National preanalytical study

OptiMeth: Optimization and standardization of ctDNA ddPCR methylation analyses

The purpose of this study is to advance the fi eld of methylation analysis in cell-free DNA, focusing on optimizing the bisulfi te treatment process for higher sensitivity. Simultaneously, the study aims to explore and compare three methods - traditional bisulfi te treatment, Methylation-Sensitive Restriction Enzymes (MSRE), and the innovative EpiDirect® platform.

Abstract

Relevance:
DNA methylation is an epigenetic mechanism that regulates biological processes in normal development as well as in diseases, including various cancers. Aberrant DNA methylation is found in most cancers including colorectal and lung cancers. Since an increasing number of clinical studies ctDNA methylation analyses, there is an increasing need for optimization and standardization.

Background:
Traditional approaches, such as bisulfi te treatment, while widely used, pose challenges including DNA degradation and irreversible changes to the DNA, prompting the exploration of alternative methods for methylation analysis in circulating tumor DNA. Methylation-Sensitive Restriction Enzymes (MSREs) off er a distinct approach by recognizing and cleaving methylated DNA sequences without the need for time consuming pre-treatment, but their applicability is hindered by the dependence on CpG site presence. In contrast, innovative methods like EpiDirect® by Pentabase present a promising solution, bypassing the drawbacks of bisulfi te treatment and off ering direct quantifi cation of methylation in untreated DNA. The challenge lies in validating the specifi city of EpiDirect® across diverse genomic regions.

Aim:
The purpose of this study is to advance the fi eld of methylation analysis in cell-free DNA, focusing on optimizing the bisulfi te treatment process for higher sensitivity. Simultaneously, the study aims to explore and compare three methods - traditional bisulfi te treatment, Methylation-Sensitive Restriction Enzymes (MSRE), and the innovative EpiDirect® platform. By addressing the limitations associated with bisulfi te treatment and evaluating alternative methods, the study seeks to enhance the accuracy and effi ciency of DNA methylation analysis in cell-free DNA. We will include methylation markers for C9orf50, KCNQ5, CLIP4 (TriMeth), NPY, MLH1 and SFRP1 in our optimization and standardization, since these are used in a number of clinical protocols with the DCCC ctDNA ResearchCenter. In addition, we will also develop process and sample quality control assays, which will enable validation of samples and analyses performance and thereby potentially lead to national standardization and improved quality of methylation analyses.

Hypotheses:
Utilizing enzymatic treatment or modifi ed PCR primers from Pentabase will increase sensitivity in ctDNA analyses.

Methods:
This study will explore three methods for DNA methylation analysis, each off ering specifi c advantages and challenges. Traditional bisulfi te treatment, a cornerstone in methylation studies, induces the conversion of unmethylated cytosines to uracil, but its drawbacks include DNA degradation and irreversible changes. Methylation-Sensitive Restriction Enzymes (MSREs) provide an alternative by selectively cleaving methylated DNA sequences, yet their dependence on methylation specifi c restriction enzyme sites may limit a widespread applicability. On the other hand, the innovative EpiDirect® platform by Pentabase off ers a direct quantifi cation of methylation in untreated DNA, avoiding bisulfi te treatment challenges. However, its specifi city across diverse genomic regions requires comprehensive validation.

Curious to read about other national clinical projects?

FUNDED PROJECTS