Clinical Immunology (2008) 128, 199–204

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y c l i m

Association of interleukin-6 (− 174G>C) promoter

polymorphism with risk of squamous cell esophageal
cancer and tumor location: An exploratory study
Rohit 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, India
b
Department of Radiotherapy, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareilly Road,
Lucknow-226014, India
c
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 2008

Available online 27 May 2008

KEYWORDS Abstract Chronic inflammation plays a role in transformation from normal cell to malignant
Interleukin-6 gene state. Interleukin-6 (IL-6) regulates inflammation and various physiological processes. IL-6
polymorphism; promoter polymorphism (−174GNC) is associated with transcription differences in vitro and in
Inflammation; vivo. High expression of IL-6 may result in oxidative DNA damage and enhance risk of
Cancer risk; carcinogenesis. Therefore, we aimed to evaluate association of IL-6 −174GNC polymorphism with
Esophageal cancer; predisposition to esophageal cancer (EC) in 369 subjects (168 patients with EC and 201 controls).
Cancer susceptibility 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) and
tumor 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 its
clinical characteristics.
© 2008 Elsevier Inc. All rights reserved.

Introduction incidence is rising [1]. Epidemiological studies have shown

Esophageal cancer (OMIM number 133239) (EC) has wide var-
iation in incidence at different geographical areas and overall
⁎ Corresponding author. Fax: +91 522 2668017x2668074.
E-mail addresses: balraj@sgpgi.ac.in, bml_pgi@yahoo.com
(B. Mittal).
a role of tobacco carcinogens and alcohol intake in etiology
of EC [2]. In addition, various low penetrance genes have
been identified to be involved in modulation of cancer risk
including EC but still many candidate genes are unexplored
[3].
Some of the low penetrance genes encoding inflammatory
proteins are believed to be associated with development
of cancer [4]. Progressive inflammation leads to activation of

1521-6616/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.clim.2008.03.519
200 R. Upadhyay et al.

inflammatory cytokines, recruitment of inflammatory cells, its clinical phenotypes and modulation of risk after
generation of free radicals, and ultimately malignant trans- interaction with environmental exposures.
formation. The degree of inflammation may be influenced by
the inter-individual genetic variations and thus the risk for Materials and methods
cancer.
Interleukin-6 (IL6) or Interferon, BETA-2; IFNB2 (OMIM
During a four year period (from August 2003 to June 2007), all
number 147620) is an immunoregulatory pleiotropic cyto-
histopathologically confirmed esophageal cancer patients
kine that activates a cell-surface signaling assembly com-
(n = 168) were recruited from the outpatient clinics of
posed of IL6, IL6RA, and the shared signaling receptor
Radiotherapy and Gastroenterology (Gastromedicine and
gp130 [5]. The cytokine is involved in different physiological
Gastrosurgery) Departments of a tertiary care hospital,
and pathophysiological processes, such as inflammation,
Sanjay Gandhi Post Graduate Institute of Medical Sciences
bone metabolism, synthesis of CRP (C-reactive protein), and
(SGPGIMS), Lucknow. Histopathology was confirmed by two
carcinogenesis (Fig. 1a) [6–7]. The gene encoding IL-6 is
independent pathologists and was found to be squamous cell
localized at chromosome 7p21–14.1. It is a glycoprotein
carcinoma (SCC) in 90% of patients. The patients' demogra-
consisting of 212 amino acids. Fishman et al. [8] identified a
phy, clinical characteristics at the time of diagnosis and
promoter region polymorphism in IL-6 gene (GNC) at position
environmental details were taken from the medical records
−174 (rs1800795) and showed that construct containing
and personnel interview through a questionnaire as described
−174C allele had 0.62 ± 0.15-fold lower expression (P b 0.005)
previously [20]. At the same time, a total of 201 unrelated,
than −174G allele. Previous studies have also reported high
age and gender-matched healthy controls were selected who
IL-6 concentrations (N 10 fold) in serum of patients with
were visiting routine check-up at hospital and residing in
primary esophageal tumor [9–10]. Furthermore, the ele-
adjoining areas of northern India. Selection criteria for con-
vated level was associated with poor prognosis of disease
trols included no evidence of any personal or family history of
[10].
cancer or other malignant conditions. All patients and con-
Association of IL-6 − 174 GNC polymorphism with risk of
trols gave written informed consent and the study protocol
several malignancies and inflammatory diseases has been
was approved by ethical committee of SGPGIMS.
reported [8,11–18]. In EC, an earlier study in high risk-
Chinese population showed b 1% IL-6 variant allele fre-
quency in control population but did not determine the risk Genotyping
for cancer patients [19]. Therefore, in view of limited data
of IL-6 polymorphism in EC, we aimed to investigate the DNA was extracted from white blood cells of all enrolled study
association of IL-6 −174GNC polymorphism with risk of EC, subjects, using salting out method [21]. Genotyping of IL-6

