Use of the monoclonal antibody DAKO-ERbeta (8D5-1) to measure oestrogen receptor beta in breast cancer cells.

BACKGROUND: Oestrogen receptor beta (ERbeta) is highly homologous with the classical ER (known now as ERalpha). The exact role of ERbeta in breast cancer and its contribution in influencing patient response to endocrine therapy remains unclear. The aim of this study was to develop and evaluate a flow cytometric method for the detection of ERbeta in breast cancer cells using the DAKO monoclonal anti-ERbeta 8D5-1 antibody. METHODS: MCF7 cells were used as a positive control and U937 as a negative control for titration of the antibody. Cell lines and tumour samples were fixed with 1% paraformaldehyde and permeabilised with 0.5% saponin prior to flow cytometric analysis. RESULTS: A ten fold difference in expression of ERbeta within the different breast cell lines studied was found. Confirmation of antibody specificity against ERbeta protein by Western blot analysis detected a single band at approximately 65kDa. ERbeta immunopositive nuclei were identified in MCF7 cells by immunohistochemistry. CONCLUSIONS: DAKO ERbeta 8D5-1 antibody is specific for ERbeta protein and does not cross react with ERalpha protein. Using this antibody, ERbeta can be detected and accurately quantified in cell lines and solid breast tumours by flow cytometry.

MUC1 expression in primary breast cancer: the effect of tamoxifen treatment.

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.

The oestrogen receptors (ER alpha and ER beta) and their role in breast cancer: a review.

One of the main prognostic markers measured in breast tumours is oestrogen receptor (ER) status. With the discovery of ER beta, it has been necessary to re-evaluate ER signalling and the role of ER in breast cancer. Preliminary reports on ER beta signalling suggest it might induce opposite effects to those of ER alpha. To further understand the biology of breast cancer, the role each ER plays in disease progression must be established. This review summarizes the current understanding of ER beta and discusses some of the published work on the role of ER alpha and ER beta in breast cancer.

Expression of oestrogen and progesterone receptors in gastric cancer: a flow cytometric study.

Increased expression of oestrogen (ER) and progesterone (PR) receptors have been reported in gastric adenocarcinoma, although results have been variable. Immunohistochemical staining methodologies, in particular in the detection of ER, have been inconsistent with many tumours being classified ER-negative. In this study we have used flow cytometry to quantify expression of ER and PR in gastric adenocarcinoma and examine their relationships with established prognostic indicators. Cytokeratin-positive cells obtained from tumour biopsies of 50 patients with gastric cancer and ten control patients were labelled with biotinylated ER or PR antibodies followed by streptavidin PE. Flow cytometry was seen to increase the detection of ER levels in gastric cancer with more receptor-positive patients in this study than in results published to date. We believe this is related to the sensitivity of the flow cytometric assay with the detection of small shifts in ER level detected using cytokeratin gating. On analysis, the data showed no significant correlations with tumour stage and grade, and no differences were seen between normal mucosa and gastric cancer samples.

Growth dysregulation and p53 accumulation in human primary colorectal cancer.

p53 accumulation is common in colorectal cancer, but effects on growth homeostasis are unclear. In this study, DNA content, cell cycle phase fractions and DNA strand-breaks consistent with apoptosis were assessed by flow cytometry in 42 fresh primary colorectal tumours and matched normal mucosa. p53 accumulation was assessed in 37 fixed tumour sections, by immunohistochemistry. In normal mucosa, 10.3 +/- 6.6% (mean +/- s.d.) cells were in DNA synthesis phase while 28.7 +/- 17.9% showed apoptosis. A relationship suggestive of growth homeostasis, was observed between these parameters (r = 0.8; P < 0.05). In cancers, a greater number of cells were in DNA synthesis phase (15.6 +/- 12.9% tumour vs mucosa 10.3 +/- 6.6%; P < 0.02) while fewer showed apoptosis than normal mucosa (18.5 +/- 17.0% tumour vs mucosa 28.7 +/- 17.9%; P < 0.01). DNA synthesis and apoptosis fractions were unrelated in cancers, suggesting growth dysequilibrium. p53 accumulation was detected in 59% (22/37) tumours and was associated with reduced apoptosis compared to p53-negative tumours or mucosa (14.8 +/- 15% p53 accumulation vs 26.3 +/- 18% p53-negative; P < 0.05; vs 28.7 +/- 17.9% mucosa; P < 0.05). p53 accumulation was unrelated to DNA synthesis phase fractions. p53 accumulation is accompanied by reduced apoptosis which may accentuate growth dysequilibrium in colorectal cancer.

