Since market-entering of the oral ER degrader elacestrant at the end of 2023, liquid biopsy testing of metastatic BC increased immensely. In the laboratory-developed NGS panel employed in this study, the four disease relevant genes ESR1, PIK3CA, ERRB2, and TP53 were tested. Detection of an ESR1 mutation in metastatic BC should be followed by changing anti-estrogenic therapy to fulvestrant or elacestrant [4, 5]. PIK3CA mutations are frequent in BC and therefore a useful biomarker to ensure that cfDNA originating from tumor cells is present. Furthermore, PIK3CA mutations are possible targets for alpelisib [9] or capivasertib [10]. ERBB2 mutations are present in bone marrow metastases from lobular BC cases, and are possibly targetable with anti-HER2 therapy [6]. TP53 mutations are associated with primary endocrine resistance and might not be adequately treated with endocrine therapy alone [11].

In a recent study on tissue biopsies from breast cancer metastases, we detected ESR1 hotspot mutations in 78 of 521 (15.0%) samples [12]. In the current study, using liquid biopsy, we detected ESR1 mutations in 25.9% of cases. In a recent meta-analysis of 16 studies with in total n = 2744 patients, the overall incidence of ESR1 mutations was 23% (ranging from 11 to 55%) [13]. Differences in the mutation frequency are possibly not caused by the testing material (tissue or liquid), but by the selection of samples in the studies. For example, in n = 194 ER-positive bone marrow BC metastasis, we found 14% ESR1 mutation frequency [14], whereas lung and liver metastasis showed 26.3% and 31.3% ESR1 mutation frequency, respectively [12]. Nearly 97% of the patient samples analyzed in the current study were collected after the EMA approval of elacestrant. This strong increase reflects the urgent clinical need of new therapeutic approaches in anti-endocrine therapy in metastatic BC.

Our comparison of cfDNA and metastatic site DNA from 25 patients shows that ESR1 hotspot mutations may be missed in both sample materials. In three patients, an ESR1 mutation was detected only in the liquid biopsy cfDNA, whereas two patients had the positive mutation call only in metastatic site DNA. False-negative results in liquid biopsy analysis can be caused by contamination with white blood cell DNA or absence of tumor DNA at time of blood collection. Further, the sample transfer from external clinics and oncology practice units to the molecular pathology laboratory is prone to errors, e.g., transfer time too long. Clear instructions and regular consultation are necessary to reduce handling errors which can lead to false-negative results [15]. Nevertheless, especially in patients with multiple metastatic sites, liquid biopsies are the preferred material for molecular analysis, because the genetic heterogeneity of metastatic sub-clones is potentially represented in the cfDNA. However, in case of negative results from a liquid biopsy, the analysis of a most recent metastasis specimen should be considered if possible.

In conclusion, our results demonstrate that ESR1 detection in liquid biopsy is feasible under routine conditions identifying important therapy-changing predictive biomarkers in a substantial portion of metastatic BC patients. To the best of our knowledge, this is the first study which demonstrates the feasibility and diagnostic gain of ESR1 mutation detection in real-world liquid biopsy outpatient samples since the approval of elacestrant.