Expression of prostate-specific membrane antigen (PSMA) on circulating tumor cells (CTCs) in castration-resistant prostate cancer.

Abstract

266 Background: Prostate-specific membrane antigen (PSMA) is expressed ubiquitously in prostate adenocarcinoma, and the intensity of expression increases with disease aggressiveness. Exploiting PSMA for therapy could potentially be aided by a minimally invasive means for measuring PSMA on tumor cells. Here we describe the development and characteristics of a novel assay for quantitating PSMA on canonical and non-canonical circulating tumor cells (CTCs). Methods: The PSMA CTC test was developed using LNCaP (high PSMA), 22Rv1 (low PSMA) and PC3 (no PSMA) cells spiked into normal blood. Nucleated blood cells were plated onto glass slides and subjected to immunofluorescent staining followed by CTC identification using the Pyxis Scanning Platform at Epic Sciences. The four-color assay evaluated PSMA expression on individual CTCs, identified as cells which are cytokeratin+, CD45-, and with an intact DAPI nucleus. Multiple antibody clones and assay conditions were evaluated, and the final PSMA CTC test has high specificity and sensitivity. Acceptance criteria included signal intensities in LNCaP versus PC3, subcellular localization of PSMA, and potential interference by a PSMA-targeted therapy. Clinical feasibility of the optimized assay was assessed on samples from CRPC patients. Results: The average PSMA signal intensity for LNCaP was 20-fold higher than that for PC3 and 10-fold higher than the minimum cutoff. PSMA displayed a predominantly membrane-localized pattern of staining that was distinct from cytokeratin. Assay performance was unaffected by the presence of PSMA ADC, a PSMA-targeted antibody-drug conjugate that is in phase II clinical testing. In feasibility tests on patient samples, the assay demonstrated utility in detecting and quantitating PSMA on individual and clustered CTCs as well as on apoptotic, cytokeratin-negative, and small cytokeratin-positive CTC candidates. Conclusions: PSMA expression was successfully detected and quantitated on diverse types of circulating cells present in the blood of patients with CRPC. Assay performance was unaffected by the presence of a PSMA-targeted therapeutic agent. PSMA CTC data are being collected in the ongoing phase II study of PSMA ADC for comparison with treatment outcomes.