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A genetic roadmap of gallbladder cancer 
disparities: a potential for development of 
targeted therapies 
Madhuri R. Kashyap1, Claudia P. Garcia1, Ana Laura Leal1, Balraj Mittal2, Mahendra K. Singh1$ 
1Division of Molecular Oncology, Department of Surgery, University of Miami Miller School of Medicine, 
Sylvester Comprehensive Cancer Center, Miami, Florida, USA.  
Email: mahendra.singh@miami.edu 
2Emeritus Professor, Ambedkar University, Lucknow, UP, India. 

$Corresponding author: mahendra.singh@med.miami.edu 

ABSTRACT 
Gallbladder cancer (GBC) is an aggressive disease with a dismal prognosis and resistance to 
chemotherapy. Due to difficulties in early diagnosis of GBC, about 90% of patients are detected 
at advanced stages with palliative care being the only viable option. While surgery remains the 
mainstay treatment for early GBC, most patients who undergo surgical resection suffer from 
the high rates of recurrence. Unfortunately for many patients, surgical resection is not 
considered a possibility due to the stage at which they are diagnosed. Adjuvant therapies in the 
form of radiation and/or embolization of the tumor have been probed for the disease with 
moderate success. While there have been multiple studies (both retrospective and pooled 
analysis) that suggest an added advantage of combining adjuvant therapy with surgical 
resection, data from prospective studies is limited. Interestingly, GBC affects women, American 
Indians, Alaska Natives, black people, and certain ethnic groups in peculiar geographic 
locations such as Chile, India, China more than other groups elsewhere. These disparities in 
gender and ethnicities demonstrate the need for better understanding of underlying genetic 
events of GBC so that molecularly targeted therapies could be developed to provide hope for 
improving treatment response and better outcome. However, for GBC not much progress has 
been made mainly due to the lack of understanding of molecular pathogenesis of this disease. 
This article presents a review of literature focused on molecular and genetic alterations in GBC, 
and as to how effective targeted therapeutic strategies can be developed with demonstrated 
survival benefit. 

KEYWORDS: gallbladder cancer, GBC, gallstone, genetic polymorphism, mutation, targeted 
therapy, molecular pathogenesis, health disparity 

Citation: Kashyap MR et al (2020) A genetic roadmap of gallbladder cancer disparities: a potential for 
development of targeted therapies. Cancer Health Disparities 4: e1-e24. doi:10.9777/chd.2019.1014 



 
 
 
 
 

 
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Introduction 

Early detection of GBC has been challenging 
despite advancements in medical imaging 
technologies such as Contrast Tomography (CT) 
and Ultrasound scanning. The majority of the 
cases are detected incidentally during surgery for 
cholelithiasis and hence detected at very advanced 
stage of the disease. GBCs are the commonest 
biliary tract cancers worldwide. The main 
associated risk factors for the development of GBC 
include, obesity, chronic asymptomatic 
cholelithiasis, female gender, smoking, chronic 
infections of the gallbladder (especially 
salmonella), diabetes and certain medications such 
as methyldopa, OCPs, and isoniazid. They are also 
known to be associated with gallbladder polyps, 
pancreaticobiliary duct abnormalities, congenital 
biliary cysts, and carcinogen exposure (Lazcano-
Ponce et al., 2001). 

GBC is indeed a cancer type with layers of 
disparities associated with it. The disease 
prevalence and incidence is extremely high in Latin 
America and Asia, and comparatively high in some 
European countries such as Hungary and Poland. 
GBC incidence in the United States, however, is 
low. Chile accounts for the highest incidence and 
mortality rates of GBC in the world, with a higher 
death rate in women (15.6 per 100,000) compared 
to men (7.0 per 100,000) (Andia et al., 2008). It is 
also the leading cause of death in these women, 
overtaking breast, lung and cervical cancers. The 
southern region of Chile exhibits a higher 
incidence and mortality of the disease where there 
is a large population of the Mapuche (Amerindian) 
tribe, known to have the highest incidence of GBC. 
Interestingly, this geographic area of Chile has 
poor access to medical and surgical facilities 
attributable to extreme poverty in this part of the 

country (Andia et al., 2008). Worldwide, incidence 
of the disease has been shown to increase with 
age and is found to be two to six times higher in 
women than in men (Singh et al., 2004a). 
Interestingly, patients with GBC in India are usually 
younger ones as compared to their counterparts 
in the west who are typically in the 5th and 6th 
decade of their age at the time of presentation 
with this disease. It is important to note that in 
India, almost 80% patients with GBC do also have 
gallstones and its presence increases the 
vulnerability of the GB to mucosal injury. The 
incidence of GBC is out of proportion to the 
prevalence of gallstones in India. On the other 
hand, in the United States, it is one of the few 
cancers that has a low incidence in the African 
Americans while the disease has the highest 
incidence rates in Hispanics, Alaskan Native (AN) 
and American Indians (AI) and association of 
gallstones with GBC is not so much common as 
compared to India (Henley et al., 2015). 

Often, patients with GBC present with severe, 
persistent abdominal pain that can be diffuse or 
localized to the right upper quadrant. More often 
than not, however, the disease is “masked” thereby 
presenting with no clinical symptoms and as 
aforementioned, detected incidentally on surgery 
for gallstone disease. Patients may also present with 
complaints of weight loss and anorexia - a sign of 
late stage disease. On examination, they may 
present with jaundice, abdominal pain in the right 
upper quadrant with or without associated 
tenderness. A non-tender palpable mass in the 
same region (a tenderness is more indicative of 
gallstone disease), periumbilical lymphadenopathy, 
and palpable left supraclavicular lymph nodes are 
also found to be associated with advanced GBC. 
Nausea, vomiting and abdominal bloating are some 
of the other vague accompanying symptoms. Prior 



 
 
 
 
 

 
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to the advent of radiographic techniques in 
diagnosing GBC, the preoperative rate for detecting 
the disease was 10-15%. Use of abdominal 
ultrasound and cross sectional imaging with 
CT/MRI, have become the mainstay diagnostic tools 
for detecting GBC. Endoscopic ultrasound (EUS) 
may also be employed in detecting pre-cancerous 
lesions. Gallbladder polyps ≥2 cm in size, or a 
calcified rim of the gallbladder termed “porcelain 
gallbladder” are indications for surgical resection as 
these are pre-malignant conditions. However, it is 
important to note that porcelain gallbladder is no 
longer thought to be as strongly associated with 
GBC as previously reported, but this belief 
continues to be propagated as dogma in the 
medical literature and textbooks. Several large 
studies from the United States (articles attached) 
have shown little to no association between 
porcelain gallbladder and GBC (Stephen and 
Berger, 2001; Khan et al., 2011). However, it is 
important to note that the population in the United 
States is very different from that of the previous 
studies from other countries which likely included 
a large demographic of Amerindians and reported 
a very high incident of GBC in patients with 
porcelain gallbladder. 

Geographical variability in the incidence of GBC is 
often found to be associated with the prevalence 
of gallstone disease in the same population. 
However, the presence of gallstones alone does 
not appear to cause cancer in the gallbladder, as 
populations that have high incidence of gallstones 
do not necessarily have high incidence of GBC. 
Chronic infection with Salmonella typhi is another 
recognized risk factor for GBC (Singh et al., 
2004b). A research group from Varanasi, India, in 
particular, has not only shown the association of 
chronic infection with gallbladder cancer but also 
has demonstrated underlying mechanism as to 

how infection of Salmonella species can help 
promote transformation of normal gallbladder 
cells (Sharma et al., 2007; Scanu et a., 2015). 
Additionally, socioeconomic status and genetic 
elements that impede access to early detection 
and surgical procedures such as cholecystectomy 
are thought to be contributory to poor outcome. 
In addition to varied geographical incidences, 
some other factors such as the differences in age, 
race and sex also contribute to the occurrence of 
the disease. Figure 1 briefly describes some 
characteristic features of GBC. 

