Clinical Cancer Research Candidate Pathways, Whole Genome Scans: Reconciling Results, Looking into the Future Translational Cancer Medicine 2008: Cancer Clinical Trials and Personalized Medicine
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Clinical Cancer Research Vol. 10, 6126-6133, September 15, 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Aberrant Promoter Hypermethylation of Multiple Genes in Gallbladder Carcinoma and Chronic Cholecystitis

Takao Takahashi1, Narayan Shivapurkar1,2, Erick Riquelme3, Hisayuki Shigematsu1, Jyotsna Reddy1, Makoto Suzuki1, Kuniharu Miyajima1, Xian Zhou6, B. Nebiyou Bekele6, Adi F. Gazdar1,2 and Ignacio I. Wistuba3,4,5

1 Hamon Center for Therapeutic Oncology Research, 2 Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas; 3 Department of Anatomic Pathology, Pontificia Universidad Católica de Chile, Santiago, Chile; and Departments of 4 Pathology, 5 Thoracic and Head and Neck Medical Oncology, and 6 Biostatistics and Applied Mathematics, M. D. Anderson Cancer Center, Houston, Texas


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Aberrant methylation of 5' gene promoter regions is an epigenetic phenomenon that is a major mechanism for silencing of tumor suppressor genes in many cancer types. There is limited information about the molecular changes involved in the pathogenesis of gallbladder carcinoma (GBC), including methylation status.

Experimental Design: We investigated the aberrant promoter methylation profile of 24 known or suspected tumor suppressor genes in 50 GBCs and compared those results with the findings in 25 chronic cholecystitis (CC) specimens without cancer. The methylation-specific polymerase chain reaction and combined restriction analysis methods were used to detect methylation, and the results were confirmed by sequencing of cloned polymerase chain reaction products.

Results: In GBC, gene methylation frequencies varied from 0% to 80%. Ten genes demonstrated relatively high frequencies of aberrant methylation: SHP1 (80%), 3-OST-2 (72%), CDH13 (44%), P15INK4B (44%), CDH1 (38%), RUNX3 (32%), APC (30%), RIZ1 (26%), P16INK4A (24%), and HPP1 (20%). Eight genes (P73, RARß2, SOCS-1, DAPK, DcR2, DcR1, HIN1, and CHFR) showed low frequencies (2–14%) of methylation, and no methylation of the remaining six genes (TIMP-3, P57, RASSF1A, CRBP1, SYK, and NORE1) was detected. In CC, methylation was detected for seven genes: SHP1 (88%), P15INK4B (28%), 3-OST-2 (12%), CDH1 (12%), CDH13 (8%), DcR2 (4%), and P16INK4A (4%). Significantly higher frequencies of methylation in GBC compared with CC were detected for eight genes (3-OST-2, CDH13, CDH1, RUNX3, APC, RIZ1, P16INK4A, and HPP1). Of those, four genes showed frequent methylation (>30%) in GBCs. The mean methylation index, an expression of the amount of methylated genes by case, was significantly higher in GBC (0.196 ± 0.013) compared with CC (0.065 ± 0.008; P < 0.001).

Conclusions: Our study constitutes the most comprehensive methylation profile report available in GBC and demonstrates that this neoplasm has a distinct pattern of abnormal gene methylation. Whereas gallbladders from healthy individual were not available, our finding of methylation in CC cases without cancer suggests that this phenomenon represents an early event in the pathogenesis of GBC.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gallbladder carcinoma (GBC) is a relatively uncommon neoplasm that demonstrates considerable geographic and gender variation in incidence (1 , 2) . Mortality rates are highest among American Indian women from the southwestern United States and Chilean and Japanese women. In Chile, GBC is one of the most frequent cancers and represents the leading cause of cancer deaths in females (1) . GBC is a highly malignant neoplasm with a poor prognosis, with most cases diagnosed at advanced stages. There is a clear worldwide association between GBC and cholelithiasis and chronic inflammation of the gallbladder [chronic cholecystitis (CC)], which are present in almost all GBC specimens (1) .

