Gallbladder disease and progression

Gallbladder carcinoma is the cancer of gallbladder epithelium with low incidence rates compared to other cancer types. Among populations at highest risk, about 1% deaths occur because of gallbladder cancer due to poor survival rates. During 2008, the cases of gallbladder cancer incidence at the global level were 145, 662 with an Age-Standardized Rate (ASR) of 2.0 per 105 person years. Incidence varies geographically with higher rates in certain areas of Latin America (Colombia, Peru, and Ecuador), Japan, and Eastern Europe (Poland, the Czech Republic, Slovakia, Hungary, and the former East Germany). High rates of gallbladder cancer have been noted in Hispanic and American Indian populations in North America. The incidence among women is approximately double than that of men in high-risk populations [1,2].

In India, during 2001, the estimated number of gallbladder cancer was 14,986 and is likely to increase to 23,750 by 2016 as a result of aging and increase in size of the population. The GBC incidence rates have been reported to be highest in women from India (21.5 out of 100,000), Chile (18.1 out of 100,000), Pakistan (13.8 out of 100,000) and Ecuador (12.9 out of 100,000) [3,4]. Thus with ever-increasing incidence and poor prognosis, early diagnosis and treatment of gallbladder carcinoma is essential. Various markers according to the stages of gallbladder cancer are yet to be experimentally documented and thus still require extensive research.

The advancement of cancer up to the stage of gallbladder carcinoma is a lengthy process. There are many risk factors currently proposed to be involved in progression of gallbladder cancer. One of the main causes is chronic cholecystisis (CC) which may lead to various molecular changes (like continuous release of inflammatory factors), therefore resulting in the progression from inflammation to malignancy. The staging towards gallbladder cancer is usually characterized by chronic cholelithiasis with inflammation, metaplasia, dysplasia, carcinoma in situ and then finally invasive carcinoma [1,2]. Based on these observations researchers propose the period for progression from dysplasia to advanced gallbladder carcinoma to be approximately 15 years. Studies examining various tumor-related genes and gene products have shown great promise as possible prognostic and diagnostic markers, which are yet to be explored and well-understood in gallbladder cancers [5].

There are a number of reasons for GBC which include a) mutation b) loss of heterozygosity c) microsatellite instability (MSI) and d) promoter methylation. GBC could be the result of any of these phenomena or due to the cumulative effect of these phenomena [6]. Identifying these alterations and mapping their regulation is of utmost importance for improved prognosis and diagnosis of gallbladder carcinoma. In this study we focus on two such parameters (i.e. MSI and gene methylation) which have shown some promising results in GBC.

Microsatellite instability as a molecular marker of gallbladder disease progression

Microsatellites are the tandemly repeated short sequence motifs that range from 1-6 base pairs. These DNA regions are simple sequences or Short Tandem Repeats (STRs) and classified as mono-, di-, tri-, tetra-, penta or hexanucleotide repeats on the basis of the number of repeating units. A typical microsatellite can be repeated up to 100 times and consists of a single repeat type only or they may consist of more than a single repeat type known as compound microsatellite. A single microsatellite locus can be amplified using specific Polymerase Chain Reaction (PCR) which is possible due to the fact that microsatellites are embedded in single copy DNA. Microsatellite stretches have often been reported to be disrupted by base substitutions which are called imperfect microsatellites or by insertions called interrupted microsatellite [7-9].

Microsatellite Instability (MSI) was initially reported in solid tumors in patients with colon cancer and Hereditary Non-Polyposis Colorectal Cancer (HNPCC) syndrome. MSI is probably caused by an aberrant system of gene repair during DNA replication. When the repair mechanism is altered, it results in mutations in the DNA sequences, further giving rise to either accumulated single nucleotide mutations, or altered length of repetitive microsatellite sequences which ultimately leads to MSI [9,10]

Failure to repair errors in repetitive sequences may be due to mutations in DNA mismatch repair genes (such as MLH1, MSH2 or MSH6) that leads to Microsatellite Instability (MSI) of the tumours. MSI can occur in tumours of many organs, but it has been largely documented in colorectal cancer. Studies have shown that among consecutive colorectal cancers, MSI is usually due to somatic MLH1 gene methylation; constitutional mutations of MLH1, MSH2 or MSH6 are the initial stage of development of MSI in the majority of HNPCC associated cancers [8,11].

