J Hepatobiliary Pancreat Surg (2006) 13:296305 DOI 10.

1007/s00534-005-1058-0

Aberrant promoter hypermethylation in biliary tract carcinoma

Naohiko Kohya, Yasuo Koga, Yoshihiko Kitajima, and Kohji Miyazaki
Department of Surgery, Saga University Faculty of Medicine, 5-1-1 Nabeshima, Saga 849-8501, Japan

Abstract Biliary tract carcinoma is a relatively rare tumor with a poor survival rate. The molecular biological mechanisms underlying the development of biliary tract carcinomas are not well understood. Promoter methylation is an important epigenetic mechanism for suppressing tumor-suppressor gene activity. There is limited information regarding the abnormal methylation of cancer-related genes in biliary tract carcinoma; however, a few insights have been obtained into the role of epigenetic silencing in the progression of biliary tract carcinoma. In this review, we summarize recent data on gene silencing by promoter hypermethylation, and we discuss the implications for biliary tract carcinomas. Key words Promoter methylation Bile duct carcinoma Gallbladder carcinoma

Introduction Biliary tract carcinoma, including extrahepatic bile duct and gallbladder carcinomas, is a relatively rare tumor.13 The mortality rate of this cancer has been increasing worldwide over the past few decades,4 and the prognosis is poor.1,2 Mortality rates are highest among American Indian women from the Southwestern United States, and among Chilean and Japanese women.5 The 5-year survival rate is less than 25% for intra-, and extrahepatic bile duct carcinoma, and 61% for gallbladder carcinoma, even after radical resection of the tumor.1,2,6 There is no effective therapy for biliary tract carcinomas, except for surgical resection. The molecular biological mechanisms underlying the development of biliary tract carcinomas are not well

understood. According to the multistep model of tumorigenesis,7,8 accumulations of mutations in protooncogenes, DNA repair genes, and tumorsuppressor genes are responsible for the malignant transformation of biliary epithelial cells. Most studies have focused on mutations of dominant oncogenes (Kras)of the tumor-suppressor genes. p53, and p16IKK4A.913 Recently, microarray analysis of around 22 000 transcripts in a series of biliary tract tumors detected a number of abnormally expressed genes, most of which had not been previously described in these tumor types.14 DNA hypermethylation is an important epigenetic mechanism for suppressing gene activity by changing the chromatin structure.15,16 It has become clear that aberrant DNA methylation of promoter region CpG islands may serve as an alternative mechanism to coding-region mutation for the inactivation of tumor-suppressor or tumor-related genes, and therefore methylation would play an important role in tumorigenesis.17,18 In biliary tract carcinomas, E-cadherin, p16, and RASSF1A promoter methylation, with loss of expression, is frequently present.1925 There is very limited information regarding the abnormal methylation of cancer-related genes in biliary tract carcinoma; however, a few insights have been obtained into the role of epigenetic silencing in the progression of biliary tract carcinoma. In this review, we present some recent rata regarding gene silencing by promoter hypermethylation (Table 1), and we discuss the implications for biliary tract carcinomas.

Promoter hypermethylation of multiple genes in biliary tract carcinoma Table 1 summarizes the reported frequencies of hypermethylation in biliary tract carcinoma.

Offprint requests to: K. Miyazaki Received: August 1, 2005 / Accepted: September 1, 2005

