Comparison of circulating tumour cell extraction methods for men with metastatic prostate cancer

Introduction & Objectives: Enumeration of circulating tumour cells (CTCs) has been found to be reflective of tumour burden, biologic activity and prognostic for clinical outcome in a variety of cancers including prostate, breast and head and neck. CTC numbers have been directly associated with poor outcome, which can be then used to assess treatment response. The technology is evolving such that the focus is now beyond enumeration, to allow isolation of cells for further investigations such as phenotypic characterisation and RNA extraction for RT-PCR, microarray and next generation sequencing. In this project, CTC technologies have been compared to determine the ability of individual technologies to enrich CTCs for, propagation, characterisation and further analysis. The three technologies compared include CellSearch™ (EPCAM positive selection), ClearCell FX™ (microfluidic spiral selection) and RosetteSep™ (CD45 negative depletion). Methods: Whole blood samples were obtained from patients during screening for Androgen Deprivation Therapy and Adjuvant Metformin (ADMET) Trial (n = 59) after informed consent. Samples were collected in CellSave™ tubes (Janssen Diagnostics), Lithium Heparin or EDTA tubes at room temperature and processed within 4 hours of collection. Enriched cells on cytospin slides or cultured cells were assessed by immunofluorescent characterisation using DAPI and CellSearch® directly conjugated antibodies (Veridex, Warren, NJ, USA) for, pan-CK and CD45. In patients with known CTCs, CellSearch™, ClearCell FX™ and RosetteSep™ were used for comparison of enrichment performance. Cells extracted from CellSearch™ and ClearCell FX™ were used for temporary propagation in a novel in vitro culture media. Results: In 3 patient samples, CellSearch™ enrichment was compared directly with RosetteSep™ negative selection technology. In all three samples, RosetteSep™ yielded more cells (average 442 vs 78 cells). RosetteSep™ yields viable cells allowing downstream