Breast Cancer Tissue Extracts Research Articles

Association of uPA and PAI-1 tumor levels and 4G/5G variants of PAI-1 gene with disease outcome in luminal HER2-negative node-negative breast cancer patients treated with adjuvant endocrine therapy

BackgroundThe aim of this study was to evaluate the prognostic potential of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) tumor tissue levels and examine the association between these biomarkers and classical prognostic factors in early node-negative luminal breast cancer patients. The clinical value of 4G/5G variants of PAI-1 gene was evaluated.Patients and methodsThis study involved 81 node-negative, estrogen receptor-positive and/or progesterone receptor-positive and human epidermal growth factor receptor 2-negative operable breast cancer patients who underwent radical surgical resection and received adjuvant endocrine therapy. Determination of uPA and PAI-1 concentrations in the breast cancer tissue extracts was performed using FEMTELLE® uPA/PAI-1 ELISA. An insertion (5G)/deletion (4G) polymorphism at position − 675 of the PAI-1 gene was detected by PCR-RFLP analysis.ResultsOur research showed that patients with uPA tumor tissue levels higher than 3 ng/mg of protein had significantly reduced disease-free survival (DFS) and overall survival (OS) when compared to patients with uPA tumor tissue levels lower or equal to 3 ng/mg of protein. Patients with PAI-1 tumor tissue levels higher than 14 ng/mg of protein had significantly decreased OS in comparison with patients with PAI-1 tumor tissue levels lower or equal to 14 ng/mg of protein. ROC analysis confirmed the uPA and PAI-1 discriminative potential for the presence/absence of relevant events in these patients and resulted in higher cut-off values (5.65 ng/mg of protein for uPA and 27.10 ng/mg of protein for PAI-1) than standard reference cut-off values for both biomarkers. The prognostic importance of uPA and PAI-1 ROC cut-off values was confirmed by the impact of uPA higher than 5.65 ng/mg of protein and PAI-1 higher than 27.10 ng/mg of protein on poorer DFS, OS and event-free survival (EFS).We observed that patients with dominant allele in PAI-1 genotype (heterozygote and dominant homozygote, − 675 4G/5G and − 675 5G/5G) had significantly increased DFS, OS and EFS when compared with patients with recessive homozygote genotype (− 675 4G/4G).ConclusionOur study indicates that uPA and PAI-1 tumor tissue levels and 4G/5G variants of PAI-1 gene might be of prognostic significance in early node-negative luminal HER2-negative breast cancer patients treated with adjuvant endocrine therapy.

AuNPs colorimetric sensor for detecting platelet-derived growth factor-BB based on isothermal target-triggering strand displacement amplification

We developed a colorimetric sensor based on gold nanoparticle (AuNP) labels and target-triggered strand displacement amplification (SDA) for the detection of platelet-derived growth factor-BB (PDGF-BB). Upon the target binding, the linear probe conformation was converted into a triple-helix conformation and then a large number of reporter oligonucleotides were produced by SDA. PDGF-BB was then determined by detecting the reporter oligonucleotides using gold nanoparticle labels. The signal output showed a linear relationship for PDGF-BB within the concentration range from 2.0 to 80nM with a detection limit of 1.1nM under the optimal conditions. An obvious AuNPs color change was provided when the concentration of PDGF-BB was as low as 4.0nM. Notably, the assay demonstrated here was free from the interference of other proteins commonly existing in the human breast cancer tissue extracts.

Integrating meta-analysis of microarray data and targeted proteomics for biomarker identification: application in breast cancer.

