HR-HPV E6/E7 mRNA In Situ Hybridization: Validation Against PCR, DNA In Situ Hybridization, and p16 Immunohistochemistry in 102 Samples of Cervical, Vulvar, Anal, and Head and Neck NeoplasiaMills, Anne M. MD; Dirks, Dawn C. MS; Poulter, Melinda D. PhD; Mills, Stacey E. MD; Stoler, Mark H. MDAmerican Journal of Surgical Pathology: May 2017 - Volume 41 - Issue 5 - p 607–615 doi: 10.1097/PAS.0000000000000800 Original Articles Abstract Author Information Dysregulated expression of oncogenic types of E6 and E7 is necessary for human papillomavirus (HPV)-driven carcinogenesis. An HPV E6/E7 mRNA in situ hybridization (ISH) assay covering 18 common high-risk types (“HR-RISH,” aka HR-HPV RNA18 ISH) has not been extensively studied in the anogenital tract or validated on automated technology. We herein compare HR-RISH to DNA polymerase chain reaction (PCR), p16 immunohistochemistry, and a previously available HPV DNA ISH assay in HPV-related anogenital and head and neck (H&N) neoplasia. A total of 102 squamous intraepithelial lesions (16 CIN1, 25 CIN3, 3 AIN1, 12 AIN3, 9 VIN3)/invasive squamous cell carcinomas (17 cervical, 2 anal, 18 H&N) as well as 10 normal and 15 reactive cervix samples were collected. HR-RISH, DNA ISH, and p16 immunohistochemistry were performed on whole formalin-fixed, paraffin-embedded sections. RNA ISH for 6 low-risk HPV types (LR-RISH) was also performed. RNA and DNA ISH assays used automated systems. HR-HPV PCR was performed on morphology-directed formalin-fixed, paraffin-embedded punches. HR-RISH was ≥97% sensitive for PCR+ and p16+ neoplasia, as well as morphologically defined anogenital high grade squamous intraepithelial lesion/invasive squamous cell carcinoma. HR-RISH was also positive in 78% of anogenital low grade squamous intraepithelial lesion, including 81% of CIN1. Furthermore, a subset of PCR-negative/invalid and p16-negative lesions was positive for HR-RISH. Only 1 problematic reactive cervix sample and no normal cervix samples stained. These results demonstrate that HR-RISH is a robust method for the detection of HR-HPV-related neoplasia and provides insight into HPV pathobiology. Performance meets or exceeds that of existing assays in anogenital and H&N lesions and may play a role in resolving diagnostically challenging CIN1 versus reactive cases. Department of Pathology, University of Virginia, Charlottesville, VA Conflicts of Interest and Source of Funding: The Authors report the following affiliation with regard to this study. This study represents a “beta-testing” series supported by Advanced Cell Diagnostics for the testing of their HPV RNA in situ hybridization assay (RNAscope) on an automated Leica platform. The authors received RNAscope reagent kits free of charge from Advanced Cell Diagnostics and were provided with temporary use of a Leica Bond RX for the completion of this study; they did not receive financial compensation. Correspondence: Anne M. Mills, MD, Department of Pathology, University of Virginia, 1215 Lee Street, Charlottesville, VA 22908 (e-mail: email@example.com). Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved.