Gene expression profiling of human adrenocortical tumors using complementary deoxyribonucleic Acid microarrays identifies several candidate genes as markers of malignancy

The aim of this study was to identify predictor sets of genes whose over- or underexpression in human sporadic adrenocortical tumors would help to identify malignant vs. benign tumors and to predict postsurgical metastatic recurrence. For this, we analyzed the expression of 230 candidate genes using cDNA microarrays in a series of 57 well-characterized human sporadic adrenocortical tumors (33 adenomas and 24 carcinomas). We identified two clusters of genes (the IGF-II cluster containing eight genes, including IGF-II, and the steroidogenesis cluster containing six genes encoding steroidogenic enzymes plus eight other genes) whose combined levels of expression appeared to be good predictors of malignancy. This predictive value was as strong as that of the pathological score of Weiss. The analysis of the population of carcinomas (13 tumors) for genes whose expression would be strongly different between recurring and nonrecurring tumors allowed identification of 14 genes meeting these criteria. Among these genes, there are probably new markers of tumor evolution that will deserve additional validation on a larger scale. Taken together, these results show that the parallel analysis of the expression levels of a selected group of genes on microgram quantities of tumor RNA (a quantity that can be obtained from fine needle aspirations) appears as a complementary method to histopathology for the diagnosis and prognosis of evolution of adrenocortical carcinomas.     

Genome-wide profiling identifies a DNA methylation signature that associates with TET2 mutations in diffuse large B-cell lymphoma

The discovery that the Ten-Eleven Translocation (TET) hydroxylases cause DNA demethylation has fundamentally changed the notion of how DNA methylation is regulated. Clonal analysis of the hematopoetic stem cell compartment suggests that TET2 mutations can be early events in hematologic cancers and recent investigations have shown TET2 mutations in diffuse large B-cell lymphoma. However, the detection rates and the types of TET2 mutations vary, and the relation to global methylation patterns has not been investigated. Here, we show TET2 mutations in 12 of 100 diffuse large B-cell lymphomas with 7% carrying loss-of-function and 5% carrying missense mutations. Genome-wide methylation profiling using 450K Illumina arrays identified 315 differentially methylated genes between TET2 mutated and TET2 wild-type cases. TET2 mutations are primarily associated with hypermethylation within CpG islands (70%; P<0.0001), and at CpG-rich promoters (60%; P<0.0001) of genes involved in hematopoietic differentiation and cellular development. Hypermethylated loci in TET2 mutated samples overlap with the bivalent (H3K27me3/H3K4me3) silencing mark in human embryonic stem cells (P=1.5×10⁻³⁰). Surprisingly, gene expression profiling showed that only 11% of the hypermethylated genes were down-regulated, among which there were several genes previously suggested to be tumor suppressors. A meta-analysis suggested that the 35 hypermethylated and down-regulated genes are associated with the activated B-cell-like type of diffuse large B-cell lymphoma in other studies. In conclusion, our data suggest that TET2 mutations may cause aberrant methylation mainly of genes involved in hematopoietic development, which are silenced but poised for activation in human embryonic stem cells.     

Array-based DNA methylation profiling in acute myeloid leukaemia

Methylation in the promoter region of many genes is involved in regulating gene expression patterns. Using the Illumina GoldenGate^© methylation assay, we examined the methylation status of 1505 CpG‐sites from 807 genes in 32 samples from patients with acute myeloid leukaemia (AML) at diagnosis, nine at relapse and 15 normal controls and performed additional pyrosequencing and semiquantitative methylation specific polymerase chain reaction (MSP) of the GNMT promoter in 113 diagnostic AML samples. We found a gain of overall methylation in AML samples with a further increase at relapse. Regional hypermethylation as assessed by array analysis could be confirmed by both MSP and pyrosequencing. Additionally, large‐scale methylation analysis identified interesting candidate genes. Cluster analysis indicated that cytogenetic subgroups seemed to be characterized by additional distinct epigenetic modifications and that basic DNA methylation patterns remain at relapse. Therefore, promoter hypermethylation is a frequent event in AML and is accentuated at relapse. Array‐based methylation analysis determined distinct methylation profiles for non‐malignant controls and AML samples with specific chromosomal aberrations and can identify target genes for further evaluation.     

