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Originally published as Ann Surg Oncol Early Release 10.1245/ASO.2003.02.014 on September 15, 2003

Annals of Surgical Oncology 10:882-889 (2003)
© 2003 Society of Surgical Oncology
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ORIGINAL ARTICLES

Progression of Gene Hypermethylation in Gallstone Disease Leading to Gallbladder Cancer

Michael G. House, MD, Ignacio I. Wistuba, MD, Pedram Argani, MD, MingZhou Guo, MD, PhD, Richard D. Schulick, MD, Ralph H. Hruban, MD, James G. Herman, MD and Anirban Maitra, MD

From the Departments of Surgery (MGH, RDS) and Pathology (PA, RHH, AM), The Johns Hopkins Medical Institutions, Baltimore, Maryland; Department of Oncology (MG, RHH, JGH), The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland; and Department of Pathology (IIW), Pontificia Universidad Catolica de Chile, Santiago, Chile.

Correspondence: Address correspondence and reprint requests to: Michael G. House, MD, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Tumor Biology, 1650 Orleans Street, Room 543, Baltimore, MD 21231-1000; Fax: 410-614-9884; E-mail: mgh{at}jhu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Background: Aberrant methylation of tumor-suppressor genes is associated with a loss of gene function that can afford selective growth advantages to sporadic neoplastic cells arising during gallbladder inflammation.

Methods: Fifty-four gallbladder neoplasms were selected from tumor banks in the United States and Chile. Each of the neoplasms was subjected to methylation-specific polymerase chain reaction to detect promoter methylation associated with six candidate tumor-suppressor genes (p16, APC, methylguanine methyltransferase, hMLH1, retinoic acid receptor beta-2, and p73) implicated in multiple human cancer types.

Results: Aberrant methylation of any of the six candidate tumor-suppressor genes was detected in 72% of the gallbladder neoplasms, 28% of the cases of chronic cholecystitis, and in only 1 of the 15 normal gallbladder controls. The four most commonly methylated genes in the gallbladder cancers were p16 (56%), p73 (28%), APC (27%), and hMLH1 (14%). Significant differences in gene methylation were discovered between US gallbladder cancers and those from Chile, where gallbladder cancer is one of the leading causes of cancer-related deaths. APC methylation was present in 42% of the US cases but in only 14% of the Chilean tumors (P = .028). p73 methylation was common among the Chilean cancers (40%) compared with those from the United States (13%; P = .034).

Conclusions: The acquisition of hypermethylation at multiple tumor-suppressor gene-promoter sites may contribute to tumor formation and progression within the chronically inflamed gallbladder. The apparent differences in methylation patterns among the Chilean and US gallbladder cases may indicate a unique biology associated with this cancer in different parts of the world.

Key Words: Methylation • Gallbladder • Cancer • Tumor-suppressor genes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Adenocarcinoma of the gallbladder is a relatively uncommon cancer in the United States, with an estimated incidence of 5,000 new cases per year (2.5 per 100,000 population).1 In contrast, the incidence of gallbladder cancer in other parts of the world, namely, Chile, Bolivia, and parts of Latin America, can be as much as 5-fold greater.2,3 Despite this marked geographical and ethnic variation in the distribution of gallbladder cancer, implying a combination of genetic and environmental etiologies, only certain predisposing factors have been linked to the development of gallbladder cancer worldwide, particularly, chronic cholelithiasis, obesity, Salmonella typhi infection, and anomalous pancreaticobiliary anatomy.4–7 Recently, a number of genetic alterations have been characterized for gallbladder cancer, including mutations of the K-ras, p53, O6-MGMT (methylguanine methyltransferase), and p16 genes, as well as overexpression of ErbB-2 and vascular endothelial growth factor.8–19 Clearly, further characterization of the molecular events specific to gallbladder cancer and not chronic cholecystitis or normal gallbladder epithelium will help to identify novel molecular targets that can be exploited for diagnostic and therapeutic strategies.

