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• W2000940387 abstract "To determine the methylation profile of multiple tumor-related genes during multistep hepatocarcinogenesis, we investigated the methylation status of CpG islands of 9 genes, using methylation-specific polymerase chain reaction for 60 paired hepatocellular carcinoma (HCC) and non-HCC liver tissue samples, 22 dysplastic nodule (DN), 30 liver cirrhosis (LC), 34 chronic hepatitis (CH) and 20 normal liver samples. The methylation status of 9 genes was correlated to the clinicopathological findings of HCC patients. All HCC samples showed methylation of at least one gene, whereas it was shown in 72.7% of DN and 40% of LC, but was not shown in CH and normal liver samples (P < 0.001). The number of genes methylated showed a stepwise increase with the progression of stages (0 for normal liver and CH, 0.5 for LC, 1.5 for DN, and 3.7 for HCC (P < 0.001)). The genes frequently methylated in HCC were APC (81.7%), GSTP1 (76.7%), RASSF1A (66.7%), p16 (48.3%), COX-2 (35%), and E-cadherin (33.3%). COX-2, p16, RASSF1A, and TIMP-3 were not methylated in LC and CH from patients without concurrent HCC. Chronic liver diseases with concurrent HCC showed higher methylation frequencies of the tested genes, and a higher number of methylated genes than those without concurrent HCC. HCC patients with methylation of E-cadherin or GSTP1 showed poorer survival than those without (P = 0.034 and 0.043, respectively). In conclusion, our results indicated that CpG island methylation of tumor-related genes is an early and frequent event, and accumulates step-by-step during a multistep hepatocarcinogenesis. CpG island methylation of E-cadherin or GSTP1 might serve as a potential biomarker for prognostication of HCC patients. To determine the methylation profile of multiple tumor-related genes during multistep hepatocarcinogenesis, we investigated the methylation status of CpG islands of 9 genes, using methylation-specific polymerase chain reaction for 60 paired hepatocellular carcinoma (HCC) and non-HCC liver tissue samples, 22 dysplastic nodule (DN), 30 liver cirrhosis (LC), 34 chronic hepatitis (CH) and 20 normal liver samples. The methylation status of 9 genes was correlated to the clinicopathological findings of HCC patients. All HCC samples showed methylation of at least one gene, whereas it was shown in 72.7% of DN and 40% of LC, but was not shown in CH and normal liver samples (P < 0.001). The number of genes methylated showed a stepwise increase with the progression of stages (0 for normal liver and CH, 0.5 for LC, 1.5 for DN, and 3.7 for HCC (P < 0.001)). The genes frequently methylated in HCC were APC (81.7%), GSTP1 (76.7%), RASSF1A (66.7%), p16 (48.3%), COX-2 (35%), and E-cadherin (33.3%). COX-2, p16, RASSF1A, and TIMP-3 were not methylated in LC and CH from patients without concurrent HCC. Chronic liver diseases with concurrent HCC showed higher methylation frequencies of the tested genes, and a higher number of methylated genes than those without concurrent HCC. HCC patients with methylation of E-cadherin or GSTP1 showed poorer survival than those without (P = 0.034 and 0.043, respectively). In conclusion, our results indicated that CpG island methylation of tumor-related genes is an early and frequent event, and accumulates step-by-step during a multistep hepatocarcinogenesis. CpG island methylation of E-cadherin or GSTP1 might serve as a potential biomarker for prognostication of HCC patients. Hepatocellular carcinoma (HCC) ranks as the fifth most common cancer in the world.1Bosch X Ribes J Borras J Epidemiology of primary liver cancer.Semin Liver Dis. 1999; 19: 271-285Crossref PubMed Scopus (857) Google Scholar In Korea, an endemic area of HCC, most HCCs are associated with chronic hepatitis B or C viral infections, and the HCC-associated death rate is high, 21.3/100,000 persons.2Ministry of Health and Welfare Republic of Korea Annual Report of Cancer Registry Program in the Republic of Korea. Ministry of Health and Welfare Republic of Korea, Seoul2000: 1-191Google Scholar The development and progression of HCC is a multistep process whereby the normal hepatocytes undergo inflammation, fibrosis by the hepatitis virus or other stimuli, followed by liver cirrhosis (LC), which then progresses to HCC or dysplastic nodule (DN) and subsequent HCC. The understanding of the molecular pathways of hepatocarcinogenesis is limited, although recent molecular biological studies have led to rapid progress in the understanding of the molecular events involved. Most previous studies have concentrated on the documentation of mutational events leading to the activation of oncogenes or the inactivation of tumor suppressor genes in hepatocarcinogenesis.3Feitelson MA Sun B Tufan NLS Liu J Pan J Lian Z Genetic mechanisms of hepatocarcinogenesis.Oncogene. 2002; 21: 2593-2604Crossref PubMed Scopus (293) Google Scholar However, recent advances in the field of epigenetics have brought an awareness, where not only genetic, but also epigenetic changes, play roles in carcinogenesis.4Jones PA Laird PW Cancer epigenetics comes of age.Nat Genet. 1999; 21: 163-167Crossref PubMed Scopus (2061) Google Scholar, 5Baylin SB Herman JG DNA hypermethylation in tumorigenesis: epigenetics joins genetics.Trends Genet. 2000; 16: 168-174Abstract Full Text Full Text PDF PubMed Scopus (1408) Google Scholar DNA methylation is one of the best-understood epigenetic mechanisms. It has been firmly established that aberrant hypermethylation of CpG islands in gene promoter regions correlate with the lack of gene transcription.6Baylin SB Herman JG Graff JR Vertino PM Issa JPJ Alterations in DNA methylation: a fundamental aspect of neoplasia.Adv Cancer Res. 1998; 72: 141-196Crossref PubMed Google Scholar In HCCs, a growing number of genes have been recognized as undergoing aberrant CpG island hypermethylation, which is associated with the transcriptional inactivation and loss of gene function, suggesting that CpG island hypermethylation is an important molecular mechanism for the development of HCC. Most studies have focused on single target genes,7Iwata N Yamamoto H Sasaki S Itoh F Suzuki H Kikuchi T Kaneto H Iku S Ozeki I Karino Y Satoh T Toyota J Satoh M Endo T Imai K Frequent hypermethylation of CpG islands and loss of expression of the 14–3-3 σ gene in human hepatocellular carcinoma.Oncogene. 2000; 19: 5298-5302Crossref PubMed Scopus (221) Google Scholar, 8Kaneto H Sasaki S Yamamoto H Itoh F Toyota M Suzuki H Ozeki I Iwata N Ohmura T Satoh T Karino Y Satoh T Toyota J Satoh M Endo T Omata M Imai K Detection of hypermethylation of the p16INK4A gene promoter in chronic hepatitis and cirrhosis associated with hepatitis B or C virus.Gut. 2001; 48: 327-377Crossref Scopus (125) Google Scholar, 9Liew CT Li HM Lo KW Leow CK Chan JY Hin LY Lau WY Lai PBS Lim BK Huang J Leung WT Wu S Lee JKC High frequency of p16INK4A gene alterations in hepatocellular carcinoma.Oncogene. 1999; 18: 789-795Crossref PubMed Scopus (166) Google Scholar, 10Kanai Y Ushijima S Hui AM Ochiai A Tsuda H Sakamoto M Hirohashi S The E-cadherin gene is silenced by CpG methylation in human hepatocellular carcinomas.Int J Cancer. 1997; 71: 355-359Crossref PubMed Scopus (243) Google Scholar, 11Tchou JC Lin X Freije D Isaacs WB Brooks JD Rashid A De Marzo AM Kanai Y Hirohashi S Nelson WG GSTP1 CpG island DNA hypermethylation in hepatocellular carcinomas.Int J Oncol. 