Abstract B073: Circulating Tumor Cell (CTC) single cell protein quantification reveals inter-patient heterogeneity in PSMA expression associated with LuPSMA response in a Metastatic Castration Resistant Prostate Cancer (mCRPC) patient cohort

Abstract Background: While treatment options for castration-resistant prostate cancer (mCRPC) patients have historically been limited, theranostic approaches targeting prostate specific membrane antigen (PSMA) have shown promise, including the FDA approval of 177Lu-PSMA radioligand therapy in mCRPC patients with PSMA positive disease on positron emission tomography scan (PET). While LuPSMA improves clinical outcomes in mCRPC, intrinsic and acquired resistance remain common, the mechanisms of which remain poorly understood. Crosstalk between the PI3K pathway and AR signaling plays a role in resistance to androgen therapies, and may also modulate PSMA expression. We hypothesized that heterogeneity in PSMA expression driven by crosstalk with PI3K pathway signaling could contribute to LuPSMA resistance. Here, we utilized in vitro models as well as circulating tumor cell (CTC) molecular profiling from mCRPC patients receiving LuPSMA to evaluate the role of PSMA protein-expression heterogeneity and PSMA-PI3K signaling crosstalk in LuPSMA resistance. Methods: 77 CTC samples were collected from 35 patients with mCRPC receiving standard of care 177Lu-PSMA. CTCs were isolated using microfluidic chip based immunomagnetic capture and protein staining followed by fluorescent imaging. CTC enumeration and single cell fluorescent quantification of PSMA and phospho-ribosomal protein S6 (S6) quantification was compared between patients with early progression (EP) on LuPSMA (within the first 3 cycles/18 weeks) versus those without EP. A mCRPC cell line, 22RV1, was exposed to PI3K pathway inhibitors, before Western blot analysis of PSMA and PI3K expression. Results: 23/35 patients had CTCs detected at baseline (median: 5, range: 1-17), with CTC PSMA fluorescent intensities (PSMA-FI) ranging from less than 100 to greater than 5000 (median: 158. 93 range: 69. 37-5449. 44). There was a significantly increased proportion of EP among patients with low CTC PSMA expression (3/3 with EP) versus high CTC PSMA expression (2/13 with EP) detected at baseline (p=0. 018). CTCs from patients with EP exhibited a significant increase in phospho-rpS6 expression from baseline to cycle 2 compared to responders (59. 11 vs -124. 87, n=10, p=0. 017). There was no difference in baseline CTC number (7 vs 5 CTCs/7. 5ml, p=0. 750) between patients with and without EP. Among PSMA-FI high patients, CTC PSMA heterogeneity (PSMA-FI median absolute deviation 0. 606 vs 0. 843, p=0. 769) did not significantly differ by EP status. In 22RV1 cells, treatment with PI3K pathway inhibitors Everolimus (mean fold change = 1. 94, n=3, p = 0. 002) and Gedatolisib (mean fold change = 2. 78, n =3, p= 0. 046), but not Ipatasertib, resulted in a significant increase in PSMA expression. Conclusions: CTC PSMA expression is variable in mCRPC patients with PSMA-PET-positive disease, low CTC PSMA expression correlates with non-response to LuPSMA. CTCs from patients with EP have increased PI3K pathway signaling compared to responders. Crosstalk between PSMA and the PI3K signaling pathway impacts PSMA expression in vitro. Citation Format: William M. Stump, Alex H. Chang, Viridiana Carreno, Matthew L. Bootsma, Jamie M. Sperger, David Gallo, Shannon R. Reese, Emily Abella, Kaitlin Durnen, Kristin Rosche, Muhammad Dar, Charlotte Linebarger, Amy K. Taylor, Kendra Marr, Katharine E. Tippins, Kyle T. Helzer, Grace C. Blitzer, John Floberg, David Kosoff, Rana R. McKay, Xiao Wei, Shuang G. Zhao, Joshua M. Lang, Marina N. Sharifi. Circulating Tumor Cell (CTC) single cell protein quantification reveals inter-patient heterogeneity in PSMA expression associated with LuPSMA response in a Metastatic Castration Resistant Prostate Cancer (mCRPC) patient cohort abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (2Suppl): Abstract nr B073.