 

Figure 1: Gallbladder cancer: basic features, risk 
factors, and therapeutic options. 

• Risk Factor: gallstones, gallbladder polyps, 
chronic cholecystitis, Chronic typhoid carrier 
state, obesity, diabetes 

• Females are at more at risk than their male 
counterparts 

• Typically presents as an incidental finding 
following cholecystectomy (localized stage) or 
with abdominal pain (advanced stage) 

• Majority of them are adenocarcinoma 

• Level 1 evidence for adjuvant chemotherapy: 
Capecitabine 

• Palliative 1st line chemotherapy: Gemcitabine 



 
 
 
 
 

 
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• No 2nd line palliative chemotherapy with a 
demonstrated survival benefit over active 
symptom control 

• Median overall survival: ~12 months 

Molecular pathogenesis 

Late 70s and early 80s was an exciting time in 
biomedical research for cancer researchers who 
invested their efforts in establishing a link between 
cancer and heredity. The role of mutated genes in 
cancer biology was beginning to be discovered 
and explored in various types of cancers. 
Mechanistic insight into discovering the molecular 
basis for the development of GBC in the western 
world started very early on and in full force. 
Reports implicating the role of genetic/hereditary 
predisposition can be traced back to as early as 
the 80s (Trajber et al., 1982; Weiss et al., 1984). 
Karyotyping techniques first were used by Hecht 
and his colleagues in 1983 to demonstrate the 
presence of a high level of aneuploidy in the form 
of missing or extra chromosomes and/or 
chromosomal rearrangement in a tissue specimen 
obtained from a Papago Indian woman with GBC 
in the United States (Hecht et al., 1983). This study 
reported the presence of double minute 
chromosomes as well as homogenously stained 
regions on chromosomes in those cancer cells. A 
decade later, another group explored the 
correlation between the DNA content and the 
cytological and histological features in the tumor 
cells obtained from the patients with GBC (Roa et 
al., 1993). These reports were the among the first 
ones to have used molecular techniques to 
decipher the molecular/genetic basis for GBC. The 
following year, a small but an important study 
conducted by Japanese researchers, demonstrated 
mutations in the p53 gene in almost one-third of 
the patients with GBC. They used PCR-single 

strand conformation polymorphism (SSCP) for the 
very first time to discover and establish their 
findings (Takagi et al., 1994). The next several years 
brought about an increase in the number of 
published studies mostly from the Japanese 
investigators. These studies involved discovering 
various mutations in K-ras (Watanabe et al., 1994; 
Hanada et al, 1996; Matsubara et al., 1996; 
Tomono et al., 1996; Saetta et al., 1996; Iwase et al., 
1997., Tanno et al., 1998) and p53 genes (Hanada 
et al, 1996 ; Ajiki et al., 1996; Fujii et al., 1996; 
Hanada et al., 1997; Jonas et al., 1997; Yokoyama 
et al., 1998) in a significant percentage of patients 
presenting with GBC. 

By late 1990s, many studies were able to detect the 
mutations and/or the altered expression of tumor 
suppressor genes and oncogenes in the patients 
with GBC. However, most of them lacked 
mechanistic explanations for their findings. In 1999, 
a study was published discussing the molecular 
mechanisms underlying tumorigenesis of 
gallbladder carcinomas, the first of its kind 
(Wistuba et al., 1999). The researchers pointed out 
that while nearly all of the gallbladder carcinomas 
evolved from dysplasia and carcinoma in situ, role 
of pre-cancerous lesions such as gallbladder 
adenomas in the pathogenesis of GBC was still 
controversial. They further analyzed DNA obtained 
from micro-dissected tissue samples of pyloric and 
intestinal-type gallbladder adenomas to compare 
the molecular abnormalities found in adenomas 
with the ones seen in carcinomas. Tissue samples 
derived from carcinoma samples displayed TP53 
mutations alongside mutations in both the K- as 
well as N-ras genes. They also demonstrated loss 
of heterozygosity (LOH) at chromosomal regions- 
5q22 APC-MCC region, 9p21-CDKN2a, TP53, RB, 
and DCC. These genetic aberrations have been 
known to be associated with and play important 



 
 
 
 
 

 
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role in tumor initiation and progression in several 
other cancer types. Since these 
mutations/molecular changes were not exhibited 
by adenoma samples, the researchers concluded 
that adenomatous changes were not similar to 
those seen in conditions of dysplasia, carcinoma in 
situ, and/or invasive carcinoma of the gallbladder. 
Similarly, it was shown that in 25% of adenomas, 
there was a mutation in the K-ras oncogene at 
codons 12 and 61, suggesting strongly that these 
adenomas were not forerunners of invasive GBC 
(Wistuba et al., 1999). Later in 2001, a Japanese 
study confirmed this hypothesis by analyzing beta-
catenin protein expression and mutation in the 
corresponding genes. Beta catenin is a regulator 
of cell-cell adhesion and intranuclear transcription 
in gallbladder carcinogenesis. The authors also 
observed that the gallbladder adenomas, in 
comparison to carcinomas, showed a remarkably 
high expression of beta-catenin protein in both 
the cytoplasm as well as the nucleus (P < 0.05 and 
P < 0.001 respectively). 62.5% of adenomas, while 
a mere 4.8% of carcinomas demonstrated exon 3 
mutations in the beta-catenin gene. It was further 
demonstrated that the adenoma to carcinoma 
sequence was a minor pathway in the 
pathogenesis of GBC using beta-catenin as a 
molecular marker) (Yanagisawa et al., 2001). 

Besides K-ras and p53, role of other cancer 
associated proteins were also explored in the 
pathogenesis of GBC. For example, two mucin 
proteins MUC1 and MUC2 expressions were 
examined both at mRNA as well as at protein level 
in carcinoma (55 cases), dysplasia (20 cases) and 
non-dysplastic epithelia of the gallbladder, in a 
Japanese study (Yamato et al., 1999). Based on 
their observation, the authors suggested that 
MUC1 expression indicated histological 
dedifferentiation, increased proliferation and 

invasion. MUC2 expression, on the other hand, 
showed a correlation to decreased proliferation 
with reflection of some differentiation into goblet 
cells. It was further suggested that expression of 
MUC1 in a dysplastic gallbladder reflected the 
potential to developing into a malignancy (Yamato 
et al., 1999). In the same year, another study from 
North Korea revealed the role of molecular 
alterations in the carcinogenesis of gallbladder. 
They analyzed 32 carcinomatous cases and 11 
dysplastic cases for LOH and microsatellite 
instability (MSI) using 17 microsatellite markers, on 
chromosomal 3p, 5q, 8p, 9p, 13q, 17p, and 18q 
sites (Chang et al., 1999). LOH on 5q indicated a 
premature transformation to carcinogenesis while 
presence of LOH on chromosomal sites 3p and 9p 
showed an association to the advancement of 
GBC. Late event of the disease was likely to be due 
to presence of LOH on chromosomal sites of 13q 
and 18q. They observed that LOH on 17p was 
present not only in dysplastic disease but also 
found to be higher in the different stages of tumor 
development. The authors further demonstrated 
that while accumulation of LOH was associated 
with carcinogenesis of the gallbladder, role of MI 
was not of much significance (Chang et al., 1999). 
A subsequent Japanese study then aimed to 
investigate the various genetic changes associated 
with gallbladder carcinogenesis and thus implied 
that the presence of LOH on chromosome 17p led 
to the progression of GBC at a fairly early stage, 
while presence of LOH on 18q and 9p played a 
crucial role in further advancement of the disease 
(Hidaka et al., 1999). Another study from Japan 
emphasizing on the etiologic context of 
pancreaticobiliary maljunction, a unique yet an 
important factor associated with GBC in Japanese 
patients, hypothesized that hyperplastic epithelium 
played an important role in carcinogenesis. Based 



 
 
 
 
 

 
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on their findings, they believed that development 
of neoplasia in a gallbladder with this anatomic 
anomaly, advanced through the processes of 
hyperproliferation and genetic variations (Obara et 
al., 1999). 