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E-cadherin The E-cadherin gene on chromosome 16q22.1 encodes a protein product important in the maintenance of the epithelial phenotype mediated by Ca2+-dependent, homotypic cell-cell adhesion. The gene has been termed a metastasis suppressor gene, because the E-cadherin protein can suppress tumor cell invasion and metastasis. E-cadherin gene expression is reduced or silenced in tissues from carcinoma of the breast and liver and in many cell lines derived from colon, stomach, and prostate cancer.26 Alterations in DNA methylation patterns are commonly found in essentially all cancers, often with concomitant changes in gene expression. In a mutation analysis of six bile duct cancer cell lines,27 there was no mutation in any of the 16 exons of the Ecadherin gene, as determined by polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis. However, the authors detected hypermethylation of the E-cadherin gene in two cell lines (SNU-478 and SNU-1079) by methylation-specic PCR (MSP) analysis. E-cadherin mRNA expression in these two cell lines was silenced by reverse transcription (RT)-PCR, and the expression of E-cadherin mRNA was restored after treatment with a demethylating agent (5-aza-2-deoxycytidine). Previously, we reported that reduced expression of E-cadherin in resected gallbladder cancer tissues was signicantly correlated with poor prognosis.28 Loss of E-cadherin expression, followed by expression of the mts1 gene, may be an important event for increasing cell proliferation, motility, and invasion activity in the progression of gallbladder cancer.29 In our study, the E-cadherin promoter was methylated in 6 of 15 (40%) bile duct carcinomas and in 9 of 22 (40.9%) gallbladder carcinomas.30 Tozawa et al.31 also reported E-cadherin promoter methylation, in 8 of 23 (34.8%) bile duct carcinomas and in 2 of 9 (22.2%) gallbladder carcinomas. Takahashi et al.32 found Ecadherin promoter methylation in 9 of 50 (18%) gallbladder carcinomas.

p14ARF and p16INK4A The p16INK4A tumor-suppressor gene, which maps to chromosome 9p21p22, is frequently inactivated in a wide variety of tumors by deletion, mutation, or CpG island methylation.3337 The p14ARF gene is present in the same region of chromosome 9p as the p16 gene.38 p16 and p14 transcripts are produced from two separate promoters and use alternative rst exons (1 and 1, respectively) joined through the same splice acceptor site to exon 2 coding sequences, but in different reading frames.39,40 p16 is a cyclin-dependent kinase (CDK) inhibitor involved in the regulation of the G1 checkpoint

in the cell cycle.41 The p16 protein blocks the G1-S transition in cells by binding to and preventing the association of CDK4 and CDK6 with cyclin D1, leading to dephosphorylation of the retinoblastoma gene product and the repression of transcription factor E2F.41,42 p16 mutations43,44 loss of heterozygosity of chromosome 9p,4447 and loss of p16 expression48 have been reported in primary biliary tract carcinomas and in cell lines derived from these carcinomas. p16 is the most frequently reported gene involved in promoter methylation in biliary tract carcinoma. In a recent study, more than half (52.2%, 12 of 23) of the specimens obtained from patients with the bile duct carcinoma had p16 promoter methylation,49 while the proportion was 60% for gallbladder carcinoma. Conversely, only 2 of 32 (6.3%) specimens obtained from patients with biliary stone disease or those without biliary disease showed p16 promoter methylation, whereas 45.6% of patients with primary sclerasing cholangitis (PSC) revealed p16 promoter methylation. Similarly, 10 of 21 (47.6%) bile duct carcinoma specimens showed p14 promoter methylation, while the proportion was 40% for gallbladder carcinoma. Furthermore, p14 promoter methylation was present in a mere 3.1% of patients without biliary disease, and was higher, at 60%, in PSC patients. In the same series of bile duct and gallbladder carcinomas, 42.9% of specimens with p16 promoter methylation showed simultaneous methylation of the adjacent p14 promoter.49 In another study, p16 promoter methylation was detected in 8 of 54 (14.8%) gallbladder carcinoma specimens, and in 5 of 31 (16.1%) bile duct carcinoma specimens.50 In a study of nine bile duct cancer cell lines (six bile duct and three gallbladder carcinoma cell lines), only one cell line (CC-LP-1) showed p16 promoter methylation, while no p14 promoter methylation was detected.24 These authors also showed that p16 promoter methylation was present in 9 of 21 (42.9%) primary tumors, all being methylated cased with loss of p16 protein expression.24 House et al.25 found p16 promoter methylation, using two-step MSP, in 18 of 30 (60%) gallbladder carcinomas in Chile and in 12 of 50 (50%) gallbladder carcinomas in the United States. Tozawa et al.31 Takahashi et al.,32 and Koga et al.30 also reported p16 promoter methylation in bile duct carcinoma and gallbladder carcinoma; however, the frequency of this promoter methylation varied widely, from 26.1% to 80.0% in bile duct carcinoma, and from 22.2% to 63.6% in gallbladder carcinoma. RASSF1A Deletion mapping and functional studies have targeted an extrahepatic cholangiocarcinoma-related tumor suppressor gene to a region at 3p21.3.51 Dammann et al., in the year 2000,52 isolated a RAS association domain fam-

