Role In Gallbladder Carcinogenesis Research Articles

Association of haplotype and linkage disequilibrium of PARP1 polymorphisms rs1136410, rs1805405 and rs3219088 with gallbladder cancer.

Previously, we have reported that PARP1 rs1136410 is significantly associated with increased the risk of gallbladder cancer. We aimed to investigate the association of PARP1 rs1805405 and rs3219088 polymorphisms with risk of GBC and also association of the haplotype and combined effect of PARP1 SNPs (rs1805405 G/A, rs3219088 G/T and rs1136410 A/G). We have also investigated the expression profile of PARP1 and its correlation with polymorphisms, clinical parameters and overall survival. PARP1 polymorphisms were genotyped by PCR-RFLP and the expression profile of PARP1 at mRNA level was analyzed by semi-quantitative PCR. Overall survival was analyzed using Kaplan-Meier plot and Cox-regression analysis. Haplotype analysis of the PARP1 polymorphisms revealed that AGG, AAG and GGT haplotypes are significantly associated with decreased risk of GBC, while AAT, AGT, GGG and GAG haplotypes are significantly associated with increased risk of GBC. Patients with T1+T2 and treated with chemotherapy having risk genotypes of rs1805405 have decreased overall survival. Upregulation of PARP1 is significantly associated with longer overall survival in patients with GBC with different clinical parameters. SNPs rs1136410 and rs1805405 genotypes are significantly associated with PARP1 expression. Haplotype analysis suggests that PARP1 may have a potential role in gallbladder carcinogenesis.

Integrative molecular characterisation of gallbladder cancer reveals micro-environment-associated subtypes

Gallbladder cancer (GBC) is the most common type of biliary tract cancer, but the molecular mechanisms involved in gallbladder carcinogenesis remain poorly understood. In this study, we applied integrative genomics approaches to characterise GBC and explore molecular subtypes associated with patient survival. We profiled the mutational landscape of GBC tumours (whole-exome sequencing on 92, targeted sequencing on 98, in total 190 patients). In a subset (n= 45), we interrogated the matched transcriptomes, DNA methylomes, and somatic copy number alterations. We explored molecular subtypes identified through clustering tumours by genes whose expression was associated with survival in 47 tumours and validated subtypes on 34 publicly available GBC cases. Exome analysis revealed TP53 was the most mutated gene. The overall mutation rate was low (median 0.82 Mut/Mb). APOBEC-mediated mutational signatures were more common in tumours with higher mutational burden. Aflatoxin-related signatures tended to be highly clonal (present in ≥50% of cancer cells). Transcriptome-wide survival association analysis revealed a 95-gene signature that stratified all GBC patients into 3 subtypes that suggested an association with overall survival post-resection. The 2 poor-survival subtypes were associated with adverse clinicopathologic features (advanced stage, pN1, pM1), immunosuppressive micro-environments (myeloid-derived suppressor cell accumulation, extensive desmoplasia, hypoxia) and T cell dysfunction, whereas the good-survival subtype showed the opposite features. These data suggest that the tumour micro-environment and immune profiles could play an important role in gallbladder carcinogenesis and should be evaluated in future clinical studies, along with mutational profiles. Gallbladder cancer is highly fatal, and its causes are poorly understood. We evaluated gallbladder tumours to see if there were differences between tumours in genetic information such as DNA and RNA. We found evidence of aflatoxin exposure in these tumours, and immune cells surrounding the tumours were associated with survival.

Abstract A10: Gallbladder cancer disparities in New Mexico: Examining the role of environmental heavy metal exposures as a driver of gallbladder epithelial signaling dysfunction