2000; 16: 663-676PubMed Google Scholar and a few have attempted to analyze the hypermethylation of multiple genes in HCCs and associated chronic liver diseases.12Kondo Y Kanai Y Sakamoto M Mizokami M Ueda R Hirohashi S Genetic instability and aberrant DNA methylation in chronic hepatitis and cirrhosis: a comprehensive study of loss of heterozygosity and microsatellite instability at 39 loci and DNA hypermethylation on 8 CpG islands in microdissected specimens from patients with hepatocellular carcinoma.Hepatology. 2000; 32: 970-979Crossref PubMed Scopus (242) Google Scholar, 13Saito Y Kanai Y Sakamoto M Saito H Ishii H Hirohashi S Expression of mRNA for DNA methyltransferases and methyl-CpG binding proteins and DNA methylation status on CpG islands and pericentromeric satellite regions during human hepatocarcinogenesis.Hepatology. 2001; 33: 561-568Crossref PubMed Scopus (233) Google Scholar, 14Shen L Ahuja N Shen Y Habib NA Toyota M Rashid A Issa JP DNA methylation and environmental exposures in human hepatocellular carcinoma.J Natl Cancer Inst. 2002; 94: 755-761Crossref PubMed Scopus (223) Google Scholar However, DNA methylation has not yet been investigated in DN. Thus, information relating to CpG island hypermethylation during multistep hepatocarcinogenesis is quite limited. In the present study, we determined the methylation status of CpG islands, including 9 genes and/or 5 MINT loci in normal liver, CH, LC, DN, and HCC, and correlated the methylation status to the clinicopathological data of HCC patients. We compared the methylation frequency of 9 genes in chronic liver diseases with respect to the association of HCC. The 9 genes were selected for their involvement in carcinogenesis and frequent epigenetic inactivation in other tumor types. The present study aimed to determine the chronological pattern of CpG island hypermethylation of multiple genes along the multistep process of hepatocarcinogenesis, and to identify useful epigenetic biomarkers for the disease progression or outcome of HCCs. A total of 226 liver samples were obtained from surgically resected (60 HCC, 10 DN, and 30 LC), or needle-biopsied (12 DN and 34 CH), specimens from patients treated at the Seoul National University Hospital, Seoul, Korea. The tissue samples were formalin-fixed, paraffin-embedded tissues, consisting of 60 paired specimens of primary HCC and non-HCC liver tissues (mean age, 53.8 years; 47 males and 13 females; 54 hepatitis B virus (HBV)-positive and 6 hepatitis C virus (HCV)-positive), 22 DN (57.8 years; 14 males and 8 females; 16 HBV-positive and 4 HCV-positive), 30 LC (46.5 years; 20 males and 10 females; 27 HBV-positive, 2 HCV-positive, and 1 autoimmune etiology), 34 CH (31.5 years; 30 males and four females; 19 HBV-positive, 13 HCV-positive, and 2 autoimmune hepatitis) and 20 normal liver tissue samples (58.4 years; seven males and 13 females). 30 LC and 34 CH, which appear in Table 2, Table 4, were obtained from patients without HCCs and paired non-tumorous liver tissues from HCC patients were divided into two groups, 29 liver cirrhosis with concurrent HCC, and 31 chronic hepatitis with concurrent HCC, which appear in Table 4. DN was classified according to the International Working Party's criteria into low-grade DN and high-grade DN.15International Working Party Terminology of nodular hepatocellular lesions.Hepatology. 