Detection of MGMT promoter methylation in glioblastoma using pyrosequencing

Recent clinical trials on patients with glioblastoma revealed that O6-Methylguanine-DNA methyltransferase (MGMT) methylation status significantly predicts patient’s response to alkylating agents. In this study, we sought to develop and validate a quantitative MGMT methylation assay using pyrosequencing on glioblastoma. We quantified promoter methylation of MGMT using pyrosequencing on paraffin-embedded fine needle aspiration biopsy tissues from 43 glioblastoma. Using a 10% cutoff, MGMT methylation was identified in 37% cases of glioblastoma and 0% of the non-neoplastic epileptic tissue. Methylation of any individual CpG island in MGMT promoter ranged between 33% and 95%, with a mean of 65%. By a serial dilution of genomic DNA of a homogenously methylated cancer cell line with an unmethylated cell line, the analytical sensitivity is at 5% for pyrosequencing to detect MGMT methylation. The minimal amount of genomic DNA required is 100 ng (approximately 3,000 cells) in small fine needle biopsy specimens. Compared with methylation-specific PCR, pyrosequencing is comparably sensitive, relatively specific, and also provides quantitative information for each CpG methylation.     

Epigenetics in the Pathogenesis of Esophageal Adenocarcinoma

Epigenetic influences, such as DNA methylation, histone acetylation, and up‐regulation/down‐regulation of genes by microRNAs, change the genetic makeup of an individual without affecting DNA base‐pair sequences. Indeed, epigenetic changes play an integral role in the progression from normal esophageal mucosa to Barrett''s esophagus to esophageal adenocarcinoma via dysplasia–metaplasia–neoplasia sequence. Many genes involved in esophageal adenocarcinoma display hypermethylation, leading to their down‐regulation. The classes of these genes include cell cycle control, DNA and growth factor repair, tumor suppressors, antimetastasis, Wnt‐related genes, and proapoptotic genes. Histone acetylation in the pathophysiology of esophageal diseases has not been thoroughly investigated, and its critical role in the development of esophageal adenocarcinoma is less defined. Many microRNAs have been associated with the development of Barrett''s esophagus and esophageal adenocarcinoma. Here, we critically addressed the specific steps most closely influenced by microRNAs in the progression from Barrett''s esophagus to esophageal adenocarcinoma. However, microRNAs can target up to hundreds of genes, making it difficult to correlate directly with a given phenotype of the disease. Esophageal adenocarcinoma progressing from premalignant condition of Barrett''s esophagus carries an extremely poor prognosis. Risk stratification for patients based on their epigenetic profiles may be useful in providing more targeted and directed treatment to patients.     

Epigenetic variation in the serotonin transporter gene predicts resting state functional connectivity strength within the salience‐network

Genetic variation in the serotonin transporter gene (SLC6A4) has been associated with psychopathology and aberrant brain functioning in a plethora of clinical and imaging studies. In contrast, the neurobiological correlates of epigenetic signatures in SLC6A4, such as DNA methylation profiles, have only recently been explored in human brain imaging research. The present study is the first to apply a resting state functional magnetic resonance imaging approach to identify changes in brain networks related to SLC6A4 promoter methylation (N = 74 healthy individuals). The amygdalae were defined as seed regions given that resting state functional connectivity in this brain area is under serotonergic control and relates to a broad range of psychiatric phenotypes. We further used bisulfite pyrosequencing to analyze quantitative methylation at 83 CpG sites within a promoter‐associated CpG island of SLC6A4 from blood‐derived DNA samples. The major finding of this study indicates a positive relation of SLC6A4 promoter methylation and amygdaloid resting state functional coupling with key nodes of the salience network (SN) including the anterior insulae and the dorsal anterior cingulate cortices. Increased intra‐network connectivity in the SN is thought to facilitate the detection and subsequent processing of potentially negative stimuli and reflects a core feature of psychopathology. As such, epigenetic changes within the SLC6A4 gene predict connectivity patterns in clinically and behaviorally relevant brain networks which may in turn convey increased disease susceptibility. Hum Brain Mapp 36:4361–4371, 2015. © 2015 Wiley Periodicals, Inc.     

Evolution and function of genomic imprinting in plants

Genomic imprinting, an inherently epigenetic phenomenon defined by parent of origin-dependent gene expression, is observed in mammals and flowering plants. Genome-scale surveys of imprinted expression and the underlying differential epigenetic marks have led to the discovery of hundreds of imprinted plant genes and confirmed DNA and histone methylation as key regulators of plant imprinting. However, the biological roles of the vast majority of imprinted plant genes are unknown, and the evolutionary forces shaping plant imprinting remain rather opaque. Here, we review the mechanisms of plant genomic imprinting and discuss theories of imprinting evolution and biological significance in light of recent findings.     

MUC5AC hypomethylation is a predictor of microsatellite instability independently of clinical factors associated with colorectal cancer

Colorectal cancers (CRC) with microsatellite instability (MSI) display unique clinicopathologic features including a mucinous pattern with frequent expression of the secreted mucins MUC2 and MUC5AC. The mechanisms responsible for this altered pattern of expression remain largely unknown. We quantified DNA methylation of mucin genes (MUC2, MUC5AC, MUC4) in colonic cancers and examined the association with clinicopathological characteristics and molecular (MSI, KRAS, BRAF, and TP53 mutations) features. A control cohort was used for validation. We detected frequent hypomethylation of MUC2 and MUC5AC in CRC. MUC2 and MUC5AC hypomethylation was associated with MUC2 and MUC5AC protein expression (p = 0.004 and p < 0.001, respectively), poor differentiation (p = 0.001 and p = 0.007, respectively) and MSI status (p < 0.01 and p < 0.001, respectively). Interestingly, MUC5AC hypomethylation was specific to MSI cancers. Moreover, it was significantly associated with BRAF mutation and CpG island methylator phenotype (p < 0.001 and p < 0.001, respectively). All these results were confirmed in the control cohort. In the multivariate analysis, MUC5AC hypomethylation was a highly predictive biomarker for MSI cancers. MUC5AC demethylation appears to be a hallmark of MSI in CRC. Determination of MUC5AC methylation status may be useful for understanding and predicting the natural history of CRC.     

The increasing roles of epigenetics in breast cancer: Implications for pathogenicity,biomarkers, prevention and treatment

Nowadays, the mechanisms governing the occurrence of cancer are thought to be the consequence not only of genetic defects but also of epigenetic modifications. Therefore, epigenetic has become a very attractive and increasingly investigated field of research in order to find new ways of prevention and treatment of neoplasia, and this is particularly the case for breast cancer (BC). Thus, this review will first develop the main known epigenetic modifications that can occur in cancer and then expose the future role that control of epigenetic modifications might play in prevention, prognostication, follow‐up and treatment of BC. Indeed, epigenetic biomarkers found in peripheral blood might become new tools to detect BC, to define its prognostic and to predict its outcome, whereas epi‐drugs might have an increasing potential of development in the next future. However, if DNA methyltransferase inhibitors and histone desacetylase inhibitors have shown encouraging results in BC, their action remains nonspecific. Thus, additional clinical studies are needed to evaluate more precisely the effects of these molecules, even if they have provided encouraging results in cotreatment and combined therapies. This review will also deal with the potential of RNA interference (RNAi) as epi‐drugs. Finally, we will focus on the potential prevention of BC through epigenetic based on diet and we will particularly develop the possible place of isothiocyanates from cruciferous vegetables or of Genistein from soybean in a dietary program that might potentially reduce the risk of BC in large populations.