Abstract Introduction: Gallbladder carcinoma (GBC) is a rare, yet deadly, malignancy of the human gut (5-year survival <8%). NM is a global hotspot for GBC. Native Americans (NA) living in New Mexico (NM) have a 5- to 8-fold higher incidence of GBC compared to Caucasians in NM. The risk factors driving GBC incidence disparities in NM are unknown. We hypothesize that heavy metal exposures such as cadmium (Cd) and uranium (U) may play a key role in gallbladder carcinogenesis in NM due to the abundance of these metals in the local New Mexican environment. Methods and Procedures: We established a retrospective clinical cohort of 24 GBC patients in NM stratified by ethnicity. Archived FFPE samples were sequenced using DNA-seq approaches to identify key mutational patterns of GBC in NM. Key changes were validated via Immunohistochemistry and FISH approaches. Next, we used primary human GB cell lines to measure effects of Cd exposures on GB cell signaling activation. In particular, we focused on effects on Akt, MAPK, and COX-2 signaling. A combination of cell viability, quantitative reactive oxygen species (ROS), and Western blot-based molecular biology assays was used to understand Cd exposure effects on cell signaling dysregulation in a gallbladder epithelial cell model. Results: DNA-seq analysis results show the mutational spectrum of GBC samples in NM for the first time. We observed an elevated incidence of mutations in the Akt and MAPK gene pathways similar to results from earlier studies in GBC patients in China. TP53 was the commonest mutation seen in the NM GBC cohort (36%). Of importance, we observed that approximately 25% of NM GBC patients have a targetable HER2 amplification (n=6/24). In our GB epithelial cell studies, we observed a dose-dependent reduction of cell viability of gallbladder epithelial cells due to Cd exposures. The measured EC50 values of gallbladder primary cells range from 11-18 μM. Importantly, we observed activation of the Akt and MAPK signaling pathways via increased phosphorylation. The ROS activity in GB epithelial cells is elevated due to Cd exposures, which are responsible for activation of Akt and MAPK pathways. We also observed that Cd exposures led to elevation of the COX-2 expression, supporting its role in chronic GB inflammation that is characteristic of gallbladder carcinogenesis. Conclusions: While there is epidemiologic evidence for a role for heavy metal exposures in GBC, the basic research evidence supporting this hypothesis is lacking thus far. Our study shows, for the first time, a definitive role for cadmium exposures in GB cell signaling activation via the Akt and MAPK signaling pathways. Additionally, Cd exposure may also lead to COX-2 activation, which is a second key feature that explains the chronic inflammatory origins of GBC. Thus, our initial studies demonstrate a role for environmental heavy metal exposures (Cd) in gallbladder carcinogenesis for the first time. Future research will seek to strengthen the role of Cd and U in particular in GB carcinogenesis in NM. Citation Format: Rama R. Gullapalli, Trevar S. Caldwell, Megan N. Rivera, Priyanka Sharma. Gallbladder cancer disparities in New Mexico: Examining the role of environmental heavy metal exposures as a driver of gallbladder epithelial signaling dysfunction [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A10.

Genetic mutational analysis of β-catenin gene affecting GSK-3β phosphorylation plays a role in gallbladder carcinogenesis: Results from a case control study

This manuscript has reported different mutations of β-catenin gene in gallbladder cancer patients which affect GSK-3β phosphorylation site. PurposeGallbladder carcinoma (GBC) is a relatively rare and fatal cancer with poor prognosis. The molecular mechanism of gallbladder carcinogenesis is still not clear. Wnt signaling pathway is a highly conserved pathway that regulates proliferation, differentiation, migration, genetic stability, apoptosis, and stem cell renewal. β-catenin plays major role in Wnt signaling and aberrations in β-catenin has found to be involved in several cancers pathogenesis. This study was carried out to document the mutations of β-catenin gene in gallbladder cancer and to evaluate its possible role in gallbladder carcinogenesis. MethodsPCR-SSCP (Single Stranded Conformation Polymorphism) for ctnnb1 was performed in 50 patients each of gallbladder cancer, cholelithiasis and 50 healthy controls. Samples that showed variation in banding pattern were sequenced. ResultsVariation in banding pattern was observed in 9 (18%) samples of GBC, 4 (8%) of cholelithiasis and 2 (4%) of control. Sequencing analysis showed 9 novel mutations of ctnnb1 in exon 3 in 18% of gallbladder cancer (χ2 = 5.778; p < 0.05). Six point mutations, 1 deletion and 1 insertion mutation were found in 9 cases of gallbladder cancer. All point mutations were mis-sense mutation that affected highly conserved serine or threonine region that is important for GSK-3β phosphorylation. ConclusionFindings of the study suggests that high frequency of non synonymous mutations of β-catenin gene (ctnnb1) occurs in patients with gallbladder cancer. As these mutations mainly effect GSK 3β phosphorylation, it may be concluded that this might be an important step in gallbladder carcinogenesis. These β-catenin mutations lead to Wnt pathway activation and appear to have a role in progression from inflammation to cancer in gallbladder.

