MS-HRM检测结直肠癌患者外周血AKAP12基因及其临床意义

目的 探讨MS-HRM法定量检测结直肠癌患者外周血中AKAP12基因甲基化程度及其临床意义.方法 采用MS-HRM法定量检测80例结直肠癌患者和20名健康人外周血中AKAP12基因的甲基化水平,并分析评价其与病理分期分级的关系.同时对MS-HRM法的重复性和与传统MSP法检测的灵敏度和检出率进行比较.结果 MS-HRM法从80例结直肠癌患者中检测到38例(47.5%)AKAP12基因启动子区域不同程度的甲基化患者(甲基化1%～20%的24例,20%～60%的12例,60%～100%的2例),20名健康人中仅检测到甲基化<10%的2名.MS-HRM法检测AKAP12基因甲基化的灵敏度(1%)优于传统的MSP法(10%).AKAP12基因甲基化水平与结直肠癌患者的年龄、性别的差异无统计学意义(x2分别为3.13、1.70,P均>0.05),但与结直肠癌的Dukes分期和分化程度的差异均有统计学意义(x2分别为5.93、8.41,P均=0.01).结论 外周血MS-HRM定量检测AKAP12基因甲基化程度的方法比传统MSP法灵敏度更高,AKAP12基因甲基化有望成为结直肠癌进展评估的分子指标.     

Methylated cyclin D2 gene circulating in the blood as a prognosis predictor of hepatocellular carcinoma

BackgroundPrognosis of hepatocellular carcinoma (HCC) remains poor because of high recurrence rate. We examined preoperatively the methylated CCND2 gene levels present in the serum following release from HCC cells as a prognosis predictor in patients undergoing curative hepatectomy.MethodsQuantitative real-time RT-PCR and quantitative methylation-specific PCR were used to measure methylated CCND2 gene and its mRNA levels.ResultsThe CCND2 mRNA levels were down-regulated in HCC with early intrahepatic recurrence (IHR) within 1 year of curative hepatectomy. We also identified that this down-regulation was due to promoter hypermethylation. In 70 HCC patients who underwent curative hepatectomy, 39 patients sero-positive for the methylated CCND2 gene (> 70 pg/ml serum) exhibited a significantly shorter disease-free survival (DFS) period (P = 0.02) than the 31 patients who were sero-negative for the methylated CCND2 gene. None of the sero-negative patients demonstrated early IHR, and this method of serum testing did not produce any false-negative predictions for early IHR. Multivariate analysis showed that the serum level of methylated CCND2 was an independent risk factor for DFS (hazard ratio of 1.866, 95% CI: 1.106–3.149).ConclusionMethylated CCND2 gene in the serum serves as a prognosis predictor of HCC after curative hepatectomy.     

Aberrant hypomethylation of SALL4 gene is associated with intermediate and poor karyotypes in acute myeloid leukemia

ObjectiveSALL4 gene has been identified to stimulate the expansion of hematopoietic stem cell (HSCs) and enhance the self-renewal of HSCs. Overexpression of SALL4 has been found in several cancers. The present study was aimed to investigate the methylation status of SALL4 promoter region in acute myeloid leukemia (AML).Designs and methodsThe methylation status of SALL4 promoter was analyzed in 84 patients with AML using methylation-specific PCR (MSP) and its clinical significance was evaluated.ResultsAberrant hypomethylation of SALL4 gene, which was correlated with SALL4 expression, was found in 17.8% (15/84) cases. The patients with SALL4 hypomethylation had significantly older age and higher WBCs than those without SALL4 hypomethylation. The incidence of SALL4 hypomethylation was higher in M1 subtype than in M2 and other subtypes (50%, 26% and 6%, respectively, P = 0.001). SALL4 hypomethylation was associated with cytogenetically intermediate and poor groups. Although survival time of the SALL4-hypomethylated AML was shorter than that of SALL4-methylated group (4 months vs 9 months), the difference was not statistically significant (P = 0.356).ConclusionsHypomethylation of SALL4 promoter is a common event and is associated with the intermediate and poor karyotypes in AML.     