There is limited information about the molecular changes involved in the pathogenesis of GBC. Most studies have focused on mutations of dominant oncogenes (K-ras) or tumor suppressor genes (TSGs) [TP53, P16INK4A, and FHIT (3, 4, 5) ]. Genome-wide and specific chromosome arm allelotyping analyses indicated that allelic losses at multiple sites of the genome are frequent in this neoplasm, suggesting the involvement of multiple TSGs in its pathogenesis (6 , 7) . Recently, Hansel et al. (8) , using a RNA-based global gene expression microarray analysis of ~22,000 transcripts in a series of biliary tract tumors including 10 GBC specimens, detected a number of abnormally expressed genes, most of which had not been described previously in these tumor types. DNA methylation of the promoter regions is emerging as the major mechanism of inactivation of TSGs, and the abnormal methylation profile of human tumors has been studied in the recent years (9 , 10) . Individual tumor types frequently have characteristic patterns of acquired aberrant methylation (10 , 11) . In many cases, aberrant methylation of the CpG island genes has been correlated with a loss of gene expression, and it is proposed that DNA methylation provides an alternative pathway to gene deletion or mutation for the loss of TSG function (11 , 12) . The understanding of the methylation profile of tumors may impact on several clinical issues, including early detection, chemoprevention, prognosis, and cancer treatment (12) .

In GBC, there is very limited information regarding abnormal methylation of cancer-related genes. There is only one report of methylation analysis of six candidate TSGs (P16INK4A, APC, MGMT, hMLH1, RARß2, and p73), which showed significant differences in gene methylation patterns between GBC cases from Chile and the United States (13) . To further investigate the TSG methylation profile in GBC, we examined the methylation profile of 24 genes frequently silenced by aberrant methylation in a number of tumor types in 50 GBCs from Chile, and we compared the findings with those present in 25 CC cases. The 24 genes were chosen for study due to their presumed or known roles in various cellular functions related to cancer development, including cell cycle regulation, tissue invasion and metastasis, Janus-activated kinase (JAK)/signal transducers and activators of transcription (STAT) and transforming growth factor ß signal pathways, key components of retinoid activity, signal transduction, apoptosis, angiogenesis, putative cytokine, mitotic stress checkpoint, methyltransferase superfamily, and O-sulfotransferase (refs. 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 ; Table 1Citation ).


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  		Table 1 Summary data of genes tested for aberrant promoter hypermethylation in GBC (n = 50) and CC specimens (n = 25)

 

   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Clinical Samples.
Fifty GBC samples and 25 CC samples were obtained from cases resected by cholecystectomy at the Catholic University Medical School Hospital (Santiago, Chile). The patients consisted of 39 women and 11 men ranging in age from 36 to 94 years (mean age, 65 years). The tumors consisted of 45 adenocarcinomas, 4 adenosquamous carcinomas, and 1 squamous cell carcinoma. Adenocarcinomas consisted of 30 tubular, 5 papillary, and 10 tubulo-papillary tumors. Most of the tumors were advanced GBCs with invasion of the gallbladder subserosa (16 cases, 32%) and serosa (29 cases, 58%), and the rest of the cases (5 cases, 10%) were early GBCs (mucosa or muscularis propia invasion). Twenty-five CC specimens obtained by cholecystectomy from 21 women and 4 men ranging in age from 34 to 80 years (mean age, 48 years) were also selected.

Methylation Assays.
Genomic DNA was isolated from frozen tissue by digestion with 100 µg/mL proteinase K followed standard phenol-chloroform (1:1) extraction and ethanol precipitation. DNA was treated with sodium bisulfite as described previously (14) . Briefly, 1 µg of genomic DNA was denatured by incubation with 0.2 mol/L NaOH for 10 minutes at 37°C. Aliquots of 10 mmol/L hydroquinone (30 µl; Sigma Chemical Co., St. Louis, MO) and 3 mol/L sodium bisulfite (pH 5.0; 520 µl; Sigma Chemical Co.) were added, and the solution was incubated at 50°C for 16 hours. Treated DNA was purified by use of the Wizard DNA Purification System (Promega, Madison, WI), desulfurated with 0.3 mol/L NaOH, precipitated with ethanol, and resuspended in water. Modified DNA was stored at –80°C until use. Gene name, gene location, function, and reference for methodology of all genes are summarized in Table 1Citation . The methylation status of 23 genes was determined by methylation-specific polymerase chain reaction (MSP). We used a combined restriction analysis for DAPK (35) . Negative control samples without DNA were included for each set of polymerase chain reaction (PCR). PCR products were analyzed on 2% agarose gels containing ethidium bromide.

