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Genetic abberations in gallbladder cancer
Surgical Oncology 21 (2012) 37e43
Contents lists available at ScienceDirect
Surgical Oncology journal homepage: www.elsevier.com/locate/suronc
Review
Genetic abberations in gallbladder cancer Sanjeev K. Maurya, Mallika Tewari, Raghvendra R. Mishra, Hari S. Shukla* Department of Surgical Oncology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, UP, India
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 6 September 2010
Gallbladder carcinoma (GBC) is the most common type of biliary tract carcinoma and the third commonest digestive tract malignancy in our region. Studies available in literature do not clearly define the molecular genetic mechanisms involved in the pathogenesis of GBC. Most of these studies are limited to protein expression analysis by immunohistochemistry and western blotting, and only a few have been done on mRNA (messenger RNA) and mutation analysis. This review aims to critically analyze all the available evidence on genetic aberrations in gallbladder carcinoma. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Genetic abnormality Oncogenes Tumor suppressor genes Gallbladder carcinoma
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37 Models of gallbladder carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Tumor suppressor genes (TSG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Oncogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 K-ras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 HER-2/neu (erbB-2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 EGFR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Cell cycle regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Cell adhesion molecules (CAM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Hormone receptor status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Angiogenesis factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Telomerase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Loss of heterozygosity (LOH) and micro satellite instability (MSI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Current clinical trails . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41 Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41 Authorship statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41
Introduction Gallbladder carcinoma (GBC) is the most common type of biliary tract carcinoma and the third commonest digestive tract malignancy in our region. GBC arises in the setting of chronic
* Corresponding author. 7 SKG Colony, Lanka, Varanasi-221005, UP, India. Tel.: þ91 9415 224400; fax: þ91 542 2368856. E-mail address: [email protected] (H.S. Shukla). 0960-7404/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.suronc.2010.09.003
inflammation and the commonest source is cholesterol gallstones (in more than 75% patients) [1,2]. Other causes of chronic inflammation include primary sclerosing cholangitis, ulcerative colitis, liver flukes, chronic Salmonella typhi and paratyphi infections, and Helicobacter infection. Many other factors have also been identified such as ingestion of certain chemicals, exposures through water pollution, heavy metals and radiation exposure. Only a small fraction of GBC are associated with hereditary syndromes like Gardner syndrome, neurofibromatosis type I and hereditary non-polyposis colon cancer [3e5].
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Studies available in literature do not clearly define the molecular genetic mechanisms involved in pathogenesis of GBC and this is an area of intense active research. Over 1281 genes are found mutated in GBC [6]. The early molecular changes include p53 mutation, cyclooxygenase type 2 (COX-2) overexpression, mitochondrial DNA mutations and abnormal hyper-methylation of promoters of various tumor suppressor genes (TSG) [7]. In the process of development of dysplasia further molecular changes take place, such as allelic loss at several chromosomal sites (in particular 3 and 4), insertion or deletion polymorphism in lipoprotein receptor associated protein (LRPAP1) gene [8], and overexpression of p16 gene [9]. Late changes in carcinoma insitu stage include inactivation of the fragile histidine triad (FHIT) [10] and cyclin-dependent kinase inhibitor 2A (CDKN) tumor suppressor genes [7] and loss at additional chromosomal regions particularly on the chromosomes 9, 18, and 22 [8] (Fig. 1). This review aims to give a bird’s eye view of the available evidence on genetic aberrations in gallbladder cancer and the likely possibilities of its knowledge in diagnosis and targeted therapy of GBC. Models of gallbladder carcinogenesis
significantly higher proportions (58.3e100%) than in adenoma (10e20%) and chronic cholecystitis [17,18] and proportions also fluctuate in different histological types of GBC e.g. 100% in intestinal type, 66% in papillary type, 83% in adenosquamous carcinomas and in 66% of giant cell cancers [19]. Mutations in p53 exons 5,6,7,8 and 9 are also reported in GBC [20]. These include missense mutation at codons 134 (Phe to Leu) and 146 (Try to Arg), transversions and mutations at CpG sites (33.3%) [21]. Elevated level of p53 and mutation in exons are crucial for gallbladder carcinogenesis. LOH in fragile histidine triad (FHIT) candidate TSG locus at 3p14.2 [22,23], methylation of the promoter [10,24], frame shift mutation [25] and elevated expression [26] are common in GBC. The retinoblastoma (Rb) gene mutation [27], down regulation [17], micro satellite instability and LOH [28] in GBC is also reported [29]. TSGs are guards which prohibit uncontrolled cell proliferation during cell division and cell differentiation. Any alteration in TSGs modifies the natural function of and leads to development in GBC. An increasing body of evidence has demonstrated that mutation and aberrant expression of p53 may contribute to GBC but its relation to prognosis of GBC remains to be determined.
Two models of gallbladder carcinogenesis have been proposed
Oncogenes
(1) Dysplasia-Carcinoma Sequence
The genes that cause the transformation of normal cells, into cancerous cells are called oncogenes. Oncogenes are generally mutated forms of normal cellular genes (proto-oncogenes). The following studies have been done in gallbladder carcinoma.
