A let-7 microRNA binding site

polymorphism in the KRAS 3′UTR is

associated with increased risk and reduced
survival for gallbladder cancer in North
Indian population
Hasan Raza Kazmi, Abhijit Chandra,
Saket Kumar, Leena Khare Satyam,
Annapurna Gupta, Jaya Nigam, Meenu
Srivastava & Balraj Mittal
Journal of Cancer Research and
Clinical Oncology

ISSN 0171-5216

J Cancer Res Clin Oncol

DOI 10.1007/s00432-016-2254-9

1 23
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 Author's personal copy
J Cancer Res Clin Oncol
DOI 10.1007/s00432-016-2254-9

ORIGINAL ARTICLE – CLINICAL ONCOLOGY

A let‑7 microRNA binding site polymorphism in the KRAS

3′UTR is associated with increased risk and reduced survival
for gallbladder cancer in North Indian population
Hasan Raza Kazmi1,2 · Abhijit Chandra1 · Saket Kumar1 · Leena Khare Satyam3 ·
Annapurna Gupta4 · Jaya Nigam1 · Meenu Srivastava1 · Balraj Mittal4

Received: 20 August 2016 / Accepted: 1 September 2016

© Springer-Verlag Berlin Heidelberg 2016

Abstract
Purpose Gallbladder cancer is a lethal malignancy of
hepato-biliary system with high incidence in North India,
especially along gangetic plain. The let-7 microRNAs play
a key role in regulating KRAS expression and a polymor-
phism in 3′ untranslated region (rs61764370, T/G) of KRAS
leads to its higher expression. This polymorphism is known
to be associated with increased risk and prognosis of various
cancers but its association with gallbladder cancer has not
been evaluated. To address this research question, we evalu-
ated whether rs61764370 variant is associated with gall-
bladder cancer susceptibility and clinical outcomes.
Methods In present case–control study, we enrolled 541
patients with gallbladder malignancy and 307 controls.
Genomic DNA was obtained from peripheral blood and
genotyping was performed using Taqman allelic discrimi-
nation assay.
Results Heterozygous (TG) individuals are at a signifi-
cant higher risk for GBC as compared with wild genotype
(TT) (p = 0.007, odds ratio = 2.56, 95 % CI 1.27–5.18).
At allelic level, allele G has significant higher risk for GBC
as compared with T allele (p = 0.008, odds ratio = 2.5,
95 % CI 1.25–5.01). Survival analysis reveals decrease in
overall survival for heterozygous genotype (p < 0.0001,
hazard ratio = 3.42, 95 % CI 1.21–4.20). Also, significant
decrease in overall survival was observed for patient carry-
ing allele G (p < 0.0001, HR = 2.89, 95 % CI 1.21–4.20)
as compared with allele C.
Conclusions We conclude that KRAS rs61764370 poly-
morphism is significantly associated with risk and progno-
sis of gallbladder malignancy in this endemic belt.
Keywords Gallbladder cancer · KRAS · let-7 · rs61764370
Introduction
Gallbladder cancer (GBC) is the most common malignancy
of the biliary tract with high incidence in Northern India
especially along the gangetic belt (Dhir and Mohandas

* Abhijit Chandra Balraj Mittal

abhijitchandra@hotmail.com bml_pgi@yahoo.com
Hasan Raza Kazmi
1
hasanrazakazmi@yahoo.co.in Department of Surgical Gastroenterology, King George’s
Medical University, Lucknow 226003, India
Saket Kumar
2
krsaketsingh@gmail.com Present Address: Department of Molecular and Human
Genetics, Banaras Hindu University, Varanasi 221005, India
Leena Khare Satyam
3
leena.satyam@gmail.com Cell and Molecular Biology Department, Aurigene Discovery
Technologies Limited, Bangalore 560095, India
Annapurna Gupta
4
annapurnagupta12@gmail.com Department of Medical Genetics, Sanjay Gandhi Post
Graduate Institute of Medical Sciences, Lucknow 226003,
Jaya Nigam
India
jayanigam27@gmail.com
Meenu Srivastava
meenu.srivastava12@gmail.com