While most of the early research in Japan was 
primarily focused on genetic alterations in GBC, a 
group of medical researchers in Varanasi, a city in 
India located on the banks of river Ganges where 
incidence of GBC is very high, started exploring 
association of various etiologic agents and their 
role in the pathogenesis of this cancer. They were 
first research group from India, which 
demonstrated the presence of Salmonella typhi in 
gallbladder cancer tissues as compared to control 
subjects and established the etiopathogenic role of 
chronic typhoid carriage in gallbladder 
carcinogenesis (Nath et al., 1997). Another 
important study from this group in Varanasi, India, 
demonstrated significantly higher level of heavy 
metals such as cadmium, chromium, and lead in 
the patients of GBC than in those with gall stones 
(Shukla et al., 1998). Role of these well-known 
chemical carcinogens was further corroborated by 
the presence of dangerously high concentrations 
of these metals in drinking water in this 
geographic location of India. Interestingly, more 
than a decade later a comparative study using 
resected gallbladder samples obtained from India 
and Japan confirmed that Indian patients with GBC 
had significantly higher level of chromium, lead, 
arsenic and zinc compared to their Japanese 
counterparts (Chhabra et al., 2012). This finding 
indicates towards a need for more focused study 
to explore the role of heavy metals derived genetic 
aberrations and relevant signaling pathways so as 
to develop targeted therapeutics. 

By the end of 1990s, it was widely believed that 
abnormalities in TP53 (17p13) and 
p16(Ink4)/CDKN2 (9p21-22) loci were regularly 
encountered and frequently associated with early 
pathogenesis of the neoplastic disease of the 
gallbladder. Mutations in the K-ras genes 
appeared to be a rare neoplastic event, the 
exceptions of which included anomaly associated 
GBC. The sequence of dysplasia to carcinoma (CIS) 
was believed to be a major route for the 
development of this disease in gallbladders with 
associated anomalies (Wistuba et al., 1999). In a 
small but significant study that examined the role 
of genetic variation in tumorigenesis and as to 
how these variations were associated with the 
clinicopathological features of GBC using multiple 
markers to study MSI and LOH, authors observed 
that there was one genetic pathway dependent on 
the MSI pathway and another on LOH pathway in 
GBC [31]. Interestingly, metastasis to the lymph 
nodes was not seen in patients with MSI- positive 
tumors. An inverse relationship was also found 
between the presence of LOH and MSI in GBC 
(Yoshida et al., 2000). Soon, a Korean study 
demonstrated that development of dysplastic 
and/or subsequent malignant features in GBC was 
associated with the presence of multiple LOH. 
Malignant changes from dysplastic gallbladder was 
associated with alterations in K-ras, p53 and p16 
genes, while role of MSI is limited in the 
development of GBC (Kim et al., 2001). However, a 
follow up report from Chile which is another 
geographic area almost endemic for GBC, 
emphasized the role of MSI in GBC (Roa et al., 
2005). This group observed MSI in equal 
proportions in early as well as late stages of GBC. 
They also demonstrated that MSI was seen in 
premalignant lesions and indicated that 
inactivation of hMLH1, hMSH2, and hMSH6, genes 



 
 
 
 
 

 
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were responsible for mismatch repair, occurred 
early in gallbladder carcinogenesis. 

In the meantime, contradicting reports on the role 
of MSI in GBC kept emerging from different parts 
of the world. A Greek study in 2006 asserted the 
role of MSI in GBC was minor (Saetta et al., 2001). 
However, it is vital to make a note of the 
differences in the results which could be due to a 
varied selection of markers required to study MSI. 
It is also imperative to note the role of different 
mechanisms due to varied etiologies in a diverse 
patient population, as a possibility. Availability of 
newer and more effective molecular methods in 
the early years of the 21st century made it possible 
for gallbladder researchers to conduct more 
studies using sophisticated analyses to detect 
molecular alterations in gallbladder carcinogenesis. 
A Japanese study using allelotyping analysis 
demonstrated that GBC associated with 
anomalous junction of pancreaticobiliary duct 
(AJPBD) exhibited a high frequency of loss of 
alleles along with the presence of two new 
regions, believed to harbor genes with putative 
tumor suppressor function (Nakayama et al., 2001). 
In the same year, a group of investigators from 
Johns Hopkins Medical Institute in the USA 
developed their interest in studying this intriguing 
disease and used genome-wide allelotyping 
analysis, a sophisticated global analytical 
technique, to reveal multiple sites of allelic loss in 
GBC (Wistuba et al., 2001). According to this study, 
frequent allele losses were seen in 21 
chromosomal regions, thereby being suggestive of 
the inactivation of the supposed tumor suppressor 
genes in the pathogenesis of GBC. This study was 
the first of its kind, that too from west, where GBC 
is a rare disease, to provide a global estimate of 
the extent to which genetic changes were involved 
in GBC development and to have the potential to 

identify new tumor suppressor genes and thus 
identifying numerous new markers in translational 
research. Another similar study followed up in the 
the same year from Europe, also demonstrated 
that alterations in the p53 gene had a likely role in 
the new pathway of gallbladder carcinogenesis 
(Saetta et al., 2001). 

One of the most significant studies reporting other 
aberrated molecular lesions (besides p53 and K-
ras) in GBC resulted from a collaborative effort 
between Chile and US based researchers. 
Presence of a tumor suppressor gene at 
chromosomal location 3p14.2 containing the 
fragile histidine triad (FHIT) was shown to be 
involved in the pathogenesis of this disease 
(Wistuba et al., 2002). Much later in 2009, another 
US based study, demonstrated that the loss of 
FHIT and a fragile gene product WWOX 
expression were part of an earlier events 
underlying pathogenesis of GBC (Bloomston et al., 
2009). The same year, a group based in India 
analyzed LOH and MSI in FHIT gene (Priya et al., 
2009). This group of investigators from India also 
looked for the expression of the p53 gene in GBC, 
Chronic Cholecystitis (CC), Xanthogranulomatous 
Cholecystitis (XGC) and the normal gallbladder to 
establish the role of the FHIT gene in the 
gallbladder cancer using microsatellite markers 
such as D3S1217, D3S1300, D3S1313, D3S1600, and 
D3S2757. This study reported 17.5% of MSI and 
27.5% of LOH in the GBC cases from India. 
Significant differences were also noted in LOH 
between GBC and CC (p=0.002) and GBC and 
normal GB (p=0.02). Their results suggested that 
CC acts as a pre-invasive lesion in the 
pathogenesis of GBC (Priya et al., 2009. It is 
important to note that some researchers believe 
that XGC, an uncommon variant of CC, is a 
precursor lesion of GBC. This was the first report to 



 
 
 
 
 

 
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study and compare genomic instability in GBC 
versus different variants of CC and normal 
gallbladder from any Asian country. However, an 
earlier report in 2001 had studied XGC at 
molecular level and confirmed that while 
etiopathologic factors of XGC could have a 
correlation to cancer, XGC might not be an 
immediate cause of cancer (Takada et al., 2002). 

