Molecular characterization of circulating tumor cells in breast cancer by a liquid bead array hybridization assay

Clin Chem. 2011 Mar;57(3):421-30. doi: 10.1373/clinchem.2010.154328. Epub 2011 Jan 18.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers, Tumor / blood
  • Biomarkers, Tumor / metabolism*
  • Breast Neoplasms / blood
  • Breast Neoplasms / diagnosis*
  • Breast Neoplasms / genetics
  • Breast Neoplasms / pathology
  • Case-Control Studies
  • Cell Line, Tumor
  • Early Detection of Cancer / methods
  • Female
  • Gene Expression Regulation, Neoplastic*
  • Humans
  • Microspheres
  • Neoplastic Cells, Circulating / chemistry
  • Neoplastic Cells, Circulating / metabolism*
  • Nucleic Acid Hybridization / methods
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sensitivity and Specificity


  • Biomarkers, Tumor
  • RNA, Messenger