Rosemary R. Millis

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.

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.

Immunohistochemical demonstration of c-erbB-2 protein in mammary ductal carcinoma in situ

The presence of the c-erbB-2 protein, demonstrated immunohistochemically with antibody 21N, has been studied in 72 cases of pure mammary ductal carcinoma in situ. Sixty-one percent of cases showed positive staining. The protein was always present in large-cell comedo type of ductal carcinoma in situ, and never in small-cell cribriform/micropapillary type of ductal carcinoma in situ. When nuclear size was measured, staining was associated with tumors containing cells with large nuclei measuring up to 20 mu, and was never present in lesions containing cells with small nuclei measuring 10 mu or less. In tumors of mixed histopathologic type of ductal carcinoma in situ, variable staining was seen; the cells with small nuclei never stained, while the majority of cells with large nuclei reacted positively. The possible relevance of these findings to the biologic behavior of DCIS is discussed.

Overexpression of the c-erbB-2 oncoprotein: Why does this occur more frequently in ductal carcinoma in situ than in invasive mammary carcinoma and is this of prognostic significance?

Overexpression of c-erbB-2 occurs in 60% of in situ and 25% of infiltrating ductal carcinomas. We have previously found very strong associations between immunohistochemical staining for c-erbB-2 and histological pattern and nuclear size in ductal carcinoma in situ (DCIS) and less strong correlation with proliferative activity. In a further study of infiltrating ductal carcinomas we have found that, in addition to tumours arising from c-erbB-2 positive, large celled, rapidly proliferating, comedo carcinomas and c-erbB-2 negative small celled cribriform/micropapillary carcinomas with a low proliferative rate, there is a third group of c-erbB-2 negative tumours with large nuclei and variable proliferative activity. These latter tumours are not seen in pure DCIS suggesting that they have a very transient in situ stage. Therefore, although in pure DCIS c-erbB-2 positively appears to be associated with tumours with a greater invasive potential, and c-erbB-2 negativity with tumours having a more favourable prognosis, the latter is not necessarily true in infiltrating disease.

The effects of chemotherapy on morphology, cellular proliferation, apoptosis and oncoprotein expression in primary breast carcinoma

The use of chemotherapy as a form of primary treatment for breast cancer is increasing and, as a result, more resection specimens contain tumours which have been exposed to cytotoxic drugs. We have studied the effects of chemotherapy on the tumour morphology and various biological features of breast carcinoma in a group of 35 patients. These were a group who responded to treatment in a clinical study of the use of primary chemotherapy designed to reduce tumour bulk prior to surgery. Characteristic morphological changes, temporally related to the administration of cytotoxic agents, are seen. The malignant cells become enlarged with vacuolated cytoplasm and vesicular nuclei containing prominent nuclei; occasionally the nuclei were angular and hyperchromatic. These features are interpreted as degenerative in nature. In 15 cases sufficient material was present in the pretreatment biopsies to compare the grade of the tumours before and after chemotherapy: changes were found in six tumours. Cytotoxic drugs do not induce a consistent pattern of change in the proliferation and apoptotic indices of individual tumours, but there is a tendency to reduce proliferative activity over all the tumours as a group. It was also found that chemotherapy is capable of modifying the expression of the oncoproteins c-erbB-2 and p53 in a minority of cases of breast cancer, usually resulting in an acquisition of immunoreactive oncoprotein. It is important to be aware of these effects when studying breast carcinomas removed after chemotherapy.

Oestrogen receptors, sites of metastatic disease and survival in recurrent breast cancer

Two hundred and seventy eight patients with advanced breast cancer who had oestrogen receptor (ER) analyses performed on primary or recurrent tumours were studied. Oestrogen receptor (ER) positive (ER ≧ 5 fmole receptor/mg cytosol protein) tumours recurred significantly more commonly in bone and ER negative (ER < 5 fmole receptor/mg cytosol protein) tumours recurred significantly more often in liver and brain. Patients with ER positive tumours had a significantly better survival after relapse. ER analysis of either primary or recurrent tumour gives some indication of the natural history of breast cancer.

PATTERNS OF EXPRESSION OF KERATIN 19 AS DETECTED WITH MONOCLONAL ANTIBODIES IN HUMAN BREAST TISSUES AND TUMOURS

The monoclonal antibodies BA 16 and BA 17 directed to different epitopes on human keratin 19 have been tested for their reaction with normal breast and with benign and malignant breast lesions and associated tissue. In Western blots of gel‐separated extracts of fibroadenomas, malignant tumours or normal mammary epithelial cells, the antibodies reacted with only one component of 40 kd molecular weight. Immunoperoxidase staining of sections of normal breast tissues showed all basal cells and a few luminal cells to be unstained by the antibodies. The distribution of the unstained (keratin 19−) luminal cells in the mammary tree is consistent with that of cells with the proliferative potential to give rise to the growth of terminal ductal lobular units (TDLU) seen at pregnancy. A total of 42 benign and 141 malignant lesions were stained with the antibodies, and a clear difference in staining pattern was seen between the benign and malignant tumours. All but 3 of the benign lesions showed a heterogeneous staining pattern with 5–50% unstained cells. In contrast, the cancer cells in 106/116 invasive primary tumours and in all 21 metastatic lesions examined showed a homogeneously positive reaction with antibodies BA16 and BA17: the malignant cells in 4 cases of Pagets disease also showed homogeneously positive staining with the antibody. In the malignant tumours, the observed homogeneity in expression of keratin 19 was confined to the malignant cells; tumour‐associated normal tissue and benign proliferative lesions contained keratin 19−cells. Seven pure in situ tumours were examined and 5 showed the homogeneous pattern of staining characteristic of invasive tumours while 2 contained a high number of keratin 19− cells. A general model is presented to explain the presence of keratin 19− cells in benign proliferation and the dominance of keratin 19− cells in invasive carcinoma.

Mammary hamartoma—a review of 35 cases

Mammary hamartomas are macroscopically well‐delineated tumours composed of a variable mixture of epithelial elements, fat and fibrous tissue. Such lesions are an under‐recognized entity and, as they can be visualized by mammography, may be seen more frequently with the advent of the UK National Breast Screening Programme. The clinical and pathological features of 35 cases of mammary hamartoma seen at the Imperial Cancer Research Fund Clinical Oncology Unit at Guys Hospital between 1979 and 1990 have been reviewed. Hormone receptor analysis on nine cases gave high progesterone with low oestrogen levels, probably reflecting their premenopausal status. Immunohistochemistry showed that the positive receptor staining was confined to the epithelial elements. In 25 cases pseudo‐angiomatous hyperplasia was evident in the stroma of the lesion. The importance of distinguishing the interanastomosing stromal spaces seen in the latter condition from low‐grade angiosarcoma is emphasized; the relationship between pseudo‐angiomatous hyperplasia and mammary hamartoma is discussed; and the possibility that the former represents a permanently dilated form of the lymphatic labyrinth suggested.