In a study conducted in Italy, expression of the p53 
gene as well as microsatellite instability (MSI) status 
were studied in tumor tissues obtained from 71 
patients with GBC. Examination of all neoplasms 
were conducted using IHC staining for hMSH2, 
hMLH1, p53 proteins and markers of GI 
differentiation along with a microsatellite analysis 
at mononucleotide locus BAT-26. The authors 
showed that although MSI was not a key factor in 
GBC development, alteration in the p53 gene 
played a critical role in a large percent of cases 
with the exclusion of mucinous and squamous 
GBC (Sessa et al., 2003). Concurrently, a small 
study at Johns Hopkins Medical Institute, found 
hypermethylated regions at various promoter sites 
of tumor suppressor genes that contributed to the 
formation of the tumor as well as its progression 
inside a GB that was chronically inflamed. Since 
this study included a large cohort of specimens 
obtained from two different populations, Chile and 
the United States, plain disparities in the patterns 
of methylation between the gallbladders obtained 
from these two countries led to the hypothesis that 
these differences indicated a varied and distinctive 
biology associated with this disease around the 
world (House et al., 2003). In a subsequent Chilean 
study, mutations in mitochondrial DNA (mtDNA) 
was observed in GBC which suggested that 
mtDNA be additionally investigated in patients 
from different geographical regions thus including 

it in a list of molecular biomarkers for early 
detection of GBC (Tang et al., 2004). 

Same year, investigators at Johns Hopkins 
demonstrated that GBC had a unique pattern of 
abnormal gene methylation in a study which 
encompassed the most inclusive methylation 
profiling. The finding of methylation in 
gallbladders with CC (and without cancer) in 
healthy individuals suggested that this was an 
important phenomenon in the early pathogenesis 
of GBC (Takahashi et al., 2004). Subsequently, a 
group from Chile observed that the high 
frequency of methylation of promoter areas for 
CDKN2A (p16), FHIT, APC, and CDH1 genes led to 
the inactivation of genes responsible for tumor 
suppression and control of cellular proliferation in 
the carcinogenesis mechanism (Roa et al., 2006). 
Another Chilean study then confirmed by 
epigenetic inactivation that abnormal methylation 
in the promoter regions of tumor suppressor 
genes such as DUTT1 (3p12), FHIT (3p14.2), BLU, 
RASSF1A, SEMA3B and hMLH1 (3p21.3) on 
chromosome 3p was a frequent occurrence 
(Riquelme et al., 2007). 

A small study from Greece reported mutations in 
the B-raf gene for the first time, which is an 
important component of Ras signaling pathway 
(RAS/RAF/MEK/ERK). They reported that almost 
one third of all patients with GBC also presented 
with mutations in the B-raf gene (Saetta et al., 
2004). It is imperative to note that the Ras pathway 
is an organized, downstream pathway in which Raf 
proteins are activated in a Ras dependent manner. 
Considering the fact that Ras signaling pathway 
could be activated by mutations at various levels 
including B-raf, it was an important finding of re-
emphasizing the role of Ras pathway in those GBC 
cases where no mutation was detected in the K-ras 



 
 
 
 
 

 
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oncogene (Saetta et al., 2004). Same year, our 
group demonstrated that almost one third of all 
the patients with GBC has mutations in codon 12 
of the K-ras oncogene (Singh et al., 2004b). While 
this study was the first of its kind from India to use 
methods to detect changes at a molecular level in 
an oncogene, it also demonstrated that detection 
of such molecular aberration in an oncogene 
could be performed on a small number of tumor 
cells retrieved from fine-needle aspirates. 
Additionally, together with cytological examination, 
this work showed a possibility to help making a 
definitive diagnosis in patients with other risk 
factors, at least in a sub-set of patients harboring 
K-ras mutations. Besides, this work also suggested 
the role of chronic inflammation in the etiology of 
gallbladder carcinogenesis (Singh et al., 2004b). 

In the dawn of 21st century, with growing 
realization of cancer being a much more complex 
disease, than it was thought to be earlier, cancer 
researchers working on GBC initiated pathway-
mechanism oriented studies in various parts of the 
world. One such study conducted in Japan cited 
factors such as homozygous deletions, promoter 
and LOH hypermethylations and multiple LOH to 
be the major mechanisms involved in inactivation 
of the p16 gene in GBC (Tadokoro et al., 2007). 
Another such study to understand geographical 
differences in genetic changes involved in GBC 
between Japan and Hungary, was conducted in a 
collaborative effort by collecting specimens 
obtained from patients living in these two 
countries. A considerable difference between the 
two populations was attributed to the presence of 
high MSI seen in Japanese patients as opposed to 
just one high MSI patient in Hungary. This further 
affirmed the notion that geographic variation 
could play a significant role in the process of 

gallbladder carcinogenesis at mechanistic level 
(Nagahashi et al., 2008). 

Investigators in the United States relying on serial 
analysis of gene expression (SAGE) created cDNA 
libraries from the tumor tissues of stage matched 
GBC patients of Native American, Caucasian and 
Hispanic/Latino descent, along with the tissues 
from normal gallbladder. RT-qPCR analysis was 
performed on the microdissected epithelia 
extracted from both GBC as well as their 
corresponding non-neoplastic mucosae. To further 
understand the complexity of the disease, 
immunohistochemistry on the GBCs was done in a 
complex tissue microarray format. The hallmark of 
this study was using SAGE to make an impartial 
assessment of the transcriptome in GBC, and 
identifying a new positive prognostic marker- 
expression of connective tissue growth factor 
(CTGF). GBC and non-neoplastic mucosae from 
the gallbladder have SAGE libraries which are 
available publicly at the Cancer Genome Anatomy 
Project that would help facilitate the research 
needed for this deadly cancer (Alvarez et al., 
2008). In another small attempt to identify 
metastasis-associated proteins in GBC, a Chinese 
study demonstrated that overexpression of 
chloride intracellular channel 1 (CLIC1) encouraged 
cell motility and inundation of GBC-SD18L (cell line 
with a low potential for metastasis) in vitro. It was 
interesting to note that inhibition of CLIC1 
expression by RNAi significantly decreased the cell 
motility as well as the invasiveness of GBC-SD18H 
(a cell line with a high potential for metastasis). 
This study thus demonstrated that CLIC1 could 
play a crucial role in GBC metastasis, and was 
hence identified to be a regulator of metastasis in 
GBC (Wang et al., 2009). An overview of some 
such key studies reporting mutational spectrum in 
GBC is provided in Table 1. In another such 



 
 
 
 
 

 
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interesting study from India, investigators 
identified E-cadherin (CDH1) genomic instability 
and demonstrated a correlation between their 
findings with CDH1 protein expression in 
conditions of GBC and other non-neoplastic 

conditions of CC, XGC, as well as with normal GB, 
to define its role in GBC carcinogenesis. More 
importantly this study provided solid evidence as 
to how CC acts as precursor lesion to GBC (Priya 
et al., 2010). 

Table 1. Important mutations during various stages of gallbladder cancer development. 

Gene and Reference Adenoma (%) Dysplasia (%) Carcinoma (%) 
K-Ras 

Hanada et al., 1999 
Watanabe et al., 1999 

Kim et al., 2001 
Wistua et al., 1999 
Singh et al., 2004 

Li et al., 2014 

 
17 
0 
0 
25 

 
15 
 
0 

 
38 
19 
20 
 

38 
8 

Beta-Catenin 
Yanagisawa et al., 2001 

Rashid et al., 2001 
Chang et al., 2002 
Kumari et al., 2014 

 
63 
57 
58 

 
 
 
0 

 
5 
9 
0 
4 

EGFR/ErbB2/ErbB3 
Nakazawa et al., 2005 

Leone et al., 2006 
Pignochino et al., 2010 

Li et al., 2014 

   
16 
15 
10 
37 

P161NK4A 
Kim et al., 2001 

Ueki et al., 2004 

 
0 

 
0 

 
31 
62 

TP53 
Wistua et al., 1999 

Kim et al., 2001 
Li et al., 2014 

Kumari et al., 2014 
Noguchietal., 2017 

 
0 
0 

 
 
0 

 
 

36 
47 
18 
64 

 
Abovementioned studies to identify genetic 
alterations in the GBC were based on conventional 
sequencing methods such as Sanger sequencing, 
which had its own limitations. In the recent past, 
altered genetic pathways involved in human 
cancer are being studied using next-generation 
sequencing (NGS) technologies which has really 
helped in understanding the disease mechanism. 

