A hitchhiker's guide to cell-free DNA biology.

Liquid biopsy provides a noninvasive window to the cancer genome and physiology. In particular, cell-free DNA (cfDNA) is a versatile analyte for guiding treatment, monitoring treatment response and resistance, tracking minimal residual disease, and detecting cancer earlier. Despite certain successes, brain cancer diagnosis is amongst those applications that has so far resisted clinical implementation. Recent approaches have highlighted the clinical gain achievable by exploiting cfDNA biological signatures to boost liquid biopsy or unlock new applications. However, the biology of cfDNA is complex, still partially understood, and affected by a range of intrinsic and extrinsic factors. This guide will provide the keys to read, decode, and harness cfDNA biology: the diverse sources of cfDNA in the bloodstream, the mechanism of cfDNA release from cells, the cfDNA structure, topology, and why accounting for cfDNA biology matters for clinical applications of liquid biopsy.