CTCs can be found in the circulation of cancer patients and serve as an important source of tumour-related information and disease monitorisation [62]. CTCs detection has been convincingly validated as a biomarker of the worse outcome in both early and metastatic BC [24]. The BEVERLY-2 study [38] is a relevant clinical trial focusing on the non-metastatic HER2-positive invasive BC, which encompassed CTC detection and pCR and provided validating evidence of their combined prognostic impact on DFS assessment. This is an open-label, single-arm, multicentre phase II study that adds neoadjuvant and adjuvant bevacizumab to the standard-of-care regimen. Of note, the prognostic value of CTCs and circulating endothelial cells in a survival analysis after 3 years of follow-up was described therein. In this study, CTC positivity was set to ≥1 CTC per 7.5 mL of blood. The detection of CTCs was not associated with patients’ clinical or pathological characteristics. Of note, 46% of the patients were CTC-positive at least once between baseline sampling to presurgery. A high percentage of patients (35%) had detectable CTCs at baseline and the positivity decreased to 7% before surgery due to NAC. CTCs detection during NAC was associated with significant reduction in DFS (CTC-positive patients had DFS of 54% compared to DFS of 83% in patients with no CTCs), but there was no association with pCR. Moreover, patients with no CTCs throughout neoadjuvant stages had a 96% OS compared to 83% for those with ≥1 CTC. When CTC detection was combined with pCR for prognostic stratification, the patients with baseline CTC counts of <1/7.5 mL (CTC negative patients), who achieved pCR, showed excellent prognosis, while those with a baseline CTC of ≥1/7.5 mL and no pCR were at higher risk of relapse. In detail, 3–4 weeks after surgery, 13.2% of included patients were CTC positive. Additionally, in an exploratory analysis, the authors observed that 3–4 weeks after surgery, CTCs were detected in 4% of patients achieving pCR and in 31% that had not. At the end of adjuvant treatments, ≥1 CTC/7.5 mL were detected in 20.7% of the patients, but the mean CTC count in the cohort was 0.3 CTC/7.5 mL. No further differences were discovered after 1 year of adjuvant therapy in the studied patients. Studying the impact of CTCs detection on DFS, the researchers observed that the 3-year DFS in patients with ≥ 1 CTC/7.5 mL at baseline was significantly lower than for patients with no CTC detected in pretreatment samples. However, they did not observe the association between CTCs and DFS in other blood time points. Additionally, patients with CTCs detected at any point during NAC presented a significant reduction in DFS (Table 1).

An additional publication describing results from the GeparQuattro study [39], which included a mixture of BC subtypes, again postulated that CTC-negative patients achieving pCR showed the best prognosis in contrast to those CTC-positive with a suboptimal response to therapy, who had higher risk of recurrence. Moreover, patients with <2 CTCs without pCR had an intermediate risk of metastatic disease. Specifically, for HER2-positive BC, the GeparQuattro trial defined that the detection of ≥2 CTCs/7.5 mL was associated with a reduced DFS in a multivariate analysis, suggesting that some of these preoperatively detected CTCs are resistant to therapies and lead to subsequent relapse (Table 1). Indeed, the presence of subclones composed of epithelial-to-mesenchymal transition (EMT)-like CTCs and stem cell-like CTCs was described to provoke resistance to conventional oncologic treatments [63,64,65], including NAC [40,66] (Table 1). Finally, the NeoALTTO phase III trial [41] also studied CTC fluctuation during the therapy of HER2-positive patients. While no significant decrease in CTC counts was achieved during NAC, a tendency towards lower pCR rates was observed in patients with detectable CTCs. This study was likely underpowered, but a meta-analysis could help to uncover interesting associations (Table 1).

In addition to the previously mentioned, CTCs detection was also employed in the adjuvant setting to correlate CTCs positivity with DFS and OS. In this context, the SUCCESS A clinical trial is a multicenter, open-label, phase III study comparing three adjuvant chemotherapy regimens and two schemes of adjuvant treatment with bisphosphonate (2 vs. 5 years of zoledronate) for early-stage high-risk BC patients [67]. CTC presence was assessed before and 2 years after chemotherapy [42]. The investigators observed notable differences in the correlation analyses between CTCs presence and DFS and OS when considering the different BC subtypes. In luminal A-like, luminal B-like and TNBC tumours, the detection of CTCs 2 years after chemotherapy was associated with decreased OS. By contrast, there was no prognostic value of CTCs detection during follow-up in patients with HER2-type tumours, contradictory to other studies (Table 1) [43,45,46]. The authors pointed to possible carryover effects, provoked by anti-HER2 targeted treatments, which can explain this phenomenon [68]. Additional information will likely be obtained through the SUCCESS B trial, an open-label, multicentre and randomised phase III study which only includes HER2-positive BC patients.

Finally, it has been demonstrated that the presence of HER2-positive CTCs in early BC patients and showed that this positivity is an independent factor from HER2-status in the primary tumour [69,70,71,72]. In an interesting study [44], it was demonstrated that patients with HER2-negative tumours but positive CTCs treated with trastuzumab showed reduced probability of relapse and improved disease-free interval. These findings could indicate that targeting HER2-positive CTCs is a suitable strategy to avoid relapses in early BC patients. In this regard, it is important to adapt isolating antibody-based platforms to specifically detect HER2-positive CTCs population. Instead, methodologies based on CTCs cell-size couple with approaches to identify HER2-positivity at protein, RNA or DNA level could represent an excellent alternative.

In the metastatic BC setting, it is crucial to decipher disease’s evolution especially related to resistance to treatments or the appearance of novel tumour clones. These processes are well documented in metastatic BC patients treated with hormone therapy, where estrogen receptor 1 (ESR1) mutations are responsible for treatment resistance [73]. CTCs detection can offer not only information regarding their quantity but also their genomic/transcriptomic characterisation [62,74]. The molecular interrogation of these cells can provide a molecular snapshot of the whole disease, even when metastatic site is not easily accessible by conventional biopsies [75].