This involves a step-wise progression from a normal gallbladder to the development of metaplasia and dysplasia and finally carcinoma insitu and invasive GBC over the years [10e15]. (Fig. 2)
K-ras
(2) Adenoma-Carcinoma Sequence At the molecular genetic level it now appears that both the models correspond to two distinct and independent biological pathways or two different lineages [16]. Tumor suppressor genes (TSG) Allele loss of tumor suppressor genes (TSGs) and loss of heterozygosity (LOH) at polymorphic loci flanking TSGs are recognized as hallmarks of cancers. Abnormal p53 protein in GBC is found in
The genes of ras family (H-ras, N-ras and K-ras) are oncogenes that mutate frequently in human cancer, especially in tumors of the biliary tract and pancreas. Different k-ras mutations in GBC have been reported on codon 12 [30]. The k-ras mutation has been reported in second nucleotide of codon 12 and 9 attributed to a G to A and G to C transition [18,31]. GBC associated with an anomalous pancreatic obiliary duct junction (APBDJ) have a high k-ras mutation rate in 50e83% of cases and it may be used as a diagnostic marker for GBC in this category of patients. Over all the K-ras mutation rates vary from 10% to 67% in different studies [30,32].
Fig. 1. Molecular profile of Gallbladder Cancer.
S.K. Maurya et al. / Surgical Oncology 21 (2012) 37e43
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Fig. 2. Progression of Gallbladder Cancer with sequential genetic aberration.
However, no correlation has yet been proved with stage, histology and survival of GBC patients [32]. HER-2/neu (erbB-2) The HER-2/neu (erbB-2) overexpression has been reported to occur in 15.7%e63.6% cases of GBC [33,34] and in 70% cases in cholangiocarcinoma [35]. c-myc oncoprotein expression is observed in less than 10% of primary GBC and its expression is significantly elevated in metastatic carcinomas [36]. HER-2/neu gene amplification is observed in nearly 70% cases. Overexpression of HER-2/Neu is important for carcinogenesis of bile duct and gallbladder cancers, and detection of the above abnormalities in bile is helpful for early diagnosis [37e39]. Kawamoto et al. [40] even stated that HER-2 gene amplification in GBC similar to that found in breast cancer. We also found 25% HER-2/neu expression in GBC and 0% in CIS and gallstone disease. EGFR The expression of epidermal growth factor (EGF) and its receptor EGFR were found higher in GBC (63.4%, 70.7%) and dysplasia (71.4%, 85.7%) than in simple hyperplasia (15.4%, 27%) and normal gallbladder (0%) [41,38]. Somatic mutations of the EGFR tyrosine kinase domain are found in approximately 15% of the biliary tract and GBC [42]. Several trials have been undertaken in the past investigating chemotherapy for advanced biliary cancers, including cancer of the gallbladder. Several studies targeting the EGFR pathway have been undertaken [43e45]. Overall, the data indicate that HER-2/neu and specific monoclonal antibodies or tyrosine kinase inhibitors may have a therapeutic role in the treatment of GBC in future. Apoptosis Apoptosis or programmed cell death is a normal component of the development and health of multicellular organisms. The average apoptotic index in GBC is found to be 0.68 þ/0.91%. It increases with progression of the neoplastic lesions of the gallbladder epithelium. The extent of apoptosis has been found to be higher in grade II and III GBC than grade I tumor or epithelial dysplasia [46]. Caspases are enzymes known as proteases, which play essential roles in apoptosis and inflammation. Caspase 3 overexpression is reported in 95% and caspase 6 and 8 expression each in 77% cases of GBC [47]. The Bcl-2 proto-oncogene encodes an inner mitochondrial membrane protein that blocks programmed cell death.
Bcl-2 overexpression is observed in 34.7% of the GBC cases [48]. Although apoptosis of cancer cells is desirable and many established and novel cancer therapies cause apoptosis both in vitro and in vivo, we still do not know how important apoptosis is in GBC. Cell cycle regulators Very limited information is available about the cell cycle regulators affecting GBC. There are a large number of cyclinproteins involved in this process. A study reported cyclin E expression in 33.3% of patients with adenocarcinoma of the gallbladder compared to 12.5% patients with adenoma [49]. Another study found that 67% adenomas and 41% adenocarcinomas demonstrated cyclin D1 overexpression, whereas all normal epithelia and adenomyoma were negative for cyclin D1 [50e52]. The expression rates of abnormal cyclin D1 were observed in 68.3% GBC and 57.1% gallbladder adenoma and these were significantly higher than those found in chronic cholecystitis (7.1%) [27]. Specimens with cyclin D1 overexpression showed a high incidence of lymphatic permeation, venous permeation, and lymph node metastasis [53]. The cyclin-dependent kinase inhibitor p27Kip1 is an important regulator of the cell cycle. Low p27Kip1 expression was observed in 65% [54] and 43% [51] in GBC in different studies. The genes that are affected can encompass any component of the various signaling pathways or their final targets in the cell cycle machinery. Mutations in these genes render cell cycle transition autonomous, less dependent on external signaling and insensitive to internal control circuits. Cell adhesion molecules (CAM) Cell Adhesion Molecules (CAMs) are proteins located on the cell surface involved with the binding with other cells or with the extracellular matrix (ECM). The CD44 protein is a cell surface glycoprotein involved in cellecell interactions, cell adhesion and migration. CD44 is a cell surface glycoprotein and expressed in all normal gallbladder cells. No difference in expression has been observed between mucosa from control samples and mucosa adjacent to the tumor or superficial or deep tumor areas. Nearly 50% of subserous GBC shows an abnormal CD44 expression [55]. Normal gallbladder mucosa shows strong, membranous staining for CD44 but not for CD44v3 or CD44v6. A study reported that in GBC, CD44 stained as strongly as in normal mucosa, but immunoreactivity for CD44v3 and CD44v6 found significantly enhanced [56]. Above study established that CD44 variant overexpression in patients with GBC is closely linked with histologic dedifferentiation rather than clinicopathologic factors, including prognosis.