The development of signature biomarkers has gained considerable attention in the past decade. Although the most well-known examples of biomarker panels stem from gene expression studies, proteomic panels are becoming more relevant, with the advent of targeted mass spectrometry-based methodologies. At the same time, the development of multigene prognostic classifiers for early stage breast cancer patients has resulted in a wealth of publicly available gene expression data from thousands of breast cancer specimens. In the present study, we integrated transcriptome and proteome-based platforms to identify genes and proteins related to patient survival. Candidate biomarker proteins have been identified in a previously generated breast cancer tissue extract proteome. A mass-spectrometry-based assay was then developed for the simultaneous quantification of these 20 proteins in breast cancer tissue extracts. We quantified the relative expression levels of the 20 potential biomarkers in a cohort of 96 tissue samples from patients with early stage breast cancer. We identified two proteins, KPNA2 and CDK1, which showed potential to discriminate between estrogen receptor positive patients of high and low risk of disease recurrence. The role of these proteins in breast cancer prognosis warrants further investigation.

Survivin expression in breast cancer predicts clinical outcome and is associated with HER2, VEGF, urokinase plasminogen activator and PAI-1

Background: Survivin, a novel inhibitor of apoptosis, is one of the most cancer-specific proteins identified to date. In this study we (a) evaluated the association between survivin and HER2, vascular endothelial growth factor (VEGF) and uPA/PAI-1 expression and (b) defined its effect on clinical outcome in a large breast cancer patient cohort.Patients and methods: Survivin expression was measured by ELISA in primary breast cancer tissue extracts from 420 patients with long-term clinical follow-up.Results: Survivin was detected in 378 (90%) of the 420 primary breast cancer cases. Increased survivin levels were significantly associated with high nuclear grade (P < 0.0001), negative hormone receptor status (P = 0.0028), HER2 overexpression (P = 0.0094), VEGF expression (P < 0.0001), high uPA (P = 0.0002) and PAI-1 levels (P = 0.0002). Using the 25th percentile (1.4 ng/mg) as a cut-off point, patients expressing elevated survivin had a significantly worse disease-free survival (DFS: P = 0.0007, RR 1.97) and overall survival (OS: P = 0.0009, RR 2.11) compared with patients expressing lower levels of survivin. In multivariate analysis, this prognostic value of survivin was independent of both traditional and novel clinicopathologic factors for both DFS (P = 0.0076, RR 1.72) and OS (P = 0.0155, RR 1.76).Conclusions: The independent prognostic relevance of survivin, when combined with previous data from model systems implicating survivin in the inhibition of apoptosis, suggests that survivin may be a suitable target for future therapeutic strategies.

Estrogen effects on the expression of Brx in the brain and pituitary of the mouse

A member of the Dbl family of oncoproteins was discovered in breast cancer tissue extracts. This novel protein, designated Brx, contains an estrogen-receptor binding motif and is highly expressed in hormone-responsive breast tissue. Due to its ability to augment ligand-dependent activation of estrogen receptors, we analyzed the expression of Brx in the adult mouse brain and pituitary. Results indicated that Brx was expressed in specific regions of the brain and pituitary. Furthermore, the results indicate that differences exist in both brain and pituitary tissue of male and female mice with greater expression in the female. However, estrogen did not influence Brx expression in ovariectomized mice. The anatomical studies support a role for Brx in its association with the estrogen receptor and that Brx may be involved in neuronal and pituitary function in a sexually dimorphic manner.

Survivin protein expression predicts poor outcome in patients with primary breast cancer

547 Introduction: Survivin plays multiple roles in malignancy including inhibition of apoptosis, stimulation of proliferation and promotion of angiogenesis, and is up regulated in malignant tissues. Survivin is currently being investigated both as a target for new anticancer treatments and as a new tumor marker. The aim of this work was to study the expression and potential clinical value of survivin in breast cancer. Methods Survivin expression was measured by ELISA in primary breast cancer tissue extracts from 399 patients with a median clinical follow up of 48 months. Results Survivin levels were significantly increased in high grade compared to low grade tumors (p<0.001), in hormone receptor-negative compared to receptor-positive tumors (p=0.011), in HER-2 positive versus HER-2 negative tumors (p<0.047) and in VEGF-positive versus VEGF-negative tumors (p<0.0001). Using Spearman correlation analysis, survivin levels correlated weakly but significantly with numbers of nodal metastases (r=0.11, p=0.024, n=388), total VEGF levels (r=0.23, p<0.0001, n=386) and VEGF165 levels (r=0.23, p<0.0001, n=386). Using the median value as a cut-off point, high levels of survivin were associated with both shortened disease-free (Log Rank test p=0.014) and overall survival (Log Rank test p=0.015). When categorized using the 25th, 50th and 75th percentiles as cut-off points, survivin was an independent predictor of disease-free survival (p=0.037), i.e., patients with the highest quartile of survivin levels had a significantly shorter disease-free survival period than those with the lowest quartile. Conclusions We conclude that increased expression of survivin is associated with adverse outcome in patients with primary breast cancer. These patients with high levels of survivin are potential candidates for treatment with new anti-survivin (e.g, antisense oligonucleotides) therapies. No significant financial relationships to disclose.

