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Clinical Immunology (2008) 128, 199–204

Association of interleukin-6 (−174G>C) promoterpolymorphism with risk of squamous cell esophagealcancer and tumor location: An exploratory studyRohit Upadhyay a, Meenu Jain a, Shaleen Kumar b,Uday Chand Ghoshal c, Balraj Mittal a,⁎

a Department of Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareilly Road, Lucknow-226014, Indiab Department of Radiotherapy, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareilly Road,Lucknow-226014, Indiac Department of Gastroenterology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareilly Road,Lucknow-226014, India

Received 28 January 2008; accepted with revision 26 March 2008Available online 27 May 2008

⁎ Corresponding author. Fax: +91 522E-mail addresses: balraj@sgpgi.ac.

(B. Mittal).

1521-6616/$ – see front matter © 200doi:10.1016/j.clim.2008.03.519

Abstract Chronic inflammation plays a role in transformation from normal cell to malignantstate. Interleukin-6 (IL-6) regulates inflammation and various physiological processes. IL-6promoter polymorphism (−174GNC) is associated with transcription differences in vitro and invivo. High expression of IL-6 may result in oxidative DNA damage and enhance risk ofcarcinogenesis. Therefore, we aimed to evaluate association of IL-6 −174GNC polymorphism withpredisposition to esophageal cancer (EC) in 369 subjects (168 patients with EC and 201 controls).We observed significant association of IL-6 −174C non-carrier genotype with risk of EC,(OR=2.29; P=0.001), with squamous cell carcinoma (SCC) histology (OR=2.26; P=0.001) andtumor at upper and lower anatomical locations (OR=5.97; P=0.009 and OR=2.34; P=0.034).Patients having IL-6 −174C non-carrier genotype were at elevated risk of metastasis (OR=2.49;P=0.005). In conclusion, IL-6 −174GNC gene polymorphism may confer high risk for EC and itsclinical characteristics.© 2008 Elsevier Inc. All rights reserved.

KEYWORDSInterleukin-6 genepolymorphism;Inflammation;Cancer risk;Esophageal cancer;Cancer susceptibility

Introduction

Esophageal cancer (OMIM number 133239) (EC) has wide var-iation in incidence at different geographical areas and overall

2668017x2668074.in, bml_pgi@yahoo.com

8 Elsevier Inc. All rights reserved

incidence is rising [1]. Epidemiological studies have showna role of tobacco carcinogens and alcohol intake in etiologyof EC [2]. In addition, various low penetrance genes havebeen identified to be involved in modulation of cancer riskincluding EC but still many candidate genes are unexplored[3].

Some of the low penetrance genes encoding inflammatoryproteins are believed to be associated with developmentof cancer [4]. Progressive inflammation leads to activation of

.

200 R. Upadhyay et al.

inflammatory cytokines, recruitment of inflammatory cells,generation of free radicals, and ultimately malignant trans-formation. The degree of inflammation may be influenced bythe inter-individual genetic variations and thus the risk forcancer.

Interleukin-6 (IL6) or Interferon, BETA-2; IFNB2 (OMIMnumber 147620) is an immunoregulatory pleiotropic cyto-kine that activates a cell-surface signaling assembly com-posed of IL6, IL6RA, and the shared signaling receptorgp130 [5]. The cytokine is involved in different physiologicaland pathophysiological processes, such as inflammation,bone metabolism, synthesis of CRP (C-reactive protein), andcarcinogenesis (Fig. 1a) [6–7]. The gene encoding IL-6 islocalized at chromosome 7p21–14.1. It is a glycoproteinconsisting of 212 amino acids. Fishman et al. [8] identified apromoter region polymorphism in IL-6 gene (GNC) at position−174 (rs1800795) and showed that construct containing−174C allele had 0.62±0.15-fold lower expression (Pb0.005)than −174G allele. Previous studies have also reported highIL-6 concentrations (N10 fold) in serum of patients withprimary esophageal tumor [9–10]. Furthermore, the ele-vated level was associated with poor prognosis of disease[10].