Figure 1 (a): Showing IL-6 pathway and hypothesis of study. (b): Representative gel picture ofIL-6 −174GNC polymorphism, Lane 1:
50 bp DNA ladder; Lane 2: G/G (C non-carrier) genotype; Lane 3: G/C genotype; Lane 4: C/C genotype.
Interleukin-6 promoter polymorphism and squamous cell esophageal cancer 201

(− 174GNC) polymorphism (rs1800795) was done using ARMS Table 1 Association of IL-6 −174GNC genotypes with risk of
(Amplification refractory mutation system) polymerase chain esophageal cancer
reaction (PCR) [22]. Briefly, the PCR conditions of IL-6
Patients Controls Odds ratio⁎
(−174GNC) amplification were as follows: initial denaturation
(95% CI), P-value
at 95 °C for 3 min, denaturation at 95 °C for 1 min, 63 °C (n = 168) (n = 201)
annealing for 1 min and 72 °C extension for 1 min for IL-6 − 174GNC genotypes
34 cycles. IL-6 alleles (−174 G and C) were separated on 10% C+ 33 70 1 (Reference)
polyacrylamide gel electrophoresis. DNA fragments were (19.6%) (34.8%)
visualized by UV illumination. Genotypes were assigned C− 135 131 2.29 (1.401–3.724)
based on band-sizes: IL-6 −174G allele (205 bp), IL-6 −174C
(80.4%) (65.2%) 0.001
allele (176 bp) and outer primer's control product (326 bp).
Since IL-6 −174C allele has been shown to be associated with IL6 −174GNC alleles
low expression, so genotypes were subsequently grouped as C+ C allele 38 76 1 (Reference)
(C carriers or C/C+C/G) and C− (C non-carriers or G/G) (Fig. (11.3%) (18.9%)
1b) [8]. G allele 298 326 1.86 (1.216–2.848)
(88.7%) (81.1%) 0.004
Statistical analysis
Males Patients Controls
χ2 (Chi-square) goodness of fit test was used for comparison
(n = 125) (n = 150)
of observed and expected genotype frequencies in controls
and to analyze the deviation from Hardy Weinberg Equili- C+ 26 51 1 (Reference)
brium. Binary logistic regression was used to explore risk (20.8%) (34.0%)
factors of EC in which the independent variables of interest C− 99 99 2.027 (1.164–3.530)
(predictors) were tested individually against the binary (79.2%) (66.0%) 0.013
dependent variable (disease outcome). Risk was defined as
Odds ratio (OR) with 95% confidence intervals (CI), using age, Females Patients Controls
and gender as covariates and IL-6 − 174C+ genotype as (n = 43) (n = 51)
reference. Gene–environment interactions were examined
between IL-6 (− 174GNC) genotypes and environmental C+ 7 (16.3%) 19 1 (Reference)
exposure using case-only analysis [20]. All tests of signifi- (37.3%)
cance were two-sided and taken as significant when P-value C− 36 32 3.230 (1.139–9.159)
was b 0.05. In case of cell size below 5, Fisher's exact test was (83.7%) (62.7%) 0.027
performed. Statistical analysis was performed using the SPSS
software version 15.0 for Windows (SPSS, Chicago, IL, USA). Squamous cell Patients Controls
carcinoma
(n = 154) (n = 201)
Results C+ 31 70 1 (Reference)
(20.1%) (34.8%)
There was no significant difference between mean age of C− 123 131 2.26 (1.37–3.73)
patients (56.8 yrs ± 12.8) and controls (53.7yrs ± 11.3). The (79.9%) (65.2%) 0.001
gender distribution was comparable among patients (males:
74.4%; females: 25.6%) and controls (males: 74.6%; females: Adenocarcinoma Patients Controls
25.4%). Among the clinical characteristics of patients, 91.7%
(154/168) had SCC and 8.3% (14/168) had adenocarcinoma (n = 14) (n = 201)
(ADC). In majority of the patients, tumors were located in C+ 2 (14.3%) 70 1 (Reference)
middle third (53.2%, 82/154) as compared to other two (34.8%)
locations (lower and upper third) of the esophagus. A large C− 12 131 3.17 (0.69–14.64)
proportion of patients (64.1%, 82/128) had lymph node (85.7%) (65.2%) 0.148£
metastasis. Majority of patients (81.5%, 121/152) used
tobacco in some form (smoking, chewing, snuff, or both).
Lymph Controls
Alcohol drinkers were 37.0% (50/135); among them 66.0%
nodes
(33/50) were frequent drinkers. About thirty one percent of
patients had occupational exposure, mainly from household (n = 82) (n = 201)
combustible fuels [23]. C+ 15 70 1 (Reference)
(18.3%) (34.8%)
Association of IL-6 (−174GNC) polymorphism with C− 67 131 2.49 (1.31–4.72)
risk of esophageal cancer (81.7%) (65.2%) 0.005
⁎Age and gender adjusted odds ratio; C− (C non-carrier) represents
In controls, IL-6 (−174GNC) genotype frequencies were in G/G genotype and C+ (C carrier) represents combination of G/C
Hardy Weinberg equilibrium (P = 0.863). Comparing the IL-6 and C/C genotypes, £Fisher's exact test P-value.
genotype distribution, frequency of − 174C non-carrier
genotype was significantly different between patients
202 R. Upadhyay et al.

