A serum-based DNA methylation assay provides accurate detection of glioma

BackgroundThe detection of somatic mutations in cell-free DNA (cfDNA) from liquid biopsy has emerged as a noninvasive tool to monitor the follow-up of cancer patients. However, the significance of cfDNA clinical utility remains uncertain in patients with brain tumors, primarily because of the limited sensitivity cfDNA has to detect real tumor-specific somatic mutations. This unresolved challenge has prevented accurate follow-up of glioma patients with noninvasive approaches.MethodsGenome-wide DNA methylation profiling of tumor tissue and serum cfDNA of glioma patients.ResultsHere, we developed a noninvasive approach to profile the DNA methylation status in the serum of patients with gliomas and identified a cfDNA-derived methylation signature that is associated with the presence of gliomas and related immune features. By testing the signature in an independent discovery and validation cohorts, we developed and verified a score metric (the “glioma-epigenetic liquid biopsy score” or GeLB) that optimally distinguished patients with or without glioma (sensitivity: 100%, specificity: 97.78%). Furthermore, we found that changes in GeLB score reflected clinicopathological changes during surveillance (eg, progression, pseudoprogression, and response to standard or experimental treatment).ConclusionsOur results suggest that the GeLB score can be used as a complementary approach to diagnose and follow up patients with glioma.     

DNA甲基化与胶质瘤

表遗传学修饰的DNA甲基化在胶质瘤的发生发展中起重要作用。大量肿瘤抑制基因、细胞周期调控基因、DNA损伤修复基因、肿瘤侵袭相关基因等启动子区域CpG岛甲基化,与胶质瘤的发生发展密切相关。不同基因的甲基化发生频率与胶质瘤的组织类型和病理分级有关。某些基因的DNA甲基化可以为胶质瘤的早期诊断和预后评价提供分子生物学标记物,同时由于DNA甲基化具有可逆转性,也可为胶质瘤的基因治疗提供新的治疗靶点。     

Epigenetic regulation of glial fibrillary acidic protein by DNA methylation in human malignant gliomas

Glial fibrillary acidic protein (GFAP) is an intermediate filament expressed in glial cells that stabilizes and maintains the cytoskeleton of normal astrocytes. In glial tumors, GFAP expression is frequently lost with increasing grade of malignancy, suggesting that GFAP is important for maintaining glial cell morphology or regulating astrocytoma cell growth. Most permanent human glioma cell lines are GFAP negative by immunocytochemistry. Given that the GFAP gene is not mutated in human glioma specimens or glioma cell lines, we considered epigenetic mechanisms, such as promoter methylation, as a cause of silencing of GFAP in these tumors. In this study, we treated known GFAP-negative glioma cell lines with 5-aza-2'-deoxycytidine to examine GFAP promoter hypermethylation. Additionally, we performed bisulfite sequencing on primary glioma samples and glioma cell lines and showed an inverse relationship between GFAP promoter methylation status and GFAP expression. Using a gene reporter assay with the GFAP promoter cloned upstream of a luciferase gene, we showed that methylation of the GFAP promoter downregulates the expression of the luciferase gene. Our results suggest that epigenetic silencing of the GFAP gene through DNA methylation of its promoter region may be one mechanism by which GFAP is downregulated in human gliomas and glioma cell lines.     

DNA甲基化对miR-133a表达及胶质瘤细胞增殖及凋亡的影响

目的:探讨miR-133a在胶质瘤细胞中的表达及DNA甲基化对其的调控机制并初步探究miR-133a对胶质瘤细胞增殖及凋亡的影响.方法:通过RT-PCR检测胶质瘤组织与正常对照脑组织中miR-133a的表达差异;MSP实验检测胶质瘤组织与正常对照脑组织中miR-133a基因甲基化程度;BSP实验检测正常胶质细胞株HEB...     

Aberrant DNA methylation occurs in colon neoplasms arising in the azoxymethane colon cancer model