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Table 1. Summary of promoter methylation in genes in biliary tract carcinoma specimens Frequency of methylation Function BD carcinoma GB carcinoma 8/23 (34.8) 6/15 (40.0) Tissue invasion and metastasis Tissue invasion and metastasis Cell-cycle regulation Cell-cycle regulation Cell-cycle regulation 10/21 (47.6) 12/23 (52.2) 5/31 (16.1) 9/21 (42.9) 6/23 (26.1) 2/9 (22.2) 9/50 (18.0) 9/22 (40.9) 22/50 (44.0) 0/50 (0) 2/5 (40.0) 22/50 (44.0) 3/5 (60.0) 8/54 (14.8) Tissue invasion and metastasis Method Country of origin of specimens Reference no.

Gene name

Gene location

E-cadherin

16q22

H-cadherin TIMP-3 p14 p15 p16

16q24 22q12-13 9p21 9p21 9p21

MSP MSP MSP MSP MSP MSP MSP MSP MSP MSP MSP Two-step MSP

31 (Tozawa) 32 (Takahashi) 30 (Koga) 32 (Takahashi) 32 (Takahashi) 49 (Klump) 32 (Takahashi) 49 (Klump) 50 (Ueki) 24 (Caca) 31 (Tozawa) 25 (House)

12/15 (80.0) Cell-cycle regulation Angiogenesis Signal transduction Signal transduction

p57 p73

11p15.5 1p36

2/9 (22.2) 18/30 (60.0) 12/24 (50.0) 12/50 (24.0) 14/22 (63.6) 0/50 (0) 7/50 (14.0) 12/30 (40.0) 3/24 (12.5) 28/33 (84.8) 7/23 (30.4) 0/50 (0) 1/9 (11.1) 15/50 (30.0) 4/30 (13.3) 10/24 (41.7)

MSP MSP MSP MSP Two-step MSP

32 (Takahashi) 30 (Koga) 32 (Takahashi) 32 (Takahashi) 25 (House)

RASSF1A

3p21

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APC

5q21

MSP MSP MSP MSP Two-step MSP

Japan Chile Japan Chile Chile Germany Chile Germany China Germany Japan Chile USA Chile Japan Chile Chile Chile USA China Japan Chile Chile Chile USA

57 (Chen) 31 (Tozawa) 32 (Takahashi) 32 (Takahashi) 25 (House)

NORE1 RUNX3 Signal transduction TGF- signal pathway 18/23 (78.3) 4/23 (17.4) 6/15 (40.0) 3/23 (13.0) 7/15 (46.7) Methyltransferase Mitotic stress checkpoint gene O-Sulfotransferase JAK-STAT pathway JAK-STAT pathway JAK-STAT pathway Key component in retinoid activity Key component in retinoid activity Methyltransferase superfamily Apoptosis Apoptosis Putative cytokine Cell-cycle regulator MSP MSP MSP MSP MSP MSP 5/15 (33.3) 4/23 (17.4) Two-step MSP MSP MSP MSP MSP MSP MSP MSP Two-step MSP Two-step MSP Two-step MSP TGF- signal pathway Proapoptotic serine/threonine kinase Mismatch repair

1q32.1 1p36

HPP1 DAP-kinase

2q32.3 9q34

hMLH1

3p21.3

MSP MSP MSP MSP MSP MSP MSP MSP Two-step MSP 32 (Takahashi) 31 (Tozawa) 32 (Takahashi) 32 (Takahashi) 31 (Tozawa) 32 (Takahashi) 30 (Koga) 31 (Tozawa) 25 (House) 30 (Koga) 25 (House)

MGMT

10q26

CHFR

12q24.33

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3-OST-2 SHP1 SOCS-1 SKY RARb2

16q12 12p13 16p13 9q22 3p21

30 (Koga) 31 (Tozawa) 32 (Takahashi) 32 (Takahashi) 32 (Takahashi) 32 (Takahashi) 32 (Takahashi) 32 (Takahashi) 25 (House) 32 (Takahashi) 32 (Takahashi) 32 (Takahashi) 32 (Takahashi) 35 (Takahashi) 83 (Takahashi)

