HPV E6/E7 RNA in situ hybridization signal patterns as biomarkers of three-tier cervical intraepithelial neoplasia grade

PLoS One. 2014 Mar 13;9(3):e91142. doi: 10.1371/journal.pone.0091142. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Adult
  • Biomarkers / metabolism*
  • Cohort Studies
  • Cyclin-Dependent Kinase Inhibitor p16 / metabolism
  • DNA-Binding Proteins / metabolism*
  • Disease Progression
  • Female
  • Genes, Viral
  • Genotype
  • Humans
  • In Situ Hybridization
  • Middle Aged
  • Oncogene Proteins, Viral / metabolism*
  • Papillomaviridae / classification
  • Papillomaviridae / genetics
  • Papillomavirus Infections / diagnosis
  • Papillomavirus Infections / virology
  • RNA, Viral / metabolism*
  • Uterine Cervical Dysplasia / diagnosis*
  • Uterine Cervical Dysplasia / virology*
  • Young Adult

Substances

  • Biomarkers
  • Cyclin-Dependent Kinase Inhibitor p16
  • DNA-Binding Proteins
  • E6 protein, Human papillomavirus type 18
  • E7 protein, Human papillomavirus type 18
  • Oncogene Proteins, Viral
  • RNA, Viral

Grants and funding

This study was supported by the University of Vermont Department of Pathology. RNAscope in situ hybridization was performed by Advanced Cell Diagnostics (ACD), Hayward, CA at no charge. Authors Xiao-Jun Ma, Xingyong Wu, Hongwei Wang and Yuling Luo are employees and shareholders of ACD. XJM and YL contributed reagents, materials and technical consultancy on the performance of RNAscope assays, which were performed by XW and HW at ACDs facility in Hayward, CA. XJM contributed text for the technical sections of the manuscript. ACD employees had no role in study design, data interpretation, control over manuscript contents or decision to publish.