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1999; Manoharan et al. 2010; Kazmi et al. 2014; Yadav
et al. 2015; Gupta et al. 2016). Complete surgical resec-
tion remains only potential curative treatment for this
malignancy but with high recurrence rate. However, at the
time of diagnosis most patients have advanced disease due
to lack of specific sign and symptoms (Shaffer 2008; Zhu
et al. 2010; Dutta 2012). The prognosis of GBC remains
extremely poor, because of late presentation and aggres-
siveness. Hence, novel diagnostic and prognostic biomark-
ers are needed for better clinical outcome.
MicroRNAs (miRNAs) are short non-coding RNAs of
20–24 nucleotides long that play an important role in gene
regulation of their target mRNAs by binding to comple-
mentary sites in the 3′ untranslated region (UTR) (John-
son et al. 2005; Hollestelle et al. 2011). A large number
of studies have confirmed miRNA binding site polymor-
phism to be novel class of polymorphic variations of clini-
cal importance and could be useful biomarkers for disease
progression, treatment response, and prognosis (Cipollini
et al. 2014). Let-7 miRNAs are genetic regulators that are
important in controlling KRAS expression and a polymor-
phism in 3′UTR (rs61764370 T/G) of the gene disrupts
let-7 miRNA binding site which ultimately leads to higher
expression of ras protein (Chin et al. 2008; Hollestelle et al.
2011). The association of rs61764370 polymorphism with
several types of cancer has been extensively studied (Chin
et al. 2008; Nelson et al. 2010; Sebio et al. 2013; Hol-
lestelle et al. 2011; Ratner et al. 2010; Christensen et al.
2009). However, its association with risk and prognosis for
gallbladder malignancy has not been evaluated. Therefore,
in present study we investigated KRAS rs61764370 poly-
morphism and its association with susceptibility, clinical
outcomes, and survival of GBC patients in Northern Indian
high risk population.
Material and methods
This present hospital based case control study was ethi-
cally approved by the Institutional Ethics Committee and
all subjects provide written inform consent before enrol-
ment. In our study, a total of 541 histologically confirmed
or fine needle aspiration cytology-proven cases of gallblad-
der cancer were included. There are 307 controls (healthy
subjects, without any malignancy or gallstones or polyp)
in this cohort from the same geographical area and ethnic-
ity. Cases and controls were mainly recruited from January
2010 to October 2015 from outpatient department (OPD)
of King George’s Medical University, Lucknow. All the
procedures were according to the Declaration of Helsinki.
Staging for gallbladder malignancy was done according
to 7th edition of American Joint Committee on Cancer—
Tumor node metastasis classification (TNM), 2010 or by
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radiological evaluation. Patients were followed after one
month of recruitment to their death or completion of the
study whichever occurred earlier. Telephonic interview was
done to obtain prognostic data (number of chemotherapy
cycles and overall survival).
Genomic DNA was obtained from 3 ml peripheral blood
(collected in EDTA) which was obtained from each subject
by intravenous puncture. Integrity of DNA was checked in
0.8 % agarose gel and quantification of DNA was assessed
