RESUMO
Circulating tumor cells (CTCs) can reliably be identified in cancer patients and are associated with clinical outcome. Next-generation "liquid biopsy" technologies will expand CTC diagnostic investigation to include phenotypic characterization and single-cell molecular analysis. We describe here a rare cell analysis platform designed to comprehensively collect and identify CTCs, enable multi-parameter assessment of individual CTCs, and retrieve single cells for molecular analysis. The platform has the following four integrated components: 1) density-based separation of the CTC-containing blood fraction and sample deposition onto microscope slides; 2) automated multiparameter fluorescence staining; 3) image scanning, analysis, and review; and 4) mechanical CTC retrieval. The open platform utilizes six fluorescence channels, of which four channels are used to identify CTC and two channels are available for investigational biomarkers; a prototype assay that allows three investigational biomarker channels has been developed. Single-cell retrieval from fixed slides is compatible with whole genome amplification methods for genomic analysis. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
Assuntos
Neoplasias/patologia , Células Neoplásicas Circulantes/patologia , Biomarcadores Tumorais/genética , Contagem de Células/métodos , Linhagem Celular Tumoral , Separação Celular/métodos , Fluorescência , Humanos , Biópsia Líquida/métodos , Neoplasias/genética , Análise de Célula Única/métodosRESUMO
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. CONCLUSION: 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.
Assuntos
Separação Celular/métodos , Células Neoplásicas Circulantes , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Hibridização Genômica Comparativa , Análise Mutacional de DNA , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Masculino , Neoplasias da Próstata/patologia , Análise de Célula ÚnicaRESUMO
The RareCyte CyteFinder instrument is an automated scanner that allows rapid identification of circulating tumor cells (CTCs) on microscope slides prepared by the AccuCyte process (see Chapter 13 ) and stained by immunofluorescence. Here, we present the workflow for CyteFinder scanning, analysis, and CyteMapper scan review which includes CTC confirmation and report generation.
Assuntos
Separação Celular/métodos , Células Imobilizadas/patologia , Processamento de Imagem Assistida por Computador/métodos , Neoplasias/diagnóstico , Células Neoplásicas Circulantes/patologia , Análise de Célula Única/métodos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Automação Laboratorial/instrumentação , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Biomarcadores Tumorais/metabolismo , Contagem de Células , Linhagem Celular Tumoral , Separação Celular/instrumentação , Células Imobilizadas/imunologia , Células Imobilizadas/metabolismo , Centrifugação/instrumentação , Centrifugação/métodos , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/imunologia , Molécula de Adesão da Célula Epitelial/metabolismo , Desenho de Equipamento , Imunofluorescência/métodos , Corantes Fluorescentes/química , Humanos , Imunoconjugados/química , Queratinas/genética , Queratinas/imunologia , Queratinas/metabolismo , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/imunologia , Antígenos Comuns de Leucócito/metabolismo , Neoplasias/sangue , Neoplasias/imunologia , Neoplasias/patologia , Células Neoplásicas Circulantes/imunologia , Células Neoplásicas Circulantes/metabolismo , Ligação Proteica , Análise de Célula Única/instrumentaçãoRESUMO
The CytePicker module built into the RareCyte CyteFinder instrument allows researchers to easily retrieve individual cells from microscope slides for genomic analyses, including array CGH, targeted sequencing, and next-generation sequencing. Here, we describe the semiautomated retrieval of CTCs from the blood processed by AccuCyte (see Chapter 13) and amplification of genomic DNA so that molecular analysis can be performed.