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Mucins are high molecular weight glycoproteins that provide a protective layer on epithelial surfaces and are involved in cellecell interactions, signaling, and metastasis. A high-level expression of MUC1 [57,58] is correlated with poor survival in GBC. The cellular distribution of MUC1 is heterogeneous among carcinomas of different depths of invasion [59,60]. Chang et al. [61] characterized the expression profile of MUC1, significantly higher than MUC5AC and MUC6 in GBC. In normal gallbladder mucosa expression of MUC1 is absent whereas MUC5AC and MUC6 is much diffused [62]. MUC1 expression and distribution pattern suggests that it could be used as a marker of malignant transformation of gallbladder epithelium and its depolarized expression could also be a marker of invasion [63]. Erythrocyte complement receptor 1 (CR1) can act as a negative regulator of the complement cascade, mediates immune adherence and phagocytosis and inhibits both the classic and alternative pathways. The number and adhering activity of CR1 is found significantly lower in patients with GBC compared to patients with chronic cholecystitis, cholelithiasis and healthy controls [64]. Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) induces apoptosis in immune cells bearing the RCAS1 receptor. High expression of RCAS1 (55%) [65e67] and 96% [68] observed in GBC and expression is possibly related to promoting tumor progression but its actual role in GBC still remains under investigation. Hormone receptor status Sex steroids play an important part in the functioning of normal gallbladder, formation of gallstones and possibly in the pathogenesis of gallbladder cancer [69]. Sumi et al. (2004) [70] immunohistochemically evaluated expression of estrogen receptor (ER) in 26 gallbladder adenocarcinoma specimens and 11 non-cancerous regions using ER a and ER b antibodies. ER a was expressed in most specimens (both cancerous and normal areas), whereas expression of ER b was significantly different between cancerous and noncancerous regions [71]. Thus several reports on estrogen receptors in gallbladder tissue support the hypothesis that estrogen influences the development of gallstones and GBC formation. Angiogenesis factors In most cancers the growth of malignant tumors is dependent upon the development of new blood vessels (angiogenesis) to provide sufficient blood flow. Thrombospondin-1 (TSP1) expression was found absent in normal cells, even T1 cancers, but 74.5% of T2 and T3 GBC and those with lymph node metastasis showed high expression. Venous involvement is frequently found in the TSP1positive cases of GBC [72]. So stromal TSP1 and stromal TSP1 immunoreactivity seems to be a good predictor of vascular involvement and lymph node metastasis. An isoform of cycloxygenase, COX-2, is induced by mitogens, cytokines, and growth factors, and it produces prostaglandins involved in inflammation and cell growth. Zhi et al. [73] reported 71.9% COX-2 immunoactivity with vascular endothelial growth factor (VEGF) expression 54.7% in GBC. This indicates that the tumor neovascularization induced by VEGF may be one of the several effects of COX-2 responsible for poor prognosis of human GBC. Kawamoto et al. [59] showed intense COX-2 staining in large percentages of hyperplastic lesions (65%), pT(2) carcinoma specimens (76%) and pT(3) and pT(4) carcinoma specimens (64%) compared to the percentages of normal epithelia and other pathological lesions (0e25%) in gallbladder. Overexpression of COX-2 in GBC has been reported and correlated with mean survival and
tumor progression [66,73e75]. In GBC tissues, VEGF mRNA levels directly correlated with its protein level. VEGF expression in neoplasms is significantly correlated with neovascularization but not to prognostic parameters. These findings suggest that VEGF is commonly expressed in human GBC and may contribute to neovascularization during carcinogenesis. Interestingly, we investigated the expression of tumor endothelial marker (TEM) 8 by immunohistochemistry and RTPCR. The tumor stage dependent TEM8 overexpression was found [our unpublished data]. Telomerase activity Telomeres are essential elements of all eukaryotic chromosomes, protecting them from exonuclease degradation and end-toend chromosomal fusions. The occasional presence of telomerase catalytic subunit (hTERT) in normal and regenerative gallbladder mucosa and bile [74] reflects their regenerative capacity. Nevertheless, significantly higher hTERT indices in low and high grade dysplastic epithelia and in gallbladder adenocarcinomas are probably a consequence of hTERT re-expression, an early event in the multistep process of gallbladder carcinogenesis [76]. The combination of cytology and hTERT mRNA analysis of gallbladder bile might be helpful for the pre-operative diagnosis of GBC. Loss of heterozygosity (LOH) and micro satellite instability (MSI) It is still unclear whether LOH plays any significant role in gallbladder carcinogenesis, but recent studies have found a high incidence of LOH at several chromosomes in GBC. GBC demonstrated a high frequency of LOH (81%) and mutation (67%), both abnormalities indicating gene inactivation in 52% of patients [77]. LOH on chromosomes 1p, 3p, 5p, 8p, 9p, 9q, 13q, 16q, and 17p has been frequently found in GBC. LOH on 3p (100%), 8p (100%), 9p (88%), 13q, 16q, 17p, 22q and 22q11.2 has been detected in preneoplastic lesions and in the early phase of GBC [78]. Two studies have reported high incidence of LOH at 1p36 (53%), 9p21 (38%), 13q14 (56%), 16q24 (61%), and 17p13 (42%) in GBC [79,18]. Nakayama et al. [80] demonstrated high frequency of allelic loss in APBDJ associated GBC on 2p (81.8%), 4p (50%), 4q (50%), 8q (60%), 9q (50%), 10p (50%), 14p (60%), 14q (50%), 16p (60%), 19p (50%), 21p (50%) and Xp (66.6%). The highest deletion regions are on chromosomes 2p24 (3/3, 100%), 14q22 (3/4, 75%) and 21q22 (3/4, 75%). Yoshida et al. [23] used micro satellite markers for the determination of MSI. LOH was found at p53 gene in 60% informative cases, at Deleted in Colorectal Cancer (DCC) gene in 45%, at Adenomatous Polyposis Coli (APC) I gene in 33%, at Retinoblastoma (RB) gene in 13%, and at NM23-H1 in 7%. Microsatellites are repeating units in DNA of 1e5 base pairs that are abundant and repeated several times in eukaryotic genomes and their presence indicates genomic instability. The literature suggests a minor role of MSI in gallbladder carcinogenesis e.g. only about 10% in Chilean patients [16]. However, in our yet unpublished data, we found high MSI (MSI-H) in 12/30 (40%) patients of GBC. Current clinical trails A few phase I and phase II trials are currently underway in the GBC and biliary tract carcinoma using targeted therapy such as VEGF neutralizing antibody (Bevacizumab), tyrosine kinase inhibitors (Erlotinib, Sorefinib), Proteosome inhibitor (Botrezomib), Cetuximab, etc. [81].
