Maturation of the breast epithelial mucin has strong correlation with prognosis for infiltrating ductal carcinoma of the breast (IDC). This maturation mechanism, or its alterations, was studied with MoAbs in immunohistochemistry of sections of paraffin-embedded blocks of the patient's primary breast lesion. Mutltivariate statistical analyses using the Cox proportional-hazards model were performed, employing the novel EGRET software program. These analyses, indicated that strong predictive correlations exist between immunohistochemical patterns representing altered mucin maturation, and both relatively poor prognosis of survival and short-relapse time. An individualized risk factor procedure for each patient was developed that merged immunohistochemical findings and traditional prognostic variables. These results support the use of molecular maturation of breast epithelial antigens (BEAs) as valid independent prognostic markers. Since MoAbs were the probe used to detect prognostic markers on BEAs and their point of interaction is specific epitopes on them, then, strictly speaking, molecular maturation implies also epitope maturation. In this renewal application we will study the use of epitopic maturation as our prognostic marker at 3 different levels: 1) At the epitope level, with recently created to BEAs that had modified epitope structure. These studies, coupled to our knowledge of epitopic structure, will indicate the interactive contribution to MoAb binding of both the amino acid component and carbohydrate moieties of the epitope. Results could provide new, simple to use prognostic markers for immunohistopathology analogous to those that we have already developed in this grant. ii) At the mRNA level, by analyzing the contribution of the core polypeptides of BEAs to prognosis. The content of mRNA for the breast epithelial mucin, 70 and 46 kDa 'core polypeptides' will be assessed in histological sections by in situ hybridization and its use as a prognostic tool will be explored. The in situ hybridization will be performed with cDNA probes that we have developed. Correlations with prognosis will be evaluated in a retrospective study. iii) At the oligosaccharide level, by investigating the contribution of oligosaccharides that are known to undergo maturational and possibly neoplasia-associated changes. We will use different markers of oligosaccharide synthesis available to include: cDNA probes for key glycosyltransferases in in situ hybridization; Abs against enzymes critical to oligosaccharide elongation (anti-mannosidase II); lectins that will identify the degree of maturation of oligosaccharide structure (L-PHA,ECA and H.pomatia); and MoAbs that identify unfinished O-linked oligosaccharides (B72.3, 1E3 and 236.1R6). These maturational changes in the oligosaccharides will be correlated to binding of specific anti-mucin, anti-70 and anti-46 kDa MoAbs for which amino acid and carbohydrate level of contribution are known. Correlations will be explored between oligosaccharide maturation, enzyme cDNA levels and prognosis for both survival and relapse. This experimentation will characterize the different maturational forms of epitopes in the glycoproteins that constitute BEAs and then dissect the elements that contribute to this phenotype. It has as a goal to obtain prognostic marker for use in immunohistopathology of breast cancer that can participate in a composite multivariate panel that can separate as many statistically distinct risk groups as possible from among a breast cancer patient population.