Similar to the multistep genetic alterations that lead to tumorigenesis in a variety of human tissues, sequential epigenetic processes have also been linked to human cancer formation. One of these epigenetic alterations, DNA methylation, is associated with a loss of gene expression in solid tumors.20 Typically, dense regions of CpG dinucleotides, termed CpG islands, within tumor-suppressor gene promoters are protected from methylation in normal mammalian cells, and transcription is unaffected. During carcinogenesis, however, aberrant promoter-region methylation accumulates in tumor-suppressor genes, resulting in blocked transcription.21,22

Aberrant hypermethylation of tumor-suppressor genes has been observed as a common mechanism leading to transcriptional silencing in a variety of human cancers. For example, renal cell carcinoma features inactivation of the VHL tumor-suppressor gene due to promoter hypermethylation in the absence of genetic mutations.23 Likewise, hMLH1 hypermethylation and subsequent loss of expression of this mismatch-repair gene leads to microsatellite instability in colorectal adenocarcinomas.24 To date, few studies have investigated the epigenetic events associated with biliary tract cancers, although frequent methylation of the p16, APC, O6-MGMT, and RASSF genes has been reported in cholangiocarcinoma.25–29 Studies on methylation abnormalities in gallbladder cancers, especially comparing the genetic differences between endemic and nonendemic geographical regions, have not been performed previously.

Epigenetic changes, although frequently associated with human malignancy, have also been discovered in chronically inflamed tissue states. Methylation of the p16/INK4a gene is present in chronic pancreatitis and sclerosing cholangitis and confers an increased risk for subsequent malignancy.25,30,31 Similarly, ulcerative colitis demonstrates methylation of multiple tumor-suppressor gene loci. Although hypermethylation of p16 and E-cadherin/CDH-1 appears to some degree in ulcerative colitis, the presence of dysplasia and, certainly, neoplasia, in the setting of chronic colitis is associated with significantly higher levels of methylation.32,33 The implications of promoter methylation in the setting of inflammation are 2-fold: First, gene inactivation, secondary to specific promoter hypermethylation, plays an important role in the progression of chronic inflammation to cancer. Second, from a diagnostic viewpoint, the presence of aberrantly methylated DNA sequences does not equate with the diagnosis of cancer, especially in a circumstance in which a malignancy arises in the setting of a chronically inflamed epithelium.

Unfortunately, gallbladder cancer carries a poor prognosis because of its inherent aggressive biology and advanced stage at the time of diagnosis.34 Increased understanding of the molecular mechanisms that underlie the development and progression of gallbladder cancer will afford opportunities to improve the outcome of this lethal disease. We sought to characterize the early epigenetic events associated with inflammatory and neoplastic diseases of the gallbladder as a way to identify potential molecular targets for early detection, therapy, and prognosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Human Tissue Samples
Formalin-fixed, paraffin-embedded tumor samples were obtained from 54 resected adenocarcinomas of the gallbladder that presented to the departments of pathology at Johns Hopkins Hospital (n = 24) and Pontificia Universidad Catolica de Chile (n = 30) between 1981 and 2001. Permission for cataloging and processing all samples for this study was obtained in accordance with the guidelines set forth by the institutions’ investigational review boards and joint committees for clinical research. Tissue samples of chronic cholecystitis (n = 18) and normal gallbladder (n = 15) were also obtained. All specimens were sectioned sequentially at a thickness of 10 µm each. Characterization of the gallbladder samples, including tumor stage and the presence of cholelithiasis or cholesterolosis, was performed by two separate gastrointestinal pathologists examining the gross specimens and histological tissue sections. Table 1 indicates the pertinent characteristics and associated patient demographics for the samples included in this study. Overall, 70% of the gallbladder tumors and 89% of the cases of chronic cholecystitis were derived from female patients. All specimens of chronic cholecystitis and normal gallbladder mucosa were procured at Pontificia Universidad Catolica de Chile. The mean duration of symptoms for the cases of chronic cholecystitis was 5 years (range, 2–7 years). The normal gallbladder specimens were obtained from patients undergoing exploratory celiotomy for traumatic, infectious, or neoplastic diseases outside the gallbladder (i.e., hydatid disease of the liver, hepatic hemangioma, rhabdomyosarcoma, gastric adenocarcinoma, and so on).


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  		TABLE 1. Tissue Samples
 
DNA Preparation
Two sequential 10-µm sections from each specimen of gallbladder adenocarcinoma, chronic cholecystitis, and normal gallbladder mucosa were deparaffinized with xylene and digested overnight at 50°C with proteinase K buffered in 1% sodium dodecyl sulfate (pH 8). DNA was isolated by phenol-chloroform extraction and ethanol precipitation.35 Approximately 4 to 5 µg of DNA was partially purified from the two 10-µm tissue sections.

