c-erbB-3 protein expression in ductal carcinoma in situ of the breast.

c-erbB-3, A recently identified member of the type I tyrosine kinase receptor family, has been shown to be overexpressed in invasive ductal carcinoma of breast. In this study, expression of the c-erbB-3 protein was examined in 57 cases of pure ductal carcinoma in situ of the breast (DCIS) by immuno-cytochemical methods. Staining was either absent (17 cases), present at levels equivalent to that found in adjacent normal tissue (20) or greater than in normal tissue (20). In most cases the pattern of staining was cytoplasmic, but in 4 cases with the most intense reaction there was also focal membrane staining. In the same series of cases, c-erbB-2 protein had previously been shown to be overexpressed in 28 of 57 cases, c-erbB-2 overexpression was correlated with normal level of c-erbB-3, and lack of c-erbB-2 expression was correlated with c-erbB-3 overexpression.

Expression of the hypervariable PUM locus in normal and malignant lung: the tumor-associated epitopes are present but masked in normal tissue.

A single highly polymorphic gene locus PUM codes for a family of mucin-type glycoproteins present in human urine. These glycoproteins can be detected after electrophoresis using a group of monoclonal antibodies which show marked tumour specificity on immunohistology and include the HMFG and Ca antibodies (Swallow et al., 1986, 1987). Here we show by electrophoretic analysis of lung specimens and urine samples from nine individuals, that the PUM locus is expressed both in malignant and in normal lung. In contrast immunohistology of frozen sections of normal lung showed very little staining using the same antibodies, occasional reactive type 2 pneumocytes alone staining, whilst the carcinoma material showed strong staining in each case. However, after formalin fixation much more staining was observed in normal lung, all type 1 and 2 pneumocytes being stained. These observations suggest a difference in accessibility of the epitopes in normal and malignant lung, rather than a difference in expression of the PUM gene.

Breast lymphomas: a clinicopathologic review.

Primary lymphoma is an uncommon tumor in the breast. Review of the literature shows two distinct clinicopathologic groups. One, which affects young women, is frequently bilateral, is often associated with pregnancy, and is a Burkitt-type lymphoma. The second group affects older women, is usually B-cell non-Hodgkins-type lymphoma, and presents clinical features identical to carcinoma of the breast. Three recent studies have suggested that up to half of the cases in the latter group belong to the category of lymphomas arising from the mucosa-associated lymphoid tissues. We have identified nine cases of primary lymphoma from the files of Guys Hospital Clinical Oncology Breast Unit in the 16-year period from 1974 to 1990. The clinical features have been reviewed and the tumors have been evaluated both on a morphologic and an immunohistochemical basis, and seven of nine of the cases have been screened for t[14;18] translocation using the polymerase chain reaction. All the tumors occurred in women older than 50 years and who presented with features of mammary carcinoma. One tumor was true histiocytic lymphoma; the remaining eight cases were B-cell lymphomas. Seven of the latter cases were high-grade B-cell lymphomas and one was a true follicular lymphoma. None of our cases showed the features of lymphoma arising in mucosa-associated lymphoid tissue.

Study of interlaboratory reliability and reproducibility of estrogen and progesterone receptor assays in Europe. Documentation of poor reliability and identification of insufficient microwave antigen retrieval time as a major contributory element of unreliable assays.

Immunohistochemical assays for estrogen receptors (ERs) and progesterone receptors (PRs) have not been surveyed for technical validity. In the present study, the reliability of the immunohistochemical assay for ER and PR was evaluated using data from 105 laboratories participating in external quality assessment (EQA) during a 2-year period. Technical variables associated with reliable immunostaining were analyzed. The efficiency of the antigen retrieval step was identified as the single most important contributory factor influencing the overall reproducibility of the assays. Reliable assays were found in 24 (36%) of 66 laboratories participating in continual EQA, including the majority of centers known to have clinically validated results. Inadequate assay sensitivity, with subsequent weak staining, was the main cause of poor and variable results by laboratories using microwave antigen retrieval; too short a heating time was identified as the principal contributory factor. Extension of the heating time resulted in significant improvement regardless of all other variables in the immunohistochemical protocol. Continual participation in EQA is an effective means for identifying and ameliorating variables that influence the reliability of immunohistochemical assays for predictive markers, thereby assisting in technical validation and standardization.

Monoclonal antibody to cytokeratin for use in routine histopathology.

CAM 5.2 is a murine monoclonal antibody, raised against the colon carcinoma cell line HT29, which recognises lower molecular weight intracellular cytokeratin proteins within secretory epithelia. Extensive indirect immunohistochemical studies have confirmed that this antibody stains formalin fixed (and freshly frozen) normal and malignant human tissue in a consistent manner. Reliable staining of conventionally processed pathological tissues provides more accurate identification and staging of human malignant epithelial diseases.

Angiogenesis and inflammation in invasive carcinoma of the breast.

