Distinct methylation profiles of glioma subtypes

Gliomas are tumors of the central nervous system with a wide spectrum of different tumor types. They range from pilocytic astrocytoma, with a generally good prognosis, to the extremely aggressive malignant glioblastoma. In addition to these 2 types of contrasting neoplasms, several other subtypes can be distinguished, each characterized by specific phenotypic, as well as genotypic features. Recently, the epigenotype, as evident from differentially methylated DNA loci, has been proposed to be useful as a further criterion to distinguish between tumor types. In our study, we screened 139 tissue samples, including 33 pilocytic astrocytomas, 46 astrocytomas of different grades, 7 oligoastrocytomas, 10 oligodendrogliomas, 10 glioblastoma multiforme samples and 33 control tissues, for methylation at CpG islands of 15 different gene loci. We used the semiquantitative high throughput method MethyLight to analyze a gene panel comprising ARF, CDKN2B, RB1, APC, CDH1, ESR1, GSTP1, TGFBR2, THBS1, TIMP3, PTGS2, CTNNB1, CALCA, MYOD1 and HIC1. Seven of these loci showed tumor specific methylation changes. We found tissue as well as grade specific methylation profiles. Interestingly, pilocytic astrocytomas showed no evidence of CpG island hypermethylation, but were significantly hypomethylated, relative to control tissues, at MYOD1. Our results show that glioma subtypes have characteristic methylation profiles and, with the exception of pilocytic astrocytomas, show both locus specific hyper- as well as hypomethylation.     

Distinct CpG island methylation profiles and BRAF mutation status in serrated and adenomatous colorectal polyps

A subset of colorectal cancers with CpG island methylator phenotype-high (CIMP-H) is frequently associated with MSI and BRAF V600E mutation. Since limited data are available on different histological types of colorectal polyps, we compared the pattern and the frequency of promoter methylation, CIMP-H, MSI, KRAS and BRAF V600E mutations and the relationship among these molecular parameters and the clinicopathologic characteristics in 110 serrated polyps (48 hyperplastic polyps, 32 sessile serrated adenomas and 30 serrated adenomas) and 32 tubular adenomas using 7 commonly used tumor-associated gene loci. No significant difference in the frequency of overall methylation frequency (86% vs. 100%) and CIMP-H (39% vs. 28%) between serrated polyps and tubular adenomas was observed, but proximally located serrated polyps showed more frequent methylation at 5 of 7 loci examined, and were more likely to be CIMP-H (62% vs. 22%). MGMT methylation was more common in tubular adenomas while MLH1 and HIC1 were more frequently methylated in serrated polyps. BRAF mutation was frequently present in all types of serrated polyps (80%), but was absent in tubular adenomas and was not associated with CIMP or MSI status. These results show comparable frequencies of promoter methylation of tumor-associated genes and CIMP-H, but distinct differences in gene-specific or colonic site-specific methylation profiles occur in serrated polyps and tubular adenomas. BRAF mutation occurs independently of CIMP and MSI in all types of serrated polyps and may serve as a marker of serrated pathway of colorectal carcinogenesis.     

Examination of a CpG island methylator phenotype and implications of methylation profiles in solid tumors

The CpG island methylator phenotype (CIMP), thoroughly described in colorectal cancer and to a lesser extent in other solid tumors, is important in understanding epigenetics in carcinogenesis and may be clinically useful for classification of neoplastic disease. Therefore, we investigated whether this putative phenotype exists in exposure-related solid tumors, where somatic gene alterations and enhanced clonal growth are selected for by carcinogens, and examined the ability of methylation profiles to classify malignant disease. We studied promoter hypermethylation of 16 tumor suppressor genes and 3 MINT loci (acknowledged classifiers of CIMP) in 344 bladder cancers, 346 head and neck squamous cell carcinomas (HNSCC), 146 non-small-cell lung cancer (NSCLC), and 71 malignant pleural mesotheliomas (MPM). We employed rigorous statistical methods to examine the distribution of promoter methylation and the usefulness of these profiles for disease classification. In bladder cancer, HNSCC, and NSCLC, there was a significant correlation (P < 0.0001) between methylation of the three MINT loci and methylation index, although the distribution of methylated loci varied significantly across these disease. Although there was a significant (P < 0.001) association between gene methylation profile and disease, rates of misclassification of each disease by their methylation profile ranged from 28% to 32%, depending on the classification scheme used. These data suggest that a form of CIMP exists in these solid tumors, although its etiology remains elusive. Whereas the gene profiles of hypermethylation among examined loci could not unequivocally distinguish disease type, the existence of CIMP and the relative preponderance of hypermethylation in these cancers suggest that methylation analysis may be clinically useful as a targeted screening tool.     

