Diana M. Barnes

c-erbB-2 protein overexpression in breast cancer is a risk factor in patients with involved and uninvolved lymph nodes.

The c-erbB-2 gene is overexpressed in about 20% of human breast cancers. Four hundred and eighty-three cases previously examined by immunohistochemical staining for c-erbB-2 expression were analysed to assess the risk associated with the elevated protein expression. Oncoprotein expression was correlated with increasing tumour grade but not with oestrogen receptor status, nodal involvement, tumour size or age. There was an increased risk of relapse and death associated with c-erbB-2 expression irrespective of nodal involvement. This marker thus appears to be a significant prognostic factor in the early as well as the late stages of breast cancer.

Cyclin D1 and prognosis in human breast cancer

We have used immunohistochemical staining to assess the expression of cyclin D1 in formalin‐fixed sections of 345 breast carcinomas, dating back 20 years. Clinical follow‐up data were available on all patients. Approximately 50% of the tumours showed excessive nuclear staining for cyclin D1 as compared with normal epithelium. Some tumours showed strong cytoplasmic staining in the absence of nuclear staining, and around 25% of the tumours were judged to be negative for nuclear cyclin D1. Contrary to expectations, moderate/strong staining for cyclin D1 was associated with improved relapse‐free and overall survival relative to patients whose tumours stained weakly or negatively. Conversely, tumours that were considered negative for cyclin D1 staining had an adverse prognosis, and the poor outcome was further accentuated if the tumours were also oestrogen receptor‐negative. A possible explanation for our findings is that tumours in which cyclin D1 levels are abnormally low may have sustained mutations in other genes, such as RB1 and that it is this abnormality that has the more significant impact on survival from breast cancer.

Expression of the ERBB3 gene product in breast cancer.

Abnormalities of the EGF receptor and/or the related ERBB2 receptor occur in a significant proportion of cases of human breast cancer and are important influences in the behaviour of this tumour type. In this report we demonstrate by nucleic acid analysis and immunohistochemistry that the recently recognised third member of this gene family, ERBB3, shows a wide range of expression in breast cancer, and shows stronger immunoreactivity than that observed in normal tissue in 43 out of 195 cases (22%) of primary breast cancer. Overexpression of ERBB3 appears to result from increased levels of gene transcription since in none of the cell lines or primary cancers analysed did we find evidence of gene amplification. High expression of ERBB3 is positively associated with the presence of lymph node metastases, but there was no demonstrable relationship with patient survival in this series.

Overexpression of c-erbB2 is an independent marker of resistance to endocrine therapy in advanced breast cancer

SummaryThe present study investigated the interaction between c-erbB2 overexpression and the response to first-line endocrine therapy in patients with advanced breast cancer. The primary tumours of 241 patients who were treated at first relapse with endocrine therapy were assessed for overexpression of c-erbB2 by immunohistochemistry. c-erbB2 was overexpressed in 76 (32%) of primary breast cancers and did not correlate with any other prognostic factor. The overall response to treatment and time to progression were significantly lower in patients with c-erbB2-positive tumours compared to those that were c-erbB2-negative (38% vs 56%, P = 0.02; and 4.1 months vs 8.7 months, P < 0.001, respectively). In multivariate analysis, c-erbB2 status was the most significant predictive factor for a short time to progression (P = 0.0009). In patients with ER-positive primary tumours treated at relapse with tamoxifen (n = 170), overexpression of c-erbB2 was associated with a significantly shorter time to progression (5.5 months vs 11.2 months, P < 0.001). In conclusion, overexpression of c-erbB2 in the primary tumour is an independent marker of relative resistance to first-line endocrine therapy in patients with advanced breast cancer. In patients with ER-positive primary tumours, the overexpression of c-erbB2 defines a subgroup less likely to respond to endocrine therapy.

Cyclin D1 in breast cancer

Cyclin D1 protein plays an important part in regulating the progress of the cell during the G1 phase of the cell cycle. The cyclin D1 gene, CCND1, is amplified in approximately 20% of mammary carcinomas, and the protein is over- expressed in approximately 50% of cases. This has led to intensive study to ascertain whether cyclin D1 is a biological marker in breast cancer; however, the clinical work has produced unexpected results. Work in cell lines and in transgenic mice indicate that CCND1 is a weak oncogene and it was expected that, like c- erbB-2, over-expression of cyclin D1 protein would be associated with a poor prognosis. Early immunohistochemical prognostic studies produced equivocal results but we, and others, have recently shown that strong staining for cyclin D1 is more likely to be seen in well differentiated, estrogen receptor positive carcinomas. Furthermore, we have found that over-expression of cyclin D1 is actually associated with a good outcome, both in terms of prognosis and response to endocrine treatment. Cyclin D1 is frequently over- expressed in ductal carcinoma in situ but not in benign breast disease, including atypical ductal hyperplasia; hence its expression appears to be closely linked with carcinogenesis. In order to help explain the apparent beneficial effects of cyclin D1 over- expression, a number of closely associated cell cycle proteins have also been evaluated, including the cyclin dependent kinase inhibitor p27, which blocks the activating effects of cyclin D1. Initial reports show that high levels of p27 are associated with a good prognosis and we have shown a positive association between p27 and cyclin D1 expression. These clinical results of cyclin D1 are an example of how information obtained from basic cell biology studies needs to be complemented by clinical studies to ascertain the true worth of a prognostic marker.

