Sixteen research groups participated in the ISOBM TD-4 Workshop in which the reactivity and specificity of 56 monoclonal antibodies against the MUC1 mucin was investigated using a diverse panel of target antigens and MUC1 mucin- related synthetic peptides and glycopeptides. The majority of antibodies (34/56) defined epitopes located within the 20-amino acid tandem repeat sequence of the MUC1 mucin protein core. Of the remaining 22 antibodies, there was evidence for the involvement of carbohydrate residues in the epitopes for 16 antibodies. There was no obvious relationship between the type of immunogen and the specificity of each antibody. Synthetic peptides and glycopeptides were analyzed for their reactivity with each antibody either by assay of direct binding (e.g. by ELISA or BiaCore) or by determining the capacity of synthetic ligands to inhibit antibody binding interactions. There was good concordance between the research groups in identifying antibodies reactive with peptide epitopes within the MUC1 protein core. Epitope mapping tests were performed using the Pepscan analysis for antibody reactivity against overlapping synthetic peptides, and results were largely consistent between research groups. The dominant feature of epitopes within the MUC1 protein core was the presence, in full or part, of the hydrophilic sequence of PDTRPAP. Carbohydrate epitopes were less easily characterized and the most useful reagents in this respect were defined oligosaccharides, rather than purified mucin preparations enriched in particular carbohydrate moieties. It was evident that carbohydrate residues were involved in many epitopes, by regulating epitope accessibility or masking determinants, or by stabilizing preferred conformations of peptide epitopes within the MUC1 protein core. Overall, the studies highlight concordance between groups rather than exposing inconsistencies which gives added confidence to the results of analyses of the specificity of anti-mucin monoclonal antibodies.
Background: The primary aim of the study was to reduce interference in an in-house two-site, two-step immunometric assay. Methods: In the running laboratory routine, 11 261 samples were tested with a carcinoembryonic antigen (CEA) assay with bovine immunoglobulin but no murine immunoglobulins in the buffer, in parallel to our routine CEA assay, using 15 mg/L heat-treated nonspecific murine immunoglobulin (MAK33) in the buffer and with the Fc fragments removed from the capture antibody. Results: The frequency of interference was estimated to be 4.0% (95% confidence interval, 3.3–4.7%). The addition of 15 mg/L native MAK33 had little effect (frequency, 3.9%; 95% confidence interval, 3.2–4.6%), whereas adding 15 mg/L heat-treated MAK33 reduced interference to 0.86% (0.61–1.12%), and adding 50 mg/L reduced it further to 0.06% (0–0.13%). Removing the Fc fragments by itself reduced interference to 0.10% (0.02–0.19%). There were no statistically significant differences for age (P <0.23) or gender (P <0.40) between patients with interference (n = 210) and a randomly selected interference-negative control group (n = 186). Interference was not constant in patients: 15 of 25 individuals positive for interference and with four or more samples screened for interference had an interference-negative sample either before or after the peak of interference. Conclusions: In a two-site, two-step immunometric assay using mouse monoclonal antibodies, use of heat-treated nonspecific murine immunoglobulin in the buffer or removal of the Fc fragment from the capture antibody could improve performance.
The serum tumor markers CA 125, MUC1 and CEA were measured in 221 breast cancer patients over a period of 2 years. Patients examined on at least three occasions were included in the study. Thirty-three patients had increasing or continuously high concentrations of CA 125. Thirty (91%) of these had involvement of the pleura, either as pleural metastasis or metastasis in surrounding tissue i.e. bone structures in the thorax cavity or lung parenchyma. MUC1 and CEA were elevated in 27 (82%) and 24 (73%) of the 33 patients, respectively. Increased concentrations of these two markers did not relate to the site of metastasis. However, the three tumor markers complemented each other in detecting early metastases. Increased CA 125 was associated with metastasis in or near the pleura, and in stage IV breast cancer it was related to poor prognosis.