1995; 22: 983-993PubMed Google Scholar Low-grade DN was composed of minimally atypical hepatocytes with slightly increased cellularity, whereas high-grade DN showed cellular atypia, with an irregular trabecular and/or pseudoglandular arrangement, but insufficient for the diagnosis of malignancy. In the present study, low-grade DN was not included in the study material because low-grade DN is not well discriminated from large regenerative cirrhotic nodule.Table 2The Frequency of CpG Island Hypermethylation in Neoplastic Liver Samples and Non-Neoplastic Liver Samples without Concurrent Hepatocellular CarcinomaDiagnosisNo. of casesNo. of cases methylated for at least one gene (%)*Analyzed by χ2 test, P < 0.001.Average no. of methylated gene†Analyzed by one-way ANOVA test, P < 0.001.Hepatocellular carcinoma6060 (100)3.7Dysplastic nodule2216 (72.7)1.5Liver cirrhosis3012 (40)0.5Chronic hepatitis3400Normal liver2000* Analyzed by χ2 test, P < 0.001.† Analyzed by one-way ANOVA test, P < 0.001. Open table in a new tab Table 4Methylation Frequency for Nine Genes and the Average Number of Methylated Genes in Chronic Hepatitis and Liver Cirrhosis Samples with or without Hepatocellular CarcinomaGeneFrequency of methylation (%)LC-HCC (n = 29)CH-HCC (n = 31)LC (n = 30)CH (n = 34)APC13 (44.8)6 (19.4)4 (13.3)0COX-26 (20.7)8 (25.8)00DAP-kinase3 (10.3)03 (10)0E-cadherin2 (6.9)2 (6.5)2 (6.7)0GSTP111 (37.9)5 (16.1)5 (16.7)0hMLH10000p165 (17.2)6 (19.4)00RASSF1A0000TIMP32 (6.9)000Average no. of methylated genes*Analyzed by two-tailed t-test, P < 0.001.2.31.81.20.4LC-HCC, liver cirrhosis with concurrent hepatocellular carcinoma; CH-HCC, chronic hepatitis with concurrent HCC.* Analyzed by two-tailed t-test, P < 0.001. Open table in a new tab LC-HCC, liver cirrhosis with concurrent hepatocellular carcinoma; CH-HCC, chronic hepatitis with concurrent HCC. After identification of the tumorous lesion on the hematoxylin-eosin-stained slides of HCC or DN patients, portions of tumors, where tumor cells comprised more than 80% of the cells, were scraped from 20-μm-thick paraffin sections. Paired non-tumorous liver tissues were scraped in the areas remote from the tumor, and free from the intravascular tumor emboli. The collected materials were dewaxed by washing in xylene, and rinsed in ethanol. The dried tissues were digested using proteinase K and subjected to classical DNA extraction using phenol/chloroform/isoamylalcohol and ethanol precipitation. Sodium bisulfite modification of the DNA from 226 samples was performed as previously described.17Tsuchiya T Tamura G Sato K Endoh Y Sakata K Jin Z Motoyama T Usuba O Kimura W Nishizuka S Wilson KT James SP Yin J Fleisher AS Zou T Silverberg SG Kong D Meltzer SJ Distinct methylation patterns of two APC gene promoters in normal and cancerous gastric epithelia.Oncogene. 2000; 19: 3642-3646Crossref PubMed Scopus (172) Google Scholar Briefly, 10 μl (5 μg) of genomic DNA was heat-denatured for 6 minutes at 97°C, followed by incubation with 0.2 mol/L NaOH for 10 minutes at room temperature. The denatured DNA was treated with 3.5 mol/L sodium bisulfite and 1 mmol/L hydroquinone (pH 5.0) for 16 hours at 55°C. The reaction mixture was purified with a JETSORB gel extraction kit (Genomed, Bad Oeynhausen, Germany), and desulphonated with 0.3 mol/L NaOH for 10 minutes at room temperature. The DNA was then precipitated with three volumes of cold ethanol, dissolved in H2O, and stored at −20°C. MSP was performed to examine the methylation status at CpG islands of APC, COX-2, DAP-kinase, E-cadherin, GSTP1, hMLH1, p16, RASSF1A, TIMP-3, MINT1, MINT12, MINT25, MINT31 and MINT32 loci. The primer sequences of each locus, for both the methylated and unmethylated reactions are described in Table 1. To amplify the bisulfite-modified promoter sequence of p16, E-cadherin, COX-2 and hMLH1, a polymerase chain reaction (PCR) mixture, containing 1X PCR buffer [10 mmol/L Tris (pH 8.3), 50 mmol/L KCl and 1.5 mmol/L MgCl2], deoxynucleotide triphosphates (each at 0.2 