Detection of circulating tumor cells in breast cancer patients utilizing multiparameter flow cytometry and assessment of the prognosis of patients in different CTCs levels

Hu, Yanjie; Fan, Lingling; Zheng, Jianming; Cui, Rui; Liu, Wei; He, Yanli; Li, Xin; Huang, Shiang

doi:10.1002/cyto.a.20838

Cited by 67 publications

(47 citation statements)

References 26 publications

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“…To improve the chances of tumor cell detection, antibodies against intracellular melanoma tumor antigens, gp100, Mart-1, S100 and tyrosinase were pooled and detected by an indirect PE stain and combined with an APC conjugated antibody against the surface antigen MCSP. Pooling of antibodies and detecting them with multiple PMTs is consistent with a study in breast cancer, where antibodies against EpCAM and cytokeratins were labeled with PE and FITC, respectively, to detect breast cancer in blood samples (16). The pooling was performed to decrease the possibility of false-negatives (16), and we saw similar data indicating improved detection of tumors when antibodies were pooled.…”

Section: Discussionsupporting

confidence: 82%

Flow cytometry assessment of residual melanoma cells in tumor‐infiltrating lymphocyte cultures

et al. 2012

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Adoptive transfer of tumor-infiltrating lymphocytes (TIL) is in development for the treatment of metastatic melanoma. In phase II clinical trials, patients with metastatic melanoma that received TIL after preconditioning had a 50-70% clinical response rate. The current approach to generate TIL is to culture melanoma enzyme digests in the presence of IL-2 for a 10-to 20-day period followed by 2 weeks of rapid expansion (REP). Prior to administration, cell therapies are characterized and tested for purity. TIL are characterized by CD3 surface marker expression, and purity is assessed by the amount of tumor remaining in culture. Evaluating TIL purity has traditionally been done by immunohistochemistry, which is often considered semiquantitative. To generate a quantitative assay, we used multiparameter flow cytometry to evaluate the presence of viable tumor cells by staining TIL populations with a viability dye and an antibody cocktail that detects intracellular tumor-antigens gp100, Mart-1, tyrosinase, S100, and surface tumor-antigen melanoma chondroitin sulfate proteoglycan (MCSP), and CD3 on T cells. Tumors were identified by gating on the viable CD3 2 population. Antigens in tumors were initially optimized with individual antibodies using both immunohistochemistry and flow cytometry. When eight different tumor cell lines were spiked into an activated T cell culture, flow cytometry was able to distinguish lymphocytes from tumors in all samples tested. Most importantly, the assay was able to detect melanoma cells in all enzyme digests (9/9) from patient samples. After IL-2-induced TIL expansion, there was a significant decrease in tumor cells; tumor cells were detected in only 2 of 12 samples. In eight IL-2-induced TIL samples that were further expanded in REP, no tumor cells were detected. We have demonstrated that flow cytometry is an alternative to immunohistochemistry for defining the purity of a TIL population. ' 2012 International Society for Advancement of Cytometry

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Section: Discussionsupporting

confidence: 82%

Flow cytometry assessment of residual melanoma cells in tumor‐infiltrating lymphocyte cultures

et al. 2012

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“…The advantages of flow cytometry (FCM) are a rapid readout of routine measurements, the fact that size information is included in the data, and the capability of multicolor analysis. Several studies have independently reported flow cytometric detection and enumeration of CTC (13)(14)(15)(16). Because the detection limit of rare cells using the cytometer is approximately 10 25 (6), preenrichment and multicolor analysis of CTC will be necessary for their detection using common methods of immunofluorescence analysis.…”

Section: Introductionmentioning

confidence: 99%

Enumeration, characterization, and collection of intact circulating tumor cells by cross contamination‐free flow cytometry