Microsatellites form a huge reservoir for polymorphic genetic markers, this is because there are several hundred microsatellites present in eukaryotic genomes, and each of the loci can be subjected to DNA replication slippage. Microsatellite alleles also differ in their number of repeats. Therefore, an efficient and cost-effective method of genotyping can be the PCR amplification of a microsatellite locus which is followed by sizing of the. Moreover, a high sample throughput analysis is nowadays possible due to the availability of capillary sequencers and mass spectroscopy [8,9].

Studies have reported Microsatellite Instability (MSI) in colon cancer, gastric cancer, endometrium cancer, ovarian cancer, hepatobiliary tract cancer, urinary tract cancer, brain cancer and skin cancers [7,8]. Thus MSI cannot be neglected as a potential biomarker and needs to be well understood in every cancer type.

Genetic basis for MSI: Alterations in the genome are normally repaired by the DNA mismatch repair (MMR) system. These proteins correspond to several MutS and MutL homologues from the prokaryotic MutHL DNA repair system, hMLH1 (human MutL Homologue 1), and hMSH2, hMSH3, and hMSH6 (human MutS homologues 2, 3, and 6). They bind the mismatched DNA, excise the desired region and repair the sequence. Due to mutation or promoter methylation of critical MMR genes, normal mismatch repair fails to function. Alterations in the repeat sequences remain unrepaired due to which alleles of different sizes will be formed at the next replication. These different sized alleles form microsatellite instability [7,8,11].

Subsets of sporadic pancreatic, endometrial, prostate, and gastric carcinomas are affected by MSI. The inactivation of genes containing repeat sequences alters the tumor biology. Transforming growth factor beta receptor type II, BAX, and E2F4 are some examples of such kind of vulnerable genes [11].

A possible correlation between aberrant expressions of certain tumor suppressor genes and co-existing MSI during gallbladder cancer, has been reported by certain researchers. Studies were conducted to check co-relation between the p53, K-ras and MSI presence in a population with gallbladder carcinoma in Japan and Hungary. It appears that the p53 mutations and MSI differ in patients with gallbladder carcinoma between two distinct high-incidence areas. But, the results were not as significant as that in case of colorectal cancer where there is a prominent inverse relation between the presence of p53 and MSI [12].

Another study screened the genomic DNA from 21 gallbladder carcinomas using PCR-SSCP followed by sequencing to check for alterations in exon 15 of the B-raf gene where. The results obtained showed abnormal bands. Other features also examined were the association of the mutational status with the presence of K-ras or p53 mutations, MSI and the clinicopathological features (namely age, gender, histological type and stage of the tumor). Sporadic MSI-H colorectal Cancers (CRCs) harbouring hMLH1 methylation but not Lynch syndrome-related CRCs has earlier been shown to be associated with the BRAF V600E mutation. This study on gallbladder carcinoma showed B-raf mutations in 7 of 21 (33%) of the gallbladder carcinomas and were all located at the hot spot codon 599 of exon 15. K-ras mutations at codon 12 had been previously found in 4 of the 23 specimens (25%) and p53 mutations in 5 of 21 cases (24%). BAT26 used as a marker for indicating the presence of MSI. Alterations in BAT26 were not observed in these specimens. Both B-raf and p53 mutations were determined whereas no K-ras and B-raf mutations were identified simultaneously in the same samples [13].

