Introduction

Liquid biopsy (LB) plays an important role in the treatment of breast cancer (BC), especially in advanced hormone receptor positive (HR+), HER2 negative (HER2−) BC. In the early setting, LB-based detection of minimal residual disease (MRD) has been tested and first encouraging results have been reported. In this review, we give an overview of current clinical applications and potential future strategies for LB.

In BC, LB is mainly used to detect circulating tumor DNA (ctDNA) in blood. There are targeted approaches and whole genome sequencing-based approaches [1]. The most suitable test depends on the clinical question. One of the main advantages of LB is its easy detection in blood samples and its repeatability over time of disease. This means invasive tissue sampling can be avoided. Moreover, LB enables a strategy to overcome the obstacle of spatial and temporal heterogeneity in the treatment of BC.

Current clinical applications

LB has its main clinical relevance in the metastatic setting of HR+, HER2− disease for detection of potential treatment targets after CDK4/6 inhibition treatment. CDK4/6 inhibition in combination with endocrine treatment is the actual standard of care with encouraging overall survival data [2]. The ESMO (European Society for Medical Oncology) guidelines recommend somatic mutation testing in tissue or plasma and furthermore testing of BRCAness or PALB2 after progress on CDK4/6 inhibitor [3]. The ASCO (American Society of Clinical Oncology) recommends testing within tissue or ctDNA for biomarkers such as PIK3CA, BRCA, and ESR1 mutations [4, 5]. One of the first trials implementing a ctDNA-guided treatment strategy in advanced BC was the plasmaMATCH trial conducted by Turner et al. [6]. In this phase 2a trial, clinically relevant activity of substances targeting alterations, like ESR1 or HER2 mutations, that were detected by LB was shown [6]. In addition, an observational multicenter trial exposed that treatment strategies targeting druggable ctDNA alterations in advanced BC significantly improved progression-free survival (PFS) and disease control rate (DCR) in heavily pretreated patients [7]. However, this trial included patients between 2016 and 2021, thus, underrepresenting actual treatment standards [7].

AKT pathway

One of the first approved substances in BC depending on mutation testing within LB was alpelisib. The α‑specific PI3K inhibitor showed a significant improvement in PFS in patients harboring a PIK3CA mutation independent of its detection in tissue or plasma [8]. Considering the increase of PIK3CA mutations under treatment, testing should be repeated to identify patients who benefit from alpelisib [9]. The Capitello-291 trial studied another agent in this setting, the AKT inhibitor capivasertib [10]. Capivasertib in combination with endocrine treatment led to an improved PFS of 7.2 versus 3.6 months (hazard ratio [HR] 0.6; 95% confidence interval [CI] 0.51–0.71; p < 0.001) compared to fulvestrant monotherapy [10]. However, in patients with BC with an alteration in the AKT pathway, PFS was 7.3 vs 3.1 months (HR 0.50; 95% CI 0.38–0.65; p < 0.001) [10]. In this phase III trial, alterations in the AKT pathway were only tested in tissue; data regarding ctDNA-based alteration testing prior to treatment start are missing [10].

ESR1 mutations

A common resistance mutation under endocrine treatment is the ESR1 mutation, mainly leading to worse response against aromatase inhibitors (AI) [9]. The most common hotspot mutations are L536Y, Y537S/N/C, and D538G [11]. So-called selective estrogen receptor degraders (SERDs) as fulvestrant or oral SERDS such as elacestrant or camizestrant can overcome endocrine resistance based on hotspot mutations in the ligand-binding domain of the ESR1. One of the first clinical trials regarding ESR1 mutations in HR+, HER2− mBC is the PADA1 trial [12]. Bidard et al. showed that the PFS can be significantly improved by switching from an AI to fulvestrant if an ESR1 mutation emerges in ctDNA under endocrine treatment in combination with CDK4/6 inhibition [12]. Elacestrant is the first US Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved oral SERD in patients after progression on endocrine treatment harboring an ESR1 hotspot mutation detected in ctDNA [13]. Camizestrant is another oral SERD and has shown encouraging results in the phase II SERENA 2 trial, especially in patients harboring only a single ESR1 mutational variant compared to patients with more than one ESR1 mutation [14]. This supports the strategy of repeated testing for ESR1 mutations in ctDNA and early application of SERDs. The ongoing SERENA 6 trial studies the strategy of the PADA-1 trial with the oral SERD camizestrant [15]. Patients under treatment with CDK4/6 inhibitor plus AI switch to the combination of camizestrant plus CDK4/6 inhibitor if ESR1 mutations are detected within ctDNA without radiological progress of disease [15].

