Abstract B40: Analytical validation and initial clinical utility of multianalyte transcriptomic biomarker profiling of circulating tumor cells using automated exclusion-based sample preparation technology

Abstract

Abstract The efficacy of androgen receptor (AR) signaling inhibitors (ARSIs) in prostate cancer is limited by both de novo and acquired resistance. Two well-described mechanisms of resistance to ARSIs are the development of constitutively activated AR splice variants (AR-Vs) or neuroendocrine prostate cancer (NEPC). Expression of NEPC markers is traditionally evaluated in solid tumor biopsies while AR-Vs can be detected in circulating tumor cells (CTCs) and have prognostic relevance in prostate cancer. Unlike solid tumor biopsy-based detection strategies, liquid biopsies can be performed periodically over the course of treatments, enabling early identification of resistance mechanisms. To evaluate for ARSI resistance, we developed a multiplex gene expression panel of AR-Vs and NEPC markers. We evaluated potential clinical utility of this assay in CTCs captured using an exclusion-based sample preparation technology. This test was evaluated with a cohort of 46 patients, showing 39% sensitivity and 94% specificity at detecting ARSI resistance. To develop a clinical-grade liquid biopsy assay with this multiplexed gene expression panel, we utilized a high-throughput automated exclusion-based sample preparation (ESP) technology for CTC capture, mRNA extraction, and gene expression analysis using RT-qPCR. Cell lines were used to evaluate the analytical performance of the methodology, and spike-in control materials were designed to monitor the efficiency of mRNA extraction for each individual patient sample. Serial dilutions of cell lines demonstrated the sensitivity to extract and quantify mRNA at the single-cell level with high precision between evaluations performed on three different days. Increasing numbers of cells resulted in the quantification of proportionally higher amounts of both AR-Vs and NEPC markers, demonstrating the analytical accuracy of the methodology (R2 values of 0.8 and above). To monitor for RNA degradation, a grape mRNA amplicon material was designed as an internal spike-in control for daily use in testing patient samples. Healthy donor PBMCs were used to confirm primers were specific for cells of cancer origin. ESP-mRNA extraction followed by RT-qPCR demonstrates single-cell resolution for the detection of AR-V/NEPC transcriptomic expression, with high precision in evaluating actual patient samples. The high analytical performance, and the promising preliminary correlation with ARSI resistance, warrant further efforts towards implementation into clinical laboratory testing environments and testing in prospective clinical trials. Citation Format: Zachery D. Schultz, Jennifer L. Schehr, Rory M. Bade, Molly M. Morgan, Madalyn S. Gill, Hannah M. Pezzi, Jaime M. Sperger, Charlotte N. Stahlfeld, Anupama Singh, Jay W. Warrick, David J. Beebe, Joshua M. Lang. Analytical validation and initial clinical utility of multianalyte transcriptomic biomarker profiling of circulating tumor cells using automated exclusion-based sample preparation technology [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B40.