Search

In August 2006, the Australian government announced a decision to subsidize trastuzumab therapy for early breast cancer, to commence 6 weeks later. It was mandated that HER2 gene amplification, determined by in situ hybridization (ISH), be shown, and that the sponsor company, Roche Products Pty Ltd, should fund this testing. This announcement potentially required provision of ISH testing for HER2 for every newly diagnosed breast cancer, where previously HER2 testing had been performed by immunohistochemistry with support from a single fluorescence ISH (FISH) reference laboratory for indeterminate cases. The Australian HER2 Testing Advisory Board, an independent expert group, responded to the challenge of rapidly providing accurate nationwide ISH testing. Bright-field ISH was selected as the testing platform and a decentralized testing model, with support from a central FISH laboratory, was adopted. An implementation plan was developed addressing standards for training, accreditation, and quality assurance. Within 6 weeks, 8 pathology laboratories were accredited for ISH testing and by September 2008, 2 years after the announcement, 22 ISH testing laboratories were taking part in the national program and almost 20,000 ISH tests had been performed. This article describes the design and rapid implementation of a nationwide program of bright-field ISH as the first-line testing platform for HER2 status in early breast cancer. We believe that this model for the coordinated and large-scale implementation of a new biomarker test has wide application, given that accurate assessment of a range of novel biomarkers is being used increasingly to determine eligibility for new targeted treatment modalities.

The tests that are currently available for the measurement of overexpression of the human epidermal growth factor-2 (HER2) in breast cancer have shown considerable problems in accuracy and interlaboratory reproducibility. Although these problems are partly alleviated by the use of validated, standardised 'kits', there may be considerable cost involved in their use. Prior to testing it may therefore be an advantage to be able to predict from basic pathology data whether a cancer is likely to overexpress HER2. In this study, we have correlated pathology features of cancers with the frequency of HER2 overexpression assessed by immunohistochemistry (IHC) using HercepTest (Dako). In addition, fluorescence in situ hybridisation (FISH) has been used to re-test the equivocal cancers and interobserver variation in assessing HER2 overexpression has been examined by a slide circulation scheme. Of the 1536 cancers, 1144 (74.5%) did not overexpress HER2. Unequivocal overexpression (3+ by IHC) was seen in 186 cancers (12%) and an equivocal result (2+ by IHC ) was seen in 206 cancers (13%). Of the 156 IHC 3+ cancers for which complete data was available, 149 (95.5%) were ductal NST and 152 (97%) were histological grade 2 or 3. Only 1 of 124 infiltrating lobular carcinomas (0.8%) showed HER2 overexpression. None of the 49 'special types' of carcinoma showed HER2 overexpression. Re-testing by FISH of a proportion of the IHC 2+ cancers showed that only 25 (23%) of those assessable exhibited HER2 gene amplification, but 46 of the 47 IHC 3+ cancers (98%) were confirmed as showing gene amplification. Circulating slides for the assessment of HER2 score showed a moderate level of agreement between pathologists (kappa 0.4). As a result of this study we would advocate consideration of a triage approach to HER2 testing. Infiltrating lobular and special types of carcinoma may not need to be routinely tested at presentation nor may grade 1 NST carcinomas in which only 1.4% have been shown to overexpress HER2. Testing of these carcinomas may be performed when HER2 status is required to assist in therapeutic or other clinical/prognostic decision-making. The highest yield of HER2 overexpressing carcinomas is seen in the grade 3 NST subgroup in which 24% are positive by IHC.

Image analysis was used to investigate the prognostic significance of immunostaining for oestrogen receptor (ER), p53 tumour-suppressor protein and tumour cell proliferation (MIB-1) in a random cohort of 200 primary breast cancer patients with between 4 and 7 years of clinical follow-up. Image measurements of the percentage of immunopositive cancer cell nuclei (% positive nuclear area) were recorded for the above tumour features for each patient. Assessment of relative risk using Cox's univariate analysis indicated that tumour size, number of cancer-involved nodes, MIB-1 and ER % positive nuclear area were significantly associated with breast cancer disease outcome, i.e., relapse-free survival and overall survival. In multivariate analysis, tumour size, number of involved nodes, ER and MIB-1 % positive nuclear area were retained as independent predictors of prognosis, depending on the image measurement cut-point used. A prognostic model, which can be used without reference to nodal involvement, was constructed for tumour size, ER cut-point of 30% positive nuclear area and MIB-1 cut-point of 10% positive nuclear area. Kaplan-Meier analysis of this image-based prognostic index identified 4 risk groups with predicted 5-year overall survival rates of 93%, 83%, 76.7% and 61.5%. We conclude that image measurements of ER and proliferative rate can be combined with tumour size to construct a prognostic index which reliably predicts disease outcome in primary breast cancer without knowledge of the nodal status of the patient. Int. J. Cancer (Pred. Oncol.) 84:203–208, 1999. © 1999 Wiley-Liss, Inc.