Mouse models of intestinal tumors have advanced our understanding of the role of gene mutations in colorectal malignancy. However, the utility of these systems for studying the role of epigenetic alterations in intestinal neoplasms remains to be defined. Consequently, we assessed the role of aberrant DNA methylation in the azoxymethane (AOM) rodent model of colon cancer. AOM induced tumors display global DNA hypomethylation, which is similar to human colorectal cancer. We next assessed the methylation status of a panel of candidate genes previously shown to be aberrantly methylated in human cancer or in mouse models of malignant neoplasms. This analysis revealed different patterns of DNA methylation that were gene specific. Zik1 and Gja9 demonstrated cancer‐specific aberrant DNA methylation, whereas, Cdkn2a/p16, Igfbp3, Mgmt, Id4, and Cxcr4 were methylated in both the AOM tumors and normal colon mucosa. No aberrant methylation of Dapk1 or Mlt1 was detected in the neoplasms, but normal colon mucosa samples displayed methylation of these genes. Finally, p19^Arf, Tslc1, Hltf, and Mlh1 were unmethylated in both the AOM tumors and normal colon mucosa. Thus, aberrant DNA methylation does occur in AOM tumors, although the frequency of aberrantly methylated genes appears to be less common than in human colorectal cancer. Additional studies are necessary to further characterize the patterns of aberrantly methylated genes in AOM tumors. © 2009 Wiley‐Liss, Inc.     

Development of a Sox2 reporter system modeling cellular heterogeneity in glioma

BackgroundMalignant gliomas are complex systems containing a number of factors that drive tumor initiation and progression, including genetic aberrations that lead to extensive cellular heterogeneity within the neoplastic compartment. Mouse models recapitulate these genetic aberrations, but readily observable heterogeneity remains challenging.MethodsTo interrogate cellular heterogeneity in mouse glioma models, we utilized a replication-competent avian sarcoma-leukosis virus long terminal repeat with splice acceptor/tumor virus A (RCAS-tva) system to generate spontaneous mouse gliomas that contained a Sox2-enhanced green fluorescent protein (EGFP) reporter. Glial fibrillary acidic protein-tva mice were crossed with Sox2–EGFP mice, and tumors were initiated that contained a subpopulation of Sox2–EGFP-high cells enriched for tumor-initiating cell properties such as self-renewal, multilineage differentiation potential, and perivascular localization.ResultsFollowing implantation into recipient mice, Sox2–EGFP-high cells generated tumors containing Sox2–EGFP-high and Sox2–EGFP-low cells. Kinomic analysis of Sox2–EGFP-high cells revealed activation of known glioma signaling pathways that are strongly correlated with patient survival including platelet-derived growth factor receptor beta, phosphoinositide-3 kinase, and vascular endothelial growth factor. Our functional analysis identified active feline sarcoma (Fes) signaling in Sox2–EGFP-high cells. Fes negatively correlated with glioma patient survival and was coexpressed with Sox2-positive cells in glioma xenografts and primary patient-derived tissue.ConclusionsOur RCAS-tva/Sox2-EGFP model will empower closer examination of cellular heterogeneity and will be useful for identifying novel glioma pathways as well as testing preclinical treatment efficacy.     

弥漫大B细胞淋巴瘤的DNA甲基化改变及治疗进展

弥漫大B细胞淋巴瘤(DLBCL)异质性较高,具有不同的临床和病理特征,是成人淋巴瘤的一种常见类型。尽管一部分DLBCL已经可以治愈,但难治和复发患者的治疗仍然具有挑战性。近年来,DNA甲基化等表观遗传修饰成为DLBCL的研究热点。调控甲基化修饰的基因突变及抑癌基因甲基化修饰在DLBCL发病及预后中起到关键作用。本文对DLBCL的DNA甲基化研究进展进行综述。     

Genome-wide association study of peripheral blood DNA methylation and conventional mammographic density measures

Age- and body mass index (BMI)-adjusted mammographic density is one of the strongest breast cancer risk factors. DNA methylation is a molecular mechanism that could underlie inter-individual variation in mammographic density. We aimed to investigate the association between breast cancer risk-predicting mammographic density measures and blood DNA methylation. For 436 women from the Australian Mammographic Density Twins and Sisters Study and 591 women from the Melbourne Collaborative Cohort Study, mammographic density (dense area, nondense area and percentage dense area) defined by the conventional brightness threshold was measured using the CUMULUS software, and peripheral blood DNA methylation was measured using the HumanMethylation450 (HM450) BeadChip assay. Associations between DNA methylation at >400,000 sites and mammographic density measures adjusted for age and BMI were assessed within each cohort and pooled using fixed-effect meta-analysis. Associations with methylation at genetic loci known to be associated with mammographic density were also examined. We found no genome-wide significant (p < 10⁻⁷) association for any mammographic density measure from the meta-analysis, or from the cohort-specific analyses. None of the 299 methylation sites located at genetic loci associated with mammographic density was associated with any mammographic density measure after adjusting for multiple testing (all p > 0.05/299 = 1.7 × 10⁻⁴). In summary, our study did not find evidence for associations between blood DNA methylation, as measured by the HM450 assay, and conventional mammographic density measures that predict breast cancer risk.     