In metastatic BC, it has been demonstrated that HER2 status may change between primary tumours and CTCs [20,47]. It has been also demonstrated that patients with HER2-positive tumours can present HER2-negative CTCs in their blood. Moreover, the detection of HER2-positive CTCs is not infrequent in HER2-negative tumours, as specified bellow, and could indicate worse clinical outcome. A small-series study, led by Flores and colleagues [48], reported that 33% of metastatic BC patients with HER2-negative disease had HER2-amplified CTCs. Similar CTC findings were observed in the CirCE T-DM1 trial [49] and in the plasmaMATCH study focused on ctDNA (Table 1) [35]

In an interesting study, Wang and colleagues investigated the impact of anti-HER2 therapy in patients with HER2-negative tumours but positive CTCs [50]. They observed that these patients with ≥2 HER2-positive CTCs showed less survival and better benefits from anti-HER2 therapies. Moreover, in a follow-up analysis, those patients changing between ≥2 to <2 HER2-positive CTCs presented better survival. Similarly, another study explored the effect of lapatinib in patients with HER2-positive CTCs but HER2-negative tumours [51]. The authors reported that 7 out of 96 patients with detected CTCs presented HER2-positivity. However, they did not observe tumour response to lapatinib in this patient population but only one patient with disease stabilisation. By contrast, Agelaki and colleagues [52] also treated patients with HER2-negative advanced tumours but HER2-positive CTCs with lapatinib. They observed a decrease in the HER2-positive CTCs number in patients with disease stabilisation but also in the median number of detected CTCs per patient. However, no objective responses were observed, probably because of the low number of patients included in the study. Moreover, the DETECT III study compared standard therapy alone or in combination with anti-HER2 targeted therapy (lapatinib) in patients with HER2-negative metastatic BC and HER2-positive CTCs [53]. Their results showed favourable outcomes after combined targeted treatment indicated by early decline of CTC counts. Still, this clearance was not significant compared to the standard arm [57.1% vs. 50.0%; p = 0.63]. Of note, patients in the lapatinib arm showed a tendency towards a better progression-free survival (PFS) and a significantly improved OS by univariate (HR 0.54; 95% CI, 0.34–0.86; p = 0.008) and multivariate (HR 0.55; 95% CI, 0.34–0.90; p = 0.016) cox regression analysis when compared to those in the standard arm. The CTC clearance at the end of treatment was not associated with OS, although better OS was observed in patients with no evidence of CTCs at first follow-up compared to patients with detectable CTCs (HR 0.36; 95% CI, 0.17–0.76; p = 0.005) (Table 1). Finally, in a very recent research article also within the DETECT program, the authors analysed the HER2 status of CTCs in patients HER2-negative tumours and its clinical significance [54]. Herein, CTCs HER2 status was analysed in 1159 CTCs-positive patients. They observed that patients with estrogen and progesterone-positive status were more prone to present strong-stained HER2 CTCs. The authors also demonstrated the association of ≥1 CTCs strong-stained for HER2 with shorter OS but not between the proportion of HER2-positive CTCs and clinical outcome. The association with OS is lost when including moderate-stained HER2 CTCs. Importantly, CTCs HER2 status was not significantly associated to PFS in this cohort.

While most of the studies reviewed thus far are based on limited sample sets, it is already clear that liquid biopsy and ctDNA detection in the HER2-positive early BC setting can provide prognostic information regarding both the response to NAC and early relapse. In this regard, ctDNA monitoring could be a tool to modulate NAC schedules and avoid overtreatment in low-risk patients or increase dosage in those with suboptimal responses. In the adjuvant setting, the detection of MRD and recurrence before it is detectable clinically could crucially improve BC patients’ survival by the timely starting of adjuvant therapy as well as by preventing overtreatment in patients with a lower risk of relapse. In this regard, large clinical studies employing ctDNA detection for clinical decision-making and personalisation are needed for the final validation of this methodology. Ideally, these studies employ ultrasensitive methodologies to detect ultralow levels of ctDNA.

Likewise, more studies are needed to determine the role of ctDNA detection in the metastatic HER2-positive BC. The development of novel methodologies to sensitively track ERBB2 amplifications and mutations in blood and characterise disease evolution, resistance and the acquisition of HER2-positivity in tumours previously HER2-negative is paramount to improve the clinical manage of BC patients.

More research also warrants to clarify the possible application of CTCs counts in clinics both in the early and metastatic setting. During NAC, certain associations between CTCs detection and worse outcomes were already observed. However, important discrepancies can be found between CTCs counts and response to NAC. In the same manner, no conclusive results exist to clarify whether CTCs detection in early HER2-positive BC during adjuvant therapy would be beneficial for patients’ stratification. In addition, a shift between HER2-negative to positive disease at advanced disease stages was demonstrated using CTCs detectable in the blood of some patients with HER2-negative tumours. Moreover, changes in CTCs counts and clinical benefits were addressed in these patients when they are treated with anti-HER2 therapies. However, there is a need to develop studies including a higher number of patients to achieve consistent conclusions. It is necessary to develop novel and highly sensitive technologies to detect as well as to characterise HER2-negative or positive CTCs in the metastatic BC setting. Overall, it is important to shed light over the variability aspect between techniques as a factor behind the contradictory observations in the different studies. On top of that, it is crucial to understand how the disease is evolving and detect early resistance to be able to efficiently treat it. The advanced disease can provide us with high CTCs numbers that, unlike ctDNA, potentially offer deep insights about the disease transcriptomics and genomics.