Methylation-Specific Polymerase Chain Reaction
The methylation status of the promoter regions for six tumor-suppressor genes (p16/INK4a, APC, O6-MGMT, hMLH1, RAR-ß-2 [retinoic acid receptor], and p73) was determined by the method of methylation-specific polymerase chain reaction (MSP), further modified as a nested two-step approach to increase the sensitivity of detecting allelic hypermethylation at targeted sequences and to facilitate the examination of multiple gene loci.35–37 Initially, 1 µg of tissue DNA was bisulfite-treated according to previously described protocols to render unmethylated cytosines to uracil.37 The bisulfite-treated DNA was column-purified over Wizard cleanup resin (Promega, Madison, WI) and ethanol-precipitated. Step 1 of the nested MSP was performed with primer sets (sense and antisense) for four individual genes in each reaction. Step 1 primers flanked the CpG-rich promoter regions of the respective targeted genes. Hence, these primers did not discriminate between methylated and unmethylated nucleotides after bisulfite treatment. Polymerase chain reaction products of step 1 were diluted 1/1000 and subjected to the second step of MSP, which incorporated one set of primers for each gene (labeled as unmethylated or methylated) and was designed to recognize bisulfite-induced modifications of unmethylated cytosines. All of the primer sequences and polymerase chain reaction conditions for this nested-MSP approach have been published previously.38 Both steps of the nested MSP used a 25-µL reaction volume, .5 µL of Jump Start Red Taq DNA polymerase (Sigma, St. Louis, MO), and 1 µL of DNA template. DNA isolated from normal peripheral lymphocytes from healthy individuals served as a negative methylation control. Human placental DNA was treated in vitro with SssI methyltransferase (NEB, Beverly, MA) to create completely methylated DNA at all CpG-rich regions. In vitro methylated DNA served as the positive methylation control. MSP products were analyzed on 6% polyacrylamide gel electrophoresis.

Statistical Analysis
Fisher’s exact probability test was used to analyze the univariate differences in the methylation status of tumor-suppressor genes among adenocarcinomas from the United States and Chile (Stata Release 6; Stata Corp., College Station, TX). Methylation differences between stages of gallbladder cancer and chronic cholecystitis were also examined. A two-tailed P value of <.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Hypermethylation in Gallbladder Cancer
Using a nested-MSP approach to study epigenetic changes involved in gallbladder cancer, we examined promoter methylation associated with six selected tumor-suppressor genes established to have a role in human neoplasia. Figure 1 displays representative MSP results for the promoter regions of two tumor-suppressor genes, APC and p73, in gallbladder specimens of normal mucosa, chronic cholecystitis, and adenocarcinoma. Although the presence of unmethylated alleles among the gallbladder cancers possessing promoter methylation likely results from contamination of the neoplastic tissues with surrounding normal mucosa, it is possible that hemimethylation affects one allele within a totally clonal population of cells in the neoplasm and would be insufficient to silence gene expression. Also, the presence of distinct clonal subpopulations within a single neoplasm (one with specific methylation and one without a methylation selection) could account for this mixed allelic methylation status.



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  		FIG. 1. The amplified products after step 2 of the nested methylation-specific polymerase chain reaction for the tumor-suppressor genes APC (A) and p73 (B). Lanes marked "U" and "M" contain products derived from unmethylated and methylated alleles, respectively. Tissue samples are labeled according to histopathologic diagnosis: normal gallbladder (NGB), chronic cholecystitis (ChrGB), and adenocarcinoma (GBCa). Three cancers from the United States and Chile are represented. In vitro methylated DNA (IVD) served as the positive control. Product sizes are consistent with primer design. U bands in samples containing methylated alleles indicate contamination by surrounding normal tissue.

 
The promoter methylation results for the APC, p16, MGMT, hMLH1, RAR, and p73 genes are summarized for each of the samples of normal gallbladder, chronic cholecystitis, and adenocarcinoma (Fig. 2). Overall, promoter methylation of any of the 6 genes was found in 72% (39 of 54) of the gallbladder cancers. A total of 71% of the US and 73% of the Chilean cancers demonstrated methylation of at least one tumor-suppressor gene. Individual promoter methylation was found frequently among the gallbladder cancers: p16 (56%), p73 (28%), APC (26%), MGMT (13%), hMLH1 (13%), and RAR (4%). Methylation of more than one tumor-suppressor gene was present in 40% (22 of 54) of the cancers, and no significant differences in multigene methylation were apparent between the US and Chilean cases. Advanced tumor-node-metastasis stage (American Joint Committee on Cancer stages II and III) was associated with a higher frequency of methylation among the three most commonly affected genes: p73, p16, and APC (Fig. 3). Although these differences did not reach statistical significance, they suggest that specific tumor-suppressor gene hypermethylation may be associated with tumor progression in addition to tumor evolution. Multigene methylation, defined as two or more genes, did not differ between stage I (20%) and II (25%) disease, although some studies have suggested that the acquisition of hypermethylation at multiple promoter regions correlates with tumor progression.39–41