CpG island methylation in carcinoid and pancreatic endocrine tumors

Carcinoid tumors and pancreatic endocrine tumors (PETs) are uncommon neuroendocrine neoplasms and their genetic alterations are not well characterized. CpG island methylation is a mechanism of gene silencing, and concordant methylation of multiple CpG islands as CpG island methylator phenotype (CIMP) has been described in tumors. The aim of this study was to evaluate CIMP in carcinoid tumors and PETs. We studied 16 carcinoid tumors, 11 PETs, and 22 associated normal mucosa or pancreas. Methylation status of the p14, p16, cyclo-oxygenase 2 (COX2), O(6)-methyl-guanine methyltransferase (MGMT), estrogen receptor (ER), thrombospondin 1 (THBS1), retinoic acid receptor beta 2 (RARbeta), T-type calcium channel (CACNA1G), and multiple endocrine neoplasia type-1 (MEN1) genes, and of MINT1, MINT2, MINT25, MINT27 and MINT31 loci was evaluated by methylation-specific-PCR (MSP) or combined bisulfite restriction analysis (COBRA). Carcinoid tumors were frequently methylated at RARbeta, MGMT, p16, COX2, p14, THBS1, and ER ranging from 25 to 63% of tumors. Other CpG islands were infrequently methylated or unmethylated. The adjoining normal mucosa was also methylated for ER, COX2, and RARbeta, but methylation at p14, p16, THBS1, and MGMT was tumor-specific. By contrast, PETs and normal pancreas were frequently methylated only at ER. Methylation was more frequent in carcinoid tumors than PETs at MGMT (25 versus 0%, p = 0.03), THBS1 (44 versus 9%, p = 0.04), p14 (44 versus 9%, p = 0.04) and RARbeta (25 versus 0%, p = 0.03). Loss of p16 protein expression correlated with methylation of p16 gene in carcinoid tumors (p = 0.006). Our study indicates that methylation profile of carcinoid tumors differs from PETs, reflecting different molecular pathogenesis.     

Correlation between CpG methylation profiles and hormone receptor status in breast cancers

Introduction

Aberrant DNA methylation has been found frequently in human breast cancers, associated with the loss of expression of a number of regulatory genes for growth and correlated to clinical outcomes. The present study was undertaken to determine whether methylation of a set of growth-suppressor genes would correlate to the expression of estrogen receptors (ERs) and progesterone receptors (PRs).

Methods

We used a pyrosequencing methylation analysis to study the methylation of 12 known growth-suppressor genes in 90 pairs of malignant/normal breast tissues. We also examined the expression of ERs and PRs in those specimens by immunohistochemistry. Mutations of p53 in tumor cells were detected by direct sequencing.

Results

Twelve tumor-suppressor genes: ARHI, RASSF1A, HIN-1, RARβ2, hMLH1, 14-3-3 σ, RIZ1, p16, E-cadherin, RIL, CDH13, and NKD2 were selected for this methylation study. Five of them (RIL, HIN-1, RASSF1A, CDH13, and RARβ2) were frequently methylated in breast cancers (57%, 49%, 58%, 44%, and 17%, respectively) but not the normal breast (0–4%). Two panels of methylation profiles were defined. The methylation of the HIN-1/RASSFIA panel strongly correlated to the expression of ERs, PRs, and hormone receptors (HRs; which were defined as 'positive' if ERs and/or PRs were positive; p < 0.001). Conversely, the methylation of the RIL/CDH13 panel strongly correlated to negative ER, PR, and HR expression (p = 0.001, 0.025, and 0.001, respectively). The subset of triple-negative breast cancers (in other words, those with negative ER, PR, and HER-2/neu status) was positively associated with the methylation of the RIL/CDH13 panel and negatively associated with the HIN-1/RASSF1A panel. Mutations of p53 were found in nine breast tumors (11%), seven of which lacked methylation in both panels.

Conclusion

We have defined two panels (HIN-1/RASSFIA, and RIL/CDH13) of methylation profiles, which correlated, either positively or negatively, to HR status.     

Distinct methylation patterns of benign and malignant liver tumors revealed by quantitative methylation profiling.