The clinical significance of vimentin intermediate filament (VIF) expression was studied in relation to other established prognostic parameters in primary breast cancer. Archival tumour samples embedded in paraffin were examined by immuno-histochemistry with monoclonal antibodies (MAbs) to VIF, p53 protein and cell proliferation marker MIB-I. The vimentin staining pattern was heterogeneous, but in vimentin-positive areas > 80% of the tumour cells were positive. There was no association between vimentin expression and tumour size or the number of axillary lymph nodes involved. Vimentin expression was significantly associated with high-grade tumours, absence of hormone receptors, increased p53 expression and high tumour proliferation fraction as estimated by MIB-I count. Despite these associations with several recognised features of tumour aggressiveness, vimentin expression was not associated with increase in risk of relapse or death from breast cancer.

Summary This paper describes immunostaining of consecutive sections from 15 cases of fibrocystic change of the breast (including 2 examples of intraductal papilloma), 4 ductal carcinomas-in-situ and 17 invasive carcinomas (4 tubular, 1 papillary, 2 lobular and 10 infiltrating ductal, NOS) with antisera to components of the basement membrane (BM), type IV collagen and laminin, and with the muscle antibodies actin and muscle-specific actin. A simple digestion technique was developed to improve the clarity of BM staining with these antibodies. The BM stains facilitated identification of small invasive foci through breaks in the BM in 2 of the cases which had been reported as pure intraductal carcinoma. Tubular carcinomas were surrounded by abnormal, fragmented, and focally discontinuous BM, a feature which could be used to distinguish this well-differentiated breast carcinoma sub-type from sclerosing adenosis, in which individual acini were invariably surrounded by a continuous BM. BM staining emphasized the fibrovascular core of intraductal papillomas, whereas the BM layer was absent in intraductal, cytologically malignant, papillary projections. Similarly, myoepithelial cells, stained with antisera to muscle actins, were identified in a continuous layer surrounding benign epithelial proliferations. These immuno-histochemical staining techniques may thus assist the diagnostic histopathologist in differentiating between benign epithelial proliferations of the breast and well-differentiated invasive breast carcinoma, and in identifying foci of microinvasive carcinoma.

Purpose: The purpose is to determine whether the levels of expression of extracellular matrix components in peritumoral stroma are predictive of disease outcome for women with node-negative breast cancer. Experimental Design: Tumor tissue from 86 patients with node-negative breast cancer was examined by immunohistochemical staining for the expression of versican, chondroitin sulfate (CS), tenascin, and hyaluronan (HA). With the exception of HA, the expression of the extracellular matrix components was measured by video image analysis. Statistical correlation of the immunohistochemical data with clinicopathological characteristics and disease outcome was performed. Results: All of the extracellular matrix components were present in the peritumoral stroma of the entire study cohort. In contrast, immunoreactivity within the cancer cell was observed in 82% of tumors for HA, 12% for CS, and 4% for tenascin; no immunostaining of cancer cells for versican was observed for any of the tumors. Cox regression and Kaplan-Meier analyses indicated that elevated expression of stromal versican predicted increased risk and rate of relapse in this cohort. Elevated expression of tenascin was predictive of increased risk and rate of death only. Although neither CS nor HA were predictive of disease outcome in this cohort, tumor size was predictive of increased risk and rate of both relapse and survival. Conclusions: Elevated expression within peritumoral stromal matrix of versican and tenascin was predictive of relapse-free and overall survival, respectively, in women with node-negative breast cancer.

Summary There is a continuing controversy regarding the value of estimating degree of intra-tumor vascularity to predict prognosis in breast cancer. In order to resolve this controversy, primary tumors from a cohort of 519 women with breast cancer were analysed to determine whether association exists between degree of vascutarity and prognosis. Tumor vascularity was estimated by immunohistochemistry using a monoclonal antibody to the antigen CD31. The tumor area showing the highest degree of vascularity was chosen to score the number of microvessels per unit area. Issues such as the reproducibility of the microvascularity score and its association with tumor parameters including size, histological grade and hormone receptor levels were investigated. Although previously agreed criteria were used, consensus between two pathologists' estimations of the degree of vascularity was only moderate. There was no statistically significant association between tumor vascularity score and other currently established parameters of prognosis. After a median follow up of 71 months for axiltary node negative patients, there was no association between tumor vascularity score and increased risk of relapse or death from breast cancer. In axillary node positive patients, tumor vascularity score was associated with increased risk of relapse and death from breast cancer. This association was not however independent of other established parameters of prognosis.

