title: HPV E6/E7 RNA in situ hybridization signal patterns as biomarkers of three-tier cervical intraepithelial neoplasia grade.
abstract: Cervical lesion grading is critical for effective patient management. A three-tier classification (cervical intraepithelial neoplasia [CIN] grade 1, 2 or 3) based on H&E slide review is widely used. However, for reasons of considerable inter-observer variation in CIN grade assignment and for want of a biomarker validating a three-fold stratification, CAP-ASCCP LAST consensus guidelines recommend a two-tier system: low- or high-grade squamous intraepithelial lesions (LSIL or HSIL). In this study, high-risk HPV E6/E7 and p16 mRNA expression patterns in eighty-six CIN lesions were investigated by RNAscope chromogenic in situ hybridization (CISH). Specimens were also screened by immunohistochemistry for p16INK4a (clone E6H4), and by tyramide-based CISH for HPV DNA. HPV genotyping was performed by GP5+/6+ PCR combined with cycle-sequencing. Abundant high-risk HPV RNA CISH signals were detected in 26/32 (81.3%) CIN 1, 22/22 (100%) CIN 2 and in 32/32 (100%) CIN 3 lesions. CIN 1 staining patterns were typified (67.7% specimens) by abundant diffusely staining nuclei in the upper epithelial layers; CIN 2 lesions mostly (66.7%) showed a combination of superficial diffuse-stained nuclei and multiple dot-like nuclear and cytoplasmic signals throughout the epithelium; CIN 3 lesions were characterized (87.5%) by multiple dot-like nuclear and cytoplasmic signals throughout the epithelial thickness and absence/scarcity of diffusely staining nuclei (trend across CIN grades: P<0.0001). These data are consistent with productive phase HPV infections exemplifying CIN 1, transformative phase infections CIN 3, whereas CIN 2 shows both productive and transformative phase elements. Three-tier data correlation was not found for the other assays examined. The dual discernment of diffuse and/or dot-like signals together with the assay's high sensitivity for HPV support the use of HPV E6/E7 RNA CISH as an adjunct test for deciding lesion grade when CIN 2 grading may be beneficial (e.g. among young women) or when 'LSIL vs. HSIL' assignment is equivocal.
authors.affiliation: Department of Pathology, University of Vermont, Burlington, Vermont, United States of America.
authors.forename: Mark F
authors.initials: MF
authors.lastname: Evans
authors.affiliation: Department of Pathology, University of Vermont, Burlington, Vermont, United States of America.
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Peng
authors.affiliation: Department of Pathology, University of Vermont, Burlington, Vermont, United States of America; Department of Pathology and Laboratory Medicine, Fletcher Allen Health Care, Burlington, Vermont, United States of America.
authors.forename: Kelli M
authors.initials: KM
authors.lastname: Clark
authors.affiliation: Department of Pathology, University of Vermont, Burlington, Vermont, United States of America.
authors.forename: Christine S-C
authors.initials: CS
authors.lastname: Adamson
authors.affiliation: Advanced Cell Diagnostics, Inc., Hayward, California, United States of America.
authors.forename: Xiao-Jun
authors.initials: XJ
authors.lastname: Ma
authors.affiliation: Advanced Cell Diagnostics, Inc., Hayward, California, United States of America.
authors.forename: Xingyong
authors.initials: X
authors.lastname: Wu
authors.affiliation: Advanced Cell Diagnostics, Inc., Hayward, California, United States of America.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Wang
authors.affiliation: Advanced Cell Diagnostics, Inc., Hayward, California, United States of America.
authors.forename: Yuling
authors.initials: Y
authors.lastname: Luo
authors.affiliation: Department of Pathology, University of Vermont, Burlington, Vermont, United States of America; Department of Pathology and Laboratory Medicine, Fletcher Allen Health Care, Burlington, Vermont, United States of America.
authors.forename: Kumarasen
authors.initials: K
authors.lastname: Cooper
mesh_terms: D000293:Adolescent; D000328:Adult; D015415:Biomarkers / Q000378:metabolism*; D015331:Cohort Studies; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018450:Disease Progression; D005260:Female; D005814:Genes, Viral; D005838:Genotype; D006801:Humans; D017403:In Situ Hybridization; D008875:Middle Aged; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D027383:Papillomaviridae / Q000145:classification / Q000235:genetics; D030361:Papillomavirus Infections / Q000175:diagnosis / Q000821:virology; D012367:RNA, Viral / Q000378:metabolism*; D055815:Young Adult; D002578:Uterine Cervical Dysplasia / Q000175:diagnosis* / Q000821:virology*
chemical_list: D015415:Biomarkers; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004268:DNA-Binding Proteins; C052603:E6 protein, Human papillomavirus type 18; C052633:E7 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D012367:RNA, Viral