Immunoassays of urokinase (uPA) and its type-1 inhibitor (PAI-1) in detergent extracts of breast cancer tissue.

Two immunoassays for quantitation of the biological markers uPA and PAI-1 were evaluated for their use with detergent extracts of breast cancer tissue. Both assays were based on murine monoclonal capture antibodies and rabbit polyclonal detector antibodies. Horseradish peroxidase-conjugated goat anti-rabbit antibodies enabled measurement of the bound antigen. The detection limit of the uPA assay was 13 pg/ml, with a linear dose-response relationship up to 350 pg/ml. The assay detected free uPA as well as uPA in complex with PAI-1 and/or with its receptor. The detection limit of the PAI-1 assay was 50 pg/ml, with a linear dose-response relationship up to 1500 pg/ml. The assay detected both free PAI-1 and uPA:PAI-1 complex. Both assays were validated for detergent extracts using immunoabsorption and recovery tests. Highly significant associations between tumour tissue uPA and PAI-1 levels and prognosis were verified in a cohort of 164 lymph node-negative primary breast cancer patients. It is concluded that the two immunoassays are well-suited for the quantitation of uPA and PAI-1 in detergent extracts of breast cancer tissues.

Automated assay for HER-2/neu in serum.

BACKGROUND: The extracellular domain of the HER-2/neu oncogene product is increased in sera of some patients with epithelial cancers. Our aim was to develop an automated serum assay for the extracellular domain of the HER-2/neu protein. METHODS: We used a monoclonal antibody labeled with fluorescein for capture and a monoclonal Fab' fragment labeled with alkaline phosphatase for detection. Separation of bound and free detection conjugate was performed with magnetizable particles coated with monoclonal antibody to fluorescein. Alkaline phosphatase activity was measured kinetically at 405 or 450 nm. RESULTS: The assay was linear from 0.1 to 250 microg/L. No hook effect was evident up to 10 000 microg/L. Within-run imprecision (CV) was 0.8-1.2%, and total imprecision was 1.1-1.7%. Cross-reactivity with human epidermal growth factor receptor, which has extensive homology with HER-2/neu extracellular domain, was <0.6%. Human anti-mouse antibodies, heterophilic antibodies, and rheumatoid factor did not interfere, nor did the therapeutic monoclonal antibody Herceptin((R)). In 51 healthy females, the mean value was 9.3 microg/L with a range of 6.4-14.0 microg/L. No reagent lot-to-lot variability was detected over four lots of reagents tested. CONCLUSION: The Bayer Immuno 1(TM) assay for HER-2/neu was precise and resistant to interferences, characteristics that are essential for longitudinal monitoring of cancer patients.

Prediction of response to antiestrogen therapy in advanced breast cancer patients by pretreatment circulating levels of extracellular domain of the HER-2/c-neu protein.

PURPOSE: Overexpression of the HER-2/c-neu/c-erbB2 proto-oncogene is associated with a worse prognosis in patients with breast cancer, perhaps due to an association of the HER-2 proto-oncogene protein with resistance to hormone and/or chemotherapy. Circulating levels of the extracellular domain (ECD) of the HER-2/c-neu-related protein (NRP) are elevated in 20% to 40% of patients with metastatic breast cancer. We investigated whether pretreatment levels of NRP predict response to hormone therapy (HT). MATERIALS AND METHODS: Circulating NRP levels were determined in 94 patients who participated in a randomized trial of three different doses of the antiestrogen, droloxifene (DRO), as first-line HT for metastatic breast cancer. RESULTS: NRP levels were elevated (> or = 5,000 U/mL) in 32 of 94 patients (34%). Only three of 32 patients (9%) with elevated NRP levels responded to DRO, compared with 35 of 62 (56%) with nonelevated NRP levels (P = .00001). Low pretreatment NRP level was the most powerful predictor of response to DRO (odds ratio of response, 22.4; P = .0001). Elevated pretreatment NRP levels were also associated with a shorter time to progression (TTP) and survival duration. CONCLUSION: Pretreatment circulating NRP levels predict a low likelihood of benefit from HT, specifically DRO, in patients with estrogen receptor (ER)-positive and/or progesterone receptor (PgR)-positive or receptor-unknown metastatic breast cancer, even when adjusted for other known predictive factors, such as ER and/or PgR levels, site of disease, disease-free interval from primary treatment to recurrence, and prior adjuvant chemotherapy. These data suggest that pretreatment NRP levels may be useful in deciding whether to treat a patient who otherwise appears to be likely to respond to HT.

Effects of steroids on mouse oocyte maturation in vitro.

Oestrone and dihydrotestosterone had no significant action. Other steroids inhibited maturation. The stage of maturation most affected and the median effective concentration (MEC) at this stage varied with different steroids. The predominant effect of pregnenolone (MEC = 6.4 microM), progesterone (MEC = 5.3 microM), androstenedione (MEC = 28.0 microM) and oestradiol-17 beta (MEC = 23.0 microM) was to block maturation after the resumption of meiosis but before completion of the first meiotic division. Testosterone was also effective at this stage (MEC = 23.5 microM) but at higher concentrations it prevented germinal vesicle breakdown (MEC = 40 microM) without causing oocyte degeneration. The inhibitory actions of all steroids were reversible in oocytes exposed for 4 or 18 h.

Effect of hypophysectomy on mouse oocyte maturation in vitro.

There was no difference in frequency of maturation of oocytes obtained from mice hypophysectomized for 2 weeks compared to those from sham-operated or untreated (control) animals of the same age. By 7 weeks, and also at 12 and 17 weeks, the incidence of polar body formation in vitro was significantly reduced. The number of oocytes which remained meiotically inactive in culture was increased at 7, 12 and 17 weeks after hypophysectomy. This decrease in spontaneous oocyte maturation in vitro could be partly overcome by administering exogenous PMSG, oestradiol-17beta or PMSG + oestradiol-17beta, but not progesteron or hCG, to hypophysectomized mice.

The effect of cold shock on mouse oocyte maturation in vitro.

Maintenance of mouse ovaries in ice-cold medium for 1, 2 or 4 h before removing the oocytes did not alter the incidence of spontaneous maturation or increase the number of oocytes degenerating, fragmenting or forming 2 equal blastomeres in culture.