NGS has numerous advantages when compared 
to the conventional sequencing methods in that it 
is a highly throughput method. It permits a large 
number of parallel sequencing simultaneously in 
multiple genomic regions of many samples to 
identify any associated mutations in the same run 
sequence. Another crucial benefit to using NGS is 
the reduction in the turnaround analysis time 



 
 
 
 
 

 
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during routine tumor sequencing, which ultimately 
will lead to short clinical reporting time. In 
addition, the amount of RNA/DNA required for 
analysis is very low in comparison to traditional 
methods. Genomic aberrations such as 
single/multiple nucleotide variants, copy number 
variations (CNVs), fusion transcripts, and small and 
large insertions and deletions can be detected 
simultaneously with high sensitivity and rate of 
accuracy. The highlight of NGS is the high rate of 
sensitivity and quantitative evaluation of the 
mutated allele that is significantly better than 
Sanger sequencing (2-10% vs 15-25%). 

A group of investigators from China were the first 
to take advantage of the newly available NGS 
technique to identify new mutations in GBC (Li et 
al., 2014). Here, they studied 57 pairs of tumor-
normal tissues and subjected them to a 
combination of exome as well as ultra deep 
targeted sequencing of cancer- related genes 
[54]. They found that there was an increase in 
C>T/G>A transition in GBC. This identification of 
somatic mutation framework of GBC was done in a 
systematic manner, discovering GBC driver genes 
in the C>T/G>A transition along with studying a 
pathway centered around ErbB signaling. They 
also pointed out that genetic mutations in the 
ErbB pathway was a poor prognostic indicator of 
the disease. This study also went on to suggest 
that patients exhibiting genetic mutations in the 
ErbB pathway could potentially benefit from 
therapies already available or in the process of 
development (Li et al., 2014). In a similar kind of 
study using NGS techniques, a Japanese group 
analyzed 29 tissues obtained from Japanese 
patients with GBC, using an integration of whole-
exome and transcriptome sequencing techniques 
to uncover molecular variations and thus, 
prospective therapeutic targets (Nakamura et al., 

2015). It was suggested in this study that APOBEC-
mediated somatic mutational signature, associated 
with APOBEC3B expression and a higher number 
of mutations, selectively contributed to GBC 
initiation and progression. The Japanese authors 
also reaffirmed the findings in the previous study 
(Nakamura et al., 2015) and suggested that 
activation of the EGFR family of genes (EGFR, 
ErbB2, ErbB3) was seen frequently in GBC and 
could be clinical important as they could be used 
as potential targets. 

A recent study by one of the authors (BM) of this 
article, determined the presence of 184 non 
synonymous somatic mutations and 60 rare 
germline mutations in SMAD family member 4 
(SMAD4), lysine methyltransferase 2C (KMT2C) 
and TP53 genes using ultra deep sequencing 
across 409 cancer related genes in GBC patients of 
Northern India (Yadav et al., 2017). Of note, 
somatic mutations of high significance within 9 
novel genes in GBC were identified. The results in 
study hinted at the presence and significance of 
rare, inherited germline mutations in the genes of 
the DNA-repair pathway in addition to acquired 
somatic mutations in the carcinogenesis of GBC. A 
high throughput sequencing based analysis of the 
mutation profile in GBC was carried out for the 
very first time by an Indian group from the 
northern part of the country. This study was 
different in the sense that they restricted the study 
to regions of common genetic aberrations so as to 
take advantage of targeting sequencing 
approaches and covering rare mutations high 
confidence. Cancer initiating as well as progressing 
events were tagged with biological pathways 
associated with 409 cancer genes which were 
analyzed in the study (Yadav et al., 2017). The 
somatic mutation spectrum in this study was found 
to be dominated by substitution of the C>T/G>A 



 
 
 
 
 

 
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transition, consistent with other GBC sequencing 
studies (Li et al., 2014; Nakamura et al., 2015). This 
study from India (Yadav et al., 2017) holds 
promises for its possible role in future precision 
medicine practice in GBC, as it suggests that both 
the somatic mutation profile as well as the 
information regarding various germline mutations 
could be beneficial for understanding the disease 
pathology as well as for developing novel 
therapeutic strategies. 

Genetic polymorphism and risk of GBC 

It is believed that majority of the cancers are 
derived from single somatic cells along with the 
cells derived by the rapid division of those parent 
cells. These new cells acquire various genetic or 
epigenetic changes within them that lead to 
altered genetic and thus to phenotypic variants. 
However, with the advent of the Human Genome 
Project in early 2000s and published evidences 
started supporting the theory that common 
genetic variations play important role in 
determining the range of individual susceptibility 
to neoplastic diseases within a particular 
population. It was thought that they may result, 
among others, from the differences in the 
metabolism of environmental carcinogens and 
mechanisms of DNA repair and the kind and rate 
of metabolism is genetically determined by 
polymorphic enzyme coding genes participating in 
the process of xenobiotic transformation. A large 
number of enzymes involved in the reaction of 
oxidation or conjugation of exo-endogenous 
xenobioties have shown genetic polymorphism. 
Gene variability may alter the expression or 
enzymatic activity of coded enzymes. First such 
study in GBC was conducted in Japan in which 
authors investigated the correlation between the 
genetic polymorphisms in cytochrome P4501A1 

(CYP1A1) gene and the risk of development of 
GBC. The frequency and relationship between the 
CYP1A1 genetic polymorphisms and the 
development of GBC was closely examined in 
order to determine the differences in susceptibility 
to developing the disease. They demonstrated that 
females with genotypes C and/or Ile/Val in CYP1A1 
gene were potentially more susceptible to 
developing GBC (Tsuchiya et al., 2002). Next was 
our own study that was performed on a large 
cohort of 153 patients with GBC from North India 
(Singh et al., 2004a). We reported that the 
frequency of the X- allele of apolipoprotein B 
(apoB) was significantly increased in GBC patients 
irrespective of the presence or absence of 
gallstones (GS). The odds ratio was found to be 
2.3 and 1.7 respectively. Apolipoprotein B (apoB) is 
a gene that is widely known to be associated with 
alteration in serum lipid levels and susceptibility to 
gallstone (GS) disease. By the abovementioned 
findings, we suggested that a polymorphism in the 
apoB-XbaI gene results in increased susceptibility 
to GBC in a favorable environment (Singh et al., 
2004a). 

With time many other similar studies started 
appearing looking for polymorphic variants 
associated with risk of developing GBC in endemic 
areas. In 2006, a Chinese study investigated the 
potential risk associated with polymorphisms in the 
Cytochrome P450 17alpha-hydroxylases-C-(17,20)-
lyase (CYP17) enzyme (involved in synthesis of sex 
hormones) in GBC. It was found that CYP17 MspA1 
polymorphism was associated with an exaggerated 
increase in GBC development and was also 
contributory to the development of biliary stones 
among the overweight and diabetic individuals. 
This finding led to the study of an intertwining 
relationship between the genetic and hormonal 
risk factors associated with gallbladder disease 



 
 
 
 
 