Analysis of Carcinogenic Heavy Metals in Gallstones and its Role in Gallbladder Carcinogenesis.

Gallstone is a high-risk factor for gallbladder pre-malignancy or malignancy (GB PM-M) but which substances of gallstones definitely assist to turn out in to GB PM-M, remains unclear. This study aimed to find out the presence of carcinogenic heavy metals in gallstones and to explore the aetiopathogenesis of gallbladder pre-malignancy and malignancy. Presence of elements in gallstones was detected by energy dispersive X-ray spectroscopy (EDS) with scanning electron microscopy (SEM) and then level of carcinogenic heavy metals was estimated in gallstones using atomic absorption spectroscopy (AAS). The experiment was carried out in gallstone samples of 46 patients with gallbladder pre-malignant and malignant condition (PM-M group) and 65 sex and age-matched patients with chronic cholecystitis (C-C group). Gallstones were also classified in to three types such as cholesterol stone, mixed stone, and black pigment stone. EDS analysis detected presence of mercury, lead, and cobalt elements in all types of gallstones of both PM-M and C-C groups. AAS analysis revealed significantly higher amount of mercury (p<0.001), lead (p<0.0001), cobalt (p<0.01), and cadmium (p<0.01) in the gallstones of PM-M than C-C groups. The presence of these heavy metals also varied among stone types of both groups. EDS phase analysis showed 'dense deposits' of these metals in gallstones. Presence of significantly higher amount of mercury, lead, cobalt, and cadmium in gallstones may play a pivotal role as risk factors in the development of gallbladder malignancy or pre-malignancy. 'Dense deposits' of these metals in the gallstones which is the first observation, may act as crucial doses of carcinogens.

Expression and clinicopathological significance of antiapoptotis protein survivin in gallbladder cancer

Clinical significance of survivin (antiapoptosis protein) in gallbladder cancer is not yet established. This study was performed to assess the expression pattern of survivin in benign and malignant gallbladder lesions using immunohistochemistry (IHC), and to assess its clinicopathological significance. Prospective study from July 2012 to July 2014 was performed as a part of intramural research project. Tissue samples from resected gallbladder for cholelithiasis (n = 27) and carcinoma gallbladder (n= 24) were evaluated for survivin expression by IHC using a scoring system. Their expression was correlated with different clinicopathological parameters. Fisher's exact test, Student's t-test, and Chi-square test were used as appropriate for data analysis. Kaplan-Meier methods were used to calculate overall and disease-free survival rates among different groups. Two-sided P< 0.05 was considered as significant. Benign group (19 females, age [mean ± standard deviation [SD] 45 ± 14 years) and malignant group (20 females, age [mean ± SD] 48.9 ± 13.4 years) were comparable with respect to menopausal status, presence, size and types of stones. However, survivin expression was significantly higher (66.7%, 95% confidence interval [CI] 24-75) in gallbladder cancer than in cholelithiasis group (33%, CI 46-83), P= 0.025). Its expression did not correlate with gender, age, menopausal status, presence of gallstones or their size, number and type, tumor differentiation, and tumor stage. Significantly higher expression of survivin protein in gallbladder cancer as compared to cholelithiasis group suggests its role in gallbladder carcinogenesis though it may not have prognostic value.