Immunohistochemical detection of p53 protein in mammary carcinoma: An important new independent indicator of prognosis?

In an immunohistochemical pilot study of 195 primary breast cancer patients with a 10-year median follow-up we found that patients with carcinomas who express p53 protein in the majority of their tumor cells (19% of the cases) have a considerably worse prognosis than those who do not. The effect of the presence of the protein is seen on disease-free interval (chi-square, 11.69; P < .001), overall survival (chi-square, 19.68; P < .001), and survival after relapse (chi-square, 4.93; P < .02), and is seen in node-negative (chi-square, 6.99; P < .009) and node-positive (chi-square, 13.05; P < .001) patients. Furthermore, the effect is most apparent in patients with infiltrating lobular and grade II infiltrating ductal carcinomas (chi-square, 27.97; P < .001) that have a rather heterogeneous clinical behaviour and are difficult to subdivide on the basis of currently available markers. Cox multivariate analysis shows that p53 majority staining is second only to node status in significance of effect on overall survival.

Immunohistochemical determination of oestrogen receptor: comparison of different methods of assessment of staining and correlation with clinical outcome of breast cancer patients

Immunohistochemical staining for oestrogen receptor (ER) has been carried out using antibody ER ID5 on 170 women who received first-line tamoxifen treatment for evaluable metastatic breast cancer. ER status had been determined some years previously, using a ligand-binding cytosol assay. The adequacy of the tissue used for the cytosol assay was always checked by histology on an adjacent block and was deemed to be typical of the tumour overall as was the block used for immunohistochemistry. Six different methods were used to assess the degree of staining and comparisons were made to determine which method gave the most clinically relevant results. Clinical outcome was assessed both in terms of duration of response to tamoxifen determined by log-rank analysis and type of response using the chi-squared test. The ER immunohistochemical assay gave superior results compared with the cytosol assay, with all of the subjective methods of assessment of staining giving statistically significant correlations with clinical outcome. The additional contribution of progesterone receptor (PR) staining with antibody NCL PGR was also studied.

An immunohistochemical evaluation of c- erbB-2 expression in human breast carcinoma

The c-erbB-2 gene codes for a putative transmembrane protein, similar in structure to the epidermal growth factor (EGF) receptor. Amplification of the gene has been described in a variety of human adenocarcinomas and is particularly well documented in breast carcinoma. It has been suggested that amplification is indicative of poor prognosis and, as such, is comparable with lymph node status as a predictor of clinical outcome. This study examines the suggestion indirectly by an immunohistochemical technique. Archival tissue from 195 patients with primary breast carcinoma was stained with the polyclonal antibody 21N, raised to amino acids 1243-1255, the C-terminus of the predicted amino acid sequence of the c-erbB-2 protein. Up to 10 year verified follow-up data were available on all patients. Staining compatible with significant amplification was observed in 17 patients. Using the chi-squared test for trend a significant correlation was found between staining and grade (P = 0.04) but not with either node or receptor status. No significant association was found between staining and clinical outcome although there was a tendency for patients with stained tumours to have a worse prognosis. A Cox regression analysis was used to adjust for node status and grade and still no correlation was revealed between staining and prognosis. However a study of this size in which only a small number of patients have been found to have stained tumours does have wide confidence limits. Comparable staining observed in in situ and infiltrating components of tumours suggests that amplification is an early event in carcinogenesis. Similar staining in primary and subsequent metastatic lesions was also noted. It is considered that further studies at both the DNA/mRNA and protein levels are required to confirm the significance of c-erbB-2 amplification in human breast carcinoma.

Abnormal expression of wild type p53 protein in normal cells of a cancer family patient

Mutations in the p53 gene are the commonest specific genetic change in human cancer. In normal tissues, p53 protein is present in such low quantities that it is not readily detectable by immunochemical techniques. However, in many tumour cells large amounts of p53 protein accumulate and can be seen by simple immunohistochemical staining; this is generally attributed to the accumulation of stabilised, mutant protein. We have found a mother and daughter, who both have a history of breast cancer, who show strong immunohistochemical staining of p53 in most of their normal epithelial and mesenchymal cells. Their family has a history of multiple cancers developing at an early age. Detailed protein analysis and gene sequencing of material obtained from cultured cells, grown from a skin biopsy taken from the daughter, suggest that her cells contained large quantities of normal (unmutated) p53. We suggest that this phenotype defines a new inherited cancer susceptibility syndrome that is distinct from the germ-line mutations in p53 found in some Li-Fraumeni families. This new syndrome affects p53 tumour suppressor function through an indirect mechanism that stabilises normal p53. It remains to be established whether this mechanism also contributes to the accumulation of p53 in sporadic cancers.

Steroid-hormone receptors and survival after first relapse in breast cancer

Oestrogen receptors were measured in the primary breast tumours of 508 patients and progesterone receptors in those of 486 patients. Survival from mastectomy was significantly longer in patients with receptor-positive tumours. There was no significant difference between patients with receptor-positive and receptor-negative tumours in the relapse-free interval, but survival from first relapse was longer in patients with receptor-positive tumours. Axillary node status and tumour size indicated the probability of relapse but did not influence the length of survival after relapse. Response to tamoxifen or ovarian ablation was known in 65 of the 137 patients who relapsed. Survival from first relapse was significantly longer in patients who both responded to hormone therapy and had receptor-positive tumours. Patients who did not respond to hormone therapy and had receptor-positive tumours had the same survival characteristics as those with receptor-negative tumours who did not respond.