Open Journal of Gastroenterology, 2013, 3, 337-343 OJGas Published Online December 2013 (
Adenocarcinomas of the gallbladder from United States
patients demonstrate less frequent molecular change for
several genetic markers than other intra-abdominal
Peter Zauber1#, Stephen Marotta2, Marlene Sabbath-Solitare2
1Department of Medicine, Saint Barnabas Medical Center, Livingston, USA
2Department of Pathology, Saint Barnabas Medical Center, Livingston, USA
Received 28 October 2013; revised 29 November 2013; accepted 12 December 2013
Copyright © 2013 Peter Zauber et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accor-
dance of the Creative Commons Attribution License all Copyrights © 2013 are reserved for SCIRP and the owner of the intellectual
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Context: The incidence of gallbladder cancer is quite
low in the US, with an estimate (2013) for new cases
of less than 10,000. The rarity suggests a possible
shared molecular pathology that might facilitate a
greater understanding of this tumor. Objective: We
wished to assess the molecular genetic profile of this
tumor, particularly KRAS gene mutations, which are
frequent in tumors associated with chronic inflame-
mation elsewhere within the abdomen. Design: We
ascertained 25 cases of gallbladder adenocarcinoma
from our pathology department records for 2000-
2012. PCR based techniques were used to evaluate the
DNA for loss of heterozygosity of the APC and DCC
genes; for point mutations in the KRAS gene, codons
12 and 13; for point mutation in the BRAF gene,
codon 600; for point mutation in the GNAS gene,
codon 201; and for microsatellite instability. Results:
Patients included 5 males and 20 females. Approxi-
mately three-quarters of cases were associated with
gallstones, inflammation and dy splasia. Microsatellite
instability and GNAS mutation, both present in just
4% of cases, and BRAF mutation present in no cases,
do not appear to be significant parts of carcinogene-
sis of gallbladder carcinoma. We detected a KRAS
gene mutation in only 8% of the cases. Loss of het-
erozygosity for the APC was detected in 16.7% of in-
formative cases; and for the DCC gene, in 34.8% of
informative cases. Conclusions: Many molecular ge-
netic changes frequently seen with tumors arising
from other intra-abdominal organs are infrequent in
this tumor type. In particular, KRAS mutations were
uncommon, in contra-distinction to other malignant
tumors developing in the setting of chronic inflamma-
Keywords: Gallbladder Carcinoma; Molecular Genetic
Changes; KRAS Mutation; GNAS Mutation; BRAF
Mutation; Microsatellite In stability; Loss of
The gross appearance and invasive characteristic of car-
cinoma of the gallbladder were first described in 1777
[1]. Almost two and one-half centuries later, typical fea-
tures continue to be with late diagn osis, limited treatment
options and poor prognosis. The incidence of gallbladder
cancer is quite low in the United States, with an esti-
mated number of new cases of gallbladder plus other
biliary cancers of just 10,300 for 201 3 [2]. Yet, this very
uniqueness suggests the possibility of a common patho-
physiology and shared molecular pathology that might
facilitate a greater understanding of this tumor, and
thereby perhaps enhance our knowledge regarding other
gastrointestinal and abdominal tumors.
There is a worldwide association between chronic in-
flammation, or cholelithiasis, and gallbladder cancer, as
well as accumulating data to support the relationship of
chronic infection with gallbladder cancer [3]. Informa-
tion about the molecular genetic changes involved in
*Financial support provided by the H. Nussbaum Foundation of Saint
Barnabas Medical Center and the Jun e B le iw ise Foundation.
#Corresponding author.
P. Zauber et al. / Open Journal of Gastroenterology 3 (2013) 337-343
gallbladder carcinogenesis is quite limited and variable,
and the relationships between chronic infection, inflame-
mation, pre-cancerous changes, and carcinoma have not
been adequately explored.
We have evaluated the surgical, histological and mo-
lecular findings in a cohort of 25 cases of gallbladder
carcinoma seen at our hospital. We evaluated each cancer
for genetic changes that have been reported previously
with regard to gallbladder cancer [4-8], as well as for
mutations in the GNAS gene, seen frequently in appen-
diceal mucinous neoplasms, an organ, like the gallblad-
der, may demonstrate inflammation [9]. Specifically, we
assayed for loss of heterozygosity for APC and DCC
genes, microsatellite instability, KRAS, BRAF and GNAS
common point mutations.
