Summary Sera from 40 patients with newly diagnosed bladder cancer (28 superficial tumours (pTa and pTI) and 12 muscle-invasive tumours) were assessed by enzyme-linked immunosorbent assay (ELISA) to determine the concentrations of soluble E-cadherin (sE-cadherin), soluble E-selectin (sE-selectin), soluble vascular cell adhesion molecule-I (sVCAM-1) and soluble intercellular adhesion molecule-I (sICAM-1). Corresponding frozen sections of primary tumour were analysed for E-cadherin expression using the monoclonal antibody, HECD-1 and standard immunohistochemistry. Patients with bladder cancer had significantly higher concentrations of sE-cadherin compared with a control group (P=0.017). No difference was found between the two groups with regard to sE-selectin (P=0.403), sVCAM-1 (P=0.942) and sICAM-1 (P=0.092). High levels of sE-cadherin were related to poor histological grade (P=0.009), number of superficial tumours at presentation (P= 0.008) and a positive 3 month check cytoscopy in superficial disease (P= 0.036). Abnormal Ecadherin expression was associated with increasing tumour stage (P = 0.009) and grade (P = 0.03). There was no correlation between high levels of soluble E-cadherin in sera and abnormal E-cadherin expression by the tumour (P= 0.077). Elevated levels of sE-cadherin are found in sera of patients with bladder cancer and correlate with known prognostic factors.
A method for the use of a biotinylated antibody (DAKO-ER 1D5) to quantify oestrogen receptors (ER) on tumour cells by flow cytometry is described. ER quantification was determined after treatment with saponin rendering cells permeable to ER antibody. Use of dual parameter labelling was performed utilizing a FITC-conjugated antibody (NCL-5D3) directed against cytokeratin 8/18. This allowed selection of breast cancer cells of epithelial origin by gating to exclude contaminating hflammatory and stromal cells. Use of such a gating technique was seen to identify cells with a higher level of ER expression. Using QC quantum bead standards, the number of ER binding sites per cell was assessed. Results were compared with conventional ER quantification using a radio-ligand binding assay.A high degree of correlation was found between the two methods. The flow cytometric method for ER quantification described is simple, rapid, and reproducible. The assay may be of particular value in measuring ER on urgent clinical samples. Advantages of this assay over the radio-ligand binding assay include reduction in use of radio-labelled iodine compounds, a decrease in analysis time, and reduced cost and quantity of material needed for assay. Key terms: Flow cytometry, oestrogen receptor, cytokeratin, radio-ligand bindingThe oestrogen receptor (ER) is a 65,000 molecular weight, oestrogen-binding protein (5) associated with the nucleus (7) and found in -60% of all breast cancers. Binding of hormones to ER results in formation of a stabilized complex that interacts with specific regions of DNA (17). This leads to increased transcription of hormone-dependant genes, translation into proteins, and eventually replication and tumour cell division and growth. In human breast cancer, ER has been associated with superior prognosis (6) and a greater likelihood of response to endocrine therapy on relapse. Reduced levels of ER correlates with high tumour grade and poorer prognosis. Thus oestrogen receptor has been used as a prognostic marker for breast cancer for many years (1 1).Oestrogen receptor quantification by flow cytometr y has remained a relatively untried methodology with early work involving the use of fluorescently labelled oestradiol (l,lO,l6). This methodology relied on the availability of a free oestradiol binding site. However, with the availability of monoclonal antibodies directed against the oestrogen receptor, experiments with cell lines have yielded promising results. The MCF7 cell line has been reported to be ER positive ( 16) and as such is likely to be a good positive control. More recently, Scheres et al. (14) have shown receptor status to vary on cell lines depending on their culture conditions. A further problem encountered is the low levels of ER expressed by both cell lines and tumours. From a flow cytometric point of view, small shifts in fluorescence are more difficult to quantify, especially when the signal contains information from non-tumour cells. Fixation and overnight incubations have been employed ( 15) to tr...
This was a non-randomised single institution retrospective study. Forty-six banked frozen tumour specimens were obtained from a group of patients who had undergone 3 weeks of neoadjuvant treatment with tamoxifen between biopsy and surgery. Fifty-one comparison specimens were randomly selected from a group of concomitantly treated primary breast cancer patients who did not receive neoadjuvant tamoxifen. Specimen selection was not based on prognostic factors: hormone receptor status, patient age, or menopausal status. MUC1 expression and cell cycle distribution were assessed by flow cytometry. S-phase fraction of MUC1 positive and MUC1 negative cells were compared. A lower percentage of cells expressed MUC1 following 3-week tamoxifen treatment 18.2% versus 28.5% (p = 0.03, Mann-Whitney) and lower levels of MUC1 expression were seen following tamoxifen treatment 31,519 molecules/cell versus 39,387 (p = 0.04, Mann-Whitney). MUC1 positive cells, irrespective of treatment group, had a greater proportion of cells in S-phase of the cell cycle 27.9% versus 16.8% (p = 0.0004, Mann-Whitney) and demonstrated more cases of aneuploidy 80.65% versus 42.6% (p < 0.0001). MUC1 levels in primary tumours treated neoadjunctively with 3 weeks of tamoxifen were lower than a comparison group which did not receive tamoxifen. MUC1 should be explored further as an intermediate biomarker for assessment of treatment and prognosis.
A method for the purification, conjugation, and use of EGF-R antibody to quantify EGF receptors on tumour cells by flow cytometry is described. The quantification of both internal and external EGF receptors was determined by treatment with saponin, rendering the cells permeable to the EGF-R antibody. Using QCS bead standards, the number of EGF-R binding sites per cell was assessed. Results were compared with conventional EGF-R quantification using a radio-ligand binding assay. A high degree of correlation was found between the two methods. The flow cytometric method for EGF-R quantification described is sim-ple, rapid, and reproducible. The assay may be of particular value in measuring EGF-R on urgent clinical samples or those that are too small (such as breast aspirates) for measurement by the radio-ligand binding assay. Advantages of this assay over the radio-ligand binding assay include reduction in use of radio-labelled iodine compounds, a decrease in analysis time, and reduced cost and quantity of material needed for assay. In addition, flow cytometry offers the possibility of selecting cell phenotypes by gating as well as live/dead cells by using multi-parameter flow cytometry. 0 1994 Wiley-Liss, Inc.
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