Clinical significance of Stratifin,ERα and PR promoter methylation in tumor and serum DNA in Indian breast cancer patients

Objectives:The objective of this study was to determine the concordance of promoter methylation of stratifin, ERα and PR in tumor and circulating DNA in breast cancer patients and their association with clinicopathological parameters and disease prognosis.Design and methods:Methylation specific PCR were carried out to investigate the promoter methylation status of stratifin, ERα and PR in tumor and circulating DNA in 100 breast cancer patients in a prospective study. The effect of promoter methylation on protein expression was evaluated by immunohistochemistry.Results:Significant association was observed between promoter methylation of stratifin in tumors (61%) and paired sera (56%) (r = 0.78; p ≤ 0.001). Loss of stratifin expression was observed in 47% tumors and was associated with poor overall survival (p = 0.05). Significant correlation was observed between methylation status of ERα with PRB (p < 0.0001, OR = 20.8, 95% CI = 7.4–58.0) and stratifin (p = 0.003, OR = 2.0, 95% CI = 0.8–4.4).Conclusion:This study underscores the potential utility of serum DNA methylation of these genes as surrogate for tumor DNA methylation as a promising tool for cancer diagnosis.     

Rapid and quantitative detection of CpG-methylation status using TaqMan PCR combined with methyl-binding-domain polypeptide

ObjectivesTo assess the medical applicability of CpG methylation as molecular markers for cancer diagnosis, we established a new system to determine DNA methylation based on TaqMan PCR combined with a methyl-binding-domain polypeptide 2.Design and methodsWe evaluated the diagnostic applicability of this approach by examining the methylation status of two tumor suppressor genes, RASSF1A and APC, in 10 paired hepatocellular carcinoma (HCC) and the corresponding non-tumor liver tissues.ResultsMethylation levels of total 20 clinical samples measured by the TaqMan PCR assay showed a significantly positive correlation (R = 0.814, P < 0.0005 for RASSF1A, R = 0.736, P < 0.00001 for APC) with those calculated by bisulfite sequencing. The methylated DNA amount measured by our TaqMan PCR system precisely replicated the methylation status estimated by direct sequencing.ConclusionsThis suggests our method may serve as a reliable and easy-to-use tool for cancer diagnosis using methylated genes as biomarkers.     

Prognostic significance of RASSF1A promoter methylation in operable breast cancer

ObjectivesThe aim of our study was to evaluate the prognostic significance of RASSF1A promoter methylation status in operable breast cancer.Design and methodsBy using Methylation Specific PCR, we evaluated the specificity of RASSF1A promoter methylation in 10 breast tumors and matching normal tissues, 10 breast fibroadenomas and 11 normal breast tissues. The prognostic significance of RASSF1A methylation was validated in 93 formalin fixed paraffin-embedded (FFPE) tissues obtained from patients with operable breast cancer.ResultsMethylation of RASSF1A promoter was observed in 1/31 (3.2%) non-cancerous breast tissues and 53/93 (57.0%) early stage breast tumors. The only positive sample in the non-cancerous breast tissues group was found in a histological normal tissue surrounding the tumor. During the follow-up period, 24/93 (25.8%) patients relapsed and 19/93 (20.4%) died. Disease-Free-Interval (DFI) was significantly associated with RASSF1A methylation (p = 0.028).ConclusionsRASSF1A promoter methylation provides important prognostic information in early stage breast cancer patients.     

Improved measurement of LINE-1 sequence methylation for cancer detection

BackgroundMethylation of long interspersed nuclear element-1 (LINE-1) sequences varies among normal cells and it is often decreased in cancer genomes and white blood cells (WBC) of cancer patients. Current measurement techniques of genome-wide level are inadequate because LINE-1 methylation is distinctive at each locus. Here, we improved the detection of cancer by combining information of LINE-1 methylation pattern and level.MethodsCombined bisulfite restriction analysis (COBRA) of LINE-1, COBRA LINE-1, was used to test cancer cell lines, two oral rinse cohorts, and WBC from normal and cancer patients. COBRA LINE-1 separated LINE-1 sequences into 4 products depending on the methylation statuses of 2 CpG dinucleotides, as follows: 2 unmethylated CpGs (^uC^uC), partial methylation (^mC^uC), 1 methylated CpG (^mC), and 1 unmethylated CpG (^uC).ResultsThe association between ^mC^uC and ^uC^uC was directly correlated in normal cells (r = 0.4895, p = 0.0009) but inversely correlated in cancer (r = ? 0.8979, p = 0.0002). Oral rinse AUC values of ^uC^uC were 0.763 and 0.926 and methylation levels were 0.707 and 0.621, respectively. ^uC^uC, but not overall methylation level, differentiated cancer WBC from normal (p = 0.0082 and p = 0.4830, respectively).ConclusionLINE-1 partial methylation represents hypomethylation in normal cells but hypermethylation in cancer cells. This information improves LINE-1 methylation detection in cancer.     