mmol/L), primers (10 pmol each), and bisulfite-modified DNA (30–50 ng), in a final volume of 25 μl, was used. For amplification of the APC, DAP-kinase, GSTP1, RASSF1A, TIMP3, MINT1, MINT12, MINT25, MINT31, and MINT32 clones, a PCR mixture containing 1X PCR buffer [16.6 mmol/L (NH4)2SO4, 67 mmol/L Tris (pH 8.8), 6.7 mmol/L MgCl2 and 10 mmol/L β-mercaptoethanol], deoxynucleotide triphosphates (each at 1 mmol/L), primers (10 pmol each), and bisulfite-modified DNA (30–50 ng), in a final volume of 25 μl, was used. The reactions were hot-started at 98°C for 5 minutes, followed by the addition of 0.75 U of Taq polymerase (Takara Shuzo Co., Kyoto, Japan). The amplifications were carried out in a thermal cycler (PerkinElmer, Foster City, CA) for 33 cycles (40 seconds at 95°C, 50 seconds at variable temperatures according to primer, and 50 seconds at 72°C), with a final 10-minute extension. The PCR products underwent electrophoresis on 2.5% agarose gels, and were visualized under UV illumination after ethidium bromide staining.Table 1Primer Sequences and PCR Conditions for MSP AnalysisPrimer namePrimer sequence (5′–3′) forwardPrimer sequence (5′–3′) reverseProduct size (bp)Annealing temp. (°C)ReferencesAPCMTATTGCGGAGTGCGGGTCTCGACGAACTCCCGACGA985517Tsuchiya T Tamura G Sato K Endoh Y Sakata K Jin Z Motoyama T Usuba O Kimura W Nishizuka S Wilson KT James SP Yin J Fleisher AS Zou T Silverberg SG Kong D Meltzer SJ Distinct methylation patterns of two APC gene promoters in normal and cancerous gastric epithelia.Oncogene. 2000; 19: 3642-3646Crossref PubMed Scopus (172) Google ScholarUGTGTTTTATTGTGGAGTGTGGGTTCCAATCAACAAACTCCCAACAA10860COX-2MTTAGATACGGCGGCGGCGGCTCTTTACCCGAACGCTTCCG1616118Akhtar M Cheng Y Magno RM Ashktorab H Smoot DT Meltzer SJ Wilson KT Promoter methylation regulates Helicobacter pylori-stimulated cyclooxygenase-2 expression in gastric epithelial cells.Cancer Res. 2001; 61: 2399-2403PubMed Google ScholarUATAGATTAGATATGGTGGTGGTGGTCACAATCTTTACCCAAACACTTCCA17161DAP-kinaseMGGATAGTCGGATCGAGTTAACGTCCCCTCCCAAACGCCGA986019Katzenellenbogen RA Baylin SB Herman JG Hypermethylation of the DAP-kinase CpG island is a common alteration in B-cell malignancies.Blood. 1999; 93: 4347-4353Crossref PubMed Google ScholarUGGAGGATAGTTGGATTGAGTTAATGTTCAAATCCCTCCCAAACACCAA9860E-cadherinMTTAGGTTAGAGGGTTATCGCGTTAACTAAAAATTCACCTACCGAC1155716Hermans JG Graff JR Myöhänen 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: 9821-9826Crossref PubMed Scopus (5240) Google ScholarUTAATTTTAGGTTAGAGGGTTATTGTCACAACCAATCAACAACACA9753GSTP1MTTCGGGGTGTAGCGGTCGTCGCCCCAATACTAAATCACGACG915920Esteller M Corn PG Urena JM Gabrielson E Baylin SB Herman JG Inactivation of glutathione s-transferase P1 gene by promoter hypermethylation in human neoplasia.Cancer Res. 1998; 58: 4515-4518PubMed Google ScholarUGATGTTTGGGGTGTAGTGGTTGTTCCACCCCAATACTAAATCACAACA9759hMLH1MTATATCGTTCGTAGTATTCGTGTTCCGACCCGAATAAACCCAA1536021Kang GH Shim YH Ro JY Correlation of methylation of the hMLH1 promoter with lack of expression of hMLH1 in sporadic gastric carcinomas with replication error.Lab Invest. 1999; 79: 903-909PubMed Google ScholarUTTTTGATGTAGATGTTTTATTAGGGTTGTACCACCTCATCATAACTACCCACA12460p16MTTATTAGAGGGTGGGGCGGATCGCGACCCCGAACCGCGACCGTAA1506516Hermans JG Graff JR Myöhänen 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: 9821-9826Crossref PubMed Scopus (5240) Google ScholarUTTATTAGAGGGTGGGGTGGATTGTCAACCCCAAACCACAACCATAA15160RASSF1AMGTGTTAACGCGTTGCGTTGCGTATCAACCCCGCGAACTAAAAACGA936022Lo KW Kwong J Hui ABU Chan SYY To KF Chan ASC Chow LSN Teo PML Johnson PJ Huang DP High frequency of promoter hypermethylation of RASSF1A in nasopharyngeal carcinoma.Cancer Res. 2001; 61: 3877-3881PubMed Google