1Pathology Division, National Cancer Center Research Institute, Tokyo, 2Department of Pathology, National Defense Medical College,Tokorozawa, Saitama,3Department of Surgery, National Cancer Center Hospital, Tokyo, and 4Epidemiology and Biostatistics Division, National Cancer Center Research Institute, East Kashiwa, Japan
Even among breast cancer patients without metastasis to axillary lymph nodes, early recurrence can occur. To determine the risk factors for early recurrence, we performed a case-control study between 32 patients with an early recurrence of breast cancer and 122 patients without recurrence, in which tumor size, age of patient and date of operation were matched. In all 154 node-negative patients, followed up over a 13.1-year median period, expression of p53, c-erbB-2 and cathepsin D in the primary tumor were studied immunohistochemically in paraffin-embedded tissues and were compared with morphologic factors such as histologic grade and invasive growth. Univariate analysis showed that nuclear p53 immunoreaction was significantly predictive of early recurrence [risk ratio (RR) 3.3]. Likewise, cathepsin D (RR 0.22) was significantly negatively associated; however, when the risk ratio was analyzed in terms of intensity of cathepsin D staining, no superior survival was found for patients with strongly positive tumors. Overexpression of c-erbB-2 protein was not associated with outcome by either univariate or multivariate analysis. As a whole, histologic grade was confirmed as being a strong predictor (RR 42.6) and multivariate analysis showed that only histologic grade was a significant risk factor for early recurrence. p53 immunoreaction was not a significant independent factor because it was closely linked to histologic grade (P = 0.002), especially to a high mitotic index (P < 0.001). Moreover, in 14 patients with `special histologic types' of invasive carcinomas and no recurrence, all were p53-negative except one medullary carcinoma. Nuclear p53 immunoreaction is useful in supporting histologic grade to detect a high-risk for early recurrence in node-negative patients who may be eligible for systemic adjuvant therapy.
Key words: p53 - histologic grade - c-erbB-2 - cathepsin D - node-negative breast cancer
Among Western females, about 25% of patients with primary breast cancer without lymph node metastasis develop and die from tumor recurrence (1,2) In Japan the ratio is around 10%, reflecting the fact that the natural history of breast cancer differs between patients in the Western countries and those in Japan (3,4). Recent prospective, randomized clinical trials in patients with node-negative breast cancer have found small but significant improvements in both disease-free and overall survival resulting from the use of adjuvant chemotherapy (5,6). In order to tailor therapy to appropriate patients on the basis of the biological characteristics of the individual tumor, to avoid excessive treatment and to decrease treatment costs for these patients, the development of new and more powerful prognostic indicators that correlate with clinical outcome would help in the selection of patients for whom systemic adjuvant chemotherapy is necessary.
Recently, p53 protein detected by immunohistochemical methods has been reported to be an independent prognostic factor and a new predictor of node-negative patients at high risk of recurrence and death (7-10). The p53 tumor suppressor gene codes for a nuclear phosphoprotein thought to regulate the proliferation of normal cells. Mutations in the p53 gene, one of the most common genetic defects in human cancer, have been shown to result in a conformational change in and prolongation of the half-life of the p53 protein. It also causes accumulation of the p53 protein in tumor cell nuclei, which appears to be involved in the development and progression of many neoplastic diseases including human breast cancer (11-13).
We are interested in evaluating p53 immunoreaction as a relative risk factor for node-negative breast cancer and comparing it with new biologic markers such as c-erbB-2 and cathepsin D as well as conventional morphologic factors. Among many prognostic factors, the size of the primary tumor is currently the best established and most reliable in node-negative patients (14). We identified 32 patients with a primary breast cancer v5 cm in size that had recurred within 2 years. In this study the features of patients with an early recurrence were demonstrated by careful comparison with patients without recurrence in order to elucidate the risk factors for early recurrence.
Our records showed that 5211 primary breast cancer patients had received standard radical or modified radical mastectomies at the National Cancer Center Hospital between 1962 and 1991. From these patients, we selected 154 in whom metastases were not detected microscopically in the axillary lymph nodes and tumor size was v5 cm in diameter on palpation. Thirty-two patients (1.4% of 2230 patients with early stage breast cancer) suffered recurrence within 2 years after surgery, and for each of these, three or four control cases were selected. These 32 patients were all Japanese women aged between 26 and 71 years (median age 46.2 years). Tumor size ranged from 3 mm to 50 mm (median size: 22.6 mm).