The impact of transurethral resection of bladder tumour on serum levels of soluble E-cadherin.

OBJECTIVE: To evaluate soluble E-cadherin (sE-cadherin) as a potential tumour marker in patients with transitional cell carcinoma (TCC) of the bladder (previously shown to correlate with tumour grade, number of Ta/T1 tumours at presentation and a positive 3-month check cystoscopy) by assessing its serum concentration in relation to transurethral resection of bladder tumour (TURBT). PATIENTS AND METHODS: Samples of venous blood were obtained from 25 patients with bladder cancer: (i) before cystoscopy/TURBT: (ii) intraoperatively, during tumour resection; and (iii) on the first day after surgery. Levels of sE-cadherin were measured using an enzyme-linked immunosorbent assay. RESULTS: Sixty-three serum samples from patients with TCC of the bladder were available for analysis (23 before, 21 during and 19 after surgery). Patients with G2/3 tumours had significantly higher median preoperative levels of sE-cadherin (16.37 and 13.03 microg/mL, respectively) than those with G1 tumours (9.493 microg/mL; P = 0.0164). There was no correlation between tumour stage and preoperative sE-cadherin concentration. The median concentrations of sE-cadherin were not significantly different before, during and after TURBT. CONCLUSIONS: This study confirmed the previous finding that higher levels of serum sE-cadherin correlate with increasing tumour grade but not with clinicopathological stage. Serum sE-cadherin levels are not significantly altered by TURBT in the immediate perioperative period.

Cyclin D3 expression, cell proliferation and pathological stage of human primary colorectal cancer.

Cyclin D3 promotes cell cycle progression but its expression and prognostic significance in human colorectal cancer is unknown. This study assayed cyclin D3 expression against cell cycle phase fraction and Duke's stage in 35 fresh human primary colorectal cancers. DNA content, cell cycle phase fraction and cyclin D3 expression were assessed by flow cytometry in disaggregated tumors. Cyclin D3 expression and S-phase fraction were independently related to Duke's stage. In Duke's stage C tumors, a higher proportion of cells expressed cyclin D3 (14.4 vs. 8.8%, mean; p < 0.05 by Mann-Whitney U test) and were in DNA synthesis (S) phase (21.1 vs. 9.7%, mean; p < 0.05 by Mann-Whitney U test). Neoplastic deregulation of cyclin D3 expression may provide a selective growth advantage which is related to stage in human colorectal cancer.

Cytokeratin expression in breast cancer: phenotypic changes associated with disease progression.

Transition from a normal to a cancerous state is marked by alterations in the cytoskeletal structure of those cells involved. We have examined such changes to determine if these transitions are markers of disease progression. Cytokeratin (CK) protein and messenger RNA (mRNA) expression were examined in malignant and benign breast tissues. Flow cytometric results demonstrated a significant correlation between cytokeratin protein expression detected by 5D3 antibody, specific for cytokeratins 8, 18, and 19 and axillary node metastasis (P = 0.01). A threshold of positivity of 338,000 molecules/cell was determined and reflected the wide range in cytokeratin levels expressed by normal or benign tissues. Examination of cytokeratins 8, 18, and 19 revealed a consistent pattern of expression with respect to tumor grade. Only cytokeratin 19 showed significant correlation with increasing tumor size (P = 0.006). mRNA expression for cytokeratin 8 was significantly higher in node-positive compared with node-negative disease (P = 0.02). Cytokeratin 18 mRNA levels were significantly lower in both node-negative (P = 0.03) and node-positive (P = 0.02) patients when compared with benign samples. Increased levels of cytokeratin 18 mRNA showed an inverse relationship with protein expression (P = 0.05). The results indicate that cytokeratin expression in breast cancer may be associated with tumor progression. Furthermore, the alteration in the expression of individual cytokeratins deserves further investigation to determine the consequences of these changes with respect to cellular function.

