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Rationale: Our objective was to develop a circulating tumor cell (CTC)-RNA assay for characterizing clinically relevant RNA signatures for the assessment of androgen receptor signaling inhibitor (ARSI) sensitivity in metastatic castration-resistant prostate cancer (mCRPC) patients. Methods: We developed the NanoVelcro CTC-RNA assay by combining the Thermoresponsive (TR)-NanoVelcro CTC purification system with the NanoString nCounter platform for cellular purification and RNA analysis. Based on the well-validated, tissue-based Prostate Cancer Classification System (PCS), we focus on the most aggressive and ARSI-resistant PCS subtype, i.e., PCS1, for CTC analysis. We applied a rigorous bioinformatic process to develop the CTC-PCS1 panel that consists of prostate cancer (PCa) CTC-specific RNA signature with minimal expression in background white blood cells (WBCs). We validated the NanoVelcro CTC-RNA assay and the CTC-PCS1 panel with well-characterized PCa cell lines to demonstrate the sensitivity and dynamic range of the assay, as well as the specificity of the PCS1 Z score (the likelihood estimate of the PCS1 subtype) for identifying PCS1 subtype and ARSI resistance. We then selected 31 blood samples from 23 PCa patients receiving ARSIs to test in our assay. The PCS1 Z scores of each sample were computed and compared with ARSI treatment sensitivity. Results: The validation studies using PCa cell line samples showed that the NanoVelcro CTC-RNA assay can detect the RNA transcripts in the CTC-PCS1 panel with high sensitivity and linearity in the dynamic range of 5-100 cells. We also showed that the genes in CTC-PCS1 panel are highly expressed in PCa cell lines and lowly expressed in background WBCs. Using the artificial CTC samples simulating the blood sample conditions, we further demonstrated that the CTC-PCS1 panel is highly specific in identifying PCS1-like samples, and the high PCS1 Z score is associated with ARSI resistance samples. In patient bloods, ARSI-resistant samples (ARSI-R, n=14) had significantly higher PCS1 Z scores as compared with ARSI-sensitive samples (ARSI-S, n=17) (Rank-sum test, P=0.003). In the analysis of 8 patients who were initially sensitive to ARSI (ARSI-S) and later developed resistance (ARSI-R), we found that the PCS1 Z score increased from the time of ARSI-S to the time of ARSI-R (Pairwise T-test, P=0.016). Conclusions: Using our new methodology, we developed a first-in-class CTC-RNA assay and demonstrated the feasibility of transforming clinically-relevant tissue-based RNA profiling such as PCS into CTC tests. This approach allows for detecting RNA expression relevant to clinical drug resistance in a non-invasive fashion, which can facilitate patient-specific treatment selection and early detection of drug resistance, a goal in precision oncology.

157 Background: Tissue-based gene signatures can predict clinical behavior in prostate cancer (PC). Our objective was to extend their application to circulating tumor cells (CTCs) and to show that changes in the signature were associated with changes in clinical behavior. Methods: Our approach combined the Thermoresponsive(TR)-NanoVelcro CTC purification system with the Nanostring nCounter system for cellular purification and transcriptomic analysis. The Prostate Cancer Classification System (PCS) panel was modified for use in CTCs. We selected 31 blood samples from 23 PC patients receiving androgen receptor signaling inhibitors (ARSI) and measured the PCS1 Z score (probability). These findings were compared with clinical outcome data (responsiveness/resistance). Results: A modified, 16-gene PCS1 signature was established and validated through a rigorous bioinformatics process. We performed analytical validation of our combined CTC-RNA system to ensure reproducibility and specificity. In patient bloods, ARSI-resistant samples (ARSI-R, n = 14) had significantly higher PCS1 Z scores as compared with ARSI-sensitive samples (ARSI-S, n = 17) (Rank-sum test, P = 0.003). The analyzed bloods contained samples from 8 patients who developed resistance to an ARSI allowing for dynamic measurement of gene expression. Our analysis found that the PCS1 Z score increased at the time that ARSI-resistance emerged (Pairwise T-test, P = 0.016). Conclusions: Using this new methodology, contemporary, clinically-relevant gene signatures such as PCS could be measured non-invasively in CTCs. These findings can be used to relate gene expression to clinical drug response. This approach also allowed for measurement of dynamic variations of gene expression in individual patients over time that correlated to ARSI sensitivity.