The aim of the present study was to establish a robust, reliable and fully automated immunofluorometric assay for the breast cancer serum marker MUC1. This would further serve as a prototype assay for evaluation of other MUC1 assays based on new antibody combinations. Using time-resolved fluorescence as tracer signal we developed an automated immunofluorometric assay for MUC1 (MUC1 IFMA). This assay was compared with two commercial assays. The CA15-3 EIA (CanAg) which use the same antibodies as the MUC1 IFMA, and the ETI-CA-15-3 K (Sorin) which use the original antibodies defining the CA 15-3 assay. The three assays showed comparable results. The coefficient of variation was below 10% from 9 to 2,400 kU/l for the MUC1 IFMA, from 15 to 250 kU/l for the CA15-3 EIA, and from 25 to 200 kU/l for the ETI-CA-15-3 K assay. At a specificity of 0.94 the overall diagnostic sensitivities for the MUC1-IFMA, CA15-3 EIA and ETI-CA-15-3 K assays were 0.40, 0.37, and 0.38, respectively. When applied to metastatic breast cancer, all assays had sensitivities close to 0.80. There was a close correlation (Spearman rank = 0.99) between results from the new assay and the CA15-3EIA. The new automated assay was not strictly immunometric as we could not achieve conditions where solid phase or tracer antibodies were in apparent excess. However, the assay performed well at a wide range of assay conditions. The automation, which minimizes imprecision in pipetting and handling of samples, and the high capacity of the AutoDELFIA instrument enabling measurement of all samples in a single run, were important aspects for establishing a reliable assay. The principle of the new automated immunofluorometric assay will be used as a rapid and reliable evaluation of a wide range of monoclonal antibody combinations in our search for the optimal MUC1 assay. This new automated immunofluorometric assay will be useful in the rapid and reliable evaluation of a wide range of monoclonal antibody combinations in our search for the optimal MUC1 assay.
Breast cancer is one of the most common sources of brain metastases. After clinical detection of such tumour spread, median survival is usually only a few months, with brain metastases being the major cause of death (Boogerd et af., 1993). Between 20 and 30% of breast-cancer patients have metastatic brain lesions at time of death (Lee, 1985). Moreover, the established modes of treatment for brain metastases give 1-year survival rates of less than 20%. Clearly there is a need to further our understanding of the biology and treatment of brain metastasis in breast cancer.It is well known that some cancers metastasise in a selective manner to organs distant from their primary tumour site. Breast-cancer metastasis to the brain is an example of this pattern of organ colonization. Our understanding of such selective metastasis has been assisted by the use of animal models, which also provide a valuable resource in developing novel forms of treatment. In the establishment of such models, the nude mouse has proved to be an excellent host for many human tumour xcnografts (Fodstad, 1991), while maintaining good conservation of histological and phenotypic characteristics of the original tumour (Fidler, 1986). However, breast tumours and breast-derived cell lines remain among the most exacting to establish and transplant successfully into cxpcrimental animals (Engel and Young, 1978;Fodstad, 1991;Murthy et al., 1995). Indeed. the transplantability or "take rate" of breast tumours is estimated to be less than 10% (Fodstad, 1991). Consequently. reproducible brain metastases from breast carcinomas have been demonstrated in only a few studies using an intracarotid injection route in mice (Schackert et al., 1989; Zhang et al., 1991, 1992). Although this injection model for brain metastases appears to be successful, there arc certain limitations. Moreover, the relatively small number of reports suggests that additional experimental models of brain metastasis from breast cancer are required. In the present report, we describe a reproducible model for brain metastases in nude mice using a new human breast-cancer cell linc, MA11. Compared with other studies, our experimental model seems to more closely reflect the clinical situation and, together with the relatively simple injection procedure, should favour its general application for biological and therapeutic research. MATERIAL AND METHODS PatientA 65-year-old Caucasian female attending the Department of Gynaecology, Heidelberg, underwent surgery for removal of a breast lump with axillae dissection in September 1993. Subsequent histological diagnosis confirmed a grade-I1 invasive lobular carcinoma (classic tubular-lobular type) 3 cm in diameter, and one axillary-lymph-node metastasis was found. TNM tumour staging was thus classified as Tz NI Mo (according to the UICC AJC clinical staging system). Cytological analyses of the primary tumour indicated a diploid-cell population with an S-phase level of 4.1%. The tumour was positive for the hormone receptors ER (50 pmol/g) and PgR (23 pm...
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