Cell-Free DNA-Methylation-Based Methods and Applications in Oncology

Abstract

Liquid biopsy based on cell-free DNA (cfDNA) enables non-invasive dynamic assessment of disease status in patients with cancer, both in the early and advanced settings. The analysis of DNA-methylation (DNAm) from cfDNA samples holds great promise due to the intrinsic characteristics of DNAm being more prevalent, pervasive, and cell- and tumor-type specific than genomics, for which established cfDNA assays already exist. Herein, we report on recent advances on experimental strategies for the analysis of DNAm in cfDNA samples. We describe the main steps of DNAm-based analysis workflows, including pre-analytics of cfDNA samples, DNA treatment, assays for DNAm evaluation, and methods for data analysis. We report on protocols, biomolecular techniques, and computational strategies enabling DNAm evaluation in the context of cfDNA analysis, along with practical considerations on input sample requirements and costs. We provide an overview on existing studies exploiting cell-free DNAm biomarkers for the detection and monitoring of cancer in early and advanced settings, for the evaluation of drug resistance, and for the identification of the cell-of-origin of tumors. Finally, we report on DNAm-based tests approved for clinical use and summarize their performance in the context of liquid biopsy.

The MAL Protein, an Integral Component of Specialized Membranes, in Normal Cells and Cancer

“…The use of different methods—the sequencing of bisulfite-treated DNA, methylation-specific PCR (MSP), quantitative MSP, pyrosequencing, etc. [ 216 , 217 , 218 ]—and the analysis of different segments of the MAL CpG island make it difficult to identify the methylation of specific CpG dinucleotides critical to the regulation of MAL gene expression. However, a common feature of most of these studies is that the methylation of the first exon and the promoter region most proximal to the transcriptional start site is important for regulating MAL gene activity.…”

Detection of Cell Types Contributing to Cancer From Circulating, Cell-Free Methylated DNA

“…There are many techniques that can be used to study DNA methylation as well as different strategies that can be applied to classify and quantify methylation status (Olkhov-Mitsel and Bapat, 2012;Kurdyukov and Bullock, 2016;Galardi et al, 2020;Zhao et al, 2020). These methodologies must be able to distinguish between methylated and unmethylated cytosines.…”

Saliva Gene Promoter Hypermethylation as a Biomarker in Oral Cancer

“…Targeted bisulfite sequencing techniques represent a cost-efficient method with high depth of sequencing coverage that allow the simultaneous analysis of multiple genomic regions by target PCR amplification or probe hybridization capture. In addition, different PCR-based techniques have been used for evaluating the DNA methylation status at a locus-specific level, such as MSP, qMSP, MethyLight and droplet digital PCR (ddPCR) [ 66 ]. Overall, the most frequently used methods for evaluating salivary DNA methylation in oral cancer were MSP and qMSP.…”