Abstract 2736: A digital droplet PCR assay for the quantitation of androgen receptor and splice variant expression in CTCs from metastatic castration resistant prostate cancer patients
Authors: Gjyrezi Ada , Galletti Giuseppe , Strati Areti , Kim Seaho , Lianidou Evi , Nanus David M. , Luo Jun , Antonarakis Emmanuel , Tagawa Scott T. , Armstrong Andrew , Giannakakou Paraskevi 
Published: July 2017

Abstract
Prostate cancer (PC) is the second leading cause of cancer death in men in the US. The aberrant functioning of androgen receptor signaling is the central driving force behind prostatic tumorigenesis and its transition into metastatic castration resistant disease. Hence, androgen deprivation therapy (ADT) is the first line of treatment for PC patients. However, many patients progress becoming resistant to ADT therapy, due to the expression of AR splice variants (AR-Vs), which lack the ligand binding domain and are constitutively active in the nucleus. Expression of the AR splice variant, AR-v7, in circulating tumor cells (CTCs) isolated from the blood of PC patients was correlated with resistance to enzalutamide and abiraterone, which are the next generation AR signaling inhibitors in CRPC. Further, there is evidence that AR-Vs may convey cross-resistance, not only to enzalutamide and abiraterone, but also to taxanes, highlighting that their assessment in the clinic may have clinical utility.
We developed a novel, specific and highly sensitive assay to measure mRNA expression of the AR full length (AR-FL) and the splice variants ARv7 and ARv567es, by using Droplet Digital PCR in CTCs isolated from CRPC patients. The analytical specificity of the assay was determined by transfecting cells with plasmids encoding AR-FL, AR-v7 and AR-v567 and showed that each probe detected signal only in cells expressing the respective transcript. No signal was detected against genomic DNA, indicating lack of non-specific binding. Also, the assay detected endogenous expression of AR-FL and AR-v7 in VCAP or 22RV1 cells, while no variant expression was detected in healthy donor blood. The analytical sensitivity of the assay was determined in a series of serial dilution experiments that showed sensitivity down to single cell.
We then used this assay to determine the clinical prevalence and expression pattern of each of these variants in CTCs from about 200 mCRPC patient samples and blood from 40 healthy donors. CTCs were enriched by EpCAM- or PSMA-based positive selection or CD45 negative depletion in an antigen-agnostic manner. AR-FL was detected in ~80% of mCRPC samples irrespective of CTC-enrichment technology. AR-v7 was expressed in 65% of the samples in which in CTCs were enriched either by PSMA-positive selection or by negative depletion. In contrast, EpCAM-based CTC enrichment showed lower AR-v7 expression both in terms of expression levels and prevalence. In addition, CTC enrichment following negative depletion showed that 30% of the samples had higher AR-v7 expression levels as compared to AR-FL. This expression pattern was not observed in the samples using EpCAM-based selection. Collectively, these data suggest distinct CTC subpopulations are present in CRPC patient samples, with differential expression of AR-Vs that could have important predictive and prognostic implications.
Citation Format: Ada Gjyrezi, Giuseppe Galletti, Areti Strati, Seaho Kim, Evi Lianidou, David M. Nanus, Jun Luo, Emmanuel Antonarakis, Scott T. Tagawa, Andrew Armstrong, Paraskevi Giannakakou. A digital droplet PCR assay for the quantitation of androgen receptor and splice variant expression in CTCs from metastatic castration resistant prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2736. doi:10.1158/1538-7445.AM2017-2736