Yu Ting Felicia Wee1, Syed Muhammad Fahmy Alkaff2, Jeffrey Chun Tatt Lim2, Josh Jie Hua Loh2, Maryam Hazly Hilmy2, Clara Ong2, Wen Long Nei3, Amit Jain3, Alvin Lim2, Angela Takano2, Rafay Azhar2, Wei Keat Wan2, Evan Newell4, Joe Yeong5, Tony Kiat Hon Lim6. 1. Department of Anatomical Pathology, Division of Pathology, Singapore General Hospital, 20 College Road, Academia, Diagnostics Tower, 169856, Singapore; Department of Anatomy, National University of Singapore (NUS), Yong Loo Lin School of Medicine, MD 10, 4 Medical Drive, 117594, Singapore. 2. Department of Anatomical Pathology, Division of Pathology, Singapore General Hospital, 20 College Road, Academia, Diagnostics Tower, 169856, Singapore. 3. National Cancer Centre Singapore, 11 Hospital Drive, 169610, Singapore. 4. Singapore Immunology Network, Agency of Science, Technology and Research, 8A Biomedical Grove, Immunos Building, Biopolis, 138648, Singapore. 5. Department of Anatomical Pathology, Division of Pathology, Singapore General Hospital, 20 College Road, Academia, Diagnostics Tower, 169856, Singapore; Singapore Immunology Network, Agency of Science, Technology and Research, 8A Biomedical Grove, Immunos Building, Biopolis, 138648, Singapore. 6. Department of Anatomical Pathology, Division of Pathology, Singapore General Hospital, 20 College Road, Academia, Diagnostics Tower, 169856, Singapore. Electronic address: lim.kiat.hon@singhealth.com.sg.
Abstract
BACKGROUND/AIM: Epstein-Barr virus (EBV) is an oncovirus that 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, CD30 and 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.
BACKGROUND/AIM: Epstein-Barr virus (EBV) is an oncovirus that 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, CD30 and 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.
Authors: Zuwena A Richardson; Claire Deleage; Candani S A Tutuka; Marzena Walkiewicz; Perla M Del Río-Estrada; Rachel D Pascoe; Vanessa A Evans; Gustavo Reyesteran; Michael Gonzales; Samuel Roberts-Thomson; Mauricio González-Navarro; Fernanda Torres-Ruiz; Jacob D Estes; Sharon R Lewin; Paul U Cameron Journal: J Immunol Methods Date: 2021-12-02 Impact factor: 2.287
Authors: Harry Ho Man Ng; Ren Yuan Lee; Siting Goh; Isabel Shu Ying Tay; Xinru Lim; Bernett Lee; Valerie Chew; Huihua Li; Benedict Tan; Sherlly Lim; Jeffrey Chun Tatt Lim; Bijin Au; Josh Jie Hua Loh; Sahil Saraf; John Edward Connolly; Tracy Loh; Wei Qiang Leow; Joycelyn Jie Xin Lee; Han Chong Toh; Fabio Malavasi; Ser Yee Lee; Pierce Chow; Evan W Newell; Su Pin Choo; David Tai; Joe Yeong; Tony Kiat Hon Lim Journal: J Immunother Cancer Date: 2020-08 Impact factor: 13.751
Authors: Shuting Han; Joshua K Tay; Celestine Jia Ling Loh; Axel Jun Ming Chu; Joe Poh Sheng Yeong; Chwee Ming Lim; Han Chong Toh Journal: Front Immunol Date: 2021-12-10 Impact factor: 7.561