Comparative analysis of promoter methylation and gene expression endpoints between tumorous and non-tumorous tissues from HCV-positive patients with hepatocellular carcinoma.
ABSTRACT: Transcriptional silencing of tumor suppressor genes and other cancer-related genes induced by promoter CpG island hypermethylation is an important epigenetic mechanism of hepatocarcinogenesis. Previous studies have established methylation profiles of hepatocellular carcinomas (HCCs) and demonstrated that methylation of several candidate genes in resected tissues may be associated with time to recurrence. The goals of our study were to test whether specific promoter methylation and mRNA levels of candidate genes, as well as global changes in DNA methylation, can be linked with time to recurrence and clinicopathological variables in a homogenous study group of HCC patients. Forty-three tumorous and 45 non-tumorous liver tissue samples from the surgical margin were obtained from HCV-positive, HBV-negative HCC patients who underwent tumor resection surgery and who were monitored for tumor recurrence thereafter (median follow-up time: 16 months (range, 0-79 months)). Methylation-specific PCR was used to assess the promoter methylation status of P16(INK4a), SOCS-1, RASSF1A, APC, GSTP1, RIZ1, and MGMT genes, while the level of LINE-1 methylation was used as marker of global DNA methylation levels. Methylation frequencies in P16(INK4a), RASSF1A, APC, GSTP1, and RIZ1 genes were significantly greater in tumorous versus non-tumorous tissues. Methylation of RIZ1 in non-tumorous tissues was significantly associated with time to recurrence. Additionally, genomic DNA was significantly more hypomethylated in tumorous tissues, and this change was associated with shorter recurrence, but not with clinicopathological features. In conclusion, this study supports the role of aberrant methylation in the pathobiology of HCV-positive HCCs. The finding that RIZ1 methylation and increased levels of LINE-1 hypomethylation in non-tumorous tissues are associated with time to recurrence underscores the importance of assessing the epigenetic state of the liver remnant.
Project description:Aberrant DNA methylation is an important epigenetic alteration in hepatocellular carcinoma (HCC). However, the molecular processes underlying the methylator phenotype and the contribution of hepatitis viruses are poorly understood. The current study is a comprehensive methylation analysis of human liver tissue specimens. A total of 176 liver tissues, including 77 pairs of HCCs and matching noncancerous liver and 22 normal livers, were analyzed for methylation. Methylation of 19 epigenetic markers was quantified, and the results were correlated with different disease states and the presence or absence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. Based on methylation profiles, the 19 loci were categorized into 3 groups. Normal liver tissues showed methylation primarily in group 1 loci (HIC-1, CASP8, GSTP1, SOCS1, RASSF1A, p16, APC), which was significantly higher than group 2 (CDH1, RUNX3, RIZ1, SFRP2, MINT31) and group 3 markers (COX2, MINT1, CACNA1G, RASSF2, MINT2, Reprimo, DCC) (P < 0.0001). Noncancerous livers demonstrated increased methylation in both group 1 and group 2 loci. Methylation was significantly more abundant in HCV-positive livers compared with normal liver tissues. Conversely, HCC showed frequent methylation at each locus investigated in all 3 groups. However, the group 3 loci showed more dense and frequent methylation in HCV-positive cancers compared with both HBV-positive cancers and virus-negative cancers (P < 0.0001).Methylation in HCC is frequent but occurs in a gene-specific and disease-specific manner. Methylation profiling allowed us to determine that aberrant methylation is commonly present in normal aging livers, and sequentially progresses with advancing stages of chronic viral infection. Finally, our data provide evidence that HCV infection may accelerate the methylation process and suggests a continuum of increasing methylation with persistent viral infection and carcinogenesis in the liver.