ScholarUTTTGGTTGGAGTGTGTTAATGTGCAAACCCCACAAACTAAAAACAA10560TIMP3MCGTTTCGTTATTTTTTGTTTTCGGTTTTCCCGAAAACCCCGCCTCG1165923Bachman KE Herman JG Corn PG Merlo A Costello JF Cavenee WK Baylin SB Graff JR 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: 798-802PubMed Google ScholarUTTTTGTTTTGTTATTTTTTGTTTTTGGTTTTCCCCCCAAAAACCCCACCTCA12259MINT1MAAAAAAAAACACCTAAAACTCACTACTTCGCCTAACCTAACG1026424Ueki T Toyota M Skinner H Walter KM Yeo CJ Issa JP Hruban RH Goggins M Identification and characterization of differentially methylated CpG islands in pancreatic carcinoma.Cancer Res. 2001; 61: 8540-8546PubMed Google ScholarUGGGGTTGAGGTTTTTTGTTAGTTTCACAACCTCAAATCTACTTCA11764MINT12MTTGGGAGTTTATTTAGGTCGACAACGATCTTCCGAATTTA1525525Lee S Kim WH Jung HY Yang MH Kang GH Aberrant CpG island methylation of multiple genes in intrahepatic cholangiocarcinoma.Am J Pathol. 2002; 161: 1015-1022Abstract Full Text Full Text PDF PubMed Scopus (134) Google ScholarUTGGGAGTTTATTTAGGTTGGAAACACAACAATCTTCCAAAT15555MINT25MGTTCGTTAGAGTAATTTTGCGTTATAACTAACGAAACACCGC1285525Lee S Kim WH Jung HY Yang MH Kang GH Aberrant CpG island methylation of multiple genes in intrahepatic cholangiocarcinoma.Am J Pathol. 2002; 161: 1015-1022Abstract Full Text Full Text PDF PubMed Scopus (134) Google ScholarUAGTAATTTTGTGGTGGAAGGACTAACAAAACACCACACCC11455MINT31MTTGAGACGATTTTAATTTTTTGCAAAACCATCACCCCTAAACG1006224Ueki T Toyota M Skinner H Walter KM Yeo CJ Issa JP Hruban RH Goggins M Identification and characterization of differentially methylated CpG islands in pancreatic carcinoma.Cancer Res. 2001; 61: 8540-8546PubMed Google ScholarUGAATTGAGATGATTTTAATTTTTTGTCTAAAACCATCACCCCTAAACA10564MINT32MGATGTTAGAGGAATTTAGGCAAAACGAACGAAACGTCCG1266424Ueki T Toyota M Skinner H Walter KM Yeo CJ Issa JP Hruban RH Goggins M Identification and characterization of differentially methylated CpG islands in pancreatic carcinoma.Cancer Res. 2001; 61: 8540-8546PubMed Google ScholarUGAGTGGTTAGAGGAATTTAGGTCTAAAAAAACAAACAAAACATCCA13362M, methylated sequence; U, unmethylated sequence. Open table in a new tab M, methylated sequence; U, unmethylated sequence. We examined the methylation status of the CpG islands of 9 tumor-related genes and/or 5 MINT loci, known to be frequently methylated in other cancers, in 60 paired HCC and non-HCC liver tissue samples, 22 DN, 30 LC, 34 CH, and 20 normal liver samples (Figure 1). Samples giving negative results in the PCR with specific primer sequences for the unmethylated forms of p16 gene were excluded from the study, because the presence of an unmethylated p16 gene was considered to ensure the integrity of the bisulfite-modified DNA in the samples. The detailed results of the methylation for 9 genes in a large series of liver samples are shown in Figure 2. All 60 HCC samples had methylation of one or more genes, ranging from 1 to 7, whereas none of the samples from the normal liver tissues and CH were methylated. CpG island methylation was detected for at least one of the tested gene in 40, 72.7, and 68.3% of LC, DN, and non-HCC liver tissue from HCC patients, respectively.Figure 2.Summary of methylation analysis of APC, COX-2, E-cadherin, DAP-kinase, GSTP1, p16, RASSF1A, TIMP3, and hMLH1 in 226 liver samples. Filled boxes indicates the presence of methylation and open boxes indicates the absence of methylation. DX, diagnosis; T, tumor; N, paired non-tumorous liver tissue; No*, case number; No†, number of genes methylated; No‡, number of MINT loci methylated; CIMP-P, CpG island methylator phenotype-positive cases; CIMP-N, CIMP-negative cases; DAP-k, DAP-kinase; E-cad, E-cadherin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) When the number of genes methylated (n = 9) in each step lesion was compared, with the exclusion