Original Paper
Dig Surg 2013;30:240–246
DOI: 10.1159/000353133
Prognostic Significance of K-ras Codon 12
Mutation in Patients with Resected
Gallbladder Cancer
Hasan Raza Kazmi a Abhijit Chandra a Jaya Nigam a M. Noushif a
Devendra Parmar b Vishal Gupta a
a
Department of Surgical Gastroenterology, King George’s Medical University, and b Developmental Toxicology
Division, Indian Institute of Toxicology Research (IITR), Lucknow, India
Key Words
K-ras point mutation · Gallbladder cancer · Overall survival ·
Prognostic factor
Abstract
Background: Point mutation of K-ras is associated with car-
cinogenesis and overall survival in various cancers. We in-
vestigated the mutational spectrum of K-ras codon 12 in re-
sected normal and gallbladder cancer tissue samples in a
Northern Indian population and correlated it with different
clinicopathological parameters. Patients and Methods:
Gallbladder tissues from normal (n = 24) and cancer patients
(n = 39) were analyzed for K-ras codon 12 mutation by re-
striction fragment length polymorphism. Statistical analysis
was carried out using the χ2 test or Fisher’s exact test. Sur-
vival was estimated using the Kaplan-Meier method, and
the difference between survival curves was analyzed by the
log-rank test. Results: The frequency of K-ras mutation was
significantly higher (p = 0.001) in gallbladder cancer tissue
samples (16/39) compared to normal samples (1/24). Pa-
tients with K-ras mutation had a significantly decreased
overall survival (p = 0.003), particularly for stage II (p = 0.021)
and III (p = 0.009) cancers. No significant correlation was ob-
served with any of the other clinicopathological factors
© 2013 S. Karger AG, Basel
0253–4886/13/0303–0240$38.00/0
E-Mail karger@karger.com
www.karger.com/dsu
Received: January 21, 2013
Accepted after revision: May 9, 2013
Published online: July 6, 2013
studied. Conclusions: Gallbladder cancer has a high fre-
quency of K-ras codon 12 mutation with poorer outcomes
in resected stage II and III disease. K-ras mutational analysis
has important prognostic implications that need to be in-
vestigated further. Copyright © 2013 S. Karger AG, Basel
Introduction
Gallbladder cancer (GBC) is the fifth most common
malignancy involving the gastrointestinal tract [1]. Prog-
nosis is dismal due to aggressive behavior and the absence
of effective treatment [2, 3]. The prevalence of GBC varies
widely in different geographic regions and among differ-
ent races and ethnic groups. Incidence and mortality rates
have been found to be very high in American-Indian and
Chilean-Mapuche populations and in the northern part
of India [4].
This paper was elected and presented at the award winning paper
session of the XXII National Conference of the Indian Association of
Surgical Gastroenterology on October 10–14, 2012, in Bengaluru,
India.
117.197.54.38 - 7/16/2013 11:56:48 AM
Postgraduate Institute of Med.
Dr. Abhijit Chandra, MCh, Professor and Head
Department of Surgical Gastroenterology
King George’s Medical University
Downloaded by:
Lucknow, 226003 (India)
E-Mail abhijitchandra @ hotmail.com
 A number of genetic and environmental factors (di-
etary factors, chronic gallbladder infections, and gall-
stone disease) have been associated with the development
of GBC. For a better understanding and to determine
valuable prognostic markers, genetic changes involved in
the development of GBC need to be investigated in areas
of high endemicity.
It is now well known that the ras oncogene plays an
important role in the pathogenesis of various cancers. A
high frequency of K-ras oncogene activation has been
recognized as an early event in pancreatic and colonic
carcinogenesis [5, 6]. K-ras point mutation exclusively
occurs in its ‘hot spot’ (codons 12, 13, and 61) [7]. Most
of the K-ras mutations in GBC have been reported in co-
don 12 of the gene, with variable incidence in different
populations [7–9]. Moreover, K-ras mutations are associ-
ated with a more advanced stage, increased metastatic po-
tential, a poor prognosis, and decreased overall survival