(80.4%, 135/168) and controls (65.2%, 131/201) (OR = 2.28; Table 3 Interaction of environmental factors with IL-6
95% CI = 1.40–3.72, P = 0.001) (Table 1). Distribution of alleles − 174GNC genotypes and modulation of risk for esophageal
also showed distinct difference in the frequency of IL-6 cancer
−174G allele between patients (88.7%, 298/336) and con-
IL6 − Tobacco users Non-users Odds Ratio⁎ (95% CI),
trols (81.1%, 326/402) (OR = 1.86, 95% CI = 1.22–2.85,
174GNC P-value
P = 0.004) (Table 1). After stratifying the study subjects
(n = 121) (n = 31)
according to gender, both male and female patients with IL-6
−174C non-carrier genotypes were at significantly high risk C+ 22 (18.2%) 7 (22.6%) 1 (Reference)
of EC (OR = 2.32; 95% CI = 1.42–3.78, P = 0.001 and OR = 3.230; C− 99 (81.8%) 24 (77.4%) 1.29 (0.46–3.66)
95% CI = 1.139–9.159, P = 0.027) (Table 1). 0.631
Smokers Non-smokers

Association of IL-6 (−174GNC) polymorphism with (n = 72) (n = 81)

clinical characteristics C+ 16 (22.2%) 13 (16.0%) 1 (Reference)
C− 56 (77.8%) 68 (84.0%) 0.57 (0.23–1.39)
After evaluation of frequency of genotypes in two histological 0.215
subtypes, SCC and ADC, significant differences were found in Tobacco- Non-tobacco
patients with SCC having IL-6 −174C non-carrier genotypes chewers chewers
(OR = 2.26; 95% CI = 1.37–3.73, P= 0.001) (Table 1). When esoph- (n = 91) (n = 62)
ageal tumor anatomical location was considered, association
C+ 16 (17.6%) 13 (21.0%) 1 (Reference)
of IL-6 −174C non-carrier genotype showed significant high
C− 75 (82.4%) 49 (79.0%) 1.34 (0.58–3.10)
risk for developing the tumor in upper and lower third locations
0.489
(OR = 5.97; 95% CI= 1.35–26.28, P = 0.009 and OR = 2.34; 95%
Drinker Non-drinker
CI= 1.06–5.121, P= 0.034) (Table 2).
(n = 50) (n = 85)