GRBP1 RIZ1 DcR1 DcR2 HIN15q35 Reprimo

3q21-22 1p36 8p22 8p22 5q35 2q23

0/50 (0) 2/9 (22.2) 16/50 (32.0) 11/55 (20.0) 2/9 (22.2) 4/50 (8.0) 5/22 (22.7) 0/9 (0) 5/30 (16.7) 2/24 (8.3) 10/22 (45.5) 3/30 (10.0) 4/24 (16.7) 13/22 (59.1) 0/9 (0) 1/50 (2.0) 36/50 (72.0) 40/50 (80.0) 6/50 (12.0) 0/50 (0) 7/50 (14.0) 1/30 (3.3) 1/24 (4.2) 0/50 (0) 14/55 (25.5) 1/50 (2.0) 3/50 (6.0) 1/50 (2.0) 31/50 (62) Chile Japan Chile Chile Japan Chile Japan Japan Chile USA Japan Chile USA Japan Japan Chile Chile Chile Chile Chile Chile Chile USA Chile Chile Chile Chile Chile Chile

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ily protein, RASSF1A, which was located in the 120-kb region of a minimal homozygous deletion at 3p21.3 in lung cancer. They demonstrated that one of the major transcripts of this gene, RASSF1A, was frequently inactivated by promoter methylation. The tumor-suppressor function of RASSF1A was indicated by reduced colony formation, suppressed anchorageindependent growth, and inhibited tumor formation in nude mice upon RASSF1A exogenous re-expression in cell lines.53 RASSF1A contains a Ras-association domain; thus, a protein function as an effector of Ras signaling in normal cells has also been suggested.53 Hypermethylation of the promoter region has been proposed as a major mechanism in the inactivation of the RASSF1A gene. Promoter methylation of RASSF1A in cholangiocarcinoma was detected in 9 of 13 cases (69%) in one study.54 Further expression analysis of these 9 cases with promoter methylation indicated substantially reduced RASSF1A expression compared to that in normal livers. A similar incidence of RASSF1A promoter methylation (around 60%) has also been reported in human bladder and breast cancers.55,56 In extrahepatic cholangiocarcinoma, 28 of 33 (84.8%) tumor samples were methylated.57 Tozawa et al.31 reported methylation in 7 of 23 (30.4%) bile duct carcinomas, and in 1 of 9 (11.1%) gallbladder carcinomas, whereas Takahashi et al.32 found that none of 50% (0%) gallbladder carcinomas was methylated in the RASSF1A promoter region. RUNX3 RUNX3, a Runt domain transcription factor involved in transforming growth factor (TGF)-beta signaling, maps to chromosome 1p36, a region frequently altered by deletions or translocations in a wide variety of human carcinomas, including those of the bile duct and the pancreas.5860 Recently, frequent inactivation of RUNX3 has been demonstrated in human gastric carcinomas.61 In one study of bile duct cell lines, seven of ten (70%) cell lines exhibited no expression of the mRNA. All these seven cell lines that did not express RUNX3 showed methylation in the promoter CpG island of the gene. Moreover, treatment with the methylation inhibitor 5-aza-2-deoxycytidine restored RUNX3 mRNA expression in all seven cancer cell lines that originally lacked RUNX3 expression.62 In cancer specimens, the frequency of methylation of the RUNX3 gene was 18 of 23 (78.3%) bile duct carcinomas, 2 of 9 (22.2%) gallbladder carcinomas, and 1 of 5 (20.0%) ampullary carcinomas.31 Moreover, the patients with methylated promoters of RUNX3 showed signicantly shorter survival than those with a non-methylated promoter.31 Takahashi et al.32 also found that 16 of 50 (32%) gallbladder carcinomas were methylated in the RUNX3 promoter region.

