The rising tide of cell-free DNA profiling: from snapshot to temporal genome analysis

Abstract

Genomes of diverse origins are continuously shed into human body fluids in the form of fragmented cell-free DNA (cfDNA). These molecules maintain the genetic and epigenetic codes of their originating source, and often carry additional layers of unique information in newly discovered physico-chemical features. Characterization of cfDNA thus presents the opportunity to non-invasively reconstruct major parts of the host- and metagenome in silico. Data from a single specimen can be leveraged to detect a broad range of disease-specific signatures and has already enabled the development of many pioneering diagnostic tests. Moreover, data from serial sampling may allow unparalleled mapping of the scantily explored landscape of temporal genomic changes as it relates to various changes in different physiological and pathological states of individuals. In this review, we explore how this vast dimension of biological information accessible through cfDNA analysis is being tapped towards the development of increasingly powerful molecular assays and how it is shaping emerging technologies. We also discuss how this departure from traditional paradigms of snapshot genetic testing may pave the way for an onrush of new and exciting discoveries in human biology.

New Perspectives on the Importance of Cell-Free DNA Biology

“…These issues are increasingly addressed [ 7 , 58 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 ], yet preanalytical optimization and standardization remains a major, evolving issue that requires ongoing surveillance and active problem solving. As reviewed elsewhere [ 130 ], ctDNA profiling is also challenged by clonal hematopoiesis (CH)-derived cfDNA that bear cancer-specific mutations. This is especially problematic as this is a common phenomenon in both cancer patients [ 131 ] and healthy subjects [ 132 , 133 , 134 , 135 ].…”

“…Significant research efforts in the last decade uncovered a rich landscape of cfDNA physico-chemical features (reviewed in [ 130 , 137 , 138 , 139 , 140 ]). Beyond hotspot mutations, numerous cfDNA features are candidate biomarkers, including (i) various genetic features, such as DNA sequence features ( Figure 4 A), chromosomal abnormalities ( Figure 4 B), and topological forms ( Figure 4 C); (ii) primary epigenetic markers such as DNA methylation ( Figure 4 D) and histone modifications ( Figure 4 E); and (iii) various secondary epigenetic features that cfDNA molecules attain intracellularly or extracellularly following disruption of the primary structure of DNA, such as binding to proteins, extracellular vesicles, or cell membranes ( Figure 4 F), and fragmentomic features ( Figure 4 G).…”

“…Not only do we currently have a weak grasp on the outlines of this problem, but also a very poor understanding of most of the individual factors. Yet, it is increasingly recognized that a holistic understanding of cfDNA biology is vital [ 1 , 3 , 6 , 59 , 130 , 137 , 253 , 494 ]. A deep and structured enquiry into the origin, physico-chemical features, and functions of cfDNA as it relates to the various biological, physiological, pathological, environmental, and lifestyle factors as well as preanalytical factors will provide new insights that impact many aspects of cfDNA research.…”

“…In contrast to traditional methods of genome analysis (e.g., cell cultures, in vivo models, and tissue biopsies), which have been valuable but limited by the inherent restraints of reductive approaches (e.g., sampling bias, loss of contextual logic, snapshot information) (reviewed in ref. [ 130 ]), data from serially profiled cfDNA represent a richer, more dynamic and much more realistic source of actual genome function, composition, and alterations.…”

Preanalytical Variables in the Analysis of Mitochondrial DNA in Whole Blood and Plasma from Pancreatic Cancer Patients

“…Although CA 19-9 is a widely used biomarker for the diagnosis of pancreatic cancer, its specificity is rather low. Up to now, blood-based nucleic acid markers have limited use in clinical settings even though they may have a major impact on early detection, monitoring, and treatment by virtue of their disease-specificity, the minimal invasiveness of venipuncture, and the possibility of longitudinal sampling [ 5 , 6 , 7 , 8 ]. In recent years, liquid biopsy (also known as liquid profiling), which refers to the analysis of various biomarkers present in human bodily fluids collected with minimally-invasive procedures emerged as a promising diagnostic, prognostic, and theranostic test [ 2 , 9 , 10 ].…”