Interaction of IL-6 (−174GNC) polymorphism with C+ 9 (18.0%) 19 (22.4%) 1 (Reference)

environmental exposures C− 41 (82.0%) 66 (77.6%) 1.16 (0.46–2.96)
0.753
Occupational Non-
In a case-only analysis, interaction of IL-6 (− 174GNC) exposure occupational
genotypes with tobacco use (smoking or smokeless tobacco); exposure
alcohol or occupational exposure was analyzed to observe
influence on EC risk. Frequency of IL-6 − 174C non-carrier (n = 42) (n = 92)
genotype was slightly increased in tobacco users than non- C+ 6 (14.3%) 18 (19.6%) 1 (Reference)
users (81.8% vs. 77.4%) with no significance (OR = 1.29; 95% C− 36 (85.7%) 74 (80.4%) 1.31 (0.439–3.933)
CI = 0.46–3.66, P = 0.631) (Table 3). Distribution of IL-6 − 0.626
174C non-carrier genotype within subgroups of tobacco ⁎Age and gender adjusted odds ratio; C− (C non-carrier) represents
G/G genotype and C+ (C carrier) represents combination of G/C
and C/C genotypes.
Table 2 Association of IL-6 −174GNC genotypes with tumor
location and risk of esophageal cancer chewers, drinkers and occupational exposure showed dis-
IL-6 Upper Controls Odds ratio⁎ (95% CI), tinct difference in patients (Odds ratios N 1) but was not
−174GNC P-value statistically significant (Table 3).
(n = 24) (n = 201)
C+ 2 (8.3%) 70(34.8%) 1 (Reference) Discussion
C− 22 (91.7%) 131(65.2%) 5.97 (1.35–26.28) 0.009£
The gastrointestinal malignancies are a heterogeneous group
Middle Controls of diseases with significant variability in etiology, genetics,
(n = 82) (n = 201) demographics, presentation, and clinical behavior. In the
present study, IL-6 promoter region polymorphism (−174GNC)
C+ 20 (24.4%) 70(34.8%) 1 (Reference)
was undertaken as it possess functional significance [8]. If we
C− 62 (75.6%) 131(65.2%) 1.79(0.99–3.26) 0.055
look at frequency distribution of IL-6 −174 genotypes in
different ethnic control populations, higher variant allele
Lower Controls
frequency was observed in UK Caucasians (40.3%), moderate in
(n = 48) (n = 201) India (15%–18%), low in Afro-Caribbean (5.0%), and Southern
Chinese population (b 1%) [19,24–26]. In the present study, IL-6
C+ 9 (18.8%) 70(34.8%) 1 (Reference)
genotype frequencies were similar to that of previously
C− 39 (81.3%) 131(65.2%) 2.34(1.06–5.121) 0.034
described Indian reports. We observed that individuals with
⁎Age and gender adjusted odds ratio; C− (C non-carrier) represents IL-6 −174C non-carrier genotype were at higher risk of EC
G/G genotype and C+ (C carrier) represents combination of G/C (OR 2.28; P= 0.001). On stratifying study subjects according to
and C/C genotypes, £Fisher's exact test P-value.
gender, both genders with IL-6 −174C non-carriers were at
Interleukin-6 promoter polymorphism and squamous cell esophageal cancer
increased risk for EC. Thus, it seems that −174C non-carriers
may not have gender specific risk. Several other studies have
also confirmed that IL-6 −174C non-carrier genotype is
associated with risk and poor prognosis of malignancies
[12,14,17]. It has been shown that IL-6 C non-carrier genotype
is associated with high levels of circulating IL-6. Elevated levels
of IL-6 in serum and malignant tissues have also been shown to
be associated with poor prognosis of EC [9,10]. The reason
behind the association of IL-6 genotype with susceptibility of
EC may be due to blocked cytotoxic function of tumor-infil-