PURPOSE: A comparative quantitative methylation profiling of hepatocellular carcinoma and the most frequent benign liver tumor, hepatocellular adenoma, was set up for the identification of tumor-specific methylation patterns. EXPERIMENTAL DESIGN: The quantitative methylation levels of nine genes (RASSF1A, cyclinD2, p16INK4a, DAP-K, APC, RIZ-1, HIN-1, GSTpi1, SOCS-1) were analyzed in hepatocellular carcinoma and adjacent normal tissue (n = 41), hepatocellular adenoma and adjacent normal tissue (n = 26), focal nodular hyperplasia (n = 10), and unrelated normal liver tissue (n = 28). Accumulated methylation data were analyzed using various statistical algorithms, including hierarchical clustering, to detect tumor-specific methylation patterns. RESULTS: Cluster analysis revealed that hepatocellular adenoma displays a methylation profile much more similar to that found in normal liver tissue and focal nodular hyperplasia than to that found in hepatocellular carcinoma. Many characteristic differences were not detected when using mere qualitative methylation assays. The cyclinD2 gene was identified as a new and frequent target for aberrant hypermethylation in hepatocellular carcinoma (68%). In the control group of 28 liver specimens from healthy donors, a clear correlation between age of patient and frequency and level of aberrant methylation was seen, which could not be detected in the group of hepatocellular carcinoma specimens. CONCLUSIONS: Methylation profiling can clearly contribute to the unequivocal classification of suspicious lesions, but only if done in a quantitative manner applying cell type and gene-specific thresholds. In hepatocellular carcinoma, the altered methylation patterns accompanying malignant transformation override the age-dependent increase in gene methylation.     

Distinct DNA methylation profiles in malignant mesothelioma, lung adenocarcinoma, and non-tumor lung

DNA methylation markers provide a powerful tool to make diagnoses based on genetic material obtained directly from tumors or from "remote" locations such as sputum, pleural fluid, or serum. In particular when limited cell numbers are available, amplifyable DNA markers can provide a very sensitive tool for cancer detection and classification. Malignant mesothelioma (MM), an aggressive cancer strongly associated with asbestos exposure, can be difficult to distinguish from adenocarcinoma of the lung when limited material is available. In an attempt to identify molecular markers for MM and adenocarcinoma, we examined the DNA methylation status of 14 loci. Analysis of methylation levels in 10 MM and 8 adenocarcinoma cell lines showed that methylation of APC was significantly elevated in adenocarcinoma compared to MM cell lines (P=0.0003), while methylation of CDH1 was higher in MM (P<0.02). Subsequent examination of the methylation status of the 14 loci in 6 MM and 7 adenocarcinoma primary tumors, which yielded similar methylation profiles, supported these observations. Comparison of methylation in MM cell lines and tumors versus non-tumor lung tissue indicated that APC exhibits less methylation in MM (P=0.003) while RASSF1, PGR1, ESR1, and CDH1 show more methylation in MM, the latter two showing the most significant difference between the two tissue types (P< or = 0.0001). Comparison of methylation in adenocarcinoma cell lines and tumors versus non-tumor lung tissue showed methylation of ESR1, PGR1 and RASSF1 to be significantly elevated in adenocarcinoma, with RASSF1 being most significant (P=0.0002). Thus, with the examination of 14 loci, we have identified 5 candidates that show potential for distinguishing between MM, adenocarcinoma and/or non-cancer lung. Our observations support the strong potential of methylation markers as tools for accurate diagnosis of neoplasms in and around the lung.     

CpG methylation in the Fhit regulatory region: relation to Fhit expression in murine tumors

To determine if: (1) 5' CpG island methylation is related to Fhit inactivation; (2) there are tumor or carcinogen-specific methylation patterns, we examined 35 CpG sites in the promoter, exon and intron 1 of the mouse Fhit gene. In primary tumors of lung, urinary bladder and tongue, induced by different carcinogens, 15-35% of sites were methylated, with specific methylation patterns associated with each cancer type, suggesting cancer- or tissue-specific methylation patterns. The methylation patterns were associated with reduced Fhit expression, as determined by immunohistochemical analyses. Methylation of rat Fhit 5' CpGs in mammary adenocarcinomas, detected by methylation specific PCR amplification, also correlated with reduced gene expression. Thus, there was an overall association between promoter/exon 1 methylation and decreased Fhit expression. In contrast, in cancer-derived cell lines 70-95% of the CpG sites were methylated. This is the first detailed study of the relationship between Fhit 5' CpG island methylation and Fhit expression in murine tumors, our main models for preclinical cancer studies, and provides evidence that loss of Fhit expression and methylation are correlated in these mouse models and these models will be useful to examine the complex relationships among gene expression, methylation patterns and organ specificity.     

