Cell-Free DNA: Features and Attributes Shaping the Next Frontier in Liquid Biopsy

Abstract

Cell-free DNA (cfDNA) is changing the face of liquid biopsy as a minimally invasive tool for disease detection and monitoring, with its main applications in oncology and prenatal testing, and rising roles in transplant patient monitoring. However, the processes of cfDNA biogenesis, fragmentation, and clearance are complex and require further investigation. Evidence suggests that cfDNA production relates to mechanisms of cell death and DNA repair, both of which further influence fragment size and its applicability as a biomarker. An emerging domain, cfDNA fragmentomics is being explored for advancing the field of diagnostics using non-mutational signatures such as fragment size ratios and methylation patterns. Thus, this review examines structural diversity in cfDNA with various fragment sizes. In examining these cfDNA subsets, we discuss their distinct biological origins and potential clinical utility. Development of sequencing methodologies has broadened the application of cfDNA in diagnosing cancers and organ-specific pathologies, as well as directing personalized therapies. This has been achieved by identifying and uncovering different subsets of cfDNA in biofluids using different methodologies and biofluids. Different cfDNA subsets provide important insights regarding genomic and epigenetic features, enhancing the understanding of gene regulation, tissue-specific functions, and disease progression. Advancement of these key areas further asserts increasing clinical relevance for the use of cfDNA as a biomarker. Continued exploration of cfDNA subsets is expected to drive further innovation in liquid biopsy and its integration into routine clinical practice.

Fig. 1

Plasma derived cfDNA possess different fragment lengths and characteristics. A Low molecular weight DNA extraction with single-stranded DNA library preparation of plasma cfDNA reveals a mononucleosomal cfDNA peak (~166 bps) along with an ultrashort cfDNA peak (40–70 bps) and short cfDNA valley (70–120 bps) (blue box). B Multiples of the nucleosomal peaks (~166 bps, ~320 bps, ~480 bps) are revealed with routine double-stranded library DNA protocols (green box). C Contrasting characteristics of different subsets of cfDNA: Genomic region mapping of called peaks differ amongst subsets of cfDNA; differences in abundance of potential secondary structures seen in cfDNA; differences in end motif cleavage for different subsets of cfDNA. Data derived from [15, 16]. Created in BioRender.com

Fig. 2

Factors affecting cfDNA characteristics: Processes and structural features that lead to cfDNA formation: biological and cellular processes, genomic structures, nucleosomal associated structures or epigenomic modifications. Source of cfDNA, biofluids, and cells of origin. Created in BioRender.com

Fig. 3

cfDNA profile in different biofluids using a single-stranded library preparation: A plasma, B saliva and C urine. Data derived from [15, 17, 76]

Fig. 4

Downstream functions of cfDNA: Immune modulatory effects, horizontal transfer of information and inducing transformation to cancer phenotype, biofilm formation. Created in BioRender.com

Fig. 5

Different technical considerations that affect cfDNA attributes. Created in BioRender.com