using nanodrop spectrophotometer. Genotyping was done
using Taqman allelic discrimination assay (Applied Biosys-
tems, USA) with a custom-designed primer probe (Forward
primer: 5′-GCCAGGCTGGTCTCGAA-3′, reverse primer:
5′-CTGAATAAATGAGTTCTGCAAAACAGGTT-3′, VIC
reporter probe: 5′-CTCAAGTGATTCACCCAC-3′, and
FAM reporter probe: 5′-CAAGTGATTCACCCAC-3′) set
using ABI PRISM 7500 software.
Statistical analysis was performed by Statistical Pack-
age for the Social Sciences (SPSS Version 16.0, Chicago,
IL) for windows or using Graph pad prism (Version 5.0).
Two-tailed p value <0.05 was considered to be statistically
significant. Kaplan–Meier survival method was used for
patient survival’s estimation and the difference was ana-
lyzed by the log-rank test. Hazard of failure was estimated
using Cox proportional hazard model.
Results
In the present case–control study, a total of 848 subjects
were recruited including 541 cases of gallbladder can-
cer while 307 subjects were healthy controls. Control
group was matched with cases by age and gender. Clini-
cal characteristics of the study population are illustrated in
Table 1. For both cases and controls, the observed geno-
type frequency was in Hardy–Weinberg equilibrium. We
observed significant difference in genotype frequency for rs
61764370 polymorphism between GBC cases and healthy
population (Table 2). However, we were unable to find any
mutant genotype (GG) in our recruited subjects. Individu-
als having heterozygous genotype (TG) have significant
higher risk for GBC as compared with wild genotype (TT)
(p = 0.007, odds ratio = 2.56, 95 % CI 1.27–5.18). Simi-
larly, at allelic estimation, allele G has significant higher
risk for GBC as compared with T allele (p = 0.008, odds
ratio = 2.5, 95 % CI 1.25–5.01).
Till the last follow-up, only 68 (12.6 %) patients were
found to be surviving, 316 patients were dead while 157
(29.0 %) were lost to follow-up. Kaplan–Meier survival
curve indicates significant decrease (p < 0.0001) in over-
all survival for patients carrying heterozygous genotype
(hazard ratio = 3.42, 95 % CI 1.21–4.20) (Fig. 1). Also,
significant decrease in overall survival was observed
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Table 1  Table showing clinical Parameters Case (n = 541) Controls (n = 307) p value
characteristics of recruited
subjects Age ± SD 50.33 ± 15.7 48.99 ± 11.28 0.19
Sex –
Male/female 153 (28.3 %)/388 (71.7 %) 100 (32.57 %)/207 (67.43 %) 0.22
Stage at diagnosis N/A –
1a/1b 1 (0.2 %)/7 (1.3 %) –
2 25 (4.6 %) –
3a/3b 57 (10.5 %)/61 (11.3 %) –
4a/4b 111 (20.5 %)/244 (45.1 %) –
Missing 35 (6.5 %) –
T N/A –
1 7 (1.3 %) –
2 84 (15.5 %) –
3 165 (30.5 %) –
4 217 (40.1 %) –
Missing 68 (12.6 %) –
N N/A –
0 114 (21.1 %) –
1 138 (25.5 %) –
2 137 (25.3 %) –
Missing 152 (28.1 %) –
M N/A –
0 285 (52.7 %) –
1 189 (34.9 %) –
Missing 67 (12.4 %) –
Gallstones N/A –
Present/absent 424 (78.4 %)/96 (17.7 %) –
Missing 21 (3.9 %) –
Jaundice N/A –
Present/absent 337 (62.3 %)/158 (29.2 %) –
Missing 46 (8.5 %) –
Palpable lump N/A –
Present/absent 363 (67.1 %)/151 (27.9 %) –
Missing 27 (5.0 %) –
Weight loss N/A –
Present/absent 390 (72.1 %)/112 (20.7 %) –
Missing 39 (7.2 %) –
Chemotherapy N/A –
Complete course 59 (10.9 %) –
Incomplete course 142 (26.3 %) –
No chemotherapy 183 (33.8 %) –
Missing 157 (29.0 %) –
Survival status N/A –
Dead/living 316 (58.4 %)/68 (12.6 %) –
Lost to follow-up 157 (29.0 %) –