Clinical material primarily reflects a suburban commu-
nity of middle economic level, with representations from
various minority groups (African-American, Asian) of
both middle and low economic status. The computer files
of the Department of Pathology were reviewed for the
years 2000 to 2011. One clinical pathologist reviewed all
histological slides and indicated the areas for molecular
study. Inflammation was graded on the basis of infiltrate,
fibrosis and activity, according to published guidelines
[10]. Dysplasia was characterized according to the crite-
ria of Sasatomi [11]. We also randomly selected cases of
acute and chronic cholecystitis from the computer files
for the years 2010 through 2011. The study was ap-
proved by the hospital Institutio nal Review Board.
2.1. DNA Extraction and Purification
All tissue specimens were formalin-fixed and paraffin-
embedded. Histological slides stained with H&E were
examined and the area of relevant tissue was identified
and marked, as was an area of normal tissue (Figure 1).
Paraffin blocks were available for all cases. Consecutive
unstained slides from the blocks were prepared and the
corresponding areas were isolated under a dissecting
microscope by manual dissection. The paraffin wax was
removed by xylene and ethanol washes. Cellular material
was lysed in a proteinase K buffer solution. DNA was
isolated and purified using the QIAamp DNA Mini Kit
(Qiagen Inc., Valencia, CA). DNA concentration was de-
termined using a DanoDrop ND- 1000 spectrophotometer
(NanoDro p Technologi es, Wilmington, DE).
2.2. Microsatellite Analysis for Loss of
Heterozygosity and for Microsatellite
In all primer sets the forward primer contained a 5’-
fluorescent label and the reverse primer contained a 5’-
Figure 1. Histological section of gallbladder. A) an area
of adenocarcinoma showing packed glandular formation;
B) adjacent normal mucosa with uniform thickness and
consistent invaginations. 40× magnification, H&E stain.
GTGTCTT tail (Applied Biosystems Custom Oligo Syn-
thesis Service, All
PCR reactions used Applied Biosystems reagents (Roche
Molecular Systems, Inc., Branchburg, NJ) with a final
1.5 mM MgCl2 concentration. Reactions were run on an
ABI 9700 thermal cycler (Applied Biosystems, Foster
City, CA) under the following conditions: 5 minutes de-
naturation at 94˚C, followed by 35 cycles of a 30 seco nd
denaturetion at 94˚C, 30 second annealing at 55˚C, and a
60 second elongation at 72˚C, with a final 30 minute
extension at 72˚C. PCR products were separated by cap-
illary electrophoresis with an ABI 3130 Genetic Analy-
zer, and the data were processed with GeneMapper soft-
ware (Applied Biosystems, Foster City, CA).
Loss of heterozygosity of the APC gene was deter-
mined by amplification of the CA repeat region within
the D5S346 locus. Samples homozygous for this locus
were analyzed using repeats within the D5S1965 and/or
D5S492 loci. Loss of heterozygosity of the DCC gene
was determined by amplification of the CA repeat mark-
ers within the D18S58, D18S61 or D18S1407 loci. Pri-
mer sets used as previously reported [12]. For all loss of
heterozygosity studies, neoplastic tissue was evaluated
simultaneously with normal colonic mucosal tissue from
the same patient. Loss of heterozygosity was defined as
previously [13].
MSI was detected using the Bethesda panel of markers
that includes two mononucleotide markers BAT25 and
BAT26, and three dinucleotide markers D2S123, D5S346,
and D17S250. A tumor was defined as “microsatellite
unstable-high” if two or more of the five markers had a
changed allele pattern, and this is referred to as “MSI”.
2.3. Sequence Analysis for KRAS, BRAF, and
The codon 12/13 region in exon 2 of the KRAS gene was
Copyright © 2013 SciRes. OPEN ACCESS
P. Zauber et al. / Open Journal of Gastroenterology 3 (2013) 337-343 339
amplified using the primer set
600 region in exon 15 of the BRAF gene was amplified
using the primer set
201 region in exon 8 of the GNAS gene was amplified
using the primer set
Hot-start PCR was performed in 50 µl volumes with
AmpliTaq Gold polymerase and ABI reagents (Applied
Biosystems, Foster City, CA) using 100 ng of template
DNA, 50 pmols of each primer, and 2.0 mM MgCl2 on a
GeneAmp PCR System 9700 (Applied Biosystems, Fos-
ter City, CA). PCR consisted of an initial 8 minute dena-
turation at 94˚C, followed by 40 total cycles of a 30 sec-
ond denaturation at 94˚C, 30 second annealing, and one
minute elongation at 72˚C, with a final 30 minute exten-
sion at 72˚C. For KRAS and BRAF, the annealing tem-
peratu r e was stepped down at 62˚C, 60˚C, and 58˚C for 5,
15, and 20 cycles, respectively. For GNAS the annealing
temperature was stepped down at 65˚C, 63˚C, and 61˚C
for 5, 15, an d 20 cycl e s, re spectively.