This paper describes an improved immunohistochemical method for demonstrating oestrogen receptor (OR) protein in paraffin-embedded sections of tissue fixed for 1.5 h in formalin. Thirty-two cases of infiltrating ductal breast carcinoma were stained with a monoclonal anti-OR antibody (H222), using a standard streptavidin-biotin method, following pretreatment with pronase. OR counts in paraffin sections were compared with those of frozen sections and with cytosolic values determined by a dextran-coated charcoal method. Twenty-seven of the carcinomas were OR-positive in paraffin sections. There was concordance between the paraffin section and the frozen section-determined receptor status in 30 cases (94 per cent) and a strong correlation was observed (r = 0.76; P less than 0.0001). Similarly, OR counts in paraffin sections correlated with cytosolic OR values (r = 0.60; P less than 0.001) and there was concordance in 97 per cent of cases. The percentage of positively-stained tumour cells in paraffin sections ranged from 0 to 94 per cent with staining intensities comparable to those seen in frozen sections. Staining of paraffin sections identified more OR-positive tumours than either frozen section staining or cytosolic assay. This study validates immunohistochemical OR analysis in formalin-fixed, paraffin-embedded breast carcinomas using a commercial anti-OR antibody.

Immunohistochemical staining for estrogen receptor (ER) was performed on :12 infiltrating duct carcinomas of the breast using formalin-fixed, paraffin-embedded sections, monoclonal anti-ER (H222, Abbott Laboratories) and a strepavldin-biotin peroxidase method. Results of ER staining in paraffin sections were compared with those obtained on frozen sections and cytosolic ER levels obtained by a dextran-coated method on the same cases. Twenty-seven of the tumours were positive for ER in paraffin sections with a concordence of 94% between staining in paraffin sections and In frozen tissue (r = 0.76; p

Measurements of the growth fraction (GF) and oestrogen receptor (OR) status were performed by immunohistochemical staining, using the monoclonal antibodies Ki-67 and anti-OR, in consecutive cryostat sections of biopsies from 74 women with primary breast cancer, and 12 women with benign breast disease. A significant inverse correlation was identified between the GF and OR status (r = -0.452, P less than 0.0001), but a group of OR-positive tumours with high GFs was also observed. There was a strong positive correlation between immunocytochemically determined OR percentages and values obtained with a cytosolic OR biochemical assay for the 70 cases in which both techniques were performed (r = 0.801, P less than 0.0001). A significant relationship was also revealed between the histological grade of infiltrating ductal carcinomas and their associated GFs (r = 0.622, P less than 0.0001). No correlation was found between the GF and the number of positive axillary nodes. Our findings suggest that GF and OR status are useful independent prognostic parameters in breast cancer, but the former probably has an overriding influence. Both parameters may be useful guides for selection of appropriate endocrine and/or adjuvant chemotherapy.

The estrogen receptor (ER) content of breast carcinoma is generally accepted as valuable in predicting clinical outcome and tumour response to hormonal manipulation. We applied a new immunocytochemical assay for estrogen receptors (Abbott ERICA Monoclonal) to 20 breast tumours, and examined the efficacy of 16 fixation procedures before immunoperoxidase staining of frozen sections and imprint preparations. Our findings indicate that the fixatives of choice are periodatelysine-paraformaldehyde at 22 degrees C, or 10% formalin followed by acetone at -10 degrees C. These fixation procedures are simpler, less time-consuming, and provide superior staining, tumour cytomorphology and higher ER values than the 3-reagent sequence recommended by Abbott Laboratories. There was a significant correlation between the ER scores in the frozen sections and the imprints. Positive ER cytosol results correlated with the staining index in the frozen sections, and the ER scores in the imprints. We conclude that imprints are suitable preparations for ER analysis by the immunoperoxidase technique, particularly for small tumour specimens.

This report describes a positive relationship between vimentin expression in infiltrating ductal breast carcinoma, and high tumour growth fraction. Vimentin expression is potentially a predictor of aggressive behaviour, and such carcinomas may benefit from early adjuvant therapy. Eighty-four malignant breast neoplasms were stained with monoclonal anti-vimentin and anti-cytokeratin antibodies. The tumour growth fractions were determined by immunostaining cryostat sections with the Ki-67 antibody. Seven (9.2 per cent) of 76 infiltrating ductal carcinomas co-expressed cytokeratin and vimentin intermediate filaments in more than 50 per cent of neoplastic cells. In each case, the corresponding Ki-67 count was much greater than 40 per cent, significantly higher than the mean growth fraction for all tumours examined (P less than 0.0001). Vimentin immunoreactivity was also positively related to the histological grade of the ductal carcinomas (P less than 0.002) and inversely related to tumour ER count (P less than 0.0002) and patient age (P less than 0.01). No relationship was observed between vimentin positivity and either the presence of axillary nodal metastases or primary tumour size.