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  		FIG. 2. Promoter methylation profile for six tumor-suppressor genes among gallbladder (GB) tissues categorized as normal, chronic cholecystitis, and adenocarcinoma. US cases are represented as samples 1 to 24; Chilean cases are samples 26 to 55. Black grids denote methylated regions. Open squares indicate unmethylated sites.

 


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  		FIG. 3. Methylation differences according to American Joint Committee on Cancer (AJCC) staging for gallbladder cancer. The frequency of methylation (as a percentage of tumors) is shown for three tumor-suppressor gene markers—p73, p16, and APC—among stage I (gray) and stage II (black) cancers. Stage I (IA/B) and stage II (IIA/B) cancers were designated according to the 2002 AJCC guidelines. NS, not significant.

 
Promoter methylation was detected in only 1 of the 15 samples of normal gallbladder and only at the hMLH1 locus. Histological re-examination of this normal specimen revealed no evidence for occult dysplasia or inflammation involving the gallbladder wall. Although scarce clinical information was available for the specimens in this retrospective study, this particular sample was obtained from a 79-year-old individual, the oldest patient sample examined. Age-dependent methylation changes in otherwise normal-appearing tissues need to be considered, as do other environmental factors that predispose human DNA to epigenetic changes. If repetitive gene transcription and cell division potentiate the widespread accumulation of methylation among promoter regions, we might anticipate such age-related methylation changes to occur despite the absence of additive initiators of cell turnover (i.e., inflammation).32

Hypermethylation in Gallstone Disease
Aberrant promoter methylation was found in 28% (5 of 18) of the gallbladder specimens diagnosed histologically as chronic cholecystitis. However, only one case of chronic cholecystitis demonstrated multigene methylation. Of the six loci studied for chronic cholecystitis, methylation was detected for p16, MGMT, and hMLH1. It is interesting to note that loss of expression of these three genes is considered a relatively early event in the adenoma-carcinoma sequence for a variety of human tumors.41 All of the chronic cholecystitis specimens were obtained from individuals who had symptomatic cholelithiasis for at least 5 years. Unlike the gallbladder cancers, two thirds of the chronic cholecystitis cases harbored histological evidence of cholesterolosis; however, cholesterolosis was not a predictor of tumor-suppressor gene methylation. Retrospective histological examination of the chronic cholecystitis cases determined to have aberrant promoter methylation did not reveal the presence of occult low-grade dysplasia or even epithelial hyperplasia.

Figure 4 shows the methylation differences between gallbladder adenocarcinoma and chronic cholecystitis for each of the gene loci. Significant differences were apparent for APC, p16, and p73. The methylation of multiple tumor-suppressor genes in gallbladder adenocarcinoma and the absence of this phenomenon in chronic cholecystitis confirm the previous observation that multigene methylation is a neoplasia-specific event, capable of predisposing sporadic abnormal cells to dysplastic and eventually neoplastic transformation.31,32



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  		FIG. 4. Gene methylation differences between gallbladder cancer and chronic cholecystitis. The frequency of methylation for each tumor-suppressor gene (APC, p16, p73, MGMT, hMLH1, and RAR) and multiple genes (two or more) is compared for adenocarcinoma (black bars) and chronic cholecystitis (gray bars). Promoter methylation involving at least two gene loci differed significantly between the two groups. Significant differences were assumed when P < .05.

 
Geographical Variation in Gallbladder Methylation
As displayed in Table 1, there were no marked differences in age, tumor-node-metastasis stage, or incidence of cholelithiasis in the gallbladder adenocarcinomas from the United States and Chile. Unfortunately, complete clinical data and environmental exposures for each of the patients were not available in several cases. Appreciating the regional differences in the incidence of gallbladder cancer worldwide, we studied the gene methylation patterns among the US and Chilean cancers and discovered significant differences for methylation of the APC and p73 genes (Fig. 5). APC gene methylation was present in 42% of the US tumors but in only 14% of the Chilean cancers (P = .028). Conversely, p73 gene methylation was a frequent finding in the Chilean cancers (40%) compared with the US cancers (13%; P = .034).