DAP-kinase The death-associated protein kinase (DAPK) gene, located on chromosome 9p34.1, was initially isolated as a calcium/calmodulin-dependent positive mediator of interferon (IFN)-induced apoptosis, and was associated with the cell cytoskeleton.6365 Loss of DAPK expression has been reported in bladder carcinoma, renal cell carcinoma,66 lung cancer,67 head and neck carcinoma,68 and B-cell malignancies,69 and is thought to lead to the immortalization of cells and the development of cancer. Promoter hypermethylation of the DAPK CpG island has also been detected in some malignancies, in which the range of methylation was variable,66,67,69,70 and the methylation status was found to be associated with silencing of the DAPK gene. Simpson et al.70 reported that the inactivation of DAPK was strongly correlated with homozygous deletion or promoter hypermethylation in pituitary tumors. Furthermore, the presence of DAPK methylation has been associated with lymph node involvement or advanced disease stage in some tumors.7173 DAP kinase was recently characterized as an upstream regulator of p53.74 The methylation frequency of DAP-kinase was 4 of 23 (17.4%) in patients with bile duct carcinoma, 2 of 9 (22.2%) in those with gallbladder carcinoma, and 2 of 5 (40%) in those with ampullary carcinoma.31 DAP-kinase methylation was more frequent in poorly differentiated tumors than in well-to-moderately differentiated ones. Furthermore, DAP-kinase methylation-positive status has been independently associated with poor survival.31 Takahashi et al.32 found that 4 of 50 (8%) bile duct carcinomas were methylated in the DAP-kinase promoter region. We also reported DAP-kinase methylation in 6 of 15 (40%) bile duct carcinomas, and in 5 of 22 (22.7%) gallbladder carcinomas.30

MGMT and hMLH1 O6-methylguanin-DNA methyltransferase (MGMT) is a DNA repair enzyme that transfers methyl groups from O6-methylguanine to itself. Alkylation of DNA at the O6 position of guanine is an important step in the induction of mutations in an organism by alkylating agents.75 Alkylating agents are principally metabolized and activated in hepatocytes and then are released into the bile duct and stored in the gallbladder. Thus, high exposure to alkylating agents occurs in the epithelium of the gallbladder and extrahepatic bile duct. O6methylguanine can pair with thymine during DNA replication, resulting in a GC-to-AT transition mutation in DNA.76 The O6-methyl G : T mismatch is recognized by the mismatch repair (MMR) pathway, and the ensuing repetitive cycle of futile MMR is believed to result in apoptosis. Of ve or more distinct proteins that are

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Fig. 1. Disease-specic Kaplan-Meier survival analysis of patients with biliary tract carcinomas according to MGMT promoter methylation status. Patients with MGMT-methylated tumors had an unfavorable prognosis compared with those who showed MGMT non-methylated status (aP = 0.024; logrank test)

involved in the MMR pathway77 MLH-1 has frequently been found to be suppressed in tumor cells.78 In a previous immunohistochemical study, we found that approximately 60% of gallbladder and extrahepatic bile duct cancers had decient MGMT expression.79 In addition, MGMT-negative staining was correlated with hepatic invasion in gallbladder carcinoma and with poor prognosis in both types of tumor. Furthermore, combined negative MGMT and hMLH1 status was shown to be a more signicant prognostic biomarker than negative status of either MGMT or hMLH1 alone in both tumor types.79 The frequency of promoter methylation was 49% for the MGMT gene and 46% for the hMLH1 gene. Furthermore, MGMT methylation status was closely correlated with MGMT protein expression, as determined by immunohistochemistry (P < 0.001). A total of 33 mutations were identied in four cancerrelated genes: p53, K-ras, b-catenin, and p16/INK4a. The most common mutation was a GC-to-AT transition (58%), which was signicantly associated with MGMT promoter methylation (P = 0.011). These ndings suggest that loss of MGMT expression by promoter methylation results in the accumulation of GC-to-AT gene mutations.30 We added nine new specimens to a previous report on methylation status in biliary tract carcinoma, bringing the total to 46 cases. Figure 1 shows disease-specic Kaplan-Meier survival analysis according to MGMT methylation status in these biliary tract carcinomas, including gallbladder and extrahepatic bile duct carcinomas. The 5-year survival rate was 58% for patients with non-methylated MGMT and 31% for patients with methylated MGMT. Log-rank tests showed that methylated MGMT was associated with poor prog-