 
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(Hou et al., 2006). Same year, a study conducted 
by one of the authors (BM) in India reported an 
association between glutathione S-transferase 
(GST) polymorphic variants (GSTT1, GSTM1, GSTP1, 
and GSTM3) with risk of gallbladder cancer 
(Pandey et al., 2006). These GSTs are a family of 
detoxifying enzymes and are thus intricately 
involved in the metabolism of many known and 
potential carcinogens. Various allelic variants of 
these polymorphic GSTs show deformed 
enzymatic activity and thus are reckoned to 
increase cancer risk and susceptibility. This was first 
study to show a correlation between Val allele of 
GSTP1 and an increased risk of GBC in the Indian 
population (Pandey et al., 2006). Following year, 
same group including one of authors of this article 
[BM] went on to show that a polymorphic variant 
of N-acetyl transferase2 (NAT2) which is also 
known as slow acetylator phenotype, influenced 
the susceptibility of GBC (Pandey et al., 2007). That 
same year a study from China demonstrated that 
Ser326Cys polymorphism in human oxoguanine 
glycosylase 1 (hOGG1), a gene involved in the 
excision repair mechanism for damaged DNA, has 
an association with GBC (Jiao et al., 2007). Another 
case- control study was undertaken in Japan in 
order to examine and determine the relationship 
between the various genetic polymorphisms of 
genetic polymorphisms of cytochrome P450 1A1 
(CYP1A1), glutathione S-transferase class mu 
(GSTM1), and tumor protein p53 (TP53) genes, and 
GBC risk, observed that the Val allele of CYP1A1 
Ile462Val and the Pro allele of TP53 Arg72Pro 
polymorphisms aided in a heightened incidence of 
GBC among Japanese women and men, 
respectively (Tsuchiya et al., 2007). Another 
collaborative study demonstrated that Val allele of 
cytochrome P4501A1 (CYP1A1) gene contributes to 
the development of GBC in women of two 

different populations of Japan and Hungary 
(Kimura et al., 2008). 

Last decade has seen a growing body of data to 
demonstrate an association between various 
polymorphic variants of many cancers associated 
genes with risk of GBC. Majority of these studies 
came from researchers including one of the 
authors of this article (BM) from North India. Some 
of the important ones are those that report a 
strong link between genetic variants of signaling 
molecules such as Tumor Necrosis Factor alpha 
(TNFA) and Interleukin 6 (IL6) (Vishnoi et al., 2007), 
A-204C of cholesterol 7alpha-hydroxylase 
(CYP7A1) (Srivastava et al., 2008a), Cholecystokinin 
receptor A gene polymorphism (Srivastava et al., 
2008b), Interleukin-1 gene polymorphism (Vishnoi 
et al., 2008), Single nucleotide polymorphism in 
ABCG8 transporter gene (Srivastava et al., 2009), 
toll-like receptor gene polymorphisms (Srivastava 
et al., 2010a), and Caspase-8 gene 
polymorphisms-- genes that regulate apoptosis in 
cancer cells (Srivastava et al., 2010b). In another 
significant study on larger sample size obtained 
from northern India from the same group led by 
one of us (BM), strong association of a haplotype 
of tumor suppressor gene DCC, Grs2229080-
Ars4078288-Crs7504990-Ars714 was reported to 
confer high risk of GBC in northern Indian 
population (Rai et al., 2013a). In a follow up 
genome wide association study (GWAS) using 
same patients’ cohort, this group also identified 
that some polymorphic variants of Phospholipase 
C epsilon 1 (PLCE1) gene which is known to play a 
critical role in both formation and progression of 
esophageal and gastric cancers and PSCA gene 
which plays an important role in inhibition of cell 
proliferation and/or inhibition of cell death. This 
study showed that these polymorphic variants 
confer susceptibility to GBC through gallstone-



 
 
 
 
 

 
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mediated inflammatory pathway and in a gender 
specific manner respectively (Sharma et al., 2013; 
Rai et al., 2013b). 

One of the authors (BM) of this article, recently 
published a GWAS study which to our knowledge 
is the largest study worldwide as it comprised of 
523 GBC patients and 274 controls all from 
northern India (Yadav et al., 2018). This study 
reaffirms the fact that the link between genetic 
polymorphism and disease risk may be very 
specific with regards to directional effect and 
histology/ethnicity by demonstrating that genetic 
variants on TERT-CLPTM1L and 8q24.21 loci affect 
GBC prognosis and predisposition. In order to 
describe the inter-relationship between genetic 
variants and GBC susceptibility in populations at 
risk, the study stressed on the need to perform 
complete gene sequencing of the 5p15.33 and 
8q24.21 loci. However, it is important to note that 
in spite of huge amount of efforts involved in 
these studies to study genetic risk factors, it is still 
unclear that which genetic pathways are actually 
important in order to make a person susceptible 
living in a geographically prone area for GBC such 
as North India. All these genes demonstrated 
above which have been shown to be associated 
with GBC as risk factor belong to different 
signaling pathways, which further complicates the 
issue of making a coherent picture of all these 
genetic susceptibility factors. Many more studies 
have been published in recent past from different 
parts of the world showing one or other type of 
polymorphic variants of different genes involves in 
metabolism, signal transduction, drug-
metabolizing enzymes related pathways, however, 
most of them are actually adding more complexity 
to the existing mechanistic insights about GBC. 

Interestingly, genetic aberrations in genes 
associated with inflammation which is an 
important aspect of gallbladder carcinogenesis has 
not been much explored in these GWAS studies. 
FOXM1, NF-kB, STAT3, Wnt/β- Catenin, HIF-1α, 
NRF2, androgen and estrogen receptors are some 
examples of the tumor suppressive as well as 
oncogenic transcription factors, the interaction 
between which, results in chronic inflammation of 
the gallbladder. The chronic inflammation may be 
either due to stones or a long standing infection of 
the organ. Studies conducted in various clinical 
models, pre-clinical samples as well as cell lines, 
show that numerous products obtained from 
natural resources namely, polyphenols, alter the 
expression and activity of many transcription 
factors in various tumor models (Agrawal et al., 
2015). Additional information regarding alterations 
in the aforementioned genes and their regulator 
pathways could help design a new and improved 
form of therapy combining these natural products 
along with the standard currently approved 
chemotherapeutic regimen. The combined 
therapy has been considered promising and the 
hope is to be able to not only treat the 
inflammation and cancer but also prevent the two 
conditions. 

Targeted Therapy in GBC 

While there were a multitude of studies by mid 
2000s that demonstrated that overexpression of 
tyrosine kinase growth factor receptors such as 
ErbB-2, epidermal growth factor receptor (EGFR), 
and Met could potentially lead to the formation of 
solid tumors, there were a group of investigators 
in Japan who studied biliary tract carcinomas. The 
investigators, with the intention of assessing novel 
chemotherapies, focused on these receptors in 
GBC and demonstrated overexpression of the 



 
 
 
 
 

 
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tyrosine kinase receptor proteins by IHC in tumor 
tissues obtained from 89 GBC patients. Another 
technique, fluorescence in situ hybridization was 
used for gene amplification in tumor tissues that 
stained positive. There was an overexpression of 
the ErbB-2 and the EGFR genes in 15.7 and 8.1% of 
GBC cases with GBC gene amplification of 79%. 
This study suggested the new adjuvant 
chemotherapies could be used in GBC in which 
ErbB-2 and EGFR are overexpressed (Nakazawa et 
al., 2005). Later, an italian study observed that in a 
subgroup of patients with GBC, somatic mutations 
of the EGFR gene in the tyrosine kinase domain 
exhibited cell signals maintaining proliferation and 
survival. Further these investigators suggested a 
small molecule inhibitor of EGFR for treatment 
(Leone et al., 2006). That same year, a Chinese 
study reported that p53 - vascular endothelial 
growth factor (VEGF), a marker for angiogenesis) 
pathway potentially played a role in regulating 
tumor angiogenesis in GBC (Tian et al., 2006). This 
study suggested that analyzing the expressions of 
the p53 as well as the VEGF genes could be useful 
in predicting the tumor vascularity in GBC and 
targeting advanced tumors with anti-VEGF 
inhibitors could be a good therapeutic strategy. 