Distinct genomic profiles in hereditary breast tumors identified by array-based comparative genomic hybridization

Mutations in BRCA1 and BRCA2 account for a significant proportion of hereditary breast cancers. Earlier studies have shown that inherited and sporadic tumors progress along different somatic genetic pathways and that global gene expression profiles distinguish between these groups. To determine whether genomic profiles similarly discriminate among BRCA1, BRCA2, and sporadic tumors, we established DNA copy number profiles using comparative genomic hybridization to BAC-clone microarrays providing <1 Mb resolution. Tumor DNA was obtained from BRCA1 (n = 14) and BRCA2 (n = 12) mutation carriers, as well as sporadic cases (n = 26). Overall, BRCA1 tumors had a higher frequency of copy number alterations than sporadic breast cancers (P = 0.00078). In particular, frequent losses on 4p, 4q, and 5q in BRCA1 tumors and frequent gains on 7p and 17q24 in BRCA2 tumors distinguish these from sporadic tumors. Distinct amplicons at 3q27.1-q27.3 were identified in BRCA1 tumors and at 17q23.3-q24.2 in BRCA2 tumors. A homozygous deletion on 5q12.1 was found in a BRCA1 tumor. Using a set of 169 BAC clones that detect significantly (P < 0.001) different frequencies of copy number changes in inherited and sporadic tumors, these could be discriminated into separate groups using hierarchical clustering. By comparing DNA copy number and RNA expression for genes in these regions, several candidate genes affected by up- or down-regulation were identified. Moreover, using support vector machines, we correctly classified BRCA1 and BRCA2 tumors (P < 0.0000004 and 0.00005, respectively). Further validation may prove this tumor classifier to be useful for selecting familial breast cancer cases for further mutation screening, particularly, as these data can be obtained using archival tissue.     

Expression and CpG methylation of the insulin-like growth factor II gene in human smooth muscle tumors.

Previously we have shown that expression of the insulin-like growth factor II (IGF-II) gene in 36 normal smooth muscle tissues (myometria) and 26 benign smooth muscle tumors (leiomyomas) was detectable by Northern blot analysis but that the RNA levels were low. In 9 of 20 malignant smooth muscle tumors (leiomyosarcomas) IGF-II gene expression was also low or absent, while in 11 of 20 the IGF-II gene was abundantly expressed. In 32 of these tissues we have now studied the DNA methylation state of the IGF-II gene. For the analysis of overall methylation of the gene the restriction endonucleases HpaII and MspI were used. In normal smooth muscle and in leiomyomas the IGF-II gene appeared to be methylated. In leiomyosarcomas with low IGF-II gene expression the DNA was partly demethylated. In leiomyosarcomas with abundant IGF-II gene expression overall methylation of the DNA tended to be low. In addition, we have studied the methylation state of one particular CpG site in the IGF-II gene with the restriction endonuclease AvaII. The results of the latter analysis confirm the analysis with HpaII and MspI. In conclusion, in malignant smooth muscle tumors the data indicate an inverse correlation between CpG methylation and expression of the IGF-II gene.     

The RNA component of telomerase as a marker of biologic potential and clinical outcome in childhood neuroblastic tumors

