Novel liquid biopsy method for detecting androgen receptor splice variant 7 in circulating tumor cells of mCRPC.

Abstract

e14542 Background: Androgen receptor splice variant 7 (AR-V7) is constitutively activated isoform of AR and has been associated with the resistance towards AR targeting therapies and development into metastatic castration-resistant prostate cancer (mCRPC). Analysis of AR-V7 expression is an important step in prostate cancer diagnosis and determining the therapeutic drug, but the detection of AR-V7 with tissues has limitations such as clinical challenge to poorly accessible tumor tissues before and after treatment. Here, we developed the method of AR-V7 detection in circulating tumor cells (CTC) after isolation with CytoGen’s. Smart Biopsy CTC isolator by droplet digital polymerase chain reaction (ddPCR) with high sensitivity and specificity, overcoming the limitations of tissue biopsy. Methods: Sensitivity and specificity were tested using AR-V7 positive cells (VCaP or 22Rv1) and negative cells (PBMC). Limit of detection (LOD) test of AR-V7 expression was performed that AR-V7 positive cells (10 and 5 cells) were spiked into human blood sample (5mL, healthy donor) to mimic CTC after isolation by CytoGen’s Smart Biopsy CTC isolator, subsequently AR-V7 expression was tested with ddPCR. All ddPCR processes included droplet making and reading for each assay were performed on the QX200 System. For the clinical evaluation, blood samples of metastatic prostate cancer patients (n = 10) and normal healthy humans (n = 3) were tested with IF assay to count CTC number and ddPCR to analyze the expression of AR-V7. Results: In in vitro spiking LOD test, AR-V7 expression was clearly detected in the AR-V7 positive cells spiked into human blood and was not detected at all in the negative controls. In clinical evaluation study, isolation of CTC from the blood of prostate cancer patients using CytoGen’s Smart Biopsy CTC isolator was confirmed from the result of IF assay, and the expression of AR-V7 in CTC was successfully detected by ddPCR. IF analysis identified more than 3 CTCs in blood from all patients, and expression of AR-V7 was verified by ddPCR in 6 out of 10 mCRPC patients based on the results of healthy humans as cut-off value . These results demonstrate that it is possible to clearly detect the presence of RNA variants is related with drug resistance in mCRPC patients using liquid biopsy, utilizing CTCs, which exist in only very small quantities in the blood. Conclusions: AR-V7 expresses only a part of cancer tissue, so it is difficult to accurately diagnose with traditional histological assays. Detection of AR-V7 (RNA signature) in intact live CTC might overcome limitations of tissue biopsy. In this study, we developed the method to detect AR-V7 in CTC, and presented the results of verifying AR-V7 expression using in vitro model and blood of mCRPC patients. Expanded clinical validation study with more patients’ samples and clinical correlation between AR-V7 expression in CTC and clinical outcome will be performed and analyzed.