Mismatch repair genes are the responsible genes for hereditary non polyposis colon cancer, and mutation of these genes causes replication error (RER). In several RER-positive colon cancer cell lines, mutations of repetitive sequences of transforming growth factor beta (TGF-β) type H receptor (RII) gene have been reported. Since TGF- β inhibits cell proliferation, loss of response to TGF- β is an important tumor progression step [14]. Two separate studies conducted by Saetta A et. al., evaluated MSI in gallbladder cancer with p53, BAX and TGF- β (RII). In this study with 20 gallbladder carcinomas from Greek patients, alterations in length of the BAT-26 mononucleotide marker (as an indicator of microsatellite instability) were correlated with the presence of p53 and ras mutations, alterations of the bax and TGF-beta RII genes and tumors' clinicopathological features. In another study by the same researchers none of the specimens showed microsatellite instability at the BAT-26 marker. BAT-26 is an indicator of high-level microsatellite instability. Thus, while determining the microsatellite instability status of gallbladder carcinoma, it may not be that sufficient, when used alone. A possible explanation for this may be that gallbladder carcinomas are characterized by low-level instability. They also stated that Bax and TGF-beta RII genes may not also be targets of instability in this type of tumors. They concluded from their study that as far as flat type of cancer is concerned Ras and p53 genes do not appear to cooperate during gallbladder cancer. Further they stated that p53 alterations might have a role in the de novo pathway of gallbladder carcinogenesis [15].

MSI as biomarker for gallbladder cancer: Gallbladder cancer is a highly aggressive disease with a poor prognosis, and has a 5 year survival rate of less than 5%. Therefore, it would be very beneficial to identify the molecular mechanisms responsible for this condition. Even though a lot of studies have been done on the presence of mutated genes for example K-ras and p53 but still more detailed genetic information relating to gallbladder cancer is yet to be done.

One of the ways to investigate the molecular mechanism is to examine the microsatellite instability (MSI). With the increase in the presence of MSI in tumors, the National Cancer Institute Workshop on Microsatellite Instability recommended that tumors be divided into three groups, depending on the frequency of MSI. These three groups are: high frequency MSI (MSI-H), characterized by more than two of the five microsatellite markers showing instability; low-frequency MSI (MSI-L), characterized by only one of the five markers showing instability; and microsatellite stable (MSS), where no genetic instability is seen [9]. There are recommendations currently provided by the NCI (National Cancer Institute) panel for evaluation of MSI-H and MSI-L. These consisted of different MSI markers studied under colon cancer eg. BAT25, BAT26, D2S123, D5S346 and D17S250 etc. These guidelines are thus also useful for checking MSI in gallbladder cancers [16]. In prior studies of gallbladder cancer, there were only few reports with small number of cases and comparison with other tumor types was difficult. Using varied numbers and types of microsatellite markers is important. Using the NCI criteria, Carlos Roa et. al., analyzed a large series of early and advanced gallbladder cancers for MSI. They also examined pre-malignant glands for the same alterations as those seen in the adjacent tumors, and correlated their results with the immunohistochemical (IHC) expression of the MMR system and the clinicopathological findings. The presence of MSI in gallbladder cancers from a high-risk population were demonstrated by these studies. They also detected MSI in intestinal metaplasia and dysplasias adjacent to tumors with MSI-H and, occasionally, adjacent to tumors with MSI-L. These results indicate that in a subset of patients MSI might be participating in the early stages of gallbladder carcinogenesis [11]. In a study by Saetta et al., a group of 37 gallbladder carcinomas was analyzed for alterations in a proposed panel of mononucleotide and dinucleotide markers of MSI. Somatic frameshift mutations at repeated sequences in the coding regions of TGF bRII, Bax, hMSH3, hMSH6 were also examined. The results thus obtained were correlated with the presence of K-ras and p53 alterations, and tumors’ clinicopathological features. In 9 gallbladder carcinomas microsatellite instability and/or LOH were observed. Cases showing microsatellite instability displayed alterations only in dinucleotide markers and were classified as MSI-L carcinomas. Based on the analysis of the above mentioned panel of markers a subset of gallbladder carcinomas is characterized by low-level instability. The study concluded that the pathway of microsatellite instability plays a minor role in the pathogenesis of gallbladder cancer [17]. Using an automated fluorescent DNA sequencer employing four microsatellite markers (p53, APC, DCC, NM23-H1) Yoshida et. al., examined loss of heterozygosity (LOH) in the p53, APC, DCC, RB, and NM23-H1generegions by polymerase chain reaction (PCR)-LOH assay. They also used five additional microsatellite markers for the determination of MSI. An inverse correlation between MSI and LOH in gallbladder carcinoma was observed. They also concluded that there is a lower incidence of lymph node metastasis in gallbladder carcinoma patients with MSI [18]. Microsatellite instability and fragile histidine triad (FHIT) loss have been seen to be involved in gallbladder carcinogenesis. Niraj Kumari et. al., studied the frequency of expression loss of MMR proteins and loss of FHIT expression increased from dysplasia to carcinoma. This suggested that both these aberrancy have a role in pathogenesis of gallbladder cancer. The study also showed the anomaly to be occurring at an early stage in carcinogenesis of gallbladder. Moreover, 53% of gallbladder cancer which express loss of MMR proteins also showed loss of FHIT expression, which was more frequently observed in advanced stage disease. All the results implied that reduced FHIT expression may be correlated with expression loss of MMR proteins on immunohistochemistry [19].

