Project Summary/Abstract The University of Wisconsin Carbone Cancer Center (UWCCC), Duke University and Memorial Sloan Kettering Cancer Center (MSKCC) seek support for establishing the analytical and clinical validity of a Circulating Tumor Cell (CTC) biomarker assay via the UH2/UH3 mechanism. This biomarker assay will evaluate a gene expression signature of treatment resistant, castration-resistant prostate cancer (CRPC). While many patients with prostate cancer benefit from Androgen Receptor Signaling Inhibitors (ARSIs), a subset of patients do not respond to this class of treatments while nearly all others develop resistance within 1-2 years. Identified mechanisms of resistance include development of Neuroendocrine Prostate Cancer (NEPC) and expression of Androgen Receptor Splicing Variants (AR-Vs). Early detection of NEPC or AR-Vs as drivers of treatment resistant prostate cancer would eliminate the need to wait for clinical manifestations of resistance, accelerating the time to administration of more suitable therapy and increasing survival. While precision medicine approaches are increasing in popularity and reliability, their ultimate capacity to improve patient care hinges on their diagnostic accuracy. Realization of a clinically relevant assay requires thorough analytical and clinical evaluation, and while many biomarker assays have successfully demonstrated analytical performance, failure to address clinical utility has left many unable to improve on existing diagnostics. By focusing our efforts on evaluation of both analytical and clinical validity, we aim to provide diagnostic accuracy in assessing an expression of NEPC and AR-Vs, building a necessary foundation for future clinical trials. To that end, we have optimized a multi-plexed gene expression assay on CTCs, that identifies these two major categories of resistance to ARSIs. This assay has shown promising initial results in a preliminary cohort of patients with aggressive CRPC. Optimization of this assay has taken into consideration the rarity of CTCs and the diversity of other blood cells in circulation; ensuring efficient RNA extraction, probe specificity, and appropriate data interpretation. The manipulation and retention of rare cells is enabled by our Exclusion-based Sample Preparation (ESP) technology, wherein centrifugation and wash steps are eliminated. This automated and commercially available platform, also called the Gilson ExtractMax, offers minimal user variability, thus maximizing precision. Our collaboration with Dr. Kaitlin Sundling at the Wisconsin State Lab of Hygiene, a CAP- approved clinical testing laboratory, provides expert oversight for planning and execution of analytical validation. In collaboration with Dr. Andrew Armstrong, Dr. Susan Halabi and Dr. Dana Rathkopf, we have assembled a team of clinical researchers and biostatisticians to rapidly validate this multi-plexed biomarker in a prospective study. This RNA-based CTC assay shows potential for iden...