The circulating proteins CEA, CA 15-3, and CA 27-29 were recommended for therapy monitoring in 2015 by the ASCO [334]. However, their long half-lives might be one of the reasons for their low sensitivity for therapy response monitoring [158].

CTC quantification was frequently shown to have monitoring value in the MBC setting, independent of the given therapy. It was shown that the CTC count itself by CellSearch evaluated 3–5 or 6–8 weeks after initiation of therapy was significantly associated with PFS and OS [168]. A decrease in CTC counts from baseline to a time point under therapy was related to an increased PFS and OS [335] and persistently high CTC counts from baseline to under therapy, despite radiologically proven therapy response, associated with worse outcome [336]. The DETECT V/CHEVENDO (NCT02344472) is currently recruiting HR-positive/HER-positive MBC patients who receive pertuzumab and trastuzumab, either in combination with chemotherapy or endocrine therapy, to evaluate CTC quantification before and during the therapy to examine the prognostic and monitoring value of this blood-based evaluation.

Based on these results, in the SWOG S0500 trial, MBC patients with persistently increased CTC counts after 21 days of therapy were randomly assigned to continue initial therapy or change to an alternative chemotherapy [337]. The data evaluation showed no significant benefit from the early switch to an alternative chemotherapy in MBC patients with persistently high CTC counts after 21 days under initial therapy [337]. In the CirCe01 trial [338], MBCs were randomized either in the CTC-driven or the standard arm. In the CTC-driven arm, CTC counts via CellSearch were assessed at baseline and after the first cycle of therapy. Response to therapy was defined by CTC counts as ≥70% decrease in CTC number from baseline to completion of the first therapy cycle or an absolute number of ≥5 CTC per 7.5 mL blood after the first therapy cycle [339]. Patients not showing these CTC-driven response criteria were exposed to an early therapy switch. Data evaluation of the CirCe01 trial, however, revealed no significant prolonged OS in the CTC-driven arm compared to the arm with standard therapy response evaluation [338].

Molecular characterization of CTCs was found to have monitoring value. Comparison of the number of apoptotic CTCs from baseline to under therapy revealed a 50% apoptotic CTC reduction to differentiate between patients showing stable versus progressive disease [340] and in case the apoptotic CTC number decreased from baseline to under therapy by less than 10%, progressive disease was identified with 74% specificity [340]. A significant decrease in HER2-positive CTCs was only detected in MBC patients responding to anti-HER2 treatment with lapatinib, but not in patients progressing under lapatinib [341]. In case of anti-RANKL therapy with Denosumab in MBC patients, the increase in RANK-positive CTCs from baseline to day 2 [342] as well as the persistence of RANK-positive CTCs was related to a longer time to progress of the bone metastasis [342]. The persistence of CTCs overexpressing EpCAM, MUC1 or HER2 transcripts under therapy in MBC patients correlated with shorter OS [174]. CTC overexpression signals were related to the staging result at the time of blood draw in MBC patients and revealed 74% of all patients with progressive disease to have CTCs overexpressing either EMT markers or the stem cell marker ALDH1 in contrast to only 10% of patients with stable disease [173]. Similarly, it was shown that the overexpression of ERBB2, ERBB3, and ERCC1 alone or in combination with AURKA in CTCs of MBCs was significantly more prevalent in patients showing progressive disease at the time of blood draw compared to patients with stable disease [343]. Identification of CTCs with overexpression of ERBB2, ERBB3, and ERCC1 alone or in combination with AURKA during therapy in MBCs was furthermore related to a shorter OS [343]. In more detail, ERBB2 overexpression in CTCs was only detected in patients not treated with anti-HER2 therapy and was related to therapy failure at the time of blood draw and to a reduced OS [343]. The same group further showed similar results using a different CTC gene expression panel [344]: patients with progressive disease at the time of blood draw were more likely to have CTC overexpression signals than patients with stable disease. Interestingly, two different gene expression pattern in CTCs were shown for patients with progressive disease (with high prevalence of ESR, MUC1, AURKA, RAD51, TOP2A, ADAM17, SCGB2A2, KRT19, and EPCAM overexpression), but a homogeneous expression pattern in patients with stable disease [344]. ERBB2 and/or ERBB3 overexpression in CTCs was significantly correlated with progressive disease at the time of blood draw [345].

In 2022, the ASCO stated that there are insufficient data to recommend CTCs to monitor response in MBCs [271]. In Germany, the AGO recommended CTC quantification to evaluate the early therapy response after three weeks in MBCs, but did not recommend the CTC quantification for therapy switch [148].

Besides CTC overexpression signals, EV overexpression signals were studied. A stronger correlation of ERBB2 and ERBB3 signals in CTCs and EVs with disease progression was identified compared to ERBB2 and ERBB3 signals in CTCs alone, revealing a synergistic value of CTCs and EVs for therapy monitoring [345]. Interestingly, mTOR overexpression signals in EVs of MBCs under therapy were related to consecutive therapy failure [345] while mTOR overexpression in CTCs was related to patients showing therapy response over at least six months.

In the TBCRC 005 study, a 9-marker cfDNA methylation assay was shown to forecast disease progression three months earlier than radiographic staging in MBC patients [346].

A more than 50% reduction in genomic instability number (GIN) from low-pass WGS of cfDNA at baseline to one week under therapy was shown to associate with the stable disease proven by staging after 3 months and also with OS in a cohort of 25 MBC patients [157]. A rise in GIN from baseline to two weeks under therapy was associated with poor response, evaluated three months after therapy initiation by staging [157]. Another approach to identifying gains or losses of chromosomal material in the cfDNA (the mFAST-SeqS), output defined as z-score, showed that the comparison of z-scores at baseline and under therapy (z-score trajectories) has monitoring value in HR-positive/HER2-negative MBC patients treated with CDK4/6i [317].