for patients with colorectal cancer [10].
In the present study, we analyzed the K-ras codon 12
mutation and its clinicopathological significance in nor-
mal (undiseased) and malignant gallbladder tissue sam-
ples in patients from Northern India which has one of the
highest worldwide incidences of GBC.
Patients and Methods
Patients
Histopathologically proven GBC (n = 39) and normal gallblad-
der (n = 24) tissue samples from patients who underwent surgery
between May 2010 and August 2012 were analyzed. Normal gall-
bladder (without gallstones, polyps, or malignancy) was obtained
from those patients in whom cholecystectomy was performed as
part of other surgeries [choledochal cyst excision (n = 11), Whip-
ple’s pancreaticoduodenectomy (n = 9), or following hepatobiliary
trauma (n = 4)]. This study was approved by the institutional eth-
ics committee, and informed consent was obtained from patients
before enrollment. Preoperative evaluation of GBC patients in-
cluded blood tests (hemogram and liver function tests) and ab-
dominal imaging (ultrasonography and computerized tomogra-
phy). GBC was diagnosed on the basis of histopathological exami-
nation and staged according to American Joint Committee on
Cancer (AJCC) tumor node metastasis (TNM) staging, 2010 [11].
Patients with stage I disease and advanced malignancy (not ame-
nable for a curative resection) were excluded.
Tissue Samples and DNA Isolation
Tissue samples were immediately put into TRIzol reagent (In-
vitrogen) and stored at –80 ° C until further processing. DNA was
isolated from homogenized tissue as per the protocol described
by Chomczynski et al. [12] with slight modifications.
K-ras Mutation in GBC
1 2 3 4
utant 10 p
77 p
il
2 p
Fig. 1. Ethidium bromide-stained agarose gel (3.5%) showing the
digested product with MvaI restriction endonuclease. Lane M: 20-
bp ladder. Lanes 1 and 2: normal samples. Lanes 3 and 4: GBC
samples.
Table 1. Primer sequences and PCR conditions for amplification
of a 106-bp fragment of codon 12 of the K-ras gene
Forward 5′-ACTGAATATAAACTTGTGGTAGTTGGACCT-3′
Reverse 5′-CTATTGTTGGATCATATTCG-3′
94°C for 5 min, 35 cycles of 94°C for 1 min, 49°C for 1 min, and 72°C for
2 min, followed by 72°C for 10 min.
Detection of K-ras Mutation Using the Restriction Fragment
Length Polymorphism Method
K-ras codon 12 mutation was detected by restriction fragment
length polymorphism analysis using the MvaI (Fermantas, USA)
restriction enzyme [13]. Twenty microliters of the polymerase
chain reaction (PCR) mixture contains 1× Taq buffer, 2.0 mM of
MgCl2, 200 µM of deoxyribonucleoside triphosphates, 0.2 µM of
forward and reverse primers, 100–200 ng of DNA, and 1.5 U of
Taq polymerase (Fermentas). The specific primer sequence and
PCR conditions are mentioned in table 1. The PCR product of
106 bp was digested by the restriction enzyme MvaI. The digested
products were subjected to a second round of PCR. A cleavage site
for the MvaI restriction enzyme was present in the normal ampli-
fied allele giving digested products of 77 and 29 bp, whereas the
restriction enzyme cleavage site was lost in the mutant allele and a
106-bp product was obtained for it. The products were visualized
in 3.5% agarose gel (fig. 1).
Statistical Analysis
Statistical analysis was carried out using the χ2 test with Yates’
correction. The effect of K-ras mutation on patient survival was
estimated using the Kaplan-Meier method, and the difference be-
tween the survival curves of the two groups was analyzed by the
log-rank test. Cox proportional hazard analysis was used for esti-
mating the hazard of failure. Two-tailed p values less than 0.05
were considered statistically significant.
117.197.54.38 - 7/16/2013 11:56:48 AM
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Dig Surg 2013;30:240–246 241
DOI: 10.1159/000353133
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 Table 2. Characteristics of normal (control) and GBC patients
50 p = 0.001a
Parameters Normal GBC
41.03
(controls)
40
Frequency of K-ras mutation

