Significance: For breast cancer patients, the extent of regional lymph node (LN) metastasis influences the decision to remove all axillary LNs. Metastases are currently identified and classified with visual analysis of a few thin tissue sections with conventional histology that may underrepresent the extent of metastases.
Aim: We sought to enable nondestructive three-dimensional (3D) pathology of human axillary LNs and to develop a practical workflow for LN staging with our method. We also sought to evaluate whether 3D pathology improves staging accuracy in comparison to two-dimensional (2D) histology.
Approach: We developed a method to fluorescently stain and optically clear LN specimens for comprehensive imaging with multiresolution open-top light-sheet microscopy. We present an efficient imaging and data-processing workflow for rapid evaluation of H&E-like datasets in 3D, with low-resolution screening to identify potential metastases followed by high-resolution localized imaging to confirm malignancy.
Results: We simulate LN staging with 3D and 2D pathology datasets from 10 metastatic nodes, showing that 2D pathology consistently underestimates metastasis size, including instances in which 3D pathology would lead to upstaging of the metastasis with important implications on clinical treatment.
Conclusions: Our 3D pathology method may improve clinical management for breast cancer patients by improving staging accuracy of LN metastases.
Improving the sensitivity of mammograms in breast cancer screening has increased the detection of suspicious findings such as calcifications and masses at the cost of a high false positive rate (55-85%). Additionally, the diagnostic interpretation of biopsies varies (75.3% concordance), leading to suboptimal treatments and poor patient outcomes. The goal of this pilot study is to investigate whether the chemical composition of breast calcifications, present in more than 80% of mammograms, can be used to improve breast lesion classification. We hypothesized that the spatial and compositional variation of breast calcifications strongly correlates with breast malignancy. To test this hypothesis, we used an advanced Raman imaging technique called hyperspectral stimulated Raman scattering (hsSRS) microscopy to study 12 patient cases (30 calcifications). We characterized unique Raman signatures of type I (calcium oxalate) and type II (calcium hydroxyapatite) calcifications in archival breast tissue at high speed and spatial resolution with hsSRS microscopy. We found that the carbonation level of hydroxyapatite decreases when comparing benign and atypical ductal hyperplasia. However, the average carbonation of hydroxyapatite was highly variable in fibroadenoma cases (3±0.6%) and DCIS (4±1.1%). In the case of DCIS, the carbonation of hydroxyapatite varied relative to the grade and the neoplastic microenvironment (nearby inflammation, necrosis, and more.) In high-grade DCIS, the carbonation was lowest around the periphery where the contact with neoplastic cells was present. Our preliminary results indicate that microcalcifications change with the neoplastic microenvironment. Further studies of neoplastic progression in association with microcalcifications can improve the statistical value of the correlation.
Intraoperative assessment of breast surgical margins will be of value for reducing the rate of re-excision surgeries for lumpectomy patients. While frozen-section histology is used for intraoperative guidance of certain cancers, it provides limited sampling of the margin surface (typically <1 % of the margin) and is inferior to gold-standard histology, especially for fatty tissues that do not freeze well, such as breast specimens. Microscopy with ultraviolet surface excitation (MUSE) is a nondestructive superficial optical-sectioning technique that has the potential to enable rapid, high-resolution examination of excised margin surfaces. Here, a MUSE system is developed with fully automated sample translation to image fresh tissue surfaces over large areas and at multiple levels of defocus, at a rate of ∼5 min / cm2. Surface extraction is used to improve the comprehensiveness of surface imaging, and 3-D deconvolution is used to improve resolution and contrast. In addition, an improved fluorescent analog of conventional H&E staining is developed to label fresh tissues within ∼5 min for MUSE imaging. We compare the image quality of our MUSE system with both frozen-section and conventional H&E histology, demonstrating the feasibility to provide microscopic visualization of breast margin surfaces at speeds that are relevant for intraoperative use.
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