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Di Raimo et al. J Cancer Metastasis Treat 2018;4:54 I http://dx.doi.org/10.20517/2394-4722.2018.50 Page 7 of 14
[166]
metastasis (OR = 0.47; 95%CI: 0.27-0.80; P = 0.01) . Hence, it is important to understand, using different
epithelial and/or mesenchymal markers, how defined other clinically relevant sub-populations of CTCs.
Accordingly, taking into account the attested probability of false-negative results, cell-surface vimentin
and EGFR were suggested as alternative markers for detecting mesenchymal transitioned CTCs [136,167,168] . To
recapitulate, the common issue underlined with positive selection procedures is to fail the capture of cells
with low expression of EpCAM and non-epithelial phenotypes such as those that have undergone EMT.
In addition, the isolated CTCs have reduced viability and this aspect represents an important obstacle to
[137]
CTCs’ biological characteristics understanding . Otherwise, immunological methods based on negative
selection are also available. The latters are commonly used to deplete cells that do not express CD45 leuco-
cyte antigen or a cocktail of antibodies direct against red and white blood cells, such as RosetteSep, Easy-
[137]
Sep, Dynabeads, mojoSort . Cells isolated with this approach are relatively more viable but, at the same
time, are highly impure. In fact, the purified cells pool contains epithelial and non-epithelial phenotypes
[137]
together with normal blood vessel, stromal cells or other cells normally present in the circulation . These
evidences, as reported by a huge number of studies, confirm that the main challenge of CTCs isolation and
characterization are the lack of specific standardized procedures that strongly restrict their use in clinical
practice [134,169,170] .

CLINICAL RELEVANCE OF CTCS
Despite progress achieved in terms of prevention, diagnosis and treatment, drug resistance and tumor re-
lapse, whose severity and probability are specific for each patient, remain one of the principal issue in breast
cancer. Therefore, as a good clinical practice, it has been established that a patient’s 5-year follow up, since
primary tumor, could lead to an early detection of recurrence or metastasis and to a more specific and ef-
[169]
ficient therapy . Canonical tissue biopsy represent on one side a costly, painful and hard to repeat proce-
dure. In addition, it is not able to provide a complete genetic or epigenetic tumor characterization in order
[171]
to identify possible tumor phenotypical alteration . In this optic, non-invasive liquid biopsies and the
measurement of specific blood-based biomarkers represent an effective alternative parameter to monitored
invasive BC patients. Cancer Antigen 15-3 (CA 15-3), carcinoembryonic antigen, tissue polypeptide antigen,
tissue polypeptide-specific antigen and the soluble form of HER2 represent the most detected serum BC
biomarkers [172-174] . Nevertheless, even if it has been demonstrated a correlation between single or combined
circulating biomarker levels and recurrence incidence, many issues need to be solved [175-178] . For instance,
there are still problems associated with the lack of a validated clinically relevant level to establish, for each
[169]
biomarker, a cut-off parameter . Furthermore, it has been demonstrated that biomarker prognostic efficacy
depends on the recurrence site. In fact, higher levels of biomarkers were detected in BC distant metastases,
[179]
such as bone or liver, than in loco-regional or lung recurrence . Additionally, these biomarkers are inap-
[169]
propriate to figure out mechanisms of therapy resistance . For these reasons, nowadays, the detection of
CTCs from patient blood samples appears as a powerful tool in the management of early and advanced BC
[138]
patients . CTC-based liquid biopsy represents a more informative tool, able to improve patients’ selection
and monitoring for target treatments, than conventional tumor tissue based- biomarkers that focused only
on the primary tumor or metastases. Indeed, in the last few years, several studies highlighted the prognos-
tic relevance of CTCs in MBC. In particular, it has been demonstrated that patients with a persistent CTCs
count > 5 cell per 7.5 mL blood had a worse patient free survival (PFS) and overall survival (OS) compared
to those that have CTCs < 5 at baseline and during follow-up [25,27,180-182] . Furthermore, due to their character-
istics and minimally invasive procedures, the use of CTCs permits to evaluate the dynamic change of tumor
over time for each patient that may impair the response to specific targeted treatments [138,183] . From this point
of view, CTC detection appears to hold promise of a better patients’ management but up to date they are
not still routinely used in clinical practice. In fact, CTC enumeration and variation during treatment were
independent from any other baseline clinical or pathological characteristics and were not associated with
pathological complete response [26,27] . Furthermore, as highlight by the SWOG S0500 randomize trial in ad-
vanced breast cancer, there is no evidence that changing or discontinuing therapy based on CTC level could