DNA methylation profiles delineate epigenetic heterogeneity in seminoma and non-seminoma

Background:

It remains important to understand the biology and identify biomarkers for less studied cancers like testicular cancer. The purpose of this study was to determine the methylation frequency of several cancer-related genes in different histological types of testicular cancer and normal testis tissues (NT).

Methods:

DNA was isolated from 43 seminomas (SEs), 14 non-SEs (NSEs) and 23 NT, and was assayed for promoter methylation status of 15 genes by quantitative methylation-specific PCR. The methylation status was evaluated for an association with cancer, and between SEs and NSEs.

Results:

We found differential methylation pattern in SEs and NSEs. MGMT, VGF, ER-β and FKBP4 were predominately methylated in NSEs compared with SEs. APC and hMLH1 are shown to be significantly more methylated in both subtypes in comparison with NT. When combining APC, hMLH1, ER-β and FKBP4, it is possible to identify 86% of the NSEs, whereas only 7% of the SEs.

Conclusions:

Our results indicate that the methylation profile of cancer-associated genes in testicular cancer correlates with histological types and show cancer-specific pattern for certain genes. Further methylation analysis, in a larger cohort is needed to elucidate their role in testicular cancer development and potential for therapy, early detection and disease monitoring.     

Genome‐wide DNA methylation analysis identifies MEGF10 as a novel epigenetically repressed candidate tumor suppressor gene in neuroblastoma

Neuroblastoma is a childhood cancer in which many children still have poor outcomes, emphasising the need to better understand its pathogenesis. Despite recent genome‐wide mutation analyses, many primary neuroblastomas do not contain recognizable driver mutations, implicating alternate molecular pathologies such as epigenetic alterations. To discover genes that become epigenetically deregulated during neuroblastoma tumorigenesis, we took the novel approach of comparing neuroblastomas to neural crest precursor cells, using genome‐wide DNA methylation analysis. We identified 93 genes that were significantly differentially methylated of which 26 (28%) were hypermethylated and 67 (72%) were hypomethylated. Concentrating on hypermethylated genes to identify candidate tumor suppressor loci, we found the cell engulfment and adhesion factor gene MEGF10 to be epigenetically repressed by DNA hypermethylation or by H3K27/K9 methylation in neuroblastoma cell lines. MEGF10 showed significantly down‐regulated expression in neuroblastoma tumor samples; furthermore patients with the lowest‐expressing tumors had reduced relapse‐free survival. Our functional studies showed that knock‐down of MEGF10 expression in neuroblastoma cell lines promoted cell growth, consistent with MEGF10 acting as a clinically relevant, epigenetically deregulated neuroblastoma tumor suppressor gene. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.     

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.     

DNA甲基化与胰腺癌发病的关系

 胰腺癌是临床常见预后不良的肿瘤之一,由于临床症状与体征缺乏特异性,许多患者就诊时已属晚期,从而失去了手术时机。虽然近年来吉西他滨的临床使用使晚期胰腺癌患者的1年生存率提高到20 %左右,但并没有取得明显进展。DNA甲基化是机体表观遗传调控的主要机制之一,其表达在胰腺癌的发病中可能起着重要作用。     

MethCancerDB--aberrant DNA methylation in human cancer

Early detection, classification and prognosis of human cancers by analysis of CpG methylation carry huge diagnostic potential. MethCancerDB collects and annotates genes and sequences from the abundance of published methylation studies and interlinks them to all methylation-relevant bioinformatical resources. MethCancerDB starts with 4720 entries from 348 sources and is freely accessible at http://www.methcancerdb.net.     

The presence of aberrant DNA methylation in noncancerous esophageal mucosae in association with smoking history

BACKGROUND:

Esophageal squamous cell carcinomas (ESCCs) tend to have multiple primary lesions, and it is believed that they arise from background mucosae with accumulation of genetic/epigenetic alterations. In this study, the objective was to elucidate the effects of smoking and drinking on the accumulation of epigenetic alterations in background mucosae.

METHODS:

Genes that are silenced in human ESCCs were searched for by treating 3 ESCC cell lines with the demethylating agent, 5‐aza‐2′‐deoxycytidine and performing oligonucleotide microarrays. Methylation levels were analyzed by quantitative methylation‐specific polymerase chain reaction analysis of 60 ESCCs and their corresponding background mucosae.