trating lymphocytes by high local IL-6 concentrations at tumor
sites [27]. Thus, it is possible that increased IL-6 production in
−174C non-carriers may lead to escape of esophageal tumor
cells from immune surveillance and promote oncogenesis [9].
In EC, clinical phenotypes manifest in diverse histology,
different anatomical site and lymph node involvement which
may be linked to certain host genetic factors [28]. Our results
showed significant risk for SCC with respect to IL-6 −174C non-
carrier genotypes. The increased risk of esophageal SCC
reflects importance of histology specific susceptibility and
may be credited to different biological behavior in two
histologies or presence of predominant SCC histology. Our
results correspond to those of Zhou et al. [29]. They have also
shown that IL-6 gene is upregulated in grade II basal cell
hyperplasia, high-grade dysplasia, carcinoma in situ, early and
advanced esophageal SCC. Furthermore, individuals with IL-6
−174C non-carrier genotype were at significantly increased
risk for developing tumor at upper and lower third location of
esophagus. This observation also reiterates role of genetics in
site specific risks as earlier reported [20,28].
Tumor progression is a concerted process including evasion
of host immune surveillance, tumor cell proliferation, killing of
the immune cell, and the invasion of neighboring tissues as
well as distant organs. The present study found that patients
having IL-6 −174C non-carrier genotype had elevated risk of
metastasis (OR 2.49, P = 0.005; Table 1). Earlier studies also
showed 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 cytokine
IL-6 response maintains chronic recruitment of lymphocytes
and mast cells which stimulates SCC invasion and metastasis.
Once carcinoma in situ penetrates the basement membrane
to involve the lamina propria, it is invasive carcinoma and
capable of widespread dissemination. Therefore, understand-
ing the interplay of genes and the pathways they utilize can
lead to the detection of novel molecular targets in the
diagnosis, prognosis, and treatment of EC.
Environmental factors like smoking and alcohol are well-
established mediators of high risk in EC [30]. In the present
study, interaction of IL-6 −174GNC polymorphism with environ-
mental factors (like tobacco usage, alcohol or household com-
bustible fuels) did not modulate the risk of EC. It seems that
IL-6 gene is not a candidate for gene–environment interactions
in patients with EC. Moreover other classes of genes like
CYP1B1 (phase I metabolism of pro-carcinogens), MAPK14 (MAP
kinase family member), ATF4 (transcription factor), SOD2 (free
radical scavenging enzyme) and SERPINB2 (primary regulator of
plasminogen activation) may be looked upon in response to
environmental exposure in EC patients [29].
Summarizing, the observations of study suggest that IL-6
(− 174GNC) polymorphism is strongly associated with risk for
EC or its clinical phenotypes. However, functional studies
203
of the polymorphism in EC especially in biopsy samples of
specific locations of tumor are required to draw definitive
conclusions. The results of the study need to be reconfirmed
in a larger sample size from other populations to establish
real association of IL-6 genotypes with risk of esophageal
cancer.
Acknowledgments