Bisulfite Sequencing.
The PCR products of MSP were cloned into plasmid vectors using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA) as described previously (36) . The inserted PCR fragments of four individual clones obtained from each sample were sequenced with M13 reverse primers using the Applied Biosystems PRISM dye terminator cycle sequencing method (Perkin-Elmer, Foster City, CA).

Data Analysis.
Frequencies of methylation of two groups were compared using the {chi}2 test or Fisher’s exact test. The methylation index (MI), a reflection of the methylation status of all of the genes tested, is defined as the total number of genes methylated divided by the total number of genes analyzed. To compare the extent of methylation for the panel of genes examined, we calculated the MI for each case and then determined the mean for the different groups. Statistical analysis of MI between two variables was performed using the Mann-Whitney nonparametric U test. For all tests, P < 0.05 was regarded as statistically significant. For identification of a predictive model for GBC compared with CC based on gene methylation status, a multivariate penalized logistic regression model was used, and sensitivity and specificity were calculated.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Frequency of Gene Methylation in Gallbladder Carcinoma and Chronic Cholecystitis Specimens.
We examined the methylation status of 24 genes (Table 1)Citation in 50 GBC and 25 CC specimens (Fig. 1)Citation . In GBC, gene methylation frequencies varied from 0% to 80%. Ten genes demonstrated a relatively high frequency of aberrant methylation: SHP1 (80%), 3-OST-2 (72%), CDH13 (44%), P15INK4B (44%), CDH1 (38%), RUNX3 (32%), APC (30%), RIZ1 (26%), P16INK4A (24%), and HPP1 (20%). Eight genes (P73, RARß2, 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; Fig. 2Citation ). In CC, methylation was detected for seven genes: SHP1 (88%), P15INK4B (28%), 3-OST-2 (12%), CDH1(12%), CDH13 (8%), DcR2(4%) and P16INK4A (4%; Fig. 2Citation ). The unmethylated form of P16INK4A, run as a control for DNA integrity, was present in all tumor and inflammatory gallbladder samples examined. To confirm our MSP findings, bisulfite sequencing analysis of two representative methylated GBC or CC samples was performed in seven genes found to be frequently positive by the MSP technique (3-OST-2, CDH13, CDH1, RUNX3, APC, SHP1, and p15). All 14 samples in 56 individual clones analyzed demonstrated methylation of the CpG sites included in the amplicons, with methylation varying from 76% to 100%.



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  		Fig. 1. Representative examples of MSP analyses of methylated form (M) or unmethylated form (UM) of five genes (3-OST-2, CDH13, RUNX3, SHP1, and p15) in GBC and CC samples. P, positive control; N, negative control.

 


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  		Fig. 2. Comparison of frequencies of aberrant methylation and mean MIs in GBC and CC samples. P values are shown when there was a significant difference between two groups.

 
Comparative analysis of the methylation frequencies between GBC and CC classifies the 24 genes examined into three groups. The first group is that of genes showing significantly higher frequencies of methylation in GBC compared with CC (n = 8; 3-OST-2, CDH13, CDH1, RUNX3, APC, RIZ1, P16INK4A, and HPP1). Of these, four genes (3-OST-2, CDH13, CDH1, and RUNX3) showed methylation frequencies of >30%, and 3-OST-2 was the most frequently (72%) methylated gene in GBCs. Because our GBC and CC patients differed in age (patients ≥ 65 years, 50% GBC versus 12% CC; P = 0.002), we were unable to establish a predictive model for GBC based on methylation status of the above-mentioned eight genes. However, we performed a predictive model analysis using patients younger than 65 years (n = 47; GBC, 25 patients; CC, 22 patients) to minimize the age difference bias as much as possible. Our multivariate penalized logistic regression model analysis identified a five-gene (3-OST-2, CDH13, RUNX3, P16INK4A, and HPP1) combination as a significant predictor of GBC compared with CC (92% sensitivity, 81% specificity). The second group was that of genes demonstrating frequent methylation in both GBC and CC specimens (n = 2; SHP1 and P15INK4B). The third group was that of genes showing low frequencies of methylation in both types of gallbladder specimens examined (the remaining 14 genes; Fig. 2Citation ).

