Abstract 1976: Circulating tumor cell (CTC) RNA sequencing and single cell protein phenotyping reveal heterogeneity of cell surface target expression in metastatic prostate cancer CTCs

Abstract Background: Metastatic prostate cancer is an androgen receptor (AR) driven disease. While AR targeted therapies (ARPIs) have led to improvements in patient outcomes, development of treatment resistance remains universal. ARPI resistance can occur through lineage state transitions that bypass AR signaling and culminate in neuroendocrine prostate cancer (NEPC). Cell surface targeted therapies are a new therapeutic class that has shown promise for the treatment of ARPI-resistant metastatic prostate cancer. However, heterogeneity in cell surface target expression during lineage state transitions could impact the efficacy of these approaches. CTCs are uniquely suited to longitudinal profiling to understand the evolution of cell surface target expression during the lineage state transition from AR-driven adenocarcinoma to NEPC. Methods: CTCs were isolated from peripheral blood from patients with ARPI-resistant metastatic prostate adenocarcinoma utilizing our automated microfluidic platform integrating immunomagnetic negative and positive selection, followed by protein staining on chip for single CTC fluorescent quantification of prostate adenocarcinoma (PSMA) and NEPC (DLL3) cell surface target expression (n=29) or RNA isolation on chip and bulk RNA sequencing (n=146). Results: In the CTC RNA-seq cohort, 112/146 (76.7%) samples had expression of FOLH1 (PSMA) only, 5/146 (3.4%) samples had expression of DLL3 only and 9/146 (6.2%) samples had expression of both DLL3 and FOLH1. Median expression of DLL3 in the double positive group was similar to that in the DLL3 only group (4.34 v 6.67 log2(tpm+1), p = 0.076), while median expression of FOLH1 in the double positive group was similar to that of the FOLH1 only group (6.80 v 7.26 log2(tpm+1), p = 0.316). In the single CTC protein phenotyping cohort, 27/29 (93%) of samples had detectable CTCs, with a mean of 18 CTCs per patient (range 1-172). 19/29 (65.5%) samples had PSMA positive CTCs, 1/29 (3.4%) samples had DLL3 positive CTCs, and 2/29 (6.9%) samples had both PSMA positive and DLL3 positive CTCs, but no single CTCs with co-expression of both proteins were identified. Conclusion: CTC RNA sequencing identifies a subset of ARPI-resistant metastatic prostate cancers with cell surface target expression associated with small cell/neuroendocrine differentiation (DLL3), including samples with co-occurring gene expression of adenocarcinoma (PSMA) and small cell (DLL3) markers. Single CTC protein phenotyping also identified patients with simultaneous detection of both PSMA positive and DLL3 positive CTCs, however no PSMA/DLL3 double positive CTCs. The presence of mixed lineage state populations with potentially mutually exclusive cell surface target expression suggests consideration of co-targeting approaches to improve therapeutic efficacy in the ARPI-resistant setting. Citation Format: Alex H. Chang, Jamie M. Sperger, Viridiana Carreno, Katherine R. Kaufmann, Matthew L. Bootsma, Shannon R. Reese, Luke A. Nunamaker, Charlotte Linebarger, Amy K. Taylor, Katharine E. Tippins, Kyle T. Helzer, David Kosoff, Shuang G. Zhao, Joshua M. Lang, Marina N. Sharifi. Circulating tumor cell (CTC) RNA sequencing and single cell protein phenotyping reveal heterogeneity of cell surface target expression in metastatic prostate cancer CTCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1976.