An In-Depth Evaluation of the Validity and Logistics Surrounding the Testing of AR-V7 mRNA Expression in Circulating Tumor Cells

Abstract

Recent reports have emphasized the clinical relevance of detecting AR-V7 in circulating tumor cells (CTCs). Our aim was to set up a validated multicenter pipeline to measure AR-V7 by quantitative RT-PCR (RT-qPCR) in RNA isolated from CellSearch-enriched CTCs to provide an AR-V7-positive or AR-V7-negative score in a clinically acceptable time range. CellSearch-enirched CTCs from patients with metastatic castration-resistant prostate cancer were characterized by RT-qPCR. After optimization, it was prospectively tested whether it was possible to report the AR-V7 status within 11 days (PRELUDE study). In the range of the RNA equivalent of 0.2 to 12 VCaP cells, the CV for AR-V7 was 9% (n = 37). The limit of detection was 0.3, and the limit of quantitation was 3 cells in the final RT-qPCR. No differences were observed between AR-V7 data generated by five technicians or in two different laboratories. For the 45 patients in PRELUDE, 13 patients were ineligible, 22 patients were AR-V7 negative, and 10 were AR-V7 positive. The median time to inform the physician of the test result was 7 days (range, 2 to 11 days). This assay can establish the AR-V7 status in CTCs from patients with metastatic castration-resistant prostate cancer. Furthermore, it was possible to provide an AR-V7 outcome within 11 days, indicating that it may be used to choose between an anti-androgen receptor or taxane-based cabazitaxel treatment.

AR splice variants in circulating tumor cells of patients with castration‐resistant prostate cancer: relation with outcome to cabazitaxel

“…Only patients with sufficient epithelial input in the sample were selected for the analyses to ascertain reliable results from the AR‐V assays. A higher median baseline CTC count of 83 vs seven CTCs/7.5 mL blood was detected in the evaluable patients vs the excluded patients with an epithelial signal too low for a reliable AR‐V assessment (Sieuwerts et al , ), respectively, which supports the robustness of our assay in measuring tumor‐driven signals.…”

“…The presence of the androgen receptor (AR) splice variant ( AR‐V ) 7 in circulating tumor cells (CTCs) was recently shown to predict resistance to new‐generation anti‐AR‐targeted treatments (abiraterone acetate and enzalutamide), but not to taxane‐based chemotherapy in patients with metastatic castration‐resistant prostate cancer (mCRPC) (Antonarakis et al , ; Antonarakis et al , ; Antonarakis et al , ; De Laere et al , ; De Laere et al , ; Nakazawa et al , ; Onstenk et al , ; Onstenk et al , ; Scher et al , ; Scher et al , ; Scher et al , ; Tagawa et al , ). If further validated, like currently ongoing in the CABA‐V7 study (NCT03050866), the AR‐V7 status of CTCs may be used in clinical care to select the best treatment for an individual patient at a specific time (Sieuwerts et al , ).…”

Androgen receptor expression in circulating tumor cells of patients with metastatic breast cancer

“…After enrichment of CTCs, RNA was isolated with the AllPrep DNA/RNA Micro Kit (Qiagen, Germantown, MD) and cDNA was generated, pre‐amplified for the targets of interest and real time amplified (RT‐qPCR) using Taqman Gene Expression Assays (Applied Biosystems, Carlsbad, CA) (Supporting Information Table 2A). A more detailed description how RT‐qPCR data were generated has been published previously …”

“…detailed description how RT-qPCR data were generated has been published previously. [21][22][23] From 52 patients matched formalin-fixed paraffin embedded (FFPE) tissue from the primary tumor with at least 30% invasive tumor cells was available (median [range]; 60% [30%-85%]). RNA from the FFPE samples was isolated with the High-Pure RNA Paraffin Kit (Roche Applied Science, Penzberg, Germany) and the quality and quantity of the RNA was checked with the Nanodrop 1,000-v.3.7 (Thermo Scientific, Wilmington), the MultiNA Microchip Electrophoresis system (Shimadzu, Kyoto, Japan), and three reference genes (GUSB, HMBS and HPRT1) as described before.…”

PIK3CA hotspot mutations in circulating tumor cells and paired circulating tumor DNA in breast cancer: a direct comparison study

“…CTC detection and enumeration are associated with progression-free survival (PFS) and overall survival (OS) in metastatic (Bidard et al, 2014;Cristofanilli et al, 2004) and early BrCa (Lucci et al, 2012;Rack et al, 2014;Stathopoulou et al, 2002). Beyond enumeration, CTC molecular characterization is very important, since it can provide significant information at the gene expression, DNA methylation, and DNA mutation level (Aktas et al, 2016;Chimonidou et al, 2011Chimonidou et al, , 2013Markou et al, 2011Markou et al, , 2014Mastoraki et al, 2018;Mostert et al, 2015;Sieuwerts et al, 2018;Strati et al, 2011). However, CTCs are highly heterogeneous, and there is a lack of a unique marker for their isolation and identification, since there are not well-defined universal surface targets among all malignant cell types (Lianidou and Hoon, 2017).…”