Excluding SHP1 and P15INK4B genes, which were frequently methylated in malignant and nonmalignant gallbladder specimens, 96% of GBCs had methylation of one or more genes, whereas most of CCs (68%) had no gene methylated. In addition, a statistically significant (P < 0.001) higher mean MI was detected in GBC (0.196 ± 0.013) compared with CC (0.065 ± 0.008; Fig. 2Citation ).

Correlation between Methylation and Clinicopathological Features in Gallbladder Carcinoma.
We compared the relationships between aberrant methylation of the eight genes (group 1) showing cancer specificity and clinicopathological features in GBCs. Interestingly, some statistically significant correlations between GBC features and gene methylation status of specific genes were established (Table 2)Citation Citation : (a) HPP1 was more frequently methylated in male patients (55%) than in female patients (10%; P = 0.004); (b) tumors displaying exclusively papillary or mixed papillary/tubular patterns demonstrated higher frequency of APC methylation than cancers exhibiting only tubular features (60% versus 13%, respectively; P = 0.004); (c) of interest, methylation of RIZ1 correlated with tumor depth of invasion in the gallbladder wall and in adjacent liver tissue (P = 0.0037 and P = 0.024, respectively); and (d) CDH1 methylation was associated with the presence of distant metastasis (P = 0.024). No significant association between methylation status and age was detected. In addition, no correlation between mean MI and clinicopathological features was noted.


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  		Table 2 Relationship between aberrant promoter hypermethylation of eight genes (group 1) and clinicopathological features in GBCs

 

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  		Table 2A Continued.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies have reported the importance of DNA methylation in human cancers and focused on regions of the genome that may have functional significance resulting from suppression of gene activity. Whereas most individual cancers have several, perhaps hundreds, of methylated genes, the methylation profiles of individual tumor types are characteristic (10 , 11) . In GBC, there is very limited information regarding abnormal methylation of cancer-related genes (13) . Thus, to further investigate the methylation profile of GBCs we tested the methylation status of a panel of 24 genes with presumed or known roles in various cellular functions related to cancer development that had been studied in many tumor types. Because of geography-related differences in the methylation profiles of GBCs, our studies were limited to tumors from Chile.

Analysis of the methylation frequencies in GBCs compared with the findings in CC specimens demonstrated a group of eight genes (3-OST-2, CDH13, CDH1, RUNX3, APC, RIZ1, P16INK4A, and HPP1) that showed specificity for GBC, suggesting that they play an important role in GBC pathogenesis. A predictive model analysis based on these eight genes delineating GBC from CC failed because of substantial differences in age between both patient groups. However, if only patients < 65 years were considered, a five-gene (3-OST-2, CDH13, RUNX3, P16INK4A, and HPP1) combination was a good predictor for GBC with 92% sensitivity and 81% specificity. Of these genes, the gene with the highest frequency (72%) of methylation in GBC was 3-OST-2. This gene encodes an O-sulfotransferase that is involved in the final modification step of glycosaminoglycan chains of heparan sulfate proteoglycans (HSPGs), including glypicans and syndecans, whose altered expressions have been reported recently in human breast cancers (37) . HSPGs are known to play major roles in cell growth, adhesion, and migration by interaction with a wide range of growth factors, morphogens, cytokines, and extracellular matrices (38) . Silencing of 3-OST-2 suggests that altered modification of HSPGs is involved in cancer development in some tumor types. Recently, silencing and aberrant promoter hypermethylation of 3-OST-2 has been demonstrated in breast cancer cell lines, as well as in several human tumors, including those of the breast (88%), colon (80%), lung (70%), and pancreas (100%; ref. 34 ). Our findings suggest that 3-OST-2 may play an important role in GBC development.

Two members of the cell adhesion cadherin family, CDH13 and CDH1, which are related to tumor invasion and metastasis, also showed frequent methylation in GBC (44% and 38%, respectively). Inactivation by promoter methylation of both genes has been described in several tumor types including lung, breast, colon, and bladder cancers (17 , 36 , 39) . Of interest, our data showed significant correlation between CDH1 methylation and the presence of distant metastasis in our GBC cases, phenomena that have been reported previously in other tumor types (40) .

