Novel method for rapid enrichment of high purity circulating tumor cells (CTCs) for prostate cancer (PCa) gene expression profiling.

271 Background: CTC RNA analysis currently involves single cell recovery that is laborious and expensive, or alternatively lysis of preserved whole blood which yields RNA predominantly from leukocytes which vastly outnumber CTCs. To effectively characterize gene expression in large patient cohorts, new enrichment methodologies are needed that yield high purity CTC populations while preserving RNA integrity. Here we describe a simple yet robust method for enrichment of prostate CTCs for gene expression analysis. Methods: Blood was drawn with informed consent under an IRB-approved protocol. For initial optimization, CFSE-stained PCa cells were spiked into healthy blood and recovered using various combinations of 2 methods: microfluidic enrichment (Parsortix™ system) and CD45 depletion. For assay qualification, a prostate-specific multiplexed qRT-PCR gene expression panel was developed. Enrichment and gene expression were tested initially using PCa cell lines spiked into healthy blood, then metastatic castrate resistant prostate cancer (mCRPC) blood samples in parallel with CellSearch enumeration. Results: Optimal enrichment of live cells was achieved with CD45 depletion followed by microfluidic enrichment, resulting in an average spiked cell recovery of 30% and approximately 100 contaminating background leukocytes. Using this enrichment method, prostate specific genes were detectable by multiplexed qRT-PCR down to 25 cells spiked into 7.5 ml whole blood, and transcripts were not measurable in matched healthy blood controls. When applied to mCRPC patient blood containing CTCs by CellSearch, multiplexed qRT-PCR successfully detected prostate specific genes in all samples. Conclusions: We developed a novel enrichment method capable of rapidly and efficiently recovering live CTCs with high purity, free of magnetic beads and with very few background leukocytes. Captured cells yielded high-quality RNA with high sensitivity and specificity for prostate-specific transcripts. This approach is applicable to high throughput gene expression profiling assays and offers an alternative to laborious single cell recovery or non-cancer-specific whole blood fixation.

Biological significance of novel populations of prostate cancer circulating tumor cells (CTC).

315 Background: Decisions regarding selection and sequencing of treatment in advanced prostate cancer (PC) are often made empirically, without information on the PC molecular profile at the time of treatment. CTC provide real time information regarding contemporaneous biologically relevant cancer subtypes. Current assays often rely on EpCAM or other epithelial markers and may underestimate or exclude other CTC populations of clinical relevance. We devised a novel, simple, rapid and cheap method for isolation of CTC subsets including those without epithelial markers. Methods: Five mL of blood was processed as follows: CD45+ depletion (RosetteSep Human CD45 Depletion Cocktail, Stemcell Technologies); Ficoll centrifugation; antibody staining (mix of EpCAM-PE, PSMA-APC, N-cadherin-AF488, E-cadherin-PerCP/Cy5.5, CD45-APC-H7); Sytox Blue live/dead cell stain; followed by sorting (Becton Dickinson FACSAria Fusion cell sorter). Live cells were sorted into 4 categories denoted E, E+P, P, N (Table). N and P are not identifiable using existing conventional CTC techniques. CTC were assayed for prostate markers ( AR, FOLH1 (PSMA), KLK3 (PSA), TMPRSS2, AMACR) and malignant markers ( PTEN copy number) using digital droplet PCR. Results: Preliminary data are derived from 62 patient samples providing 47 sorted populations. Details are in the Table. Spiking experiments reliably identified as few as 10 CTC / 7.5 mL of blood. P cells were detected in all patients, often outnumbering sum of E and E+P; E and N were never co-expressed. P and N samples frequently expressed prostate markers. Only 1/27 showed no expression of any of these five prostate markers. PTEN deletion assays are in progress. Conclusions: “Unconventional” EpCAMneg CTC appear to be derived from PC and can be detected using broader cell selection criteria. These CTC may provide information on treatment resistance or escape mechanisms. [Table: see text]