An integrated automated multispectral imaging technique that simultaneously detects and quantitates viral RNA and immune cell protein markers in fixed sections from Epstein-Barr virus-related tumours

Abstract Background/aim Epstein-Barr virus (EBV) is an oncovirusthat is commonly associated with the development of lymphomas and epithelial carcinomas. In the era of immunotherapy, histological evaluation of EBV-related cancers is currently a multi-sample, multi-technique process requiring separate time-consuming detection of EBV-encoded small RNAs by in situ hybridisation (ISH), and parallel labelling of sections for cancer-associated protein markers. Methods Using EBV-associated tumours as proof-of-concept for feasibility, here we developed an approach that allows simultaneous detection of EBV RNAs and multiple protein markers such as PD-L1, EBV-LMP, CD8, CD4, CD20, CD30and CD15on a single tissue section based on our recently reported automated staining protocol. Results We successfully combined multiplex immunofluorescence (mIF) to detect 3 abovementioned protein markers involved in cancer, with ISH, and applied the protocol to f tissue samples from patients diagnosed with EBV-associated pulmonary lymphoepithelioma-like carcinoma(LELC), gastric carcinoma and Hodgkin's Lymphoma. Empowered by the Vectra 3 Automated Quantitative Pathology Imaging System, we demonstrate the utility and potential of this integrated approach to concurrently detect and quantitate viral RNA and protein biomarkers of immune and tumour cells. Conclusion This study represents an important step forward in the research and diagnosis of EBV-associated cancers, and could be readily modified to include other proteins and RNA markers to apply to other malignancies. More importantly, the novel automated ISH-mIF protocol that we detailly described here could also be readily reproduced by most of the diagnostic and research lab to future projects that aim to look at both RNA and protein markers.