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 cancer12-14 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 manufacturers 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 Spearmans 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.
|
Clinicopathologic
Features and Results of the
Immunohistochemical Stains in 26 Breast Cancers
|
| |
|
|
|
Tumor
|
|
|
Immunostain |
Treatment |
|
| Case |
Age |
Tumor
Type |
Tumor
Size (cm) |
Grade
|
Stage |
Progesterone
Receptors |
Estrogen
Receptors |
Bcl-2 |
p53 |
Chemo-therapy |
Radiation
Therapy |
Follow-up
After Surgery (mos) |
| 1 |
71 |
D |
1.5 |
2 |
IV |
|
+ |
2 |
2 |
+ |
+ |
AWD (48) |
| 2 |
41 |
M |
1.0 |
2 |
I |
+ |
+ |
1 |
0 |
|
|
NED (20) |
| 3 |
88 |
D |
2.0 |
2 |
IIA |
+ |
+ |
2 |
2 |
+ |
|
NED (52) |
| 4 |
44 |
D |
1.8 |
2 |
IIA |
+ |
+ |
1 |
3 |
|
|
NED (8) |
| 5 |
47 |
M |
1.5 |
2 |
I |
|
+ |
0 |
3 |
|
|
NED (29) |
| 6 |
54 |
D |
2.5 |
2 |
IV |
|
|
3 |
3 |
+ |
|
DOD (36) |
| 7 |
42 |
M |
1.5 |
1 |
I |
+ |
+ |
0 |
3 |
|
|
NED (60) |
| 8 |
50 |
M |
2.0 |
1 |
I |
|
+ |
0 |
3 |
|
|
NED (156) |
| 9 |
47 |
D |
1.5 |
3 |
I |
|
|
3 |
0 |
|
|
LTF |
| 10 |
58 |
D |
3.5 |
1 |
IIA |
|
|
0 |
0 |
|
|
NED (72) |
| 11 |
45 |
M |
1.5 |
1 |
I |
+ |
+ |
0 |
0 |
|
|
NED (60) |
| 12 |
65 |
M |
1.4 |
1 |
I |
|
|
2 |
0 |
|
|
NED (60) |
| 13 |
75 |
D |
1.5 |
2 |
IIA |
+ |
+ |
3 |
0 |
|
|
NED (24) |
| 14 |
65 |
D |
0.7 |
2 |
I |
+ |
+ |
2 |
3 |
|
|
NED (13) |
| 15 |
46 |
D |
1.4 |
1 |
I |
+ |
+ |
2 |
3 |
|
|
NED (23) |
| 16 |
63 |
D |
2.0 |
2 |
I |
|
+ |
2 |
0 |
|
|
NED (19) |
| 17 |
57 |
D |
2.8 |
2 |
IV |
|
+ |
3 |
0 |
+ |
+ |
AWD (14) |
| 18 |
42 |
D |
1.5 |
3 |
IIA |
|
|
2 |
2 |
+ |
+ |
NED (52) |
| 19 |
38 |
D |
2.5 |
2 |
IIB |
+ |
+ |
1 |
3 |
+ |
+ |
NED (53) |
| 20 |
60 |
D |
1.3 |
3 |
I |
+ |
+ |
1 |
3 |
+ |
+ |
NED (65) |
| 21 |
65 |
D+L |
1.2 |
3 |
IIA |
+ |
+ |
1 |
3 |
+ |
+ |
LTF |
| 22 |
56 |
D |
4.0 |
3 |
IIA |
|
|
1 |
0 |
|
|
NED (39) |
| 23 |
78 |
D |
1.5 |
2 |
I |
+ |
+ |
1 |
0 |
|
|
NED (22) |
| 24 |
75 |
D |
2.7 |
2 |
IIB |
+ |
+ |
3 |
0 |
+ |
+ |
NED (23) |
| 25 |
69 |
D |
2.5 |
3 |
IV |
+ |
+ |
3 |
0 |
+ |
+ |
AWD (16) |
| 26 |
58 |
D |
3.2 |
3 |
IV |
+ |
+ |
3 |
0 |
+ |
+ |
AWD (33) |
|
M = medullary features
D = ductal carcinoma
L = lobular carcinoma
1 = weak staining
2 = moderate staining
3 = strong staining
NED = no evidence of disease
AWD = alive with disease
DOD = dead of disease
LTF = 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).

Fig 1. Infiltrating ductal carcinoma. p53
immunostain decorates the nuclei of the majority of the neoplastic cells.
|

Fig 2. Strong Bcl-2 immunostain decorating
the cytoplasm of cells lining normal as well as hyperplastic ducts.
|
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