The LOTUS and INSPIRE trials documented that the mean allele frequency dynamics from baseline to a time point under therapy related to therapy response at the time of blood draw or to PFS and OS in MBCs treated with different therapy regimens [347,348,349,350,351,352]. In the POSEIDON and SUMMIT trials, early evaluation of ctDNA changes forecasted the radiologic treatment response and the emergence of specific mutations correlated with clinical drug resistance [251,353]. More specifically, allele frequency of HER2 mutations in cfDNA decreased under pan-HER inhibitor neratinib in the SUMMIT trial, but increased upon radiographically proven progression [251]. In HER2-positive MBCs with brain metastases, the dynamic changes in ctDNA in CSF and plasma under therapy revealed decreased allele frequencies in the plasma to be consistent with extra-CNS disease control and increased allele frequencies in the CSF to be related to poor treatment benefit in CNS [354]. In the INSPIRE trial, TNBC patients, among patients with other tumor entities, were treated with pembrolizumab and ctDNA level changes from baseline to six weeks under treatment forecasted the therapy benefit [355]. In all patients who responded to therapy, ctDNA clearance was detected before the visible radiological response [355].

The PALOMA-3 trial demonstrated that the cfDNA level decrease, in general, was not valuable to forecast PFS or OS. cfDNA ESR1 mutations were also a weak marker for monitoring whereas cfDNA PIK3CA mutation dynamics had significant monitoring value [259]. A decrease in PIK3CA mutations in the cfDNA of palbociclib-treated patients from baseline to two weeks correlated significantly with increased PFS and long-term clinical benefit [259]. It was questioned whether a persistently high PIK3CA mutation level in cfDNA after two weeks of CDK4/6i might indicate a PI3Ki to be more effective [259]. However, a corresponding intervention trial has not been initiated. In the ALCINA trial, cfDNA evaluation at day 15 under palboclib plus fulvestrant showed a decrease in all patients independent of their PFS [356]. However, on day 30, undetectable cfDNA mutations (PIK3CA, TP53 and AKT1 studied) were associated with improved PFS [356]. In another evaluation of the ALCINA trial, the decline in cfDNA ESR1 mutations from baseline to day 15 under palbociclib plus fulvestrant was validated [357] and the informative value regarding PFS forecast of cfDNA mutation analysis at day 30, in comparison to baseline, was shown for ESR1 mutations [357]. ESR1 mutation detection in the plasma under first-line AI treatment revealed a direct association with progressive disease with 100% specificity [358]. ESR1 mutations were detectable prior to progression with a median lead time of 110 days [358].

In the interventional PADA-1 trial, cfDNA ESR1 mutations were questioned as both predictive and monitoring markers. Rising allele frequencies of cfDNA ESR1 mutations were used to identify patients with no radiographically proven progressive disease under palbociclib and AI suitable for therapy switch to fulvestrant plus palbociclib [276]. Data evaluation demonstrated a significant clinical benefit with regard to PFS in case the therapy switch was conducted from AI plus palbociclib to fulvestrant plus palbociclib in patients with rising ESR1 mutations detectable under therapy [276]. In accordance with the PADA-1 trial, the SERENA-6 trial (NCT04964934) is currently recruiting patients to receive letrozole and CDK4/6i, who will be confronted with a switch to the SERD AZD9833 plus CDK4/6i in case no progression is visible by radiographic staging but rising ESR1 mutations in the plasma are detectable.

Before the publication of the PADA-1 trial results, the ESMO did not recommend ctDNA analysis in general for monitoring purposes [256] and also rejected ESR1 mutation detection for monitoring or switch from AI to fulvestrant [256]. In the same year as the publication of the PADA-1 trial, ASCO and ESMO did not recommend monitoring MBC therapy responses by (ESR1) cfDNA detection [271,332]. The ESMO reported the need for validating whether cfDNA dynamics have clinical utility, the need to show improved OS, the need to define optimal time points for blood draw and optimal thresholds [332].

Monitoring on the basis of ctDNA evaluation was shown to have a higher sensitivity and higher correlation with tumor burden compared to CA 15-3 and CTC evaluations in MBCs [359]. A multimodal approach evaluating CTC mRNA, EV mRNA and cfDNA mutations emphasized the additive value of these analytes in MBC treatment monitoring, noting unique features of each analyte for disease surveillance [360]. In more detail, it was shown that the presence of either ERBB3 overexpression signals or ERBB2 overexpression signals in CTCs were related significantly to the staging result at the time of blood draw [360]. Additionally, the combined evaluation of ERBB3 in all three analytes associated with therapy response at the time of blood draw [360]. Dynamics from one time point to the next time point were more informative than single time point evaluations. In this regard, the overexpression signals in EVs were the most dynamic ones during therapy and newly occurring ERCC1 overexpression signals in EVs from one time point to the next had a specificity of 97% but sensitivity of 18% to determine therapy response [360]. The accuracy for detecting disease progression was 70% and 66% for PIK3CA and ESR1 variant appearances and the combined evaluation of ESR1 or PIK3CA allele frequency development was significantly correlated with disease progression [360]. Analysis of index patients indicated that the multimodal approach might cover the range of inter-patient heterogeneity. The three blood analytes complement each other, as specific EV signals were shown to be the most dynamic markers, the most accurate monitoring markers originated from the CTC fraction and the actionability of detected cfDNA variants might enrich the monitoring value by their predictive value relevant for therapy switch to a specific targeted therapy in the next line [360].