Coexistence of HER2 over-expression and p53 protein accumulation is a strong prognostic molecular marker in breast cancer
© Yamashita et al., licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. 2004
Received: 21 July 2003
Accepted: 15 October 2003
Published: 7 November 2003
Many laboratories are currently evaluating the usefulness of determination of HER2, p53, and Ki67 proliferation indices using immunohistochemical techniques in cancer. Although the available studies suggest that these factors might indeed be helpful in making treatment decisions in cancer patients, their clinical usefulness is still controversial.
Expression of HER2, p53, and Ki67 was examined by immunohistochemistry in samples of breast tissue from 506 patients with invasive ductal carcinoma, obtained between 1981 and 1999 (median follow up period 82 months), and their significance for prognosis was analyzed.
Of the 506 carcinoma tissue samples, 20.1%, 29.0%, and 53.6% were positive for HER2 over-expression, p53 protein accumulation, and Ki67 expression, respectively. Over-expression of HER2 significantly reduced disease free (P = 0.02) and overall survival (P = 0.005). Accumulation of p53 protein significantly decreased disease free (P = 0.01) and overall survival (P = 0.01). Patients with tumors that were positive for both HER2 and p53 relapsed and died within a significantly shorter period of time after surgery (P = 0.0001 and P < 0.0001, respectively). In multivariate analysis, patients with both HER2 and p53 positive tumors had considerably decreased overall survival (P = 0.04), as did patients with larger tumor size and positive lymph node status.
The findings of the present study indicate that the coexistence of HER2 over-expression and p53 protein accumulation is a strong prognostic molecular marker in breast cancer.
KeywordsHER2 immunohistochemistry invasive ductal carcinoma p53
Prognostic biomarkers in a disease provide information regarding outcome irrespective of therapy. Candidate prognostic biomarkers in breast cancer include elevated levels of expression of proliferation indices such as Ki67 and proliferating cell nuclear antigen; expression of estrogen receptor (ER) and progesterone receptor; amplification and over-expression of HER2, cyclin D1, and c-myc; p53 nuclear protein accumulation; bcl-2 expression; and alteration in angiogenesis proteins such as vascular endothelial growth factor [1–5]. In particular, review of the literature suggests that over-expression of HER2 and p53 may have prognostic significance in breast cancer. HER2 (c-erbB2) encodes a membrane protein (p185) that is tyrosine phosphorylated after interaction with its ligands. Over-expression of HER2 occurs through either amplification of the gene or mRNA over-expression. p53 is involved in regulating cell proliferation, inducing apoptosis, and in promoting chromosomal stability. Disruption of these functions appears to play an important role in carcinogenesis. There is evidence that over-expression of HER2 and p53 is involved in breast cancer progression . This hypothesis is based on the high frequency of HER2 and p53 over-expression among invasive and noninvasive breast cancers and among benign breast diseases [7–9]. This suggests that HER2 and p53 play roles in the early stages of breast tumorigenesis.
In the present study we examined the expression of HER2, p53, and Ki67 in samples of breast tissue from 506 patients with invasive ductal carcinoma, obtained between 1981 and 1999, and analyzed their significance for prognosis. Our results indicate that the coexistence of HER2 over-expression and accumulation of p53 protein is a strong prognostic molecular marker in breast cancer.
Patients and breast cancer tissues
Clinicopathologic characteristics of patients with invasive ductal carcinoma
Value (n [%])
Age at diagnosis (years)
Age range (years)
Tumor size (cm)
Number of positive lymph nodes
Follow up (months)
Immunohistochemical analysis for estrogen receptor-α, HER2, p53, and Ki67
One 4-μm section from each submitted paraffin block was first stained with hematoxylin and eosin in order to verify that an adequate number of invasive ductal carcinoma cells were present and that quality of fixation was sufficient for immunohistochemical analysis. Serial sections (4-μm) were prepared from selected blocks and float mounted on adhesive coated glass slides for ER-α, HER2, p53, or Ki67 staining. Primary antibodies included monoclonal mouse antihuman estrogen receptor antibody (1D5; DAKO, Glostrup, Denmark) at 1 : 100 dilution for ER-α, rabbit antihuman c-erbB2 oncoprotein antibody (DAKO) at 1 : 200 dilution for HER2, monoclonal mouse antihuman p53 protein antibody (PAb1801; Novocastra, Newcastle, UK) at 1 : 50 dilution for p53, and monoclonal mouse anti-human Ki67 antibody (MIB-1; DAKO) at 1 : 100 dilution for Ki67. The DAKO EnVision system (DAKO EnVision labelled polymer, peroxidase) was used as the detection system for ER-α, HER2, and Ki67. The streptavidin-biotin system (SAB-PO kit; Nichirei Co., Inc., Tokyo, Japan) was applied for detection of the bound antibody of p53.
