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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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Number of CTCs in blood samples of epithelial cancer and melanoma patients.
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Figure 2: Number of CTCs in blood samples of epithelial cancer and melanoma patients.

Mentions: CTCs were enriched by CD45 depletion and then analyzed by flow cytometry in 84 blood samples from 48 epithelial cancer patients (10 breast, 11 colon, 3 gastric, 6 ovarian, 7 cervix, 3 NSCLC and 8 SCCHN) and in 32 samples from 22 metastatic melanoma patients. Results were shown in Figure 2. CTCs could be found in 56% (47/84) of peripheral samples drawn from epithelial cancer patients, and in 53% (17/32) sample from patients with melanoma. The median number of CTCs was 3 (range: 1-55)/10 mL blood in epithelial cancer patients and 9 (range: 1-551)/10 mL blood in melanoma patients. The overall count of CTCs in melanoma patients was significantly higher than in carcinoma patients (p = 0.005).


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)

Number of CTCs in blood samples of epithelial cancer and melanoma patients.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Number of CTCs in blood samples of epithelial cancer and melanoma patients.
Mentions: CTCs were enriched by CD45 depletion and then analyzed by flow cytometry in 84 blood samples from 48 epithelial cancer patients (10 breast, 11 colon, 3 gastric, 6 ovarian, 7 cervix, 3 NSCLC and 8 SCCHN) and in 32 samples from 22 metastatic melanoma patients. Results were shown in Figure 2. CTCs could be found in 56% (47/84) of peripheral samples drawn from epithelial cancer patients, and in 53% (17/32) sample from patients with melanoma. The median number of CTCs was 3 (range: 1-55)/10 mL blood in epithelial cancer patients and 9 (range: 1-551)/10 mL blood in melanoma patients. The overall count of CTCs in melanoma patients was significantly higher than in carcinoma patients (p = 0.005).

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