Association of IL-6 −174 GNC polymorphism with risk ofseveral malignancies and inflammatory diseases has beenreported [8,11–18]. In EC, an earlier study in high risk-Chinese population showed b1% IL-6 variant allele fre-quency in control population but did not determine the riskfor cancer patients [19]. Therefore, in view of limited dataof IL-6 polymorphism in EC, we aimed to investigate theassociation of IL-6 −174GNC polymorphism with risk of EC,

Figure 1 (a): Showing IL-6 pathway and hypothesis of study. (b): R50 bp DNA ladder; Lane 2: G/G (C non-carrier) genotype; Lane 3: G

its clinical phenotypes and modulation of risk afterinteraction with environmental exposures.

Materials and methods

During a four year period (from August 2003 to June 2007), allhistopathologically confirmed esophageal cancer patients(n=168) were recruited from the outpatient clinics ofRadiotherapy and Gastroenterology (Gastromedicine andGastrosurgery) Departments of a tertiary care hospital,Sanjay Gandhi Post Graduate Institute of Medical Sciences(SGPGIMS), Lucknow. Histopathology was confirmed by twoindependent pathologists and was found to be squamous cellcarcinoma (SCC) in 90% of patients. The patients' demogra-phy, clinical characteristics at the time of diagnosis andenvironmental details were taken from the medical recordsand personnel interview through a questionnaire as describedpreviously [20]. At the same time, a total of 201 unrelated,age and gender-matched healthy controls were selected whowere visiting routine check-up at hospital and residing inadjoining areas of northern India. Selection criteria for con-trols included no evidence of any personal or family history ofcancer or other malignant conditions. All patients and con-trols gave written informed consent and the study protocolwas approved by ethical committee of SGPGIMS.

Genotyping

DNAwas extracted from white blood cells of all enrolled studysubjects, using salting out method [21]. Genotyping of IL-6

epresentative gel picture ofIL-6 −174GNC polymorphism, Lane 1:/C genotype; Lane 4: C/C genotype.

Table 1 Association of IL-6 −174GNC genotypes with risk ofesophageal cancer

Patients Controls Odds ratio⁎(95% CI), P-value(n=168) (n=201)

IL-6 −174GNC genotypesC+ 33

(19.6%)70(34.8%)

1 (Reference)

C− 135(80.4%)

131(65.2%)

2.29 (1.401–3.724)0.001

IL6 −174GNC allelesC allele 38

(11.3%)76(18.9%)

1 (Reference)

G allele 298(88.7%)

326(81.1%)

1.86 (1.216–2.848)0.004

Males Patients Controls

(n=125) (n=150)

C+ 26(20.8%)

51(34.0%)

1 (Reference)

C− 99(79.2%)

99(66.0%)

2.027 (1.164–3.530)0.013

Females Patients Controls

(n=43) (n=51)

C+ 7 (16.3%) 19(37.3%)

1 (Reference)

C− 36(83.7%)

32(62.7%)

3.230 (1.139–9.159)0.027

Squamous cellcarcinoma

Patients Controls

(n=154) (n=201)

C+ 31(20.1%)

70(34.8%)

1 (Reference)

C− 123(79.9%)

131(65.2%)

2.26 (1.37–3.73)0.001

Adenocarcinoma Patients Controls

(n=14) (n=201)

C+ 2 (14.3%) 70(34.8%)

1 (Reference)

C− 12(85.7%)

131(65.2%)

3.17 (0.69–14.64)0.148£

Lymphnodes

Controls

(n=82) (n=201)

C+ 15(18.3%)

70(34.8%)

1 (Reference)

C− 67(81.7%)

131(65.2%)

2.49 (1.31–4.72)0.005

⁎Age and gender adjusted odds ratio; C− (C non-carrier) representsG/G genotype and C+ (C carrier) represents combination of G/Cand C/C genotypes, £Fisher's exact test P-value.