BACKGROUND: Telomerase is a ribonucleoprotein enzyme associated with cellular immortality that may be useful in determining the biologic potential of a tumor. Neuroblastoma (NB), ganglioneuroblastoma (GNB), and ganglioneuroma (GN) are neuroblastic tumors (NTs) that exhibit a spectrum of histologic features and are often associated with unpredictable behavior and clinical outcome. METHODS: The authors investigated the expression of the RNA component of human telomerase (hTR) by in situ hybridization in 32 cases of NTs (including 24 NBs, 4 GNBs, and 4 GNs), using [35S]-UTP labeled single stranded sense and antisense RNA probes. Eight NBs were early stage, 12 NBs were advanced stage, and 4 NBs were Stage IVS, a widely metastatic variant associated with an excellent clinical prognosis. Four NBs had N-myc amplification. In addition, the authors compared a proliferation marker, MIB-1, with hTR expression in a subset of tumors. RESULTS: Thirty of 32 NTs expressed hTR, with expression varying from weak (1+) to intense (4+). Most advanced stage NBs (9 of 12) and only 2 of 8 early stage NBs had moderate to intense (2 to 4-) expression of hTR. The remaining early stage tumors (6 of 8) and 3 of 12 advanced stage NBs had absent or weak expression of hTR (0 to 1+). There was no disease progression in any of the patients with absent or weak expression of hTR. In contrast, 8 tumors (from 7 patients) with moderate to intense expression of hTR in the tumor sections had adverse clinical outcomes, including recurrence, persistent disease, or death. hTR expression in all the Stage IVS tumors was weak, despite the fact that the patients had widely metastatic disease at presentation. The mean hTR score of 3.1 for NBs associated with an adverse outcome (n = 8) was significantly different from the mean hTR score of 1.3 for NBs associated with a favorable outcome (n = 16), P < 0.001. hTR expression in the GNB/GNs was limited to the ganglion cells only; Schwann cells were negative for hTR expression. Stage IVS tumors, which are associated with an excellent outcome, had high MIB-1 but weak hTR expression, indicating that the latter may be a better discriminator of true biologic potential and that hTR levels do not always correlate with cell proliferation. CONCLUSIONS: Increased hTR expression may reflect the potential for aggressive behavior within the spectrum of NTs; conversely, down-regulation of hTR may be useful in identifying subsets with limited capacity for progression and a favorable prognosis.     

CpG methylation of viral DNA in EBV-associated tumours

In EBV-immortalized lymphoblastoid cell lines (LCLs) a small number of "latent" proteins are expressed. These are the EBV nuclear antigens, EBNAs 1-6, and a latent membrane protein, LMP. We have investigated the expression of these proteins in a variety of EBV-associated tumours and cell lines. Whereas transplant and B-cell lymphomas from cotton-top tamarins appear to express the full range of antigens found in LCLs, we and others have found that in Burkitt's lymphomas (BL) and a nasopharyngeal carcinoma (NPC) isolate, EBNA expression is restricted to EBNA-I. (In NPC, but not in BL, LMP may also be expressed). In order to ask what restricts the expression of EBNA 2-6 in NPC and BL cells it seemed reasonable to consider the possibility that the DNA sequences normally regulating expression of these antigens could be chemically modified. In this analysis, a tight inverse correlation between methylation of CpG dinucleotides in the 5' flanking region of the EBNA-2 gene and the expression of EBNAs 2-6 has been revealed. In the NPC tumour, CpG methylation within the gene is also observed, as is specific methylation over the EBNA-I region I and II binding sites (in oriP). The significance of these observations is considered.     

新疆哈萨克族食管癌survivin启动子区CpG岛甲基化状态及其临床意义

目的： 检测消化道肿瘤早期相关基因survivin mRNA在哈萨克族食管癌组织中的表达及其启动子区CpG岛甲基化状态,并进一步探究survivin启动子甲基化是否参与了食管癌的发生。方法：提取哈萨克族食管癌患者癌组织及远端无癌组织RNA,RT-PCR检测组织中survivin mRNA的表达水平,甲基化特异性PCR (methylation specific PCR,MSP)检测survivin启动子区CpG 岛的甲基化状态。结果：在20对食管癌组织标本中,癌组织中survivin mRNA阳性表达率 (95%)明显高于远端无癌组织 (65%) (P＜0.05)。癌组织及远端无癌组织中survivin基因启动子区CpG岛多为杂合型甲基化,且两种组织间甲基化状态无明显差异 (P＞0.05)。结论：survivin mRNA表达水平的增高在哈萨克族食管癌的发生、发展过程中起到了一定的作用,但其表达与启动子区CpG岛甲基化无关,提示甲基化并未直接调控该基因的表达并促进哈萨克族食管癌的发生发展。     

Different molecular profiles characterize well-differentiated endocrine tumors and poorly differentiated endocrine carcinomas of the gastroenteropancreatic tract.