Immunostained slides were scored after the entire slide had been evaluated by light microscopy. The expression of ER-α was scored by assigning a proportion score and an intensity score according to Allred's procedure . Any brown nuclear staining in invasive breast epithelium was counted toward the proportion score. Tumors with scores of 3 or greater were considered to be positive for ER-α expression. HER2 immunostaining was evaluated using the same method as is employed by the HercepTest (DAKO). To determine the score of HER2 expression the membrane staining pattern was estimated and scored on a scale of 0 to 3+. Tumors with scores of 2 or greater were considered to be positive for HER2 over-expression. The expression status of p53 and Ki67 was assessed according to the estimated proportion of nuclear staining of tumor cells that were positively stained. Scoring criteria for p53 were as follows (in the form proportion of nuclear staining = score): none = 0, < 1/10 = 1, 1/10-1/2 = 2, and > 1/2 = 3. Scoring criteria for Ki67 were as follows (in the form proportion of nuclear staining = score): none = 0, < 1/100 = 1, 1/100-1/10 = 2, 1/10-1/2 = 3, and > 1/2 = 4. Tumors with a score of 1 or greater for p53 were considered to be positive for p53 protein accumulation, and tumors with a score of 2 or greater for Ki67 were considered to be positive for Ki67 expression.
The χ2 test was used to compare immunohistochemical results for molecular markers with clinicopathologic characteristics. Estimation of disease free and overall survival was performed using the Kaplan–Meier method, and differences between survival curves were assessed with the log-rank test. Cox's proportional hazards model was used for univariate and multivariate analyses of prognostic values.
Relationship between HER2, p53, and Ki67 expression and clinicopathologic factors
Correlation between clinicopathologic factors and molecular markers
Positive/total (n [%])
Tumor size (cm)
Number of positive lymph nodes
Correlation between HER2 over-expression, p53 protein accumulation, and Ki67 expression
Correlation between HER2, p53, and Ki67 expression
Disease free and overall survival categorized by HER2, p53, and Ki67 expression
Prognostic analysis of disease free survival
Prognostic factors in 470 invasive ductal carcinomas compared with disease free survival
RR (95% CI)
Number of positive lymph nodes
Prognostic analysis of overall survival
Prognostic factors in 470 invasive ductal carcinomas compared with overall survival
RR (95% CI)
Number of positive lymph nodes
The present study indicates that the coexistence of HER2 over-expression and p53 protein accumulation has strong prognostic significance in invasive ductal carcinoma of the breast after a median follow-up period of 82 months.
HER2 encodes a 185-kDa transmembrane glycoprotein with intracellular tyrosine kinase activity that belongs to the epidermal growth factor receptor family . Although no ligand has been identified for HER2, several peptide growth factors bind to the other members of the family. Amplification or over-expression of HER2 is observed in 20–40% of human breast cancers. The prognostic significance of HER2 over-expression was first reported in 1987 . Subsequently, over 200 studies have been reported in which the role of amplification/over-expression of HER2 was investigated as a prognostic marker in breast cancer. Also, we previously reported that HER2 amplification was strongly associated with both disease free and overall survival in breast cancer . In the present study we extended our analysis of HER2 over-expression to more than 500 invasive ductal tumors, and showed that over-expression of HER2 was associated with poor prognosis.
Nearly one-third of breast cancers have mutations in the p53 gene, which are associated with high histological grade and clinical aggressiveness . Immunohistochemical assays generally detect nuclear accumulation of the protein, which is often related to conformational alterations and a prolonged half-life of the encoded protein [13, 14]. Accumulation of p53 protein was significantly associated with poor prognosis in our study and in other studies of patients with breast cancer [15, 16]. These studies suggest both a prognostic and a predictive role for p53 .
Tumors with both HER2 over-expression and p53 protein accumulation were reported in several studies, and patients with such tumors were found to have poor prognosis [17–22]; the findings reported here also indicate that both HER2 over-expression and p53 protein accumulation are associated with markedly poorer disease free and overall survival. On the other hand, some studies have shown a better prognosis in patients with breast cancers with HER2 overexpression and p53 protein accumulation . These differences may reflect the effect of various therapeutic regimens.
Most patients with early breast cancer receive adjuvant treatment, and the identification of predictive factors may help in selecting the optimal therapeutic strategy for individual patients. HER2 over-expression may be associated with reduced efficacy of adjuvant endocrine therapy with tamoxifen [24–26]. The role of p53 mutations in the efficacy of endocrine therapy is still under evaluation. On the other hand, previous data suggested that HER2 positive tumors might be resistant to adjuvant treatment with CMF [27, 28]. There is evidence that women whose tumors over-express HER2 are likely to derive greater benefit from therapy with anthracycline-containing regimens than from alkylating agents [25, 29–31]. It was also reported that patients with both HER2 and p53 positive tumors had an improved 10-year survival when treated with a high dose FAC (fluorouracil, doxorubicin, cyclophosphamide) regimen . The patients included in the present study were treated with tamoxifen, fluorouracil, or a CMF regimen, and anthracycline based chemotherapy was not used. Further studies are needed to determine which endocrine or chemotherapeutic agents should be used in breast cancers with different expression profiles, especially in patients with poor prognosis.
We examined the expression of HER2, p53, and Ki67 in 506 invasive ductal carcinoma tissue samples. The results indicate that the coexistence of HER2 over-expression and p53 protein accumulation is a strong prognostic molecular marker in breast cancer.
- CMF = cyclophosphamide:
methotrexate, and fluorouracil
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture in Japan 13671248.
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