S.K. Maurya et al. / Surgical Oncology 21 (2012) 37e43
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Table 1 Summary of genetic alterations in Gallbladder Cancer. Gene
Major Normal Function
Alteration in GBC
TP53
It binds DNA, which in turn stimulates to produce p21 that interacts with a cell divisionstimulating protein (cdk2) The FHIT encodes a cytoplasmic Mr 16,800 protein with diadenosine triphosphate (Ap3A) hydrolase activity. Inactivation of Rb results in unscheduled cell proliferation, apoptosis and widespread developmental defects, leading to embryonic death A number of these proto-oncogenes especially c-abl, pim-l, c-mos, int-1, c-raf, c-ras, c-fos, c-jun and c-myc are expressed in a stage-specific pattern during spermatogenesis in the testis suggesting that they may have an important function in germ-cell development. It is a potential cell surface receptor of the tyrosine kinase gene family c-Src functions at the hub of a vast array of signal transduction cascades that influence cellular proliferation, differentiation, motility, and survival. Small mitogenic protein that is thought to be involved in mechanisms such as normal cell growth, oncogenesis, and wound healing
Up regulation, point, frame shift mutation. Down regulation, MSI and LOH
FHIT Rb Ras-family
HER-2/neu Src EGF and EGFR CA 125 Caspases BCL-2 Cyclin E Cyclin D Kinase inhibitor CD44 MUC CR1 RCAS1 Ep-CAM ER PR EGFR
It have essential roles in apoptosis and inflammation play a role in neoplasia by inhibiting tumor cell apoptosis It is a protein that is involved in the regulation of cellular replication Specifically regulates the transition from the G1 phase to the S phase of the cell cycle Kinase inhibitor p27Kip1 is an important regulator of the cell cycle cell surface glycoprotein involved in cellecell interactions, cell adhesion and migration cellecell interactions, signaling, and metastasis negative regulator of the complement cascade, mediate immune adherence and phagocytosis and inhibit both the classic and alternative pathways Induces apoptosis in immune cells bearing the RCAS1 receptor mediated adhesions did not resolve any junction-type contacts, such as the adherens junctions mediated by cadherins Hormone receptors Hormone receptors Growth factor receptor
Conclusions Despite intensive research, the etiopathogenesis of GBC remains obscure and the dismal disease continues to be rampant in certain pockets across the globe. Several factors play a contributory role in the gallbladder carcinogenesis and all eventually result in various aberrations at the molecular level. The accumulation of these genetic changes leads to disruption in cell cycle regulation and results in continuous cell proliferation and carcinogenesis (Table 1). Hence, it is prudent to further understand the deranged molecular pathways in GBC to eventually help in early diagnosis; development of prognostic and predictive markers; cancer specific therapies such as replacing mutant TSG, suppressing activated proto-oncogenes, antiangiogenic strategies, proteasome inhibitors, histone deacetylase inhibitors, etc and to identifying the high risk individuals resulting in primary prevention of this dreaded disease. Conflict of interest statement The authors have no conflict of interests. Acknowledgments The authors would like to thank jointly to the Centre of Experimental Medicine & Surgery, Institute of Medical Sciences, Banaras Hindu University and University Grant Commission, New Delhi, for the fellowship grant to conduct study related to this review. Authorship statement Guarantor of the integrity of the study: Hari S. Shukla Study concepts: Hari S. Shukla Study design: Hari S. Shukla Definition of intellectual content: Mallika Tewari, Sanjeev K. Maurya Literature research: Mallika Tewari, Sanjeev K. Maurya
Down regulation, MSI and LOH Mutation
Overexpression Hyper-active Up regulation Up regulation Overexpression Overexpression Overexpression Overexpression Down expression Overexpression Overexpression Low activity Overexpress Overexpress Overexpress Overexpress Overexpress, Mutation
Clinical studies: None Experimental studies: None Data acquisition: Mallika Tewari, Sanjeev K. Maurya Data analysis: None Statistical analysis: None Manuscript preparation: Hari S. Shukla, Mallika Tewari, Sanjeev K. Maurya Manuscript editing:Hari S. Shukla, Mallika Tewari, RR Mishra
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Contents lists available at ScienceDirect
Surgical Oncology journal homepage: www.elsevier.com/locate/suronc
Review
Genetic abberations in gallbladder cancer Sanjeev K. Maurya, Mallika Tewari, Raghvendra R. Mishra, Hari S. Shukla* Department of Surgical Oncology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, UP, India
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 6 September 2010
Gallbladder carcinoma (GBC) is the most common type of biliary tract carcinoma and the third commonest digestive tract malignancy in our region. Studies available in literature do not clearly define the molecular genetic mechanisms involved in the pathogenesis of GBC. Most of these studies are limited to protein expression analysis by immunohistochemistry and western blotting, and only a few have been done on mRNA (messenger RNA) and mutation analysis. This review aims to critically analyze all the available evidence on genetic aberrations in gallbladder carcinoma. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Genetic abnormality Oncogenes Tumor suppressor genes Gallbladder carcinoma