RUNX3 has been suggested as a putative tumor suppressor involved in the development of gastric cancers (29) . In this tumor type, hypermethylation combined with hemizygous deletion of the RUNX3 correlates with a significant reduction in expression, and the tumorigenicity of cell lines in nude mice is inversely related to their level of RUNX3 expression (29) . A recent report showed that hypermethylation of the RUNX3 gene promoter also occurs frequently (75%) in testicular yolk sac tumors of infants (41) . Our result of frequent methylation of RUNX3 (32%) in GBC expands the spectrum of tumor types in which this putative TSG may play an important role.

Selective methylation and silencing of the APC1A promoter and its specific products have been detected frequently in several human tumors (42) . In GBC, we found methylation of this gene in 30% of the cases examined. Although the APC gene locus (5q21) allele losses were a rare event in GBCs (3) , our methylation data are similar to other published findings (13) . It is somewhat intriguing that we detected a substantially higher frequency of APC methylation in GBCs with papillary features.

The retinoblastoma protein-interacting zinc finger gene, RIZ1, a putative TSG, is a member of a nuclear histone/protein methyltransferase superfamily. RIZ1 inactivation has been commonly found in many types of human cancers (43 , 44) . RIZ1 mapped within a chromosomal region frequently deleted in many types of human tumors (1p36.1), including GBC (6) . RIZ1 was recently reported to be frequently methylated in several tumor types, such as breast (44%), liver (62%), and nasopharyngeal (60%) tumors (33 , 45) . In addition to the relatively (26%) high frequency of RIZ1 methylation in our GBC cases, methylation of this gene was significantly associated with increasing depth of invasion and tumor-node-metastasis (TNM) stage of our GBCs.

P16INK4A gene abnormalities, including allelic losses at 9p21 (50%; ref. 3 ), gene mutations (20%; ref. 46 ), and methylation (50%; ref. 13 ), as well as loss of protein immunohistochemistry expression (75%; ref. 47 ), have been detected frequently in GBC. Although our finding of 24% methylation of P16INK4A in fresh frozen tumors is lower than the previously detected percentage of methylation (60%) in formalin-fixed, paraffin-embedded gallbladder tumors from Chilean patients, our data still support P16INK4A as an important TSG in gallbladder tumorigenesis.

The HPP1 gene was initially discovered because of its frequent hypermethylation in hyperplastic colon polyps, but it is also hypermethylated in colorectal adenomas and carcinomas (48) and gastric tumors (30) . Our finding of 20% methylation in gallbladder tumors indicates that inactivation of HPP1 may play an important role in the pathogenesis of a subset of GBCs.

Epidemiologic and clinical data support the current concept of chronic cholelithiasis and subsequent inflammation as established risk factors for GBC (1) . Several molecular abnormalities, including allelic losses at TSG loci (7) and mitochondrial DNA mutations (49) , have been demonstrated in a subset (up to 25%) of CC specimens, suggesting that they harbor clones of epithelial cells with malignant potential. Recently, House et al. (13) detected gene promoter methylation in three (P16INK4A, 10%; hMLH1, 10%; MGMT, 15%) of six genes examined in formalin-fixed, paraffin-embedded CC specimens. Our finding that 7 of 24 known and candidate TSGs exhibited methylation in CC supports the concept that CC is a preneoplastic lesion for GBC. Unfortunately, it is virtually impossible to obtain normal gallbladder specimens (without gallstones and CC) because the organ is almost never surgically removed from disease-free individuals. Thus, our findings have to be interpreted in the context of not knowing the methylation status of normal gallbladder epithelium. Of those seven genes, four demonstrated substantially higher frequencies of methylation in GBCs compared with CCs. However, the two genes (SHP1 and P15INK4B) demonstrating the highest frequencies of methylation in CC specimens did not show any significant difference in methylation frequencies in GBC, suggesting that methylation of these genes is an early event. Gallbladder preneoplastic epithelium, such as dysplasia and carcinoma in situ, needs to be examined to investigate the stage of the sequential pathogenesis of GBC in which methylation of the above-mentioned genes occurs. SHP1 is a negative regulator of the JAK/STAT signaling pathway (50) . A high frequency of silencing of SHP1 by promoter methylation was detected in most leukemias and lymphomas (20) . SHP1 may play a role in both GBC and CC. Similar to our findings, methylation of several TSGs has been detected in nonneoplastic epithelium from colon and stomach in patients without cancer (51 , 52) , indicating that early gene methylation could be a widespread phenomenon in human carcinoma pathogenesis.

