Failure pattern and survival after breast conserving therapy. Long-term results of the Danish Breast Cancer Group (DBCG) 89 TM cohort.

UNLABELLED: Based on the results from the DBCG 82 trial, breast conserving therapy (BCT) has been implemented as standard in Denmark since 1989, and today constitutes more than 70% of the primary treatment. Our aim was to evaluate the implementation of BCT as a routine procedure in patients treated according to the DBCG 89 program and compare recurrence pattern and survival both overall and when separated in age groups, with the results from the randomized DBCG 82 TM trial. MATERIAL AND METHODS: A total of 1847 patients treated between 1989 and 1999 were included in a retrospective population-based cohort study. Data from the DBCG database were completed via search through the Danish Pathology Data Bank and medical records. RESULTS: Median follow-up time was 17 years. At 20 years the cumulative incidences of local recurrence (LR) and disease-specific mortality (DSM) were 15.3% and 25.8%, respectively. Twenty-year overall survival (OS) and recurrence-free survival were 63.7% and 43.1%, respectively. Subdivided by age groups cumulative incidences at 20 years were LR: 18.9%, 10.5% and 12.4%, and DSM: 28.9%, 18.9% and 28.4% in young (≤45 years), middle-aged (46-55 years) and older (≥56 years) women, respectively. In an adjusted analysis age maintained a significant and independent effect on both LR and DSM. CONCLUSION: The DBCG 82 TM program was successfully implemented. The women treated with BCT in the DBCG 89 program displayed equal failure pattern and improved survival in comparison with women from the DBCG 82 TM protocol. Occurrence of first failure and mortality varied with age; demonstrated by increased risk of LR, DM and DSM in the young patients and increased risk of DM and DSM in the older patients, compared to the middle-aged patients.

Stereological quantification of mast cells in human synovium.

Mast cells participate in both the acute allergic reaction as well as in chronic inflammatory diseases. Earlier studies have revealed divergent results regarding the quantification of mast cells in the human synovium. The aim of the present study was therefore to quantify these cells in the human synovium, using stereological techniques. Different methods of staining and quantification have previously been used for mast cell quantification in human synovium. Stereological techniques provide precise and unbiased information on the number of cell profiles in two-dimensional tissue sections of, in this case, human synovium. In 10 patients suffering from osteoarthritis a median of 3.6 mast cells/mm2 synovial membrane was found. The total number of cells (synoviocytes, fibroblasts, lymphocytes, leukocytes) present was 395.9 cells/mm2 (median). The mast cells constituted 0.8% of all the cell profiles present in the synovium. A significantly positive correlation was demonstrated between the number of mast cells and the total number of cells. Thus, the present study reports stereological quantification of the mast cells and the total number of cells in synovium from patients with osteoarthritis. A possible link between the mast cell and osteoarthritis is discussed upon obtaining a precise estimate of cell profiles in human synovium.

Purification of human colonic and gastric mast cells.

We have developed a method to purify mast cells from enzymatic isolates of human colonic mucosa (HCM) and submucosa/muscle (HCS), and gastric mucosa (HGM) and submucosa/muscle (HGS). The purification of mast cells from these enzymatic isolates involves positive affinity-magnetic selection of mast cells using a monoclonal antibody specific for the c-kit receptor tyrosine kinase (CD117). The monoclonal antibody is coupled to Dynabeads for positive affinity selection of c-kit receptor positive cells which includes mast cells. This selection procedure generates preparations of mast cells from HCM, HCS, HGM and HGS that are 80% pure. The purified mast cells were microscopically normal and viable (> 85%). The functionality of purified mast cells was examined by studying the effect of anti-human IgE, Concanavalin A (Con A) and calcium ionophore A23187 on histamine release. These results show that this purification procedure generates microscopically normal, viable and functional mast cells. This method of purifying human gastrointestinal tissue mast cells may be a valuable tool for the further study of mast cell heterogeneity and the role of mast cells in the gastrointestinal tract.

Mast cells and atopic dermatitis. Stereological quantification of mast cells in atopic dermatitis and normal human skin.