The effect of 3-week tamoxifen treatment on oestrogen receptor levels in primary breast tumours: a flow cytometric study.

The effect of 3-week, preoperative tamoxifen treatment on oestrogen receptor (ER) levels, expressed by primary breast tumours, was examined. Patients (age-matched) with breast cancer, confirmed by fine-needle aspiration, were either treated with 20 mg ml(-1) oral tamoxifen per day or received no medication in the 3-week interval between assessment and surgery. Quantification of ER using flow cytometry was performed on the surgically removed tumour samples from tamoxifen-treated (n = 40) and control (n = 38, untreated) patient groups. The tumours were mechanically disaggregated, and saponin treatment rendered these cells permeable to antibodies. Using dual-parameter labelling with a FITC-conjugated antibody (NCL-5D3) directed against cytokeratin 8/18/19 and a biotinylated antibody (DAKO-ER 1D5) directed against the oestrogen receptor, ER quantification was determined on a number of receptors per cell basis. Using QC quantum bead standards, ER levels in the epithelial cell population, the non-epithelial cell population and the whole-cell population (ER+) were calculated. ER levels were significantly lower in the total cell population than tamoxifen-treated patients (P = 0.002) when compared with the control (untreated) group. By using a gating procedure using 5D3 antibody positivity, a significantly lower level was detected on examining the cytokeratin-positive population alone (P = 0.006). Using a complementary gating technique, ER levels were quantified in the cytokeratin-negative cell population. Examination of this group of cells showed no significant difference between the levels of oestrogen receptor found in the tamoxifen-treated and untreated groups (P = 0.4). We have demonstrated that ER levels can be monitored by flow cytometry. ER levels in patients treated with tamoxifen 3 weeks before operation are significantly lower than in a comparative group of patients who received no drug. Furthermore, the most significant difference in receptor levels is seen by quantification of total ER levels expressed by all the tissue.

Examination of multidrug resistance in cell lines and primary breast tumours by flow cytometry.

The aim of this study was to measure multidrug resistance (MDR) by flow cytometry and quantify the expression of P-glycoprotein (using antibody) glutathione transferase (using alpha-GSTpi antibody) in alpha-JSB-1 and alpha-GSTpi of a series of cell lines and primary breast cancers, and to assess the relationship between these MDR proteins and a selection of oncogene and prognostic markers in breast cancer. Flow cytometry was performed using permeabilised cells stained with fluorescent antibodies using well-established methods. Antibody staining was confirmed for JSB1, but not GSTpi by use of known positive and negative controls. No correlation was seen when comparing the number of molecules of alpha-JSB-1 with alpha-GSTpi (P = 0.1, r2 = 0.4, n = 14) using a selection of cell lines. Examination of 45 breast tumours for expression of JSB-1 and GSTpi revealed a significant association between these two antibodies (P < 0.00001, r2 = 0.5, n = 45). On examining the breast tumours, alpha-JSB-1 showed a positive association with c-erbB-2 (P = 0.003), c-myc (P = 0.0004) and c-jun (P = 0.02) but not ER or EGF-R expression. alpha-GSTpi showed a positive association with c-erbB-2 (P = 0.03) and c-myc (P = 0.0004) but not ER, EGF-R or c-jun. Flow cytometric MDR levels were not related to tumour grade or axillary node status. In solid tumours, a relationship between the two antibodies used has been clearly demonstrated, however, specificity of alpha-GSTpi is questioned. Both antibodies show an association with c-erbB-2, which is associated with poor prognosis and with c-myc which is involved in cell cycling and differentiation. Monitoring MDR markers (Pgp) using this methodology may be useful for evaluation of prognosis in breast cancer.

Cell adhesion molecules in bladder cancer: soluble serum E-cadherin correlates with predictors of recurrence.

Sera from 40 patients with newly diagnosed bladder cancer (28 superficial tumours (pTa and pT1) 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-1 (sVCAM-1) and soluble intercellular adhesion molecule-1 (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-selection (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 E-cadherin 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 flow cytometric study of c-erbB-3 expression in breast cancer.

