Observer agreement comparing the use of virtual slides with glass slides in the pathology review component of the POSH breast cancer cohort study.

AIMS: (1) To compare the use of scanned virtual slide images (virtual microscopy) with glass slides (conventional microscopy) in the assessment of morphological characteristics of breast cancers within the setting of the Prospective study of Outcomes in Sporadic versus Hereditary breast cancer (POSH), involving a cohort of women under 40 years of age, presenting with breast cancer. (2) To assess the acceptability to histopathologists of the use of virtual slide images. METHODS: 13 histopathologists from the UK and Australia participated in the POSH pathology review. The observers were asked to assess multiple morphological features such as tumour grade and type. Comparisons were made for a single observer using both virtual images and glass slides. Intra- and inter-observer variability was calculated using the κ statistic and a comparison was made between the use of each image modality. RESULTS: Diagnostic performance with virtual slides was comparable to conventional microscopic assessment, with the measurement of agreement best for vascular invasion, necrosis and the presence of a central scar (κ=0.37-0.78), and poor for more subjective parameters such as pleomorphism, stroma, the nature of the tumour border and the degree of lymphocytic infiltrate (κ=0.1). CONCLUSION: Virtual slides represent an acceptable methodology for central review of breast cancer histopathology and can circumvent the need for either travel to view material, or the potential problems of sending it by post.

Cyclin D-1 protein over-expression is not associated with gene amplification in benign and atypical apocrine lesions of the breast.

Cyclin D-1 protein over-expression and/or gene amplification have been shown to be frequent events in subsets of breast carcinomas. Cyclin D-1 is generally considered as a weak oncogene, and its over-expression has been shown to occur in occasional benign breast lesions. In a previous series, we have shown that cyclin D-1 was over-expressed in subsets of apocrine metaplasia (APM) and apocrine adenosis (AA) of the breast and that such over-expression was mostly associated with a significant increase in the proliferative capacity of these lesions. We examined the mechanisms involved in cyclin D-1 over-expression in apocrine lesions. A total of 41 cases were analysed in this study. The cases were divided into: 18 cases of APM and 23 cases of AA. All cases analysed had been previously analysed by immunohistochemistry, and all showed over-expression of the cyclin D-1 protein. Fluorescence in situ hybridisation (FISH) was performed using a dual cyclin D-1 (spectrum orange)/chromosome 11 centromere (spectrum green) DNA probe. The results showed that none of the cases studied had concomitant gene amplification. These results suggest that other post-transcriptional mechanisms might be responsible for cyclin D-1 protein over-expression in benign apocrine lesions. Further studies are needed to understand the mechanisms involved in abnormal cyclin D-1 expression in these lesions.

Distinction of isolated tumour cells and micrometastasis in lymph nodes of breast cancer patients according to the new Tumour Node Metastasis (TNM) definitions.

Isolated tumour cells and micrometastases represent two different staging categories and are often dealt with differently when identified in sentinel lymph nodes of breast cancer patients. The reproducibility of these categories was found to be suboptimal in several studies. The new edition of the TNM (Tumour Node Metastasis) is expected to improve the reproducibility of these categories. Fifty cases of possible low-volume nodal involvement were represented by one to four digital images and were analysed by members of the European Working Group for Breast Screening Pathology (EWGBSP). The kappa value for interobserver agreement of the pN (TNM) staging categories and of the isolated tumour cells category were 0.55 and 0.56 reflecting moderate reproducibility, and the kappa of the micrometastatic category (0.62) reflected substantial reproducibility. This is an improvement over the results gained on the basis of the previous edition of the TNM. Maximal adherence to the category definitions supplemented by explanatory texts in the staging manual should result in more homogeneous nodal staging of breast cancer.

Cell turnover in apocrine metaplasia and apocrine adenosis of the breast.

