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Detection of EpCAM-Negative and Cytokeratin-Negative Circulating Tumor Cells in Peripheral Blood.

Mikolajczyk SD, Millar LS, Tsinberg P, Coutts SM, Zomorrodi M, Pham T, Bischoff FZ, Pircher TJ - J Oncol (2011)

Bottom Line: Higher recovery of CTCs was demonstrated using antibody mixtures compared to anti-EpCAM.In addition, CK-positive breast cancer cells were found in 15 of 24 samples (63%; range 1-60 CTCs), while all samples contained additional CE-positive cells (range 1-41; median = 11; P = .02).Thus, antibody mixtures against a range of cell surface antigens enables capture of more CTCs than anti-EpCAM alone and CE staining enables the detection of CK-negative CTCs.

View Article: PubMed Central - PubMed

Affiliation: Research and Development, Biocept Inc., 5810 Nancy Ridge Drive, Suite 150, San Diego, CA 92121, USA.

ABSTRACT
Enrichment of rare circulating tumor cells (CTCs) in blood is typically achieved using antibodies to epithelial cell adhesion molecule (EpCAM), with detection using cytokeratin (CK) antibodies. However, EpCAM and CK are not expressed in some tumors and can be downregulated during epithelial-to-mesenchymal transition. A micro-fluidic system, not limited to EpCAM or CK, was developed to use multiple antibodies for capture followed by detection using CEE-Enhanced (CE), a novel in situ staining method that fluorescently labels the capture antibodies bound to CTCs. Higher recovery of CTCs was demonstrated using antibody mixtures compared to anti-EpCAM. In addition, CK-positive breast cancer cells were found in 15 of 24 samples (63%; range 1-60 CTCs), while all samples contained additional CE-positive cells (range 1-41; median = 11; P = .02). Thus, antibody mixtures against a range of cell surface antigens enables capture of more CTCs than anti-EpCAM alone and CE staining enables the detection of CK-negative CTCs.

No MeSH data available.


Related in: MedlinePlus

Clinical breast cancer samples sequentially stained with anti-CK and with CEE-Enhanced. The antibody mixture was used to capture CTCs. The dark bars on the bottom represent the number of CK positive cells detected in a sequential series of stage IV breast cancer samples. The location of these cells was recorded and then the channel was restained with CE. The light bars on top represent the newly detected CTCs after CE stain. All cells designated as positive were CD45 negative and DAPI positive.
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fig7: Clinical breast cancer samples sequentially stained with anti-CK and with CEE-Enhanced. The antibody mixture was used to capture CTCs. The dark bars on the bottom represent the number of CK positive cells detected in a sequential series of stage IV breast cancer samples. The location of these cells was recorded and then the channel was restained with CE. The light bars on top represent the newly detected CTCs after CE stain. All cells designated as positive were CD45 negative and DAPI positive.

Mentions: Following CTC enumeration of the breast cancer samples in Figure 7, the CEE microchannels were processed for multi-color FISH using the FDA approved PathVysion HER-2 DNA Probe Kit (centromere 17 specific probe (CEP 17-Spectrum Green) and locus-specific HER2 probe (Spectrum orange)) and a centromere-specific probe to chromosome 8 (CEP 8-Spectrum Aqua, Abbott Molecular). Each of the microchannels was first dehydrated before the addition of the probe mixture. Codenaturation of the probe mixture was performed on a ThermoBrite unit (Abbott Laboratories) at 95°C followed by hybridization at 37°C overnight. Postwash was performed at 74°C in 0.4× saline-sodium citrate (SSC) buffer containing 0.3% IPEGAL (Sigma-Aldrich, St. Louis, MO.) followed by 2× SCC wash containing 0.1% IPEGAL and then DAPI (blue). The CEE channels were imaged on the Olympus BX51 fluorescence microscope equipped with filters to view DAPI, SpectrumAqua, SpectrumOrange, and SpectrumGreen (Olympus America Inc.). Images were analyzed with use of the ISIS imaging system v5.2 (Metasystems, Waltham, Mass.). Evaluation of FISH signal patterns was performed on both CK-positive and CE-positive cells in the microchannel. CTCs were identified. The ratio of HER2 : CEP 17 was calculated and a ratio >2.2 was regarded as positive for HER2 gene amplification.