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37 Models of gallbladder carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Tumor suppressor genes (TSG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Oncogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 K-ras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 HER-2/neu (erbB-2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 EGFR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Cell cycle regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Cell adhesion molecules (CAM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Hormone receptor status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Angiogenesis factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Telomerase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Loss of heterozygosity (LOH) and micro satellite instability (MSI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Current clinical trails . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41 Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41 Authorship statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41
Introduction Gallbladder carcinoma (GBC) is the most common type of biliary tract carcinoma and the third commonest digestive tract malignancy in our region. GBC arises in the setting of chronic
* Corresponding author. 7 SKG Colony, Lanka, Varanasi-221005, UP, India. Tel.: þ91 9415 224400; fax: þ91 542 2368856. E-mail address: [email protected] (H.S. Shukla). 0960-7404/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.suronc.2010.09.003
inflammation and the commonest source is cholesterol gallstones (in more than 75% patients) [1,2]. Other causes of chronic inflammation include primary sclerosing cholangitis, ulcerative colitis, liver flukes, chronic Salmonella typhi and paratyphi infections, and Helicobacter infection. Many other factors have also been identified such as ingestion of certain chemicals, exposures through water pollution, heavy metals and radiation exposure. Only a small fraction of GBC are associated with hereditary syndromes like Gardner syndrome, neurofibromatosis type I and hereditary non-polyposis colon cancer [3e5].
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Studies available in literature do not clearly define the molecular genetic mechanisms involved in pathogenesis of GBC and this is an area of intense active research. Over 1281 genes are found mutated in GBC [6]. The early molecular changes include p53 mutation, cyclooxygenase type 2 (COX-2) overexpression, mitochondrial DNA mutations and abnormal hyper-methylation of promoters of various tumor suppressor genes (TSG) [7]. In the process of development of dysplasia further molecular changes take place, such as allelic loss at several chromosomal sites (in particular 3 and 4), insertion or deletion polymorphism in lipoprotein receptor associated protein (LRPAP1) gene [8], and overexpression of p16 gene [9]. Late changes in carcinoma insitu stage include inactivation of the fragile histidine triad (FHIT) [10] and cyclin-dependent kinase inhibitor 2A (CDKN) tumor suppressor genes [7] and loss at additional chromosomal regions particularly on the chromosomes 9, 18, and 22 [8] (Fig. 1). This review aims to give a bird’s eye view of the available evidence on genetic aberrations in gallbladder cancer and the likely possibilities of its knowledge in diagnosis and targeted therapy of GBC. Models of gallbladder carcinogenesis
significantly higher proportions (58.3e100%) than in adenoma (10e20%) and chronic cholecystitis [17,18] and proportions also fluctuate in different histological types of GBC e.g. 100% in intestinal type, 66% in papillary type, 83% in adenosquamous carcinomas and in 66% of giant cell cancers [19]. Mutations in p53 exons 5,6,7,8 and 9 are also reported in GBC [20]. These include missense mutation at codons 134 (Phe to Leu) and 146 (Try to Arg), transversions and mutations at CpG sites (33.3%) [21]. Elevated level of p53 and mutation in exons are crucial for gallbladder carcinogenesis. LOH in fragile histidine triad (FHIT) candidate TSG locus at 3p14.2 [22,23], methylation of the promoter [10,24], frame shift mutation [25] and elevated expression [26] are common in GBC. The retinoblastoma (Rb) gene mutation [27], down regulation [17], micro satellite instability and LOH [28] in GBC is also reported [29]. TSGs are guards which prohibit uncontrolled cell proliferation during cell division and cell differentiation. Any alteration in TSGs modifies the natural function of and leads to development in GBC. An increasing body of evidence has demonstrated that mutation and aberrant expression of p53 may contribute to GBC but its relation to prognosis of GBC remains to be determined.
Two models of gallbladder carcinogenesis have been proposed
Oncogenes
(1) Dysplasia-Carcinoma Sequence
The genes that cause the transformation of normal cells, into cancerous cells are called oncogenes. Oncogenes are generally mutated forms of normal cellular genes (proto-oncogenes). The following studies have been done in gallbladder carcinoma.
