A real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to detect breast carcinoma cells in peripheral blood

Aerts, Joeri L.; Wynendaele, Wim; Paridaens, Robert; Christiaens, Marie Rose; Bogaert, Walter Van den; Oosterom, A.T. van; Vandekerckhove, F

doi:10.1016/s0959-8049(99)80717-5

Cited by 24 publications

(47 citation statements)

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“…Positive cells were identified in the blood of four out of 20 (20%) healthy volunteers compared with 34 out of 40 (85%) patients with metastatic breast cancer (Figure 1). In common with a previous study, the upper 95% confidence interval of mean cell numbers in controls was used as a cutoff to define positive samples in patients with metastatic breast cancer (Aerts et al, 2001). Using such a threshold, 19 out of 40 (48%) samples from patients with metastatic breast cancer were positive.…”

Section: Detection By Lsc Following Imsmentioning

confidence: 99%

“…The detection of such cells could have significant clinical utility in risk stratification in early breast cancer, in early detection of relapse and in monitoring response to treatment. Cytometric techniques based on immunohistochemical analyses and nucleic acid-based approaches to cell detection have been described (Racila et al, 1998;Lambrechts et al, 1999;Smith et al, 2000;Aerts et al, 2001;Stathopoulou et al, 2002;Witzig et al, 2002). However, there is considerable variability in the reported sensitivities and specificities of existing techniques with putative carcinoma cells reported to be present in between 0 and 100% of blood samples from patients with metastatic breast cancer (Ring et al, 2004).…”

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confidence: 99%

“…The PCR assay was developed to detect cytokeratin 19 (CK19), mammaglobin and prolactin-inducible peptide (PIP) (Clark et al, 1999), which are expressed in breast epithelial cells but at low levels in normal blood components. CK19 was chosen as it is expressed in the majority of breast cancers (Bartek et al, 1986;Moll, 1994), and has been extensively used in studies in this area (Slade et al, 1999;Smith et al, 2000;Aerts et al, 2001;Stathopoulou et al, 2002Stathopoulou et al, , 2003. Mammaglobin gene expression has previously been used to detect circulating breast cancer micrometastases with no false positives in control populations (Zach et al, 1999;Silva et al, 2002).…”

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Detection of circulating epithelial cells in the blood of patients with breast cancer: comparison of three techniques

et al. 2005

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This study compares the sensitivities and specificities of three techniques for the detection of circulating epithelial cells in the blood of patients with breast cancer. The number of circulating epithelial cells present in the blood of 40 patients with metastatic breast cancer and 20 healthy volunteers was determined by: immunomagnetic separation (IMS) and laser scanning cytometry (LSC), cell filtration and LSC and a multimarker real-time RT -PCR assay. Numbers of cytokeratin-positive cells identified and expression of three PCR markers were significantly higher in the blood of patients with breast cancer than in healthy volunteers. Using the upper 95% confidence interval of cells detected in controls to determine positive patient samples: 30% of patients with metastatic breast cancer were positive following cell filtration, 48% following IMS, and 60, 45 and 35% using real-time RT -PCR for cytokeratin 19, mammaglobin and prolactin-inducible peptide. Samples were significantly more likely to be positive for at least one PCR marker than by cell filtration (83 vs 30%, Po0.001) or IMS (83 vs 48%, Po0.001).The use of a multimarker real-time RT -PCR assay was therefore found to be the most sensitive technique for the detection of circulating epithelial cells in the blood of patients with breast cancer.

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Section: Detection By Lsc Following Imsmentioning

confidence: 99%

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Detection of circulating epithelial cells in the blood of patients with breast cancer: comparison of three techniques

et al. 2005

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“…1 Our preliminary results based on this assay (Protocol A) prompted us to optimize the experimental conditions. Here we report an improved assay (Protocol B), which more sensitively and specifically detects CTC in breast cancer patients.…”

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“…In Protocol A, we used the sequence and concentration of the Ck19 primers (primers A and B, Figure 1) and Ck19 TaqMan probe as reported by Aerts et al (Figure 1). 1 The Ck19 TaqMan probe is modified at the 5 0 -end with FAM dye and at the 3 0 -end with TAMRA dye. In all, 2 1 2 ml of the cDNA solution was used in a 25 ml PCR reaction containing the following: 1 Â PCR buffer (Qiagen, Valencia, CA, USA), 200 mM of each dNTP (Roche, Indianapolis, IN, USA), 900 nM each of primers A and B, 200 nM Ck19 TaqMan probe, and 2.5 U HotStarTaq DNA polymerase (Qiagen, Valencia, CA, USA).…”

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Improved method for the detection of cytokeratin 19-positive cells in the peripheral blood of breast cancer patients

Alvero¹,

Burtness

Ercan

et al. 2004

Laboratory Investigation

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A relevant immunomagnetic assay to detect and characterize epithelial cell adhesion molecule‐positive cells in bone marrow from patients with breast carcinoma

Choesmel

Anract

Høifødt

et al. 2004

Cancer

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BACKGROUND The efficient detection and characterization of micrometastatic cells in the bone marrow of patients with breast carcinoma are of prognostic and therapeutic importance. The technique used must overcome the challenges that result from the small number of target cells (1 per 1 million hematopoietic cells) and the heterogeneous expression of micrometastatic cell markers. In this study, the authors assessed and improved the current methods for purifying and characterizing rare disseminated carcinoma cells. METHODS The authors developed a single‐step assay that does not require density‐gradient separation. This assay can be performed directly on crude human bone marrow aspirates and is based on the use of immunomagnetic beads coated with an antibody that recognizes an epithelial cell‐surface epitope, the epithelial cell adhesion molecule (EpCAM). To determine the specificity of the assay, the authors evaluated bone marrow specimens from 46 control patients. RESULTS The novel method was highly reproducible and was capable of detecting as few as 10 carcinoma cells among 50 million hematopoietic cells. The yield was nearly 100%, with only 0.01% nonspecific cell draining. The authors found that 68 ± 51 cells were trapped per 50 million cells in control crude aspirates and that density‐gradient separation increased this number by 2‐fold to 29‐fold. These trapped cells expressed EpCAM, represented 1.4 × 10−4 % of the sample, and were characterized as of hematopoietic cell origin (CD45 positive) or progenitor cell origin (CD34 positive). CONCLUSIONS The authors developed a highly efficient and reproducible, single‐step immunomagnetic assay that may be performed directly on crude human bone marrow aspirates. The authors believe the current study is the first to demonstrate that some rare bone marrow cells (CD45‐positive or CD34‐positive cells) may express EpCAM and, to some extent, may contaminate the purified micrometastatic cell fraction. Thus, a universal marker for micrometastatic cells remains to be discovered. Cancer 2004. © 2004 American Cancer Society.

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A real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to detect breast carcinoma cells in peripheral blood

Cited by 24 publications

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Detection of circulating epithelial cells in the blood of patients with breast cancer: comparison of three techniques

Detection of circulating epithelial cells in the blood of patients with breast cancer: comparison of three techniques

Improved method for the detection of cytokeratin 19-positive cells in the peripheral blood of breast cancer patients

A relevant immunomagnetic assay to detect and characterize epithelial cell adhesion molecule‐positive cells in bone marrow from patients with breast carcinoma

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