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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 7 of 18

infiltration, lymphatic metastasis, progression-free survival, and the decrease of median survival time;
[26]
consequently, they are proposed as good markers for endometrial carcinoma and metastasis . Other
studies have proposed mRNA multi-markers based on the isolation of CTCs or by CellSearch followed
[28]
by RT-qPCR analysis. Bao et al. proposed the following pan: CK20, CEA, AGR2, MGB2, DLL4, EphA2,
[32]
Her3, and PDGFRα. Obermayr et al. proposed another pan focused on: CCNE2, DKFZp762E1312,
EMP2, MAL2, PPIC, and SLC6A8.
Another study explored the presence of specific mutations EC-related genes such as CTNNB1, STS, GDF15,
[24]
RELA, RUNX1, BRAF, and PIK3CA, which may be suggested as potential therapeutic targets .

A consensus panel profile based on DNA or mRNA CTC analysis has not been defined, due to the low
number of studies performed, the different technologies used for CTC isolation and biomarker analysis,
and the different biomarkers analyzed and procedures used for identification. Moreover, another challenge
is represented by the need of specific databases and algorithms able to integrate several sources of
information derived from analyzing multiparametric data as well as multiple biospecimens (e.g., CTC and
ctDNA) .
[33]
Count and biomarkers of CTCs
Several studies have analyzed both the count of CTCs and their molecular characterization, in which
the antigen for isolation was used as a marker as well as for enumeration, e.g., EpCAM and stathmin [as
confirmed by immunohistochemistry (IHC)], showing an increase in CTC number and stathmin IHC in
non-endometrioid versus endometrioid histology, tumor size ≥ 5 cm vs. < 5 cm, higher-stage disease, and
[28]
worse survival .
An investigation performed on patients affected by EC with and without recurrence, ranging from
Grade 3 Stage IB to Stage IV carcinomas and recurrences, by EpCAM-based immunoisolation using the
TM
CELLection Epithelial Enrich kit (Invitrogen, Dynal) followed by RT-qPCR analysis, associated the
presence of CTC with high-risk EC, evidencing the CTC-plasticity phenotype with stemness and EMT
[22]
gene-expression profile .
CTC in vitro for drug response model
The ability to isolate live CTCs by specific device (e.g., MetaCell®), as reported by Kolostova, might be
[34]
useful to test in vitro specific drug treatments .

Single CTC analysis
Although limited in sample size and number of studies, due to the highly technical and expensive
procedures, for characterizing the profile of CTCs, it may be useful to perform genetic and expression
profiles of single CTC, which have demonstrated superior diagnostic accuracy in defining lineage identity
[35]
in other tumors such as multiple myeloma and prostate cancer . The main procedure adopted is the
[20]
evaluation of genetic profile by NGS after whole genome amplification .
ctDNA
Biology of ctDNA
The presence of ctDNA was first described more than 30 years ago, even if intensive research began only
in the 2010s. Active and passive mechanisms were proposed for the origin of cell-free DNA (cfDNA).
Through passive mechanism, cfDNA is released into the blood via apoptosis and necrosis performed by
macrophages and phagosomes from hematopoietic cells. Through active mechanism, tumor cells secrete
cytoplasmic fragmented DNA to communicate with distant tissue through exosomes.