High-recovery visual identification and single-cell retrieval of circulating tumor cells for genomic analysis using a dual-technology platform integrated with automated immunofluorescence staining

BMC Cancer

Technical Advance

C Anthony Blau¹  Michael Dorschner²  Daniel E Sabath³  Alisa C Clein³  Amy Breman⁴  Sibel Blau⁵  Jackie L Stilwell⁶  Joshua J Nordberg⁶  Eric P Kaldjian⁶  Steve Quarre⁶  Daniel E Campton⁶  Paulina Varshavskaya⁶  Arturo B Ramirez⁶  Barry H Friemel⁶  Nick Drovetto⁶ 

[1] Center for Cancer Innovation, University of Washington, Washington, USA;Department of Pathology, University of Washington, Washington, USA;Departments of Laboratory Medicine and Medicine, University of Washington, Washington, USA;Medical Genetics Laboratories, Baylor College of Medicine, Houston, USA;Rainier Hematology-Oncology, Northwest Medical Specialties, Washington, USA;RareCyte, Inc, Seattle, WA, USA;

关键词: Circulate Tumor Cell;    Buffy Coat;    Glyph;    SKBR3 Cell;    Whole Genome Amplification;   

DOI  :  10.1186/s12885-015-1383-x

 received in 2014-10-01, accepted in 2015-04-28,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundCirculating tumor cells (CTCs) are malignant cells that have migrated from solid cancers into the blood, where they are typically present in rare numbers. There is great interest in using CTCs to monitor response to therapies, to identify clinically actionable biomarkers, and to provide a non-invasive window on the molecular state of a tumor. Here we characterize the performance of the AccuCyte® – CyteFinder® system, a comprehensive, reproducible and highly sensitive platform for collecting, identifying and retrieving individual CTCs from microscopic slides for molecular analysis after automated immunofluorescence staining for epithelial markers.MethodsAll experiments employed a density-based cell separation apparatus (AccuCyte) to separate nucleated cells from the blood and transfer them to microscopic slides. After staining, the slides were imaged using a digital scanning microscope (CyteFinder). Precisely counted model CTCs (mCTCs) from four cancer cell lines were spiked into whole blood to determine recovery rates. Individual mCTCs were removed from slides using a single-cell retrieval device (CytePicker™) for whole genome amplification and subsequent analysis by PCR and Sanger sequencing, whole exome sequencing, or array-based comparative genomic hybridization. Clinical CTCs were evaluated in blood samples from patients with different cancers in comparison with the CellSearch® system.ResultsAccuCyte – CyteFinder presented high-resolution images that allowed identification of mCTCs by morphologic and phenotypic features. Spike-in mCTC recoveries were between 90 and 91%. More than 80% of single-digit spike-in mCTCs were identified and even a single cell in 7.5 mL could be found. Analysis of single SKBR3 mCTCs identified presence of a known TP53 mutation by both PCR and whole exome sequencing, and confirmed the reported karyotype of this cell line. Patient sample CTC counts matched or exceeded CellSearch CTC counts in a small feasibility cohort.ConclusionThe AccuCyte – CyteFinder system is a comprehensive and sensitive platform for identification and characterization of CTCs that has been applied to the assessment of CTCs in cancer patient samples as well as the isolation of single cells for genomic analysis. It thus enables accurate non-invasive monitoring of CTCs and evolving cancer biology for personalized, molecularly-guided cancer treatment.

【 授权许可】

Unknown   
© Campton et al.; licensee BioMed Central. 2015. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.