The presence of cell-free DNA in circulation has been widely documented. All living cells release discrete amounts of free DNA into circulation. Following normal cell turnover, cells in apoptosis release DNA fragments into the circulation. Under physiological conditions, apoptotic and necrotic cells are rapidly removed, and cfDNA levels are relatively low. More than 90% of healthy individuals have less than 25 ng of cfDNA per mL. Under certain pathological conditions, including inflammation, exercise, tis sue injury, or surgery, cfDNA levels can increase significantly. Generally, cfDNA levels can increase up to magnitude even in patients with tumors. More specifically, ctDNA refers to the proportion of free DNA released from cancer cells. Thus, in patients with cancer, a fraction of cfDNA derives from the tumor and is referred to as ctDNA. The ctDNA can originate from primary tumors, metastatic lesions, or CTCs. The fraction of ctDNA relative to the total circulating free DNA can range from 10%. The mechanisms of ctDNA release have not yet been elucidated. Proposed hypotheses include passive mechanisms, such as cell necrosis, and controlled release mechanisms, such as apoptosis. The hypothesis that ctDNA may result from cell necrosis is supported by the observation that more advanced stage tumors, characterized by a higher degree of necrosis, are associated with higher levels of ctDNA. The hypothesis that ctDNA is released following apoptosis is supported by its high degree of fragmentation. Indeed, ctDNA is typically fragmented into segments of the same length, 160–180 bp, as the nucleosome-protected DNA observed in apoptotic cells. CtDNA can also be released within exosomes, although this mode appears more appropriate for low-molecular-weight nucleic acids such as miRNAs (Fig. 1).

Fig1. Primary tumor and/or metastasis release ctDNA into the circulation by secretion, necrosis, and apoptosis. Circulating tumor cells (CTCs), another source of ctDNA, are also released. The ctDNA has the same alterations as the tumor cell of origin (point mutations, large rearrangements, insertions, deletions) and can be subjected to molecular analysis. (Copyright EDISES 2021. Reproduced with permission)

The clearance of ctDNA occurs in the spleen, kidney, and liver. Its half-life is very short, 16 min according to some Authors, about 1 h for the rapid phase, and 13 h for the late phase, according to others. In any case, the half-life is much shorter than many of the protein markers.

The ctDNA can provide quantitative information by measuring the number of copies in the circulation, and qualitative information, by searching for tumor-specific mutations. In the first case, given the extreme interindividual variability of ctDNA levels, it would seem to be the kinetics of ctDNA, and therefore its variations over time, rather than its absolute value, to provide clinically useful information about the effectiveness of therapy or the onset of relapse. In the second case, i.e., ctDNA mutational analysis, diagnostic applications are based on the fact that mutations in ctDNA correspond exactly to mutations in the primary tumor, including both point mutations and copy number variations and rearrangements.

Technologies for ctDNA Isolation

Theoretically, since the tumor-specific mutations sought in ctDNA are not present in normal cells, it could represent an excellent opportunity to identify the presence of a tumor mass noninvasively. However, ctDNA analysis to date is not helpful for diagnosis, detection of minimal residual disease, or prognostication because the sensitivity of currently available methods is often insufficient to identify ctDNA molecules, which in some cases represent little more than 0.01% of total cfDNA.

Isolation of ctDNA is far more feasible than that of CTCs; however, two factors make manipulation difficult:

– The extreme interindividual variability in ctDNA concentration;

– The “dilution” effect of circulating ctDNA because a large amount of cfDNA from healthy cells is present

The most variable stage is preanalytical, particularly sample collection, handling, and storage. It is preferable to use EDTA-plasma rather than serum since coagulation may result in the breakdown of leukocytes with subsequent release of wild-type DNA and further dilution of ctDNA. Commercially available methods typically require 1.5–2 mL of plasma. The stability of ctDNA is limited by the presence of circulating DNA activity, so sample processing should occur within hours of collection. Subsequent quantification is also not without criticality. In addition, other non- oncologic diseases may affect cfDNA levels.

ctDNA Mutational Analysis

 The mutational analysis of ctDNA can be carried out basically by two approaches: a targeted one, i.e., aimed at finding known mutations that fall in mutational hot spots of specific genes known to be associated with cancer (e.g., KRAS, EGFR, and BRAF in lung cancer, colon, and melanoma, respectively), and a non-targeted one, based on NGS techniques. In the latter case, knowledge of specific tumor- associated mutations is not required However, any molecular alteration in the ctDNA is identified within the limits of the analytical sensitivity of the method. Among the disadvantages of massive sequencing methods are the limited analytical sensitivity, the still high costs, and the difficulties in interpreting the results for clinical purposes.

Initially, the analysis of known point mutations of ctDNA (targeted approach) was performed by end-point Polymerase Chain Reaction (PCR) and real-time PCR. More recently, digital-PCR, ARMS-PCR, or BEAMing technology have been developed to achieve higher analytical sensitivity. Most of these methods, however, allow the analysis of only a few loci; mutations in genes that do not present mutational hot spots, such as oncosuppressors, are not detected.

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