Abstract 1588: Single cell analysis of AR N terminal, AR C terminal and the ARv7 splice variant in the CTCs of metastatic castration resistant prostate cancer (mCRPC) patients
Authors: Kelvin James , Lu David , Packer Davin , Bambury Richard , Rathkopf Dana , Schreiber Nicole , Arslan Zaina , Prigozhina Natalie , Brown David , Krupa Rachel , Swangren Edward , Landers Mark , Lee Florence , Marrinucci Dena , Dittamore Ryan , Scher Howard I. 
Published: August 2015

Background: Androgen signaling directed therapies, including Abiraterone (A) and Enzalutamide (E), prolong survival in patient (pts) with mCRPC and are FDA approved. Resistance to A and E is associated with the presence of the ARv7 splice variant which, along with other AR ligand binding domain (LBD) alterations, may constitutively activate AR. Previous work showed marked heterogeneity in cell types and protein expression in CTCs of mCRPC pts mandating single cell analysis to assess the AR LBD to understand passenger vs. driving clonal subtypes. We have developed and begun clinical validation for AR N terminal (AR N), AR C terminal (AR C), ARv7 protein and ARv7 mRNA enabling single CTC analysis.

Methods: Cell lines (VCaP, LnCaP, LnCaP-95, 22RV1, & PC3) expressing varying levels of AR LBD alterations were spiked into healthy donor blood. 47 pt samples were collected for CTC analysis utilizing the Epic Sciences platform which 24 pts (6 treatment naïve, 10 post A or E, and 8 post taxane) were selected for AR N/C/v7 analysis due to CTC prevalence across clinical decision point where change in therapy was needed. Epic analysis included ID of traditional CTCs, CK- CTCs, small CTCs, and CTC clusters. Cell lines & pts were analyzed for AR N, AR C and ARv7 IF, and a subset of patients CTC for ARv7 mRNA by RNA FISH.

Results: Immunofluorescent protein expression of AR N, AR C and ARv7 in cell lines were consistent with published profiles. AR C & ARv7 were detected only in AR N+ CTCs indicating assay specificity for both targets. ARv7 expression was observed in traditional, CK-, small CTCs and CTC clusters, but no association was seen between ARv7+ and any CTC subtype. AR v7 RNA FISH was specific to AR v7 IF+ pts.

Conclusions: AR C loss is more sensitive in detecting AR LBD alterations than ARv7. Variable ARv7/AR N ratios in different cell types suggest intrapatient AR heterogeneity. The low prevalence of ARv7+ CTCs of total and AR N+ CTCs suggests many pts may have other driving resistance mechanisms.

ARv7+ and AR C Loss by Clinical Decision Point1st Line CRPC TherapyA or E as 2nd line TherapyTaxane Post A, E, or A+En = 6n = 10n = 8# pts with v7+ CTCs0/6 (0%)5/10 (50%)2/8 (25%)# pts with C loss2/6 (33%)5/10 (50%)3/8 (38%)% CTCs which are ARv7+ (only in pts with ARv7+ cells)1st Line CRPC TherapyA or E as 2nd line TherapyTaxane Post A, E, or A+En = 0n = 5n = 2v7+ pts:% v7+ CTCs [range (median)]n/a1.4-35 (9.8)5.1-11 (8.1)v7+ pts: v7/N-term [range (median)]n/a2-71 (14)5-13 (9)

Citation Format: James Kelvin, David Lu, Davin Packer, Richard Bambury, Dana Rathkopf, Nicole Schreiber, Zaina Arslan, Natalie Prigozhina, David Brown, Rachel Krupa, Edward Swangren, Mark Landers, Florence Lee, Dena Marrinucci, Ryan Dittamore, Howard I. Scher. Single cell analysis of AR N terminal, AR C terminal and the ARv7 splice variant in the CTCs of metastatic castration resistant prostate cancer (mCRPC) patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1588. doi:10.1158/1538-7445.AM2015-1588