for patient carrying allele G as compared with allele T node involvement, and distant metastasis) in respect
(p < 0.0001, HR = 2.89, 95 % CI 1.21–4.20). We do not with KRAS genotype (Table 3). Also, no difference in
observe change in mean overall survival for any of the mean survival was observed for presence or absence of
pathological parameters (stage of tumor, tumor invasion, gallstones, jaundice, gallbladder mass, weight loss, and

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Table 2  Genotype and allele frequency distribution of KRAS (rs61764370) gene polymorphism in gallbladder cancer patients and healthy con-
trols
Polymorphism Cases (n = 541)
rs61764370
Wild (TT) 498
Variant (heterozygous + mutant) (TG + GG) 43
Allele carriage
T allele 1039
G allele 604
Differences in KRAS-variant allele frequencies between cases and controls were tested for significance by a χ2-test. Case–control odds ratios
and their confidence intervals were calculated using Woolf approximations
p < 0.05 was considered as statistically significant
Fig. 1  Kaplan–Meier overall survival curve of Gallbladder cancer
patients (n = 541) for KRAS (rs61764370) gene polymorphism. Sta-
tistical analysis using the log-rank test indicates significant difference
between the survival curves
status of adjuvant chemotherapy clinical parameters
(Table 4).
Discussion
To the best of our knowledge, this is the first study evalu-
ating the association of rs61764370 polymorphism with
susceptibility and prognosis of gallbladder malignancy in
this endemic belt. The study results show that heterozy-
gous genotype (TG) and mutant allele (G) is associated
with increased risk of GBC with worse outcomes in North
Indian population. Heterozygous carrier carries signifi-
cant higher risk (odds ratio = 2.56, 95 % CI 1.27–5.18,
p = 0.007) of GBC with respect to homozygous wild geno-
type (TT). Similarly, at allelic level, patients with allele G
have higher risk of malignancy (odds ratio = 2.5, 95 % CI
1.25–5.01, p = 0.008) in comparison with allele T. Sub-
sequent limitation of the current study is of sample size,
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Control (n = 307) Odds ratio (95 % confidence interval) p value
297 1 Ref 1 Ref
10 2.56 (1.27–5.18) 0.007
604 1 Ref 1 Ref
10 2.5 (1.25–5.01) 0.008
and these findings require further validation. In our cohort
of 838 subjects, we didn’t find mutant genotype (GG) and
hence all comparison is done with reference to heterozy-
gous genotype (TG). Our results are consistent with Cerne
et al. (2012) who have made a similar observation in a large
sample pool. Absence of detection of mutant genotype
(GG) in this study may be due to its very low frequency in
North Indian population. However, this hypothesis needs to
be validated in further studies.
The association of KRAS rs61764370 polymorphism
with susceptibility to head and neck, ovarian, oral, breast,
colorectal and non-small cell lung cancer has been widely
studied (Cipollini et al. 2014). Paranjape et al. (2011) iden-
tified variant genotype as a genetic marker for triple-neg-
ative breast cancer in premenopausal women. In addition,
altered gene expression and lower level of let-7 miRNA has
been observed for KRAS-variant tumors. Dai et al. (2015)
observed a functional role of rs61764370 polymorphism in
colorectal cancer development and oxaliplatin-based chem-
otherapy response. Significant association is also observed
in females who develop both breast and ovarian cancer with
uninformative BRCA sequencing results (Pilarski et al.
2012). In line with these results, we have also observed sig-
nificant association of rs61764370 polymorphism with sus-
ceptibility of GBC. These findings indicate possible asso-
ciation of the variant genotype with the risk of malignancy.
An account of this association may be lowered cellular lev-
els of let-7 which may increase cell growth or altered let-
7 binding in the 3′UTR which may lead to KRAS overex-
pression. However, Caiola et al. (2012) have not observed
significant association of rs61764370 polymorphism with
ovarian cancer. Also, in a large case control study, no sig-
nificant association with ovarian or breast cancer has been
observed (Ovarian Cancer Association Consortium, Breast
Cancer Association Consortium, and Consortium of Modi-
fiers of BRCA1 and BRCA2). These contradictory results
may be due to population and patient selection bias and
need further evaluation.
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Table 3  Table showing association between various pathological characteristics with overall survival of GBC patients

Tumor stage (n = 367) Mean survival in months ± SD p value

Wild genotype (TT) (n = 297) Heterozygous + mutant (TG + GG) (29)

