Brian Angus

EPIDERMAL GROWTH-FACTOR RECEPTOR (EGFR) AS A MARKER FOR POOR PROGNOSIS IN NODE-NEGATIVE BREAST-CANCER PATIENTS - NEU AND TAMOXIFEN FAILURE

Analysis of EGFr and ER was performed on tumour samples from 231 patients with operable breast cancer followed for up to 6 yr after surgery. The median duration of follow-up in patients still alive at the time of analysis was 45 months. Thirty-five percent of patients (82) had tumours greater than 10 fmol/mg 125I-EGF binding (EGFr+) and 47% (109) had cystolic ER concentration greater than 5 fmol/mg (ER+), with a marked inverse relationship between EGFr and ER (P less than 0.00001). EGFr was second only to axillary node status as a prognostic marker for all patients both in terms of relapse-free and overall survival (P less than 0.001, logrank EGFr+ vs EGFr-). For patients with histologically negative axillary nodes EGFr was superior to ER in predicting relapse and survival (P less than 0.01 and P less than 0.005, respectively, compared to P less than 0.1 and P less than 0.1, logrank). In a multivariate (Cox model) analysis only EGFr, out of EGFr, ER, size and grade, was predictive for either relapse-free or overall survival for patients with node-negative disease (P = 0.052 and P = 0.026, respectively). The correlation of neu expression with response to tamoxifen in patients with recurrent disease was assessed immunochemically. Response rate was reduced in the presence of neu from 50 to 17% for ER+ cases and from 26 to 0% for ER- cases.

Prognostic significance of the estrogen-regulated protein, cathepsin D, in breast cancer. An immunohistochemical study

Expression of the estrogen‐regulated lysosomal protease, cathepsin D, was studied in a series of 94 breast cancers using an immunohistochemical technique. Granular staining of tumor cell cytoplasm was detected in 62 cases. Positive staining was associated with a significant increase in overall time to relapse and when survival was analyzed in terms of intensity of cathepsin D staining there was a significant trend for both increased time to relapse and increased length of survival. The presence of estrogen receptor was associated with positive cathepsin D immunostaining, and in the subgroup of estrogen receptor‐positive tumors cathepsin D staining was associated with significantly prolonged survival; this was not the case for estrogen receptor‐negative tumors. Positive cathepsin D immunostaining was associated with significant prognostic advantage in patients with confirmed lymph node metastasis but not in node‐negative patients. It is suggested that cathepsin D expression reflects the functional integrity of the estrogen response pathway. Cathepsin D may prove a clinically useful adjunct to assessment of estrogen receptor status.

New monoclonal antibodies to oestrogen and progesterone receptors effective for paraffin section immunohistochemistry.

Assessment of oestrogen and progesterone receptors (ER and PgR) in breast cancer is widely used for the prediction of response to endocrine therapy and as a prognostic marker. Cytosolic assays have been replaced in many centres by immunochemical techniques, which have many advantages including applicability to small samples, simplicity, and cost‐effectiveness. This study describes the generation and characterisation of two novel murine monoclonal antibodies recognizing ER and PgR, designated NCL‐ER‐6F11 and NCL‐PGR respectively, which are effective in heat‐treated formalin‐fixed, paraffin‐embedded tissue. The antibodies have been characterized by Western blotting and by immunohistochemistry on normal and pathological breast and other tissues. NCL‐ER‐6F11 has been shown to compare favourably with a currently available ER antibody. These antibodies may prove of value in the assessment of hormone receptor status in human breast cancer.

c-erbB-2 oncoprotein expression in mammary and extramammary Paget's disease: an immunohistochemical study

Over‐expression of the c‐erbB‐2 oncogene occurs in a proportion of human adenocarcinomas and in breast carcinoma is associated with poorer prognosis. Sections of formalin‐fixed, paraffin‐embedded tumour tissue from 22 patients with mammary and extramammary Pagets disease have been stained immunohistochemically using a monoclonal antibody (NCL‐CB11) raised against a synthetic peptide from the C‐terminal end of the predicted equence of the c‐erbB‐2 protein product. All 12 cases of mammary Pagets disease showed membrane staining of intra‐epidermal cells, indicating c‐erbB‐2 over‐expression. Sections of underlying ductal breast carcinoma were available in nine cases; all nine tumours were c‐erbB‐2 positive and in eight the in situ component was of comedo or solid type. There was membrane staining of tumour cells in four of the 10 cases of extramammary Pagets disease; staining intensity was generally weaker than that observed in the cases of mammary disease. The possible implications of these findings for the histogenesis of both mammary and extramammary Pagets disease are discussed.

Prognostic value of Ki67 antigen expression in basal cell carcinomas

Summary Recurrence of basal cell carcinoma (BCC) following treatment is a common event and long‐term follow‐up of all patients presenting with a primary BCC has been recommended. Proliferation indices have been recognized as important prognostic factors in several tumour types in a variety of cancer systems, being significantly elevated in more aggressive lesions. We have examined 51 BCCs (17 non‐recurrent tumours [group 1]. 17 original tumours which later recurred [group 2‐0]. and the corresponding 17 recurrent specimens [group 2‐R] for Ki67 antigen expression, a proliferalionassociated antigen using immunohistochemistry with the monoclonal antibody MIB1. There was a significant increase in the percentage positive for MIB1 in the Group 2‐0 as compared with the group 1 BCCs (P

Monoclonal antibodies recognizing CD5, CD10 and CD23 in formalin-fixed, paraffin-embedded tissue: production and assessment of their value in the diagnosis of small B-cell lymphoma

Assessment of the expression of antigens CD5, CD10 and CD23 can be of value in the differential diagnosis of small B‐cell lymphoma. Correct subclassification is important since optimal treatment regimes differ between the subtypes. The aim of this study was to generate monoclonal antibodies recognizing these antigens in paraffin‐embedded tissue and to assess their efficacy using a panel of cases of small B‐cell lymphoma of various subtypes.