Detection of EpCAM-Negative and Cytokeratin-Negative Circulating Tumor Cells in Peripheral Blood.

Mikolajczyk SD, Millar LS, Tsinberg P, Coutts SM, Zomorrodi M, Pham T, Bischoff FZ, Pircher TJ - J Oncol (2011)

Clinical breast cancer samples sequentially stained with anti-CK and with CEE-Enhanced. The antibody mixture was used to capture CTCs. The dark bars on the bottom represent the number of CK positive cells detected in a sequential series of stage IV breast cancer samples. The location of these cells was recorded and then the channel was restained with CE. The light bars on top represent the newly detected CTCs after CE stain. All cells designated as positive were CD45 negative and DAPI positive.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3090615&req=5

fig7: Clinical breast cancer samples sequentially stained with anti-CK and with CEE-Enhanced. The antibody mixture was used to capture CTCs. The dark bars on the bottom represent the number of CK positive cells detected in a sequential series of stage IV breast cancer samples. The location of these cells was recorded and then the channel was restained with CE. The light bars on top represent the newly detected CTCs after CE stain. All cells designated as positive were CD45 negative and DAPI positive.
Mentions: Following CTC enumeration of the breast cancer samples in Figure 7, the CEE microchannels were processed for multi-color FISH using the FDA approved PathVysion HER-2 DNA Probe Kit (centromere 17 specific probe (CEP 17-Spectrum Green) and locus-specific HER2 probe (Spectrum orange)) and a centromere-specific probe to chromosome 8 (CEP 8-Spectrum Aqua, Abbott Molecular). Each of the microchannels was first dehydrated before the addition of the probe mixture. Codenaturation of the probe mixture was performed on a ThermoBrite unit (Abbott Laboratories) at 95°C followed by hybridization at 37°C overnight. Postwash was performed at 74°C in 0.4× saline-sodium citrate (SSC) buffer containing 0.3% IPEGAL (Sigma-Aldrich, St. Louis, MO.) followed by 2× SCC wash containing 0.1% IPEGAL and then DAPI (blue). The CEE channels were imaged on the Olympus BX51 fluorescence microscope equipped with filters to view DAPI, SpectrumAqua, SpectrumOrange, and SpectrumGreen (Olympus America Inc.). Images were analyzed with use of the ISIS imaging system v5.2 (Metasystems, Waltham, Mass.). Evaluation of FISH signal patterns was performed on both CK-positive and CE-positive cells in the microchannel. CTCs were identified. The ratio of HER2 : CEP 17 was calculated and a ratio >2.2 was regarded as positive for HER2 gene amplification.

Bottom Line: Higher recovery of CTCs was demonstrated using antibody mixtures compared to anti-EpCAM.In addition, CK-positive breast cancer cells were found in 15 of 24 samples (63%; range 1-60 CTCs), while all samples contained additional CE-positive cells (range 1-41; median = 11; P = .02).Thus, antibody mixtures against a range of cell surface antigens enables capture of more CTCs than anti-EpCAM alone and CE staining enables the detection of CK-negative CTCs.

View Article: PubMed Central - PubMed

Affiliation: Research and Development, Biocept Inc., 5810 Nancy Ridge Drive, Suite 150, San Diego, CA 92121, USA.

ABSTRACT
Enrichment of rare circulating tumor cells (CTCs) in blood is typically achieved using antibodies to epithelial cell adhesion molecule (EpCAM), with detection using cytokeratin (CK) antibodies. However, EpCAM and CK are not expressed in some tumors and can be downregulated during epithelial-to-mesenchymal transition. A micro-fluidic system, not limited to EpCAM or CK, was developed to use multiple antibodies for capture followed by detection using CEE-Enhanced (CE), a novel in situ staining method that fluorescently labels the capture antibodies bound to CTCs. Higher recovery of CTCs was demonstrated using antibody mixtures compared to anti-EpCAM. In addition, CK-positive breast cancer cells were found in 15 of 24 samples (63%; range 1-60 CTCs), while all samples contained additional CE-positive cells (range 1-41; median = 11; P = .02). Thus, antibody mixtures against a range of cell surface antigens enables capture of more CTCs than anti-EpCAM alone and CE staining enables the detection of CK-negative CTCs.

No MeSH data available.


Related in: MedlinePlus