Apocrine metaplasia (APM) is a common finding in the breast of postmenopausal women and is seen in a broad spectrum of lesions ranging from microscopic cysts to invasive apocrine carcinoma. Apocrine metaplasia within sclerosing adenosis is known as apocrine adenosis (AA) and is considered a benign lesion of the breast. Apocrine metaplasia and AA have been the subject of many studies; however, little is known about the dynamics of cell turnover in these lesions. Recent studies have shown that some forms of AA may show altered degree of proliferation along with altered expression of bcl-2 and bax proteins. In the current study, we investigate further aspects of apoptosis to help understand the mechanisms of cell turnover in AA and APM. To investigate cell turnover in APM and AA, immunohistochemistry was used to study the expression of the apoptotic markers Bak, Mcl-1, Bcl-x, and Bcl-x(L) in 45 cases of APM (13 cases of nonpapillary APM, 21 cases of simple papillary APM, and 11 cases of complex papillary APM). Also, 34 cases of AA (23 cases of non-atypical AA [NAA] and 11 cases of atypical AA [AAA]) were included in the study. The expression of hTERT and the proliferation marker Ki-67 were also determined. The TdT-mediated dUTP nick-end labeling (TUNEL) technique was used to study the apoptotic status in 28 cases of APM (12 cases nonpapillary APM and 16 cases of papillary APM including simple and complex forms) and 22 cases of AA (15 cases of NAA and 7 cases of AAA). The results showed that all cases studied by immunohistochemistry were positive for the expression of Bak, Mcl-1, Bcl-x, and Bcl-x(L) showing a pattern of staining similar to that seen in the normal breast epithelium. There was no relation between hTERT positivity and the degree of proliferation in any of the lesions studied. The TUNEL results revealed an apoptotic index (AI) of 0.4% and 0.2% in the papillary and nonpapillary groups of APM, respectively. There was no statistical significance between the AI of these 2 groups and that of the normal breast epithelium (0.3%). The average Ki-67 index in the nonpapillary group was 0.7%, whereas in the papillary group, a value of 4% was recorded. In the cases of AA, an AI of 0.4% and 0.3% in NAA and AAA, respectively, was seen. There was no statistical significance between the AI of these 2 groups and that of the normal breast epithelium (0.3%). The Ki-67 index was 5.2% and 6.6% in the NAA and AAA, respectively. The current results show that apoptosis is not a common event in APM and AA even in the presence of increased proliferation, which may render some of these lesions more susceptible to oncogenic changes. Further studies are needed to study other apoptotic pathways that may be involved in cell turnover in these lesions.

Alterations of the cell cycle regulators cyclin D1, cyclin A, p27, p21, p16, and pRb in apocrine metaplasia of the breast.

G1/S transition defects have been a proposed requirement for tumor development. Apocrine metaplasia (APM) in the breast has been held as a sign of benignity. Yet, a number of studies have reported the presence of molecular abnormalities in some forms of APM suggesting a possible oncogenic potential for some of these lesions. We currently investigate the role of some of the cell cycle proteins, previously reported to be de-regulated in breast cancer, in an attempt to assess their significance in APM. Using immunohistochemistry, the expression of cyclin D1, cyclin A, p27, p21, p16, pRb and Ki-67 was examined in 93 cases of APM. The cases were divided into nonpapillary (NAPM) (30 cases) and papillary metaplasia (PAPM) (63 cases). PAPM was further subdivided into simple papillary (SPAPM) (29 cases), complex papillary (28 cases) and highly complex papillary (six cases). For statistical analysis, the last two groups were merged together (CPAPM). The results showed that increased histological complexity was associated with significant increase of proliferative capacity and alterations of the cell cycle control. The median Ki-67 index was 1.5% in SPAPM and 4.8% in the CPAPM. Also, alterations of the cell cycle regulators were significantly higher in the CPAPM than in the SPAPM. NAPM was generally similar to normal breast epithelium. Apocrine cells were negative for p16 while pRb was expressed in all cases. These findings suggest that in complex forms of APM, there is a considerable degree of cellular unrest. This may contribute to increased susceptibility to carcinogenesis.

Cell cycle alterations and their relationship to proliferation in apocrine adenosis of the breast.

AIMS: Apocrine adenosis (AA) is generally considered a benign disease of the breast. However, recent studies have suggested a precancerous potential for some of these lesions. The aim was to investigate the status of cell cycle proteins previously reported to be deregulated in breast cancer to identify their possible role in AA. METHODS AND RESULTS: The cases were categorized into AA without atypia (NAA) and atypical AA (AAA). Using immunohistochemistry, the expression of cyclin D1, cyclin A, p27, p21, p16, pRb and Ki67 was determined in 29 NAA and 16 AAA cases. Cyclin D1, p21 and cyclin A were overexpressed in 58.6%, 51.7% and 31.8% of the NAA cases, respectively, whereas 81.3%, 62.5% and 41.7% of the AAA cases showed overexpression of cyclin D1, p21 and cyclin A, respectively. All cases were negative for p16, whereas pRb was expressed in all cases. Furthermore, proliferation in AA (4.5%) was significantly higher than that of normal breast epithelium (1%). There was no statistical significance in the degree of proliferation between the NAA (3.7%) and AAA (4.8%) groups. CONCLUSIONS: The study indicates that NAA and AAA are biologically similar. A subset of AA defined by increased proliferation and significant cell cycle alterations may be susceptible to oncogenesis.

