Deep learning-enabled realistic virtual histology with ultraviolet photoacoustic remote sensing microscopy.

The goal of oncologic surgeries is complete tumor resection, yet positive margins are frequently found postoperatively using gold standard H&E-stained histology methods. Frozen section analysis is sometimes performed for rapid intraoperative margin evaluation, albeit with known inaccuracies. Here, we introduce a label-free histological imaging method based on an ultraviolet photoacoustic remote sensing and scattering microscope, combined with unsupervised deep learning using a cycle-consistent generative adversarial network for realistic virtual staining. Unstained tissues are scanned at rates of up to 7 mins/cm2, at resolution equivalent to 400x digital histopathology. Quantitative validation suggests strong concordance with conventional histology in benign and malignant prostate and breast tissues. In diagnostic utility studies we demonstrate a mean sensitivity and specificity of 0.96 and 0.91 in breast specimens, and respectively 0.87 and 0.94 in prostate specimens. We also find virtual stain quality is preferred (P = 0.03) compared to frozen section analysis in a blinded survey of pathologists.

Fast hybrid optomechanical scanning photoacoustic remote sensing microscopy for virtual histology.

A rapid scanning microscopy method for hematoxylin and eosin (H&E) like images is sought after for interoperative diagnosis of solid tumor margins. The rapid observation and diagnosis of histological samples can greatly lower surgical risk and improve patient outcomes from solid tumor resection surgeries. Photoacoustic remote sensing (PARS) has recently been demonstrated to provide images of virtual H&E stains with excellent concordance with true H&E staining of formalin-fixed, paraffin embedded tissues. By using PARS with constant velocity and 1D galvanometer mirror scanning we acquire large virtual H&E images (10mm x 5mm) of prostate tissue in less than 3.5 minutes without staining, and over two orders of magnitude faster data acquisition than the current PARS imaging speed.

Genome profiles of pathologist-defined cell clusters by multiregional LCM and G&T-seq in one triple-negative breast cancer patient.

Pathological examination is the gold standard for cancer diagnosis, and breast tumor cells are often found in clusters. We report a case study on one triple-negative breast cancer (TNBC) patient, analyzing tumor development, metastasis, and prognosis with simultaneous DNA and RNA sequencing of pathologist-defined cell clusters from multiregional frozen sections. The cell clusters are isolated by laser capture microdissection (LCM) from primary tumor tissue, lymphatic vessels, and axillary lymph nodes. Data are reported for a total of 97 cell clusters. A combination of tumor cell-cluster clonality and phylogeny reveals 3 evolutionarily distinct pathways for this patient, each associated with a unique mRNA signature, and each correlated with disparate survival outcomes. Hub gene analysis indicates that extensive downregulation of ribosomal protein mRNA is a potential marker of poor prognosis in breast cancer.

Virtual histopathology with ultraviolet scattering and photoacoustic remote sensing microscopy.

Realistic label-free virtual histopathology has been a long sought-after goal not yet achieved with current methods. Here, we introduce high-resolution hematoxylin and eosin (H&E)-like virtual histology of unstained human breast lumpectomy specimen sections using ultraviolet scattering-augmented photoacoustic remote sensing microscopy. Together with a colormap-matching algorithm based on blind stain separation from a reference true H&E image, we are able to produce virtual H&E images of unstained tissues with close concordance to true H&E-stained sections, with promising diagnostic utility.

Virtual hematoxylin and eosin histopathology using simultaneous photoacoustic remote sensing and scattering microscopy.

Hematoxylin and eosin (H&E) staining is the gold standard for most histopathological diagnostics but requires lengthy processing times not suitable for point-of-care diagnosis. Here we demonstrate a 266-nm excitation ultraviolet photoacoustic remote sensing (UV-PARS) and 1310-nm microscopy system capable of virtual H&E 3D imaging of tissues. Virtual hematoxylin staining of nuclei is achieved with UV-PARS, while virtual eosin staining is achieved using the already implemented interrogation laser from UV-PARS for scattering contrast. We demonstrate the capabilities of this dual-contrast system for en-face planar and depth-resolved imaging of human tissue samples exhibiting high concordance with H&E staining procedures and confocal fluorescence microscopy. To our knowledge, this is the first microscopy approach capable of depth-resolved imaging of unstained thick tissues with virtual H&E contrast.

Flat epithelial atypia: are we being too aggressive?

PURPOSE: The malignant upgrade rate of flat epithelial atypia (FEA) diagnosed on core needle biopsy varies between 0 and 30%. Excision versus observation with radiological follow-up for these lesions remains controversial. We hypothesize that the local rate of FEA is low and that close radiological surveillance is a reasonable treatment option for patients diagnosed with pure FEA on breast needle core needle biopsy. METHODS: This study was a retrospective review of a prospectively collated provincial pathology database. Patients diagnosed with FEA alone on needle core biopsy between 2006 and 2016 were included in our analysis. Patients who had FEA present together with either in situ or invasive carcinoma within the same biopsy cores were excluded. Along with patient demographics, the size of the lesion on preoperative imaging, the method of extraction, and the presence of co-existing benign and malignant pathology on final excision biopsy were analyzed. An independent pathological review was performed to confirm our results and help reduce inter-observer bias. RESULTS: The local rate of malignant upgrade when pure FEA is diagnosed on a breast needle core biopsy is 12%. Age at time of diagnosis, size of original lesion on mammogram, presence of atypical ductal hyperplasia (ADH) or atypical lobular hyperplasia on core needle biopsy, the use of vacuum-assisted biopsy (VAB), or concordant imaging did not significantly correlate with malignant upgrade risk. None of the patients who were managed with radiological follow-up had malignant upgrade during follow-up. Patients undergoing radiological follow-up alone were more likely to have a VAB, concordant imaging, and no concurrent ADH. CONCLUSION: Our local malignant upgrade rate is consistent with published literature. We suggest radiological follow-up is a safe alternative in patients with pure flat epithelial atypia and concordant imaging, particularly those patients with small lesions in which microcalcifications can be removed completely with vacuum-assisted biopsy.

Merkel cell carcinoma with squamous and sarcomatous differentiation.

Merkel cell carcinoma is an aggressive neuroendocrine tumor historically thought to arise from neural crest-derived cutaneous neuroendocrine cells. Recent evidence supports an epidermal origin. We present a case of Merkel cell carcinoma arising on the upper arm of a 94-year-old woman that had multiple morphologic patterns: small cells typical of Merkel cell carcinoma, malignant cells with squamous differentiation and malignant poorly differentiated spindle cells. Subsequent metastatic disease in regional lymph nodes showed only the small cells and the malignant spindle cells. To our knowledge, this is the first case of Merkel cell carcinoma showing these three patterns of differentiation at first presentation. This morphology raises the possibility that Merkel cell carcinomas may arise from epidermal stem cells that can differentiate along different lines.