Page 96 - Read Online

D’Amico et al. J Cancer Metastasis Treat 2021;7:3 I http://dx.doi.org/10.20517/2394-4722.2020.93 Page 11 of 16

Although offering a reasonable balance, these approaches have a potential downside. In fact, adjuvant and
neoadjuvant treatments may select new mutant clones that would be therefore missed, with consequently
false-negative results.

To overcome the mentioned drawbacks, hybrid capture-based NGS was developed to improve the detection
without prior knowledge of primary tumor alterations. In this approach, biotinylated probes, designed
against recurrently mutated genomic regions in breast cancer, select relevant DNA sequences for NGS
analysis. These technologies allowed the implementation of error-correction strategies (such as integrated
[66]
[15]
digital error suppression) that pushed the ctDNA detection limit to ~ 0.001% (CAPP-Seq , TEC-Seq ).
Commercial assays that utilizes hybrid capture-based NGS technology, as Guardant360®, PlasmaSELECT®
and FoundationOne® Liquid, are available for clinical use. However, despite the high self-reported accuracy,
specificity and sensitivity, a comparative study of two of these platforms demonstrated a low concordance
[67]
between the two assays .
CAVEATS AND PIRFALLS OF CTCs IN EARLY-STAGE BREAST CANCER
CTC deployment in early-stage breast cancer has encountered several analytical and pre-analytical
obstacles. The most challenging limitation encountered by CTC detection in early disease is that CTCs
are slightly less represented in blood, compared to physiological cellular components (approximately
one cancer cell among a billion normal blood cells, in the metastatic setting). Therefore, considering this
setting, even highly sensitive technologies could require a huge amount of blood to detect few CTCs,
threatening the clinical feasibility.

Thus, to overcome blood volume limitations, new detection approaches were developed. Inserting a 2 cm
long steel wire with a hydrogel functional tip coated with EpCAM antibody, the GILUPI CellCollector is a
method that allows to expose the collection tube to approximately 1 L of blood, instead of the canonical 7.5 ml
[68]
analyzed by CellSearch . However, even if this promising approach has shown a higher CTC capture rate
from stage IV and III lung cancer patients compared to the FDA cleared CellSearch, these results have not
yet been achieved in a breast cancer patients’ cohort. Furthermore, this technology requires the insertion of
a noodle in the cubic vein of the patient for 30 min, resulting to be slightly more invasive than blood draw-
based analysis.

CTCs are highly heterogenous and dynamically change their shape and morphology. For instance,
downregulation of EpCAM during epithelial-mesenchymal transition (EMT) is a known phenomenon .
[69]
Accordingly, the inability to select CTCs that underwent to mesenchymal transition represents a further
limitation of the EpCAM based CTCs detection approaches. Since the transition of adherent epithelial
cells to a migratory mesenchymal state has been implicated in tumor metastasis, as well as in the process
[70]
in which CTCs acquire greater ability to metastasize , new approaches that allow the detection of not-
epithelial CTCs are being developed .
[71]
Among them, is worth of mention the use of carbon nanotubes for adherence-based capture and isolation
of CTCs of different phenotypes. This technology, called nanotube-CTCchip, has shown the potential to
overcome both selective phenotype detection and size-dependent loss of cells, proposing itself as a versatile
[72]
platform to capture CTCs of different phenotype . Nevertheless, considering that it has only been tested
in a very small cohort of breast cancer patients (7 pts with different clinical staging and 2 healthy controls),
this technique is far from clinical deployment.

An additional point of weakness of the literature produced so far in this setting of BC is the arbitrary
nature of the positivity cut-off adopted among the different trials. Indeed, in contrast with the well-