Variations in sentinel node isolated tumour cells/micrometastasis and non-sentinel node involvement rates according to different interpretations of the TNM definitions.

Breast cancers with nodal isolated tumour cells (ITC) and micrometastases are categorised as node-negative and node-positive, respectively, in the tumour node metastasis (TNM) classification. Two recently published interpretations of the TNM definitions were applied to cases of low-volume sentinel lymph node (SLN) involvement and their corresponding non-SLNs for reclassification as micrometastasis or ITC. Of the 517 cases reviewed, 82 had ITC and 435 had micrometastasis on the basis of one classification, and the number of ITC increased to 207 with 310 micrometastases on the basis of the other. Approximately 24% of the cases were discordantly categorised. The rates of non-SLN metastases associated with SLN ITCs were 8.5% and 13.5%, respectively. Although the second interpretation of low-volume nodal stage categories has better reproducibility, it may underestimate the rate of non-SLN involvement. The TNM definitions of low-volume nodal metastases need to be better formulated and supplemented with visual information in the form of multiple sample images.

Improving the reproducibility of diagnosing micrometastases and isolated tumor cells.

BACKGROUND: The latest edition of the tumor-lymph node-metastasis (TNM) classification of malignant tumors distinguishes between isolated tumor cells (pN0) and micrometastases (pN1mi). The reproducibility of these categories has not been assessed previously. METHODS: Digital images from 50 cases with low-volume lymph node involvement from axillary sentinel lymph nodes were circulated twice for evaluation (Evaluation Rounds 1 and 2) among the members of the European Working Group for Breast Screening Pathology, and the members were asked to categorize lesions as micrometastasis, isolated tumor cells, or something else and to classify each case into a pathologic lymph node (pN) category of the pathologic TNM system. Methods for improving the low reproducibility of the categorizations were discussed between the two evaluation rounds. kappa Statistics were used for the assessment of interobserver variability. RESULTS: The kappa value for the consistency of categorizing low-volume lymph node load into micrometastasis, isolated tumor cells, or neither of those changed from 0.39 to 0.49 between Evaluation Rounds 1 and 2, but it was slightly lower for the pN categories (0.35 and 0.44, respectively). Interpretation of the definitions of isolated tumor cells (especially with respect to their localization within the lymph node), lack of guidance on how to measure them if they were multiple, and lack of any definitions for multiple simultaneous foci of lymph node involvement were listed among the causes of discordant diagnoses. CONCLUSIONS: The results of the current study indicated that the definitions available have minor contradictions and do not permit a reproducible distinction between micrometastases and isolated tumor cells. Refinement of these definitions, therefore, is required. One refinement that may improve reproducibility is suggested in this report.

Expression of c-erbB2, p53, Bcl-2, Bax, c-myc and Ki-67 in apocrine metaplasia and apocrine change within sclerosing adenosis of the breast.

Molecular evidence has recently suggested a number of different pathways leading to the development of ductal carcinoma of the breast. The links between atypical ductal hyperplasia and low-grade ductal carcinoma in situ and lobular neoplasia and lobular carcinoma are well known pathologically, but high-grade in situ and invasive carcinomas appear to have a different biological oncogenetic pathway. Morphologically there is a similarity between apocrine cells and some cases of high-grade ductal carcinoma. In order to investigate this possibility a number of different biological markers known to occur in high-grade breast carcinomas were assessed in both apocrine metaplasia (APM) and a putative premalignant lesion called apocrine change within sclerosing adenosis (AA). In 64 cases of APM and 18 cases of AA we examined for expression of c-erbB2, p53, Bcl-2, Bax, c-myc and Ki-67 proteins using immunocytochemistry. c-erbB2 expression was seen in 55.6% of AA cases and in 10.9% of APM cases. p53 expression was detected in 27.8% of AA cases but only 1.6% of APM cases. All cases of AA and APM were negative for the anti-apoptotic protein Bcl-2, but all the APM and 33.3% of AA cases showed cytoplasmic positivity for Bax, a pro-apoptotic protein. All the cases of AA and APM were positive for c-myc oncoprotein, however, the mean percentage of nuclear positivity was 50% in AA and 37% in cases of APM cases. The mean percentage positivity for Ki-67, a proliferation associated antigen, was 3.6% in AA and 1.3% in APM. The results indicate that a subset of breast lesions containing APM epithelium show abnormal oncoprotein and apoptosis-related protein expression and have a higher proliferation rate.