Takao

Takeda

2011

Cytometry Pt A

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Circulating tumor cells (CTC) are an important biomarker for several solid cancers. Most of the commercially available systems for enumeration of CTC are based on immunomagnetic enrichment of epithelial cell adhesion molecule (EpCAM/CD326)-expressing CTC before microscopic cell imaging or reverse-transcription PCR (RT-PCR). The aim of this study was to establish a practical method for enumeration of CTC using a novel flow cytometer that has a disposable microfluidic chip, which is designed to realize absolute cross contamination-free measurements and to collect the analyzed cell sample. Although the process of enumeration and labeling of CTC was optimized for this device, the simplified protocol described here could be applied to other flow cytometers. Cultured cancer cells spiked into normal blood were enriched using MACS 1 EpCAM-MicroBeads following cell labeling with an allophycocyanin (APC)-conjugated EpCAM mAb, instead of by intracellular staining of cytokeratins (CK). The EpCAM double-positive selection/labeling method allows enumeration of intact CTC, maintenance of cellular integrity, and the concomitant performance of a CTC viability test. The combination of the fine-tuned CTC enrichment process and the cytometric multicolor analysis resulted in a linear relationship between the output cell count and the input cell number from zero to hundreds of cells. In particular, a satisfactory signal/noise ratio was obtained by gate-exclusion of leukocyte signals using an anti-CD45 mAb. The entire process had little influence on the viability of the spiked lung cancer cell PC-9. Measured PC-9 and breast cancer MCF-7 cells bearing EpCAMMicroBeads, APC-conjugated EpCAM mAb, and the DNA staining dye SYTO9 grew normally, demonstrating the potential usefulness of the collected samples for further studies. This intact CTC enumeration and analysis procedure (iCeap) would be of great benefit to clinicians by providing them with rapid stratification of antitumor therapy, and to basic researchers by permitting further molecular and cellular characterization of CTC. ' 2011 International Society for Advancement of Cytometry

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“…In the study model, tumor cells from the ovarian cancer cell line CaOV-3 were spiked into leukapheresates prior to separation by elutriation, and tumor cell recovery was determined by flow cytometry. Since flow cytometry has an expected resolution of one epithelial tumor cell in 10 5 nucleated cells (46,47), a spike ratio of 26 tumor cells per million PBMNCs ($50 tumor cells per mL of blood) was specified to ensure their detection. The large number of cells harvested by leukapheresis necessitated the physical pre-enrichment of CTCs to allow the practical use of immunological based methods for their isolation thereafter.…”

Section: Discussionmentioning

confidence: 99%

Enrichment of circulating tumor cells from a large blood volume using leukapheresis and elutriation: Proof of concept

Eifler

Lind

Falkenhagen

et al. 2010

Cytometry Part B Clinical

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Background: The aim of this study was to determine the applicability of a sequential process using leukapheresis, elutriation, and fluorescence-activated cell sorting (FACS) to enrich and isolate circulating tumor cells from a large blood volume to allow further molecular analysis.Methods: Mononuclear cells were collected from 10 L of blood by leukapheresis, to which carboxyfluorescein succinimidyl ester prelabeled CaOV-3 tumor cells were spiked at a ratio of 26 to 10 6 leukocytes. Elutriation separated the spiked leukapheresates primarily by cell size into distinct fractions, and leukocytes and tumor cells, characterized as carboxyfluorescein succinimidyl ester positive, EpCAM positive and CD45 negative events, were quantified by flow cytometry. Tumor cells were isolated from the last fraction using FACS or anti-EpCAM coupled immunomagnetic beads, and their recovery and purity determined by fluorescent microscopy and real-time PCR.Results: Leukapheresis collected 13.5 3 10 9 mononuclear cells with 87% efficiency. In total, 53 to 78% of spiked tumor cells were pre-enriched in the last elutriation fraction among 1.6 3 10 9 monocytes. Flow cytometry predicted a circulating tumor cell purity of $90% giving an enrichment of 100,000-fold following leukapheresis, elutriation, and FACS, where CaOV-3 cells were identified as EpCAM positive and CD45 negative events. FACS confirmed this purity. Alternatively, immunomagnetic bead adsorption recovered 10% of tumor cells with a median purity of 3.5%.Conclusions: This proof of concept study demonstrated that elutriation and FACS following leukapheresis are able to enrich and isolate tumor cells from a large blood volume for molecular characterization.

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Detection of circulating tumor cells in breast cancer patients utilizing multiparameter flow cytometry and assessment of the prognosis of patients in different CTCs levels

Cited by 67 publications

References 26 publications

Flow cytometry assessment of residual melanoma cells in tumor‐infiltrating lymphocyte cultures

Flow cytometry assessment of residual melanoma cells in tumor‐infiltrating lymphocyte cultures

Enumeration, characterization, and collection of intact circulating tumor cells by cross contamination‐free flow cytometry

Enrichment of circulating tumor cells from a large blood volume using leukapheresis and elutriation: Proof of concept

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