Prognostic significance of survivin in resected gallbladder cancer

BackgroundSurvivin, a novel inhibitor of apoptosis, plays a role in oncogenesis and has been correlated with poor prognosis. We investigated its expression in gallbladder tissues of control, cholelithiasis, and gallbladder cancer (GBC). Survivin expression was correlated with different clinicopathologic parameters including prognosis in patients with GBC. Materials and methodsGallbladder tissue samples were collected from GBC (n = 39), cholelithiasis (n = 30), and control (n = 25). Expression of survivin messenger RNA (mRNA) was evaluated by real time polymerase chain reaction. Protein quantification was done by enzyme-linked immunosorbent assay. ResultsSignificantly higher expression of survivin mRNA was observed in GBC (2.9-fold) and cholelithiasis (1.85-fold) as compared with control (P < 0.0001). In GBC, increased survivin expression (mRNA and protein) was significantly associated with higher tumor stage (stage III versus stage II) (P < 0.0001) and poor tumor differentiation (poor and moderate versus well) (P < 0.0001). No significant correlation was observed with any of the other clinicopathologic factors studied. Increased expression of survivin was associated with shorter survival (median survival 11.5 mo versus 18 mo). ConclusionsDifferential expression of survivin in GBC suggests its possible role in gallbladder carcinogenesis. Its overexpression is associated with poor prognosis. Assessment of survivin might be used to stratify GBC patients for optimal treatment modalities, including targeted therapy.

Role of aberrant PI3K pathway activation in gallbladder tumorigenesis

The PI3K/AKT pathway governs a plethora of cellular processes, including cell growth, proliferation, and metabolism, in response to growth factors and cytokines. By acting as a unique lipid phosphatase converting phosphatidylinositol-3,4,5,- trisphosphate (PIP3) to phosphatidylinositol-4,5,-bisphosphate (PIP2), phosphatase and tensin homolog (PTEN) acts as the major cellular suppressor of PI3K signaling and AKT activation. Recently, PI3K mutations and loss/mutation of PTEN have been characterized in human gallbladder tumors; whether aberrant PTEN/PI3K pathway plays a causal role in gallbladder carcinogenesis, however, remains unknown. Herein we show that in mice, deregulation of PI3K/AKT signaling is sufficient to transform gallbladder epithelial cells and trigger fully penetrant, highly proliferative gallbladder tumors characterized by high levels of phospho-AKT. Histopathologically, these mouse tumors faithfully resemble human adenomatous gallbladder lesions. The identification of PI3K pathway deregulation as both an early event in the neoplastic transformation of the gallbladder epithelium and a main mechanism of tumor growth in Pten heterozygous and Pten mutant mouse models provides a new framework for studying in vivo the efficacy of target therapies directed against the PI3K pathway, as advanced metastatic tumors are often addicted to "trunkular" mutations.

Cyclin D1, Retinoblastoma and p16 Protein Expression in Carcinoma of the Gallbladder

Cancer of the gallbladder is a relatively rare neoplasm with a poor prognosis. The exact mechanisms of its genesis are not known and very little information is available on molecular events leading to labeling this as an orphan cancer. In this prospective case control study we evaluated the expression of p16, pRb and cyclin D1 by immunohistochemistry to study the G1-S cell-cycle check point and its possible role in gallbladder carcinogenesis. A total of 25 patients with gallbladder carcinoma (group I), 25 with cholelithiasis (group II) and 10 normal controls. were enrolled. Cyclin D1 expression was seen in 10 (40%) patients each with carcinoma and cholelithiasis while only in 2 (20%) of the normal gallbladders but differences were not statistically significant (p value=0.488). p16 was expressed in 12% patients of carcinoma of the gallbladder and 28% of cholelithiasis, however this difference was not statistically significant (p value=0.095). Retinoblastoma protein was found to be expressed in 50% of normal gallbladders and 6 (24%) of carcinoma and 8 (32%) of gallstones. The present study failed to demonstrate any conclusive role of cyclin D1/RB/ p16 pathway in carcinoma of the gallbladder. The positive relation observed between tumor metastasis and cyclinD1 expression and p16 with nodal metastasis suggested that higher cyclin D1/p16 expression may act as a predictive biomarker for aggressive behavior of gallbladder malignancies.

