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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
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...
Autores principales: | , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4430903/ https://www.ncbi.nlm.nih.gov/pubmed/25944336 http://dx.doi.org/10.1186/s12885-015-1383-x |
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author | Campton, Daniel E Ramirez, Arturo B Nordberg, Joshua J Drovetto, Nick Clein, Alisa C Varshavskaya, Paulina Friemel, Barry H Quarre, Steve Breman, Amy Dorschner, Michael Blau, Sibel Blau, C Anthony Sabath, Daniel E Stilwell, Jackie L Kaldjian, Eric P |
author_facet | Campton, Daniel E Ramirez, Arturo B Nordberg, Joshua J Drovetto, Nick Clein, Alisa C Varshavskaya, Paulina Friemel, Barry H Quarre, Steve Breman, Amy Dorschner, Michael Blau, Sibel Blau, C Anthony Sabath, Daniel E Stilwell, Jackie L Kaldjian, Eric P |
author_sort | Campton, Daniel E |
collection | PubMed |
description | 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12885-015-1383-x) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-4430903 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-44309032015-05-15 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 Campton, Daniel E Ramirez, Arturo B Nordberg, Joshua J Drovetto, Nick Clein, Alisa C Varshavskaya, Paulina Friemel, Barry H Quarre, Steve Breman, Amy Dorschner, Michael Blau, Sibel Blau, C Anthony Sabath, Daniel E Stilwell, Jackie L Kaldjian, Eric P BMC Cancer Technical Advance 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12885-015-1383-x) contains supplementary material, which is available to authorized users. BioMed Central 2015-05-06 /pmc/articles/PMC4430903/ /pubmed/25944336 http://dx.doi.org/10.1186/s12885-015-1383-x Text en © 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. |
spellingShingle | Technical Advance Campton, Daniel E Ramirez, Arturo B Nordberg, Joshua J Drovetto, Nick Clein, Alisa C Varshavskaya, Paulina Friemel, Barry H Quarre, Steve Breman, Amy Dorschner, Michael Blau, Sibel Blau, C Anthony Sabath, Daniel E Stilwell, Jackie L Kaldjian, Eric P 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 |
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 |
title_full | 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 |
title_fullStr | 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 |
title_full_unstemmed | 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 |
title_short | 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 |
title_sort | 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 |
topic | Technical Advance |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4430903/ https://www.ncbi.nlm.nih.gov/pubmed/25944336 http://dx.doi.org/10.1186/s12885-015-1383-x |
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