The study was supported by research grants from DST and
Indian Council of Medical Research, New Delhi.
References
[1] J.C. Layke, P.P. Lopez, Esophageal cancer: a review and
update, Am. Fam. Phys. 73 (2006) 2187–2194.
[2] A. Gallo, C. Cha, Updates on esophageal and gastric cancers,
World J. Gastroenterol. 12 (2006) 3237–3242.
[3] A. Kotnis, R. Sarin, R. Mulherkar, Genotype, phenotype and
cancer: role of low penetrance genes and environment in tumour
susceptibility, J. Biosci. 30 (2005) 93–102.
[4] F. Balkwill, A. Mantovani, Inflammation and cancer: back to
Virchow? Lancet 357 (2001) 539–545.
[5] M.J. Boulanger, D.C. Chow, E.E. Brevnova, K.C. Garcia, Hexame-
ric structure and assembly of the interleukin-6/IL-6 alpha-
receptor/gp130 complex, Science 300 (2003) 2101–2104.
[6] S. Diehl, M. Rincón, The two faces of IL-6 on Th1/Th2
differentiation, Mol. Immunol. 39 (2002) 531–536.
[7] J.G. Asschert, E. Vellenga, M.H. Ruiters, E.G. de Vries,
Regulation of spontaneous and TNF/IFN-induced IL-6 expres-
sion in two human ovarian-carcinoma cell lines, Int. J. Cancer
82 (1999) 244–249.
[8] D. Fishman, G. Faulds, R. Jeffery, V. Mohamed-Ali, J.S. Yudkin, S.
Humphries, P. Woo, The effect of novel polymorphisms in the
interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6
levels, and an association with systemic-onset juvenile chronic
arthritis, J. Clin. Invest. 102 (1998) 1369–1376.
[9] M. Oka, K. Yamamoto, M. Takahashi, M. Hakozaki, T. Abe, N.
Iizuka, S. Hazama, K. Hirazawa, H. Hayashi, A. Tangoku, K.
Hirose, T. Ishihara, T. Suzuki, Relationship between serum levels
of interleukin 6, various disease parameters and malnutrition in
patients with esophageal squamous cell carcinoma, Cancer Res.
56 (1996) 2776–7270.
[10] L.S. Wang, K.C. Chow, C.W. Wu, Expression and up-regulation
of interleukin-6 in oesophageal carcinoma cells by n-sodium
butyrate, Br. J. Cancer 80 (1999) 1617–1622.
[11] J.M. Fernández-Real, M. Broch, J. Vendrell, C. Richart, W. Ricart,
Interleukin-6 gene polymorphism and lipid abnormalities in
healthy subjects, J. Clin. Endocrinol. Metab. 85 (2000) 1334–1339.
[12] C.B. Foster, T. Lehrnbecher, S. Samuels, S. Stein, F. Mol, J.A.
Metcalf, K. Wyvill, S.M. Steinberg, J. Kovacs, A. Blauvelt, R.
Yarchoan, S.J. Chanock, An IL6 promoter polymorphism is
associated with a lifetime risk of development of Kaposi sarcoma
in men infected with human immunodeficiency virus, Blood 96
(2000) 2562–2567.
[13] C. Belluco, F. Olivieri, M. Bonafè, S. Giovagnetti, E. Mammano, R.
Scalerta, A. Ambrosi, C. Franceschi, D. Nitti, M. Lise, −174 GNC
polymorphism of interleukin 6 gene promoter affects interleukin
6 serum level in patients with colorectal cancer, Clin. Cancer Res.
9 (2003) 2173–2176.
[14] L.A. Hefler, C. Grimm, S. Ackermann, S. Malur, A.R. Radjabi-
Rahat, S. Leodolter, M.W. Beckmann, R. Zeillinger, H. Koelbl, C.B.
Tempfer, An interleukin-6 gene promoter polymorphism influ-
ences the biological phenotype of ovarian cancer, Cancer Res. 63
(2003) 3066–3068.
204
[15] W. Cozen, P.S. Gill, S.A. Ingles, R. Masood, O. Martínez-Maza, M.G.
Cockburn, W.J. Gauderman, M.C. Pike, L. Bernstein, B.N.
Nathwani, M.T. Salam, K.L. Danley, W. Wang, J. Gage, S. Gundell-
Miller, T.M. Mack, IL-6 levels and genotype are associated with risk
of young adult Hodgkin lymphoma, Blood 103 (2004) 3216–3221.
[16] D. Campa, S. Zienolddiny, V. Maggini, V. Skaug, A. Haugen, F.