BRCA 1/2 mutations

The PARP inhibitor olaparib represents a treatment option in early or metastatic HER2− BC in patients with germline BRCA 1/2 mutations [16, 17]. Olaparib led to a significant improved PFS of 7.0 vs. 4.2 months (HR 0.58; 95%CI 0.43–0.80; p < 0.001) compared to chemotherapy [16]. Median overall survival (OS) was 19.3 months vs. 17.7 months after a median follow-up of 18.9 and 15.5 months [17]. The phase II trial TBCRC 042 enrolled 54 patients with germline PALB2, ATM, CHEK2 or BRCA 1/2 mutations (cohort 1) or somatic BRCA 1/2 mutations (cohort 2) [18]. Olaparib showed efficacy in patients with a germline PALB2 or somatic BRCA 1/2 mutation, with overall response rates (ORR) of 82% and 50%, respectively [18].

Further potential treatment targets in advanced or metastatic BC

The occurrence of HER2 mutations represents another potential target and was tested in the basket trial SUMMIT [19]. In one substudy of this trial, neratinib in combination with fulvestrant and trastuzumab was administered in patients with HER2− tumors harboring a HER2 mutation after progression on CDK4/6-based treatment [19]. An ORR of 39% and a median PFS of 8.3 months was reported for 57 patients [19]. Alterations in FGFR are often associated with endocrine resistance; in the RAGNAR basket trial, 13 patients with FGFR alterations were included. In the few cases of BC, the ORR was 31% and the DCR 69% [20]. However, more data regarding alterations and treatment strategies are needed.

Future clinical strategies

Early prediction of progression in advanced or metastatic BC

Several studies showed that LB can be useful for disease monitoring, especially for early detection of progress [21, 22]. Dandachi et al. implemented a model for longitudinal untargeted measurement of cell free DNA using z‑score in order to predict patients with high risk of clinical progress [21]. Chiu et al. monitored ctDNA fraction in patients with advanced HR+, HER2− BC under treatment with ribociclib and endocrine treatment [22]. Although it was a small sample size with 87 patients, statistically significant differences were found between the median ctDNA fractions in patients with PFS > 24 months and patients with PFS < 6 months (p = 0.034) and PFS 12–24 months (p = 0.016) [22]. A dynamic change of ctDNA fraction could be detected before radiological progress, with a median time to detecting progress of 83 days (range 14–309 days) in 13 patients [22].

Prediction of residual disease in early BC

The application of LB in early BC has not been routinely implemented in clinical practice. However, the first trials have shown the impact of ctDNA clearance during neoadjuvant treatment on response in patients [23,24,25]. This might be useful for treatment escalation and de-escalation strategies in early BC. Magbanua et al. showed that an early ctDNA clearance (3 weeks after treatment start) was significantly associated to pathological complete response (pCR) and residual cancer burden (RCB) 0 in triple negative breast cancer (TNBC) [23]. In contrast, in HR+, HER2− BC patients no association between ctDNA clearance during neoadjuvant chemotherapy and pCR or RCB was detected [23]. However, lack of ctDNA clearance at all time points, especially at 12 weeks and before surgery, was significantly associated with shorter distant-recurrence-free survival (DRFS) in both subgroups [23]. Moreover, patients with ctDNA positivity before surgery had shorter DRFS and OS independent of the RCB status [23]. Patients who were ctDNA negative at any time point had the best outcome [23]. However, the study cohort including 283 patients with HER2-BC was quite small and had heterogeneous biology and treatment [23]. Parson et al. showed a strong association between ctDNA clearance during neoadjuvant chemotherapy and RCB0 rates in a small cohort of patients with TNBC [24]. In both trials, personalized ctDNA assays for treatment monitoring were used to increase sensitivity.

Prediction of relapse in early BC

As in metastatic setting, elevation of ctDNA might be a signal for recurrence of disease. Garcia-Murillas et al. showed a significant correlation between ctDNA detection and detection of relapses in patients with early BC in follow-up after treatment [26]. In all, 79.3% of the patients with disease relapse were ctDNA positive, whereas 20.7% of patients had no detectable ctDNA. Interestingly, these 6 patients had solitary relapses (3 brain, 1 ovary, 2 locoregional recurrence of disease) [26]. In the c‑TRAK TN trial, Turner et al. conducted a prospective ctDNA surveillance trial with at risk patients diagnosed with early TNBC [27]. In the intervention cohort, administration of pembrolizumab was planned in patients with ctDNA positivity and lack of clinical relapse. However, only 9 of 32 patients remained and only 5 of them received pembrolizumab including only 1 patient with ctDNA falling under treatment with pembrolizumab [27]. Thus, most patients with ctDNA had metastatic disease, with a median time between ctDNA detection to disease recurrence of 1.6 months (95% CI 1.2–4.9 months) in the intervention cohort and 4.1 months (95% CI 3.2 months–not defined) in the observation cohort [27].

Conclusion

Liquid biopsy is currently used in clinical practice to identify druggable alterations within ctDNA. Further applications like ctDNA-based surveillance in early and advanced disease and prediction of response under neoadjuvant treatment leading to treatment escalation or de-escalation could be promising future strategies. However, clinical trials are needed before future implementation in clinical routine.