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Methylation analysis of circulating cell-free DNA (cirDNA), as a liquid biopsy, has a significant potential to advance the detection, prognosis, and treatment of cancer, as well as many genetic disorders. The role of epigenetics in disease development has been reported in several hereditary disorders, and epigenetic modifications are regarded as one of the earliest and most significant genomic aberrations that arise during carcinogenesis. Liquid biopsy can be employed for the detection of these epigenetic biomarkers. It consists of isolation (pre-analytical) and detection (analytical) phases. The choice of pre-analytical variables comprising cirDNA extraction and bisulfite conversion methods can affect the identification of cirDNA methylation. Indeed, different techniques give a different return of cirDNA, which confirms the importance of pre-analytical procedures in clinical diagnostics. Although novel techniques have been developed for the simplification of methylation analysis, the process remains complex, as the steps of DNA extraction, bisulfite treatment, and methylation detection are each carried out separately. Recent studies have noted the absence of any standard method for the pre-analytical processing of methylated cirDNA. We have therefore conducted a comprehensive and systematic review of the important pre-analytical and analytical variables and the patient-related factors which form the basis of our guidelines for analyzing methylated cirDNA in liquid biopsy., (© 2021. The Author(s).)

Purpose: Recently, 'solid tumor biopsies' have been challenged by the emergence of 'liquid biopsies', which are aimed at the isolation and detection of circulating cell-free tumor DNA (ctDNA) in body fluids. Here, we developed and optimized a method for selective capture of ctDNA on magnetic beads (SCC-MAG) for mutation detection in plasma of patients with colorectal cancer (CRC)., Methods: Blood and tissue samples from 28 CRC patients were included for the detection of KRAS mutations. For the tissue samples, mutation analysis was conducted by high resolution melting (HRM) analysis and sequencing. For the SCC-MAG method, ctDNA was isolated from 200 µl plasma from patients with a mutant KRAS gene. For comparison, ctDNA extraction was carried out using a silica membrane-based method, after which mutations were detected using Intplex allele-specific PCR., Results: The mean ctDNA integrity index in plasma samples of cancer patients was 1.03, comparable with that of silica membrane-derived ctDNA (1.011). Notably, the limit of detection for the SCC-MAG approach was lower than that of the silica membrane method and measured 2.25 pg/ml ctDNA in plasma. Our analyses showed that while the silica membrane-based approach was capable of collecting ctDNA from two out of six CRC patient samples (average Cq 34.23), the SCC-MAG captured ctDNA from all samples with an average Cq of 29.76., Conclusions: We present a robust, reproducible, and highly sensitive method for the analysis of mutation statuses in liquid biopsies. The SCC-MAG method can readily be applied to any nucleic acid target for diagnostic purposes upon careful design of the specific capture probes, and can be multiplexed by several probes to identify multiple targets.

Colorectal cancer (CRC), the second leading cause of cancer mortality, constitutes a significant global health burden. An accurate, noninvasive detection method for CRC as complement to colonoscopy could improve the effectiveness of treatment. In the present study, SureSelectXT Methyl-Seq was performed on cancerous and normal colon tissues and CLDN1, INHBA and SLC30A10 were found as candidate methylated genes. MethyLight assay was run on formalin-fixed paraffin-embedded (FFPE) and fresh case and control tissues to validate the methylation of the selected gene. The methylation was significantly different (p-values < 2.2e-16) with a sensitivity of 87.17%; at a specificity cut-off of 100% in FFPE tissues. Methylation studies on fresh tissues, indicated a sensitivity of 82.14% and a specificity cut-off of 92% (p-values = 1.163e-07). The biomarker performance was robust since, normal tissues indicated a significant 22.1-fold over-expression of the selected gene as compared to the corresponding CRC tissues (p-value < 2.2e-16) in the FFPE expression assay. In our plasma pilot study, evaluation of the tissue methylation marker in the circulating cell-free DNA, demonstrated that 9 out of 22 CRC samples and 20 out of 20 normal samples were identified correctly. In summary, there is a clinical feasibility that the offered methylated gene could serve as a candidate biomarker for CRC diagnostic purpose, although further exploration of our candidate gene is warranted.

Innovative diagnostics are becoming an essential component in personalized cancer medicine. These diagnostics are increasingly based on cell-free nucleic acids and membrane vesicles. Isolating and sequencing cell free circulating DNA (cfDNA) in plasma may progressively substitute tumor biopsies. A small albeit now detectable fraction of cfDNA correspond to circulating tumor DNA (ctDNA). In this review, we describe the pre-analytical procedures for collecting ctDNA from plasma, since these procedures should be optimized within laboratories depending on the available infrastructures. We also provide an overview of the technological breakthrough in ctDNA Isolation for instance digital PCR methods and next generation sequencing techniques and discuss their key challenges. The clinical implementations of liquid biopsy and more specifically ctDNA in cancer management are reviewed. We predict in the near future, ctDNA will be used more routinely to guide cancer treatment and provide a new approach to personalize treatment in precision medicine., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: In the present study, we investigated different simple and cost effective methods to evaluate and validate cell free DNA (cfDNA) isolation. The ability of the QIAamp DNA Blood Mini Kit method to extract cfDNA was assessed by several approaches, including purification of endogenous cfDNA and exogenous spike-in control material, prior to plasma extraction, and followed by quantitative-PCR., Results: Using QIAamp DNA Blood Mini kit, nearly 27% (380 bp) to 35% (173 bp) cfDNA was recovered with a higher recovery of smaller size cfDNA (173 bp) in comparison to larger ones (380 bp). These simple laboratory methods can be used to assess the efficiency of any cfDNA isolation method.