Project description:It has been shown in some articles that genetic and epigenetic abnormalities cannot only be found in tumor tissues but also in adjacent regions that appear histologically normal. This phenomenon is metaphorically called field cancerization or field defect. Field cancerization is regarded as clinically significant because it is assumed to be an important factor in local recurrence of cancer. As the field showing these molecular abnormalities may not be removed completely by surgery, these changes might lead to neoplasms and subsequent transformation to a tumor. We aimed to investigate the applicability of the methylation status of six tumor suppressor genes as biomarkers for detecting field cancerization in breast cancer.The promoter methylation status of CCND2, DAPK1, GSTP1, HIN-1, MGMT and RASSF1A was determined by methylation-sensitive high-resolution melting (MS-HRM) analysis. MS-HRM methods for CCND2, MGMT and RASSF1A were developed in-house, primer sequences for DAPK1, GSTP1 and HIN-1 have already been published. Biopsy samples were taken from tumor, tumor-adjacent and tumor-distant tissue from 17 breast cancer patients. Normal breast tissues of four healthy women served as controls.All MS-HRM methods proved to be very sensitive. LODs were in the range from 0.1 to 1.5 %, LOQs ranged from 0.3 to 5.3 %. A total of 94 %, 82 % and 65 % of the tumors showed methylation of RASSF1A, HIN-1 and MGMT promoters, respectively. The methylation status of these promoters was significantly lower in tumor-distant tissues than in tumor tissues. Tumor-adjacent tissues showed higher methylation status of RASSF1A, HIN-1 and MGMT promoters than tumor-distant tissues, indicating field cancerization. The methylation status of the HIN-1 promoter in tumor-adjacent tissues was found to correlate strongly with that in the corresponding tumors (r?=?0.785, p?<?0.001), but not with that in the corresponding tumor-distant tissues (r?=?0.312, p?=?0.239).Among the gene promoters investigated, the methylation status of the HIN-1 promoter can be considered the best suitable biomarker for detecting field cancerization. Further investigation is needed to test whether it can be used for defining surgical margins in order to prevent future recurrence of breast cancer.
Project description:BACKGROUND:The functional and prognostic significance of Ras association domain family 1A gene (RASSF1A) on hepatocellular carcinoma (HCC) has not been well characterized. OBJECTIVES:This study aimed to investigate the association between Ala133Ser polymorphism or promoter methylation in RASSF1A and the prognosis of HCC in Nantong City, one of the areas with the highest incidence of cancer in China. PATIENTS AND METHODS:Using peripheral blood plasma, the incidence rate of RASSF1A Ala133Ser in 235 controls and subjects with 260 HCC was analyzed by the polymerase chain reaction and sequencing. We further investigated the RASSF1A methylation status in HCC and corresponding peri-tumorous normal tissues using the methylation-specific polymerase chain reaction approach. RESULTS:It was found that the frequency of the RASSF1A Ala133Ser T allele (Ala/Ser and Ser/Ser) genotype in HCC cases was observably higher than that of normal subjects (P < 0.001). In comparison to the Ala/Ala genotype, the T allele genotype improved the susceptibility to HCC. The study also found that RASSF1A methylation improves the risk of HCC. Furthermore, in contrast with the corresponding peri-tumorous normal tissues, we observed that the RASSF1A methylation status was markedly higher in HCC tissues (P < 0.001). The Kaplan-Meier and multivariate analyses suggested that the poor survival of HCC patients was closely connected with hepatocirrhosis, Barcelona Clinic Liver Cancer stage, Edmondson division, RASSF1A methylation and Ala133Ser polymorphism (P < 0.001). CONCLUSIONS:The polymorphism and promoter methylation of RASSF1A may be a significant factor in HCC, and can be an indicator for poor prognosis in patients with HCC.
Project description:Gene methylation is an epigenetic alteration in hepatocellular carcinoma (HCC), and hepatitis B virus (HBV) plays a crucial role in carcinogenesis of HCC. However, the association between gene methylation and HBV infection in HCC remains unclear. In our study, we conducted a comprehensive meta-analysis to evaluate the association. A total of 1,148 studies were initially retrieved from some literature database. After a four-step filtration, we obtained 69 case-control studies in this meta-analysis. Our results showed six genes (p16, RASSF1A, GSTP1, APC, p15 and SFRP1) in HBV-positive carcinoma tissues, one gene (GSTP1) in HBV-positive adjacent tissues and two gene (p16 and APC) in HBV-positive carcinoma serums, which were significantly hypermethylated. Subgroup meta-analysis by geographical populations revealed that GSTP1 methylation was significantly higher in HBV-positive carcinoma tissues in China and Japan. In addition, p16 and RASSF1A methylation was significantly higher in China but not in Japan. Our study indicated that HBV infection could induce DNA methylation in HCC and DNA methylation could lead to the development of HBV-related HCC.
