Introduction

The p53 protein is the product of a tumor suppressor gene located on the human chromosome 17, thought to regulate the proliferation of normal cells. Mutations of p53 gene have been reported in human breast carcinoma,^1-4 especially in more advanced and/or more aggressive tumors.^5-7 Molina et al⁸ recently observed p53 positivity in 37.3% of 655 human breast carcinomas. These authors reported no significant correlation between p53 expression and tumor size, nodal involvement, or histologic type. However, in another study, Falette et al⁹ found that tumors carrying p53 alterations had a highly aggressive behavior and that the presence of altered p53 was an independent prognostic marker of early relapse and death. The p53 immunoreactivity correlated with tumor type in only a few instances, with 40% to 50% of medullary breast carcinomas expressing p53 oncoprotein.^10,11

The expression of Bcl-2 protein has also been reported in breast cancer^12-14 without correlation to tumor type. This protein is capable of preventing apoptosis and promoting tumor development. Leek et al¹⁵ found a direct correlation between Bcl-2 expression and the presence of estrogen receptors in breast carcinoma. These authors also noticed no correlation between Bcl-2 protein expression and nodal status, tumor size, or differentiation. Others have reported strong Bcl-2 expression in small, estrogen-receptor-positive, slowly proliferating, and p53-negative tumors.¹⁶ Interestingly, it has been demonstrated that in human breast carcinoma cell lines, a mutated and/or wild-type p53 downregulates Bcl-2 expression.¹⁷

To further investigate the role of these biomarkers in the progression of breast ductal neoplasia, we analyzed the expression levels of p53 and Bcl-2 by immunohistochemistry in a group of 26 invasive human breast carcinomas. We also evaluated the relationship between these markers and the expression of estrogen and progesterone receptors.

Materials and Methods

Twenty-six human breast carcinoma specimens from surgical resections performed at H. Lee Moffitt Cancer Center & Research Institute (Tampa, Fla) and Cooper University Hospital (Camden, NJ) were obtained. All the tumors were infiltrating ductal carcinomas. One case had focal lobular features, and seven exhibited medullary features. Clinical information including age, sex, tumor type, grade, stage, and therapy were also obtained from the medical records.

Immunohistochemical detection of p53 and Bcl-2 proteins was performed on formalin-fixed, paraffin-embedded sections using the avidin biotin peroxidase complex technique (Vectastatin Elite ABC Kit, Vector Laboratories, Inc, Burlingame, Calif) following the manufacturer’s instructions. For the detection of p53, a murine monoclonal antibody, clone DO1

(Santa Cruz Laboratories, Calif), directed against a denaturation-resistant epitope of human p53 located between amino acid 37 and 45 was used at a 1:500 dilution in all cases. The primary antibody used for the detection of Bcl-2 product was Bcl-2 DAKO M887, 124 (Dako Corp, Carpenteria, Calif) (dilution 1:500 in phosphate-buffered saline with 1% bovine serum albumin). This antibody was applied to sections after microwave antigen retrieval, as previously described.

Positive and negative controls were performed at the same time for each section. Controls for specificity included incubation of the tissue sections with unrelated primary mouse monoclonal antibodies, with unrelated secondary antimouse monoclonal antibodies, and with phosphate-buffered saline. Reactions were observed and evaluated by two of us (D.C. and E.C.) according to the intensity and percentage of cell staining independently. Nuclear or cytoplasmic staining was assigned a numerical value of 3 for strong staining, 2 for moderate staining, or 1 for weak staining. The percentage of positively stained cells were segregated into the following groups: 0 to 33% into group 1, 34% to 66% into group 2, and 67% to 100% into group 3.

As the variables analyzed were ordinal, associations were assessed by Spearman’s Rho, a nonparametric analog to the Pearson correlation coefficient. All P values reported are two-sided.

Results

The Table indicates the clinicopathologic features and results of the immunohistochemical stains. The age of the patients ranged between 38 to 88 years (mean = 57 years; median = 58 years). The size of their tumors ranged from 0.7 cm to 4.0 cm (mean = 1.9 cm; median = 1.7 cm). Following the Bloom and Richardson grading system, 6 invasive carcinomas were low grade, 13 were intermediate grade, and 7 were high grade. Disease stage was as follows: 12 patients had stage I, 7 patients had stage IIA, 2 patients had stage IIB, and 5 patients had stage IV. Three patients received chemotherapy after surgery; in 9 patients, both radiation and chemotherapy were given after resection. Fifteen tumors expressed both estrogen and progesterone receptors, 5 tumors expressed estrogen receptors only, and 6 tumors were negative for both. At follow-up, only one patient died of disease 36 months after surgery. Of the others, 4 were alive with disease (mean survival = 28 months; median = 31 months), and 19 were alive with no evidence of disease (mean survival = 38 months; median = 82 months). Two patients were lost to follow-up.

Moderate to strong (2 to 3) nuclear p53 immunoreactivity was present in 13 (50%) of 26 infiltrating ductal carcinomas (Fig 1), the other 13 tumors being p53 negative. Similarly, Bcl-2 cytoplasmic protein expression was moderate to strong (2 to 3) in 14 (54%) of 26 tumors. Seven carcinomas were only weakly Bcl-2 positive, and this stain was negative in 5 cases (Fig 2). An inverse correlation between p53 and Bcl-2 proteins expression was noted in 12 cases but was not statistically significant (rho = –0.35; P = .08).

In 7 cases (27%), negative or weak (1) Bcl-2 stain was detected in the presence of strong p53 positivity, but in 7 other cases (27%), the reverse was true. Furthermore, moderate to high expression of both oncoproteins was present in 6 (23%) of the 26 cases. The normal mammary duct epithelium and the hyperplastic ducts, when present adjacent to the tumor, were intensely decorated with Bcl-2 protein antibody (Fig 3) but were p53