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Paraffin wax sections of 70 surgically resected colorectal adenocarcinomas were examined for the overexpression of HER2/ c-erbB-2 oncoprotein using three different specific antibodies and the avidinbiotin immunoperoxidase technique. The patients included 38 men and 32 women aged between 47 and 80 years. The tumours were derived from various parts of the large intestinal tract, and represented all three stages of Dukes' classification and the three histological grades of differentiation. Many tumour sections also included adjacent normal or transitional mucosa. Eight tubular adenomas found in the colectomy specimens in association with some carcinomas were also examined. No positive membrane staining was seen in any of the 70 carcinomas, four adenomas, two hyperplastic polyps, nor in the adjacent normal or transitional mucosa.

Summary The expression of pS2 was examined histochemically in paraffin sections taken from biopsy material from patients diagnosed with ductal carcincoma in situ (DCIS). Often intense immunoreactivity, to an anti-pS2 monoclonal antibody, was observed in comedo, solid, cribriform and micropapillary types of DCIS, with significant positivity found in 63-67% of cases. In 15 samples analysed, we found a good correlation between pS2 expression and presence of progesterone receptor positive cells, but not with estrogen receptor. There was only a limited degree of correspondence between the cells staining with these anti-sera. Some pS2 positive cells were also seen in normal acini in areas adjacent to cancer but much less frequently in sections of normal breast from reduction mammoplasty. Most normal areas were negative, as were cysts. In benign proliferative conditions (seen in sections with and without DCIS) such as adenosis, sclerosing adenosis, mild and florid ductal epithelial hyperplasia, significant pS2 positivity was seen in about 50% of cases.These results suggest that there is a progressive increase in pS2 from normal to benign to cancer cells and that this gene is expressed in both the invasive and pre-invasive forms of breast cancer.The pS2 gene, which was originally isolated by virtue of its estrogen-inducibility (Masiakowski et al., 1982), has been shown to be predominantly associated with estrogen receptor (ER) positive breast cancers (Rio et al., 1987;Skilton et al., 1989;Henry et al., 1989). Limited pS2 immunostaining has also been reported in normal breast and in parts of the ileum (Piggott et al., 1991;Luqmani et al., 1992), as well as more extensive expression in the stomach, but is otherwise absent from the vast majority of normal tissues. It is however, widely expressed in other epithelial cancers Luqmani et al., 1989;Rio et al., 1988; Luqmani et al., 1991;Wysocki et al., 1990) as well as in inflammatory conditions of the gastro-intestinal tract Seitz et al., 1992). pS2 has significant sequence homology with the (pancreatic) spasmolytic polypeptide (hSP) Wright et al., 1990) with which it is co-expressed in gastric mucosa (Theisinger et al., 1991) and is also secreted into the gastric fluid (Rio et al., 1988).In a small study we have recently (Shousha et al., manuscript submitted) found pS2 to have no prognostic significance in patients with colo-rectal cancer, but high pS2 levels were predictive of both longer survival (Foekens et al., 1990) and favourable response of breast cancer patients to endocrine therapy (Henry et al., 1989;Skilton et al., 1989) and, in this regard, may be superior to ER (Schwartz et al., 1991). Little is known of the expression of pS2 in early breast cancer. We therefore carried out an immunohistochemical study using archival material obtained from patients who had been diagnosed with ductal carcinoma in situ (DCIS), a neoplastic condition in which the malignant cells are confined within the basement membranes of the mammary ducts. This is believed to constitute a fore...

In the preliminary part of the study, paraffin wax sections of two progesterone receptor strongly positive tumours and one progesterone receptor negative tumour were used. Six sections from each case were investigated. Two were pretreated with trypsin, two with DNAse,2 and two were not pretreated. One section from each group was used for the demonstration of progesterone receptors by the peroxidase-antiperoxidase (PAP) method, and the other section was used for the demonstration of the receptor by the avidin-biotin-peroxidase complex (ABC) technique. The ABC technique, with no pretreatment, was the only method that gave good results that could be clearly visualised and was adopted for the rest of the investigation.In the next phase of the investigation 10 cases of breast carcinoma were studied. These included five progesterone receptor positive and five negative cases, assayed biochemically.Three paraffin wax sections from each case were studied. The receptors were investigated in two sections by the ABC technique, but the final staining result was developed in one by diaminobenzidine (DAB) and in the other by a mixture of DAB and imidazole.3 The third section was used as a negative control.Briefly, the sections were dewaxed by placing the slides in two changes of xylene, for two minutes each, and then hydrated through graded alcohols. Endogenous peroxidase activity was blocked by applying 3% hydrogen peroxide in methanol for 30 minutes. After rinsing in three changes for five minutes each of 0-2M TRIS-buffered-saline (TBS), pH 7-6, the sections were covered with normal goat serum for 30 minutes. This was then tipped off and the sections incubated with a few drops of PgR-ICA monoclonal antibody for 16-20 hours at 4°C. After rinsing in three changes of TBS for five minutes each sections were covered for two hours by biotinylated anti-rat IgG (Vector Laboratories), diluted 1/100 in TBS. Sections were then rinsed in TBS as before and incubated for two hours with avidin-biotin complex (Dakopatts). After rinsing in TBS, the sites of peroxidase activity were visualised by incubating the sections in 0-05% DAB (Sigma) and 0-01% hydrogen peroxide, either with or without added 0-05% imidazole, for six and 15 minutes, respec-

The presence of oestrogen and progesterone receptors was studied in paraffin sections of 81 screen-detected breast carcinomas using the monoclonal antibodies ER-ICA and PgR-ICA (Abbott) and the immunoperoxidase technique. The immunohistological results were compared with the results of the standard dextran-coated charcoal biochemical assay in 28 tumours which were big enough to provide tumour tissue for this assay. Sixty-three cases (78%) were oestrogen receptor positive and 62 (77%) were progesterone receptor positive. There was no statistical difference between receptor positivity in palpable or impalpable, in situ or invasive tumours. In the 28 cases where the biochemical assay was carried out, the two methods gave similar results in 23 (82%) and 21 (75%) tumours for oestrogen and progesterone receptors respectively. The majority of the remaining tumours, with one exception, were positive with immunohistology and negative with biochemistry. A good correlation was also present between the mean numerical biochemical values and the semiquantitative histological scores for both receptors. It is concluded that assessment of receptor status of small screen-detected carcinomas is feasible using routinely processed paraffin sections. There is reasonably good correlation with the results obtained by the standard dextran-coated charcoal biochemical assay, but more genuine receptor positive cases are detected by immunohistology.