Efficient detection of hepatocellular carcinoma by a hybrid blood test of epigenetic and classical protein markers

BackgroundThere are few blood tests for an efficient detection of hepatocellular carcinoma (HCC) associated with hepatitis C virus (HCV) infection.MethodsThe abilities of quantitative analyses of 7 genes hypermethylation in serum DNA, α-fetoprotein (AFP) and prothrombin-induced vitamin K absence II (PIVKA-II), and various combinations to detect HCC were evaluated in a training cohort of 164 HCV-infected patients (108 HCCs; 56 non-HCCs). An optimal hybrid detector, built using data for 2 methylated genes (SPINT2 and SRD5A2), AFP, and PIVKA-II, achieved the most satisfactory ability to detect HCC in the training cohort. We evaluated the ability of the optimal hybrid detector to detect HCC in an independent validation cohort of 258 consecutive HCV-infected patients (112 HCCs; 146 non-HCCs) who were newly enrolled in 4 distinct institutes.ResultsIn the validation cohort of 258 patients, accuracy, sensitivity, and specificity of the hybrid detector for detection of HCC were 81.4%, 73.2%, and 87.7%, respectively. Notably, even when detecting HCC ≤ 2 cm in diameter, the hybrid detector maintained markedly high abilities (84.6% accuracy, 72.2% sensitivity, 87.7% specificity). Youden's index (sensitivity + specificity ? 1) for HCC ≤ 2 cm was 0.60, vastly much superior to the 0.39 for AFP at a cut-off value of 20 ng/ml and the 0.28 for PIVKA-II at a cut-off value of 40 mAU/ml.ConclusionsThese results show that the optimal hybrid blood detector can detect HCV-related HCC more accurately.     

应用MSP方法检测非小细胞肺癌RASSFlA基因启动子甲基化

目的探讨应用甲基化特异性PCR(MSP)方法检测非小细胞肺癌(NSCLC)RASSFlA基因启动子甲基化情况。方法留取58例非小细胞肺癌患者手术标本及正常肺组织,甲基化特异性PCR分析RASSFlA基因启动子甲基化情况。结果58例非小细胞肺癌中RA SSFlA基因启动子甲基化阳性率为34.5%,甲基化与各临床参数之间无显著相关性。结论原发性非小细胞肺癌中存在着较高比例的RASSFlA启动子过度甲基化,提示RASSFlA在非小细胞肺癌的发生中起着重要作用。     

应用MSP方法检测非小细胞肺癌RASSFlA基因启动子甲基化

目的探讨应用甲基化特异性PCR（MSP）方法检测非小细胞肺癌（NSCLC）RASSF1A基因启动子甲基化情况。方法留取58例非小细胞肺癌患者手术标本及正常肺组织，甲基化特异性PCR分析RASSF1A基因启动子甲基化情况。结果58例非小细胞肺癌中RASSF1A基因启动子甲基化阳性率为34．5％，甲基化与各临床参数之间无显著相关性。结论原发性非小细胞肺癌中存在着较高比例的RASSF1A启动子过度甲基化，提示RASSF1A在非小细胞肺癌的发生中起着重要作用。     

Promoter methylation and expression of SOCS-1 affect clinical outcome and epithelial-mesenchymal transition in colorectal cancer