Quantitative assessment of HER-2/neu protein concentration in breast cancer by enzyme-linked immunosorbent assay.

The HER-2/neu protein (p185) has become a promising target for antibody therapy in breast cancer. We tested the feasibility of a quantitative approach for HER-2/neu testing based on the analysis of tumor tissue extracts by an enzyme-linked immunosorbent assay (ELISA). Tumor tissue extracts of primary human breast cancers (n=124) were prepared using a triton-based buffer. HER-2/neu concentration was quantified by ELISA. Paraffin-embedded tissue sections of the same tumors were analyzed by immunohistochemical staining applying the monoclonal HER-2/neu antibody TAB 250 (n=124) and by chromogenic in situ hybridization (CISH) (n=73). Concentrations of p185 in tissue extracts determined by ELISA varied from 1 to 927 ng per mg protein with a median of 25 ng/mg protein, whereas normal breast tissue showed values from 0.4 to 5.5 ng/mg with a median of 2.2 ng/mg (p<0.0001, Mann-Whitney U test). A significant correlation between p185 concentration and immunohistochemical staining was observed (p<0.0001, Kruskal-Wallis test). In addition, p185 concentration measured by ELISA was correlated with the degree of HER-2/neu gene amplification determined by CISH. HER-2/neu-amplified tumors had significantly higher p185 concentrations (median value 181 ng/mg protein) than non-amplified tumors (median value 20 ng/mg; p<0.0001, Mann-Whitney U test). ELISA-based measurement of HER-2/neu protein concentration in breast cancer tissue extracts is feasible and provides quantitative results for p185 protein concentrations that correlate closely with HER-2/neu immunoscore and gene amplification.

Quantitative assessment of HER-2/neu protein concentration in breast cancer by enzyme-linked immunosorbent assay

The HER-2/neu protein (p185) has become a promising target for antibody therapy in breast cancer. We tested the feasibility of a quantitative approach for HER-2/neu testing based on the analysis of tumor tissue extracts by an enzyme-linked immunosorbent assay (ELISA). Tumor tissue extracts of primary human breast cancers (n=124) were prepared using a triton-based buffer. HER-2/neu concentration was quantified by ELISA. Paraffin-embedded tissue sections of the same tumors were analyzed by immunohistochemical staining applying the monoclonal HER-2/neu antibody TAB 250 (n=124) and by chromogenic in situ hybridization (CISH) (n=73). Concentrations of p185 in tissue extracts determined by ELISA varied from 1 to 927 ng per mg protein with a median of 25 ng/mg protein, whereas normal breast tissue showed values from 0.4 to 5.5 ng/mg with a median of 2.2 ng/mg (p<0.0001, Mann-Whitney U test). A significant correlation between p185 concentration and immunohistochemical staining was observed (p<0.0001, Kruskal-Wallis test). In addition, p185 concentration measured by ELISA was correlated with the degree of HER-2/neu gene amplification determined by CISH. HER-2/neu-amplified tumors had significantly higher p185 concentrations (median value 181 ng/mg protein) than non-amplified tumors (median value 20 ng/mg; p<0.0001, Mann-Whitney U test). ELISA-based measurement of HER-2/neu protein concentration in breast cancer tissue extracts is feasible and provides quantitative results for p185 protein concentrations that correlate closely with HER-2/neu immunoscore and gene amplification.