AIM: To investigate the relation between angiogenesis and inflammation in invasive carcinoma of the breast. METHODS: Sections from 75 invasive carcinomas of the breast were stained using immunohistochemistry for von Willebrand factor, CD3, CD8, CD45RO, CD45RA, CD20, CD68, and c-erbB-2. Tumour vascularity was assessed by counting vessels in the three most vascular areas, and calculating the average (x 400 magnification, field 0.168 mm2). Each pattern of inflammation was scored semiquantitatively. RESULTS: The main pattern of inflammation was a diffuse infiltrate of macrophages, and to a lesser extent T cells. Perivascular and perilobular clusters of B and T cells were noted at the edge of the carcinomas, but were less prominent than the diffuse inflammation. Diffuse inflammation, particularly macrophages, was associated with high tumour grade, tumour necrosis, large tumour size, and c-erbB-2 expression. Perivascular and perilobular inflammation also increased with tumour grade. Tumour vascularity increased slightly with intensity of diffuse inflammation (Spearman's rank correlation coefficient rs = 0.17, p = 0.08), and was inversely related to perilobular inflammation (rs = -0.23, p = 0.03). CONCLUSIONS: The correlations between inflammation and vascularity were weak in this study (r2 about 0.04) and thus there was no evidence of an important relation. Discrepancies between this and other studies may be resolved by studying expression of angiogenic cytokines and proteolytic enzymes by tumour infiltrating inflammatory cells, and their relation to tumour vascularity.

Reliability of immunohistochemical demonstration of oestrogen receptors in routine practice: interlaboratory variance in the sensitivity of detection and evaluation of scoring systems.

AIMS: To investigate interlaboratory variance in the immunohistochemical (IHC) detection of oestrogen receptors so as to determine the rate of false negatives, which could adversely influence the decision to give adjuvant tamoxifen treatment. METHODS: To ensure that similar results are obtained by different institutions, 200 laboratories from 26 countries have joined the UK national external quality assessment scheme for immunocytochemistry (NEQAS-ICC). Histological sections from breast cancers having low, medium, and high levels of oestrogen receptor expression were sent to each of the laboratories for immunohistochemical staining. The results obtained were evaluated for the sensitivity of detection, first by estimating threshold values of 1% and 10% of stained tumour cells, and second by the Quick score method, by a panel of four assessors judging individual sections independently on a single blind basis. The results were also evaluated using participants' own threshold values. RESULTS: Over 80% of laboratories were able to demonstrate oestrogen receptor positivity on the medium and high expressing tumours, but only 37% of laboratories scored adequately on the low expressing tumour. Approximately one third of laboratories failed to register any positive staining in this tumour, while one third showed only minimal positivity. CONCLUSIONS: There is considerable interlaboratory variability, especially in relation to the detection of breast cancers with low oestrogen receptor positivity, with a false negative rate of between 30% and 60%. This variability appears to be caused by minor differences in methodology that may be rectified by fine adjustment of overall technique.

A monoclonal antibody that detects HLA-D region antigen in routinely fixed, wax embedded sections of normal and neoplastic lymphoid tissues.

We describe the use of a monoclonal antibody (TAL-IB5) to HLA-D region alpha-chains that reacts well with HLA-D positive cells in normal and neoplastic lymphoid tissues fixed in routine fixatives and embedded in paraffin wax in the conventional fashion. This antibody should prove to be useful in routine histological investigations of lymphoid and possibly other neoplasms as well as other non-neoplastic conditions where the immune system plays an important part.

The classification of ductal carcinoma in situ and its association with biological markers.

One hundred five cases of pure ductal carcinoma in situ (DCIS) seen in the Guys Hospital breast unit between 1975 and 1991 were reviewed and reclassified using a modified histologic classification based on cytological features as well as histological architecture. The expression of p53 protein, cerbB2 protein, progesterone receptor, and a proliferation antigen KiS1, all factors reported to be of prognostic significance in invasive ductal carcinoma, was also evaluated using immunohistochemical methods. The mode of presentation of these cases was noted, and its relationship to biological markers and histologic type was also assessed. Good interobserver agreement was achieved by two independent observers using the modified histologic classification. Strong correlation was seen between histologic pattern and biological markers as well as between the individual markers. Poorly differentiated DCIS was associated with a high proliferation rate, the presence of cerbB2 and p53 protein and the absence of progesterone receptors. Well-differentiated DCIS showed the reverse, and the intermediate group showed an intermediate pattern. Paget's disease of the nipple was only seen in association with poorly differentiated DCIS, but no other significant association was noted between mode of presentation and DCIS type.

Peroxidase labelling immunocytochemistry: a comparison of eleven commercially-available avidin-biotin systems.

Eleven commercially produced avidin-biotin peroxidase labelling systems employed in immunocytochemistry were compared by titrating a monoclonal and a polyclonal antibody onto routinely prepared formalin-fixed paraffin wax sections of tonsil and appendix. The cost per test for each labelling system was also calculated.

Pathological and mammographic prognostic factors for screen detected cancers in a multi-centre randomised, controlled trial of mammographic screening in women from age 40 to 48 years.