The post-PCR products were quality checked by aga-
rose gel and then purified using the QIAquick PCR Puri-
fication Kit (Qiagen Inc., Valencia, CA) prior to se-
quencing. The sequencing reactions were performed in
20 µl volumes using 0.5X BigDye Terminator Cycle Se-
quencing Reagents (Applied Biosystems, Foster City,
CA), 5.0 pmol of either the reverse KRAS primer, the for-
ward BRAF primer, or the reverse GNAS primer and 1.0
µl of the purified PCR reaction. Reactions were run on a
GeneAmp PCR System 9700 (Applied Biosystems, Fos-
ter City, CA) for 25 cycles using a 2 minute extension
time. The sequencing reaction fragments were cleaned
using isopropanol precipitation. Sequencing products
were separated by capillary electrophoresis with an ABI
3130 Genetic Analyzer and the data was processed with
Sequencing Analysis (Applied Biosystems, Foster City,
CA) software.
2.4. Statistical Methods
Descriptive statistics were used to characterize the gall-
bladder cancer cases. We used mean and range to de-
scribe continuous variables such as age, and frequency
distributions to describe categorical variables.
We identified a total of 25 cases of primary cancer of the
gallbladder through the computer search. Slides and par-
affin blocks were available for all 25 cases. The patients
included 5 males and 20 females. The average age for the
group was 73.7 years, with a span from 49 to 89 years.
The average for males was 74.6 years and 73.5 years for
females. Twenty-three of the patients were Caucasians,
one female was of Korean ancestry and one female was
of Japanese ancestry.
All cancers were adenocarcinomas. Histological find-
ings are summarized in Ta bl e 1. We attempted to evalu-
ate the case material for the presence of bacteria by
staining newly cut slides with the Brown and Brenn
Gram stain. No definite clusters of bacteria could be
identified in any of the cases, despite extensive histologic
areas of acute and chronic inflammation. Cultures at the
time of surgery were not possible for these retrospective
Loss of heterozygosity for the APC gene was de-
Ta b le 1 . Histologic findings in 25 cases of adenocarcinoma of
the gallbladder.
Histologic feature No. (%)
Gallstone 25
present 19 (76)
absent 6 (24)
Differentiation 25
Well differentiated 5 (20)
Moderately differentiated 13 (52)
Poorly differentia ted 7 (28)
Inflammatory activity* 25
None 5 (20)
Grade 1 0
Grade 2 8 (32)
Grade 3 12 (48)
Dysplasia 25
None 8 (32)
Mild 2 (8)
Moderate 4 (16)
Severe 11 (44)
Tumor invasion 25
Nerve 10 (40)
Blood vessel 10 (40)
Lymphatics 10 (40)
Peri-gallbladder lymph node 5 (22)
*Inflammation grading based on Barcia. (ref); Dysplasia grading based on
Sasatomi. (re f) ; Lymph node status u nknown for two cancers.
Copyright © 2013 SciRes. OPEN ACCESS
P. Zauber et al. / Open Journal of Gastroenterology 3 (2013) 337-343
tected in 4 of 24 (16.7%) informative cases, and for the
DCC gene in 8 of 23 (34.8%) informative cases. Micro-
satellite instability was found in just one of 25 (4%)
tumors. A KRAS mutation was present in 2 of 25 (8%)
tumors, both were c.35G > A. All tumors were BRAF
wild type and one tumor (4%) contained a GNAS muta-
tion in codon 201 (Table 2). We also studied 7 cases of
acute cholecystitis and 26 cases of chronic cholecystitis.
Gallstones were present in 28 of these 33 (84.8%) cases,
and 28 (84.8%) had moderate or severe inflammatory
changes. However, none of these 33 cases contained any
molecular genetic change of loss of heterozygosity for
APC or DCC, KRAS, BRAF or GNAS mutation, or micro-
satellite instability. Additionally, no molecular genetic
changes were detected in one case each of a gallbladder
adenoma, bile duct carcinoma, gallbladder dysp lasia, and
gallbladder mucinous metaplasia.