New ELISA for quantitation of human urokinase receptor (CD87) in cancer.

The serine protease urokinase-type plasminogen activator (uPA), its inhibitor (PAI-1), and its receptor (uPAR; CD87) facilitate cancer cell invasion and metastasis. Whereas uPA and PAI-1 antigen levels determined in tumor tissue extracts of breast cancer patients correlate with disease recurrence and overall survival, the prognostic relevance of uPAR is still a matter of debate. We established two new sandwich-type enzyme-linked immunosorbent assay (ELISA) formats (HU/IIIF10-ELISA and HU/HD13-ELISA) using the epitope-defined monoclonal antibody (mAb) IIIF10 or the conformation-dependent mAb HD13.1, a polyclonal chicken antibody (HU277), and recombinant soluble uPAR (CHO-suPAR) as the standard. The lower detection limit of the assays was at 0.16 ng/ml, with a linear dose-response up to 5 ng/ml of uPAR antigen. Both ELISA formats showed good reproducibility and recovery. The intra-assay and the inter-assay variation coefficients were respectively 4.3% and 11.7% (HU/IIIF10-ELISA) and 4.0% and 10.7% (HU/HD13-ELISA). The recovery rate of uPAR in cell lysates spiked with CHO-suPAR was above 82% and 88%, respectively. With these new ELISA formats, uPAR antigen content in breast cancer tissue extracts and tumor cell lysates was determined and compared to a commercially available ELISA (ADI-ELISA). By all of the three uPAR ELISA formats CHO-suPAR and uPAR present in lysates of non-malignant epithelial cells and stimulated monocytes were quantified with similar sensitivity. Interestingly, in breast cancer cell lines of epitheloid origin a higher uPAR antigen content was determined by the HU/IIIF10-ELISA than the HU/HD13- or ADI-ELISA formats. In lysates of fibroblastic breast cancer cell lines similar uPAR values were obtained with the HU/IIIF10- and ADI-ELISA formats, whereas with the HU/HD13-ELISA significantly lower uPAR concentrations were determined. The prognostic relevance of tumor uPAR antigen was evaluated in 199 primary breast cancer patients with a median follow-up of 24 months. uPAR antigen values above the cut-off of 3.33 ng/mg protein as determined by the HU/IIIF10-ELISA were significantly correlated with short disease-free survival (p=0.025). Results obtained by the other two ELISA formats (HU/HD13-ELISA and ADI-ELISA) were not associated with prognosis. Our findings stress the need of well-characterized antibodies, which detect both uPAR of non-malignant and tumor cells, in setting up a uPAR-ELISA useful for assessing breast cancer patient prognosis.

Plasminogen activator inhibitor-1 (PAI-1) gene 4G/5G promoter polymorphism is not associated with breast cancer.

The antigen content of plasminogen activator inhibitor-1 (PAI-1) in primary breast cancer tissue extracts may be of strong prognostic value: high levels of PAI-1 in tumors predict poor prognosis for patients. The gene encoding PAI-1 is highly polymorphic and an insertion (5G)/deletion (4G) polymorphism in the PAI-1 gene promoter (the 4G/5G polymorphism), may have functional significance in PAI-1 expression. In the present work the distribution of genotypes and frequency of alleles of the 4G/5G polymorphism in subjects with breast cancer were investigated. Tumor tissues were obtained from 100 postmenopausal women with node-negative and node-positive ductal breast carcinoma with uniform tumor size. Blood samples from age matched healthy women served as control. The 4G/5G polymorphism was determined by PCR amplification using the allele specific primers. The distribution of the genotypes of the 4G/5G polymorphism in both control and patients did not differ significantly (P > 0.05) from those predicted by the Hardy-Weinberg distribution. There were no differences in the genotype distributions and allele frequencies between node-positive and node-negative patients. The 4G/5G polymorphism may not be linked with elevated level of PAI-1 observed in breast cancer and therefore may not be associated with appearance and/or progression of breast cancer.

