## 4 Discussion  
Mutations detected in cancer can help to choose an appropriate targeted therapy. In the advanced disease, analysis of a liquid biopsy can facilitate to identify such druggable targets. Various studies reported suitable tools for the analysis of single CTCs' WGA-products, either by investigation of single genomic positions by e.g., Sanger sequencing [22; 23] or by sequencing multiple genomic positions by, e.g., NGS panels [24; 25]. However, most available assays are either cost intensive, not adapted to single cell whole genome amplified DNA, or cover only single or not all relevant genomic positions for therapy selection in breast cancer. Here, we developed and provided the protocol for a multiplex PCR-based, Ampli1 WGA-adapted NGS panel that covers mutation hotspots in the _PIK3 CA_, the _ESR1_, the _AKT1_, and the _ERBB2_ genes that are most relevant for a liquid biopsy-based targeted therapy in breast cancer. The assay was validated with spiked cell line cells and finally applied to CTCs from metastatic breast cancer patients. Mutations that provide the basis for potential targeted therapy were detected in 10 out of 13 patients. Although one CTC from patient 4 was found to harbor the _ERBB2_ A775T mutation, it was not considered as clinically targetable since this mutation has not been characterized in breast cancer, yet. For the treatment of hormone receptor-positive, HER2-negative advanced breast cancers harboring _PIK3 CA_ variants such as N345K, E545K, H1047L, or H1047R, alpelisib plus fulvestrant is indicated [26]. Furthermore, HER2-positive tumors of patients with this variant exhibited resistance to trastuzumab [27]. The detected _ESR1_ L536P mutation leads to a conformational change of the protein and to a ligand-independent ER\(\alpha\) activation, thereby conferring resistance to aromatase inhibition [13]. However, treatment with second-generation selective ER\(\alpha\) degraders such as fulvestrant is recommended [28]. The _AKT1_ E17K mutation hyperactivates the mTOR pathway that could be targeted by mTOR inhibitors, such as everolimus, or AKT-inhibitors, such as ipatasertib and capivasertib [29; 30]. Mutations such as S310F, L755S, or V777L in the _ERBB2_ gene offer to be treated with the tyrosine kinase inhibitor neratinib [16]. The frequency of _PIK3 CA_ and _AKT1_ mutations in our patient cohort exceeded the frequencies published in literature. _AKT1_ E17K mutation frequencies between 1.4% and 8.2% have been described in breast cancer tissue [31] and mutations of the _PIK3 CA_ gene are found in 25%-40% of all breast cancers [32; 33]. One reason for that is that such mutations can be acquired during the metastasis process leading to a discrepancy of the mutation status of the primary tumor and a liquid biopsy [34]. Furthermore, mutations in the _PIK3 CA_ and the _AKT1_ gene lead to an increased proliferation of the tumor combined with reduced apoptosis [10; 15] and might thereby cause a higher probability of CTCs detected in general and of CTCs with a high genome integrity in particular. Most of the mutations we detected were located in well-described hotspot regions. Some mutations, however, are novel and have not been reported yet, such as the _PIK3 CA_ M318T, _ERBB2_ L313P, or the _ERBB2_ A775T mutations. These mutations were only detected in single WGA products and due to prior WGA, a false positive result cannot be totally excluded. To further investigate these mutations, tools to predict the effect on the protein structure have been applied [35; 36; 37]. Although they are not fully consistent, the results indicate that these mutations might affect the function of the protein (Table S3). Heterogeneity was observed within CTCs from almost all patients. Such heterogeneity indicates that different subclones exist within the tumor and that the patient might therefore benefit from the combination of treatments targeting different proteins.