Technical Advances in Circulating Cell-Free DNA Detection and Analysis for Personalized Medicine in Patients' Care

Abstract

Circulating cell-free DNA (cfDNA) refers to small fragments of DNA molecules released after programmed cell death and necrosis in several body fluids such as blood, saliva, urine, and cerebrospinal fluid. The discovery of cfDNA has revolutionized the field of non-invasive diagnostics in the oncologic field, in prenatal testing, and in organ transplantation. Despite the potential of cfDNA and the solid results published in the recent literature, several challenges remain, represented by a low abundance, a need for highly sensitive assays, and analytical issues. In this review, the main technical advances in cfDNA analysis are presented and discussed, with a comprehensive examination of the current available methodologies applied in each field. Considering the potential advantages of cfDNA, this biomarker is increasing its consensus among clinicians, as it allows us to monitor patients' conditions in an easy and non-invasive way, offering a more personalized care. Nevertheless, cfDNA analysis is still considered a diagnostic marker to be further validated, and very few centers are implementing its analysis in routine diagnostics. As technical improvements are enhancing the performances of cfDNA analysis, its application will transversally improve patients' quality of life.

Keywords: cell-free DNA; digital PCR; liquid biopsy; next-generation sequencing; non-invasive diagnostics.

Figure 1

List of the NGS-based and non-NGS methods for cfDNA analysis described in the review. The different methodologies are divided according to their technological approaches. The main methods are highlighted in blue, while derived methods are indicated by arrows. References are listed by application field [27,29,33,34,35,39,46,47,51,56,57,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84]. NGS: Next-generation Sequencing; TAm-Seq: Tagged-amplicon Deep Sequencing; CAPP-Seq: Cancer Personalized Profiling by Deep Sequencing; WGBS-Seq: Whole Genome Bisulfite Sequencing; WES: Whole Exome Sequencing; WGS: Whole Genome Sequencing; qPCR: quantitative PCR; ARMS-PCR: Amplification Refractory Mutation System PCR; PNA Clamp PCR: Peptide Nucleic Acid Clamp PCR; COLD-PCR: Co-amplification at Lower Denaturation Temperature-based PCR; dPCR: digital PCR; BEAMing: Beads, Emulsion, Amplification, Magnetics PCR.