Circulating Tumor Cells: Clinically Relevant Molecular Access Based on a Novel CTC Flow Cell

Abstract

BackgroundContemporary cancer diagnostics are becoming increasing reliant upon sophisticated new molecular methods for analyzing genetic information. Limiting the scope of these new technologies is the lack of adequate solid tumor tissue samples. Patients may present with tumors that are not accessible to biopsy or adequate for longitudinal monitoring. One attractive alternate source is cancer cells in the peripheral blood. These rare circulating tumor cells (CTC) require enrichment and isolation before molecular analysis can be performed. Current CTC platforms lack either the throughput or reliability to use in a clinical setting or they provide CTC samples at purities that restrict molecular access by limiting the molecular tools available.Methodology/Principal FindingsRecent advances in magetophoresis and microfluidics have been employed to produce an automated platform called LiquidBiopsy®. This platform uses high throughput sheath flow microfluidics for the positive selection of CTC populations. Furthermore the platform quantitatively isolates cells useful for molecular methods such as detection of mutations. CTC recovery was characterized and validated with an accuracy (<20% error) and a precision (CV<25%) down to at least 9 CTC/ml. Using anti-EpCAM antibodies as the capture agent, the platform recovers 78% of MCF7 cells within the linear range. Non specific recovery of background cells is independent of target cell density and averages 55 cells/mL. 10% purity can be achieved with as low as 6 CTCs/mL and better than 1% purity can be achieved with 1 CTC/mL.Conclusions/SignificanceThe LiquidBiopsy platform is an automated validated platform that provides high throughput molecular access to the CTC population. It can be validated and integrated into the lab flow enabling CTC enumeration as well as recovery of consistently high purity samples for molecular analysis such as quantitative PCR and Next Generation Sequencing. This tool opens the way for clinically relevant genetic profiling of CTCs.

Circulating tumor cell technologies

“…Many affinity-based enrichment technologies use epithelial markers, which are down-regulated during EMT, to capture CTCs. In response, researchers have expanded the antigen repertoire used in affinity-based capture to include stem cell markers (e.g., CD133), mesenchymal markers (e.g., cellsurface vimentin, CSV) (Satelli et al, 2014), and cancerspecific antigens (e.g., HER2, PSMA) (Galletti et al, 2014;Kirby et al, 2012;Winer-Jones et al, 2014 Another commercially available platform for CTC detection is AdnaTest (Adnagen AG). AdnaTest enriches CTCs through the use of magnetic, antibody-coated beads.…”

“…LiquidBiopsy uses a multilayer sheath flow to minimize nonspecific binding to magnetized surfaces (Winer-Jones et al, 2014). The fixed sample, labeled with magnetic nanoparticles, enters the microfluidic device through a central channel between two density-adjusted buffer streams.…”

“…Although not originally developed for CTC isolation, researchers have used Ficoll-Paque Ò in this application. By using a reverse transcription-PCR (RT-PCR) assay to amplify cytokeratin 20 transcripts, Weitz et al detected CTCs in 41% of patients undergoing colorectal resections with a sensitivity of 10 CTCs per 10 ml blood (Weitz et al, 1998). Designed for CTC isolation, OncoQuick Ò (Grenier BioOne, Frickenhausen, Germany) has combined density-based gradient centrifugation and filtration by integrating a porous barrier into the system above the separation media, which captures CTCs while allowing erythrocytes and some leukocytes to pass through.…”

Opportunities and Challenges for Pancreatic Circulating Tumor Cells

“…Researchers have recently described an integrated magnetic CTC isolation system that combines inertial microfluidics and on-chip CTC labeling with antibodies and magnets to select for CTCs, while depleting most leukocytes from the sample. 39 Several other integrated microfluidic platforms for CTC isolation have also been reported, some of which allow for onchip automated detection of CTCs [40][41][42] .…”

The Next Generation of Antibody Drug Conjugates

“…In one recent study, a flow cytometrybased detection of the rare CTCs in the peripheral blood was validated, providing a proof of concept for the future use of such assays in a clinical setting. 99 …”