201Interleukin-6 promoter polymorphism and squamous cell esophageal cancer

(−174GNC) polymorphism (rs1800795) was done using ARMS(Amplification refractory mutation system) polymerase chainreaction (PCR) [22]. Briefly, the PCR conditions of IL-6(−174GNC) amplification were as follows: initial denaturationat 95 °C for 3 min, denaturation at 95 °C for 1 min, 63 °Cannealing for 1 min and 72 °C extension for 1 min for34 cycles. IL-6 alleles (−174 G and C) were separated on 10%polyacrylamide gel electrophoresis. DNA fragments werevisualized by UV illumination. Genotypes were assignedbased on band-sizes: IL-6 −174G allele (205 bp), IL-6 −174Callele (176 bp) and outer primer's control product (326 bp).Since IL-6 −174C allele has been shown to be associated withlow expression, so genotypes were subsequently grouped asC+(C carriers or C/C+C/G) and C− (C non-carriers or G/G) (Fig.1b) [8].

Statistical analysis

χ2 (Chi-square) goodness of fit test was used for comparisonof observed and expected genotype frequencies in controlsand to analyze the deviation from Hardy Weinberg Equili-brium. Binary logistic regression was used to explore riskfactors of EC in which the independent variables of interest(predictors) were tested individually against the binarydependent variable (disease outcome). Risk was defined asOdds ratio (OR) with 95% confidence intervals (CI), using age,and gender as covariates and IL-6 −174C+ genotype asreference. Gene–environment interactions were examinedbetween IL-6 (−174GNC) genotypes and environmentalexposure using case-only analysis [20]. All tests of signifi-cance were two-sided and taken as significant when P-valuewas b0.05. In case of cell size below 5, Fisher's exact test wasperformed. Statistical analysis was performed using the SPSSsoftware version 15.0 for Windows (SPSS, Chicago, IL, USA).

Results

There was no significant difference between mean age ofpatients (56.8 yrs±12.8) and controls (53.7yrs±11.3). Thegender distribution was comparable among patients (males:74.4%; females: 25.6%) and controls (males: 74.6%; females:25.4%). Among the clinical characteristics of patients, 91.7%(154/168) had SCC and 8.3% (14/168) had adenocarcinoma(ADC). In majority of the patients, tumors were located inmiddle third (53.2%, 82/154) as compared to other twolocations (lower and upper third) of the esophagus. A largeproportion of patients (64.1%, 82/128) had lymph nodemetastasis. Majority of patients (81.5%, 121/152) usedtobacco in some form (smoking, chewing, snuff, or both).Alcohol drinkers were 37.0% (50/135); among them 66.0%(33/50) were frequent drinkers. About thirty one percent ofpatients had occupational exposure, mainly from householdcombustible fuels [23].

Association of IL-6 (−174GNC) polymorphism withrisk of esophageal cancer

In controls, IL-6 (−174GNC) genotype frequencies were inHardy Weinberg equilibrium (P=0.863). Comparing the IL-6genotype distribution, frequency of −174C non-carriergenotype was significantly different between patients

Table 3 Interaction of environmental factors with IL-6−174GNC genotypes and modulation of risk for esophagealcancer

IL6 −174GNC

Tobacco users Non-users Odds Ratio⁎ (95% CI),P-value

(n=121) (n=31)

C+ 22 (18.2%) 7 (22.6%) 1 (Reference)C− 99 (81.8%) 24 (77.4%) 1.29 (0.46–3.66)

0.631Smokers Non-smokers

(n=72) (n=81)

C+ 16 (22.2%) 13 (16.0%) 1 (Reference)C− 56 (77.8%) 68 (84.0%) 0.57 (0.23–1.39)

0.215Tobacco-chewers

Non-tobaccochewers

(n=91) (n=62)

C+ 16 (17.6%) 13 (21.0%) 1 (Reference)C− 75 (82.4%) 49 (79.0%) 1.34 (0.58–3.10)

0.489Drinker Non-drinker

(n=50) (n=85)

C+ 9 (18.0%) 19 (22.4%) 1 (Reference)C− 41 (82.0%) 66 (77.6%) 1.16 (0.46–2.96)

0.753Occupationalexposure

Non-occupationalexposure

(n=42) (n=92)