All these studies reveal the biomarker capabilities of MSI in order to detect gallbladder carcinomas. Also the importance of this marker has been evaluated in early stages of gallbladder disease. A study by Yanagisawa et al revealed MSI in 30% of severe chronic cholelithiasis cases suggesting it may play a key role in early stages of gallbladder carcinogenesis [20].

The correlation studies with known tumor suppressor genes as well as oncogenes report some significant data with a requirement for extensive analysis. Figure 1 depicts the overall relation between various molecular changes and MSI that occur during gallbladder cancer.

Figure 1: Molecular mechanisms contributing to Microsatellite instability during gallbladder carcinogenesis

DNA methylation in gallbladder cancer

DNA methylation causes ‘turning off’ of the genes and occurs at the CpG islets of the DNA, residing in the promoters, first exons and 5’ untranslated regions. Tumor Suppressor Genes (TSGs) are said to the ‘Gatekeeper’ that regulate cell proliferation by providing check points in the cell cycle. Aberrant promoter methylation silences TSGs in many cancers. This methylation process of CpG islets is known as hypermethylation [21]. When loss of methylation occurs in case of oncogenes, they get activated. This process is known as hypomethylation. Methylation of TSGs is one of the main causes for GBC. This epigenetic modification is also observed in chronic cholecystisis (CC) during gallbladder disease [21-23]. Figure 2 highlights some important genes showing increasing frequency in methylation status during progression of gallbladder disease.

Figure 2: Increasing methylation patterns seen in candidate genes during progression of gallbladder carcinoma