The estimate for new cases of gallbladder cancer for
Table 2. Molecular findings in 25 cases of adenocarcinoma of
the gallbladder.
Molecular change No. (%)
KRAS mutation 25
Mutated 2 (8)
Wild type 23 (92)
Mutated 0
Wild type 25 (100)
Mutated 1 (4)
Wild type 24 (96)
APC 25
Loss of heterozygosity 4 (16)
Not studied 1 (4)
Normal 20 (80)
DCC 25
Loss of heterozygosity 8 (32)
Homozygous 2 (8)
Normal 15 (60)
Microsatellite instability 25
Present 1 (4)
Absent 24 (96)
2013 in the United States is approximately one seventh
that of pancreatic carcinoma and one-twentieth that of
colorectal cancer [2]. More than 90% of gallbladder can-
cers are adenocarcinomas. During embryological devel-
opment, the liver, biliary apparatus and pancreas arise as
diverticula from the foregut, suggesting possibly some
commonality to the tissues of the gallbladder and pan-
creas. Two pathways for the development of gallbladder
carcinoma have been identified. The first is associated
with a specific congenital abnormality of the pancreatic-
bile duct junction, in which the pancreatic and common
bile ducts join together before reaching the duodenal
wall, allowing reflux of pancreatic secretions into the
gallbladder. This anomaly is particularly common in Ja-
pan [14].
The more common pathway for the development of
gallbladder carcinoma is associated with gallbladder
disease, usually in the form of cholelithiasis combined
with cholecystitis [3]. Similar to other epithelial cancers,
such as colon cancer, a series of pre-malignant changes
defined as metaplasia, dysplasia and carcinoma in situ
has been described for gallbladder carcinoma [15,16].
These precursor changes may be detected in the mucosa
adjacent to carcinomas, and they may incidentally be
detected in gallbladders removed for cholelithiasis. Gall-
stones are found with cancer in about 80% of cases, and
diffuse calcification of the gallbladder (“porcelain gall-
bladder”) is found in about 15% of cases.
A link has been proposed between chronic infection of
the gallbladder with Salmon ella typhi and gallbladder
cancer [17], but whether the link is specific to this or-
ganism, and whether the link is specifically modulated
by chronic inflammation and specific molecular genetic
changes, has not b een clarified. Additionally, an associa-
tion has been reported between Helicobacter bilis and
gallbladder cancers in 3 of 11 cases, in which bacterial
DNA was detected in the cancer tissues [18]. Gallbladder
cancer is more common in areas of the world reporting
mixed gallstone disease, rather than pure cholesterol
stones. The mixed type of gallstone is more likely to
originate from a nidus of infection. Our in ability to dem-
onstrate the presence of bacteria by histology is con-
sistent with the findings of others [19]. Gallbladder car-
cinoma is more frequent in females than males in all
populations evaluated. The female/male ratio of 4:1 for
our cases is slightly higher than most reported ratios.
Information about the molecular genetic changes in-
volved in gallbladder carcinogenesis is quite limited and
inconsistent. Most of the reported molecular studies are
of cases from Chile and Japan. Gallbladder carcinoma is
approximately five times more prevalent in Japan than in
the US [20], and findings in Japanese cases may not be
representative of cases in the United States. Nagahashi et
Copyright © 2013 SciRes. OPEN ACCESS
P. Zauber et al. / Open Journal of Gastroenterology 3 (2013) 337-343 341
al studied Japanese and Hungarian cases of gallbladder
cancer and reported a KRAS mutation in just 1 of 42
(2.4%) tumors [4]. A second Japanese study involving
different patients found KRAS mutations in 30 of 51
(59%) cases of gallbladder cancer. [5] A study from
Chile found only 1 of 21 (4.8%) gallbladder cancers to
have a KRAS mutation [6]. A more recent study from
China detected a KRAS gene mutation in just 2 of 75
(2.7%) gallbladder cancers [21]. One study of 29 US
cases found 2 (6.9%) with a KRAS mutation and none
with a BRAF mut at i on [22].