C+ 6 (14.3%) 18 (19.6%) 1 (Reference)C− 36 (85.7%) 74 (80.4%) 1.31 (0.439–3.933)

0.626

⁎Age and gender adjusted odds ratio; C− (C non-carrier) represents

202 R. Upadhyay et al.

(80.4%, 135/168) and controls (65.2%, 131/201) (OR=2.28;95% CI=1.40–3.72, P=0.001) (Table 1). Distribution of allelesalso showed distinct difference in the frequency of IL-6−174G allele between patients (88.7%, 298/336) and con-trols (81.1%, 326/402) (OR=1.86, 95% CI =1.22–2.85,P=0.004) (Table 1). After stratifying the study subjectsaccording to gender, both male and female patients with IL-6−174C non-carrier genotypes were at significantly high riskof EC (OR=2.32; 95% CI=1.42–3.78, P=0.001 and OR=3.230;95% CI= 1.139–9.159, P=0.027) (Table 1).

Association of IL-6 (−174GNC) polymorphism withclinical characteristics

After evaluation of frequency of genotypes in two histologicalsubtypes, SCC and ADC, significant differences were found inpatients with SCC having IL-6 −174C non-carrier genotypes(OR=2.26; 95%CI=1.37–3.73,P=0.001) (Table 1).Whenesoph-ageal tumor anatomical location was considered, associationof IL-6 −174C non-carrier genotype showed significant highrisk for developing the tumor in upper and lower third locations(OR=5.97; 95% CI=1.35–26.28, P=0.009 and OR=2.34; 95%CI=1.06–5.121, P=0.034) (Table 2).

Interaction of IL-6 (−174GNC) polymorphism withenvironmental exposures

In a case-only analysis, interaction of IL-6 (−174GNC)genotypes with tobacco use (smoking or smokeless tobacco);alcohol or occupational exposure was analyzed to observeinfluence on EC risk. Frequency of IL-6 −174C non-carriergenotype was slightly increased in tobacco users than non-users (81.8% vs. 77.4%) with no significance (OR=1.29; 95%CI=0.46–3.66, P=0.631) (Table 3). Distribution of IL-6 −174C non-carrier genotype within subgroups of tobacco

Table 2 Association of IL-6 −174GNC genotypes with tumorlocation and risk of esophageal cancer

IL-6−174GNC

Upper Controls Odds ratio⁎ (95% CI),P-value(n=24) (n=201)

C+ 2 (8.3%) 70(34.8%) 1 (Reference)C− 22 (91.7%) 131(65.2%) 5.97 (1.35–26.28) 0.009£

Middle Controls

(n=82) (n=201)

C+ 20 (24.4%) 70(34.8%) 1 (Reference)C− 62 (75.6%) 131(65.2%) 1.79(0.99–3.26) 0.055

Lower Controls

(n=48) (n = 201)

C+ 9 (18.8%) 70(34.8%) 1 (Reference)C− 39 (81.3%) 131(65.2%) 2.34(1.06–5.121) 0.034

⁎Age and gender adjusted odds ratio; C− (C non-carrier) representsG/G genotype and C+ (C carrier) represents combination of G/Cand C/C genotypes, £Fisher's exact test P-value.

G/G genotype and C+ (C carrier) represents combination of G/Cand C/C genotypes.

chewers, drinkers and occupational exposure showed dis-tinct difference in patients (Odds ratios N1) but was notstatistically significant (Table 3).

Discussion

The gastrointestinal malignancies are a heterogeneous groupof diseases with significant variability in etiology, genetics,demographics, presentation, and clinical behavior. In thepresent study, IL-6 promoter region polymorphism (−174GNC)was undertaken as it possess functional significance [8]. If welook at frequency distribution of IL-6 −174 genotypes indifferent ethnic control populations, higher variant allelefrequencywas observed in UK Caucasians (40.3%), moderate inIndia (15%–18%), low in Afro-Caribbean (5.0%), and SouthernChinese population (b1%) [19,24–26]. In the present study, IL-6genotype frequencies were similar to that of previouslydescribed Indian reports. We observed that individuals withIL-6 −174C non-carrier genotype were at higher risk of EC(OR 2.28; P= 0.001). On stratifying study subjects according togender, both genders with IL-6 −174C non-carriers were at

