Transcriptomic profiling of metastatic cancer can illuminate mechanisms of progression and lead to new therapies, but standard biopsy is invasive and reflects only a single metastatic site. In contrast, circulating tumor cell (CTC) profiling is noninvasive and repeatable, reflecting the dynamic and systemic nature of advanced disease. To date, transcriptomic profiling of CTCs has not delivered on its full potential, because white blood cells (WBCs) vastly outnumber CTCs. Current profiling strategies either lack cancer sensitivity and specificity or require specialized CTC capture protocols that are not readily scalable to large patient cohorts. Here, we describe a new strategy for rapid CTC enrichment and transcriptomic profiling using commercially available WBC depletion, microfluidic enrichment and RNA sequencing. When applied to blood samples from patients with advanced prostate cancer (PC), transcriptomes from enriched samples cluster with cancer positive controls and previously undetectable prostate‐specific transcripts become readily measurable. Gene set enrichment analysis reveals multiple significantly enriched signaling pathways associated with PC, as well as novel pathways that merit further study. This accessible and scalable approach yields cancer‐specific transcriptomic data and can be applied repeatedly and noninvasively in large cancer patient cohorts to discover new therapeutic targets in advanced disease.

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.

e16587 Background: CTCs have the potential to reflect not only genomic alterations but also cancer-relevant transcriptomic phenotypes. However, CTC gene expression has been hampered by signal-to-noise: rare CTC-derived transcripts are drowned out by abundant leukocyte-derived RNA. To date, a few specialized labs have achieved CTC RNAseq by capturing and analyzing single cells, a laborious and expensive approach not suitable for routine analysis of numerous samples. To address this need, we developed and validated a simple, rapid method for enrichment of live CTCs for RNAseq. Methods: Blood was drawn with informed consent under an IRB-approved protocol. Prostate cancer cell line spike-in samples were used to optimize live CTC enrichment by sequential leukocyte depletion (RosetteSep, Stem Cell Technologies) and size-based enrichment (Parsortix, Angle). Cancer-specific gene expression was first measured by multiplexed prostate specific qRT-PCR and subsequently by whole transcriptome amplification (WTA, SMARTer V2, Clontech) and RNAseq. Four patient samples were similarly analyzed by enrichment and RNAseq, along with spike-in positive controls and matched unenriched buffy coat negative controls. Results: Processing “from patient to RNA” took < 3 hrs. and achieved mean CTC recovery of 30% (range 28-33%) and mean leukocyte background of 100 (range 47-179), a 100,000-fold enrichment. Prostate specific genes (AR, PSA, PSMA) were consistently detected by qRT-PCR from enriched samples but not from unenriched samples. When analyzed by RNAseq, patient samples clustered with spike-in positive controls and away from matched buffy coat controls by principle component analysis and by unsupervised hierarchical clustering. Differential gene expression (enriched vs. matched buffy coat) identified prostate cancer-relevant transcripts. Conclusions: We developed a simple and efficient method for live CTC enrichment and expression profiling, applicable to large numbers of patient samples. This approach can be used serially over time to detect known cancer-specific transcripts and to discover new gene expression signatures that reflect tumor biology and inform disease management.