RESULTS:

Forty‐seven genes were identified as methylation‐silenced in at least 1 of the 3 ESCC cell lines, and 14 of those genes (claudin 6 [CLDN6]; G protein‐coupled receptor 158 [GPR158]; homeobox A9 [HOXA9]; metallothionein 1M [MT1M]; neurofilament, heavy polypeptide 200 kDa [NEFH]; plakophilin 1 [PKP1]; protein phosphatase 1, regulatory [inhibitor] subunit 14A [PPP1R14A]; pyrin domain and caspase recruitment domain containing [PYCARD]; R‐spondin family, member 4 [RSPO4]; testis‐specific protein, Y‐encoded–like 5 [TSPYL5]; ubiquitin carboxyl‐terminal esterase L1 [UCHL1]; zinc‐finger protein 42 homolog [ZFP42]; zinc‐finger protein interacting with K protein 1 homolog [ZIK1]; and zinc‐finger and SCAN domain containing 18 [ZSCAN18]) were used as markers. In the background mucosae, methylation levels of 5 genes (HOXA9, MT1M, NEFH, RSPO4, and UCHL1) had significant correlations with smoking duration (ρ = .268; P = .044; ρ = .405; P = .002; ρ = .285; P = .032; ρ = .300; P = .024; and ρ = .437; P = .001, respectively). In contrast, an inverse correlation between PYCARD methylation levels and alcohol intake was observed (ρ = ?.334, P = .025) among individuals with the inactive aldehyde dehydrogenase 2 (ALDH2) genotype.

CONCLUSIONS:

The current results suggested that ESCCs developed from an epigenetic field for cancerization, which was induced by exposure to carcinogenic factors, such as tobacco smoking. The epigenetic field defect will be a novel target for risk diagnosis and prevention of ESCCs. Cancer 2009. © 2009 American Cancer Society.     

DNA异常甲基化在前列腺癌发生发展及诊疗中的研究进展

前列腺癌是全球男性常发的恶性肿瘤之一。近年来研究发现前列腺癌的发生发展很大程度上是由表观遗传修饰所驱动的,其中最重要的当属DNA异常甲基化。DNA启动子的异常甲基化会造成基因的异常表达,从而调控着前列腺癌的发生发展过程。此外,目前前列腺癌的诊断仍然依靠侵入性的前列腺穿刺活检,而基于DNA异常甲基化的无创性液体活检有望成为未来前列腺癌分子诊断的发展趋势,同时也为前列腺癌提供了新的治疗靶标。本文就DNA甲基化在前列腺癌发生发展、早期诊断、预后评估以及治疗中的研究进展进行综述。     

Efficient induction of differentiation and growth inhibition in IDH1 mutant glioma cells by the DNMT Inhibitor Decitabine

Mutation in the IDH1 or IDH2 genes occurs frequently in gliomas and other human malignancies. In intermediate grade gliomas, IDH1 mutation is found in over 70% of tumors. These mutations impart the mutant IDH enzyme with a neomorphic activity – the ability to synthesize 2-hydroxyglutarate (2-HG). This ability leads to a reprogramming of chromatin state, a block in differentiation, and the establishment of the glioma hypermethylator phenotype (G-CIMP). It has been hypothesized but not proven that the extensive DNA methylation that occurs in G-CIMP tumors helps maintain and “lock in” glioma cancer cells in a dedifferentiated state. Here, we tested this hypothesis by treating patient derived IDH1 mutant glioma initiating cells (GIC) with non-cytotoxic, epigenetically targeted doses of the DNMT inhibitor decitabine. Global methylome analysis of treated IDH1 mutant GICs showed that DAC treatment resulted in reversal of DNA methylation marks induced by IDH and the re-expression of genes associated with differentiation. Accordingly, treatment of IDH1 mutant glioma cells resulted in a dramatic loss of stem-like properties and efficient adoption of markers of differentiation, effects not seen in decitabine treated IDH wild-type GICs. Induction of differentiation was much more efficient than that seen following treatment with a specific inhibitor of mutant IDH enzyme (Agios). Decitabine also decreased replicative potential and tumor growth in vivo. Reexpression of polycomb regulated genes accompanied these DAC-induced phenotypes. In total, our data indicates that targeting the pathologic DNA methylation in IDH mutant cells can reverse mutant IDH induced hypermethylation and block in differentiation and promote tumor control. These findings have substantial impact for exploring new treatment strategies for patients with IDH mutant gliomas.     

DNA甲基化与鼻咽癌

癌基因和抑癌基因表达失常是鼻咽癌发病的关键。鼻咽癌与细胞凋亡和细胞周期调控基因、细胞黏附分子编码基因、应激反应基因、DNA错配修复基因、维甲酸信号通路相关基因、Wnt信号通路相关基因以及RIZ1基因、DLC1基因、H19和COX-2基因等基因异常甲基化相关。