This involves a step-wise progression from a normal gallbladder to the development of metaplasia and dysplasia and finally carcinoma insitu and invasive GBC over the years [10e15]. (Fig. 2)
K-ras
(2) Adenoma-Carcinoma Sequence At the molecular genetic level it now appears that both the models correspond to two distinct and independent biological pathways or two different lineages [16]. Tumor suppressor genes (TSG) Allele loss of tumor suppressor genes (TSGs) and loss of heterozygosity (LOH) at polymorphic loci flanking TSGs are recognized as hallmarks of cancers. Abnormal p53 protein in GBC is found in
The genes of ras family (H-ras, N-ras and K-ras) are oncogenes that mutate frequently in human cancer, especially in tumors of the biliary tract and pancreas. Different k-ras mutations in GBC have been reported on codon 12 [30]. The k-ras mutation has been reported in second nucleotide of codon 12 and 9 attributed to a G to A and G to C transition [18,31]. GBC associated with an anomalous pancreatic obiliary duct junction (APBDJ) have a high k-ras mutation rate in 50e83% of cases and it may be used as a diagnostic marker for GBC in this category of patients. Over all the K-ras mutation rates vary from 10% to 67% in different studies [30,32].
Fig. 1. Molecular profile of Gallbladder Cancer.
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Fig. 2. Progression of Gallbladder Cancer with sequential genetic aberration.
However, no correlation has yet been proved with stage, histology and survival of GBC patients [32]. HER-2/neu (erbB-2) The HER-2/neu (erbB-2) overexpression has been reported to occur in 15.7%e63.6% cases of GBC [33,34] and in 70% cases in cholangiocarcinoma [35]. c-myc oncoprotein expression is observed in less than 10% of primary GBC and its expression is significantly elevated in metastatic carcinomas [36]. HER-2/neu gene amplification is observed in nearly 70% cases. Overexpression of HER-2/Neu is important for carcinogenesis of bile duct and gallbladder cancers, and detection of the above abnormalities in bile is helpful for early diagnosis [37e39]. Kawamoto et al. [40] even stated that HER-2 gene amplification in GBC similar to that found in breast cancer. We also found 25% HER-2/neu expression in GBC and 0% in CIS and gallstone disease. EGFR The expression of epidermal growth factor (EGF) and its receptor EGFR were found higher in GBC (63.4%, 70.7%) and dysplasia (71.4%, 85.7%) than in simple hyperplasia (15.4%, 27%) and normal gallbladder (0%) [41,38]. Somatic mutations of the EGFR tyrosine kinase domain are found in approximately 15% of the biliary tract and GBC [42]. Several trials have been undertaken in the past investigating chemotherapy for advanced biliary cancers, including cancer of the gallbladder. Several studies targeting the EGFR pathway have been undertaken [43e45]. Overall, the data indicate that HER-2/neu and specific monoclonal antibodies or tyrosine kinase inhibitors may have a therapeutic role in the treatment of GBC in future. Apoptosis Apoptosis or programmed cell death is a normal component of the development and health of multicellular organisms. The average apoptotic index in GBC is found to be 0.68 þ/0.91%. It increases with progression of the neoplastic lesions of the gallbladder epithelium. The extent of apoptosis has been found to be higher in grade II and III GBC than grade I tumor or epithelial dysplasia [46]. Caspases are enzymes known as proteases, which play essential roles in apoptosis and inflammation. Caspase 3 overexpression is reported in 95% and caspase 6 and 8 expression each in 77% cases of GBC [47]. The Bcl-2 proto-oncogene encodes an inner mitochondrial membrane protein that blocks programmed cell death.
Bcl-2 overexpression is observed in 34.7% of the GBC cases [48]. Although apoptosis of cancer cells is desirable and many established and novel cancer therapies cause apoptosis both in vitro and in vivo, we still do not know how important apoptosis is in GBC. Cell cycle regulators Very limited information is available about the cell cycle regulators affecting GBC. There are a large number of cyclinproteins involved in this process. A study reported cyclin E expression in 33.3% of patients with adenocarcinoma of the gallbladder compared to 12.5% patients with adenoma [49]. Another study found that 67% adenomas and 41% adenocarcinomas demonstrated cyclin D1 overexpression, whereas all normal epithelia and adenomyoma were negative for cyclin D1 [50e52]. The expression rates of abnormal cyclin D1 were observed in 68.3% GBC and 57.1% gallbladder adenoma and these were significantly higher than those found in chronic cholecystitis (7.1%) [27]. Specimens with cyclin D1 overexpression showed a high incidence of lymphatic permeation, venous permeation, and lymph node metastasis [53]. The cyclin-dependent kinase inhibitor p27Kip1 is an important regulator of the cell cycle. Low p27Kip1 expression was observed in 65% [54] and 43% [51] in GBC in different studies. The genes that are affected can encompass any component of the various signaling pathways or their final targets in the cell cycle machinery. Mutations in these genes render cell cycle transition autonomous, less dependent on external signaling and insensitive to internal control circuits. Cell adhesion molecules (CAM) Cell Adhesion Molecules (CAMs) are proteins located on the cell surface involved with the binding with other cells or with the extracellular matrix (ECM). The CD44 protein is a cell surface glycoprotein involved in cellecell interactions, cell adhesion and migration. CD44 is a cell surface glycoprotein and expressed in all normal gallbladder cells. No difference in expression has been observed between mucosa from control samples and mucosa adjacent to the tumor or superficial or deep tumor areas. Nearly 50% of subserous GBC shows an abnormal CD44 expression [55]. Normal gallbladder mucosa shows strong, membranous staining for CD44 but not for CD44v3 or CD44v6. A study reported that in GBC, CD44 stained as strongly as in normal mucosa, but immunoreactivity for CD44v3 and CD44v6 found significantly enhanced [56]. Above study established that CD44 variant overexpression in patients with GBC is closely linked with histologic dedifferentiation rather than clinicopathologic factors, including prognosis.
