title: 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.
abstract: BACKGROUND
  Circulating 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.
  
  
  METHODS
  All 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.
  
  
  RESULTS
  AccuCyte--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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: RareCyte, Inc, Seattle, WA, USA. dcampton@rarecyte.com.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Campton
authors.affiliation: RareCyte, Inc, Seattle, WA, USA. aramirez@rarecyte.com.
authors.forename: Arturo B
authors.initials: AB
authors.lastname: Ramirez
authors.affiliation: RareCyte, Inc, Seattle, WA, USA. jnordberg@rarecyte.com.
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Nordberg
authors.affiliation: RareCyte, Inc, Seattle, WA, USA. ndrovetto@rarecyte.com.
authors.forename: Nick
authors.initials: N
authors.lastname: Drovetto
authors.affiliation: Departments of Laboratory Medicine and Medicine, University of Washington, Washington, USA. aclein@uw.edu.
authors.forename: Alisa C
authors.initials: AC
authors.lastname: Clein
authors.affiliation: RareCyte, Inc, Seattle, WA, USA. pvarshavskaya@rarecyte.com.
authors.forename: Paulina
authors.initials: P
authors.lastname: Varshavskaya
authors.affiliation: RareCyte, Inc, Seattle, WA, USA. bfriemel@rarecyte.com.
authors.forename: Barry H
authors.initials: BH
authors.lastname: Friemel
authors.affiliation: RareCyte, Inc, Seattle, WA, USA. squarre@rarecyte.com.
authors.forename: Steve
authors.initials: S
authors.lastname: Quarre
authors.affiliation: Medical Genetics Laboratories, Baylor College of Medicine, Houston, USA. breman@bcm.edu.
authors.forename: Amy
authors.initials: A
authors.lastname: Breman
authors.affiliation: Department of Pathology, University of Washington, Washington, USA. mod@uw.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Dorschner
authors.affiliation: Rainier Hematology-Oncology, Northwest Medical Specialties, Washington, USA. sibelblau@msn.com.
authors.forename: Sibel
authors.initials: S
authors.lastname: Blau
authors.affiliation: Center for Cancer Innovation, University of Washington, Washington, USA. tblau@uw.edu.
authors.forename: C Anthony
authors.initials: CA
authors.lastname: Blau
authors.affiliation: Departments of Laboratory Medicine and Medicine, University of Washington, Washington, USA. dsabath@u.washington.edu.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Sabath
authors.affiliation: RareCyte, Inc, Seattle, WA, USA. jstilwell@rarecyte.com.
authors.forename: Jackie L
authors.initials: JL
authors.lastname: Stilwell
authors.affiliation: RareCyte, Inc, Seattle, WA, USA. ekaldjian@rarecyte.com.
authors.forename: Eric P
authors.initials: EP
authors.lastname: Kaldjian
mesh_terms: D001943:Breast Neoplasms / Q000473:pathology; D045744:Cell Line, Tumor; D002469:Cell Separation / Q000379:methods*; D015179:Colorectal Neoplasms / Q000473:pathology; D055028:Comparative Genomic Hybridization; D004252:DNA Mutational Analysis; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008297:Male; D009360:Neoplastic Cells, Circulating; D011471:Prostatic Neoplasms / Q000473:pathology; D059010:Single-Cell Analysis