There are many genes involved in GBC which get methylated at variable frequencies. They are: SHP1, 3-OST-2, CDH13, P15INK4B, CDH1, RUNX3, APC, RIZ1, P16INK4A, HPP1, P73, RAR_2, SOCS-1, DAPK, DcR2, DcR1, HIN1, CHFR, TIMP-3, P57, RASSF1A, CRBP1, SYK, NORE1, DLC1, RARb2, MGMT, FHIT, RASSF1, hMLH1, GSTP1, SEMA3B, BLU, DUTT1. Some of these genes are also methylated during chronic cholelithiasis and thus may be of prognostic significance in gallbladder disease progression. For instance, in one study by Takahsi et al., it was observed that ten genes showed a relatively high frequency of abnormal methylation: SHP1 (80%), 3-OST-2 (72%), CDH13 (44%), P15INK4B (44%), CDH1 (38%), RUNX3 (32%), APC (30%), RIZ1 (26%), P16INK4A (24%), and HPP1 (20%) [22,24]. In the same study, eight genes (P73, RAR_2, SOCS-1, DAPK, DcR2, DcR1, HIN1, and CHFR) showed a low frequency (2-14%) of methylation, and no methylation was detected for the remaining six genes (TIMP-3, P57, RASSF1A, CRBP1, SYK, and NORE1. Similarly, methylation in CC was observed in seven genes: SHP1 (88%), P15INK4B (28%), 3-OST-2 (12%), CDH1 (12%), CDH13 (8%), DcR2 (4%) and P16INK4A (4%) [22,24]. Table 1 summarizes some of the important genes methylated in gallbladder cancers with variable frequencies.

Table 1: Methylation pattern of some of the significant genes in gallbladder cancer.

Important genes involved in methylation and could serve as biomarkers

Since genetic instability in the form of MSI has shown to be present in early stages of gallbladder cancer and is also co related with other genetic markers eg. tumor suppressor genes or oncogenes the utilization of MSI as a diagnostic marker is promising. Further studies in context to the fact that whether MSI positive gallbladder cancer follow the classical pathways of gallbladder cancer. If yes, then in what frequency and if not, then whether it forms a new group for gallbladder cancer progression model. Since, MSI is a permanent change in the DNA it may be inherited and thus there is increased chances of cancer being hereditarily transferred to offspring. More intensive research in this regard is required to gain knowledge about gallbladder cancer and to prepare molecules for early detection and therapeutics.

The methylation of RASSF1A, CDH13, APC and DLC1 can be promising biomarkers for GBC. Since CDH13 is important for cell-cell adhesion, its alteration leads to metastasis which is the leading cause of mortality in case of GBC. It is involved in EMT and MET during metastasis. On the other hand CDH13 is also involved in signal transduction. Patients with APC methylation generally have a poor survival chance and therefore as a therapeutic step, this gene can be targeted with de-methylating agents with specificity. Furthermore APC is involved in other cancers as well. The APC protein is a negative regulator controlling Beta-catenin concentrations and also interacts with E-cadherin, which are involved in cell adhesion. Mutations in the APC gene mainly results in colorectal cancer [37]. DLC1 is said to be a candidate tumor suppressor gene for human liver cancer, as well as for prostate, lung, colorectal, and breast cancers. The main function of DLC1 is its Rho-GAP activity. It enhances activated GTP-bound Rho-GTPases' specifically, RhoA and Cdc42, to convert their GTP into GDP, thus leaving them inactive. Rho-GTPases are members of the Ras superfamily, and are involved in cell adhesion and cell polarity. Thus it finally leads the way to further migration of tumor cells. It regulates apoptosis as well. It is also responsible for negatively regulating angiogenesis. Its loss/inactivation results in upregulation of VEGF through EGFR-MEK-HIF1 signalling pathway. VEGF upregulation is a highly significant event in prostate cancers wherein DLC1 is downregulated. Thus, this strongly suggests that its loss may serve as a “second hit” in inducing angiogenesis in a paracrine fashion during tumor progression [38,39]. Thus an anti-angiogenic agent may upregulate DLC1 and thus help in preventing metastasis and angiogenesis as well as enhance apoptosis. It has been also shown to induce the expression of E-cadherin (regulated by CDH1 gene) in prostrate cancers through Rho pathway [39]. By this way it also contributes to EMT. DLC1 can be a potential diagnostic marker for CC and GBC. Patients with CC can be checked for the methylation of this gene and then prognosis can be determined based on the level of its expression. Then the chances of progression from CC to advanced carcinoma can be estimated by the data. There have been satisfactory results with epigenetic therapy and more therapeutic approaches are being evaluated.