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Mucins are high molecular weight glycoproteins that provide a protective layer on epithelial surfaces and are involved in cellecell interactions, signaling, and metastasis. A high-level expression of MUC1 [57,58] is correlated with poor survival in GBC. The cellular distribution of MUC1 is heterogeneous among carcinomas of different depths of invasion [59,60]. Chang et al. [61] characterized the expression profile of MUC1, significantly higher than MUC5AC and MUC6 in GBC. In normal gallbladder mucosa expression of MUC1 is absent whereas MUC5AC and MUC6 is much diffused [62]. MUC1 expression and distribution pattern suggests that it could be used as a marker of malignant transformation of gallbladder epithelium and its depolarized expression could also be a marker of invasion [63]. Erythrocyte complement receptor 1 (CR1) can act as a negative regulator of the complement cascade, mediates immune adherence and phagocytosis and inhibits both the classic and alternative pathways. The number and adhering activity of CR1 is found significantly lower in patients with GBC compared to patients with chronic cholecystitis, cholelithiasis and healthy controls [64]. Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) induces apoptosis in immune cells bearing the RCAS1 receptor. High expression of RCAS1 (55%) [65e67] and 96% [68] observed in GBC and expression is possibly related to promoting tumor progression but its actual role in GBC still remains under investigation. Hormone receptor status Sex steroids play an important part in the functioning of normal gallbladder, formation of gallstones and possibly in the pathogenesis of gallbladder cancer [69]. Sumi et al. (2004) [70] immunohistochemically evaluated expression of estrogen receptor (ER) in 26 gallbladder adenocarcinoma specimens and 11 non-cancerous regions using ER a and ER b antibodies. ER a was expressed in most specimens (both cancerous and normal areas), whereas expression of ER b was significantly different between cancerous and noncancerous regions [71]. Thus several reports on estrogen receptors in gallbladder tissue support the hypothesis that estrogen influences the development of gallstones and GBC formation. Angiogenesis factors In most cancers the growth of malignant tumors is dependent upon the development of new blood vessels (angiogenesis) to provide sufficient blood flow. Thrombospondin-1 (TSP1) expression was found absent in normal cells, even T1 cancers, but 74.5% of T2 and T3 GBC and those with lymph node metastasis showed high expression. Venous involvement is frequently found in the TSP1positive cases of GBC [72]. So stromal TSP1 and stromal TSP1 immunoreactivity seems to be a good predictor of vascular involvement and lymph node metastasis. An isoform of cycloxygenase, COX-2, is induced by mitogens, cytokines, and growth factors, and it produces prostaglandins involved in inflammation and cell growth. Zhi et al. [73] reported 71.9% COX-2 immunoactivity with vascular endothelial growth factor (VEGF) expression 54.7% in GBC. This indicates that the tumor neovascularization induced by VEGF may be one of the several effects of COX-2 responsible for poor prognosis of human GBC. Kawamoto et al. [59] showed intense COX-2 staining in large percentages of hyperplastic lesions (65%), pT(2) carcinoma specimens (76%) and pT(3) and pT(4) carcinoma specimens (64%) compared to the percentages of normal epithelia and other pathological lesions (0e25%) in gallbladder. Overexpression of COX-2 in GBC has been reported and correlated with mean survival and
tumor progression [66,73e75]. In GBC tissues, VEGF mRNA levels directly correlated with its protein level. VEGF expression in neoplasms is significantly correlated with neovascularization but not to prognostic parameters. These findings suggest that VEGF is commonly expressed in human GBC and may contribute to neovascularization during carcinogenesis. Interestingly, we investigated the expression of tumor endothelial marker (TEM) 8 by immunohistochemistry and RTPCR. The tumor stage dependent TEM8 overexpression was found [our unpublished data]. Telomerase activity Telomeres are essential elements of all eukaryotic chromosomes, protecting them from exonuclease degradation and end-toend chromosomal fusions. The occasional presence of telomerase catalytic subunit (hTERT) in normal and regenerative gallbladder mucosa and bile [74] reflects their regenerative capacity. Nevertheless, significantly higher hTERT indices in low and high grade dysplastic epithelia and in gallbladder adenocarcinomas are probably a consequence of hTERT re-expression, an early event in the multistep process of gallbladder carcinogenesis [76]. The combination of cytology and hTERT mRNA analysis of gallbladder bile might be helpful for the pre-operative diagnosis of GBC. Loss of heterozygosity (LOH) and micro satellite instability (MSI) It is still unclear whether LOH plays any significant role in gallbladder carcinogenesis, but recent studies have found a high incidence of LOH at several chromosomes in GBC. GBC demonstrated a high frequency of LOH (81%) and mutation (67%), both abnormalities indicating gene inactivation in 52% of patients [77]. LOH on chromosomes 1p, 3p, 5p, 8p, 9p, 9q, 13q, 16q, and 17p has been frequently found in GBC. LOH on 3p (100%), 8p (100%), 9p (88%), 13q, 16q, 17p, 22q and 22q11.2 has been detected in preneoplastic lesions and in the early phase of GBC [78]. Two studies have reported high incidence of LOH at 1p36 (53%), 9p21 (38%), 13q14 (56%), 16q24 (61%), and 17p13 (42%) in GBC [79,18]. Nakayama et al. [80] demonstrated high frequency of allelic loss in APBDJ associated GBC on 2p (81.8%), 4p (50%), 4q (50%), 8q (60%), 9q (50%), 10p (50%), 14p (60%), 14q (50%), 16p (60%), 19p (50%), 21p (50%) and Xp (66.6%). The highest deletion regions are on chromosomes 2p24 (3/3, 100%), 14q22 (3/4, 75%) and 21q22 (3/4, 75%). Yoshida et al. [23] used micro satellite markers for the determination of MSI. LOH was found at p53 gene in 60% informative cases, at Deleted in Colorectal Cancer (DCC) gene in 45%, at Adenomatous Polyposis Coli (APC) I gene in 33%, at Retinoblastoma (RB) gene in 13%, and at NM23-H1 in 7%. Microsatellites are repeating units in DNA of 1e5 base pairs that are abundant and repeated several times in eukaryotic genomes and their presence indicates genomic instability. The literature suggests a minor role of MSI in gallbladder carcinogenesis e.g. only about 10% in Chilean patients [16]. However, in our yet unpublished data, we found high MSI (MSI-H) in 12/30 (40%) patients of GBC. Current clinical trails A few phase I and phase II trials are currently underway in the GBC and biliary tract carcinoma using targeted therapy such as VEGF neutralizing antibody (Bevacizumab), tyrosine kinase inhibitors (Erlotinib, Sorefinib), Proteosome inhibitor (Botrezomib), Cetuximab, etc. [81].
