Figure 3

Workflow of ctDNA analyses. (a) ctDNA (light blue DNA fragments) are present in the circulation of cancer patients together with DNA fragments released from non-malignant cells (most frequently from cells of the hematogenous system, orange). The latter are often the majority, and the percentage of ctDNA may vary depending on various parameters, such as the tumor burden. (b) The entire DNA is isolated from plasma and can be subjected directly to an array for copy number analysis, or a library can be prepared for NGS, allowing assessment of both copy number changes and mutations at the nucleotide level. (c) After alignment, DNA fragments (here shown for one chromosome) are counted relative to their position in the genome. In theory, DNA fragments from normal cells should be present in identical numbers across the entire genome, as indicated by the equal number of orange fragments. In contrast, tumor-specific fragments may vary and reflect the status of copy number changes of cells releasing material into the circulation at the time of analysis, illustrated by the variable number of blue fragments. Using bioinformatics tools, the number of different fragments at a given locus is converted to a copy number (blue line). Similarly, the percentage of ctDNA determines the allele fraction for the identification of tumor-specific somatic mutations.