Abstract 6562: Comprehensive liquid biopsy: Multi-analyte evaluation of cellular and plasma components of blood in prostate cancer from a single blood collection tube
Authors: Ladd Jon , Bayer Erin , Bartels Brock , Tischner Arista , Ponting Rachel , Kaldjian Eric , Ramirez Arturo 
Published: March 2024

Abstract
Background Liquid biopsy samples offer a non-invasive method for investigating drug targets and biomarkers in solid tumors. In this study, we describe a comprehensive liquid biopsy analysis of whole blood to identify circulating tumor cells (CTCs), large extracellular vesicles and CTC fragments, and CTC-white blood cell hybrids; quantify protein biomarker expression on CTCs; perform targeted mutation profiling on CTCs and cell-free DNA (cfDNA) from plasma; and analyze protein expression on white blood cells (WBCs) from a single 7.5 mL blood sample. This array of tests was applied to blood samples from patients with Stage IV prostate cancer to demonstrate clinical feasibility.
Methods A novel dual biomarker CTC assay was developed to measure protein expression of PSMA and ARv7 on CTCs from stage IV prostate cancer patients. Nucleated cells from clinical samples were processed to microscope slides using AccuCyte®, an unbiased, density-based separation method that also allows for plasma collection for downstream analysis. Slides were fixed and stained with antibodies to cytokeratin, EpCAM, and CD45 to detect CTCs and exclude WBCs, and with PSMA and ARv7 to evaluate protein biomarker expression on identified CTCs. The slides were imaged with the CyteFinder® automated immunofluorescence scanning microscope that identifies CTCs for confirmation by trained reviewers and quantitatively measures protein mean fluorescence intensity (MFI) on any cells detected on the slides (CTCs, CTC-WBC hybrids, large extracellular vesicles and CTC fragments and WBCs). Individual CTCs and control pools of WBCs from clinical samples with at least 4 CTCs were picked using CytePicker®, a needle-based system used to isolate single cells. cfDNA was isolated from plasma removed during the AccuCyte® process using a QIAamp MinElute ccfDNA kit. Picked CTCs, WBC pools, and isolated ctDNA were sequenced using the OncoZoom Cancer HotSpot panel from Paragon Genomics.
Results Nine progressing Stage IV prostate cancer patient blood samples were stained with a novel PSMA/ARv7 dual biomarker assay. CTCs were identified in all clinical samples (median 4, range 1 - 384). PSMA positivity in clinical samples ranged from 0% to 75%, and ARv7 positivity ranged from 33% to 100%, and. Additional analysis using QuPath was performed to determine PSMA/ARv7 protein expression on WBCs and CTC fragments. ctDNA analysis from 5 individual samples was compared to sequencing results from individual CTCs from the same samples.
Conclusions Using a single 7.5 mL blood liquid biopsy, we can measure protein expression on enumerated CTCs and quantify these same protein biomarkers on WBCs and CTC fragments/oncosomes, determine the genetic makeup of the tumor through the sequencing of cfDNA, and monitor genetic mutations in intact CTCs in circulation.
Citation Format: Jon Ladd, Erin Bayer, Brock Bartels, Arista Tischner, Rachel Ponting, Eric Kaldjian, Arturo Ramirez. Comprehensive liquid biopsy: Multi-analyte evaluation of cellular and plasma components of blood in prostate cancer from a single blood collection tube [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6562.