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Table 1 Summary of genetic alterations in Gallbladder Cancer. Gene
Major Normal Function
Alteration in GBC
TP53
It binds DNA, which in turn stimulates to produce p21 that interacts with a cell divisionstimulating protein (cdk2) The FHIT encodes a cytoplasmic Mr 16,800 protein with diadenosine triphosphate (Ap3A) hydrolase activity. Inactivation of Rb results in unscheduled cell proliferation, apoptosis and widespread developmental defects, leading to embryonic death A number of these proto-oncogenes especially c-abl, pim-l, c-mos, int-1, c-raf, c-ras, c-fos, c-jun and c-myc are expressed in a stage-specific pattern during spermatogenesis in the testis suggesting that they may have an important function in germ-cell development. It is a potential cell surface receptor of the tyrosine kinase gene family c-Src functions at the hub of a vast array of signal transduction cascades that influence cellular proliferation, differentiation, motility, and survival. Small mitogenic protein that is thought to be involved in mechanisms such as normal cell growth, oncogenesis, and wound healing
Up regulation, point, frame shift mutation. Down regulation, MSI and LOH
FHIT Rb Ras-family
HER-2/neu Src EGF and EGFR CA 125 Caspases BCL-2 Cyclin E Cyclin D Kinase inhibitor CD44 MUC CR1 RCAS1 Ep-CAM ER PR EGFR
It have essential roles in apoptosis and inflammation play a role in neoplasia by inhibiting tumor cell apoptosis It is a protein that is involved in the regulation of cellular replication Specifically regulates the transition from the G1 phase to the S phase of the cell cycle Kinase inhibitor p27Kip1 is an important regulator of the cell cycle cell surface glycoprotein involved in cellecell interactions, cell adhesion and migration cellecell interactions, signaling, and metastasis negative regulator of the complement cascade, mediate immune adherence and phagocytosis and inhibit both the classic and alternative pathways Induces apoptosis in immune cells bearing the RCAS1 receptor mediated adhesions did not resolve any junction-type contacts, such as the adherens junctions mediated by cadherins Hormone receptors Hormone receptors Growth factor receptor
Conclusions Despite intensive research, the etiopathogenesis of GBC remains obscure and the dismal disease continues to be rampant in certain pockets across the globe. Several factors play a contributory role in the gallbladder carcinogenesis and all eventually result in various aberrations at the molecular level. The accumulation of these genetic changes leads to disruption in cell cycle regulation and results in continuous cell proliferation and carcinogenesis (Table 1). Hence, it is prudent to further understand the deranged molecular pathways in GBC to eventually help in early diagnosis; development of prognostic and predictive markers; cancer specific therapies such as replacing mutant TSG, suppressing activated proto-oncogenes, antiangiogenic strategies, proteasome inhibitors, histone deacetylase inhibitors, etc and to identifying the high risk individuals resulting in primary prevention of this dreaded disease. Conflict of interest statement The authors have no conflict of interests. Acknowledgments The authors would like to thank jointly to the Centre of Experimental Medicine & Surgery, Institute of Medical Sciences, Banaras Hindu University and University Grant Commission, New Delhi, for the fellowship grant to conduct study related to this review. Authorship statement Guarantor of the integrity of the study: Hari S. Shukla Study concepts: Hari S. Shukla Study design: Hari S. Shukla Definition of intellectual content: Mallika Tewari, Sanjeev K. Maurya Literature research: Mallika Tewari, Sanjeev K. Maurya
Down regulation, MSI and LOH Mutation
Overexpression Hyper-active Up regulation Up regulation Overexpression Overexpression Overexpression Overexpression Down expression Overexpression Overexpression Low activity Overexpress Overexpress Overexpress Overexpress Overexpress, Mutation
Clinical studies: None Experimental studies: None Data acquisition: Mallika Tewari, Sanjeev K. Maurya Data analysis: None Statistical analysis: None Manuscript preparation: Hari S. Shukla, Mallika Tewari, Sanjeev K. Maurya Manuscript editing:Hari S. Shukla, Mallika Tewari, RR Mishra
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