nosis compared with non-methylated MGMT (P = 0.024). Figure 2a,b shows summaries of the methylation proles of ve gene promoter regions (p16INK4A, MGMT, hMLH1, E-cadherin, and DAPK) in 24 gallbladder cancers and 19 extrahepatic bile duct cancers. Both MGMT and hMLH1 methylated status showed signicantly poorer prognosis compared with other statuses (P = 0.034; data not shown). Furthermore, there was a signicant correlation between MGMT methylation combined with a GC-to-AT transition mutation and poor prognosis in biliary tract carcinoma (P = 0.012; data not shown). These data indicate that MGMT methylation status, as well as hMLH1 methylation status and GC-to-AT transition mutation, play an important role in carcinogenesis and progression in biliary tract carcinomas. House et al.25 also reported hMLH1 methylation, at frequencies of 5 of 30 (16.7%) gallbladder cancers in Chile and 2 of 24 (8.3%) gallbladder cancers in the United States, and they reported MGMT methylation frequencies of 3 of 30 (10%) gallbladder carcinomas in Chile and 4 of 24 (16.7%) gallbladder carcinomas in the United States, using a two step-methylation specic polymerase chain reaction (MSP) method. However, Tozawa et al.31 reported a lower frequency of hMLH1 methylation, at 3 of 23 (13%) bile duct carcinomas, and 0 of 9 (0%) gallbladder carcinomas.

Multiple gene analysis Recently, Takahashi et al.32 showed aberrant promoter hypermethylation of multiple genes in the proles of 24 known or suspected tumor suppressor genes in 50 gallbladder carcinoma and 25 chronic cholecystitis specimens. In the gallbladder carcinomas, 10 genes demonstrated relatively high frequencies of aberrant methylation: SHP1 (80%), 3-OST-2 (72%), CDH13 (44%), P15INK4B (44%), CDH1 (38%), RUNX3 (32%), APC (30%), RIZI (26%), p16INK4A (24%), and HPP1 (20%). Eight genes (p73, RAR2, SOCS-1, DAPK, DcR2, DcR1, HIN1, and CHFR) showed low frequencies (2%14%) of methylation, and no methylation was detected in the remaining 6 genes (TIMP-3, p57, RASSF1A, CRBP1, SYK, and NORE1). In chronic cholecystitis, methylation was detected in 7 genes: SHP1 (88%), p15INK4B (28%), 3-OST-2 (12%), CDH1 (12%), CDH13 (8%), DcR2 (4%), and p16INK4A (4%). Signicantly higher frequencies of methylation in gallbladder carcinoma compared with chronic cholecystitis were detected for 8 genes (3-OST-2, CDH13, CDH1, RUNX3, APC, RIZ1, p16INK4A, and HPP1). This study constitutes the most comprehensive methylation prole report available for gallbladder carcinoma and demonstrates that this neoplasm has a distinct pattern of abnormal gene methylation.

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Fig. 2. Methylation proles of ve gene promoter regions (p16/INK4A, MGMT, hMLH1, Ecadherin, and DAPK) in 24 gallbladder cancers (a) and 19 extrahepatic bile duct cancers (b); 9 new cases were added to the original report.30 Filled boxes denote positive methylation. Open boxes denote no detectable methylation

Clinical implications In biliary tract carcinomas, preoperative pathological diagnosis by biopsy is difcult, due to the anatomical location. In bile duct carcinoma, cytology of bile juice can be obtained through endoscopic or bile drainage tube analysis; however, obtaining an accurate diagnosis remains difcult. Although there is no established molecular marker for a preoperative diagnosis, a few molecular markers, such as carcinoembryonic antigen (CEA), basic broblast growth factor (bFGF), plateletderived growth factor (PDGF), human epidermal growth factor receptor type 2 (HER2/Neu), and the presence of

K-ras mutation in bile, have been investigated to improve the preoperative diagnosis in biliary tract carcinoma.8082 Genes such as p16, which is frequently methylated in bile duct carcinoma, could be potential diagnostic molecular markers. The clinical implication of epigenetic alterations in biliary tract carcinoma is not well understood. Knowing whether the presence or absence of certain epigenetic changes in tumor-suppressor genes or oncogenes affects the prognosis is highly important, and this knowledge must be pursued. This information may also help in determining initial patient treatment options and in monitoring response to therapy, such as anticancer drug therapy or radiation therapy.

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Although the prognostic role of promoter methylation is unclear in bile duct carcinoma, methylation of the RUNX3 and the DAPK genes has been associated with poor prognosis.31 We have also demonstrated that patients with MGMT methylation-positive tumors in biliary tract carcinoma have a poor prognosis compared with those with MGMT methylation-negative tumors30 (Fig. 1). Thus, the suppression of tumor-suppressor genes or DNA repair genes by promoter methylation most likely plays an important role in the evolution or progression of biliary tract cancers.

Discussion In this review, we have summarized the data for genes involved in promoter methylation in biliary tract carcinoma of the extrahepatic bile ducts and gallbladder. Biliary tract carcinoma is a relatively uncommon disease, with poor survival. The molecular biological mechanisms underlying the evolution and progression of biliary tract carcinomas are not well understood. Promoter hypermethylation is an important epigenetic mechanism for suppressing tumor suppressor gene activity. We have summarized the characteristics of 28 genes previously reported as showing promoter methylation in biliary tract carcinoma (Table 1). The known promoter-methylated genes, such as E-cadherin, p16INK4A, RASSF1A, DAPK, RUNX3, and hMLH1, have been well documented in other cancers. Promoter methylation in the p16INK4A tumorsuppressor gene in biliary tract carcinoma has been widely reported;24,25,3032,49,50 however, the frequency of promoter methylation varies widely, from 16.1% to 80.0% in bile duct carcinoma, and from 14.8% to 63.6% in gallbladder carcinoma. One reason for this variation could be ethnic differences,5 while another possible reason for the variation could be differences in MSP methodology. House et al.25 and Koga et al.30 used a two-step MSP method, using nested PCR to increase sensitivity, and the sensitivity in their studies tended to be higher than that in other studies that used single-step MSP for p16INK4A, hMLH1, and p73. However, for APC and RARb2, the methylation frequencies differed despite the same ethnic background of the patients. The primer in the MSP method is highly sensitive, due to the CpG island, and the success of the MSP method depends on the primer sequence. MSP is a new technology that can easily analyze promoter methylation, and a reliable MSP method must be established. Two hypotheses could explain how decient MGMT expression leads to poor outcomes in patients with biliary tract carcinoma. One is that MGMT deciency leads to an accumulation of GC-to-AT mutations in critical molecules such as oncogenes and tumor-

suppressor genes. These mutations then enhance the malignant phenotype of biliary tract carcinoma. The other hypothesis is that MGMT deciency allows the promoter regions of tumor-suppressor genes and other repair genes to become simultaneously hypermethylated, and this results in the poor prognosis for patients with biliary tract carcinoma. To determine which of these two molecular pathways is associated with cancer progression in biliary tract carcinoma, we analyzed mutations and the promoter methylation status of several genes. On the basis of the genetic and epigenetic alterations that we investigated, it appears that reduced expression of MGMT may increase the malignant potential of biliary tract carcinoma through both types of molecular alterations. GC-to-AT mutations, which are triggered by epigenetic gene silencing of MGMT, may accumulate in multiple cancerrelated genes, resulting in the progression of biliary tract carcinoma. Moreover, the silencing of multiple cancer-related genes by promoter hypermethylation may also occur simultaneously with MGMT methylation. These epigenetic and genetic gene alterations may lead to the poor outcomes in biliary tract carcinoma patients with MGMT methylation. Thus, epigenetic silencing by MGMT promoter hypermethylation may play an important role in the progression of biliary tract carcinoma. Recently, several reports have described the abnormal methylation of cancer-related genes in biliary tract carcinoma, and some new interesting genes, including 3OST-2, SHP1,32 and reprimo,83 which show high methylation frequency, with promoter methylation of over 60% in biliary tract carcinoma, have been reported. Further efforts are required to investigate the molecular biological mechanisms of promoter hypermethylation, and to evaluate the clinical implications of this hypermethylation for biliary tract carcinoma.

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