Total 24 39
Male 8 (33.33) 12 (30.77)
30 Female 16 (66.67) 27 (69.23)
Mean age ± SD, years 41.41±13.42 43.87±12.39
Range 19–65 21–65
20 Presence of gallstones 0 28 (71.79)
Stage N/A
II 22 (56.41)
10 III 17 (43.59)
4.17
Values represent numbers (%) unless otherwise stated. N/A =
0
Not applicable.
Normal GBC

Fig. 2. Frequency of K-ras codon 12 mutation in normal and GBC

patients. a Fischer’s exact test. Table 3. Correlation between K-ras mutation and various clinico-
pathological factors for GBC patients

Clinicopathological factors K-ras p

mutation value
Results
Age 0.99
≥45 years 9/22
Sixty-three patients (normal controls = 24; GBC = 39) <45 years 7/17
were included in the study. The mean age (years ± SD) of Sex 0.96
normal and GBC patients was 41.41 ± 13.42 (range 19– Male 5/12
65) and 43.87 ± 12.39 (range 22–67), respectively. Table 2 Female 11/27
shows the characteristic profiles of normal and GBC pa- Stage 0.99
II 9/22
tients. K-ras codon 12 mutation was observed in 1/24 III 7/17
(4.17%) and 16/39 (41.03%) normal and GBC tissue sam- Cellular differentiation 0.65
ples, respectively, with a statistically significant difference Poorly and moderately differentiated 7/20
(p = 0.001) (fig. 2). When correlated with different clini- Well differentiated 9/19
copathological parameters for GBC patients, K-ras muta- Gallstones 1.0
tion was not found to be associated with age (<45 vs. ≥45 Present 12/28
Absent 4/11
years; p = 0.99), sex (p = 0.96), presence of gallstones (p =
1.0), tumor differentiation (p = 0.65), or stage of tumors
(stage II vs. III) (p = 0.99) (table 3).
The overall survival time was significantly shorter (p =
0.003) in patients having K-ras mutation (fig. 3). The me- III GBC with K-ras mutation was 8 months, with a sig-
dian survival for GBC patients was shorter with K-ras nificant decrease in overall survival (p = 0.009) compared
mutation compared to patients without K-ras mutation with 11 months for patients without K-ras mutation
(12.5 vs. 17 months). The overall survival time of GBC (fig. 5). The hazard of failure for patients with K-ras mu-
patients was stratified by tumor stage. A statistically sig- tation was estimated by Cox proportional hazard analy-
nificant difference in overall survival was observed for sis. Overall, K-ras mutation significantly affects the prog-
stage II patients with K-ras mutation in comparison to nosis for GBC (HR = 3.54; 95% CI = 1.54–8.14). For stage
patients without K-ras mutation (p = 0.012) (fig. 4). The II GBC, K-ras-positive patients carry a 4.31 times higher
median survival for stage II GBC with K-ras mutation was risk of failure compared to K-ras-negative cases (95%
15 months compared with 18 months for patients without CI = 1.37–13.55). Similarly, the HR for stage III disease
K-ras mutation. Similarly, the median survival for stage was 7.42 (95% CI = 1.66–33.11).
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242 Dig Surg 2013;30:240–246 Kazmi/Chandra/Nigam/Noushif/Parmar/

DOI: 10.1159/000353133 Gupta
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 150 150
K-ras mutation pre ent K-ras mutation pre ent
K-ras mutation a ent K-ras mutation a ent

100 100

ur i al
ur i al

50
50

0
0 0 10 20 30
0 10 20 30 ime mont
ime mont

Fig. 3. Kaplan-Meier overall survival curve for all GBC patients Fig. 4. Kaplan-Meier overall survival curve for stage II GBC pa-
(n = 39) with K-ras codon 12 mutation. Statistical analysis using tients (n = 22) with K-ras codon 12 mutation. Statistical analysis
the log-rank test indicates a significant difference (p = 0.003) be- using the log-rank test indicates a significant difference (p = 0.012)
tween the survival curves. between the survival curves.