DNA methylation of developmental genes in pediatric medulloblastomas identified by denaturation analysis of methylation differences

DNA methylation might have a significant role in preventing normal differentiation in pediatric cancers. We used a genomewide method for detecting regions of CpG methylation on the basis of the increased melting temperature of methylated DNA, termed denaturation analysis of methylation differences (DAMD). Using the DAMD assay, we find common regions of cancer-specific methylation changes in primary medulloblastomas in critical developmental regulatory pathways, including Sonic hedgehog (Shh), Wingless (Wnt), retinoic acid receptor (RAR), and bone morphogenetic protein (BMP). One of the commonly methylated loci is the PTCH1-1C promoter, a negative regulator of the Shh pathway that is methylated in both primary patient samples and human medulloblastoma cell lines. Treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-dC) increases the expression of PTCH1 and other methylated loci. Whereas genetic mutations in PTCH1 have previously been shown to lead to medulloblastoma, our study indicates that epigenetic silencing of PTCH1, and other critical developmental loci, by DNA methylation is a fundamental process of pediatric medulloblastoma formation. This finding warrants strong consideration for DNA demethylating agents in future clinical trials for children with this disease.     

Hepatitis C virus and liver disease: global transcriptional profiling and identification of potential markers

Microarray analysis of RNA from hepatitis C virus (HCV)-infected cirrhotic livers was performed to identify a gene expression signature of liver disease. The expression levels of approximately 13600 genes were analyzed using surgical material and core biopsy specimens from HCV-infected cirrhotic liver explants in comparison with reference samples of normal nondiseased liver. In addition, normal liver samples were compared with each other to determine normal physiologic variation in gene expression. A set of genes, including some associated with stress, acute-phase immune response, and hepatic stellate cell activation, had variable expression levels in normal livers. These genes were subtracted from the sets of genes differentially expressed in cirrhotic livers. To exclude cancer-related genes from our marker sets, we subtracted genes that also were expressed differentially in hepatocellular carcinomas. The resultant HCV- and liver disease-associated gene set provided a molecular portrait of several processes occurring in the HCV-infected liver. It included (1). genes expressed in activated lymphocytes infiltrating the cirrhotic liver, and activated liver macrophages; (2). genes involved in remodeling of extracellular matrix-cell and cell-cell interactions associated with cytoskeleton rearrangements; (3). genes related to the anti-apoptotic pathway of Bcl-2 signaling; and (4). genes involved with the interferon response and virus-host interactions. In conclusion, our microarray analysis identified several potential gene markers of HCV-associated liver disease and contributed to our rapidly expanding database of experiments describing HCV pathogenesis.     

Pathogenesis of benign adrenocortical tumors

Most adrenocortical tumors (ACT) are benign unilateral adrenocortical adenomas, often discovered incidentally. Exceptionally, ACT are bilateral. However bilateral ACT have been very helpful to progress in the pathophysiology of ACT. Although most ACT are of sporadic origin, they may also be part of syndromic and/or hereditary disorders. The identification of the genetics of familial diseases associated with benign ACT has been helpful to define somatic alterations in sporadic ACT: for example, identification of PRKAR1A mutations in Carney complex or alterations of the Wnt/β-catenin pathway in Familial Adenomatous Polyposis Coli. Components of the cAMP signaling pathway-for example, adrenocorticotropic-hormone receptors and other membrane receptors, Gs protein, phosphodiesterases and protein kinase A-can be altered to various degrees in benign cortisol-secreting ACT. These progress have been important for the understanding of the pathogenesis of benign ACT, but already have profound implications for clinical management, for example in unraveling the genetic origin of disease in some patients with ACT. They also have therapeutic consequences, and should help to develop new therapeutic options.     

