Use Of Methylation Biomarkers Research Articles

Leveraging locus-specific epigenetic heterogeneity to improve the performance of blood-based DNA methylation biomarkers

BackgroundVariation in intercellular methylation patterns can complicate the use of methylation biomarkers for clinical diagnostic applications such as blood-based cancer testing. Here, we describe development and validation of a methylation density binary classification method called EpiClass (available for download at https://github.com/Elnitskilab/EpiClass) that can be used to predict and optimize the performance of methylation biomarkers, particularly in challenging, heterogeneous samples such as liquid biopsies. This approach is based upon leveraging statistical differences in single-molecule sample methylation density distributions to identify ideal thresholds for sample classification.ResultsWe developed and tested the classifier using reduced representation bisulfite sequencing (RRBS) data derived from ovarian carcinoma tissue DNA and controls. We used these data to perform in silico simulations using methylation density profiles from individual epiallelic copies of ZNF154, a genomic locus known to be recurrently methylated in numerous cancer types. From these profiles, we predicted the performance of the classifier in liquid biopsies for the detection of epithelial ovarian carcinomas (EOC). In silico analysis indicated that EpiClass could be leveraged to better identify cancer-positive liquid biopsy samples by implementing precise thresholds with respect to methylation density profiles derived from circulating cell-free DNA (cfDNA) analysis. These predictions were confirmed experimentally using DREAMing to perform digital methylation density analysis on a cohort of low volume (1-ml) plasma samples obtained from 26 EOC-positive and 41 cancer-free women. EpiClass performance was then validated in an independent cohort of 24 plasma specimens, derived from a longitudinal study of 8 EOC-positive women, and 12 plasma specimens derived from 12 healthy women, respectively, attaining a sensitivity/specificity of 91.7%/100.0%. Direct comparison of CA-125 measurements with EpiClass demonstrated that EpiClass was able to better identify EOC-positive women than standard CA-125 assessment. Finally, we used independent whole genome bisulfite sequencing (WGBS) datasets to demonstrate that EpiClass can also identify other cancer types as well or better than alternative methylation-based classifiers.ConclusionsOur results indicate that assessment of intramolecular methylation density distributions calculated from cfDNA facilitates the use of methylation biomarkers for diagnostic applications. Furthermore, we demonstrated that EpiClass analysis of ZNF154 methylation was able to outperform CA-125 in the detection of etiologically diverse ovarian carcinomas, indicating broad utility of ZNF154 for use as a biomarker of ovarian cancer.

Potential of the use of methylation biomarkers for diagnostics and prognosis of hepatocellular carcinoma in liquid biopsy

Hepatocellular carcinoma (HCC) is one of the most common types of liver cancer that is mainly diagnosed at advanced stages. Since molecular markers that are currently used in clinical practice are not sensitive enough for effective diagnosis of HCC, active search of new biomarkers is underway. Us е of minimally invasive liquid biopsy allows to detect specific markers of tumor growth and particularly alterations of gene methylation patterns in cell-free DNA distinctive for HCC, in biological liquids of patients. In the present review the most promising diagnostic and prognostic methylation biomarkers that were detected in cell-free DNA of HCC patients are being considered.

Abstract 4359: Systematic approach to methylation biomarker development in chronic lymphocytic leukemia, breast and lung cancers

Abstract There is no doubt that testing for disease specific methylation changes can guide disease risk assessment, facilitate detection of the disease, support prognostication and personalization of treatment as well as guide post treatment patient care (1). Nevertheless, the use of methylation biomarkers in standard patient care is still marginal. Difficulties in implementation of methylation biomarker testing in standard in-vitro diagnostics are mainly attributed to challenges in establishment of the systematic approach allowing for efficient discovery and subsequent validation of clinical utility of potential disease specific methylation biomarkers. We have combined state-of-the-art genome wide methylation screening technologies (including: newest Illumina MethylationEPIC 850k BeadChip) enabling methylation biomarker discovery with the cost and time efficient locus specific technologies to streamline the development, validation and implementation of methylation biomarkers for clinical disease management. With the use of technologies that in an affordable fashion enable screening for genome wide methylation changes in a substantial number of clinical samples, we were able to discover clinically relevant disease specific methylation signatures e.g. in CLL. The high technical complexity of the genome wide screen technologies prevents them for being straightforward applicable in diagnostic settings. Thus we used techniques such as Methylation-Sensitive High Resolution Melting (MS-HRM) (2) to develop assays fulfilling the requirements for diagnostic applications. Subsequently, we evaluated the clinical relevance of the most promising biomarker candidates in chronic lymphocytic leukaemia, breast and lung cancers, using these assays (3, 4). Overall, our workflow allows fast and efficient validation of not only already existing biomarker candidates but also discovery of new disease specific methylation changes that show promise for clinical implementation.

DNA methylation analysis

DNA methylation, the addition of a methyl group to cytosines and adenosines, regulates gene expression on a level that is usually referred to as epigenetic, that is, stably maintained during cell divisions. In humans, aberrant DNA methylation is associated with several malignancies, including cancer and so-called imprinting disorders, making it an attractive target for diagnostic purposes. Here we give a brief introduction to the biology of DNA methylation and present the use of methylation biomarkers in laboratory medicine. DNA methylation assays have become the standard procedure in the diagnosis of imprinting disorders, and they are about to shift cancer diagnostics and prognostics to the next level of molecular medicine. However, there is evidence of problems associated with the introduction of such cancer assays in routine diagnostics. We review several assays that have been proposed for DNA methylation analysis. The assays presented analyse the methylation status of single loci and are based either on a bisulphite-treatment or on methylation-sensitive restriction of the DNA under investigation.