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.
Highlights
Cancer metastasis involves the dissemination of primary tumor cells through the bloodstream and lymphatics
The implicit relationship between cancer metastasis and circulating tumor cells (CTC) has long been postulated [3]; the specific identity of the cells found in the circulation of cancer patients and normal healthy volunteers has been clouded by assumptions and technical limitations [4]
Epithelial derived cancers account for 80–90% of malignancies, and it has been observed that CTCs found in patients with solid epithelial tumors express epithelial markers such as the epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK)
Summary
Cancer metastasis involves the dissemination of primary tumor cells through the bloodstream and lymphatics. Rare cells have been observed, recovered and described as circulating tumor cells (CTC) [1,2]. One overlooked aspect of this processing is that each different method applies a technology-related biased definition of the CTC. These subjective definitions have consequences for the enumerative, phenotypic, or in some cases prognostic association with the disease state [11]. Limiting the scope of these new technologies is the lack of adequate solid tumor tissue samples. 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
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