203Interleukin-6 promoter polymorphism and squamous cell esophageal cancer

increased risk for EC. Thus, it seems that −174C non-carriersmay not have gender specific risk. Several other studies havealso confirmed that IL-6 −174C non-carrier genotype isassociated with risk and poor prognosis of malignancies[12,14,17]. It has been shown that IL-6 C non-carrier genotypeis associatedwith high levels of circulating IL-6. Elevated levelsof IL-6 in serum and malignant tissues have also been shown tobe associated with poor prognosis of EC [9,10]. The reasonbehind the association of IL-6 genotype with susceptibility ofEC may be due to blocked cytotoxic function of tumor-infil-trating lymphocytes by high local IL-6 concentrations at tumorsites [27]. Thus, it is possible that increased IL-6 production in−174C non-carriers may lead to escape of esophageal tumorcells from immune surveillance and promote oncogenesis [9].

In EC, clinical phenotypes manifest in diverse histology,different anatomical site and lymph node involvement whichmay be linked to certain host genetic factors [28]. Our resultsshowed significant risk for SCC with respect to IL-6 −174C non-carrier genotypes. The increased risk of esophageal SCCreflects importance of histology specific susceptibility andmay be credited to different biological behavior in twohistologies or presence of predominant SCC histology. Ourresults correspond to those of Zhou et al. [29]. They have alsoshown that IL-6 gene is upregulated in grade II basal cellhyperplasia, high-grade dysplasia, carcinoma in situ, early andadvanced esophageal SCC. Furthermore, individuals with IL-6−174C non-carrier genotype were at significantly increasedrisk for developing tumor at upper and lower third location ofesophagus. This observation also reiterates role of genetics insite specific risks as earlier reported [20,28].

Tumor progression is a concerted process including evasionof host immune surveillance, tumor cell proliferation, killing ofthe immune cell, and the invasion of neighboring tissues aswell as distant organs. The present study found that patientshaving IL-6 −174C non-carrier genotype had elevated risk ofmetastasis (OR 2.49, P=0.005; Table 1). Earlier studies alsoshowed association of elevated serum IL-6 level with esopha-geal tumor stage, lymph node metastasis and with poor prog-nosis [10]. The persistence of the pro-inflammatory cytokineIL-6 response maintains chronic recruitment of lymphocytesand mast cells which stimulates SCC invasion and metastasis.Once carcinoma in situ penetrates the basement membraneto involve the lamina propria, it is invasive carcinoma andcapable of widespread dissemination. Therefore, understand-ing the interplay of genes and the pathways they utilize canlead to the detection of novel molecular targets in thediagnosis, prognosis, and treatment of EC.

Environmental factors like smoking and alcohol are well-established mediators of high risk in EC [30]. In the presentstudy, interaction of IL-6 −174GNC polymorphismwith environ-mental factors (like tobacco usage, alcohol or household com-bustible fuels) did not modulate the risk of EC. It seems thatIL-6 gene is not a candidate for gene–environment interactionsin patients with EC. Moreover other classes of genes likeCYP1B1 (phase I metabolism of pro-carcinogens),MAPK14 (MAPkinase family member), ATF4 (transcription factor), SOD2 (freeradical scavenging enzyme) and SERPINB2 (primary regulator ofplasminogen activation) may be looked upon in response toenvironmental exposure in EC patients [29].

Summarizing, the observations of study suggest that IL-6(−174GNC) polymorphism is strongly associated with risk forEC or its clinical phenotypes. However, functional studies

of the polymorphism in EC especially in biopsy samples ofspecific locations of tumor are required to draw definitiveconclusions. The results of the study need to be reconfirmedin a larger sample size from other populations to establishreal association of IL-6 genotypes with risk of esophagealcancer.

Acknowledgments

The study was supported by research grants from DST andIndian Council of Medical Research, New Delhi.

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