Magnetic Resonance Imaging-Guided Transurethral Ultrasound Ablation of Prostate Cancer: A Systematic Review.

Purpose: MRI-guided transurethral ultrasound ablation (TULSA) uses real-time MR thermometry feedback to target prostate disease. We systematically review the literature to synthesize efficacy, functional, and safety outcomes and assess the influence of planned ablation fraction on outcome. Materials and Methods: PubMed, Embase, and the Cochrane Library were searched from inception to June 2021 following Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. Studies reporting at least one efficacy, functional, or safety outcome after a single TULSA treatment were included. The relationship of freedom from salvage treatment and potency preservation with planned ablation volume was modeled. Results: Two hundred twenty-four patients were treated in 10 studies with up to a 5-year follow-up, mainly for primary localized prostate cancer (PCa) plus smaller cohorts with recurrent PCa, and locally advanced PCa (LAPC). The prostate-specific antigen decline from baseline up to 2 years, including focal to whole-gland ablation plans, was 54% to 97%. The rate of salvage treatment after one TULSA treatment for primary PCa was 7% to 17%. Continence and potency preservation were from 92% to 100% and from 75% to 98%. Urinary symptoms were stable in men with good voiding function at baseline, and 85% of men with concurrent PCa and lower urinary tract symptoms met the criteria for improvement. Symptom relief in a small cohort of men with LAPC was observed. Grade III adverse events were incurred by 13/224 men (6%), with no rectal injury/fistula or Grade IV complication. The planned ablation fraction was linearly related to salvage-free survival. The relationship between potency preservation and planned ablation fraction followed a sigmoid curve. Conclusions: As an alternative to conventional treatments, TULSA is safe and effective for prostate tissue ablation in men with primary PCa. There is also evidence that TULSA delivers effective relief of urinary symptoms while treating PCa in a single, low-morbidity procedure. The likelihood of freedom from additional treatment or potency preservation is associated with the planned ablation fraction.

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.

Ki-67 Membranous Staining: Biologically Relevant or an Artifact of Multiplexed Immunofluorescent Staining.

In the process of developing a multiplex of 8 common breast cancer biomarkers (Her2/neu, estrogen receptor, progesterone receptor, Ki-67, aldehyde dehydrogenase-1, NaK-ATPase, cytokeratin 8/18, and myosin smooth muscle) on a single formalin-fixed paraffin-embedded slide using a sequential staining, imaging, and dye bleaching technology developed by General Electric Company, membranous Ki-67 staining was observed and colocalized with Her2/neu staining. Using immunohistochemistry as gold standards, we discovered that membranous Ki-67 was an artifact caused by the binding of cyanine 5-conjugated rabbit polyclonal Ki-67 antibody to a secondary cyanine 3-conjugated donkey anti-rabbit antibody which was previously applied and bound to rabbit Her2/neu antibody in our multiplexing experiment. After blocking with rabbit serum, a successful protocol for 8 biomarker multiplexing without cross-reactivity of antibodies from the same species was developed.

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.

Increasing specimen coverage using digital whole-mount breast pathology: implementation, clinical feasibility and application in research.

Conventional histopathological evaluation is performed on breast specimens using a highly limited sampling of tissues visualized in a two-dimensional (2D) manner although important tumor measurements are three-dimensional. Here we describe a '3D' technique for whole-mount, whole-specimen processing which reduces conformational change and dramatically increases specimen coverage, based on digitizing whole-specimen, whole-mount (up to 12.7cm×17.8cm) serial sections. We describe hardware and software tools for acquiring, viewing and processing the large image datasets (up to 400GB), validation studies investigating the clinical significance of the additional information gleaned from the 3D approach, and application to radiologic-pathologic correlation and biomarker visualization.

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.

Design and characterization of a digital image acquisition system for whole-specimen breast histopathology.

We have developed a digital histopathology imaging system capable of producing a three-dimensional (3D) representation of histopathology from an entire lumpectomy specimen. The system has the potential to improve the accuracy of surgical margin assessment in the treatment of breast cancer by providing finer sampling and 3D visualization. A scanning light microscope was modified to allow digital photomicrography of a stack of large (up to 120x170 mm2) histology slides cut serially through the entire specimen. The images are registered and displayed in 2D and 3D. The design of the system, which reduces or eliminates the appearance of 'tiling' and 'seam' artefacts inherent in the scanning method, is described and its resolution, contrast/noise and coverage properties are characterized through measurements of the modulation transfer function (MTF), depth of field (DOF) and signal difference to noise ratio (SDNR). The imaging task requires a lateral resolution of 5 microm, an SDNR of 5 between relevant features, 'tiling artefact' at a level below the detectability threshold of the eye, and 'seam artefact' of less than 5-10 microm. The tests demonstrate that the system is largely adequate for the imaging task, although further optimizations are required to reduce the degradation of coverage incurred by seam artefact.