1a (n = 1) 24.0 (n = 1) – –
1b (n = 5) 18.75 ± 7.89 (n = 4) 24.0 (n = 1) 0.349
2 (n = 19) 14.53 ± 4.56 (n = 19) – –
3a (n = 42) 11.04 ± 8.0 (n = 40) 4.39 ± 5.66 (n = 2) 0.349
3b (n = 48) 8.09 ± 3.1 (n = 46) 6.05 ± 8.41 (n = 2) 0.399
4a (n = 89) 3.23 ± 2.24 (n = 76) 2.44 ± 1.44 (n = 13) 0.224
4b (n = 163) 1.97 ± 1.18 (n = 141) 1.48 ± 0.94 (n = 22) 0.063
T status (n = 349) Mean survival in months ± SD p value
Wild genotype (TT) (n = 310) Heterozygous + mutant (TG + GG) (n = 39)

T1 (n = 4) 15.75 ± 9.53 (n = 4) – –
T2 (n = 67) 7.84 ± 4.91 (n = 65) 7.95 ± 5.73 (n = 2) 0.976
T3 (n = 115) 5.85 ± 5.73 (n = 104) 2.37 ± 3.42 (n = 11) 0.051
T4 (n = 163) 3.54 ± 3.62 (n = 137) 2.78 ± 4.49 (n = 26) 0.345
N status (n = 296) Mean survival in months ± SD p value
Wild genotype (TT) (n = 266) Heterozygous + mutant (TG + GG) (n = 30)

N0 (n = 85) 9.91 ± 6.18 (n = 77) 4.06 ± 3.43 (n = 8) 0.01

N1 (n = 102) 4.86 ± 3.66 (n = 93) 3.09 ± 3.52 (n = 9) 0.166
N2 (n = 109) 2.62 ± 2.54 (n = 96) 1.4 ± 1.04 (n = 13) 0.092
M status (n = 344) Mean survival in months ± SD p value
Wild genotype (TT) (n = 306) Heterozygous + mutant (TG + GG) (n = 38)

M0 (n = 217) 6.95 ± 5.64 (n = 196) 4.22 ± 5.55 (n = 21) 0.04

M1 (n = 127) 2.51 ± 1.38 (n = 110) 2.36 ± 0.81 (n = 17) 0.05

Statistical analysis done using independent Student t test

p < 0.05 was considered as statistically significant

Most of the studies have revealed significant associa-
tion of rs61764370 polymorphism with patient survival.
Christensen et al. (2009) observed worse clinical outcome
for oral cancer in relation to variant genotype. Similarly,
patients with colorectal cancer with G allele (mutant) have
a shorter survival and a higher risk of relapse or metasta-
sis when compared to those carrying the wild allele (Dai
et al. 2015). We also observed significant association of
heterozygous genotype (TG) and mutant allele (G) with
decreased overall survival of patients with gallbladder
malignancy. In addition, survival of GBC patients in rela-
tion to rs61764370 polymorphism was found to be inde-
pendent of any prognostic factor (stage of tumor, presence
or absence of jaundice, gallbladder mass, chemotherapy,
and weight loss). It is possible that a decrease in let-7
miRNA binding leads to a corresponding increase in KRAS
protein expression which results in activated GTPase activ-
ity which in turn leads to poor prognosis in these patients.
These results indicate that KRAS genotype (rs61764370) is
an independent prognostic biomarker for gallbladder can-
cer for North Indian population.
In a study by Ganzinelli et al. (2015), no evidence of
association was observed for overall survival and pro-
gression-free survival in non-small cell lung cancer in
regards to this polymorphism. Also, better prognosis was
observed for patients treated with docetaxel instead of
erlotinib for both overall survival and progression-free
survival. A study by Caiola et al. (2012) also has not dem-
onstrated an association of rs61764370 polymorphism
with outcome or physiopathological characteristic in
ovarian cancer. These differences may be due to hetero-
geneity of the disease and impact of several confounding
factors on the results.
We are now beginning to understand the importance of
3′ untranslated region miRNA binding site polymorphism
in cancer biology (Pelletier and Weidhaas 2010). Better

