title: p16 and MIB1 improve the sensitivity and specificity of the diagnosis of high grade squamous intraepithelial lesions: methodological issues in a report of 447 biopsies with consensus diagnosis and HPV HCII testing.
abstract: OBJECTIVE
  Many investigators are studying the additional value of biomarkers to improve histopathologic agreement, but few are using the same methodologies. Our objectives in this analysis to differentiate High-grade Squamous Intraepithelial lesions (HGSIL) from Low Grade Squamous Intraepithelial Lesions (LGSIL), atypia, and normal were: (1) to examine the rate of Human Papilloma Virus High-Risk positivity (HPV HR+), (2) to compare and grade the basal, parabasal, intermediate, and superficial layer staining of each marker, (3) to determine the optimal qualitative threshold for markers, (4) to compare p16 and MIB1 agreement, and (5) to examine the sensitivities and specificities using each markers alone and together.
  
  
  METHODS
  A sample of biopsies from 208 patients were chosen from a total of 1850 patients and 3735 biopsies obtained during the course of ongoing optical trials. At least two independent blinded reviews were performed for each biopsy. A third review was performed if there was a disagreement between the two reviews. Both endocervical and ectocervical samples were stained for p16 and MIB1. A grading system that is delineated in the text ranged from 0 to 3 for both markers and each biopsy was scored by each cell layer. Frequencies, sensitivities, and specificities were calculated using Statistica. An ANOVA was used to compare p16 and MIB1 staining in the epithelial layers. Finally the sensitivity and specificity of each marker alone and together were examined.
  
  
  RESULTS
  453 specimens from 208 patients whose final diagnoses were normal (n=244), low-grade (LG) (n=59), and high-grade (HG) (n=144) were selected for analysis. 447 of 453 specimens were available for staining. Most LG and HG lesions were HPV HR positive. Endocervical samples stained positive less often than ectocervix and often results were discordant from ectocervical results. The analysis by layers showed pronounced increases in staining of both p16 and MIB1 as lesions progressed from normal to LG to HG. The cutoff or threshold for p16 was 0 versus 1-3 while for MIB1 it was 0-1 versus 2-3. Using the intermediate epithelial layer measurement of both p16 and MIB1 in HPV High-Risk Positive separated the normal tissue from LGSIL, normal from HGSIL, and LGSIL from HGSIL by a statistically significant margin (p<0.05). Each marker had sensitivities and specificities for the diagnosis of HGSIL versus LGSIL and normal of approximately 85-90% and this improved by 5% for both sensitivity and specificity when used together (p16 sensitivity 90%, specificity 85%; MIB1 sensitivity 89%, specificity 87%; together sensitivity 94%, specificity 90%).
  
  
  CONCLUSIONS
  Several important methodological issues have been studied. Overall, p16 and MIB1 are promising markers to help pathologists differentiate HG lesions from all else. The staining of the endocervix and the ectocervix do not always agree, and the ectocervix more often stains positive with the presence of HGSIL. Each marker is helpful and both are helpful together. In conclusion, both markers are useful for the confirmation of HG lesions.
authors.affiliation: Department of Cancer Imaging, British Columbia Cancer Agency, Vancouver, British Columbia, Canada.
authors.forename: Dirk
authors.initials: D
authors.lastname: Van Niekerk
authors.forename: Martial
authors.initials: M
authors.lastname: Guillaud
authors.forename: Jasenka
authors.initials: J
authors.lastname: Matisic
authors.forename: John L
authors.initials: JL
authors.lastname: Benedet
authors.forename: J Adrian
authors.initials: JA
authors.lastname: Freeberg
authors.forename: Michele
authors.initials: M
authors.lastname: Follen
authors.forename: Calum
authors.initials: C
authors.lastname: MacAulay
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000378:metabolism*; D001706:Biopsy; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism*; D005260:Female; D006801:Humans; D008875:Middle Aged; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D012680:Sensitivity and Specificity; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D002583:Uterine Cervical Neoplasms / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology / Q000821:virology; D002578:Uterine Cervical Dysplasia / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology / Q000821:virology