Retinoblastoma protein in human breast carcinoma : Immunohistochemical study using a new monoclonal antibody effective on routinely processed tissues

Cyclic phosphorylation/dephosphorylation of the retinoblastoma gene product (pRB) has been found to play a central role in the progression of the normal cell cycle, through modulation of the activity of the E2F family of transcription factors. Mutations of the retinoblastoma gene have been described in a wide variety of human malignancies including carcinomas of the breast. The present investigation reports the production and application of a new monoclonal antibody in an immunohistochemical study of pRB expression in 233 primary breast carcinomas, allowing an assessment of the contribution made by this tumour suppressor gene to tumour development and progression. Overall, there was loss of pRB expression in 21 per cent of breast tumours. Although high‐grade tumours were found to lack detectable pRB more frequently than low‐grade tumours, the difference did not prove statistically significant. In addition, pRB immunostaining was not related significantly to relapse or survival. No significant correlations were observed between apparent loss of pRB and tumour size, parity, patient lymph‐node status, p53, c‐erbB‐2, c‐jun, EGFR or steroid hormone receptor expression. Preliminary findings, however, did suggest a relationship between pRB expression and response to endocrine therapy.

p53 expression measured by flow cytometry. A comparison of three monoclonal antibodies and the relationship with grade and DNA ploidy in breast cancer

The aim of this study was to quantify p53 expression by flow cytometry. A panel of three monoclonal antibodies: NCL-p53-240, NCL-p53-1801 and NCL-p53-DO7, was tested on breast cell lines and primary breast cancers. The relationships between ploidy, tumour grade and p53 expression for each antibody, were examined. Methodology was assessed using a variety of breast cell lines. Staining patterns were confirmed and the quantification technique qualified. Cytokeratin-positive cells from 58 samples obtained from patients with breast cancer were assayed for DNA content and p53 expression. p53 quantification was performed using calibrated fluorescent beads on cytokeratin-positive cells. Bloom and Richardson grading revealed 20 grade I and 38 grade II/III breast cancers. Examination of fluorescence thresholds showed a positive correlation between grade and DO7 (P=0.003) at a level of 8900 molecules, 240 (P=0.005) at a level of 2900 molecules and 1801 (P=0.005) at a level of 1850 molecules. These levels equated with 34% (DO7), 43% (240) and 43% (1801) of the samples being classified as p53-positive. Examination of ploidy revealed 23 diploid and 35 aneuploid breast cancers. Application of p53 threshold levels on diploid and aneuploid tumours showed correlation between aneuploidy and p53 expression for DO7 at a level of 9000 molecules, 240 at a level of 1900 molecules and 1801 at a level of 1800 molecules. These levels equated with 34% (DO7), 52% (240) and 52% (1801) of the samples being classified as p53-positive. We conclude that measurement of p53 by flow cytometry may be of clinical importance by indicating levels of positivity using fluorescence thresholds. p53 expression has been shown to correlate with both grade and ploidy. Flow-cytometric measurement of p53 may be a useful prognostic assay.

A flow cytometric study of c-erbB-3 expression in breast cancer

In order to assess the specificity of biotinylated anti-c-erbB-3 antibody, screening was performed on a series of tumour cell lines and lymphocytes. Staining was found to be consistent, with good reproducibility. Twenty-nine consecutive breast cancer samples were obtained from women treated with tamoxifen and undergoing elective mastectomy. Twenty-eight invasive ductal carcinomas and 1 DCIS were stained for c-erbB-3 expression: 2 were grade I (Bloom and Richardson), 15 grade II, and 11 grade III tumours, 1 being unclassified; 16 were axillary node positive and 10 node negative; in 2 cases no nodes were sampled. Tumours examined by flow cytometry were stained with cytokeratin FITC antibody and the cytokeratin-positive population gated. Using Mann-Whitney analysis no association was seen between c-erbB-3 expression and Bloom and Richardson grade or axillary node status. In the tumour samples c-erbB-3 expression was found to show an association with EGF-R (P=0.021r2=0.16), PgR (P=0.02,r2=0.16), c-myc (P<0.0001,r2=0.5), c-jun (P=0.001,r2=0.4) and c-fos (P=0.001,r2=0.5) but not with c-erbB-2 (P=0.2,r2=0.06), ER (P=0.4,r2=0.02) or p53 1801 (P=0.05,r2=0.2). Expression of c-erbB-3 may not be an independent marker of prognosis, but it is associated with other markers of poor prognosis and early cellular events linked with aberrant growth and differentiation.

Immunohistochemical Assessment of ER-P31, a Mouse Anti-Oestrogen Receptor Protein Monoclonal Antibody in Human Breast Cancers: Comparison with ER-ICA (Abbott) and Radioligand Assays

A new mouse anti-oestrogen receptor (ER) monoclonal antibody, ER-P31, has been developed. Comparisons of immunohistochemical detection of ER in human breast cancers using ER-P31 with anti-ER from ER-immunocytochemical assay (ER-ICA; Abbott Diagnostic Division) and radioligand dextran-coated charcoal (DCC) assays were carried out. A total of 63 breast cancers, both ER-negative and -positive, were tested. A highly significant degree of correlation between immunostaining for ER-P31 and both ER-ICA and DCC assays was obtained. It is hoped that once ER-P31 is widely available commercially, determination of ER status in breast cancers should be routinely and economically available to all women with breast cancer. Moreover, with the introduction and implementation of a screening programme for detecting breast cancers, immunocytochemical determination of ER status in unequivocal and equivocal positive fine-needle aspirates of breast lesions can be readily performed.