Discussion
150 K-ras mutation pre ent
K-ras mutation a ent
Northern India is one of the highest-risk geographic
zones for GBC [14, 15]. GBC has a female preponderance
(about 75% globally) and the mean age of presentation in
100
various reports is around 55 years [14, 16–18]. In the
present study, the mean age of GBC patients was 43.87
ur i al

years with a male-to-female ratio of 1:2.25. This differ-

ence in age distribution could be explained by the fact that
people in endemic areas like Northern India have early
exposure to the risk factors like gallstones.
The ras gene family is a group of three closely related
proto-oncogenes (H-ras, K-ras, and N-ras) that are in-
volved in the pathogenesis of many cancers [19]. When a
ras gene is mutated, p21-ras loses its capability to hydro-
lyze GTP to its inactive configuration. As a result, there is
a continued and inappropriate growth signal to the cell
nucleus resulting in uncontrolled cell division. K-ras has
been reported to contribute to carcinogenesis in many
malignancies, including biliary tract cancers [20–22]. For
GBC, the most common point mutation of the K-ras gene
is that of codon 12, which is variably reported worldwide
(table 4) and hence evaluated in our study. The current
report, which is one of the largest series for K-ras muta-
tion in GBC, has shown prevalence similar to that in a
previous report from India by Singh et al. [9] (41.03 vs.
38%).
50
0
0 5 10 15
ime mont
Fig. 5. Kaplan-Meier overall survival curves for stage III GBC pa-
tients (n = 17) with K-ras codon 12 mutation. Statistical analysis
using the log-rank test indicates a significant difference (p = 0.009)
between the survival curves.
Genetic studies from other high-risk populations like
Japan and Chile may not hold true for GBC in India,
where the possibility of an entirely different mechanism
of etiopathogenesis (gallstones) has been proposed [9].
Gallstones and GBC frequently coexist (78–85%), sug-
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K-ras Mutation in GBC Dig Surg 2013;30:240–246 243

DOI: 10.1159/000353133
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Table 4. Literature review of K-ras mutation in gallbladder lesions in various populations

First author, year Population Normal GBC Methods

studied
n incidence n incidence
% %

Tada, 1990 [24] Japan – – 9 0 direct sequencing

Imai, 1994 [25] Japan – – 23 39 PCR-DGGE
Watanabe, 1994 [22] Japan – – 11 55 direct sequencing
Wistuba, 1995 [23] Chile 2 0 21 10 designed RFLP method
Ajiki, 1996 [26] Japan 10 0 51 59 RFLP and direct sequencing
Hanada, 1996 [13] Japan – – 22 18 RFLP
Iwase, 1997 [21] Japan 11 0 6 83 PCR-SSCP and direct sequencing
Hanada, 1999 [27] Japan 4 0 39 38 PCR-SSCP and direct sequencing
Tanno, 1998 [28] Japan – – 6 67 RFLP and direct sequencing
Rashid, 2002 [29] China – – 75 2 direct sequencing
Singh, 2004 [9] India – – 21 38 RFLP
Deshpande, 2011 [30] USA – – 32 0 MSG
Nagahashi, 2008 [31] Japan – – 22 0 direct sequencing
Hungary – – 20 5
Current study India 24 4 39 41 RFLP

PCR-DGGE = Polymerase chain reaction denaturing gradient gel electrophoresis; PCR-SSCP = polymerase chain reaction single-
strand confirmation polymorphism; RFLP = restriction fragment length polymorphism; MSG = mass spectrometric genotyping.

gesting that the former may function as a cofactor for
the latter [32, 33]. In our study, 28 cases of GBC had gall-
stones and mutation was observed in 42.86% of these
patients, indicating that presence of long-standing gall-
stones may lead to genetic abnormalities in the gallblad-
der epithelium which in turn may lead to its carcinogen-
esis. However, no significant difference (p = 1.0) was ob-
served in the mutation status of K-ras in GBC patients
with or without gallstones. This suggests that develop-
ment of GBC, with or without stones, may not involve
K-ras mutation. Further, Wistuba et al. [8] demonstrat-
ed that K-ras mutations are late events, probably related
to invasive tumors. In the current study, mutation was
detected in poorly as well as well-differentiated tumors,
although this difference was not found to be statistically
significant (p = 0.65). These variations possibly suggest
different behaviors of this cancer in various high-risk
populations. None of the previous studies have found K-
ras codon 12 mutations in normal gallbladder mucosa
[21, 23, 26, 27]. In the present study, K-ras mutation was
detected in a single normal gallbladder tissue sample,
which is difficult to explain and suggest that the patient
might be at high risk.
Gallbladder carcinogenesis involves accumulation of a
series of genetic alterations during the mucosal changes
from normal to dysplasia to in situ carcinoma to invasive
carcinoma [34]. The current study showed a significantly
higher incidence of K-ras mutation in GBC, similar to
what has been described earlier [21, 23, 26, 27]. This is an
expected finding as K-ras mutation occurs late in gall-
bladder carcinogenesis when it becomes invasive. The
differential mutation pattern of K-ras not only reiterates
its late involvement in gallbladder carcinogenesis but also
helps to better understand the GBC biology when such
observation comes from a high-incidence region suggest-
ing the common occurrence of a mutation pattern across
different geographical regions. The current study is also
the first to compare the mutation pattern of K-ras be-
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244 Dig Surg 2013;30:240–246 Kazmi/Chandra/Nigam/Noushif/Parmar/

