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ctDNA AND DETECTION OF MINIMAL RESIDUAL DISEASE: EARLY DIAGNOSIS OF
RECURRENCE
Patients with potentially curable localized disease will receive surgical treatment and/or eventually
radiation and neoadjuvant/adjuvant systemic therapy. The choice of the systemic treatment and the
selection of patients that are candidates to receive more aggressive systemic therapies are research fields
in constant evolution. Despite the optimal multidisciplinary treatment and the use of appropriate subtype
specific systemic therapies, a portion of these patients will relapse as a result of undetected micrometastatic
disease. The minimal residual disease (MRD) is the presence of cancer cells that remain in the tissues
and/or in the blood after treatment. Considering that the amount of ctDNA, intended as clonal plasma
[30]
variant allele frequency (VAF), directly correlates to pathological tumor size , the detection of ctDNA
was hypothesized to be suggestive of MRD. Therefore, several prospective and retrospective trials have
addressed the potential of ctDNA to determine those patients who, even if they are considered “disease-free”
[27]
after surgery, will relapse. In a prospective, multicenter, validation study reported by Garcia-Murillas et al.
(2019) after 35.5 months of median follow-up of early breast cancer patients, the detection of ctDNA after
surgery was associated with relapse-free survival (HR = 5.8; 95%CI: 1.2-27.1; P = 0.01), with a median lead
time of 10.7 months compared with clinical relapse. In this study, the detection of ctDNA trough dPCR
assays, based on somatic mutations identified from diagnostic biopsy samples, reached a sensibility of 96%
for extracranial distant metastatic relapses. In contrast, brain-only relapses were unlikely to be detected
[31]
(17%). Similar results were presented by Coombes et al. . In this prospective trial, serial blood draws
were performed during a 6-month follow-up, subsequent to surgery and adjuvant treatments, showing
a correlation between ctDNA detection in the first post-surgical plasma sample and poorer prognosis
(HR = 11.8; 95%CI: 4.3-32.5). In this case, using upfront exome profiling of tumor tissue followed by a
personalized targeted multiplex plasma sequencing, ctDNA was detected ahead of metastatic relapse with
a median lead-time of 8.9 months. It is worth mentioning that a similar prognostic correlation was shown
for ctDNA detection in the successive follow-up plasma samples, giving rise to new question about the
potential additional meaning of seriate multiple testing.

[26]
Consistent results were reported by Garcia-Murillas et al. (2015), with a median lead time over clinical
relapse of 7.9 months. In addition, the authors addressed the above-mentioned question if several repeated
sampling after surgery (“mutational tracking”) might outperform the single post-surgical blood draw,
showing that repeated samples achieved a higher sensitivity detecting recurrences in estrogen receptor
positive (ER+) breast cancer patients. Moreover, in the former study, the alterations highlighted in
the ctDNA were more aligned with the alterations found in metastatic biopsy than in primary tumor,
supporting the hypothesis that detecting MRD through ctDNA is both a potential prognostic factor and a
reliable tool for the molecular characterization of the upcoming metastatic disease.

Despite these promising achievements, sensibility in detecting low concentration of ctDNA in patients
apparently “disease-free”, or at least bearing a minimal tumor burden, remains an issue. New technologies
are currently being developed and others have been already tested. Among them, in a small exploratory
cohort of early breast cancer patients, the targeted digital sequencing (TARDIS) achieved up to a 100-fold
[32]
improvement beyond the current limit of ctDNA detection .

CLINICAL ROLE OF CTCS IN EARLY BREAST CANCER
The amount of circulating tumor cells in peripheral blood before the administration of systemic treatment
has been proven to be an independent predictor of progression-free survival (PFS) and OS in metastatic
[33]
[4]
breast cancer . However, around 20% early-stage breast cancer patients have CTCs detected in blood . In
contrast with the current CTCs positivity cut-off used in metastatic setting, considering the lower detection
rate in the earlier stages, a cut-off of 1 or more CTCs was proposed and subsequently adopted in the vast
[34]
majority of the investigations .