The present study constitutes the most comprehensive methylation profile report available for GBC and demonstrates that this neoplasm has a distinct pattern of abnormal gene methylation. Most of the genes showing high frequencies of abnormal methylation in GBC have not been studied previously in this neoplasm. Whereas gallbladders from healthy individual were not available, our finding of gene methylation in CC without cancer suggests that this phenomenon represents an early event in the pathogenesis of GBC.


   FOOTNOTES
 
Grant support: Supported in part by Grant FONDECYT Chile 1020960 (I. Wistuba).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Ignacio I. Wistuba, Department of Pathology, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 85, Houston, TX 77030-4009. Phone: 713-563-9184; Fax: 713-563-1848; E-mail: iiwistuba{at}mdanderson.org

Received 3/25/04; revised 6/ 4/04; accepted 6/10/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Lazcano-Ponce EC, Miquel JF, Munoz N, et al Epidemiology and molecular pathology of gallbladder cancer. CA-Cancer J Clin 2001;51:349-64.[Abstract/Free Full Text]
  2. Misra S, Chaturvedi A, Misra NC, Sharma ID Carcinoma of the gallbladder. Lancet Oncol 2003;4:167-76.[CrossRef][Medline]
  3. Wistuba II, Sugio K, Hung J, et al Allele-specific mutations involved in the pathogenesis of endemic gallbladder carcinoma in Chile. Cancer Res 1995;55:2511-5.[Abstract/Free Full Text]
  4. Ajiki T, Fujimori T, Onoyama H, et al K-ras gene mutation in gall bladder carcinomas and dysplasia. Gut 1996;38:426-9.[Abstract/Free Full Text]
  5. Wistuba II, Ashfaq R, Maitra A, et al Fragile histidine triad gene abnormalities in the pathogenesis of gallbladder carcinoma. Am J Pathol 2002;160:2073-9.[Abstract/Free Full Text]
  6. Wistuba II, Tang M, Maitra A, et al Genome-wide allelotyping analysis reveals multiple sites of allelic loss in gallbladder carcinoma. Cancer Res 2001;61:3795-800.[Abstract/Free Full Text]
  7. Wistuba II, Maitra A, Carrasco R, et al High resolution chromosome 3p, 8p, 9q and 22q allelotyping analysis in the pathogenesis of gallbladder carcinoma. Br J Cancer 2002;87:432-40.[CrossRef][Medline]
  8. Hansel DE, Rahman A, Hidalgo M, et al Identification of novel cellular targets in biliary tract cancers using global gene expression technology. Am J Pathol 2003;163:217-29.[Abstract/Free Full Text]
  9. Takahashi T, Shivapurkar N, Reddy J, et al DNA methylation profiles of lymphoid and hematopoietic malignancies. Clin Cancer Res 2004;10:2928-35.[Abstract/Free Full Text]
  10. Esteller M, Corn PG, Baylin SB, Herman JG A gene hypermethylation profile of human cancer. Cancer Res 2001;61:3225-9.[Abstract/Free Full Text]
  11. Costello JF, Fruhwald MC, Smiraglia DJ, et al Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nat Genet 2000;24:132-8.[CrossRef][Medline]
  12. Herman JG, Baylin SB Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 2003;349:2042-54.[Free Full Text]
  13. House MG, Wistuba II, Argani P, et al Progression of gene hypermethylation in gallstone disease leading to gallbladder cancer. Ann Surg Oncol 2003;10:882-9.[Abstract/Free Full Text]
  14. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996;93:9821-6.[Abstract/Free Full Text]
  15. Li Y, Nagai H, Ohno T, et al Aberrant DNA methylation of p57KIP2 gene in the promoter region in lymphoid malignancies of B-cell phenotype. Blood 2002;100:2572-7.[Abstract/Free Full Text]
  16. Graff JR, Herman JG, Myohanen S, Baylin SB, Vertino PM Mapping patterns of CpG island methylation in normal and neoplastic cells implicates both upstream and downstream regions in de novo methylation. J Biol Chem 1997;272:22322-9.[Abstract/Free Full Text]
