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Aims: Technical limitations in conventional pathological evaluation of breast lumpectomy specimens may reduce diagnostic accuracy in the assessment of margin and focality. A novel technique based on whole-mount serial sections enhances sampling while preserving specimen conformation and orientation. The aim of this study was to investigate assessment of focality and margin status by the use of whole-mount serial sections versus simulated conventional sections in lumpectomies., Methods and Results: Two pathologists interpreted whole-mount serial sections and simulated conventional sections for 58 lumpectomy specimens by reporting the closest margin and focality. Measurements were compared by the use of McNemar's chi-squared test. Statistically significant differences were observed in the assignment of both margin positivity (P = 0.014) and multifocality (P = 0.021). A positive margin or multifocal disease was identified by the use of whole-mount serial sections but missed in the simulated conventional assessment in 10.3% and 17.2% of all cases, respectively. There was no case in which a positive margin was detected only in the simulated conventional assessment., Conclusions: The whole-mount technique is more sensitive than conventional assessment for identifying a positive margin or multifocal disease in breast lumpectomy specimens. Undersampling in conventional sections was implicated in almost all cases of discordance. The majority of positive margins or secondary foci identified only in whole-mount serial sections concerned in-situ disease., (© 2015 John Wiley & Sons Ltd.)

Aims: Multiplexed immunofluorescence is a powerful tool for validating multigene assays and understanding the complex interplay of proteins implicated in breast cancer within a morphological context. We describe a novel technology for imaging an extended panel of biomarkers on a single, formalin-fixed paraffin-embedded breast sample and evaluating biomarker interaction at a single-cell level, and demonstrate proof-of-concept on a small set of breast tumours, including those which co-express hormone receptors with Her2/neu and Ki-67., Methods and Results: Using a microfluidic flow cell, reagent exchange was automated and consisted of serial rounds of staining with dye-conjugated antibodies, imaging and chemical deactivation. A two-step antigen retrieval process was developed to satisfy all epitopes simultaneously, and key parameters were optimized. The imaging sequence was applied to seven breast tumours, and compared with conventional immunohistochemistry. Single-cell correlation analysis was performed with automated image processing., Conclusions: We have described a novel platform for evaluating biomarker co-localization. Expression in multiplexed images is consistent with conventional immunohistochemistry. Automation reduces inconsistencies in staining and positional shifts, while the fluorescent dye cycling approach dramatically expands the number of biomarkers which can be visualized and quantified on a single tissue section., (© 2013 John Wiley & Sons Ltd.)

Aims: Increasing the sectioning rate for breast sentinel lymph nodes can increase the likelihood of detecting micrometastases. To make serial sectioning feasible, we have developed an algorithm for computer-assisted detection (CAD) with digitized lymph node sections., Methods and Results: K-means clustering assigned image pixels to one of four areas in a colourspace (representing tumour, unstained background, counterstained background and microtomy artefacts). Four filters then removed 'false-positive' pixels from the tumour cluster. A set of 43 sections containing tumour (a total of 259 foci) and 59 sections negative for malignancy was defined by two pathologists, using light microscopy, and CAD was applied. For the clinically relevant task of identifying the largest focus in each section (micrometastasis in 22/43 sections), the sensitivity and specificity were 100%. Isolated tumour cells (ITCs) were identified in one slide initially considered to be negative. Identification of all 259 foci yielded sensitivities of 57.5% for ITCs (<0.200 mm), 89.5% for micrometastases, and 100% for larger metastases, with one false-positive. Reduced sensitivity was ascribed to variable staining. Nine additional metastases (<0.01-0.3 mm) that were not initially identified were detected by CAD., Conclusions: This algorithm is well suited to the task of sentinel lymph node evaluation and may enhance the detection of occult micrometastases., (© 2011 Blackwell Publishing Limited.)

Aims: To develop a method for preparing diagnostic-quality, whole-mount serial sections of breast specimens while preserving 3-D conformation. This required supporting the fresh specimen prior to breadloafing and refining the conventional tissue processing method. The overall goal is to use digital images of whole-specimen histopathology to improve the estimation of extent of disease., Methods and Results: To maintain a 3-D conformation, the specimen is suspended in 3.5% agar at 55 degrees C. The block is sliced at 5-mm intervals. Sectioning is performed after extended fixation in 4% formaldehyde from paraformaldehyde in 0.1 m Millonig's buffer, followed by paraffin processing using a non-routine schedule and extended paraffin infiltration. Whole-mount serial breast sections are produced with features of equal or superior quality to that which can be achieved using conventional methods. The method is compatible with some immunohistochemical stains but requires further optimization for others., Conclusions: The technique is currently suitable for research applications. With the reduction in processing time achievable with microwave-assisted processing, there is the potential for its use as a routine clinical method. This tool may improve the accuracy of margin estimates and identification of multifocality in breast cancer; further evaluation is necessary.