External quality assessment of trans-European multicentre antigen determinations (enzyme-linked immunosorbent assay) of urokinase-type plasminogen activator (uPA) and its type 1 inhibitor (PAI-1) in human breast cancer tissue extracts.

High levels of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) in breast cancer tissue extracts have been associated with rapid disease progression. In these studies, different enzyme-linked immunosorbent assay (ELISA) kits have been applied for the quantification, and consequently the ranges of uPA and PAI-1 levels reported differ considerably. Therefore, the Receptor and Biomarker Study Group (RBSG) of the European Organization for Research and Treatment of Cancer (EORTC) and a consortium of the BIOMED-1 project 'Clinical Relevance of Proteases in Tumor Invasion and Metastasis' initiated three collaborative between-laboratory assessment trials aimed at controlling uPA and PAI-1 antigen analyses. For this purpose, two control preparations were produced from different sources: pooled human breast cancer specimens (QC-240893) and human breast cancer xenografts raised in nude mice (QC-101094). The lyophilized preparations were stable for prolonged times (at least 3 and 27 months respectively) at 4 degrees C. Furthermore, a good parallelism following dilution was found for uPA and PAI-1. The data from QC trial no. 1 clearly indicated that acceptable between-laboratory coefficients of variation (CVs) for uPA (<8.2%) and PAI-1 (<16.6%) in QC-240893 could be achieved when the same type of ELISA kit (American Diagnostica) was used. From the second trial, in which ten EORTC laboratories each received five identical lyophilized QC-101094 samples, it appeared that the within-laboratory variations for uPA and PAI-1 determinations obtained by 'experienced' laboratories were lower (<12.9%) than those from non-experienced laboratories (<36.4%). In a third QC trial, five BIOMED-1 laboratories, all of which employed ELISA procedures for uPA and PAI-1, participated in six subsequent quality assessment rounds receiving five samples of QC-101094. Although for each laboratory the within-run CVs for uPA as well as for PAI-1 were low (<7.8%), the between-run CVs were found to be considerably higher (up to 56.2% for uPA and to 27.6% for PAI-1). Consequently, because of the different ELISA formats used, the absolute analyte values measured in the different laboratories varied substantially. The use of 'common external standards' in the different ELISAs resulted in a significant reduction of the between-laboratory CVs from 61.3% to 15.7% (uPA) and from 42.1% to 19.1% (PAI-1). The present data demonstrate that in multicentre studies the same ELISA kit should be used, and that external quality assurance (QA) is mandatory. Furthermore, it appears from the present study that standardization of the protein assay as a tissular parameter is imperative.

Urokinase-type receptor in breast cancer tissue extracts. Enzyme-linked immunosorbent assay with a combination of mono- and polyclonal antibodies

Urokinase-type receptor in breast cancer tissue extracts. Enzyme-linked immunosorbent assay with a combination of mono- and polyclonal antibodies

Leukocyte Migration Inhibition in Relation to Nuclear Pleomorphism and Lymphoid Infiltration in Breast Cancer

Leukocyte migration inhibition (LMI) in patients with breast cancer was examined by using 3 M-KCl tissue extracts of breast tumors. Tumor extracts were obtained from 12 breast carcinomas of various histological types. In 20 of 65 (30.8%) leukocyte preparations from 25 patients with breast cancer migration was inhibited by breast cancer tissue extracts. In 26 leukocyte preparations from eight healthy persons and 22 preparations from eight patients with benign diseases of the breast migration was not inhibited by the breast cancer extracts. In only two of 47 (4.3%) leukocyte preparations from 12 patients with other cancers migration was inhibited by the extracts. The occurrence of LMI was the highest in leukocytes from the patients with breast cancer showing marked nuclear pleomorphism (10/19, 52.6%) or showing marked mononuclear cell infiltration (6/12, 50.0%). These results suggest that 3 M-KCl tissue extracts of breast cancer may contain tumor-associated antigens in solubilized form and that there may be a correlation between LMI and nuclear pleomorphism of cancer cells of the leukocyte donor as well as mononuclear cell infiltration in the tumor of the leukocyte donor.