Isolation of salivary cell-free DNA for cancer detection.

Saliva is an emerging source of disease biomarkers, particularly for cancers of the head and neck. Although analysis of cell-free DNA (cfDNA) in saliva holds promise as a liquid biopsy for cancer detection, currently there are no standardized methodologies for the collection and isolation of saliva for the purposes of studying DNA. Here, we evaluated various saliva collection receptacles and DNA purification techniques, comparing DNA quantity, fragment size, source, and stability. Then, using our optimized techniques, we tested the ability to detect human papillomavirus (HPV) DNA- a bona fide cancer biomarker in a subset of head and neck cancers- from patient saliva samples. For saliva collection, we found that the Oragene OG-600 receptacle yielded the highest concentration of total salivary DNA as well as short fragments <300 bp corresponding to mononucleosomal cell-free DNA. Moreover, these short fragments were stabilized beyond 48 hours after collection in contrast to other saliva collection receptacles. For DNA purification from saliva, the QIAamp Circulating Nucleic Acid kit yielded the highest concentration of mononucleosome-sized DNA fragments. Freeze-thaw of saliva samples did not affect DNA yield or fragment size distribution. Salivary DNA isolated from the OG-600 receptacle was found to be composed of both single and double-stranded DNA, including mitochondrial and microbial sources. While levels of nuclear DNA were consistent over time, levels of mitochondrial and microbial DNA were more variable and increased 48 hours after collection. Finally, we found that HPV DNA was stable in OG-600 receptacles, was reliably detected within the saliva of patients with HPV-positive head and neck cancer, and was abundant among mononucleosome-sized cell-free DNA fragments. Our studies have defined optimal techniques for isolating DNA from saliva that will contribute to future applications in liquid biopsy-based cancer detection.

Cell-free DNA topology depends on its subcellular and cellular origins in cancer.

Cancer cells release large quantities of cell-free DNA (cfDNA) into the surrounding tissue and circulation. As cfDNA is a common source of biomarkers for liquid biopsy and has been implicated as a functional mediator for intercellular communication, fundamental characterization of cfDNA topology has widespread biological and clinical ramifications. Whether the topology of cfDNA is such that it exists predominantly in membrane-bound extracellular vesicles (EVs) or in nonvesicular DNA-protein complexes remains poorly understood. Here, we employed a DNA-targeted approach to comprehensively assess total cfDNA topology in cancer. Using preclinical models and patient samples, we demonstrate that nuclear cfDNA is predominantly associated with nucleosomal particles and not EVs, while a substantial subset of mitochondrial cfDNA is membrane protected and disproportionately derived from nontumor cells. In addition, discrimination between membrane-protected and accessible mitochondrial cfDNA added diagnostic and prognostic value in a cohort of head and neck cancer patients. Our results support a revised model for cfDNA topology in cancer. Due to its abundance, nuclear cfDNA within nucleosomal particles is the most compelling liquid biopsy substrate, while EV-bound and accessible mitochondrial cfDNA represent distinct reservoirs of potential cancer biomarkers whose structural conformations may also influence their extracellular stability and propensity for uptake by recipient cells.

Bioactive DNA from extracellular vesicles and particles.

Extracellular vesicles (EVs) and particles (EPs) have recently emerged as active carriers of molecular biomarkers and mediators of intercellular communication. While most investigations have focused exclusively on the protein, lipid and RNA constituents of these extracellular entities, EV/EP DNA remains poorly understood, despite DNA being found in association with virtually all EV/EP populations. The functional potential of EV/EP DNA has been proposed in a number of pathological states, including malignancies and autoimmune diseases. Moreover, the effectiveness of cell-free DNA as the biomarker of choice in emerging liquid biopsy applications highlights the role that EV/EP DNA may play as a novel disease biomarker. In this review, we provide a comprehensive overview of EV/EP DNA studies conducted to date, with a particular focus on the roles of EV/EP DNA as a functional mediator and molecular biomarker in various pathologic states. We also review what is currently known about the origins, structure, localisation and distribution of EV/EP DNA, highlighting current controversies as well as opportunities for future investigation.