In order to assess the specificity of biotinylated anti-c-erbB-3 antibody, screening was performed on a series of tumour cell lines and lymphocytes. Staining was found to be consistent, with good reproducibility. Twenty-nine consecutive breast cancer samples were obtained from women treated with tamoxifen and undergoing elective mastectomy. Twenty-eight invasive ductal carcinomas and 1 DCIS were stained for c-erbB-3 expression: 2 were grade I (Bloom and Richardson), 15 grade II, and 11 grade III tumours, 1 being unclassified; 16 were axillary node positive and 10 node negative; in 2 cases no nodes were sampled. Tumours examined by flow cytometry were stained with cytokeratin FITC antibody and the cytokeratin-positive population gated. Using Mann-Whitney analysis no association was seen between c-erbB-3 expression and Bloom and Richardson grade or axillary node status. In the tumour samples c-erbB-3 expression was found to show as association with EGF-R (P = 0.021 r2 = 0.16), PgR (P = 0.02, r2 = 0.16), c-myc (P < 0.0001, r2 = 0.5), c-jun (P = 0.001, r2 = 0.4) and c-fos (P = 0.001, r2 = 0.5) but not with c-erbB-2 (P = 0.2, r2 = 0.06), ER (P = 0.4, r2 = 0.02) or p53 1801 (P = 0.05, r2 = 0.2). Expression of c-erbB-3 may not be an independent marker of prognosis, but it is associated with other markers of poor prognosis and early cellular events linked with aberrant growth and differentiation.

Are fine-needle breast aspirates representative of the underlying solid tumour? A comparison of receptor levels, ploidy and the influence of cytokeratin gates.

Fifty-three solid and 33 fine-needle aspirate (FNA) samples (20 paired) of human breast carcinomas were examined by flow cytometry. Experiments were conducted to assess whether FNA samples were phenotypically representative of the solid tumour. Quantification of oestrogen receptor (ER), epidermal growth factor receptor (EGFR), c-erbB-2 receptor levels and ploidy were examined on the total and cytokeratin-positive cell populations. The absolute number of molecules of cytokeratin per cell expressed on the FNA (n = 33) and solid tumour (n = 53) samples showed no significant difference, but, on a proportional basis, there was a significant difference between the two samples (P = 0.004), with lower expression exhibited by the FNAs. Examination of paired data showed no significant difference in the percentage of cytokeratin-positive cells (P = 0.51) or in the number of cytokeratin molecules expressed (P = 0.25). While the correlation for ER expression between paired tumour and FNA samples in the absence of cytokeratin gating was P = 0.06, r2 = 0.18, clear correlation was shown when a cytokeratin gate was used (P = 0.005, r2 = 0.4). Repeating this experiment for EGFR, it was found that no correlation was seen between FNA and solid tumour (P = 0.2, r2 = 0.14) in ungated populations, but use of the cytokeratin gate improved the correlation (P = 0.05, r2 = 0.3). A similar finding was seen with c-erbB-2 expression (P = 0.2, r2 = 0.1) without cytokeratin gating and when it was employed (P = 0.05, r2 = 0.4). Ploidy data showed concordance in 18/20 cases. Three cases of aneuploidy were missed by FNA, and this was because of an insufficient number of cells for analysis. The presented data suggest that FNAs are representative of solid tumours and may be useful for measuring receptor levels on clinical material when cytokeratin gating is used. However, observation by light microscopy is still necessary to confirm the presence of tumour cells in FNAs subjected to flow cytometry.

p53 expression measured by flow cytometry. A comparison of three monoclonal antibodies and the relationship with grade and DNA ploidy in breast cancer.