Project description:BACKGROUND: Parathyroid adenomas constitute the most common entity in primary hyperparathyroidism, and although recent advances have been made regarding the underlying genetic cause of these lesions, very little data on epigenetic alterations in this tumour type exists. In this study, we have determined the levels of promoter methylation regarding the four tumour suppressor genes APC, RASSF1A, p16(INK4A) and RAR-beta in parathyroid adenomas. In addition, the levels of global methylation were assessed by analyzing LINE-1 repeats. METHODOLOGY/PRINCIPAL FINDINGS: The sample collection consisted of 55 parathyroid tumours with known HRPT2 and/or MEN1 genotypes. Using Pyrosequencing analysis, we demonstrate APC promoter 1A and RASSF1A promoter hypermethylation in the majority of parathyroid tumours (71% and 98%, respectively). Using TaqMan qRT-PCR, all tumours analyzed displayed lower RASSF1A mRNA expression and higher levels of total APC mRNA than normal parathyroid, the latter of which was largely conferred by augmented APC 1B transcription levels. Hypermethylation of p16(INK4A) was demonstrated in a single adenoma, whereas RAR-beta hypermethylation was not observed in any sample. Moreover, based on LINE-1 analyses, parathyroid tumours exhibited global methylation levels within the range of non-neoplastic parathyroid tissues. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that APC and RASSF1A promoter hypermethylation are common events in parathyroid tumours. While RASSF1A mRNA levels were found downregulated in all tumours investigated, APC gene expression was retained through APC 1B mRNA levels. These findings suggest the involvement of the Ras signaling pathway in parathyroid tumorigenesis. Additionally, in contrast to most other human cancers, parathyroid tumours were not characterized by global hypomethylation, as parathyroid tumours exhibited LINE-1 methylation levels similar to that of normal parathyroid tissues.
Project description:The global increase in lung cancer burden, together with its poor survival and resistance to classical chemotherapy, underscores the need for identification of critical molecular events involved in lung carcinogenesis. Here, we have applied quantitative profiling of DNA methylation states in a panel of five cancer-associated genes (CDH1, CDKN2A, GSTP1, MTHFR, and RASSF1A) to a large case-control study of lung cancer. Our analyses revealed a high frequency of aberrant hypermethylation of MTHFR, RASSF1A, and CDKN2A in lung tumors as compared with control blood samples, whereas no significant increase in methylation levels of GSTP1 and CDH1 was observed, consistent with the notion that aberrant DNA methylation occurs in a tumor-specific and gene-specific manner. Importantly, we found that tobacco smoking, sex, and alcohol intake had a strong influence on the methylation levels of distinct genes (RASSF1A and MTHFR), whereas folate intake, age, and histologic subtype had no significant influence on methylation states. We observed a strong association between MTHFR hypermethylation in lung cancer and tobacco smoking, whereas methylation levels of CDH1, CDKN2A, GSTP1, and RASSF1A were not associated with smoking, indicating that tobacco smoke targets specific genes for hypermethylation. We also found that methylation levels in RASSF1A, but not the other genes under study, were influenced by sex, with males showing higher levels of methylation. Together, this study identifies aberrant DNA methylation patterns in lung cancer and thus exemplifies the mechanism by which environmental factors may interact with key genes involved in tumor suppression and contribute to lung cancer.
Project description:OBJECTIVES: The aim of this study was to evaluate the characteristic power Doppler sonographic images of buccal space tumorous and non-tumorous lesions. METHODS: 48 patients with buccal space lesions were evaluated with greyscale sonography followed by power Doppler sonography with a 12 MHz linear transducer. On greyscale sonography, buccal space lesions were assessed for the boundary (clear or unclear), echogenicity (hypoechoic or isoechoic) and internal architecture (homogeneous or heterogeneous). Power Doppler sonography was performed to evaluate the vascular signals within the buccal space lesions. RESULTS: 48 lesions were found in the 48 patients; of these 48 lesions, 28 were tumourous and 20 were non-tumourous. In the 28 tumours, 15 cases showed clear boundaries, 15 cases were hypoechoic relative to adjacent tissues and 22 cases presented with a heterogeneous appearance on greyscale sonography. The internal vascularity of 19 tumours was shown using power Doppler sonography. In the 20 non-tumorous lesions, 11 cases showed clear boundaries, 17 cases were hypoechoic relative to adjacent tissues and 13 cases presented with a homogeneous appearance on greyscale sonography. 18 non-tumorous lesions showed no internal vascularity using power Doppler sonography. Logistic multivariate regression analysis between the tumour group and the non-tumorous lesions group demonstrated that the internal architecture (odds ratio = 8.270, p = 0.029) and vascular signals (odds ratio = 17.533, p = 0.003) were significant variables. CONCLUSIONS: Power Doppler sonography is a useful technique for the differential diagnosis of tumorous and non-tumorous buccal space lesions.