Integrated DNA copy number and methylation profiling of lymphoid neoplasms using a single array

Changes in DNA copy number (CN) and DNA methylation represent important aberrations for lymphomas and other cancers. Here, for the first time, we show that the Illumina Infinium Methylation (IIM) assay, although not originally designed for CN profiling, is able to estimate CN changes. We compared the IIM CN profiles to those obtained with a standard technique in a series of diffuse large B-cell lymphomas: the profiles showed a high degree of consensus. The demonstration of CN profiling as an additional function of the IIM assay may impact the choice of platform for methylation profiling of haematological and solid tumours.     

抑制素/激活素与肾上腺皮质肿瘤

研究显示,组成抑制素/激活素的亚单位α、βA、βB在正常肾上腺皮质有表达,在肾上腺皮质肿瘤中亦有表达。抑制素α亚单位基因突变与肾上腺皮质肿瘤有关。对抑制素基因敲除小鼠的研究显示,激活素可能是肾上腺皮质肿瘤发生的抑制因子,故认为它们可能与肾上腺皮质肿瘤的发生有关。抑制素、激活素通过影响类固醇生成及细胞凋亡,与骨形态生成蛋白相互作用调节醛固酮的生成,从而参与肾上腺皮质肿瘤的发生。抑制素与Calretinin合用可鉴别肾上腺皮质肿瘤与嗜铬细胞瘤,抗抑制素α亚单位抗体也可鉴别肾上腺皮质肿瘤与肾细胞癌。     

Genome-wide DNA methylation profiling in nonalcoholic fatty liver reveals predictive aberrant methylation in PRKCE and SEC14L3 promoters

BackgroundOptimal non-invasive biomarkers for diagnosis and treatment of nonalcoholic fatty liver disease (NAFLD) remain to be identified.AimsTo identify potential DNA methylation biomarkers for NAFLD.MethodsGenome-wide DNA methylation profiling was performed to identify differentially methylated CpG sites in peripheral blood leukocytes. Differentially methylated regions were validated using the MassCLEAVE assay. The expression levels of candidate genes were explored by Gene Expression Omnibus database.ResultsThe hypomethylation of PRKCE CpG 4.5 and CpG 18.19 was associated with nonalcoholic fatty liver (NAFL), the odds ratio (OR) and 95% confidence interval (CI) were 0.129 (0.026–0.639) and 0.231 (0.069–0.768). The methylation level of CpG 1.2 and average methylation level of SEC14L3 were correlated with NAFL, with OR (95% CI) being 0.283 (0.093–0.865) and 0.264 (0.087–0.799). PRKCE CpG 4.5 and cg17802464 of SEC14L3 were correlated with body mass index, waist circumference, total triglyceride, high-density lipoprotein cholesterol, alanine aminotransferase and aspartate aminotransferase. All selected datasets showed high expression levels of PRKCE and SEC14L3 in patients with NAFLD.ConclusionsOur findings suggest that the hypomethylation of PRKCE and SEC14L3 promoters represent attractive biomarkers for NAFLD. Further studies are warranted to validate these biomarkers as molecular tools for diagnosis of NAFLD and therapeutic targets.     

Dysregulation of microRNAs in adrenocortical tumors

MicroRNAs (miRNAs) are short non-coding RNAs that are involved in the epigenetic regulation of cellular processes. Different malignancies are often associated with the deregulation of specific sets of miRNAs. The prognosis of adrenocortical cancers (ACCs) is very poor as compared to adrenocortical adenomas (ACAs), and even within ACCs there are cases with better disease specific survival. An improved understanding of the pathobiology of this disease will therefore be useful in facilitating better management of ACCs as well as distinguishing high risk versus low risk subgroups. One third of coding genes are regulated by miRNAs and therefore changes in miRNA expression may be associated with cancer development and progression. In this review we summarize the current understanding of miRNAs in adrenocortical tumors, and highlight their potential in differentiating between ACCs and ACAs, risk stratification and prognosis.