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  		FIG. 5. Gene methylation differences between Chilean and US gallbladder cancers. The frequency of methylation for each locus is compared for tumors from Chile (gray bars) and the United States (black bars).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
In this study, we generated one of the first epigenetic profiles of promoter methylation associated with well-defined tumor-suppressor genes in gallbladder adenocarcinoma. Although it is well recognized that carcinogenesis is a multistage process that results from the accumulation of sequential genetic and, likely, epigenetic alterations, the exact histological changes reflecting these events remain undetermined for gallbladder neoplasia. The classic adenoma-carcinoma sequence that is widely accepted for colorectal cancer may not be applicable to gallbladder cancer.41,42 For instance, gallbladder adenomas demonstrate distinct molecular features and genetic abnormalities that are not commonly found among gallbladder carcinomas.43,44 Instead, several histological and genetic studies have linked the potential progression of epithelial dysplasia alone to carcinoma in situ and finally to infiltrative carcinoma within the human gallbladder.8,17,45,46

Worldwide, chronic cholelithiasis and subsequent inflammation are established risk factors for gallbladder cancer.4,6 On the basis of epidemiological data, certain ethnic and regional factors predispose patients to lithogenic bile, but the contribution of genetic factors to gallstone formation remains largely unknown.47 Our finding of specific tumor-suppressor gene methylation in chronic cholecystitis, in the absence of histological dysplasia, supports a postulate that epigenetic changes serve as early molecular events during neoplastic transformation of the gallbladder mucosa. The dramatic accumulation of promoter methylation at the six specific loci in this study and the significant increase in multigene methylation from chronic cholecystitis to adenocarcinoma support a sequential multihit epigenetic process underlying tumor evolution within the chronically inflamed gallbladder. In fact, the appearance of specific tumor-suppressor gene methylation in chronic cholecystitis may mimic or even parallel the early genetic mutations that characterize the transition from mucosal hyperplasia to dysplasia within the gallbladder.17

Although our data did not show a statistically significant difference in methylation between early- and advanced-stage adenocarcinoma, tumor progression seems to be associated with the acquisition of promoter methylation at multiple tumor-suppressor genes, especially those implicated in cell cycle control: APC, p73, and p16.

Despite little variation in the incidence of extrahepatic biliary tumors worldwide, there are tremendous geographical, sex, and ethnic differences for gallbladder cancer.1,3 For example, gallbladder cancer is the leading cause of cancer-related mortality among females in Chile.2 Whether this dramatic variation in gallbladder cancer incidence reflects genetic or environmental factors directly or indirectly, through an increased susceptibility to cholelithiasis, remains to be discovered. Our findings of significant differences in the methylation status of two cell-cycle regulatory genes, APC and p73, may help to characterize the currently inapparent biological variation in gallbladder cancer in different regions. In addition, epigenetic differences associated with early dysplastic and later neoplastic changes may identify potential etiological factors that contribute to the development of gallbladder cancer in the presence or absence of chronic inflammation.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
The methylation of tumor-suppressor genes contributes to the evolution of adenocarcinoma in the chronically inflamed gallbladder. Differences in the methylation profile for gallbladder cancers from the United States and Chile may reflect variations in epigenetic susceptibility to environmental exposures.


   ACKNOWLEDGMENTS
 
The acknowledgments are available online at www.annalssurgicaloncology.org.

Supported by the Niarchos Surgical Research Fund (MGH) of the Stavros S. Niarchos Foundation, National Cancer Institute grant CA-84986 (JGH) of the Early Detection Research Network, and grant 1020960 (IIW) from the Fondo Nacional de Desarrollo Cientifico y Tecnologico, Chile. The authors thank Craig M. Hooker, MPH, from The Johns Hopkins School of Public Health, for his assistance with the statistical analyses.


   FOOTNOTES
 
Presented at the 56th Annual Cancer Symposium of the Society of Surgical Oncology, Los Angeles, California, March 5–9, 2003.

Tumor-suppressor gene methylation contributes to the evolution of adenocarcinoma in the chronically inflamed gallbladder. Methylation profiles for gallbladder cancer show significant geographical variation worldwide.

Received for publication February 20, 2003. Accepted for publication June 6, 2003.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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