Enzymatic and non-enzymatic treatments for antigen unmasking on formalin-fixed, paraffin-embedded, dewaxed sections were optimized and compared by the use of a panel of antibodies of diagnostic relevance (anti-cytokeratins, vimentin, S-100, T- and B-cell receptors, Ki-67/MIB 1, muscle actin). Non-enzymatic unmasking was obtained by boiling the slides in a microwave oven in 0.01 M salt solution (pH 6) or in 6 M urea. Trypsin or pronase digestion was used for comparison and found to be necessary for some of the reagents. The investigation was then extended to 256 antibodies; the epitopic amino acid sequence was known for 48 of them. We found that enzymatic and non-enzymatic antigen unmasking are not dependent on the epitope sequence, but some antigens benefit selectively from one treatment but not from the other. Denaturation of proteins is the likely mechanism which leads to immunodetection on microwave oven-boiled slides; this suggestion is supported by the use of denaturating solutions and by the observation that endogenous enzymes were inactivated and a few antigens were no longer immunodetectable after boiling. Non-enzymatic methods for antigen unmasking are a powerful new tool for broadening the use of antibodies for immunostaining formalin-fixed, paraffin-embedded sections and should be used in parallel with the traditional enzymatic methods.

Immunohistochemical evaluation of 200 primary breast cancers with the anti-p53 mouse monoclonal antibody (MAb) PAb421 showed positivity in nuclei of malignant cells in 31 cases (15.5%). PAb421+ cases were significantly more frequently epidermal growth factor receptor (EGF-R)-positive (67.7%; p less than 0.001) and estrogen receptor (ER)-negative (73.3%; p less than 0.001); they displayed surface histocompatibility class-1 (80.6%; p less than 0.01) and 11 (74.2%; p less than 0.05) antigens. Low values for progesterone receptor (mean 67.20 +/- 25.2 fmol/mg; p less than 0.05) and a high number of cells positive for the proliferation-associated antigen Ki-67 (log mean 6.88 +/- 0.33; p less than 0.01) were found in PAb421+ tumors as well as a high number of grade-3 infiltrating duct carcinomas (70%; p = 0.01). Of the 200 cases of mammary carcinoma, 88 were further analyzed using another human specific anti-p53 MAb PAb1801, and 40 (45.5%) were found positive. This MAb stained all the PAb421+ cases and was significantly associated with negative ER status (39.5%; p less than 0.05) and high Ki-67 scores (log mean 6.93 +/- 0.24; p = 0.001). Analysis of PAb1801+/Pab421- cases for HLA antigens, EGF-R and ER showed a phenotype similar to that of the p53-ve/ER+ carcinomas, except for the high Ki-67 score. No differences in age of the patient, number of involved nodes, tumor size, ploidy or labelling index scores were evident between p53+ and carcinomas. We concluded that p53 in mammary carcinomas is associated with ER-negative, growth factor receptor-positive, high-grade tumors, and is a promising new parameter to evaluate the cellular biology and prognosis of breast cancer.

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