###### Abstract  
Targeted therapy has become the preferred approach to treat most cancers, including metastatic breast cancer. Using liquid biopsies, which can act as a dynamic diagnostic tool, is an appealing concept to identify effective therapies. In order to identify mutations from circulating tumor cells (CTCs) on single cell level, we have developed a multiplex PCR-based next generation sequencing-panel. The CTCs were enriched using the CellSearch system and isolated by micromanipulation followed by whole genome amplification of their DNA. Afterwards, mutation hotspot regions in the _PIK3 CA_, the _ESR1_, the _AKT1_, and the _ERBB2_ genes were amplified and barcoded. Sequencing was performed on a MiSeq system. The assay was validated with cells from various cell lines displaying the expected mutations. Mutations that provide the basis for potential targeted therapies were detected in 10 out of 13 patients in all analyzed genes. In four patients, mutations in more than one gene were observed--either in the same cell or in different cells, suggesting the presence of different tumor cell clones, which might be targeted with combination therapies. This assay is a time and cost effective tool to investigate the most relevant genomic positions indicative for targeted therapies in metastatic breast cancer. It can support therapy decision to improve the treatment of cancer patients.  
breast cancer; circulating tumor cells; liquid biopsy; metastatic breast cancer; panel sequencing; targeted therapy; single cell analysis +
Footnote †: journal: Medical  
Potential biomarkers are circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). CTCs are shed into the blood by tumor tissue and are commonly considered as precursor cells for metastasis formation [6; 7]. ctDNA as part of cell free DNA describes tumorous DNA circulating in the blood, which has been released from degraded tumor cells [8].  
In breast cancer, clinical utility has been demonstrated for the therascreen _PIK3 CA_ RGQ PCR kit, which detects _PIK3 CA_ mutations in a tissue biopsy and/or inctDNA obtained from a liquid biopsy in patients with an advanced or metastatic tumor [9]. The _PIK3 CA_ gene encodes for the p110\(\alpha\) subunit of the phosphoinositide-3-kinase (PI3K) which promotes cell proliferation and survival by activation of the PI3K/AKT signaling pathway [10]. Mutations, mostly located in its catalytic domain, can lead to an activation of downstream pathways [11]  
Further mutations that could act as targets for therapy in breast cancer are located in the \(ESR1\), the _AKT1_ and the _ERBB2_ genes: the \(ESR1\) gene codes for the estrogen receptor \(\alpha\) (ER\(\alpha\)), a nuclear hormone receptor that is involved in the regulation of gene expression affecting cellular proliferation and differentiation [12]. Mutations in the ligand binding domain of ER\(\alpha\) lead to a conformational change of the protein and to a ligand-independent ER\(\alpha\) activation, mostly acquired in resistance to estrogen deprivation therapy such as aromatase inhibition [13; 14]. AKT1 is a member of the serine-threonine kinase class and plays a key role in cellular processes, including growth, proliferation, survival, and angiogenesis. Certain _AKT1_ mutations lead to hyperactivation of the mTOR pathway [15]. The receptor tyrosine-protein kinase ERBB2 (HER2/neu (HER2)) activates the RAS/MAPK, PI3K/AKT, and JAK/STAT signaling pathways to promote cell proliferation and survival. Mutations in the hotspot regions coding for the extracellular domain and the kinase domain lead to an activation of the protein and its downstream pathways [16].  
In order to identify mutations for targeted therapy in breast cancer using CTCs, which have been enriched using the CellSearch system, we developed a multiplex PCR-based next generation sequencing (NGS)-panel. The CellSearch system has been approved by the Food and Drug Administration (FDA) for detection of CTCs in patients with metastatic breast, prostate, and colorectal cancer.