Other molecular changes have been evaluated in
gallbladder carcino ma. A point mutation in the p53 gene
has been demonstrated in 50% or more of the Japanese
and Hungarian cases mentioned above [4,23]. Increased
expression of p53 in 13 of 20 (65%) gallbladder adeno-
carcinomas was recently reported from Australia [24]. A
BRAF mutation was found in 7 of 21 (33.3%) gallbladder
carcinomas from Greece [8]. A recent study of 30 cases
of biliary intraepithelial neoplasia and intrahepatic cho-
langiocarcinomas found none with a GNAS mutation
Nagahashi et al. determined microsatellite instability
to be present in 8 of 19 (42.1%) Japanese cases and just 1
of 15 (6.7%) Hungarian cases [4]. A 1995 study of 25
gallbladder cancers from Chile found loss of heterozy-
gosity for 5q21 (APC and MCC genes) in 5 of 23 (21.7%)
cases tested; loss of heterozygosity for 9p21 (CDKN2
gene) in 4 of 10 (40%) cases, and loss of heterozygosity
for 18q21 (DCC gene) in 4 of 13 (30.8%) cases [6]. In a
study of 32 gallbladder cancers from Korea, 20 (62.5%)
demonstrated LOH for chromosome 5q, and 10 (31.2%)
demonstrated LOH for 18q [7]. In a more recent study of
cases from Chile, using high resolution analysis of 81
microsatellite markers, Wistuba et. al. found a frequency
of loss of heterozygosity of 88% to 100% for chromo-
somal regions 3p, 8p, 9q and 22q [26]. They also de-
monstrated allelic losses in normal and dysplastic gal-
lbladder epithelium. A recent study of cases from nor-
thern India, another highly endemic area, suggested that
genetic variants in certain germ line DNA repair path-
ways might be involved in gallbladder carcinogenesis.
Odds ratios of about 2.0 were found for variants of the
genes EERCC2, MSH2, and OSGGI [27].
There are two associations that might suggest a high
frequency of KRAS gene mutations in cases of gallblad-
der carcinoma. First, both the gallbladder and the pan-
creas arise from a foregut cell lineage, and KRAS muta-
tions are quite frequent in pancreatic cancer, reported as
occurring in 73 of 79 (92.4%) cases [28]. The second
association involves the relationship of gallbladder car-
cinoma with inflammation. KRAS mutations have been
found in a high percentage of other intra-abdominal
processes associated with inflammation, such as muci-
nous cystadenomas of the appendix [29] and chronic
gastritis associated with H. pylori [30]. However, only
3.8% of the intestinal-type gastric cancers related to H.
pylori contained a KRAS mutation [29]. The low inci-
dence of KRAS mutation in H. pylori-associated gastric
cancer is similar to our finding of 8% in gallbladder
cancers. This pattern of a high incidence of KRAS muta-
tion in the precursor benign tissue, but not in the malign-
nant tissue, was also reported for gallbladder tu mors [22],
and is worthy of further systematic study of paired tissue
samples from the same patients.
Our study has several limitations. The number of cases
is just 25, but this represented thirteen years of material.
Evaluation of this tumor would benefit from pooling of
pathological tissue. We did not study every possible gene,
but we focused on several genes important in intra-ab-
dominal tumorigenesis. Evaluation with newer techni-
ques should facilitate an ex panded molecular study.
Our results suggest that microsatellite instability, pre-
sent in just 4% of cases, does not appear to be a signifi-
cant part of the carcinogenesis for gallbladder carcinoma.
None of our cases demonstrated a BRAF mutation and
we found only one GNAS mutation. As BRAF is located
“downstream” from KRAS, it appears that at least two
specific members of this pathway are not contributing to
gallbladder carcinogenesis. In conclusion, our results
show that in a series of United States cases of gallbladder
carcinoma, approximately three-quarters are associated
with gallstones, inflammation and d ysplasia. Almost half
show local nerve, blood vessel and lymphatic invasion.
However, many molecular genetic changes commonly
seen in tumors arising from other in tra-abdominal organs
are found infrequently in this tumor type. Additionally,
KRAS gene mutations, frequent in some other tumors
associated with chronic inflammation, are infrequent in
gallbladder adenocarcinomas. Further study will be nec-
essary to understand these differences and to clarify
which molecular genetic changes are critical for the de-
velopment of gallbladder cancer.
The authors thank Dr. Errol Berman and Dr. Lauren Zabala for review
of histological slides. Funding provided by the Nussbaum Foundation
of Saint Barnabas Medical Center and the June Bleiwise Foundation.
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