of the non-tumorous tissue samples of HCC patients (Table 2), the average number of genes methylated showed a stepwise increase during the progression of the lesion (0 for normal liver or CH, 0.5 for LC, 1.5 for DN, and 3.7 for HCC), and the differences between each step lesions were statistically significant (P < 0.001, one-way analysis of variance test). The number of methylated genes was remarkably higher in HCC than in the corresponding non-HCC liver samples (average 3.7 vs. 1.2, P < 0.001, analyzed by 2-tailed t-test). The frequency of aberrant methylation for each gene is summarized in Table 3. Of the 9 genes tested in this study, the genes most frequently methylated in HCCs were APC (81.7%), GSTP1 (76.7%), RASSF1A (66.7%), and p16 (48.3%). COX-2 and E-cadherin were methylated at a frequencies of 35% and 33.3%, respectively. DAP-kinase and TIMP3 were methylated in less than 10% of HCC samples, and hMLH1 was not methylated at all.Table 3Methylation Frequency for Nine Genes in Hepatocellular Carcinoma and Preneoplastic LesionsGeneFrequency of methylation (%)HCC (n = 60)DN (n = 22)LC (n = 30)CH (n = 34)Significance*Analyzed by Fisher's exact test.HCC vs. DNDN vs. LCHCC vs. LCAPC49 (81.7)10 (45.5)4 (13.3)00.0020.013<0.001COX-221 (35)4 (18.2)00NS0.027<0.001DAP-kinase6 (10)3 (13.6)3 (10)0NSNSNSE-cadherin20 (33.3)02 (6.7)00.001NS0.008GSTP146 (76.7)7 (31.8)5 (16.7)0<0.001NS<0.001hMLH10000p1629 (48.3)6 (27.3)000.1300.004<0.001RASSF1A40 (66.7)2 (9.1)00<0.001NS<0.001TIMP38 (13.3)000NS0.048HCC, hepatocellular carcinoma; DN, dysplastic nodule; LC, liver cirrhosis; CH, chronic hepatitis; NS, not significant.* Analyzed by Fisher's exact test. Open table in a new tab HCC, hepatocellular carcinoma; DN, dysplastic nodule; LC, liver cirrhosis; CH, chronic hepatitis; NS, not significant. When the methylation frequency of an individual gene in lesions of various steps was compared, with exclusion of the HCC-associated non-tumorous liver samples, the tested genes, with the exception of the hMLH1 and DAP-kinase, generally showed an increase in the methylation frequency and different methylation behaviors along the multistep carcinogenesis. hMLH1 was not methylated in any of the samples of the four step lesions, and DAP-kinase was methylated at a similar frequency in HCC, DN, and LC, but not in CH. APC, E-cadherin, and GSTP1 were methylated in LC, as well as in the neoplastic lesions. A significant difference in the methylation frequency between HCC and DN, or between HCC and LC, was found in all three genes, but only APC showed a significant difference between DN and LC (P = 0.013; Fisher's exact test). COX-2, p16 and RASSF1A were methylated in the neoplastic lesions (HCC and DN), but not in chronic liver diseases (LC and CH), and TIMP-3 was methylated in HCC only. The methylation frequency of RASSF1A was significantly different between HCC and DN (P < 0.001; Fisher's exact test), but that of COX-2 or p16 was not (P > 0.05; Fisher's exact test). A temporal order was noted in the timing of the methylation of the tested individual genes along the multistep hepatocarcinogenesis; the methylation of APC, DAP-kinase, E-cadherin, or GSTP1 preceded that of COX-2, p16, RASSF1A, or TIMP-3, along the multistep hepatocarcinogenesis (Figure 3). Table 4 shows the difference in the methylation frequency of the tested genes between LC or CH samples, associated with and without HCC. The number of genes methylated was significantly higher in LC (n = 29) or CH samples (n = 31) obtained from the patients with HCC than in LC or CH without HCC (average number of methylated genes, 1.5 vs. 0.5, and 0.9 vs. 0, respectively, P < 0.001, analyzed by two-tailed t-test). While CH without concurrent