title: 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 Neoplasia.
abstract: 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.
authors.affiliation: Department of Pathology, University of Virginia, Charlottesville, VA.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Mills
authors.forename: Dawn C
authors.initials: DC
authors.lastname: Dirks
authors.forename: Melinda D
authors.initials: MD
authors.lastname: Poulter
authors.forename: Stacey E
authors.initials: SE
authors.lastname: Mills
authors.forename: Mark H
authors.initials: MH
authors.lastname: Stoler
mesh_terms: D001005:Anus Neoplasms / Q000737:chemistry / Q000235:genetics* / Q000473:pathology / Q000821:virology; D014408:Biomarkers, Tumor / Q000032:analysis / Q000235:genetics; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000032:analysis*; D005260:Female; D006258:Head and Neck Neoplasms / Q000737:chemistry / Q000235:genetics* / Q000473:pathology / Q000821:virology; D061809:Human Papillomavirus DNA Tests; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D060787:Neoplasm Grading; D015588:Observer Variation; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D050725:Papillomavirus E7 Proteins / Q000235:genetics*; D030361:Papillomavirus Infections / Q000235:genetics* / Q000473:pathology / Q000821:virology; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D012333:RNA, Messenger / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D015203:Reproducibility of Results; D002583:Uterine Cervical Neoplasms / Q000737:chemistry / Q000235:genetics* / Q000473:pathology / Q000821:virology; D014846:Vulvar Neoplasms / Q000737:chemistry / Q000235:genetics* / Q000473:pathology / Q000821:virology; D002578:Uterine Cervical Dysplasia / Q000737:chemistry / Q000235:genetics* / Q000473:pathology / Q000821:virology
chemical_list: D014408:Biomarkers, Tumor; C000614131:CDKN2A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012333:RNA, Messenger; D012367:RNA, Viral