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From the Departments of Pathology* and Internal Medicine, Seoul National University College of Medicine, Seoul; and the Cancer Research Institute, Seoul, Korea
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Abstract |
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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,7-11 and a few have attempted to analyze the hypermethylation of multiple genes in HCCs and associated chronic liver diseases.12-14 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.
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Materials and Methods |
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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 Tables 2 and 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.15
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.
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Sodium Bisulfite Modification and Methylation-Specific (MSP) PCR
Sodium bisulfite modification of the DNA from 226 samples was performed as previously described.17 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 ß-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.
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Results |
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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.
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, 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). Frequency of Methylation of Each Gene in Different Stages of Multistep Hepatocarcinogenesis
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.
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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.
Clinicopathological Correlations and Survival Analysis
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.
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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.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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There have been previous studies investigating CpG island hypermethylation in chronic liver diseases associated with HCCs, but only few on CpG island hypermethylation in chronic liver diseases from patients without HCCs.7-14 In the present study, we found that the methylation profiles in chronic liver diseases associated with HCC were quite different from those free of HCC. Compared with chronic liver diseases without an associated HCC, those with a concurrent HCC showed significant increases in the methylation frequency of an individual gene, and the number of genes methylated. The occurrence of CpG island hypermethylation of APC, COX-2, E-cadherin, GSTP1 or p16 in CH with a concurrent HCC suggests that these methylation markers could be used for the risk assessment of a malignant transformation. In LC, the methylation of COX-2, p16 or TIMP-3 may serve as such biomarkers, although the methylation frequency of TIMP-3 was low (less than 10%), and therefore, less informative.
The significant difference of genes methylated between chronic liver diseases with and without an associated HCC suggests that HCC may arise in the liver with a methylation field defect. However, the possibility that the methylation events in chronic liver diseases with a concurrent HCC might be due to contamination of adjacent malignant cells cannot be excluded, although most of the non-tumorous samples obtained were more than 3 cm from HCC, and were confirmed to be free of microscopic tumor emboli with microscopic examination. Against the possibility of contamination are the findings of the discordant methylation patterns of the tested genes, where the specific genes were methylated in non-tumorous liver samples, but not in the corresponding HCC. This discordance can be seen in the results of Zöchbauer-Müller et al,26 who studied CpG island hypermethylation of multiple genes in non-small cell lung cancers, and described that the genes methylated in the nonmalignant lung tissues were not methylated in the corresponding lung cancers from the same patients. A possible explanation for this discordance is that hepatocytes of CH or LC might have genetic or epigenetic changes,12 but individual hepatocytes from the same samples might be heterogeneous with respect to these changes,27,28 and a methylation-negative-clone for a certain gene among heterogeneous hepatocytes may acquire a growth advantage, and develops into HCC.
E-cadherin mediates homotypic cell-cell adhesion of epithelial cells and reduction of E-cadherin may be related to the acquisition of cell motility and tumor cell invasion.29 The down-regulation of E-cadherin is related to allelic deletion of the gene or CpG island hypermethylation of the promoter CpG islands and both mechanisms have been demonstrated in HCC.29-31 In the present study, E-cadherin was methylated in premalignant lesions at a frequency less than 7% and the methylation frequency increased markedly to 30% in HCC, the difference being statistically significant. The results suggest that E-cadherin methylation may participate in malignant transformation of HCC. Reduced expression of E-cadherin decreases intercellular adhesiveness, which may result in initiation of invasion and destruction of normal tissue morphology. Recently, two groups of researchers have analyzed the methylation status of E-cadherin for the non-tumorous liver tissues from patients with HCC using restriction analysis,29,30 but the methylation frequency was quite different from each other (5% vs. 81%). The former lower frequency was consistent with the result of the present study. This discrepancy may be related to the difference of the sensitivity of the methods detecting methylation or the difference of CpG sites analyzed in restriction analysis of these researchers and MSP of the present study because a striking variability between the methylation level of individual CpG site has been demonstrated in the normal epithelial cell or cancer cell line with reduced expression of E-cadherin.27,32
The findings of the present study indicate that HCC is one of the tumors with a high frequency of CpG island hypermethylation. Although the underlying mechanism of aberrant methylation in cancer cells remains unclear, a series of studies have suggested a relationship between viral oncogenesis and aberrant methylation, which was identified in SV40-positive malignant mesotheliomas and EBV-positive gastric carcinomas.33,34 Such a relationship is supported by in vitro studies that have demonstrated frequent methylation of both viral DNA and adjacent host DNA segment following viral integration into the host DNA.35-37 However, viral integration into the host DNA does not seem to be necessary for the activation of aberrant methylation because HCV does not insert into the host DNA, in contrast to HBV, and there was no difference in the methylation frequency between HBV-positive and HCV-positive HCCs in our study (data not shown). The relationship between hepatitis virus and aberrant methylation was suggested in a recent study13 that demonstrated a higher frequency of CpG island methylation in hepatitis-virus-positive HCCs than in hepatitis-virus-negative ones.
To summarize, we studied the CpG island hypermethylation of nine tumor-related genes, and determined the frequency and chronology of methylation events of specific genes during the multistep hepatocarcinogenesis from CH to HCC. Our results demonstrated that CpG island hypermethylation occurs in the premalignant stages, and tends to accumulate during multistep hepatocarcinogenesis. Our data suggested that the CpG island hypermethylation of COX-2 or p16 might be potential molecular markers for the identification of patients with chronic liver disease at high risk for progression into HCC, and the CpG island hypermethylation of E-cadherin or GSTP1 might serve as potential biomarker for the prognostication of HCC.
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Footnotes |
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Supported in part by grant number 03–2001-012 from the Seoul National University Hospital Research Fund, and by the 2002 BK21 project for Medicine, Dentistry, and Pharmacy, Seoul, Korea.
Accepted for publication June 19, 2003.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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gene in human hepatocellular carcinoma. Oncogene 2000, 19:5298-5302[Medline]
) DNA is associated with enhanced methylation of cellular DNA segments. Proc Natl Acad Sci USA 1995, 92:5515-5519This article has been cited by other articles:
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