Critical Factors in the Analytical Work Flow of Circulating Tumor DNA-Based Molecular Profiling

Liquid biopsy testing, especially molecular tumor profiling of circulating tumor DNA (ctDNA) in cell-free plasma, has received increasing interest in recent years as it serves as a reliable alternative for the detection of tumor-specific aberrations to guide treatment decision-making in oncology. Many (commercially available) applications have been developed, however, broad divergences in (pre)analytical work flows and lack of universally applied guidelines impede routine clinical implementation. In this review, critical factors in the blood-based ctDNA liquid biopsy work flow are evaluated.

In the preanalytical phase, several aspects (e.g., blood collection tubes [BCTs], plasma processing, and extraction method) affect the quantity and quality of the circulating cell-free DNA (ccfDNA) applicable for subsequent molecular analyses and should meet certain standards to be applied in diagnostic work flows. Analytical considerations, such as analytical input and choice of assay, might vary based on the clinical application (i.e., screening, primary diagnosis, minimal residual disease [MRD], response monitoring, and resistance identification). In addition to practical procedures, variant interpretation and reporting ctDNA results should be harmonized. Collaborative efforts in (inter)national consortia and societies are essential for the establishment of standard operating procedures (SOPs) in attempts to standardize the plasma-based ctDNA analysis work flow.

Development of universally applicable guidelines regarding the critical factors in liquid biopsy testing are necessary to pave the way to clinical implementation for routine diagnostics.