In the control patients, primary tumor size (v2.0 cm, or 2.1-5.0 cm ), absence of axillary lymph-node metastasis, the date of the operation (+5 years) and the age at operation (+5 years) were matched to those of the early-recurrence patients. None of the control patients developed a recurrence of breast cancer within 5 years after surgery. The mean follow-up period for the controls was 13.1 years.
Twenty-six of the 32 patients (81.3%) died due to cancer between 10 months and 14 years after mastectomy (median survival period: 3 years 8 months). The other six living patients were recent cases, except one who had had chest wall excision for a local recurrence in 1970. This indicates that early recurrence in node-negative patients is a strong predictor of death from the tumor.
Histologic grading of the primary tumor was performed according to a system based on a modification of the WHO classification (15). Briefly, the modified histologic grade of the primary lesion was a summed score comprising (a) the degree of structural atypia, (b) the degree of nuclear atypia and (c) the number of mitotic figures. Individual cases were categorized as grade 1 when the sum of scores was 3 or 4; as grade 2 when the sum was 5, 6 or 7; and as grade 3 when the sum was 8 or 9. The number of mitotic figures was counted per 10 high-power fields (y400).
Immunohistochemistry
Formalin-fixed paraffin-embedded tissue blocks containing the breast carcinoma lesions were sliced into 3 mm-thick sections. The sections were deparaffinized in xylene, and rehydrated in a descending series of ethanol concentrations, ending in phosphate buffered saline (PBS). As an antigen retrieval procedure, the sections were placed in a hot water bath set at 90_C for 15 min and left to cool for 15 min. They were incubated for 30 min in 0.3% hydrogen peroxide in methanol and then incubated for 10 min in 2% normal swine serum in PBS in order to abolish any endogenous peroxidase activity and to diminish any non-specific antibody binding. The sections were incubated at 4_C overnight with anti-p53 antibody RSP53 at a dilution of 1:10 000. RSP53 was a rabbit polyclonal antibody against a synthetic peptide corresponding to amino acid residues 54-69 (C-F-T-E-D-P-G-P-D-E-A-P-R-M-P-E-A) (Nichirei Inc., Tokyo) of p53. After thorough washing with PBS, the sections were incubated for 30 min with 1:200 diluted biotinylated horse anti-rabbit immunoglobulin as a secondary antibody (Vector Laboratories Inc., Burlingame, CA) then for an additional 30 min with 1:100 diluted avidin-biotinyl-peroxidase complex (Vectastain, Vector, Burlingame, CA). The peroxidase reaction was performed using 0.02% 3,34-diaminobenzidine tetrahydrochloride-hydrogen peroxidase as a chromogen, with 0.01 M sodium azide as an endogenous peroxidase inhibitor, in Tris buffer (pH 7.6) for 5 to 10 min. Nuclear counterstaining was performed with 2% methyl green (Chroma, Kongen, Germany). Before each step, the sections were washed three times (for 5 min each time) with PBS.
Commercially available polyclonal antibodies against c-erbB-2 protein (Nichirei Inc., Tokyo) and cathepsin D (Novocastra Laboratories Ltd, Newcastle, UK) were used for immunohistochemistry at dilutions of 1:200 and 1:600 respectively. Immunohistochemical staining was carried out according to the method described above but without the antigen retrieval procedure.
When the surface of the cell membrane was distinctly stained for c-erbB-2, the intensity of staining was categorized as strongly positive (16). The weakly positive group showed faint staining in which the difference between membrane and cytoplasm was often unclear. The intensity of staining for cathepsin D was categorized as follows: strongly positive when positive staining for cathepsin D occurred in greater than 10% of cancer cells; weakly positive when 1% to 10% of the cells were positive for cathepsin D.
Statistical analysis
The c2 test was used to compare nuclear p53 immunoreaction and other clinicopathologic factors (histologic type, histologic grade, and immunohistochemical staining for c-erbB-2 protein and cathepsin D ). Conditional logistic regression analysis was used to calculate the risk factor against recurrence and 95% confidence intervals (CI) for each prognostic factor (17). All calculations were performed using the SAS statistical software package (SAS Institute Inc., Cary, NC).
The cancer patients were classified into two groups according to the percentages of nuclei distinctly stained with the anti-p53 antibody; a p53-positive group in which w10 % of the nuclei were stained for p53 , and a p53-negative group in which <10 % of the nuclei exhibited p53 accumulation (Fig. 1). The negative group included patients with only one or two cancer cells stained faintly in the nuclei.