In order to better understand the underlying 
mechanisms of tumor development and 
progression and to improve the prognosis of GBC 
patients, an Austrian study observed an increased 
expression in the HER2/neu gene which clinically 
and statistically correlated with advanced disease 
(Puhalla et al., 2007). In various cancer types, it has 
been shown that p110alpha catalytic subunit of 
phosphatidylinositol 3-kinase (PI3K), encoded by 
the PIK3CA gene harbor somatic mutations. Exons 
9 and 20 which encode the helical and kinase 
domains of the p110alpha are the major hotspots 
for clusters of genetic mutations. Another group 

based in Switzerland also reported that somatic 
mutations in the PIK3CA gene resulted in recurrent 
activations of the PI3K/AKT pathway in a very small 
sub-set of carcinomas of the gallbladder (Riener et 
al., 2008). 

Having realized the limitations in treatment 
options other than surgical resection (which was 
an unfavorable prognostic marker), a group of 
surgeons in Japan identified cellular targets that 
were GBC specific and which could potentially be 
used as a therapeutic approach for the disease. 
They identified a cell cycle-related gene, 
topoisomerase IIalpha (TOPO IIalpha) as one of 
the highly upregulated gene in GBC tissue which 
they further confirmed as a potential 
chemotherapeutic target, because those cells 
strongly positive for TOPO IIalpha had shown 
increased sensitivity against etoposide, as well as 
doxorubicin and idarubicin (Washiro et al., 2008). 
In the subsequent year, another Japanese group 
demonstrated genomic instability due to 
amplification in Myc oncogene which resulted in 
specific amplification of EGFR and/or ERBB2 in 
GBC (Ooi et al., 2009). Another in-vitro study used 
a combination of a histone deacetylase inhibitor 
(SAHA) with repression of EZH2 by siRNA 
treatment which revealed an increased sensitivity 
of the GBC cells to SAHA as opposed to the 
normal cells. This was then considered to be 
indicative of the efficacy of the new anti- cancer 
agent (Yamaguchi et al., 2010). 

In a significant study to evaluate the response rate 
by Response Evaluation Criteria in Solid Tumors 
(RECIST) of targeted therapy in biliary cancers 
which included GBC, eligible patients (10 patients 
with GBC) were treated with bevacizumab (a VEGF 
inhibitor) and erlotinib (EGFR inhibitor) in 
combination with chemotherapy. Using a 



 
 
 
 
 

 
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combination chemotherapy of bevacizumab with 
erlotinib resulted in clinical activity with infrequent 
grade 3 and 4 adverse effects in advanced GBC 
cases. Preliminary molecular analysis showed that 
presence of a mutation in the k-ras oncogene 
altered the efficacy of Erlotinib. Though a small 
study in sample size (only 10 cases of GBC), results 
clearly warrant a larger study in future on a bigger 
cohort of GBC patients to investigate the 
efficacious performance of Bevacizumab and 
erlotinib when used together, as an alternative 
treatment option for patients with advanced GBC 
(Lubner et al., 2010). In an independent but 
encouraging small case report, the idea of using 
EGFR-tyrosine kinase inhibitor (TKI) in combination 
with conventional chemotherapy was tested to 
treat gallbladder cancer in the US based hospital 
(Mody et al., 2010). In this case report, a patient 
with stage IV GBC was shown to have complete 
and prolonged response, a not so common 
observation in GBC, to oral EGFR-TKI plus 
chemotherapy regimen. Of note, this study 
mentions that this rare response to the treatment 
for this patient with GBC was due to an absence of 
a mutation in the EGFR gene. This discovery 
should again reiterate the need for clinical trials 
using EGFR-TKIs to treat GBC. This study also 
suggest that future clinical trials should not always 
consider the mutation status of the EGFR gene as 
inclusion criteria (Mody et al., 2010). 

In an Italian study, investigators analyzed 
mutations, amplifications and over-expression of 
EGFR, HER2, and their molecular transducers in 
biliary tract cancers so that they could explore 
possibilities of combining standard therapies with 
or without molecular targeting (Pignochino et al., 
2010). They reported that EGFR was expressed in 
~38% of patients with GBC. Activated forms of 
cancer relevant signaling proteins such as p-MAPK 

and p-AKT were also highly expressed in little less 
than 46% of GBC, hinting at an activation of the 
EGFR pathway. With the aid of genomic 
amplification, about 10% of GBCs showed an 
overexpression of HER2 gene. They went on to 
conduct a preclinical in-vitro study using TGBC1-
TKB, a GBC cell line (deleted on PTEN and 
negative for Her2 expression) for testing the 
efficacy of the drugs either alone or in 
combination with gemcitabine, targeting the 
aforementioned molecules. This study also 
demonstrated that the HER2 and EGFR pathways 
could pose to be potential therapeutic targets for 
biliary tract cancers (BTCs). The combination of 
gemcitabine with Gefitinib and Lapatinib (both of 
which are reversible selective inhibitors of the 
tyrosine kinase domain of EGFR and drugs known 
to target HER2 and EGFR pathways) appeared to 
have encouraging results and thus warrants for 
further clinical studies for testing the expression 
levels and mutations in these signaling molecules 
in future clinical studies (Pignochino et al., 2010). 

While developing strategies to target GBC 
molecularly, it is important to understand the 
subtle changes at the molecular/genetic level 
between GBC and other related cancers. Although 
they are histologically similar, they are 
anatomically different in terms of the origin of the 
tumor. For example, cholangiocarcinomas arises 
from within the liver parenchyma, peri-hilar 
regions, or the distal biliary tree, and these tumors 
are collectively known as BTCs. Although these 
tumors share an anatomic origin in the biliary 
system, they have very different disease patterns, 
molecular profiles and also respond differently to 
various therapies. Historically, GBC has a tendency 
to initially be sensitive to chemotherapy but the 
survival rate is much shorter when compared to 
cholangiocarcinoma (Eckel et al., 2007). 



 
 
 
 
 

 
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Traditionally, treatment of BTCs with cytotoxic 
chemotherapy have not taken into account the 
anatomic origin of tumor or its molecular profile. 
However, in light of emerging molecular 
techniques, such as cost effective sequencing 
platforms, it will be worthwhile to have a detailed 
molecular genetic profiling of patients with GBC 
before considering a regimen of targeted therapy 
for GBC. 

Increasingly, molecular tests to detect changes at 
genetic level are being applied regularly (in 
developed countries where advance health 
facilities are available to common people) to aid in 
the forming of therapeutic decisions in cancer 
treatment. Routine testing such as genetic 
amplification of HER2/NEU, and/or genetic 
mutations involving EGFR and Kras are done in 
clinics so as to determine treatment benefit with 
targeted specific anti-cancer therapies (McDermott 
et al., 2009). Patients in which molecular genetic 
profiling has been conducted that indicate a 
potential benefit with EGFR inhibitors and other 
molecularly targeted drugs, BRAF inhibitors are 
also being tested at the earliest phases of the 
disease (Brower T, 2010). Discovering patterns of 
genetic changes within GBC is very critical in order 
to not only gain insight into the disease biology 
but also to bring out about improvised treatment 
options, especially in light of emerging and 
established studies showing that tumor genetics 
determine drug sensitivity. In another study carried 
out at Massachusetts General Hospital in the US, 
almost 13% of patients with GBC were identified 
with activating mutations in PIK3CA (Deshpande et 
al., 2011). Activating mutations in the PIK3CA 
pathways has dual advantages in it being helpful 
for cancer diagnosis as well as discovering new 
targets for therapies such as PI3 kinase inhibitors. 