AIM: To assess pathological and radiological prognostic factors for cancers detected by screening within a multi-centre RCT trial of mammographic screening of younger women. METHOD: The survival of 232 women with screen detected invasive cancer was ascertained. Data on invasive cancer size, histological grade, nodal status, vascular invasion, mammographic spiculation, comedo calcification and mammographic background were assessed. Kaplan-Meier and Cox proportional hazards methods were used to examine survival. RESULTS: Univariate analysis indicated that women with cancers with the following features had poorer survival; ≥ 30 mm, histologically grade 3, heavily node positive (4 or more positive nodes), vascular invasion positive and displaying mammographic comedo calcification. In a multivariate model survival remained poorer in women with four or more nodes positive (HR 8.36, 95% CI 2.31, 30.17) and in those with comedo calcification (HR 3.00,95% CI 1.13, 7.99). CONCLUSION: Nodal status and the presence of mammographic comedo calcification have independent prognostic significance in young women with screen detected cancer.

The value of immunohistochemistry in sentinel lymph node histopathology in breast cancer.

The optimal protocol for the histopathological examination of sentinel lymph nodes (SLNs) in breast cancer has not been determined. The value of more detailed examination using immunohistochemistry (IHC) is controversial. A total of 476 SLNs from 216 patients were reviewed. Sentinel lymph nodes were sectioned at three levels at 100 mum intervals and stained with haematoxylin and eosin (H&E). If the H&E sections showed no evidence of metastasis, then the three serial sections were stained with a murine monoclonal anti-cytokeratin antibody (CAM 5.2). Metastatic deposits were classified as macrometastasis (> 2.0 mm), micrometastasis (0.2-2.0 mm) or isolated tumour cells (ITC, < 0.2 mm). Of the 216 patients, 56 (26%) had metastasis as identified by H&E. Immunohistochemistry detected metastatic deposits in a further nine patients (4%), of whom four (2%) had micrometastasis and five (2%) had ITC only. Those cases with micrometastases were all, on review, visible on the H&E sections. Immunohistochemistry detects only a small proportion of metastasis in SLNs. All metastatic deposits identified by IHC were either micrometastasis or ITC. Until the prognostic significance of these deposits has been determined, IHC may be of limited value in the histopathological examination of SLNs.

Intermediate filaments cytokeratin and vimentin in ovarian sex cord-stromal tumours with correlative studies in adult and fetal ovaries.

The expression of the intermediate filaments cytokeratin and vimentin were studied immunohistochemically in a series of ovarian sex cord-stromal tumours (26 adult and juvenile granulosa cell tumours, 11 thecomas, six fibromas, three Sertoli-Leydig cell tumours and 1 sex cord tumour with annular tubules). Contrary to previous reports, granulosa cell tumours expressed cytokeratins as well as vimentin. Thecomas and fibromas expressed vimentin only. In Sertoli-Leydig cell tumours and the sex cord tumour with annular tubules, both cytokeratins and vimentin were detected. Correlative studies in adult ovaries showed that patterns of expression in non-neoplastic granulosa, thecal and stromal cells correspond to their neoplastic counterparts. Investigation of fetal ovaries demonstrated that these patterns of intermediate filament expression exist from relatively early stages of development. Ovarian surface epithelium and rete ovarii, like granulosa cells, co-expressed cytokeratin and vimentin. The demonstration of cytokeratins in granulosa cells and the reported presence of desmosomes and tonofilaments, suggests the epithelial nature of these cells although not clarifying their histogenesis. The presence of both these intermediate filaments in granulosa and Sertoli-Leydig cell tumours as well as in some ovarian carcinomas which may mimic them, limits their value in differential diagnosis between these tumour groups.

Can immunohistology improve detection of lymph-node metastases in large-bowel cancer?

Employing the monoclonal antibody CAM5.2, the sensitivity of immunohistologic staining was compared with conventional hematoxylin and eosin staining for detection of lymph-node metastases from large-bowel cancer. Ten patients who died unexpectedly early from recurrent disease were selected; 86 lymph nodes were examined. Where metastases were located they were readily identified by both methods but when the original lymph-node sections were reviewed, attention was drawn to two metastatic deposits that had been overlooked previously. These results suggest that immunohistologic techniques offer no advantage in the identification of lymph-node metastases in large-bowel cancer.

S100 protein in human mammary tissue--immunoreactivity in breast carcinoma, including Paget's disease of the nipple, and value as a marker of myoepithelial cells.

The expression of S100 protein, as assessed by immunohistochemistry, has been evaluated in 101 mammary carcinomas of various histological types, including Paget's disease of the nipple. S100 immunoreactivity was seen in 44 of 101 primary carcinomas, including in situ lesions. It was present in all histological types, with the exception of mucoid carcinoma. In the 33 cases with associated Paget's disease of the nipple, S100 expression was seen in the Paget's cells in six cases. S100 immunoreactivity has been suggested as a marker of myoepithelial cells, but in our hands staining of these cells is less consistent using the S100 antibody than with antibodies to actin. Furthermore, S100 protein is also expressed by some luminal epithelial cells. Therefore, in contrast to actin immunoreactivity, S100 immunoreactivity is not a reliable means of differentiating between luminal epithelial and myoepithelial cells. The possibility that staining with antibody to S100 protein may be affected by methods of fixation and immunohistochemical technique is discussed.