Wendy A. Raymond

Nucleolar organizer regions relate to growth fractions in human breast carcinoma

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.

Global Levels of Specific Histone Modifications and an Epigenetic Gene Signature Predict Prostate Cancer Progression and Development

Background: Epigenetic alterations are common in prostate cancer, yet how these modifications contribute to carcinogenesis is poorly understood. We investigated whether specific histone modifications are prognostic for prostate cancer relapse, and whether the expression of epigenetic genes is altered in prostate tumorigenesis. Methods: Global levels of histone H3 lysine-18 acetylation (H3K18Ac) and histone H3 lysine-4 dimethylation (H3K4diMe) were assessed immunohistochemically in a prostate cancer cohort of 279 cases. Epigenetic gene expression was investigated in silico by analysis of microarray data from 23 primary prostate cancers (8 with biochemical recurrence and 15 without) and 7 metastatic lesions. Results: H3K18Ac and H3K4diMe are independent predictors of relapse-free survival, with high global levels associated with a 1.71-fold (P < 0.0001) and 1.80-fold (P = 0.006) increased risk of tumor recurrence, respectively. High levels of both histone modifications were associated with a 3-fold increased risk of relapse (P < 0.0001). Epigenetic gene expression profiling identified a candidate gene signature (DNMT3A, MBD4, MLL2, MLL3, NSD1, and SRCAP), which significantly discriminated nonmalignant from prostate tumor tissue (P = 0.0063) in an independent cohort. Conclusions: This study has established the importance of histone modifications in predicting prostate cancer relapse and has identified an epigenetic gene signature associated with prostate tumorigenesis. Impact: Our findings suggest that targeting the epigenetic enzymes specifically involved in a particular solid tumor may be a more effective approach. Moreover, testing for aberrant expression of epigenetic genes such as those identified in this study may be beneficial in predicting individual patient response to epigenetic therapies. Cancer Epidemiol Biomarkers Prev; 19(10); 2611–22. ©2010 AACR.

Immunoperoxidase staining in the detection of lymph node metastases in stage i breast cancer

&NA; 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.

Assessment of invasion in breast lesions using antibodies to basement membrane components and myoepithelial cells

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.

Gastric HER2 testing study (GaTHER): An evaluation of gastric/ gastroesophageal junction cancer testing accuracy in Australia

Trastuzumab provides a survival benefit in patients with human epidermal growth factor receptor 2 (HER2)-amplified/overexpressed advanced gastric and gastroesophageal junction cancers (GC/GJCs). However, the optimal method for testing is unclear. The aim of this study was to assess interlaboratory agreement on HER2 scoring in GC/GJC to aid the development of a robust testing algorithm for diagnostic practice in Australia. Nine laboratories assessed the HER2 status of 100 GC/GJC tissue samples by immunohistochemistry (IHC) and in situ hybridization (ISH) [chromogenic (CISH) or silver (SISH)] using both HER2 copy number and HER2:chr17 (chromosome 17) ratio. Results were compared with reference fluorescence ISH (FISH). Interlaboratory agreement on IHC3+ scoring was good (&kgr;=0.76), and there was good/very good agreement between IHC (positivity defined as IHC3+) and ISH when HER2 copy number was used (&kgr;=0.72 to 0.87). Agreement on CISH/SISH scoring was good/very good when HER2 copy number was used (&kgr;=0.68 to 0.86), and agreement between CISH/SISH and FISH using HER2 copy number was very good (&kgr;=0.88 to 0.91). Agreement was reduced when HER2:chr17 ratio was used. The good agreement for HER2 copy number determined by bright-field ISH suggests that this is the optimal method for testing in GC/GJC cases. An IHC3+ score was strongly predictive of a positive ISH result, although agreement for all IHC scores was only moderate, suggesting that IHC triage before ISH testing would be the most cost-effective strategy. However, because of the unique features of GC/GJC samples and the difficulty of ensuring consistent HER2 staining in the community setting, it is recommended that HER2 status in advanced GC/GJC be determined by both IHC and ISH in the same laboratory.

Vimentin expression is not associated with poor prognosis in breast cancer

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 immunohistochemistry with monoclonal antibodies (MAbs) to VIF, p53 protein and cell proliferation marker MIB‐1. 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‐1 count. Despite these associations with several recognised features of tumour aggressiveness, vimentin expression was not associated with increases in risk of relapse or death from breast cancer.

A simple index using video image analysis to predict disease outcome in primary breast cancer

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 Coxs 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.

An evaluation of potentially suitable fixatives for immunoperoxidase staining of estrogen receptors in imprints and frozen sections of breast carcinoma

&NA; 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 periodate‐lysine‐paraformaldehyde at 22°C, or 10% formalin followed by acetone at −10°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.

Prognostic Parameters in Breast Cancer

&NA; 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.

Establishment of the Australian in situ hybridization program for the assessment of HER2 amplification in breast cancer: A model for the introduction of new biomarkers into clinical practice

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.