Whole-mount pathology of breast lumpectomy specimens improves detection of tumour margins and focality.

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.

A novel, automated technology for multiplex biomarker imaging and application to breast cancer.

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.

Development and evaluation of a robust algorithm for computer-assisted detection of sentinel lymph node micrometastases.

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.

An improved processing method for breast whole-mount serial sections for three-dimensional histopathology imaging.

To develop and validate improved processing methods for producing diagnostic-quality, whole-mount serial sections for 3-dimensional imaging of whole-breast histopathologic studies, we subjected 4-mm-thick whole-specimen slices to a 38-hour microwave-assisted protocol. Morphologic features, antigenicity, and tissue shrinkage were evaluated. A schedule using the tissue processor was optimized by evaluating the serial section yield for 3 schedules. The microwave-based processing schedule is adequate for producing diagnostic-quality whole-mount breast serial sections of an area up to 6,000 mm(2) and is compatible with a variety of immunohistochemical stains. A mean +/- SE total tissue shrinkage of 8.4% +/- 0.2% resulted. For the tissue processor, optimal results are obtained using a 59-hour schedule. Total fixation and processing time for whole-mount serial breast sections has been reduced from 21 days to 38 hours, with microwave assistance, and to 59 hours without. No adverse effects of microwaves on morphologic features, antigenicity, or gross tissue dimensions were observed.

Spatial resolution requirements for acquisition of the virtual screening slide for digital whole-specimen breast histopathology.

We examined the effect of lateral spatial resolution and reader specialty on the accuracy of detection of breast cancer. The motivation for this pilot study was the need to acquire and display very large data sets in whole-specimen 3D digital breast histopathology imaging. The ultimate goal is to determine the minimum resolution adequate for detection of malignancy. Twenty-three histologic slides were selected from breast pathology cases and digitized at 2 sampling distances (3.2 and 1.9 microm pixels). Images were viewed by 14 pathologists, of whom 5 had breast pathology as their primary specialty. The readers assessed the likelihood of malignancy on a 5-point Likert scale, and provided a provisional diagnosis. For the detection task, sensitivity, specificity, overall accuracy of detection, and area under the receiver-operator curve were calculated. An overall diagnostic score, and scores grouped by malignancy type, were also computed. Outcome measures were examined for significant resolution and specialty effects. Increasing the lateral resolution significantly improved accuracy in diagnosis (P=.004) but no effect was found for detection. Breast specialists achieved significantly higher scores for all outcome measures except specificity. Differences in performance between the 2 groups of readers tended to be greater for the diagnostic task compared to detection, especially at the higher resolution. However, specimen coverage may also be a significant factor. Factors related to the readers may have also affected performance in this study. Based on these results, a more comprehensive study should examine pixel sizes between 0.7 and 1.9 microm.

Surveillance of BRCA1 and BRCA2 mutation carriers with magnetic resonance imaging, ultrasound, mammography, and clinical breast examination.

CONTEXT: Current recommendations for women who have a BRCA1 or BRCA2 mutation are to undergo breast surveillance from age 25 years onward with mammography annually and clinical breast examination (CBE) every 6 months; however, many tumors are detected at a relatively advanced stage. Magnetic resonance imaging (MRI) and ultrasound may improve the ability to detect breast cancer at an early stage. OBJECTIVE: To compare the sensitivity and specificity of 4 methods of breast cancer surveillance (mammography, ultrasound, MRI, and CBE) in women with hereditary susceptibility to breast cancer due to a BRCA1 or BRCA2 mutation. DESIGN, SETTING, AND PARTICIPANTS: A surveillance study of 236 Canadian women aged 25 to 65 years with BRCA1 or BRCA2 mutations who underwent 1 to 3 annual screening examinations, consisting of MRI, mammography, and ultrasound at a single tertiary care teaching hospital between November 3, 1997, and March 31, 2003. On the day of imaging and at 6-month intervals, CBE was performed. MAIN OUTCOME MEASURES: Sensitivity and specificity of each of the 4 surveillance modalities, and sensitivity of all 4 screening modalities vs mammography and CBE. RESULTS: Each imaging modality was read independently by a radiologist and scored on a 5-point Breast Imaging Reporting and Data System scale. All lesions with a score of 4 or 5 (suspicious or highly suspicious for malignancy) were biopsied. There were 22 cancers detected (16 invasive and 6 ductal carcinoma in situ). Of these, 17 (77%) were detected by MRI vs 8 (36%) by mammography, 7 (33%) by ultrasound, and 2 (9.1%) by CBE. The sensitivity and specificity (based on biopsy rates) were 77% and 95.4% for MRI, 36% and 99.8% for mammography, 33% and 96% for ultrasound, and 9.1% and 99.3% for CBE, respectively. There was 1 interval cancer. All 4 screening modalities combined had a sensitivity of 95% vs 45% for mammography and CBE combined. CONCLUSIONS: In BRCA1 and BRCA2 mutation carriers, MRI is more sensitive for detecting breast cancers than mammography, ultrasound, or CBE alone. Whether surveillance regimens that include MRI will reduce mortality from breast cancer in high-risk women requires further investigation.