DOI: 10.1159/000353133 Gupta
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tween normal and GBC mucosa in this high-incidence
belt.
The association of K-ras mutation with worsened pa-
tient survival has been observed previously in colorectal
and lung cancers [35, 36]. A similar association for GBC
in the Northern Indian population is unknown and may
be particularly important as a high frequency of K-ras
mutation has been reported in this part of the world [9].
In the present study, the presence of K-ras mutation ad-
versely affected overall survival, especially for stage II (p =
0.012) and III (p = 0.009) patients. However, one of the
previous population-based studies from China did not
find a significant association between K-ras mutation and
the survival of GBC patients [29].This difference may be
due to geographical and racial variations. To the best of
our knowledge this is the first study evaluating the rela-
tionship of K-ras mutation and survival of GBC patients
in this endemic belt.
In the present study, no significant association of K-
ras mutation was observed with clinicopathological vari-
ables like age, sex, gallstones, tumor differentiation, and
stages for GBC patients. Our current findings are in line
with the results of previous studies [7, 26].
Substantial limitations of the current study are the
sample size and lack of patients with stage I and IV tu-
mors. Stage I cancer is usually detected incidentally during
cholecystectomy performed for benign diseases. At our
center, a tertiary care referral and teaching hospital, pa-
tients usually present with advanced disease. A small pro-
portion of patients with stage I cancer are referred from
other centers after the GBC is detected incidentally after
cholecystectomy for benign disease (i.e. incidental GBC).
Retrieving tissue samples from these cases for review at
our center remains difficult as these patients are usually
operated in the periphery and present to us with the his-
topathology report only. Similarly, patients with stage IV
disease who did not undergo surgery were excluded due
to the nonavailability of tissue samples. In such cases, the
diagnosis of GBC is confirmed by FNAC that provides few
cells and the PCR amplification success rate in such cases
is low [9]. Therefore, we selected only those patients who
were operated upon with a curative intent where we could
get sufficient tissue for analysis. Further, mutational anal-
ysis using direct sequencing or mass spectrometry was
performed. Apart from this, we studied only one point
mutation (codon 12) in the K-ras gene and further re-
search is warranted with other point mutations in relation
to metaplasia, dysplasia, and carcinoma to establish its sig-
nificance in gallbladder carcinogenesis.
Advances in tumor biology have made possible the de-
velopment of new targeted therapeutic agents including
ras which is under trial [37, 38]. Further, silencing mutant
K-ras through RNAi in pancreatic cancer has been shown
to alter tumor cell behavior in vitro, suggesting the effec-
tiveness of targeting mutant K-ras in vivo [39]. A similar
approach of gene-targeted treatment may prove useful
and lead to new insights for GBC therapy.
To conclude, a high frequency of K-ras codon 12 mu-
tation in GBC is present in the Northern Indian popula-
tion. Its presence indicates poorer outcomes in patients
undergoing surgery for this disease. The strong associa-
tion of K-ras mutation with shorter survival in patients
with stage II and III GBC has important clinical implica-
tions that need to be investigated further.
Acknowledgments
This work was supported by the Council of Science and Tech-
nology, Lucknow, Uttar Pradesh, India (grant No. CST/
SERPD/D-3429) and the Indian Council of Medical Research,
New Delhi, India (grant No. 3/2/2/60/2011/NCD-III).
Disclosure Statement
The authors declare no potential conflicts of interest.

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