BackgroundAbnormal DNA methylation can cause gene silencing in colorectal cancer (CRC) patients. A gene that is suspected to have a crucial role in various types of cancers is the suppressor of cytokine signaling 1 (SOCS-1). Thus, this study will analyze the ramifications of SOCS-1 promoter methylation in CRC patients. This study will also test the therapeutic effects of hypomethylation as a possible CRC therapy.MethodsFirst, 97CRC patients’ tumor and adjacent normal tissues were collected. Next, the methylation status of the SOCS-1 promoter region was assessed by methylation-specific polymerase chain reaction (MS-PCR); SOCS-1 protein and mRNA expression were also measured. A 48-month median follow-up period was used for the survival analysis of research participants. Lastly, to analyze the changes in cell invasion and migration in conjunction with protein and mRNA expression, the demethylating agent 5-azacytidine was applied in vitro to human CRC cells.ResultsThe results showed increased SOCS-1 hypermethylation in CRC samples compared to controls. Methylated SOCS-1 was associated with significant suppression of SOCS-1 expression in tumors. Additionally, SOCS-1 hypermethylation was significantly correlated with lymph node metastasis and TNM stage. The study also found a poor overall survival rate to be significantly correlated with reduced expression of SOCS-1. After 5-azacytidine treatment, reduced in vitro DNA methylation and increased SOCS-1 expression were observed, and decreased cell migration and epithelial-mesenchymal transition biomarker expression alteration were further confirmed.ConclusionsIn colorectal cancer tissues, the rate of methylation in the SOCS-1 promoter region is high. Through promoter hypermethylation, the SOCS-1 gene was severely down-regulated in the CRC tissue samples, thereby revealing a plausible therapeutic target for CRC therapy.     

Validation of methylation-sensitive high resolution melting for the detection of DNA methylation in cholangiocarcinoma

ObjectivesTo validate methylation-sensitive high resolution melting (MS-HRM) for detection of DNA methylation.Design and MethodsMethylation of two independent loci, OPCML and DcR1, was analyzed in cholangiocarcinoma and adjacent normal samples by using MS-HRM, methylation-specific PCR and pyrosequencing.ResultsThere was significant agreement between methods at both loci.ConclusionsMS-HRM represents the excellent potential and reliability for quantifying DNA methylation levels in clinical samples.     

An analysis of image texture,tumor location,and MGMT promoter methylation in glioblastoma using magnetic resonance imaging

In glioblastoma (GBM), promoter methylation of the DNA repair gene O₆-methylguanine-DNA methyltransferase (MGMT) is associated with benefit from chemotherapy. Correlations between MGMT promoter methylation and visually assessed imaging features on magnetic resonance (MR) have been reported suggesting that noninvasive detection of MGMT methylation status might be possible. Our study assessed whether MGMT methylation status in GBM could be predicted using MR imaging. We conducted a retrospective analysis of MR images in patients with newly diagnosed GBM. Tumor texture was assessed by two methods. First, we analyzed texture by expert consensus describing the tumor borders, presence or absence of cysts, pattern of enhancement, and appearance of tumor signal in T2-weighted images. Then, we applied space–frequency texture analysis based on the S-transform. Tumor location within the brain was determined using automatized image registration and segmentation techniques. Their association with MGMT methylation was analyzed. We confirmed that ring enhancement assessed visually is significantly associated with unmethylated MGMT promoter status (P = 0.006). Texture features on T2-weighted images assessed by the space–frequency analysis were significantly different between methylated and unmethylated cases (P < 0.05). However, blinded classification of MGMT promoter methylation status reached an accuracy of only 71%. There were no significant differences in the locations of methylated and unmethylated GBM tumors. Our results provide further evidence that individual MR features are associated with MGMT methylation but better algorithms for predicting methylation status are needed. The relevance of this study lies on the application of novel techniques for the analysis of anatomical MR images of patients with GBM allowing the evaluation of subtleties not seen by an observer and facilitating the standardization of the methods, decreasing the potential for interobserver bias.     

巢式MSP法检测恶性血液病细胞株p16基因启动子甲基化状态的研究

本研究应用改进的甲基化特异性PCR（MSP）法，即巢式甲基化特异性PCR法检测6种肿瘤细胞株p16基因启动子的甲基化状态效率，探讨其在筛选p16基因启动子高甲基化的肿瘤细胞株，及将其作为研究基因甲基化与表达关系的理想细胞模型中的应用。6种肿瘤细胞株基因组DNA经碱变性后以亚硫酸盐修饰，再用巢式甲基化特异性聚合酶链反应扩增，分析检测其p16启动子区CpG岛的甲基化状态。结果表明：CA46、U266都有不同程度的p16基因启动子区甲基化，而Molt4、K562、HL-60、Jurkat的p16基因启动子区均未甲基化。结论：用巢式甲基化特异性PCR可以准确的检测出恶性血液病细胞株p16基因的甲基化状态，方法简单，灵敏且重复性强，可以广泛用于筛选各种p16基因启动子区甲基化的恶性血液病细胞株以及恶性血液病诊断。     

