Background: Recently a succession of new hormonal therapies has emerged, highlighting the need for biomarkers to guide the management of HSPC. Biomarker development in HSPC has been hampered by the absence of primary tumor tissue in men who undergo radiation or present with metastatic disease. CTCs can address this challenge by providing real-time cancer tissue for biomarker analysis in HSPC. To test this approach we conducted a pilot of CTC capture and targeted NGS in HSPC. Methods: Under IRB approval, blood samples from patients with HSPC were labeled with EpCAM ferrofluid and placed into the LiquidBiopsy platform (Cynvenio Biosystems, Inc.), an immunoaffinity-based microfluidic device tailored to query genomic events. CTCs were identified by CK, CD45 and DAPI expression. A matched WBC pellet served as a control representing germline sequence. Amplicon libraries were generated using Life Technologies AmpliSeq 2.0 and sequenced on an Ion Torrent platform. Somatic single nucleotide variants (SNV) present in CTCs but not in WBC were identified. Results: CTCs were detected in all 8 patients with HSPC (CTC median 64.5, range 17-217). Germline variants were consistently detected in matched CTC and WBC samples. Significant SNVs (occurring in > 1% of DNA in a sample) were found in 4 of 8 CTC samples (range 1-5 SNVs/sample, frequency 1.2%-11.9% with 620X-14,422X coverage). Notably, 3 patients had biochemical recurrence only (no clinical metastases) yet still yielded CTCs associated with SNVs in KIT, APC, RET, SMAD4 and PTEN. One patient who had untreated metastatic disease had the highest number of CTCs which harbored 4 SNVs. Conclusions: This pilot demonstrates the feasibility of using CTCs as real-time disease relevant substrate for NGS to identify personalized genomic targets in HSPC. A high number of CTCs were detectable in all patients and CTC germline variants correlated with matched WBC controls. Encouragingly, even with a relatively narrow, primary tumor-derived AmpliSeq platform, cancer relevant SNVs were detected in half of the patients including those with only biochemical recurrence, making targeted NGS of CTCs a promising approach for biomarker discovery and validation in HSPC.