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Table 4  Table showing association of various clinicopathological parameters with overall survival of GBC patients

Gallstone (n = 365) Mean survival in months ± SD p value

Wild genotype (TT) Heterozygous + mutant (TG + GG)

Present (n = 302) 5.28 ± 5.19 (n = 269) 2.69 ± 4.41 (n = 33) 0.09

Absent (n = 63) 4.83 ± 4.83 (n = 54) 3.27 ± 3.43 (n = 9) 0.36
Jaundice (n = 371) Mean survival in months ± SD p value
Wild genotype (TT) (n = 331) Heterozygous + mutant (TG + GG) (n = 40)

Present (n = 261) 3.09 ± 2.96 (n = 228) 1.78 ± 1.29 (n = 33) 0.01

Absent (n = 110) 9.76 ± 5.79 (n = 103) 9.76 ± 5.79 (n = 103) 0.39
Palpable gallbladder Mean survival in months ± SD p value
mass (n = 374)
Wild genotype (TT) (n = 334) Heterozygous + mutant (TG + GG) (n = 40)

Present (n = 270) 3.17 ± 2.8 (n = 234) 2.04 ± 2.12 (n = 36) 0.02

Absent (n = 104) 9.77 ± 6.15 (n = 100) 9.87 ± 10.58 (n = 4) 0.98
Weight loss (n = 373) Mean survival in months ± SD p value
Wild genotype (TT) (n = 332) Heterozygous + mutant (TG + GG) (n = 41)

Present (n = 288) 3.69 ± 3.53 (n = 250) 1.83 ± 1.26 (n = 38) 0.001

Absent (n = 85) 9.63 ± 6.39 (n = 82) 16.00 ± 6.93 (n = 3) 0.09
Chemotherapy (n = 382) Mean survival in months ± SD p value
Wild genotype (TT) (n = 340) Heterozygous + mutant (TG + GG) (n = 42)

Complete (n = 59) 14.35 ± 4.29 (n = 56) 16.0 ± 6.93 (n = 3) 0.53

Incomplete (n = 142) 5.42 ± 2.48 (n = 132) 3.41 ± 0.81 (n = 10) 0.01
No chemotherapy (n = 181) 1.49 ± 0.76 (n = 152) 1.24 ± 0.84 (n = 29) 0.11

Statistical analysis done using independent Student t test

p < 0.05 was considered as statistically significant

understanding of miRNA, miRNA binding site polymor-
phism along with its functional role will be very useful
towards clinical utilization of these new genetic variations.
Also, validation of these variants as a diagnostic, prognos-
tic, and predictive biomarker for gallbladder cancer would
provide a valuable clinical tool (Kim and Slack 2014).
To conclude, in our pilot study, we have demonstrated
that KRAS rs61764370 polymorphism is significantly asso-
ciated with risk and prognosis of gallbladder malignancy
in this endemic belt. We hope that our results will help in
characterizing these variants as potential biomarker of can-
cer outcome, treatment response, and disease management,
heading towards personalized medicine.
Acknowledgments The authors are thankful to the Council of Sci-
ence and Technology, Government of Uttar Pradesh, Lucknow, India
for financially supporting the study (Grant No. CST/SERPD/D-3429).
Hasan Raza Kazmi is thankful to Indian Council of Medical Research,
Government of India, New Delhi, India for providing fellowship
(Grant No. 3/2/2/60/2011/NCDIII).
Funding The study was funded by Council of Science and Tech-
nology, Government of Uttar Pradesh, India (Grant No. CST/
SERPD/D-3429) and Indian Council of Medical Research, Govern-
ment of India, India (Grant No. 3/2/2/60/2011/NCDIII).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Human and animals rights All procedures performed in studies
involving human participants were in accordance with the ethical
standards of the institutional research committee and with the 1964
Helsinki declaration and its later amendments or comparable ethical
standards.
Informed consent Informed consent was obtained from all individual
participants included in the study.
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