Most recently, in a large whole-exome and 
targeted gene sequencing based study, a Chinese 
chohort of GBC patients, several recurrent 
mutations in erbB pathway were identified (Li et 
al., 2014). In this study, it was observed that genes 
like TP53, K-ras, and ErbB3 had a significant 
frequency of non-silent mutations of 47.1%, 7.8%, 
and 11.8% respectively with a false discovery rate 
(FDR) of <0.0.5. Furthermore, it was also 
discovered that the ErbB signaling pathway 
including EGFR, ErbB2, ErbB3 and ErbB4 along 
with their downstream genes, affected 36.8% of 
GBC samples, making it the most extensively 
mutated pathway. Mutations in the ErbB pathway 
genes were also found to be associated with a 
dismal prognosis in GBC patients, thus suggesting 
that targeted therapies, that are presently in 
development or already in use in clinics, could be 
of major benefit (Li et al., 2014). Role of ErbB 
pathway in GBC was further confirmed in few 
other independent studies from different cohorts 
of patients. In one of such study, retrospectively 
conducted in the patients with GBC in the United 
States, HER2/neu blockade was found to be a 
promising treatment strategy to treat GBC patients 
who have gene amplification (Javle et al., 2015). In 
another significant study conducted in Japan, in a 
large cohort of biliary tract cancers, EGFR family 
genes such as ErbB2 and ErbB3 were found to be 
activated while PTEN and TSC1 genes were found 
to be inactivated, in GBC. Frequent genetic 
variations in the TP53 and RB cell cycle modules 
were also reported in GBC (Nakamura et al., 2015). 

An interesting study from India using integrated 
genomic and proteomic analysis provides a 
compelling evidence that ERBB2 is an important 
therapeutic target under neo-adjuvant or adjuvant 
settings for treatment of patients with GBC. In 
addition, this study also shows that presence of K-



 
 
 
 
 

 
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ras mutations may preclude patients with GBC to 
respond to anti-EGFR treatment, similar as to how 
a clinical algorithm is often used to opt for anti-
EGFR treatment in patients with colorectal cancer 
(Iyer et al. 2018). Most recently, in a collaborative 
study between investigators from India and the US, 
PIM1 kinase has been demonstrated to promote 
cell proliferation of gallbladder cancer cells via 
inhibition of PRAS40 (Subbannayya et al., 2019). It 
is important to note that several small molecule 
inhibitors of PIM1 kinase have been developed, 
some of which are currently being tested for their 
efficacy in clinical trials for several types of cancers. 
Currently, some of the authors of this article (CPG, 
ALL and MKS) are investigating the role of Myc-
Aurora kinase signaling in the gallbladder cancer 
and as to how this signaling axis can be 
therapeutically targeted (unpublished work). 

Conclusion 

Recent studies employ large cohorts of GBC to 
emphasize the differences between the various 
GBC subtypes at a basic genetic/molecular level. 
Functional studies and clinical trials only including 
patients with specific subtypes of GBC, and 
stratifying them based on their genetic drivers, 
were the next steps to accomplishing these goals. 

For instance, use of inhibitors of the EGFR were to 
be examined only in the patients with GBC 
carrying mutations/genetic amplifications of the 
EGFR gene. Constructing a precise approach to 
genomic medicine for the treatment of this disease 
uses a combination of biological studies, drug 
research and development, technological 
advancements in genomics as well as research that 
determines health outcomes. Based on the 
genomic information as summarized in Table 2, 
several clinical trials are currently underway to 
examine the efficacy of targeted therapies in 
patients. This, in the long run, is going to help 
build a staircase to developing a targeted therapy 
for GBC. Based on the outcome of these studies, 
we should be able to offer effective therapies 
based on their genomic profiles to the patients 
with deadly GBC in the future. 

 

 

 

 

 

 

Table 2: Ongoing clinical trials using targeted therapies in gallbladder cancer. 

Drug(s) Target In combination 
with 

Phase Clinical Trials 
Identifier 

Cetuximab, Gefitinib 
Trastuzumab, 

Lapatinib, Everolimus 
Sorafenib 
Crizotinib 

EGFR, HER2, HER2, EGFR mTOR 
RAF kinase, VEGFR-2/PDGFR-beta 

ALK and ROS1 

Gemcitabine, 
Oxaliplatin (GEM 

OX) 
II NCT02836847 

Sorafenib 
RAF kinase, VEGFR- 

2/PDGFR-beta 
Gemcitabine and 

Cisplatin P II NCT00919061 

Guadecitabine 
Durvalumab 

DNA methyltransferase 
Programmed cell death-1 ligand 1 

- I NCT03257761 



 
 
 
 
 

 
www.companyofscientists.com/index.php/chd                   e19                                              Cancer Health Disparities 

RESEARCH 

Sorafenib 
RAF kinase, VEGFR- 

2/PDGFR-beta 

Gemcitabine, 
Oxaliplatin 
(GEMOX) 

II NCT00955721 

Regorafenib Receptor tyrosine kinase, VEGFR2 - II NCT02053376 

Pazopanib 
Receptor tyrosine kinases including 

VEGFR-1, VEGFR 2, VEGFR-3, 
PDGFR-α and –β, FGFR -1 and -3 

Gemcitabine II NCT01855724 

Ramucirumab VEGFR2  II NCT02520141 

Lapatinib HER2, EGFR - II NCT00107536 

Durvalumab, 
Tremelimumab 

Programmed cell death-1 ligand 1 
CTLA-4 

Gemcitabine, 
Cisplatin 

II NCT03473574 

Pembrolizumab Programmed cell death protein 1 
Gemcitabine, 

Cisplatin II NCT03260712 

 
In patients with advanced stages of unresectable 
GBC, treatment options are limited because of no 
so well defined molecular targets. Complete 
surgical resection remains the only curative 
modality to treat patients with GBC, offering 
benefit only for patients with localized disease. As 
evident from the past studies on GBC, most 
research efforts have focused on identifying 
genetic mutations, which did not provide much 
insight into signaling molecules that could be 
targeted therapeutically. GBC still continues to 
have a poor prognosis worldwide. It is widely 
accepted that cancer is primarily a signaling 
disease. Therefore, one strategy to identify 
functional therapeutic targets in GBC is to get 
deeper insights into major signaling mechanisms 
underlying initiation and progression of 
gallbladder cancer, which will have potential to be 
used in clinical decision-making as well as 
conducive to the design of personalized cancer 
treatments. Given the ethnic, geographic, and 
gender disparities associated with gallbladder 
cancer, lack of funding and resources to initiate 
prospective studies using cutting edge omics 
studies to decipher signaling events underlying 
gallbladder cancer is a major impediment. In 

recent years, with identification of few molecular 
targets in GBC such as ERBB2, ERBB3, and 
MYC amplifications, it is becoming apparent that 
development of effective therapies will be 
benefitted by focusing on personalized therapy 
such as by identification and targeting of genetic 
mutations specific to an individual patient. 
Investigation of epidermal growth factor receptors 
(EGFR) such as Cetuximab and Erlotinib in a clinical 
trial for GBC patients’ cohorts will in turn help 
decide whether or not such targeted therapeutics 
can be recommended as standard-of-care. 

Acknowledgements 
Authors acknowledge support by Sylvester 
Comprehensive Cancer Center startup funds (to 
MKS) as well as departmental support by NIH 
grants R01 CA124723 and R01 CA170946. Special 
thanks to Anthony Ferrantella, a gastrointestinal 
surgeon at University of Miami Miller School of 
Medicine, for reading the manuscript and 
correcting it where ever needed. 

Conflict of interest 
The authors declare no conflict of interest. 



 
 
 
 
 

 
www.companyofscientists.com/index.php/chd                   e20                                              Cancer Health Disparities 

RESEARCH 

Authors’ contributions 
This invited review article was conceptualized and 
designed by BM and MKS. Review of literature and 
other data was collected by CPG, ALL, and MKS. 
Article was written, reviewed, and edited by MRK 
and MKS. 

 

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www.companyofscientists.com/index.php/chd                   e24                                              Cancer Health Disparities 

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