CDX2 protein expression in gallbladder carcinoma

ABSTRACT Background and aim: CDX2 is a caudal‐type homeobox gene encoding a transcription factor that has an important role in proliferation and differentiation of the intestinal epithelium. Although CDX2‐mediated intestinal metaplasia and its association with gastric and esophageal carcinoma have been well described, its function in gallbladder carcinoma remains unclear. Methods: CDX2, MUC2 and MUC5AC expression were examined in carcinoma, intestinal metaplasia and dysplasia of the gallbladder by immunohistochemical staining. Results: CDX2 was not observed in the normal epithelium of the gallbladder but was expressed in 71.4% (20/28) of gallbladder carcinomas including 10 of 18 adenocarcinomas, not otherwise specified, 9 papillary adenocarcinomas and 1 intestinal‐type adenocarcinoma. In adenocarcinomas, CDX2 was observed more frequently in well differentiated types than in moderately and poorly differentiated types (P= 0.009). MUC2 and MUC5AC was expressed in 53.6% (15/28) and 71.4% (20/28) of gallbladder carcinomas, respectively. Both CDX2 and MUC2 were observed in 50.0% (14/28) and CDX2 expression was related to MUC2 (P= 0.006) rather than MUC5AC (P= 0.234) in gallbladder carcinomas. CDX2 was also expressed in intestinal metaplasia and dysplasia, which are considered to be premalignant conditions. Conclusions: These results suggest that CDX2 may play an important role in gallbladder carcinogenesis with intestinal differentiation.

Genetic Alterations in Gallbladder Carcinoma

Gallbladder carcinoma is an aggressive cancer associated with a poor prognosis. Unfortunately, the precise molecular mechanisms of development and progression of this highly malignant tumor remain unknown. It is still unclear whether loss of heterozygosity (LOH) plays a significant role in gallbladder carcinogenesis, but recent studies have found a high incidence of LOH at several chromosomes in gallbladder carcinoma. In particular, LOH on chromosomes 1p, 3p, 5p, 8p, 9p, 9q, 13q, 16q, and 17p has been highlighted and LOH on 3p, 9p, 13q, 16q, and 17p has been detected in preneoplastic lesions and in the early phase of gallbladder carcinoma during multistep carcinogenesis. The proto-oncogene, K-ras, is the best known genetic alteration in several human neoplasms, including gallbladder carcinoma. The accumulation of these genetic changes leads to a disruption in cell-cycle regulation and also continuous cell proliferation. We present an overview of K-ras alteration and LOH at several chromosome loci in gallbladder carcinoma. Further studies of the molecular mechanism in gallbladder carcinoma and the delineation of the genetic influence involved should promote our understanding of gallbladder carcinogenesis.

Expression of homeodomain protein CDX2 in gallbladder carcinomas

Caudal-related homeobox protein CDX2 plays an important role in the regulation of cell proliferation and differentiation of the intestinal epithelium. CDX2 is associated with intestinal metaplasia and carcinomas of the stomach, but the role of CDX2 in gallbladder carcinogenesis remains unknown. We analyzed the expression of CDX2 and intestinal apomucin MUC2 in gallbladder cancer cell lines at the mRNA level by the RT-PCR method. We also investigated the expression of CDX2 and MUC2 in 68 primary gallbladder carcinomas by the immunohistochemical staining method and compared the expression of CDX2 with the clinicopathological factors in the gallbladder carcinoma cases. Expression of CDX2 and MUC2 was found in three of four gallbladder cancer cell lines at the mRNA level. In addition, we found that CDX2 was absent in the normal gallbladder epithelium, but the CDX2 protein was expressed in 25 of the 68 (36.8%) gallbladder carcinomas. Interestingly, in the tubular type gallbladder carcinomas, the frequency of CDX2 expression was much higher in the well-differentiated type than the moderately and poorly differentiated types, the difference being statistically significant (P<0.01). CDX2 expression showed a relationship with expression of MUC2 (P<0.04) in the gallbladder carcinomas. CDX2 was expressed in intestinal metaplasia and dysplasia, which are hypothesized to be premalignant conditions. These results imply that CDX2 plays an important role in gallbladder carcinogenesis with intestinal differentiation.