  17. Sato M, Mori Y, Sakurada A, Fujimura S, Horii A The H-cadherin (CDH13) gene is inactivated in human lung cancer. Hum Genet 1998;103:96-101.[CrossRef][Medline]
  18. Bachman KE, Herman JG, Corn PG, et al Methylation-associated silencing of the tissue inhibitor of metalloproteinase-3 gene suggests a suppressor role in kidney, brain, and other human cancers. Cancer Res 1999;59:798-802.[Abstract/Free Full Text]
  19. Yoshikawa H, Matsubara K, Qian GS, et al SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet 2001;28:29-35.[CrossRef][Medline]
  20. Oka T, Ouchida M, Koyama M, et al Gene silencing of the tyrosine phosphatase SHP1 gene by aberrant methylation in leukemias/lymphomas. Cancer Res 2002;62:6390-4.[Abstract/Free Full Text]
  21. Yuan Y, Mendez R, Sahin A, Dai JL Hypermethylation leads to silencing of the SYK gene in human breast cancer. Cancer Res 2001;61:5558-61.[Abstract/Free Full Text]
  22. Esteller M, Guo M, Moreno V, et al Hypermethylation-associated inactivation of the cellular retinol-binding-protein 1 gene in human cancer. Cancer Res 2002;62:5902-5.[Abstract/Free Full Text]
  23. Virmani AK, Rathi A, Zochbauer-Muller S, et al Promoter methylation and silencing of the retinoic acid receptor-beta gene in lung carcinomas. J Natl Cancer Inst (Bethesda) 2000;92:1303-7.[Abstract/Free Full Text]
  24. Burbee DG, Forgacs E, Zochbauer-Muller S, et al Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression. J Natl Cancer Inst (Bethesda) 2001;93:691-9.[Abstract/Free Full Text]
  25. Hesson L, Dallol A, Minna JD, Maher ER, Latif F NORE1A, a homologue of RASSF1A tumour suppressor gene is inactivated in human cancers. Oncogene 2003;22:947-54.[CrossRef][Medline]
  26. Tsuchiya T, Tamura G, Sato K, et al Distinct methylation patterns of two APC gene promoters in normal and cancerous gastric epithelia. Oncogene 2000;19:3642-6.[CrossRef][Medline]
  27. van Noesel MM, van Bezouw S, Salomons GS, et al Tumor-specific down-regulation of the tumor necrosis factor-related apoptosis-inducing ligand decoy receptors DcR1 and DcR2 is associated with dense promoter hypermethylation. Cancer Res 2002;62:2157-61.[Abstract/Free Full Text]
  28. Corn PG, Kuerbitz SJ, van Noesel MM, et al Transcriptional silencing of the p73 gene in acute lymphoblastic leukemia and Burkitt’s lymphoma is associated with 5' CpG island methylation. Cancer Res 1999;59:3352-6.[Abstract/Free Full Text]
  29. Li QL, Ito K, Sakakura C, et al Causal relationship between the loss of RUNX3 expression and gastric cancer. Cell 2002;109:113-24.[CrossRef][Medline]
  30. Shibata DM, Sato F, Mori Y, et al Hypermethylation of HPP1 is associated with hMLH1 hypermethylation in gastric adenocarcinomas. Cancer Res 2002;62:5637-40.[Abstract/Free Full Text]
  31. Krop IE, Sgroi D, Porter DA, et al HIN-1, a putative cytokine highly expressed in normal but not cancerous mammary epithelial cells. Proc Natl Acad Sci USA 2001;98:9796-801.[Abstract/Free Full Text]
  32. Mizuno K, Osada H, Konishi H, et al Aberrant hypermethylation of the CHFR prophase checkpoint gene in human lung cancers. Oncogene 2002;21:2328-33.[CrossRef][Medline]
  33. Du Y, Carling T, Fang W, et al Hypermethylation in human cancers of the RIZ1 tumor suppressor gene, a member of a histone/protein methyltransferase superfamily. Cancer Res 2001;61:8094-9.[Abstract/Free Full Text]
  34. Miyamoto K, Asada K, Fukutomi T, et al Methylation-associated silencing of heparan sulfate D-glucosaminyl 3-O-sulfotransferase-2 (3-OST-2) in human breast, colon, lung and pancreatic cancers. Oncogene 2003;22:274-80.[CrossRef][Medline]
