Microsatellite instability (MSI) and frameshift mutations in genes containing nucleotide repeats have been reported in a subset of colorectal and gastric carcinomas. This study describes the analysis of MSI-positive colorectal (39 cases) and gastric carcinomas (36 cases) for the presence of frameshift mutations of the six genes known to be involved in DNA repair and containing mononucleotide repeats in their coding region. Our mutational study of the 75 MSI-positive tumors revealed frequent mutations in hRAD50 (23 cases, 31%), BLM (16 cases, 21%), and hMSH6 (16 cases, 21%); rare mutations in BRCA1 (1 case, 1%) and ATM (3 cases, 4%); and no mutation in NBS1. In contrast, no frameshift mutation was found in 60 MSI-negative colorectal and gastric carcinomas. The mutation of hRAD50, a gene that is involved in the response to cellular DNA damage and forms a complex with hMRE11 and NBS1, has not been reported previously. Our results suggest that frameshift mutations of hRAD50, BLM, and hMSH6 are selected and play a role in the tumorigenesis of colorectal and gastric carcinomas with MSI. The MSI targeting of the hRAD50 and BLM genes represents an additional link between MSI and DNA repair because alteration of these genes could accelerate defective DNA repair.

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 PDTRAPAP. 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 antimucin monoclonal antibodies.

We have tested the reactivity of the monoclonal antibodies submitted to the ISOBM TD-4 Workshop for reactivity with a hybrid molecule consisting of the bacterial beta-galactosidase and most of the extracellular nonrepeat domain of MUC1/episialin. Two monoclonal antibodies submitted to the Workshop, 232A1 and M29, were directed against the protein moiety of this domain as shown by immunoblotting and immunoprecipitation.

Episialin, also designated MUC1, CA 15-3 antigen and PEM, is an established serum marker for breast cancer. Its function and possible involvement in tumor progression has not yet been completely established. The molecule is an extended rod-like molecule protruding high above the cell surface. It is often highly overexpressed in breast cancer relative to normal breast epithelium cells. Overexpression of episialin on cells in vitro reduces cell-cell and cell-extracellular matrix adhesion, because the rod-like molecule masks the adhesion receptors. Episialin also exerts its anti-adhesion effect in vivo. In certain human tumors, where episialin was present at the basal side of the cell, abnormal contacts between the plasma membrane and the stroma were observed. As a consequence of its anti-adhesion properties, episialin overexpression reduces the sensitivity of the cells for cytotoxic lymphocytes. This might be one of the reasons why episialin transfected cells are more potent to form experimental metastases after i.v. injection into nude mice.

We have investigated the role of episialin, a surface bound mucin also known as EMA, PEM, CA 15-3 etc. encoded by the MUCI gene, in tumor progression. The molecule has an extended rod-like structure protruding more than 200 nm above the plasma membrane. The expression of the molecule is usually more than ten times that in normal epithelia as determined by in situ hybridization and can be extremely high on metastatic cells present in pleural effusions. Episialin overexpression strongly reduces cell-cell and cell-matrix adhesion. The anti-adhesion properties of the molecule are due to the extreme length of the molecule since genetically modified molecules with a reduced length did not exhibit the anti-adhesion effect. E-cadherin/episialin double transfectants showed that episialin can prevent E-cadherin mediated cell-cell adhesion. Decreased E-cadherin mediated cell-cell interactions are known to promote invasion. Episialin overexpression is expected to have the same effect. Indeed, episialin overexpression promoted invasion in matrigel. Episialin overexpression at the cell-stroma boundary in primary breast cancers caused large "clefts" between the stroma and tumor cells. These results suggest that episialin has the same anti-adhesion properties in vivo. Episialin also interfered with immune recognition. Melanoma transfectants expressing high levels of episialin were less susceptible to lysis by LAK cells and allogeneically stimulated T-lymphocytes. The same transfectants had a significantly higher propensity to form lung metastases after i. v. injection in nude mice than episialin negative revertants of the same clones. Episialin may protect the tumor cells against NK cells and/or episialin expressing cells are more likely to metastasize as a result of the decreased cell-cell interactions. Our results strongly suggest that episialin overexpression is an important factor in tumor progression. We have investigated the role of episialin, a surface bound mucin also known as EMA, PEM, CA 15-3 etc. encoded by the MUCI gene, in tumor progression. The molecule has an extended rod-like structure protruding more than 200 nm above the plasma membrane. The expression of the molecule is usually more than ten times that in normal epithelia as determined by in situ hybridization and can be extremely high on metastatic cells present in pleural effusions. Episialin overexpression strongly reduces cell-cell and cell-matrix adhesion. The anti-adhesion properties of the molecule are due to the extreme length of the molecule since genetically modified molecules with a reduced length did not exhibit the anti-adhesion effect. E-cadherin/episialin double transfectants showed that episialin can prevent E-cadherin mediated cell-cell adhesion. Decreased E-cadherin mediated cell-cell interactions are known to promote invasion. Episialin overexpression is expected to have the same effect. Indeed, episialin overexpression promoted invasion in matrigel. Episialin overexpression at the cell-stroma boundary in primary breast cancers caused large "clefts" between the stroma and tumor cells. These results suggest that episialin has the same anti-adhesion properties in vivo. Episialin also interfered with immune recognition. Melanoma transfectants expressing high levels of episialin were less susceptible to lysis by LAK cells and allogeneically stimulated T-lymphocytes. The same transfectants had a significantly higher propensity to form lung metastases after i. v. injection in nude mice than episialin negative revertants of the same clones. Episialin may protect the tumor cells against NK cells and/or episialin expressing cells are more likely to metastasize as a result of the decreased cell-cell interactions. Our results strongly suggest that episialin overexpression is an important factor in tumor progression.