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Page 8 of 18 Malentacchi et al. J Cancer Metastasis Treat 2020;6:34 I http://dx.doi.org/10.20517/2394-4722.2020.34

The fragment size of cfDNA is 170-200 bp, reflecting the structure of nucleosomes, sustained by nuclease
digestion of genomic DNA (gDNA) during apoptosis. The fragment size profile differs between cfDNA and
ctDNA from tumor cells. Shorter (< 100 bp) or longer (> 10,000 bp) fragments are frequently observed in
ctDNA from cancer patients as the majority of the cfDNA from normal cells is released after apoptosis,
while ctDNA can also be released by necrosis. Necrosis-based fragmentation appears to create longer or
shorter DNA fragments in comparison to physiological apoptotic origin in healthy subjects. Short ctDNA
fragments are more frequently observed in patients with metastatic disease than in those with early-stage
cancer.

After surgical resection, the half-life of ctDNA was estimated to be 114 min. The cfDNA amount was
[36]
estimated to be three-fold higher in cancer patients compared with that in healthy individuals . However,
pregnancy and some disorders, such as infectious and autoimmune diseases, stroke, infarction, and trauma,
[37]
induce an increase in cfDNA . The ctDNA amount shows wide variation among cancers, differs among
[36]
stages, and may correlated with tumor burden . The evaluation cfDNA in term of amount and integrity as
well as studying specific mutations is cheaper and easier than CTC evaluation.

ctDNA has already been implemented in routine clinical practice after European Medicines Agency
approval of the EGFR mutation test (Therascreen EGFR Plasma, Qiagen) in plasma of patients with non-
[24]
small cell lung cancer .
Nevertheless, no consensus or standardized analytical procedures have been defined to harmonize cfDNA
procedures (although there is agreement concerning the isolation of cfDNA and ctDNA from plasma, being
serum preferred) or the analytical phase (methodologies, reference genes for integrity, and total amount
evaluation).

Levels of cfDNA in serum are higher than in plasma due to contamination of gDNA from leucocytes
during the clotting process; therefore, plasma is preferable. For the same reason, cfDNA needs to be isolated
from standard EDTA-blood tubes within 2-4 h. Alternatively, tubes containing stabilizers that prevent cell
[38]
lysis should be used for blood collection . However, two centrifugations should be performed, the first
within 2-4 h for separating plasma from whole blood at 1000-2000 g and 4 °C for 10-15 min and the second
performed on plasma to separate plasma form platelets or cellular debris for the same time and temperature
[38]
but at about 15,000 g . ctDNA assays can identify tumor-specific genetic alterations (e.g., somatic point
mutations, loss of heterozygosity, gene fusions, gene copy number variations, and DNA methylation
changes) [3,36,39,40] , allow the monitoring of cancer evolution and the setting of specific personalized targeted
[42]
[41]
therapy , and provide suggestions for immunotherapy ; they can allow detect minimal residual disease,
monitor mutations that are related to tumor burden, and define the appearance of resistance to targeted
[43]
therapy .
Methodologies for detection of ctDNA
The methodologies depend on the purpose of ctDNA [44-48] :
(1) Quantitative PCR (qPCR): This procedure is used for the evaluation of total cfDNA amount and/or
cfDNA integrity. To be sure to identify properly ctDNA coming from EC (and not from others tumors or
pathologies), it is necessary to refer to specific mutations present in the tumor. Instead, for the evaluation of
total cfDNA amount and integrity, it is necessary to use a specific gene that is not amplified or differentially
fragmented in other tumors or pathologies.
(2) ddPCR: This procedure is mainly related to the evaluation of the presence of specific tumor-related
mutations. The concept of this procedure is based on Poisson distribution and allows the absolute
quantification of each allele or mutation considering the number of wells containing the target genes in
comparison to wild type (wt) taking into consideration that a single molecule is present in each well.