HCC showed methylation for none of the genes tested, CH with concurrent HCC showed methylation for COX-2, APC, p16, GSTP1, and E-cadherin in a decreasing order of the methylation frequency. LC without an associated HCC harbored no methylation of COX-2, p16, or TIMP-3, which were methylated in LC with a concurrent HCC. RASSF1A was methylated in the neoplastic lesions only (HCC and DN), but not in chronic liver diseases, regardless of the association of HCC. We tried to explore the clinicopathological significance of the methylation status of the genes tested. Small HCC, defined as 2 cm or less (n = 4), showed less frequent methylation than HCC larger than 2 cm (n = 55). The average number of genes methylated in small and large HCCs was 2.5 and 4.7, respectively (P = 0.004, two-tailed t-test). The methylation of GSTP1 was closely associated with tumor size (average 6.8 cm vs. 4.7 cm in methylation-positive and -negative samples, respectively, P = 0.025, two-tailed t-test). No other associations were found between the methylation status of specific genes and the clinicopathological findings, including age, sex, type of hepatitis virus, gross type, histological differentiation, and pTNM stage. We explored the survival of HCC patients, and tried to find whether the methylation status of a specific gene is significantly associated. Survival data were available for 55 of 60 HCC patients. The follow-up period ranged from 8 to 36 months (mean, 24 months). Of the 55 patients, 14 (23%) died of disease (mean, 10 months), and 41 (68%) were still alive (mean, 23 months). Overall, the patients survived between 1 and 36 months, with a mean of 20 months. The follow-up results revealed a significant difference between the overall survival and the methylation-positive or methylation-negative cases for the E-cadherin or GSTP1 (Figure 4). Thirty-six patients, negative for methylation of the E-cadherin in the HCC samples showed favorable outcomes compared to the 19 patients with methylation of this gene (mean survival time, 32 vs. 23 months, respectively; P = 0.034, log rank test). For GSTP1, 44 methylation-positive patients had a shorter survival period than the 11 methylation-negative patients (P = 0.043, log rank test), and the methylation-negative patients were all still living at the time of the follow-up. To determine the CIMP in a subset of HCCs, we investigated the methylation status of 5 cancer-specific MINT loci in HCC samples. The methylation frequencies were 46.7, 40, 66.7, 23.3, and 18.3% for the MINT31, MINT1, MINT12, MINT32, and MINT25, respectively. 20 (33.3%) of HCCs constituted CIMP-positive tumors when these were arbitrarily defined as cases where three or more loci of the 5 MINT loci tested were methylated. With respect to the methylation of the 9 genes tested, CIMP+ HCCs had a greater number of methylated genes than CIMP-negative tumors (4.4 vs. 3.3, respectively; P = 0.004, two-tailed t-test). There was no significant association between the CIMP status and the clinicopathological findings, including age, sex, gross type, histological differentiation, type of hepatitis virus infected and condition of the adjacent non-cancerous liver tissue. CpG island hypermethylation is an important mechanism for loss of function of tumor suppressor genes in human cancer. A growing number of genes have been re" @default.
• W2000940387 created "2016-06-24" @default.
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• W2000940387 date "2003-10-01" @default.
• W2000940387 modified "2023-10-14" @default.
• W2000940387 title "Aberrant CpG Island Hypermethylation Along Multistep Hepatocarcinogenesis" @default.
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