The aim of this study was to quantify p53 expression by flow cytometry. A panel of three monoclonal antibodies: NCL-p53-240, NCL-p53-1801 and NCL-p53-DO7, was tested on breast cell lines and primary breast cancers. The relationships between ploidy, tumour grade and p53 expression for each antibody, were examined. Methodology was assessed using a variety of breast cell lines. Staining patterns were confirmed and the quantification technique qualified. Cytokeratin-positive cells from 58 samples obtained from patients with breast cancer were assayed for DNA content and p53 expression. p53 quantification was performed using calibrated fluorescent beads on cytokeratin-positive cells. Bloom and Richardson grading revealed 20 grade I and 38 grade II/III breast cancers. Examination of fluorescence thresholds showed a positive correlation between grade and DO7 (P = 0.003) at a level of 8900 molecules, 240 (P = 0.005) at a level of 2900 molecules and 1801 (P = 0.005) at a level of 1850 molecules. These levels equated with 34% (DO7), 43% (240) and 43% (1801) of the samples being classified as p53-positive. Examination of ploidy revealed 23 diploid and 35 aneuploid breast cancers. Application of p53 threshold levels on diploid and aneuploid tumours showed correlation between aneuploidy and p53 expression for DO7 at a level of 9000 molecules, 240 at a level of 1900 molecules and 1801 at a level of 1800 molecules. These levels equated with 34% (DO7), 52% (240) and 52% (1801) of the samples being classified as p53-positive. We conclude that measurement of p53 by flow cytometry may be of clinical importance by indicating levels of positivity using fluorescence thresholds. p53 expression has been shown to correlate with both grade and ploidy. Flow-cytometric measurement of p53 may be a useful prognostic assay.

The relationship between flow-cytometric and immunohistochemically detected c-erbB-2 expression, grade and DNA ploidy in breast cancer.

Quantification of c-erbB-2 and its relationship with other prognostic markers using flow cytometry has been examined. In this study a level for c-erbB-2 expression above which tumours are classified as positive by flow cytometry has been determined by employment of positive cut-off threshold levels. c-erbB-2 expression by both flow cytometry and immunohistochemistry was studied using the monoclonal antibody NCL-CBII. The relationship of c-erbB-2 quantification by flow cytometry was then compared with ploidy, axillary node status, tumour size and grade. Increased c-erbB-2 expression was seen using flow cytometry. Correlation between immunohistochemistry and flow-cytometry methods just failed to reach significance (P = 0.06). Immunohistochemistry revealed a significant relationship between c-erbB-2 expression and aneuploidy (P = 0.04). Cytokeratin-positive cells from 110 samples obtained from patients with breast cancer were assayed for DNA content and c-erbB-2 expression by flow cytometry. No correlation was seen between these parameters upon application of Mann Whitney analysis. However, examination of fluorescence thresholds showed a positive correlation between grade and c-erbB-2 expression at a level of more than 3200 molecules (P < or = 0.03). At the level of 3600 molecules significance was increased (P = 0.004). These levels equated with between 15% and 19% of the samples being classified as c-erbB-2-positive. Application of these cut-off points showed no correlation between c-erbB-2 expression and ploidy, tumour size or axillary node status. Comparison of ploidy and grade showed a significant association (P = 0.0015), increased grade correlating with aneuploidy.

Use of the biotinylated antibody DAKO-ER 1D5 to measure oestrogen receptor on cytokeratin positive cells obtained from primary breast cancer cells.

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 inflammatory 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.

Flow cytometric method for the measurement of epidermal growth factor receptor and comparison with the radio-ligand binding assay.

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 simple, 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.

Production of immunosuppressive factors by a cultured tumour cell line and their effect on lymphocyte proliferation and cell cycle response.

Immunosuppression observed in patients with malignancy may be due to factors released by tumour cells. Medium conditioned by COLO 205 cells was found to inhibit mitogen-stimulated lymphocyte proliferation. Examination of CD25 and Class II MHC induction on PBMC incubated in complete, or COLO 205 conditioned, medium was not significant. The prevalence of lymphocytes in the S-phase of the cell cycle was enhanced after mitogenic stimulation and addition of COLO 205 conditioned medium. This was balanced by a concomitant fall in proportion of cells in the G0/G1 phases of the cell cycle. The immunosuppressive properties of COLO 205 conditioned medium was abrogated by heating to 60 degrees C for 30 min and by digestion with trypsin. Fractionation of the medium by gel filtration yielded two immunosuppressive fractions with relative molecular weights of 60,000 and below 20,000. It was concluded that cultured COLO 205 cells produce immunosuppressive protein/peptide factors which block cell proliferation during DNA synthesis. These factors fail to prevent upregulation of membrane-associated markers of cell activation.