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Malentacchi et al. J Cancer Metastasis Treat 2020;6:34 I http://dx.doi.org/10.20517/2394-4722.2020.34 Page 9 of 18

Standard PCR or qPCR instead evaluate the median distribution of a mixed mutated and wt solution,
which may decrease the accuracy and sensitivity for the detection of rare mutations. Several instruments
allow ddPCR, which can be distinguished depending on the size of generated droplets.
(3) NGS: This procedure is mainly related to the evaluation of the presence of specific tumor-related
mutations, although it can also be used to evaluated copy number variation (CNV) and quantification of
total amount. Deep massive paralleling allows the evaluation of multiple mutations (several mutations in
[49]
the same gene and several genes) and more than one sample in the same run .
Mutations of ctDNA
Several studies have evidenced the presence of ctDNA in gynecological malignancies and the detection of
specific mutations related to EC.

[50]
One of the first studies was performed by Dobrzycka et al. , using PCR-restriction fragment length
polymorphism in a cohort of 109 patients with EC to analyze TP53 and KRAS mutations, confirming the
data reported for primary tumors that there is a higher percentage of TP53 mutation in serous carcinomas
[37]
as well as a high frequency of KRAS mutations in grade 2 endometrioid tumors. Pereira et al. observed
the presence of mutations in TP53 PTEN, PIK3CA, MET, KRAS, FBXW7 and BRAF in ctDNA, and these
detections were useful to predict the tumor recurrence, with an average of seven months, before the
radiologic evidence.

[51]
Moreover, Bolivar et al. evidenced the presence of mutations on CTNNB1, KRAS, PTEN, or PIK3CA
genes, independently of total cfDNA amount, in association with advanced stage, deep myometrial
invasion, lymphatic/vascular invasion, and primary tumor size.

As discussed below, even for ctDNA of EC, there is evidence of discrepancy between the mutations found
in plasma compared to those evaluated in primary tissue sample with an agreement of only 33% .
[52]
Integrity index of ctDNA
Only one study has evaluated the integrity index performed on ctDNA extracted from serum and based on
evaluation of Alu repeats. The authors registered an increase in ctDNA integrity and amount in high grade
[53]
compared to G1 ECs, suggesting a role of cfDNA as potential prognostic biomarker in EC .

Copy number variation in ctDNA
[54]
The same findings were obtained for the evaluation of CNV in ECs by NGS by Nakabayashi et al. on
three patients on the following loci: 1p36-p31 and 1q12-q44 or 8q24.

Total amount of cell free DNA
Regarding the total cfDNA evaluation, the two reported articles are based on the evaluation of Alu repeats:
[55]
[53]
the previous citation suggests a relationship between integrity and EC grade, while another reports
no significant difference in cell-free DNA among stage or histological grade of EC, as well as no significant
change in cell-free DNA before and after operation.

Mitochondrial ctDNA
Recent studies have demonstrated a potential link between circulating mitochondrial cell-free DNA
(cfmtDNA) content and cancer. In particular, the evaluation of aberrant changes and altered content of
cfmtDNA represents an important approach for early cancer diagnosis with some unique advantages
over circulating nuclear cell free DNA (cfnDNA), such as the much shorter and more simply organized
mitochondrial genome and the higher number of mtDNA copy. These characteristics make screening
much easier and more cost-effective, with respect to the nuclear genome one, in body fluid samples such as