Fenggang Yu1, Yanan Lu1, Fredrik Petersson2, De-Yun Wang1, Kwok Seng Loh1,3. 1. Department of Otolaryngology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. 2. Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. 3. Head and Neck Tumor Group, National Cancer Institute of Singapore, Head and Neck Surgery, National University Health System (NUHS), Singapore.
Abstract
BACKGROUND: Chromogenic Epstein-Barr virus-encoded RNA (EBER) in situ hybridization (EBER-ISH) is the gold standard to detect Epstein-Barr virus (EBV) but it is difficult to use in conjunction with immunohistochemistry (IHC). In this study, our purpose was to validate the sensitivity and specificity of RNAscope in detection of EBV infection in nasal epithelium and its stroma. METHODS: Fluorescence-based RNAscope EBER-ISH, BRLF1-ISH, and lineage marker-IHC were performed on archived formalin-fixed paraffin-embedded tissues from normal nasal cavity (n = 5), nasopharynx (n = 8), and nasopharyngeal carcinoma (NPC) specimens (n = 10). RESULTS: The EBERs were detected in 10 of 10 NPC samples but was absent in all normal tissues from the nasal cavity and nasopharynx. The EBERs were exclusively located in pan-cytokeratin (pan-CK)-positive tumor epithelial cells but not in CD45-positive leukocytes and vimentin-positive stromal fibroblasts. The level of EBER expression varied in tumor cells within patient and between patients as well. Additionally, 5 of 10 patients had positive BRLF-ISH. CONCLUSION: We developed a simple and reproducible method to simultaneously detect mRNA and protein in formalin-fixed paraffin-embedded tissues of NPC. As a single staining, traditional EBER continues to be useful; however, for interpretation of the phenotype of EBV-infected cells, RNAscope is superior. Significantly, we showed that lytic EBV infection took place in NPC tumors.
BACKGROUND: Chromogenic Epstein-Barr virus-encoded RNA (EBER) in situ hybridization (EBER-ISH) is the gold standard to detect Epstein-Barr virus (EBV) but it is difficult to use in conjunction with immunohistochemistry (IHC). In this study, our purpose was to validate the sensitivity and specificity of RNAscope in detection of EBV infection in nasal epithelium and its stroma. METHODS: Fluorescence-based RNAscope EBER-ISH, BRLF1-ISH, and lineage marker-IHC were performed on archived formalin-fixed paraffin-embedded tissues from normal nasal cavity (n = 5), nasopharynx (n = 8), and nasopharyngeal carcinoma (NPC) specimens (n = 10). RESULTS: The EBERs were detected in 10 of 10 NPC samples but was absent in all normal tissues from the nasal cavity and nasopharynx. The EBERs were exclusively located in pan-cytokeratin (pan-CK)-positive tumor epithelial cells but not in CD45-positive leukocytes and vimentin-positive stromal fibroblasts. The level of EBER expression varied in tumor cells within patient and between patients as well. Additionally, 5 of 10 patients had positive BRLF-ISH. CONCLUSION: We developed a simple and reproducible method to simultaneously detect mRNA and protein in formalin-fixed paraffin-embedded tissues of NPC. As a single staining, traditional EBER continues to be useful; however, for interpretation of the phenotype of EBV-infected cells, RNAscope is superior. Significantly, we showed that lytic EBV infection took place in NPC tumors.
Authors: Phillip Ziegler; Yarong Tian; Yulong Bai; Sanna Abrahamsson; Alan Bäckerholm; Alex S Reznik; Anthony Green; John A Moore; Stella E Lee; Michael M Myerburg; Hyun Jung Park; Ka-Wei Tang; Kathy Ho Yen Shair Journal: PLoS Pathog Date: 2021-04-29 Impact factor: 6.823
Authors: Frans J Mulder; Faisal Klufah; Famke M E Janssen; Farzaneh Farshadpour; Stefan M Willems; Remco de Bree; Axel Zur Hausen; Mari F C M van den Hout; Bernd Kremer; Ernst-Jan M Speel Journal: Front Oncol Date: 2021-01-20 Impact factor: 6.244