Abstract 600: Liquid biopsy for neuroendocrine differentiation: Validation of a circulating tumor cell assay for synaptophysin

Abstract

Protein expression of synaptophysin (SYP) is characteristic of neuroendocrine subtype tumors. In prostate cancer, neuroendocrine differentiation is correlated with disease progression, poor prognosis, and treatment resistance. Analysis of circulating tumor cells (CTCs) by multiparameter immunofluorescence (IF) microscopy allows non-invasive characterization of cancer cell biomarker expression in real time. This information can be helpful in prognosis, treatment selection, and patient stratification. Here we describe the validation of a biomarker IF assay for enumeration of CTCs and characterization of their SYP expression. The 0920-VB SYP CTC assay workflow includes processing blood samples to slides (AccuCyte® Sample Preparation System), staining slides with a panel of fluorescent markers (RarePlex® Staining Kit and Ventana® DISCOVERY® ULTRA immunostaining system), and multiparameter imaging and analysis (CyteFinder® Instrument). The panel consists of a nuclear dye, and antibodies against cytokeratins and EpCAM (to identify epithelial CTCs), CD45 (to exclude white blood cells) and SYP. Both clinical and spike-in samples were used for assay validation. The model CTCs used to generate spike-in samples included 22Rv1 (prostate carcinoma, SYP positive) and BT-474 (breast carcinoma, SYP negative). Performance metrics for the assay included accuracy, sensitivity, specificity, repeatability, and intermediate precision of SYP detection and CTC enumeration. Single-cell SYP mean fluorescence intensities (MFI) were analyzed to determine protein expression levels. An MFI threshold for SYP positivity was established by maximizing the classification accuracy of the positive and negative cell lines. Using this threshold, 98.7% of 22Rv1 cells were correctly classified as SYP-positive, and 99.7% of BT474 cells were correctly classified as SYP-negative with an inter-run coefficient of variation of 13.9% for the 22Rv1 cell line. In clinical prostate, breast and colon cancer patient samples, subsets of CTCs were found to be SYP positive with the expected cytoplasmic localization of the marker. In summary, this liquid biopsy assay provides an analytically sensitive and specific method for CTC enumeration and SYP biomarker expression analysis, allowing non-invasive detection of neuroendocrine differentiation. Citation Format: Jennifer Chow, A Anders Tevis, Edward Lo, Alisa Clein, Daniel E. Sabath, Arturo B. Ramirez, Eric P. Kaldjian, Tad George. Liquid biopsy for neuroendocrine differentiation: Validation of a circulating tumor cell assay for synaptophysin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 600.