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Mucinous type of epithelial ovarian cancer (MuOC) is a unique subtype with a poor survival outcome in recurrent and advanced stages. The role of type-specific epigenomics and its clinical significance remains uncertain. We analyzed the methylomic profiles of 6 benign mucinous adenomas, 24 MuOCs, 103 serous type of epithelial ovarian cancers (SeOCs) and 337 nonepithelial ovarian cancers. MuOC and SeOC exhibited distinct DNA methylation profiles comprising 101 genes, 81 of which exhibited low methylation in MuOC and were associated with the response to glucocorticoid, ATP hydrolysis-coupled proton transport, proteolysis involved in the cellular protein catabolic process and ion transmembrane transport. Hierarchical clustering analysis showed that the profiles of MuOC were similar to colorectal adenocarcinoma and stomach adenocarcinoma. Genetic interaction network analysis of differentially methylated genes in MuOC showed a dominant network module is the proteasome subunit beta (PSMB) family. Combined functional module and methylation analysis identified PSMB8 as a candidate marker for MuOC. Immunohistochemical staining of PSMB8 used to validate in 94 samples of ovarian tumors (mucinous adenoma, MuOC or SeOC) and 62 samples of gastrointestinal cancer. PSMB8 was commonly expressed in MuOC and gastrointestinal cancer samples, predominantly as strong cytoplasmic and occasionally weak nuclei staining, but was not expressed in SeOC samples. Carfilzomib, a second-generation proteasome inhibitor, suppressed MuOC cell growth in vitro. This study unveiled a mucinous-type-specific methylation profile and suggests the potential use of a proteasome inhibitor to treat MuOC.

BACKGROUND Altered DNA methylation in CpG islands of gene promoters has been implicated in prostate cancer (PCa) progression and can be used to predict disease outcome. In this study, we determine whether methylation changes of androgen biosynthesis pathway (ABP)-related genes in patients' plasma cell-free DNA (cfDNA) can serve as prognostic markers for biochemical recurrence (BCR). METHODS Methyl-binding domain capture sequencing (MBDCap-seq) was used to identify differentially methylated regions (DMRs) in primary tumors of patients who subsequently developed BCR or not, respectively. Methylation pyrosequencing of candidate loci was validated in cfDNA samples of 86 PCa patients taken at and/or post-radical prostatectomy (RP) using univariate and multivariate prediction analyses. RESULTS Putative DMRs in 13 of 30 ABP-related genes were found between tumors of BCR (n = 12) versus no evidence of disease (NED) (n = 15). In silico analysis of The Cancer Genome Atlas data confirmed increased DNA methylation of two loci—SRD5A2 and CYP11A1, which also correlated with their decreased expression, in tumors with subsequent BCR development. Their aberrant cfDNA methylation was also associated with detectable levels of PSA taken after patients' post-RP. Multivariate analysis of the change in cfDNA methylation at all of CpG sites measured along with patient's treatment history predicted if a patient will develop BCR with 77.5% overall accuracy. CONCLUSIONS Overall, increased DNA methylation of SRD5A2 and CYP11A1 related to androgen biosynthesis functions may play a role in BCR after patients' RP. The correlation between aberrant cfDNA methylation and detectable PSA in post-RP further suggests their utility as predictive markers for PCa recurrence. Prostate 75:1790–1801, 2015. © 2015 Wiley Periodicals, Inc.

BACKGROUND. Emerging evidence shows that nanomechanical phenotypes of circulating tumor cells (CTC) could become potential biomarkers for metastatic castration resistant prostate cancer (mCRPC). METHODS. To determine the nanomechanical phenotypes of CTCs we applied atomic force microscopy (AFM) employing the PeakForce quantitative nanomechanical (QNM) imaging. We assessed biophysical parameters (elasticity, deformation, and adhesion) of 130 CTCs isolated from blood samples from five castration sensitive (CS) and 12 castration resistant prostate cancer (CRPCa) patients. RESULTS. We found that CTCs from CRPCa patients are three times softer, three times more deformable, and seven times more adhesive than counterparts from CSPCa patients. Both nonsupervised hierarchical clustering and principle component analysis show that three combined nanomechanical parameters could constitute a valuable set to distinguish between CSPCa and CRPCa. CONLUSIONS. Our study indicates that nanomechanical phenotypes of CTCs may serve as novel and effective biomarkers for mCRPC. Prostate

BACKGROUND Targeting tumor metabolism by energy restriction-mimetic agents (ERMAs) has emerged as a strategy for cancer therapy/prevention. Evidence suggests a mechanistic link between ERMA-mediated antitumor effects and epigenetic gene regulation.

