Autofluorescence Virtual Staining System for H&E Histology and Multiplex Immunofluorescence Applied to...

Virtual staining for digital pathology has great potential to enable spatial biology research, improve efficiency and reliability in the clinical workflow, as well as conserve tissue samples in a nondestructive manner. In this study, we demonstrate the feasibility of generating virtual stains for hematoxylin and eosin (H&E) and a multiplex immunofluorescence (mIF) immuno-oncology panel (DAPI, PanCK, PD-L1, CD3, and CD8) from autofluorescence (AF) images of unstained non–small cell lung cancer tissue by combining high-throughput hyperspectral fluorescence microscopy and machine learning. Using domain-specific computational methods, we evaluated the accuracy of virtual H&E staining for histologic subtyping and virtual mIF for cell segmentation–based measurements, including clinically relevant measurements such as tumor area, T-cell density, and PD-L1 expression (tumor proportion score and combined positive score). The virtual stains reproduce key morphologic features and protein biomarker expressions at both tissue and cell levels compared with real stains, enable the identification of key immune phenotypes important for immuno-oncology, and show moderate to good performance across various evaluation metrics. This study extends our previous work on virtual staining from AF in liver disease and prostate cancer, further demonstrating the generalizability of this deep learning technique to a different disease (lung cancer) and stain modality (mIF).

We extend the capabilities of virtual staining from AF to a different disease and stain modality. Our work includes newly developed virtual stains for H&E and a multiplex immunofluorescence panel (DAPI, PanCK, PD-L1, CD3, and CD8) for non-small cell lung cancer, which reproduce the key features of real stains.