Abstract 5230: Development of a multi-parameter immunofluorescence assay for simultaneous detection of androgen receptor and androgen receptor variant 7 in prostate cancer circulating tumor cells
Authors: Sun Yao , Campton Daniel , Ramirez Arturo , Mojica Tanisha , Clein Alisa , Higano Celestia , Sabath Daniel , Plymate Stephen , Kaldjian Eric 
Published: August 2018

Abstract
Background. Androgen receptor (AR) signaling is the primary driver of prostate cancer. Expression of the AR splice variant AR-V7, is predictive of resistance to anti-androgen therapies enzalutimide and abiratirone. There is great interest to non-invasively identify circulating tumor cells (CTCs) and investigate their expression of AR and AR-V7 and monitor them over time. RareCyte has developed a platform for automated visual identification of CTCs by immunofluorescence (IF) that allows up to 6-parameter assessment. We have developed a set of prototype assays using tyramide amplification to detect AR and AR-V7 individually and in combination with prostate membrane-specific antigen (PSMA). Using a novel method, we also developed an assay to identify AR and AR-V7 simultaneously. Methods. Normal human whole blood samples were spiked with prostate cancer lines serving as model CTCs (mCTCs). Prostate cancer blood from a patient with advanced and known high CTC count were collected under an IRB-approved protocol. Blood was processed onto microscope slides and stained on an automated stainer with CTC detection assays incorporating 4 core parameters (nuclear dye, CD45, pan-cytokeratin, EpCAM) plus the following markers: (1) AR; (2) AR-V7; (3) AR and PSMA; (4) AR-V7 and PSMA; (5) AR and AR-V7. Each assay was applied to the cancer cell lines PC3, LNCaP, and 22RV1 and to the patient sample. An antibody denaturing process was used to sequentially amplify two rabbit monoclonals for the assay containing both AR and AR-V7. No-primary (diluent only) staining controls were run to confirm success of the denaturing step. Results. Staining of spike-in CTC models confirmed the reported AR and AR-V7 phenotype of the cell lines, supporting the specificity of the assays. When applied to an equivalent volume of the patient sample, the assays identified a mean (SD) of 36 (11) CTCs. In the assays staining AR, the mean percent of AR+ CTCs was 70% (range 61 - 86) and for the assays staining AR-V7, the percent of AR-V7+ CTCs was 30% (range 26 - 39). In the assay staining both AR and AR-V7, 10 of 38 CTCs were AR-V7+ (26%); all of these were AR+; 15 CTCs were only AR+ (39%). Conclusions. We have developed multi-parameter IF assays for detection of AR and AR-V7 in prostate CTCs, including the first assay we are aware of to simultaneously visualize AR and AR-V7. It has been validated in prostate cancer CTC models. The percentages of AR and AR-V7-positive CTCs identified in a patient sample with the assay are consistent with the percentages observed when AR and AR-V7 are detected in independent assays, supporting the accuracy of the simultaneous detection assay. All AR-V7-positive CTCs were also AR-positive, supporting specificity of AR-V7 staining.
Citation Format: Yao Sun, Daniel Campton, Arturo Ramirez, Tanisha Mojica, Alisa Clein, Celestia Higano, Daniel Sabath, Stephen Plymate, Eric Kaldjian. Development of a multi-parameter immunofluorescence assay for simultaneous detection of androgen receptor and androgen receptor variant 7 in prostate cancer circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5230.