Abstract B066: Analytical validation of a multiplexed CTC-mRNA assay for androgen receptor pathway inhibitors
Authors: Jennifer L Schehr, Rory M Bade, Anupama Singh, Jacob C Caceres, Nasya A Ramli, Matthew C Mannino, Diego Eyzaguirre, Alyssa M Hintz, Manushi N Vatani, Serena K Wolfe, Kayo Miyazaki, Elizabeth Haas, Joshua Faulkes, Dana E Rathkopf, Susan Halabi, Andrew J Armstrong, Shuang G Zhao, Joshua M Lang
Journal: Clinical Cancer Research (2024)

Abstract Introduction: Androgen receptor pathway inhibitors (ARPIs) have been shown to improve survival for men with metastatic prostate cancer, however, the use of 2nd line ARPIs has limited efficacy. There is a critical need to develop biomarker-driven treatment strategies for this clinical need. We have previously reported on a novel circulating tumor cell (CTC) multiplexed mRNA biomarker that has prognostic and predictive capability in clinical utility trials to detect AR- driven resistance and Neuroendocrine Prostate Cancer (NEPC). However, before these biomarkers can be tested in prospective clinical validation trials and clinical care, they must undergo thorough analytical validation. We report a novel approach to the analytical validation of rare cell assays for gene expression analysis and the testing of this CTC mRNA assay. Methods: Analytical validation for assays with single-cell level detection is particularly challenging in that results may be categorical as well as quantitative: sometimes measured at higher or lower levels, and other times resulting in no detectable signal, precluding the use of standard measures of precision. The inter-metastatic cellular heterogeneity composition of a liquid biopsy further complicates efforts to demonstrate clinical-grade precision at the single-cell level. To address this, we applied logistic regression to replicates of patient samples, cell lines and unique DNA and mRNA oligonucleotides to define the minimum amount of analyte to achieve high precision for the underlying PCR and exclusion-based sample preparation (ESP) mRNA extraction technologies. Results: We demonstrated clinical-grade precision at the single-cell level, comparable to the sensitivity previously demonstrated for qPCR, and analyte quantities of 15 total transcripts. Equivalent analyte yield was obtained from the extraction of single-cell quantities of cell line cells with or without a maximum of 10,000 background cells, demonstrating robustness to a range of sample purities. Extractions from 10 healthy donors demonstrate analytical specificity of the assay, with a synthetic mRNA spike-in control used to confirm successful extractions. Replicates of cell line extractions at different concentrations were compared to 44 replicate patient samples to delineate technical precision from biological heterogeneity. Discussion: The automated CTC-mRNA extraction technology demonstrates high precision equivalent to PCR with single-cell sensitivity. We can further reproducibly detect as low as 15 transcripts. We have achieved a semi-qualitative assay read-out that maximizes the analytical sensitivity of the assay, allowing the clinical reporting of multiplexed results for clinical utility in prostate cancer. This validation approach also serves as a model for clinical implementation of other single-cell liquid biopsy assays. Currently, this assay is being prospectively evaluated in the ARCTIC trial for patients with prostate cancer treated with an ARPI (NCT06141993). Citation Format: Jennifer L Schehr, Rory M Bade, Anupama Singh, Jacob C Caceres, Nasya A Ramli, Matthew C Mannino, Diego Eyzaguirre, Alyssa M Hintz, Manushi N Vatani, Serena K Wolfe, Kayo Miyazaki, Elizabeth Haas, Joshua Faulkes, Dana E Rathkopf, Susan Halabi, Andrew J Armstrong, Shuang G Zhao, Joshua M Lang. Analytical validation of a multiplexed CTC-mRNA assay for androgen receptor pathway inhibitors [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B066.