Genome-wide DNA methylation profiles in both precancerous conditions and clear cell renal cell carcinomas are correlated with malignant potential and patient outcome

To clarify genome-wide DNA methylation profiles during multistage renal carcinogenesis, bacterial artificial chromosome array-based methylated CpG island amplification (BAMCA) was performed. Non-cancerous renal cortex tissue obtained from patients with clear cell renal cell carcinomas (RCCs) (N) was at the precancerous stage where DNA hypomethylation and DNA hypermethylation on multiple bacterial artificial chromosome (BAC) clones were observed. By unsupervised hierarchical clustering analysis based on BAMCA data for their N, 51 patients with clear cell RCCs were clustered into two subclasses, Clusters AN (n = 46) and BN (n = 5). Clinicopathologically aggressive clear cell RCCs were accumulated in Cluster BN, and the overall survival rate of patients in Cluster BN was significantly lower than that of patients in Cluster AN. By unsupervised hierarchical clustering analysis based on BAMCA data for their RCCs, 51 patients were clustered into two subclasses, Clusters AT (n = 43) and BT (n = 8). Clinicopathologically aggressive clear cell RCCs were accumulated in Cluster BT, and the overall survival rate of patients in Cluster BT was significantly lower than that of patients in Cluster AT. Multivariate analysis revealed that belonging to Cluster BT was an independent predictor of recurrence. Cluster BN was completely included in Cluster BT, and the majority of the BAC clones that significantly discriminated Cluster BN from Cluster AN also discriminated Cluster BT from Cluster AT. In individual patients, DNA methylation status in N was basically inherited by the corresponding clear cell RCC. DNA methylation alterations in the precancerous stage may generate more malignant clear cell RCCs and determine patient outcome

RASSF1A hypermethylation in pretreatment serum DNA of neuroblastoma patients: a prognostic marker

The tumour suppressor gene RASSF1A is known to be frequently silenced by promoter hypermethylation in neuroblastoma tumours. Here we explored the possible prognostic significance of aberrant promoter hypermethylation of RASSF1A in serum DNA samples of patients with neuroblastoma as a surrogate marker for circulating tumour cells. We analysed the methylation status of the RASSF1A gene in matched tumour and pretreatment serum DNA obtained from 68 neuroblastoma patients. Hypermethylation of RASSF1A in tumour samples was found in 64 patients (94%). In contrast, serum methylation of RASSF1A was observed in 17 patients (25%). Serum methylation of RASSF1A was found to be statistically associated with age ⩾12 months at diagnosis (P=0.002), stage 4 (P<0.001) and MYCN amplification (P<0.001). The influence of serum RASSF1A methylation on prognosis was found to be comparable with that of the currently most reliable marker, MYCN amplification on univariate analysis (hazard ratio, 9.2; 95% confidence interval (CI), 2.8–30.1; P<0.001). In multivariate analysis of survival, methylation of RASSF1A in serum had a hazard ratio of 2.4 (95% CI, 0.6–9.2), although this association did not reach statistical significance (P=0.194). These findings show that the methylation status of RASSF1A in the serum of patients with neuroblastoma has the potential to become a prognostic predictor of outcome

Functional epigenomics approach to identify methylated candidate tumour suppressor genes in renal cell carcinoma

Promoter region hypermethylation and transcriptional silencing is a frequent cause of tumour suppressor gene (TSG) inactivation in many human cancers. Previously, to identify candidate epigenetically inactivated TSGs in renal cell carcinoma (RCC), we monitored changes in gene expression in four RCC cell lines after treatment with the demethylating agent 5-azacytidine. This enabled us to identify HAI-2/SPINT2 as a novel epigenetically inactivated candidate RCC TSG. To identify further candidate TSGs, we undertook bioinformatic and molecular genetic evaluation of a further 60 genes differentially expressed after demethylation. In addition to HAI-2/SPINT2, four genes (PLAU, CDH1, IGFB3 and MT1G) had previously been shown to undergo promoter methylation in RCC. After bioinformatic prioritisation, expression and/or methylation analysis of RCC cell lines±primary tumours was performed for 34 genes. KRT19 and CXCL16 were methylated in RCC cell lines and primary RCC; however, 22 genes were differentially expressed after demethylation but did not show primary tumour-specific methylation (methylated in normal tissue (n=1); methylated only in RCC cell lines (n=9) and not methylated in RCC cell lines (n=12)). Re-expression of CXCL16 reduced growth of an RCC cell line in vitro. In a summary, a functional epigenomic analysis of four RCC cell lines using microarrays representing 11 000 human genes yielded both known and novel candidate TSGs epigenetically inactivated in RCC, suggesting that this is valid strategy for the identification of novel TSGs and biomarkers

Genome-scale screen for DNA methylation-based detection markers for ovarian cancer.