Inactivación del gen CDKN2A (p16) en cáncer de la vesícula biliar

The CDKN2A gene encodes a cyclin dependent kinase inhibitor, p16, which promotes cell cycle arrest. Methylation of the promoter region transcriptionally inactivates the gene. To study the relationship between methylation status of the prometer region of p16 gene, the immunohistochemical expression of p16 and clinical and morphological features of gallbladder carcinoma. We analyzed the methylation status of the promoter region of the CDKN2A gene in gallbladder adenocarcinomas using methylation specific PCR (MSP). We also used microsatellite markers near the CDKN2A gene to detect allelic imbalance (AI) and examined the tumors by immunohistochemistry (IHC) for p16 expression. Of 38 gallbladder adenocarcinomas analyzed by IHC, 11 cases (29%) were negative for p16 protein. Nine (24%) had methylation of the promoter region of the CDKN2A gene. Twenty nine cases were negative for methylation, but four (14%) of these 29 exhibited AI at one or more of the microsatellite markers. CDKN2A promoter methylation was not associated with microsatellite instability (MSI-H). The inactivation of CDKN2A by methylation and/or deletion might play an important role in gallbladder carcinogenesis.

Mutational analysis of BRAF in gallbladder carcinomas in association with K-ras and p53 mutations and microsatellite instability.

Little is known about the genetic changes involved in the pathogenesis of gallbladder cancer. The aim of this study was to examine the presence of mutations in exon 15 of the B-raf gene to investigate its role in gallbladder carcinogenesis. We examined the mutational status in exon 15 of B-raf gene in 21 gallbladder carcinoma specimens and investigated its association with the presence of K-ras and p53 alterations, microsatellite instability and the clinicopathological features of tumors. B-raf mutations were observed in 7 of 21 (33%) gallbladder carcinomas examined, and all were located at the hot spot codon 599 of exon 15. K-ras and B-raf mutations were never in the same specimens. B-raf gene mutations seem to be a quite common event in gallbladder carcinomas, implying that B-raf may play an important role in the pathogenesis of this tumor.

DNA content and gene expression in adenoma-carcinoma sequence of gallbladder carcinogenesis

To study the proliferative change of epithelium and gene expression in adenoma-carcinoma sequence of gallbladder carcinogenesis. Mutations of K-ras gene and APC gene were detected by PCR-RFLP, and the expression of p53 protein was detected by ABC immunohistochemistry. DNA contents were detected by using a TJTY-300 automatic image analyzer. Cell DNA contents and atypia were increased gradually in the adenoma-carcinoma sequence of gallbladder. Aneuploidy in gallbladder adenoma was associated with the size of adenoma. When the diameter of adenoma was more than 1 cm, the aneuploidy was significantly increased (P<0.05). APC gene and K-ras gene mutations were detected in the adenoma-carcinoma pathway. and p53 protein overexpression was not detected in this pathway. Adenoma-carcinoma sequence pathway plays an important role in gallbladder carcinogenesis. Gallbladder adenoma has an obvious tendency to malignant transformation, especially in the patients with adenoma of 1 cm in diameter or above. It has molecular abnormalities different from those de novo carcinomas.

Mutation and altered expression of beta-catenin during gallbladder carcinogenesis.

Gallbladder carcinoma has two main morphologic developmental pathways: a dysplasia-carcinoma sequence and an adenoma-carcinoma sequence. beta-Catenin is a key regulator of the cadherin-mediated cell adhesion system, and altered expression and mutation of beta-catenin have been identified in many human malignancies. To clarify its role in gallbladder carcinogenesis, we investigated mutation and immunohistochemical expression of beta-catenin in adenomas, dysplasias, and carcinomas. We classified adenomas according to the expression of apomucins and cytokeratin and compared the mutational and expression pattern among each type. beta-Catenin mutations were identified in 58% (14 of 24) of the adenomas, and they were absent from all carcinomas (37 cases) and dysplasias (13 cases). Altered expression of beta-catenin, such as nuclear or cytoplasmic expression and loss of membranous expression, was also significantly higher in adenomas than in dysplasias or carcinomas (p <0.001). Of the adenomas, papillary adenomas and tubular adenomas of intestinal type showed infrequent beta-catenin abnormality, which was similar to the carcinomas. The cytoplasmic and nuclear expression of beta-catenin in carcinomas was correlated with less aggressive tumor behavior; in particular, cytoplasmic expression was associated with improved patient outcome (p = 0.028). Gallbladder adenoma may be a heterogeneous entity, and the majority of adenomas are not responsible for carcinoma progression.

