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Page 2 of 16 D’Amico et al. J Cancer Metastasis Treat 2021;7:3 I http://dx.doi.org/10.20517/2394-4722.2020.93

Keywords: Liquid biopsy, early breast cancer, circulating tumor cells, circulating tumor DNA, clinical trials, screening,
early detection, minimal residual disease

INTRODUCTION
About 276,480 new cases of invasive breast cancer will be diagnosed among American women within
[1]
2020 . This incidence means breast cancer is the second most common cancer, showing a slightly
increasing trend in recent years (by 0.3% per year). Around 6% of these patients will present with metastatic
disease at diagnosis, while, among the remaining, approximately 40% will develop a systemic recurrence,
[2]
despite the surgery and eventual use of systemic and radiation therapy . The 5-year overall survival (OS)
rate among patients with localized disease ranges between 86% and 99%. Conversely, only 27% of patients
[3]
with metastatic breast cancer (mBC) will reach the 5th year after diagnosis . In consideration of these data,
it is fundamental that efforts are made to ensure that diagnosis of both primary and potential recurrence is
made as early as possible.

The current evidence suggests that mammography remains the only standard of care diagnostic imaging
for breast cancer screening (with the exception of high-risk patients, in which case the use of Magnetic
Resonance Imaging may be preferred) and, performing a biopsy of the lesion is mandatory to confirm the
diagnosis and provide further tumor pathological and molecular characteristics. The standard surveillance
methods evaluated for early detection of recurrence of disease are mostly similar to those used for the
primary diagnosis. Moreover, some serological markers (such as CA15-3 and CEA) are available for clinical
use, although none of these were demonstrated to improve the detection ability of the standard procedures
and improve outcome. Tissue biopsies are invasive, time-consuming, quite expensive and for those reasons
not often replicable within the same patient. Furthermore, the tissue biopsies appear to be too shortsighted
to capture the true spatial and temporal tumor heterogeneity.

The terms liquid biopsy mainly refers to several different diagnostic tests performed on biological fluids
(i.e., blood, saliva, urine, etc.) with the aim of investigating the presence of circulating cancer cells (CTCs)
or fragments of DNA [circulating tumor DNA (ctDNA)] that can be shed from the tumor. Despite the
development and testing of several enrichment techniques to identify, isolate and characterize CTCs in
the blood of cancer patients, the only Food and Drug Administration (FDA) approved method is the
TM
CellSearch (Menarini Silicon Biosystems, Bologna, Italy), that allows the identification and enumeration
of CTCs using an immunomagnetic enrichment of epithelial cells by anti-EpCAM antibodies. With
this method, the detection of 5 CTCs or more in the peripheral blood of patients with metastatic breast
cancer is an independent indicator of worse prognosis . In a retrospective study analyzing data from 18
[4]
international centers, the same cutoff identified a group of mBC patients with more aggressive disease
irrespective of disease subtype, recurrence site, type and line of therapy suggesting the need to use this
test to stratify mBC, Stage IV aggressive vs. Stage IV indolent . In addition, CTCs often bear the same genomic
abnormalities detected in metastatic tumor tissue with some additional peculiar phenotypic changes that
are responsible for systemic disease spread (epithelial-mesenchymal transition and stem cell markers),
or drug resistance (PI3KCA-AKT signaling pathway) , suggesting that CTCs may have a key role in the
[5]
evolution of the disease and can be considered as a sensitive, quantitative and qualitative surrogate of the
overall systemic disease.

The median level of circulating DNA is approximately five-fold higher in plasma of breast cancer patients
[6]
compared to healthy controls . In a sub-cohort of breast cancer patients, ctDNA was detected in both
metastatic (90%) and localized disease (55%), and its presence was correlated overall with each stage
[7]
of the disease and its prognosis . Moreover, ctDNA levels in metastatic breast cancer patients have