| Molecular Characterization of Circulating Tumor Cells in Breast Cancer by a Liquid Bead Array Hybridization Assay. | |
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MedLine Citation:
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PMID: 21245367 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
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BACKGROUND: Molecular characterization of circulating tumor cells (CTCs) is crucial to identify novel diagnostic and therapeutic targets for individualized therapies. We developed a multiplexed PCR-coupled liquid bead array to detect the expression of multiple genes in CTCs. METHODS: mRNA isolated from immunomagnetically enriched CTCs was subjected to multiplex PCR for KRT19 (keratin 19; also known as CK19), ERBB2 [v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian); also known as HER2], SCGB2A2 (secretoglobin, family 2A, member 2; also known as MGB1, mammaglobin A), MAGEA3 (melanoma antigen family A, 3), TWIST-1 [twist homolog 1 (Drosophila)], and HMBS (hydroxymethylbilane synthase; also known as PBGD). Biotinylated amplicons were hybridized against fluorescent microspheres carrying gene-specific capture probes and incubated with streptavidin- phycoerythrin. We quantified the captured labeled amplicons and decoded the beads by Luminex flow cytometry. The assay was validated for limit of detection, specificity, and comparison with reverse-transcription quantitative PCR (RT-qPCR), and its clinical performance was evaluated in 64 patients with operable breast cancer, 20 patients with metastasis, and 17 healthy individuals. RESULTS: The assay was specific for each gene in complex multiplexed formats and could detect the expression of each gene at the level of a single SK-BR-3 cell. The assay produced results comparable to those for RT-qPCR for each gene. None of the genes tested was detected in the CTC fraction of healthy donors. We detected KRT19, ERBB2, MAGEA3, SCGB2A2, and TWIST1 in 26.6%, 12.5%, 18.7%, 10.9%, and 31.2% of operable breast cancer patients, respectively, and detected the corresponding genes in 65%, 20%, 30%, 20%, and 20% of patients with verified metastasis, respectively. CONCLUSIONS: The expression of 6 genes in CTCs can be measured simultaneously and reliably, thereby saving precious sample and reducing the costs and time of analysis. |
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Authors:
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Athina Markou; Areti Strati; Nikos Malamos; Vasilis Georgoulias; Evi S Lianidou |
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Publication Detail:
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Type: JOURNAL ARTICLE Date: 2011-1-18 |
Journal Detail:
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Title: Clinical chemistry Volume: - ISSN: 1530-8561 ISO Abbreviation: - Publication Date: 2011 Jan |
Date Detail:
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Created Date: 2011-1-19 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 9421549 Medline TA: Clin Chem Country: - |
Other Details:
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Languages: ENG Pagination: - Citation Subset: - |
Affiliation:
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Laboratory of Analytical Chemistry, University of Athens, Athens, Greece; |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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