Epithelial cell proliferation and gene mutation in the mucosa of gallbladder with pancreaticobiliary malunion and cancer.

The significant association between pancreaticobiliary malunion (PBM), especially undilated-type PBM, and a high risk of gallbladder cancer is known. Reflux and stasis of pancreatic juice induce various epithelial changes in the gallbladder. Recently, epithelial hyperplasia of the gallbladder was shown to be significantly and frequently associated with undilated-type PBM, and it is suggested that the majority of epithelial hyperplasia may exist at birth or be acquired in early childhood, and thereafter present throughout the lives of PBM patients. Cell kinetic studies demonstrated a significant stepwise increase in cellular proliferative activity from normal gallbladder mucosa, through epithelial hyperplasia to cancer. Epithelial hyperplasia with increased proliferative activity may predispose the mucosa to mutational events, thereby increasing cancer risk in PBM patients. K-ras mutations were frequently detected in gallbladder cancer in PBM patients and in epithelial hyperplasia as well. Epithelial hyperplasia is demonstrated to be an important premalignant lesion of gallbladder cancer. A multistep process of carcinogenesis as a consequence of multiple genetic alterations of oncogenes and tumor suppressor genes has been demonstrated in various organs; however, there is limited information on the molecular mechanism in gallbladder carcinogenesis with PBM. Recent findings support the idea that epithelial hyperplasia plays an important role in gallbladder carcinogenesis with PBM and also support the concept that neoplastic development in gallbladder with PBM also evolves through a multistep process associated with hyperproliferation and genetic alterations.

Metallothionein expression in carcinoma of the gallbladder.

Metallothioneins (MT) are sulphur-rich, low molecular-weight, intracellular metal-binding proteins with a possible role in carcinogenesis of some human tumours. Metallothionein expression in gallbladder cancer has not been studied previously. Immunohistochemical expression of metallothionein was studied in 42 gallbladders (27 cases of carcinoma of the gallbladder, eight chronic cholecystitis and seven cases of normal gallbladder). Metallothionein expression was significantly higher in cases with carcinoma of the gallbladder (70.37%) as compared to chronic cholecystitis (25%) and normal gallbladders (0%). There was a trend suggestive of increasing MT expression with increasing histological dedifferentiation of carcinoma of the gallbladder. The increased expression of MT in cases of carcinoma of the gallbladder may represent an increased exposure to heavy metals, which are known carcinogens, and may have a role in gallbladder carcinogenesis. Metallothionein over-expression in carcinoma of the gallbladder may be relevant to the poor prognosis and chemoresistance seen in these cases.

A New Model for Pancreaticobiliary Maljunction without Bile Duct Dilatation: Demonstration of Cell Proliferation in the Gallbladder Epithelium

Patients with pancreaticobiliary maljunction without bile duct dilatation (PBMWBDD) develop gallbladder carcinoma frequently. No models of PBMWBDD exist, and no previous studies have clearly demonstrated changes in the gallbladder relating to carcinogenesis. We, therefore, examined the cell kinetics of the gallbladder epithelium in a new experimental model of PBMWBDD. A cat model was produced by performing choledocho-pancreatic side-to-side ductal anastomosis in nine animals. Five cats, in which the choledocho-pancreatic ducts were exposed only, served as controls. After 6 months, the gallbladders of these cats were removed and stained with anti-proliferating cell nuclear antigen (PCNA) antibody (PC10, Dako). The labeling index (LI) was determined from the percentage of positive nuclei in three microscopic fields. The diameter of the common bile duct was not different between the models and the controls. In the models, the number of PCNA positive cells was significantly increased. The mean (± standard deviation) PCNA LI was 28.1 ± 12.2% in the models and 4.3 ± 1.6% in the controls (P< 0.01). These studies clearly indicate that this model is suitable for studying PBMWBDD and that PBMWBDD has a prominent proliferating effect on the gallbladder epithelium which may play an important role in gallbladder carcinogenesis.