Project description:BACKGROUND: Aberrant DNA methylation of regulatory genes has frequently been found in human breast cancers and correlated to clinical outcome. In the present study we investigate stage specific changes in the DNA methylation patterns in order to identify valuable markers to understand how these changes affect breast cancer progression. METHODS: Quantitative DNA methylation analyses of 12 candidate genes ABCB1, BRCCA1, CDKN2A, ESR1, GSTP1, IGF2, MGMT, HMLH1, PPP2R2B, PTEN, RASSF1A and FOXC1 was performed by pyrosequencing a series of 238 breast cancer tissue samples from DCIS to invasive tumors stage I to IV. RESULTS: Significant differences in methylation levels between the DCIS and invasive stage II tumors were observed for six genes RASSF1A, CDKN2A, MGMT, ABCB1, GSTP1 and FOXC1. RASSF1A, ABCB1 and GSTP1 showed significantly higher methylation levels in late stage compared to the early stage breast carcinoma. Z-score analysis revealed significantly lower methylation levels in DCIS and stage I tumors compared with stage II, III and IV tumors. Methylation levels of PTEN, PPP2R2B, FOXC1, ABCB1 and BRCA1 were lower in tumors harboring TP53 mutations then in tumors with wild type TP53. Z-score analysis showed that TP53 mutated tumors had significantly lower overall methylation levels compared to tumors with wild type TP53. Methylation levels of RASSF1A, PPP2R2B, GSTP1 and FOXC1 were higher in ER positive vs. ER negative tumors and methylation levels of PTEN and CDKN2A were higher in HER2 positive vs. HER2 negative tumors. Z-score analysis also showed that HER2 positive tumors had significantly higher z-scores of methylation compared to the HER2 negative tumors. Univariate survival analysis identifies methylation status of PPP2R2B as significant predictor of overall survival and breast cancer specific survival. CONCLUSIONS: In the present study we report that the level of aberrant DNA methylation is higher in late stage compared with early stage of invasive breast cancers and DCIS for genes mentioned above.
Project description:When assessing outcome in hepatocellular carcinoma (HCC), it is important to consider prognostic factors in background non-tumorous liver tissue as well as in the tumor, since multiple occurrence is associated with background liver status such as hepatitis. The current study aimed to elucidate molecular prognostic predictors that have an association with HCC background non-tumorous tissue. Microarray expression profiling identified aldo-keto reductase family 1, member B10 (AKR1B10) as a putative non-tumorous prognostic factor, and AKR1B10 gene expression was investigated in 158 curatively resected HCC cases by reverse transcription-quantitative polymerase chain reaction. AKR1B10 expression (AKR1B10 value/GAPDH value × 1,000) was significantly higher in tumor tissue (median, 9.2200; range, 0.0003-611.0200; n=158) than in the corresponding non-tumorous tissue (median, 0.5461; range, 0.0018-69.0300; n=158) (P<0.001). When the samples were grouped according to AKR1B10 expression in tumor tissue relative to non-tumorous tissue, tumor<non-tumorous expression (n=26) significantly correlated with poor recurrence-free survival (P=0.0074) and overall survival (OS) (P<0.0001), and was an independent prognostic factor for OS (P=0.0011) in a multivariate analysis. The ratio of AKR1B10 messenger RNA levels in HCC and corresponding non-tumorous tissues may predict prognosis after curative hepatectomy, with low expression in HCC tissue relative to non-tumorous tissue indicative of poor prognosis.
Project description: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).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.Twelve tumor-suppressor genes: ARHI, RASSF1A, HIN-1, RARbeta2, hMLH1, 14-3-3 sigma, RIZ1, p16, E-cadherin, RIL, CDH13, and NKD2 were selected for this methylation study. Five of them (RIL, HIN-1, RASSF1A, CDH13, and RARbeta2) 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.We have defined two panels (HIN-1/RASSFIA, and RIL/CDH13) of methylation profiles, which correlated, either positively or negatively, to HR status.