The identification of sensitive biomarkers for the detection of ovarian cancer is of high clinical relevance for early detection and/or monitoring of disease recurrence. We developed a systematic multi-step biomarker discovery and verification strategy to identify candidate DNA methylation markers for the blood-based detection of ovarian cancer

Investigation of Griffithsin's Interactions with Human Cells Confirms Its Outstanding Safety and Efficacy Profile as a Microbicide Candidate

Many natural product-derived lectins such as the red algal lectin griffithsin (GRFT) have potent in vitro activity against viruses that display dense clusters of oligomannose N-linked glycans (NLG) on their surface envelope glycoproteins. However, since oligomannose NLG are also found on some host proteins it is possible that treatment with antiviral lectins may trigger undesirable side effects. For other antiviral lectins such as concanavalin A, banana lectin and cyanovirin-N (CV-N), interactions between the lectin and as yet undescribed cellular moieties have been reported to induce undesirable side effects including secretion of inflammatory cytokines and activation of host T-cells. We show that GRFT, unlike CV-N, binds the surface of human epithelial and peripheral blood mononuclear cells (PBMC) through an exclusively oligosaccharide-dependent interaction. In contrast to several other antiviral lectins however, GRFT treatment induces only minimal changes in secretion of inflammatory cytokines and chemokines by epithelial cells or human PBMC, has no measureable effect on cell viability and does not significantly upregulate markers of T-cell activation. In addition, GRFT appears to retain antiviral activity once bound to the surface of PBMC. Finally, RNA microarray studies show that, while CV-N and ConA regulate expression of a multitude of cellular genes, GRFT treatment effects only minimal alterations in the gene expression profile of a human ectocervical cell line. These studies indicate that GRFT has an outstanding safety profile with little evidence of induced toxicity, T-cell activation or deleterious immunological consequence, unique attributes for a natural product-derived lectin

Identification of candidate tumour suppressor genes frequently methylated in renal cell carcinoma

Promoter region hyermethylation and transcriptional silencing is a frequent cause of tumour suppressor gene (TSG) inactivation in many types of human cancers. Functional epigenetic studies, in which gene expression is induced by treatment with demethylating agents, may identify novel genes with tumour-specific methylation. We used high-density gene expression microarrays in a functional epigenetic study of 11 renal cell carcinoma (RCC) cell lines. Twenty-eight genes were then selected for analysis of promoter methylation status in cell lines and primary RCC. Eight genes (BNC1, PDLIM4, RPRM, CST6, SFRP1, GREM1, COL14A1 and COL15A1) showed frequent (30% of RCC tested) tumour-specific promoter region methylation. Hypermethylation was associated with transcriptional silencing. Re-expression of BNC1, CST6, RPRM and SFRP1 suppressed the growth of RCC cell lines and RNA interference knock-down of BNC1, SFRP1 and COL14A1 increased the growth of RCC cell lines. Methylation of BNC1 or COL14A1 was associated with a poorer prognosis independent of tumour size, stage or grade. The identification of these epigenetically inactivated candidate RCC TSGs can provide insights into renal tumourigenesis and a basis for developing novel therapies and biomarkers for prognosis and detection. © 2010 Macmillan Publishers Limited.Published versio

The evidence base for circulating tumour DNA blood-based biomarkers for the early detection of cancer: a systematic mapping review

Background: The presence of circulating cell-free DNA from tumours in blood (ctDNA) is of major importance to those interested in early cancer detection, as well as to those wishing to monitor tumour progression or diagnose the presence of activating mutations to guide treatment. In 2014, the UK Early Cancer Detection Consortium undertook a systematic mapping review of the literature to identify blood-based biomarkers with potential for the development of a non-invasive blood test for cancer screening, and which identified this as a major area of interest. This review builds on the mapping review to expand the ctDNA dataset to examine the best options for the detection of multiple cancer types. Methods: The original mapping review was based on comprehensive searches of the electronic databases Medline, Embase, CINAHL, the Cochrane library, and Biosis to obtain relevant literature on blood-based biomarkers for cancer detection in humans (PROSPERO no. CRD42014010827). The abstracts for each paper were reviewed to determine whether validation data were reported, and then examined in full. Publications concentrating on monitoring of disease burden or mutations were excluded. Results: The search identified 94 ctDNA studies meeting the criteria for review. All but 5 studies examined one cancer type, with breast, colorectal and lung cancers representing 60% of studies. The size and design of the studies varied widely. Controls were included in 77% of publications. The largest study included 640 patients, but the median study size was 65 cases and 35 controls, and the bulk of studies (71%) included less than 100 patients. Studies either estimated cfDNA levels non-specifically or tested for cancer-specific mutations or methylation changes (the majority using PCR-based methods). Conclusion: We have systematically reviewed ctDNA blood biomarkers for the early detection of cancer. Pre-analytical, analytical, and post-analytical considerations were identified which need to be addressed before such biomarkers enter clinical practice. The value of small studies with no comparison between methods, or even the inclusion of controls is highly questionable, and larger validation studies will be required before such methods can be considered for early cancer detection

The SEQC2 epigenomics quality control (EpiQC) study

BACKGROUND: Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA's Epigenomics Quality Control Group. RESULTS: Each sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), enzymatic deamination method (EMSeq), targeted methylation sequencing (Illumina Methyl Capture EPIC), single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies, and 850k Illumina methylation arrays. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, bwa-meth, and BitMapperBS), we find overall high concordance between assays, but also differences in efficiency of read mapping, CpG capture, coverage, and platform performance, and variable performance across 26 microarray normalization algorithms. CONCLUSIONS: The data provided herein can guide the use of these DNA reference materials in epigenomics research, as well as provide best practices for experimental design in future studies. By leveraging seven human cell lines that are designated as publicly available reference materials, these data can be used as a baseline to advance epigenomics research