Autofluorescence Virtual Staining System for H&E Histology and Multiplex Immunofluorescence Applied to Immuno-Oncology Biomarkers in Lung Cancer

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 non-destructive 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, CD8) from autofluorescence 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 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 to 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 autofluorescence 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). ### Competing Interest Statement This work was supported by Verily Life Sciences LLC and Genmab US, Inc. Verily Life Sciences LLC reports patent applications on virtual staining and alignment. JL, MR, CM, TY, CS, CJS, SV, HP, TCW, RF, SR, MG, YW, ACS, RY, VV, JSS, SSW, ER, RG, PC, and PFW are current or former employees with equity interests during tenure at Verily Life Sciences LLC. DFS is a current employee with equity interests at Google LLC. PCDS, HC, LS, CWL, and SSC are current or former employees at Genmab US, Inc. All authors performed work for this study during their respective tenures. ### Funding Statement This work was supported by Verily Life Sciences LLC and Genmab US, Inc. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: WCG IRB waived ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Due to the nature of this research, data is not available.