy (intravenous versus oral versus not received). The hazard ratio and 95% confidence interval were also estimated. A cut-off P value of 0.05 was adopted for all the statistical analyses. The statistical data were obtained using an SPSS software package (SPSS 11.5 Inc., Chicago, IL, USA) or SAS Genetic software (SAS Institute, Cary, NC, USA).
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RESULTS |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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Patient Characteristics
The median age of the patients was 60 (range, 25–83) years, and 315 (67.0%) patients were male. Curative resections (gastrectomy with more than D1 lymph node dissection) were performed in all patients. The pathologic stages after the surgical resection were as follows: stage 0 (n = 6, 1.3%), stage IA (n = 169, 35.8%), stage IB (n = 104, 22.0%), stage II (n = 104, 22.0%), stage IIIA (n = 46, 9.7%), stage IIIB (n = 25, 5.3%) and stage IV (n = 18, 3.8%). Among the 193 patients with stage II to IV diseases, 147 (76.2%) patients received adjuvant chemotherapy with four cycles of 5-fluorouracil + epirubicin (n = 59) or nimustine (n = 7) followed by oral 5-fluorouracil for 1 year, or just oral 5-fluorouracil for 1 year (n = 81). At the time of last analysis, 88 patients had experienced a disease relapse and 82 patients had died as a result of gastric cancer. The estimated 5-year OS and DFS for all the patients was 82.9 ± 1.7% and 81.4 ± 1.8%, respectively.
Correlation Between Genotype and Pathologic Characteristics or Survival
The NFKB1 –94 insertion/deletion ATTG polymorphism was successfully amplified in 97.7% of the cases, and the genotype frequency (ATTG1/ATTG1, ATTG1/ATTG2 and ATTG2/ATTG2) is shown in Table 1. The genotype distribution of the polymorphism followed the Hardy–Weinberg equilibrium, and the allele frequency of –94 insertion and deletion ATTG in NFKB1 promoter among the patients was 0.681 (ATTG1) and 0.319 (ATTG2), respectively. There were no sexual differences in relation to the genotype and allele. No correlation was observed between the frequency of the genotype or allele and the T, N or M stage (Table 1). The univariate and multivariate survival analyses showed no association between the NFKB1 –94 insertion/deletion promoter polymorphism and the DFS or OS of the patients with gastric cancer (Fig. 1).
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DISCUSSION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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The current study demonstrated that the NFKB1 insertion/deletion promoter polymorphism had no prognostic impact in quite a large population of patients with surgically resected gastric adenocarcinoma. Considering the homogenous ethnic background of Korean patients, any potential confounding effect due to ethnicity is likely to be small in the present study.
NF-B plays an important role in complicated pathogenetic regulation of apoptosis, differentiation and senescence in addition to tumorigenesis (14,15). Inhibition of NF-B abrogated tumor cell survival and tumorigenesis, and in a recent study, inhibition of NF-B activity in melanocytes results in reduction of melanoma tumor growth in vivo (15,16). Given these results, the association of NFKB1 promoter gene polymorphism with the risk or prognosis of several solid tumors, such as oral squamous cell carcinoma (8), nasopharyngeal carcinoma (9) and colorectal cancer (11), has already been reported.
The genotype and allele frequencies of –94 insertion/deletion ATTG polymorphism in the present study were significantly different when compared with those previously reported for the Chinese (8,17,18) and European populations (19,20), with higher frequency of ATTG1 allele (68.1% versus 34.2–56.0%). However, these results might be caused by somatic alteration in the process of gastric carcinogenesis because the paraffin-embedded tissue was used for DNA source of patients group in the current study, although data on the difference between germline and somatic SNP in NFKB1 promoter have not been reported.
Although the survival was not different according to the –94 insertion/deletion polymorphism in NFKB1 promoter in patients with curatively resected gastric cancer, recent case–control studies reported that this polymorphism was associated with the risk of solid tumors (8,9,18). For example, Lin et al. (8) assessed the relationship between the –94 insertion/deletion ATTG polymorphism in NFKB1 promoter and the risks of oral squamous cell carcinoma occurring on older male areca chewers, with the odds ratio in oral squamous cell carcinoma carrying ATTG2 allelotype were 1.78 relative to control subjects in individuals >50 years old. Zhou et al. (9) also reported that individuals with ATTG2 allelotype have 1.453 times greater risks for nasopharyngeal carcinoma, compared with those carrying ATTG1 allelotype (P = 0.015). In gastric cancer, Lo et al. (18) demonstrated that ATTG2 allelotype was significantly higher in aged cancer patients than controls, whereas the polymorphism was not correlated with clinicopathologic factors and survival. They suggested that the promoter sequence containing ATTG2 allele had activity two times higher than comparable sequences containing ATTG1 allele, a detrimental role of NF-B activation in nasopharyngeal carcinoma initiation and development. Meanwhile, a previous study by Riemann et al. (11) investigated the associations of this polymorphism with susceptibility and survival of patients with different types of cancer, no correlation between –94 insertion/deletion ATTG alleles and the survival of patients with colorectal carcinoma (n = 139), clear cell renal cell carcinoma (n = 140) and B-cell chronic lymphocytic leukemia (n = 72).
In conclusion, the functional NFKB1 promoter polymorphism was not found to be a prognostic marker for Korean patients with surgically resected gastric adenocarcinoma. However, since genetic polymorphisms often vary between ethnic groups, more studies are warranted to clarify the association between the NFKB1 promoter gene polymorphism and risk or prognosis of gastric cancer in diverse ethnic populations.
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Conflict of interest statement |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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None declared.
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References |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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