Loss of heterozygosity and allelic imbalance in apocrine metaplasia of the breast: microdissection microsatellite analysis.

Loss of heterozygosity (LOH) and allelic imbalance (AI) at loci reported to show allele loss and/or imbalance in preinvasive and invasive breast cancer were examined in 41 cases of apocrine metaplasia (APM) of the breast using a microdissection technique, polymorphic microsatellite markers, and the polymerase chain reaction (PCR). Occasional examples of LOH and/or AI were identified in 2/28 (7.1%) informative cases at 1p (MYCL1), 2/14 (14.3%) at 11q (INT2), 1/15 (6.7%) at 13q (D13S267), 3/22 (13.6%) at 16q (D16S539), 2/23 (8.7%) at 17p (TP53), and 1/11 (9.1%) at 17p (D17S513) and 3/16 (18.8%) at 17q (D17S250). The finding of LOH/AI in cases of APM indicates that a subset of APM appears clonal, but the significance of allelic loss or imbalance in the pathogenesis of APM or its possible subsequent progression to carcinoma is not yet clear and requires further investigation. Clinical follow-up of these particular cases of APM showing LOH/AI would be of further value.

Telepathology in a routine clinical environment: implementation and accuracy of diagnosis by robotic microscopy in a one-stop breast clinic.

The concept of using telepathology as a way of increasing the efficiency of pathologists is not new. There have been many studies attempting to evaluate the role of telepathology diagnosis, predominantly using transmission of still digital images. This study evaluates the potential value of remote diagnosis using robotic microscopy in the setting of a one-stop breast clinic. A Zeiss Axiopath telepathology system was used. The aim was to identify deficiencies in software and the minimum specifications for the computer hardware and network capability needed for reproducible pathological diagnosis with a view to developing a system that can preclude the need for an on-site pathologist. Forty-seven fine needle aspiration smears were diagnosed simultaneously by a pathologist in the breast clinic and by a different pathologist operating a robotic microscope situated in the clinic in a different wing of the hospital from the pathology department. The diagnoses, the time taken for clinic and remote diagnosis, and difficulties in using the system were recorded. Absolute correlation was achieved in 80.9% of cases. There was one false-positive diagnosis of cancer and no false negatives. The mean time taken for diagnosis per case was 2.39 min in clinic and 10.81 min by remote control robotic microscopy. However, as the pathologist did not have to leave the department, remote diagnosis was overall more economical of pathologists' time. Image quality was generally found to be good and not an obstacle to diagnosis. There were operational and technical problems that made remote diagnosis tedious and lengthy. Although at present the system is not capable of replacing an on-site pathologist, the results were encouraging and suggest that remote control remote diagnosis has the potential to increase the efficiency of pathologists.

C-myc oncoprotein expression and gene amplification in apocrine metaplasia and apocrine change within sclerosing adenosis of the breast.

Overexpression and/or amplification of c-myc oncogene are known to occur in human breast carcinomas, particularly those of high grade. Apocrine metaplasia (APM) is a common finding within fibrocystic change, and in some cases appears to be associated with an elevated risk of subsequent breast cancer. It has been suggested that apocrine metaplasia within sclerosing adenosis of the breast, also called apocrine adenosis (AA), has a premalignant potential. Little, however, is known about cellular level genetic alterations in either APM or AA of the breast. Because of this, c-myc expression and amplification in APM and AA were studied. Fluorescence in situ hybridisation (FISH) is a methodological approach to detecting these genetic alterations. In this study, APM and AA were studied immunohistochemically to detect c-myc oncoprotein expression, and FISH was employed using a DNA probe for the c-myc gene in archival tissue sections of cases of APM and AA of the breast. Nuclear immunostaining for c-myc was seen in all APM and AA cases studied, but amplification of the c-myc gene was not seen in any cases with APM or AA. The results of this study indicate that c-myc overexpression appears to occur early in breast oncogenesis. Amplification of the c-myc gene does not occur in APM or AA of the breast, however, suggesting that this particular genetic alteration constitutes a late event in the pathogenesis of breast carcinomas.