An argyrophil stain for nucleolar organizer regions (NORs) has recently been applied to paraffin sections of human tissues. This report describes a positive relationship between the mean numbers of AgNOR sites per nucleus and tumor growth fraction, as determined by immunostaining with the monoclonal antibody Ki-67, in 83 malignant breast tumors (P less than .01). This relationship supports recent suggestions that the NOR count may reflect cell synthetic activity and hence, proliferation. AgNOR counts correlated inversely with immunocytochemically assessed estrogen receptor content (P less than .002), but there was no relationship between the AgNOR count and primary tumor size, histologic grade, axillary node status, or patient age. A significant difference (P less than .00001) was found between the AgNOR counts in 64 benign breast lesions (mean, 2.05) and 85 malignant breast neoplasms (mean, 5.46). The limitations of the silver staining technique and the problems of reproducibility in AgNOR counting are detailed.

Until recently, [3H]-thymidine incorporation, DNA analysis by flow cytometry, and cell doubling times have been the main methods of studying tumour cell kinetics. All these techniques are laborious, expensive, and difficult to perform in a routine diagnostic laboratory. This study examined fresh frozen sections from 31 prostatic biopsy specimens with the hybridoma antibody Ki-67, a marker of proliferating cells, using a modified avidin-biotin-peroxidase complex technique. The percentage of glandular cells decorated by this antibody, representing the growth fraction, was determined for both benign and malignant samples. Benign prostatic glands showed an average Ki-67 score of 4 per cent, significantly less than the 16.3 per cent mean growth fraction found in prostatic carcinomas. There was a significant correlation between the tumour growth fraction as assessed by Ki-67 staining, and the histological grade. A positive correlation was also found between the Ki-67 score and the intensity of staining, and a definite trend was noted between the Ki-67 score and the tumour clinical stage. Ki-67 promises to be a useful marker in determining the prognosis of prostatic cancer.

This paper identifies another neoplasm of epithelial origin which may express vimentin in addition to cytokeratins, thereby adding to the expanding list of tumours which demonstrate intermediate filaments (IFs) other than those of their reputed cell of origin. Twenty examples of benign breast disease and 66 carcinomas were examined for vimentin and cytokeratin IFs using an avidin–biotin–peroxidase complex technique. Co-expression of these IF proteins was found in 35 per cent of cases of benign breast tissue and in 60 per cent of the carcinomas. In 8 (16 per cent) of 50 cases of infiltrating ductal carcinoma, vimentin and cytokeratin immunostaining was observed in more than 60 per cent of the tumour cells. These carcinomas were predominantly of a high histological grade. In benign breast disease and well-differentiated carcinoma, vimentin was distributed in the basal and perinuclear regions of the cells, with sparing of the apical portions. In those cases in which large numbers of tumour cells expressed vimentin, cytoplasmic staining was diffuse, and often exhibited distinctive perinuclear and subplasmalemmal accentuation. We propose that a knowledge of the list of carcinomas which may co-express vimentin and cytokeratin IFs might be helpful in the assessment of undifferentiated tumours and metastatic deposits.

Carcinoma of the breast is the most common cancer in Australian women. Current methods of treatment and refinements in therapeutic regimens are based on our understanding of the biological behaviour of the disease. Several prognostic parameters have been identified which predict survival and allow the selection of patients who may benefit from adjuvant therapies. The more important of these parameters include tumour size, histologic type, histologic grade, axillary lymph node status, estrogen receptor status and tumour growth fraction. Diagnostic pathologists who are responsible for the evaluation of these parameters should be cognizant of their relative prognostic values and also of other factors such as cellular antigens, lectin binding and oncogenes which may have potential roles in predicting survival and therapeutic responses. This review provides an update of prognostic parameters which are assessed through examination of the excised specimen.

Axillary lymph node involvement by tumour metastasis is of major prognostic significance in breast carcinoma. In an immunocytochemical study using monoclonal antibodies to cytokeratins, 7 (23%) of 30 cases of node-negative breast carcinoma were found to contain metastases, resulting in upstaging of the disease. The metastatic foci were missed on routine screening of H&E stained sections. There was no significant association between the presence of metastases and the location, diameter, type or grade of the primary tumour. These findings emphasize the contribution of immunocytochemical staining for the identification and detection of metastatic breast carcinoma in axillary lymph nodes.