PURPOSE: The molecular pathogenesis of gastroenteropancreatic endocrine tumors (ETs) is still largely unknown. The purpose of this work was a molecular characterization of 38 gastroenteropancreatic ETs with respect to the primary site and to the morphofunctional profile, pointing out useful diagnostic or prognostic molecular markers. EXPERIMENTAL DESIGN: Twenty-four well-differentiated ETs or carcinomas (WDET/Cs; 11 pancreatic, 3 gastric, and 10 intestinal) and 14 poorly differentiated endocrine carcinomas (1 pancreatic, 6 gastric, and 7 colorectal) were microallelotyped using 38 polymorphic microsatellite markers covering chromosomes 1, 3, 5q, 6, 11, 17, and 18. RESULTS: Regardless of the primary site, a significantly higher percentage of allelic imbalances (AIs) was observed in poorly differentiated endocrine carcinomas than in WDET/Cs (P = 0.012), except for 3 of 8 nonfunctioning pancreatic endocrine tumors and 1 colorectal WDEC, exhibiting multiple AIs on chromosomes 1, 3, 6, and 11. A strong positive correlation between AI percentage and Ki-67 proliferation index was detected considering both the whole series of ETs (P = 0.004) and the group of WDET/Cs alone (P = 0.011). The survival analysis showed a positive correlation between low percentage of AI and longer survival (P = 0.01). No recurrent AIs at specific chromosomal regions were identifiable with respect to the primary site. CONCLUSIONS: The malignant progression of endocrine tumors seems to be associated with complex allelotypes and chromosomal instability. Although no specific molecular markers of malignancy can be defined with certainty, the ploidy status and the degree of chromosomal derangements appear to be the most informative genetic factors with prognostic significance.     

BRCA1 promoter methylation in sporadic breast tumors: relationship to gene expression profiles

BRCA1 is a tumor suppressor gene that functions in DNA repair. Basal-like tumors are a distinctive subtype of breast cancer defined by gene expression profiles. Hereditary BRCA1 breast tumors and basal-like sporadic tumors have a similar phenotype and gene expression signature, suggesting involvement of BRCA1 in the pathogenesis of sporadic basal-like cancer. This study evaluates the role of BRCA1 in sporadic breast tumorigenesis. BRCA1 protein expression and promoter methylation are compared to tumor histopathology and gene expression profiles. We find BRCA1 protein expression correlates with tumor mitotic rate, consistent with normal cell-cycle regulation of the BRCA1 gene. Methylation is found in 21% of tumors and is associated with lower BRCA1 protein, but not with specific pathologic features. Basal-like tumors, defined by hierarchical clustering of gene expression, have infrequent BRCA1 methylation and high levels of BRCA1 protein expression consistent with their high mitotic rate. Tumors with BRCA1 promoter methylation are present in all expression clusters; however, a subgroup of ER-positive high-grade tumors has a significantly greater number of BRCA1 methylated tumors. Absence of BRCA1 promoter methylation and high levels of BRCA1 expression in basal-like sporadic tumors suggest alternate explanations for the phenotypic similarities of these tumors to hereditary BRCA1 tumors.     

Promoter CpG methylation of tumor suppressor genes in colorectal cancer and its relationship to clinical features

Aberrant promoter methylation of CpG islands of tumor suppressor genes inhibits expression of the genes and may lead to tumorigenesis. We investigated the aberrant methylation profile of potential tumor suppressor genes of p15, p16, SOCS-1, and Wnt signaling pathway in colorectal cancers and correlated the data with clinical findings. Cancerous and nearby non-cancerous tissues of 185 sporadic colorectal cancer samples were studied. Methylation specific PCR was performed to explore the mechanism of inactivation in p15, p16, SOCS-1, E-cadherin, APC, GSK-3beta, and Axin1 genes. Aberrant promoter methylation in p15, p16, SOCS-1, E-cadherin, APC, GSK-3beta, and Axin1 genes were 5.9, 7.0, 3.8, 5.9, 12.4, 2.2, and 0% for cancerous tissues, respectively, whereas the frequencies were 3.8, 0, 0, 7.0, 2.7, 0.5, and 0% for nearby non-cancerous tissues, respectively. The frequency of aberrant promoter methylation of cancerous tissues was significant higher than non-cancerous tissues in p16, SOCS-1, and APC genes (p<0.05) and methylation status of these genes had no clear relationship with clinical parameters. Of the 66 patients who showed at least one aberrant promoter methylation in the tumor-suppressor genes, 5 (7.6%) patients demonstrated multiple methylation phenotype (methylation > or =3) and associated with increased lymph node metastasis (p=0.036). Our findings suggest that inactivation of some tumor suppressor genes through aberrant promoter methylation of CpG islands may play a role in the development of colorectal cancer and methylation inactivation of these genes except p16 and SOCS1 may occur at the precancerous stage. Multiple methylation pathways may be involved in the tumorigenesis of colorectal cancer and associated with aggressiveness of clinical disease.