Canzian, Association of a common polymorphism in the
cyclooxygenase 2 gene with risk of non-small cell lung cancer,
Carcinogenesis 25 (2004) 229–235.
[17] D. Tan, X. Wu, M. Hou, S.O. Lee, W. Lou, J. Wang, B. Janarthan,
S. Nallapareddy, D.L. Trump, A.C. Gao, Interleukin-6 poly-
morphism is associated with more aggressive prostate cancer,
J. Urol. 174 (2005) 753–756.
[18] M. Libra, S.S. Signorelli, Y. Bevelacqua, P.M. Navolanic, V.
Bevelacqua, J. Polesel, R. Talamini, F. Stivala, M.C. Mazzarino,
G. Malaponte, Analysis of G(−174)C IL-6 polymorphism and
plasma concentrations of inflammatory markers in patients with
type 2 diabetes and peripheral arterial disease, J. Clin. Pathol. 59
(2006) 211–215.
[19] S.A. Savage, C.C. Abnet, K. Haque, S.D. Mark, Y.L. Qiao, Z.W.
Dong, S.M. Dawsey, P.R. Taylor, S.J. Chanock, Polymorphisms in
interleukin -2, -6, and -10 are not associated with gastric cardia
or esophageal cancer in a high-risk Chinese population, Cancer
Epidemiol. Biomark. Prev. 13 (2004) 1547–1549.
[20] M. Jain, S. Kumar, P. Lal, A. Tiwari, U.C. Ghoshal, B. Mittal, Role of
GSTM3 polymorphism in the risk of developing esophageal cancer,
Cancer Epidemiol. Biomark. Prev. 16 (2007) 178–181.
[21] S.A. Miller, D.D. Dykes, H.F. Polesky, A simple salting out
procedure for extracting DNA from human nucleated cells,
Nucleic Acids Res. 16 (1988) 215.
[22] S. Ye, S. Dhillon, X. Ke, A.R. Collins, I.N Day, An efficient
procedure for genotyping single nucleotide polymorphisms,
Nucleic Acids Res. 29 (2001) E88.
R. Upadhyay et al.
[23] M. Jain, S. Kumar, N. Rastogi, P. Lal, U.C. Ghoshal, A. Tiwari, M.C.
Pant, M.Q. Baiq, B. Mittal, GSTT1, GSTM1 and GSTP1 genetic
polymorphisms and interaction with tobacco, alcohol and
occupational exposure in esophageal cancer patients from
North India, Cancer Lett. 242 (2006) 60–67.
[24] G. Kaur, C.C. Rapthap, N. Kumar, S. Kumar, S. Neolia, N.K. Mehra,
Frequency distribution of cytokine gene polymorphisms in
the healthy North Indian population, Tissue Antigens 69 (2007)
113–120.
[25] C.S. Lim, S. Zheng, Y.S. Kim, C. Ahn, J.S. Han, S. Kim, J.S. Lee,
D.W. Chae, The − 174 G to C polymorphism of interleukin-6 gene
is very rare in Koreans, Cytokine 19 (2002) 52–54.
[26] R. Zhai, G. Liu, C. Yang, C. Huang, C. Wu, D.C. Christiani, The G
to C polymorphism at − 174 of the interleukin-6 gene is rare in a
Southern Chinese population, Pharmacogenetics 11 (2001)
699–701.
[27] J. Tanner, G. Tosato, Impairment of natural killer functions by
interleukin 6 increases lymphoblastoid cell tumorigenicity in
athymic mice, J. Clin. Invest. 88 (1991) 239–247.
[28] E. Monteiro, G. Varzim, A.M. Pires, M. Teixeira, C. Lopes,
Cyclin D1 A870G polymorphism and amplification in laryngeal
squamous cell carcinoma: implications of tumor localization
and tobacco exposure, Cancer Detect Prev. 28 (2004)
237–243.
[29] J. Zhou, L.Q. Zhao, M.M. Xiong, X.Q. Wang, G.R. Yang, Z.L. Qiu,
M. Wu, Z.H. Liu, Gene expression profiles at different stages of
human esophageal squamous cell carcinoma, World J. Gastro-
enterol. 9 (2003) 9–15.
[30] D.M. van Leeuwen, E. van Agen, R.W. Gottschalk, R. Vlietinck,
M. Gielen, M.H. van Herwijnen, L.M. Maas, J.C. Kleinjans, J.H.
van Delft, Cigarette smoke-induced differential gene expres-
sion in blood cells from monozygotic twin pairs, Carcinogenesis
28 (2007) 691–697.