Stereological quantification of mast cell numbers was applied to sections of punch biopsies from lesional and nonlesional skin of atopic dermatitis patients and skin of healthy volunteers. We also investigated whether the method of staining and/or the fixative influenced the results of the determination of the mast cell profile numbers. The punch biopsies were taken from the same four locations in both atopic dermatitis patients and normal individuals. The locations were the scalp, neck and flexure of the elbow (lesional skin), and nates (nonlesional skin). Clinical scoring was carried out at the site of each biopsy. After fixation and plastic embedding, the biopsies were cut into 2 microns serial sections. Ten sections, 30 microns apart, from each biopsy were examined and stained alternately with either toluidine blue or Giemsa stain and mast cell profile numbers were determined. The study yielded the following results: (1) in atopic dermatitis lesional skin an increased number of mast cell profiles was found as compared with nonlesional skin, (2) comparing atopic dermatitis skin with normal skin, a significantly increased number of mast cell profiles per millimetre squared was found in specimens from the neck, (3) staining with toluidine blue yielded a lower number of mast cell profiles than Giemsa staining, (4) the use of Carnoy's fixative resulted in a lower mast cell profile count than the use of formaldehyde, and (5) there was no statistically significant correlation between the clinical score and the number of mast cell profiles per millimetre squared. Using stereological techniques, this study indicated that mast cells might participate in the inflammatory process in skin leading to atopic dermatitis.

Histamine releasing cells of the newborn. Mast cells from the umbilical cord matrix and basophils from cord blood.

The cellular branch of the immune defence in the newborn has been shown to differ from adults in a number of ways. This report presents new data on the functions of the histamine-secreting cells of the newborn. Mast cells of the newborn were obtained from the human umbilical cord by enzymatic dispersion. The granules of the mast cells of the umbilical cord were found to contain both chymase and tryptase by immunohistochemical staining, and the presence of cell-bound IgE on the mast cell surface was demonstrated by staining sections of umbilical cord with peroxidase-conjugated anti-IgE. The enzymatic dispersion yielded 12,660 mast cells per gram umbilical cord (median), range 2,500-60,300 (n = 48). The mast cells were found to constitute 3.1% of the total nucleated cells in the dispersate (median), range 1.5-3.8%. The histamine release from these cells was measured using a glass microfibre-based method. Both the umbilical cord mast cells and the cord blood basophils released histamine stimulated with anti-IgE, concanavalin A and the calcium ionophore A23187. In contrast to mast cells from adult tissue, the phorbol ester TPA was found to be an efficient secretagogue in both mast cells and basophils from the newborn. After maximal stimulation with anti-IgE and phorbol ester the quantity of histamine released per millilitre of blood was significantly higher in cord blood than in adult blood. The spontaneous histamine release from cord blood basophils was also significantly higher than from adult blood basophils. The mast cells found in the umbilical cord matrix and the cord blood basophils represent a readily available source of metabolically active histamine releasing cells for exploration of the role of histamine-secreting cells in newborn immune defence.

Demonstration of immunogenic keratan sulphate in commercial chondroitin 6-sulphate from shark cartilage. Implications for ELISA assays.

The prototype monoclonal keratan sulphate (KS) antibody 5D4 that is widely used for detection of KS in tissues and biological fluids reacts strongly with commercial low grade shark cartilage chondroitin 6-sulphate. Characterization of the immunogenic material by chondroitinase ABC digestion, ELISA inhibition studies, immunoblotting and HPLC analyses confirmed the presence of substantial amounts of KS, probably as a large proteoglycan (> 120 kDa). Commercial and heterogenic glycosaminoglycan preparations therefore must be used with great caution in immunological analyses. On the other hand the shark cartilage chondroitin 6-sulphate is an easy accessible source of immunogenic KS that can be used as a reference standard and as coating antigen in KS-ELISAs. The concentration of immunogenic KS in synovial fluid measured with an ELISA based solely on reagents of shark cartilage chondroitin 6-sulphate correlated well (r = 0.90) with the concentrations obtained with a traditional KS-ELISA that uses purified aggrecan as standard and coating antigen, and KS in both serum and synovial fluid could be measured with sufficient linearity.