应用改进MSP法检测非小细胞肺癌患者血清DAPK基因甲基化

目的通过分析非小细胞肺癌(N SCLC)患者血清中死亡相关蛋白激酶(DAPK)启动子区域甲基化的改变状况及其临床意义,寻找一种切实可行的检测方法及肿瘤的生物标志物。方法运用改良的甲基化特异性PCR(M SP)技术,检测80例N SCLC患者血清DAPK基因启动因子区域甲基化的改变情况,并分析与临床病理资料的关系。结果N SCLC患者血清DAPK基因甲基化检出率为31.3%(25/80),而正常对照组和良性肺部疾病组血清未检出DAPK基因甲基化,DAPK基因甲基化检出率与N SCLC病理类型、分期及转移状态无明显关系。结论运用改进的M SP法检测血清中DAPK基因启动因子区域异常甲基化是一种行之有效的方法,检测DAPK基因甲基化可作N SCLC早期辅助诊断的分子标志物之一。     

p16基因甲基化对肺癌早期诊断的临床研究

目的检测肺癌患者支气管灌洗液及全DNA中p16基因甲基化,并探讨其在肺癌早期诊断中的作用。方法利用半巢式甲基化特异性PCR技术,检测26例肺癌患者支气管灌洗液及相应全血中DNA p16基因的异常甲基化状态。结果34.6%(9/26)肺癌组织出现p16基因甲基化,出现甲基化组织对应的血液标本中88.9%(8/9)出现p16基因甲基化。支气管灌洗液中p16基因的甲基化状况与全血中是否出现p16基因甲基化关系密切(P<0.01)。结论肺癌患者支气管灌洗液及血液标本中p16基因甲基化的检测可能有助于肺癌的早期诊断。     

Folate and choline metabolism gene variants and development of uterine cervical carcinoma

ObjectiveIt has been reported that aberrant DNA methylation can be associated with HPV infection and cervical tumorigenesis. The aim of this study was to evaluate the possibility that polymorphic variants of genes that may affect DNA methylation status are associated with the risk of cervical cancer in the Polish population.Design and methodEmploying PCR-RFLPs and HRM analyses, we examined the prevalence of BHMT Arg239Gln (rs3733890), MTR Asp919Gly (rs1805087), MTHFR Ala222Val (rs1801133), MTHFD1 Arg653Gln (rs2236225) and MTRR Ile22Met (rs1801394) genotypes and alleles in patients with advanced cervical cancer (n = 124) and controls (n = 168).ResultsThe odds ratio (OR) for BHMT Gln/Gln genotype was 0.433 (95% CI = 0.1780–1.054; p = 0.0602). The OR for patients having the BHMT Arg/Gln or Gln/Gln genotypes was 0.579 (95% CI = 0.3622–0.924; p = 0.0216). We also observed a significantly higher frequency of the BHMT 239Gln allele in controls than in patients, p = 0.0165. The genotype and allele frequencies of the MTR Asp919Gly, MTHFR Ala222Val, MTHFD1 Arg653Gln and MTRR Ile22Met gene variants did not display statistical differences between patients with cervical cancer and controls. We also did not find a significant association between the distribution of any genotypes or alleles and cancer characteristics.ConclusionOur results might suggest the protective role of the BHMT 239Gln variant in cervical cancer incidence.     

Expression of chloride intracellular channel protein 1 (CLIC1) and tumor protein D52 (TPD52) as potential biomarkers for colorectal cancer

ObjectivesUnequivocal biomarkers are needed to predict susceptibility and progression of colorectal cancer.Design and methodsPaired samples of tumor and normal tissue from six patients with colorectal cancer of different localization, pTNM stage and grade were employed in the present study. MS analysis was used to identify differentially regulated proteins after 2-DE separation and densitometric analysis.ResultsDensitometric analysis revealed differential abundance of 55 spots in tumor as compared to normal tissues. Thirty nine out of 55 spots were unambiguously identified by MS representing 32 different proteins. CLIC1, TPD52 and FABPL were consistently overexpressed (> 3-fold, P < 0.05) in all tumor tissue samples, while TPM1, TPM2, TPM3, TAGL and MLRN were consistently down-regulated (> 3-fold, P < 0.05) compared to normal tissue.ConclusionsCLIC1 and TPD52 were significantly (P < 0.05) up-regulated in all cases of colorectal cancer investigated, irrespective of localization, pTNM stage and grade of colon cancer highlighting their potential to serve as new biomarkers.