  35. Toyooka S, Toyooka KO, Miyajima K, et al Epigenetic down-regulation of death-associated protein kinase in lung cancers. Clin Cancer Res 2003;9:3034-41.[Abstract/Free Full Text]
  36. Toyooka S, Toyooka KO, Harada K, et al Aberrant methylation of the CDH13 (H-cadherin) promoter region in colorectal cancers and adenomas. Cancer Res 2002;62:3382-6.[Abstract/Free Full Text]
  37. Matsuda K, Maruyama H, Guo F, et al Glypican-1 is overexpressed in human breast cancer and modulates the mitogenic effects of multiple heparin-binding growth factors in breast cancer cells. Cancer Res 2001;61:5562-9.[Abstract/Free Full Text]
  38. Perrimon N, Bernfield M Specificities of heparan sulphate proteoglycans in developmental processes. Nature (Lond) 2000;404:725-8.[CrossRef][Medline]
  39. Toyooka KO, Toyooka S, Virmani AK, et al Loss of expression and aberrant methylation of the CDH13 (H-cadherin) gene in breast and lung carcinomas. Cancer Res 2001;61:4556-60.[Abstract/Free Full Text]
  40. Oka H, Shiozaki H, Kobayashi K, et al Expression of E-cadherin cell adhesion molecules in human breast cancer tissues and its relationship to metastasis. Cancer Res 1993;53:1696-701.[Abstract/Free Full Text]
  41. Kato N, Tamura G, Fukase M, Shibuya H, Motoyama T Hypermethylation of the RUNX3 gene promoter in testicular yolk sac tumor of infants. Am J Pathol 2003;163:387-91.[Abstract/Free Full Text]
  42. Virmani AK, Rathi A, Sathyanarayana UG, et al Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter 1A in breast and lung carcinomas. Clin Cancer Res 2001;7:1998-2004.[Abstract/Free Full Text]
  43. He L, Yu JX, Liu L, et al RIZ1, but not the alternative RIZ2 product of the same gene, is underexpressed in breast cancer, and forced RIZ1 expression causes G2-M cell cycle arrest and/or apoptosis. Cancer Res 1998;58:4238-44.[Abstract/Free Full Text]
  44. Chadwick RB, Jiang GL, Bennington GA, et al Candidate tumor suppressor RIZ is frequently involved in colorectal carcinogenesis. Proc Natl Acad Sci USA 2000;97:2662-7.[Abstract/Free Full Text]
  45. Chang HW, Chan A, Kwong DL, et al Detection of hypermethylated RIZ1 gene in primary tumor, mouth, and throat rinsing fluid, nasopharyngeal swab, and peripheral blood of nasopharyngeal carcinoma patient. Clin Cancer Res 2003;9:1033-8.[Abstract/Free Full Text]
  46. Yoshida S, Todoroki T, Ichikawa Y, et al Mutations of p16Ink4/CDKN2 and p15Ink4B/MTS2 genes in biliary tract cancers. Cancer Res 1995;55:2756-60.[Abstract/Free Full Text]
  47. Parwani AV, Geradts J, Caspers E, et al Immunohistochemical and genetic analysis of non-small cell and small cell gallbladder carcinoma and their precursor lesions. Mod Pathol 2003;16:299-308.[CrossRef][Medline]
  48. Young J, Biden KG, Simms LA, et al HPP1: a transmembrane protein-encoding gene commonly methylated in colorectal polyps and cancers. Proc Natl Acad Sci USA 2001;98:265-70.[Abstract/Free Full Text]
  49. Tang M, Baez S, Pruyas M, et al Mitochondrial DNA mutation at the D310 (displacement loop) mononucleotide sequence in the pathogenesis of gallbladder carcinoma. Clin Cancer Res 2004;10:1041-6.[Abstract/Free Full Text]
  50. O’Shea JJ, Gadina M, Schreiber RD Cytokine signaling in 2002: new surprises in the Jak/Stat pathway. Cell 2002;109(Suppl):S121-31.
  51. Waki T, Tamura G, Sato M, Motoyama T Age-related methylation of tumor suppressor and tumor-related genes: an analysis of autopsy samples. Oncogene 2003;22:4128-33.[CrossRef][Medline]
  52. Kang GH, Lee HJ, Hwang KS, et al Aberrant CpG island hypermethylation of chronic gastritis, in relation to aging, gender, intestinal metaplasia, and chronic inflammation. Am J Pathol 2003;163:1551-6.[Abstract/Free Full Text]



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