Investigating aberrant DNA methylation in the cancer genome may identify genes that play an important role in tumor progression. In this study, we combined differential methylation hybridization and a CpG microarray platform to characterize methylation profiles and identify novel candidate genes associated with hepatocellular carcinoma (HCC). The genomic DNA of 21 paired adjacent normal and HCC samples was used, and results were analyzed by hierarchical clustering. Twenty-seven hypermethylated candidates and 38 hypomethylated candidates were obtained. Six candidate genes from the hypermethylated group were validated by combined bisulfite restriction analysis; two genes, human kallikrein 10 gene (KLK10) and oxoglutarate (alpha-ketoglutarate) receptor 1 gene (OXGR1), were further analyzed by bisulfite sequencing. The DNA hypermethylation status of KLK10 and OXGR1 were subsequently examined in HCC cell lines and clinical samples using methylation-specific PCR. In 49 HCC samples, 46 (94%) showed that at least one of these two genes was highly methylated. Moreover, KLK10 and OXGR1 mRNA levels were inversely correlated (r = -0.435 and -0.497, P < 0.05) with DNA methylation as examined in paired adjacent normal and tumor samples. Statistical analyses further indicated that KLK10 hypermethylation was significantly associated with cirrhosis (P = 0.042) and HCV infection (P = 0.017) as well as inversely associated with HBV infection (P = 0.023). Furthermore, restoration of KLK10 and OXGR1 expression reduced the ability of anchorage-independent growth, and sensitized HCC cells to doxorubicin- or 5-fluorouracil-induced cytotoxicity. Our results suggest that the hypermethylated KLK10 and OXGR1 are frequent in HCC and may be useful as markers for clinical application.

We describe two brothers and a cousin with common clinical features, including mild mental retardation, motor delays, hypotonia with truncal ataxia, esotropia, and mild facial and hand dysmorphia. The initial routine chromosome study failed to detect any abnormality in the proband. Based on a high index of clinical suspicion, high-resolution chromosome studies were performed on the proband's parents. A small reciprocal translocation t(10;14) (q26.1;q32.3) was detected in the father. The breakpoint on the derivative chromosome 14 was further placed telomeric to the immunoglobulin heavy-chain gene cluster at the band q32.33 by fluorescence in situ hybridization. Studies of the proband and two affected paternal cousins revealed that each had inherited the same derivative chromosome 10 from their carrier parents. This unbalanced karyotype resulted from an adjacent-1 segregation of the 10;14 translocation.

Silencing of tumor suppressor genes (TSGs), by DNA methylation, is well known in adult cancers. However, based on the "stem cell" theory of tumorigenesis, the early epigenetic events arising in malignant precursors remain unknown. A recent report demonstrates that, while pluripotent embryonic stem cells lack DNA methylation and possess a "bivalent" pattern of activating and repressive histone marks in numerous TSGs, analogous multipotent malignant cells derived from germ cell tumors (embryonic carcinoma cells) gain additional silencing modifications to those same genes. These results suggest a possible mechanism by which aberrant differentiation, mediated by histone and DNA methylation, instigates tumor progression.

Methyl-CpG binding proteins (MBDs) mediate histone deacetylase-dependent transcriptional silencing at methylated CpG islands. Using chromatin immunoprecitation (ChIP) we have found that gene-specific profiles of MBDs exist for hypermethylated promoters of breast cancer cells, whilst a common pattern of histone modifications is shared. This unique distribution of MBDs is also characterized in chromosomes by comparative genomic hybridization of immunoprecipitated DNA and immunolocalization. Most importantly, we demonstrate that MBD association to methylated DNA serves to identify novel targets of epigenetic inactivation in human cancer. We combined the ChIP assay of MBDs with a CpG island microarray (ChIP on chip). The scenario revealed shows that, while many genes are regulated by multiple MBDs, others are associated with a single MBD. These target genes displayed methylation- associated transcriptional silencing in breast cancer cells and primary tumours. The candidates include the homeobox gene PAX6, the prolactin hormone receptor, and dipeptidylpeptidase IV among others. Our results support an essential role for MBDs in gene silencing and, when combined with genomic strategies, their potential to ‘catch’ new hypermethylated genes in cancer.

