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Negative enrichment by immunomagnetic nanobeads for unbiased characterization of circulating tumor cells from peripheral blood of cancer patients.

Liu Z, Fusi A, Klopocki E, Schmittel A, Tinhofer I, Nonnenmacher A, Keilholz U - J Transl Med (2011)

Bottom Line: EpCAM-CK+ cells have been successfully cultured and passaged longer than six months suggesting their neoplastic origin.This was confirmed by CGH.If detection of CTCs without a classical epithelial phenotype has clinical relevance need to be determined.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Hematology and Medical Oncology, Charité, Berlin, Germany.

ABSTRACT

Background: A limitation of positive selection strategies to enrich for circulating tumor cells (CTCs) is that there might be CTCs with insufficient expression of the surface target marker which may be missed by the procedure. We optimized a method for enrichment, subsequent detection and characterization of CTCs based on depletion of the leukocyte fraction.

Methods: The 2-step protocol was developed for processing 20 mL blood and based on red blood cell lysis followed by leukocyte depletion. The remaining material was stained with the epithelial markers EpCAM and cytokeratin (CK) 7/8 or for the melanoma marker HMW-MAA/MCSP. CTCs were detected by flow cytometry. CTCs enriched from blood of patients with carcinoma were defined as EpCAM+CK+CD45-. CTCs enriched from blood of patients with melanoma were defined as MCSP+CD45-. One-hundred-sixteen consecutive blood samples from 70 patients with metastatic carcinomas (n = 48) or metastatic melanoma (n = 22) were analyzed.

Results: CTCs were detected in 47 of 84 blood samples (56%) drawn from carcinoma patients, and in 17 of 32 samples (53%) from melanoma patients. CD45-EpCAM-CK+ was detected in pleural effusion specimens, as well as in peripheral blood samples of patients with NSCLC. EpCAM-CK+ cells have been successfully cultured and passaged longer than six months suggesting their neoplastic origin. This was confirmed by CGH. By defining CTCs in carcinoma patients as CD45-CK+ and/or EpCAM+, the detection rate increased to 73% (61/84).

Conclusion: Enriching CTCs using CD45 depletion allowed for detection of epithelial cancer cells not displaying the classical phenotype. This potentially leads to a more accurate estimation of the number of CTCs. If detection of CTCs without a classical epithelial phenotype has clinical relevance need to be determined.

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Calibration curve obtained by CD45 depletion in spiking experiments (n = 3) using SW620 cells at different dilutions.
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Figure 1: Calibration curve obtained by CD45 depletion in spiking experiments (n = 3) using SW620 cells at different dilutions.

Mentions: The linear regression equation obtained by enriching spiked SW620 cells by means of CD45 depletion was calculated according to the median recovery obtained in three different experiments (Figure 1). The recovery ranged from 57% to 94% (median 69%). CD45 depletion decreased leukocyte numbers from 3 × 107 to 4~6 × 104 cells which, depending on the number of tumor cells spiked, corresponded to relative CTC level, ranging from 0.1% to 1% of all events. The enrichment process was linear for the tested concentrations (R2 = 0.996). No EpCAM and CK double-positive cells could be detected in the control samples (0 cells spiked).


Negative enrichment by immunomagnetic nanobeads for unbiased characterization of circulating tumor cells from peripheral blood of cancer patients.

Liu Z, Fusi A, Klopocki E, Schmittel A, Tinhofer I, Nonnenmacher A, Keilholz U - J Transl Med (2011)

Calibration curve obtained by CD45 depletion in spiking experiments (n = 3) using SW620 cells at different dilutions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3119001&req=5

Figure 1: Calibration curve obtained by CD45 depletion in spiking experiments (n = 3) using SW620 cells at different dilutions.
Mentions: The linear regression equation obtained by enriching spiked SW620 cells by means of CD45 depletion was calculated according to the median recovery obtained in three different experiments (Figure 1). The recovery ranged from 57% to 94% (median 69%). CD45 depletion decreased leukocyte numbers from 3 × 107 to 4~6 × 104 cells which, depending on the number of tumor cells spiked, corresponded to relative CTC level, ranging from 0.1% to 1% of all events. The enrichment process was linear for the tested concentrations (R2 = 0.996). No EpCAM and CK double-positive cells could be detected in the control samples (0 cells spiked).

Bottom Line: EpCAM-CK+ cells have been successfully cultured and passaged longer than six months suggesting their neoplastic origin.This was confirmed by CGH.If detection of CTCs without a classical epithelial phenotype has clinical relevance need to be determined.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Hematology and Medical Oncology, Charité, Berlin, Germany.

ABSTRACT

Background: A limitation of positive selection strategies to enrich for circulating tumor cells (CTCs) is that there might be CTCs with insufficient expression of the surface target marker which may be missed by the procedure. We optimized a method for enrichment, subsequent detection and characterization of CTCs based on depletion of the leukocyte fraction.

Methods: The 2-step protocol was developed for processing 20 mL blood and based on red blood cell lysis followed by leukocyte depletion. The remaining material was stained with the epithelial markers EpCAM and cytokeratin (CK) 7/8 or for the melanoma marker HMW-MAA/MCSP. CTCs were detected by flow cytometry. CTCs enriched from blood of patients with carcinoma were defined as EpCAM+CK+CD45-. CTCs enriched from blood of patients with melanoma were defined as MCSP+CD45-. One-hundred-sixteen consecutive blood samples from 70 patients with metastatic carcinomas (n = 48) or metastatic melanoma (n = 22) were analyzed.

Results: CTCs were detected in 47 of 84 blood samples (56%) drawn from carcinoma patients, and in 17 of 32 samples (53%) from melanoma patients. CD45-EpCAM-CK+ was detected in pleural effusion specimens, as well as in peripheral blood samples of patients with NSCLC. EpCAM-CK+ cells have been successfully cultured and passaged longer than six months suggesting their neoplastic origin. This was confirmed by CGH. By defining CTCs in carcinoma patients as CD45-CK+ and/or EpCAM+, the detection rate increased to 73% (61/84).

Conclusion: Enriching CTCs using CD45 depletion allowed for detection of epithelial cancer cells not displaying the classical phenotype. This potentially leads to a more accurate estimation of the number of CTCs. If detection of CTCs without a classical epithelial phenotype has clinical relevance need to be determined.

Show MeSH
Related in: MedlinePlus