Epigenetic regulation of gene expression has been observed in a variety of tumor types. We have used microarray technology to evaluate the predisposition of drug response by aberrant methylation in ovarian cancer. Results indicate that loss of gene activity due to hypermethylation potentially confers a predisposition in certain cancer types and is an early event in disease progression. Methylation profiles of ovarian cancer might be useful for early cancer detection and prediction of chemotherapy outcome in a clinical context.

Gene silencing via promoter CpG island hypermethylation offers tumor cells growth advantages. This epigenetic event is pharmacologically reversible, and uncovering a unique set of methylation-silenced genes in tumor cells can bring a new avenue to cancer treatment. However, high-throughput tools capable of surveying the methylation status of multiple gene promoters are needed for this discovery process. Herein we describe an oligonucleotide-based microarray technique that is both versatile and sensitive in revealing hypermethylation in defined regions of the genome. DNA samples are bisulfite-treated and PCR-amplified to distinguish CpG dinucleotides that are methylated from those that are not. Fluorescently labeled PCR products are hybridized to arrayed oligonucleotides that can discriminate between methylated and unmethylated alleles in regions of interest. Using this technique, two clinical subtypes of non-Hodgkin's lymphomas, mantle cell lymphoma, and grades I/II follicular lymphoma, were further separated based on the differential methylation profiles of several gene promoters. Work is underway in our laboratory to extend the interrogation power of this microarray system in multiple candidate genes. This novel tool, therefore, holds promise to monitor the outcome of various epigenetic therapies on cancer patients.

Tertiary trisomy, or double trisomy, is a rare occurrence. We present two individuals with a previously unreported tertiary trisomy for chromosomes 5p and 21q in an eight-generation pedigree. Their phenotypes are compared with other partial trisomies of either 5p or 21q from the literature. The propositus was diagnosed with trisomy 21 at 2 years of age after a karyotype study for short stature and developmental delay. His phenotype was described as atypical for Down syndrome. He presented at 9 years of age because of pervasive behavioral problems and obesity. He was brachycephalic with a flattened nasal bridge, but he lacked other characteristics of trisomy 21. Because of lack of phenotypic evidence of Down syndrome, a repeat karyotype was obtained and showed 47,XY, +der(21)t(5;21)(p15.1; q22.1), incorporating partial trisomies of both chromosomes 5 and 21. Mother had a balanced translocation, 46, XX,t(5;21)(p15.1; q22.1); 8 other relatives were examined. The translocation originated from the maternal great-grandmother, but only the propositus and his mentally retarded aunt had a similar phenotye and the derivative chromosome. Fluorescence in situ hybridization showed absence of band 21q22.2 in the derivative chromosome of the propositus and his aunt, indicating that neither had trisomy for the Down syndrome critical region. These cases represent a unique double partial trisomy of chromosome arms 5p and 21q that occurred because of 3:1 malsegregation of a reciprocal translocation. These cases further demonstrate that phenotypic discordance with cytogenetic results dictate further investigation using advanced cytogenetic hybridization.

Aberrant DNA methylation is considered a major mechanism for silencing tumor suppressor genes in gastric cancer. We used CpG microarray and differential methylation hybridization strategies to identify potential tumor suppressor genes and recovered glutamate receptor, ionotropic, kainate 2 (GRIK2) as a novel epigenetic target in gastric cancer. Additional experiments showed that the promoter region of GRIK2 was hypermethylated in 3 of the 4 tested gastric cancer cell lines, and its expression was restored by treatment of cells with the DNA methylation inhibitor, 5 0 -aza-dC. In clinical samples, the GRIK2 promoter was differentially hypermethylated in tumor tissues compared with adjacent normal tissues (p < 0.001), and this methylation was inversely correlated with the expression level of GRIK2 mRNA (r 52 0.44). Functional studies further showed that GRIK2-expressing gastric cancer cell lines showed decreased colony formation and cell migration. Taken together, these results suggest that GRIK2 may play a tumor-suppressor role in gastric cancer. Future studies are warranted to examine whether DNA hypermethylation of the GRIK2 promoter can be used as a potential tumor marker for gastric cancer.

A family in which 6 males have X-linked mental retardation has been studied with polymorphic DNA probes. The males differ from unaffected males only in impaired intellect and in smaller head size. The gene that causes mental retardation in the family appears to be located in band Xq21 on the basis of linkage with 3 markers: DXS250, DXS345 and DXS3 (theta max = 0.00; Zmax = 1.6). A multipoint lod score of 2.36 was obtain with no recombination relative to DXS326 in Xq21. This family is considered to have nonspecific X-linked mental retardation and has been given the designation MRX8.