p16/ki67 and E6/E7 mRNA Accuracy and Prognostic Value in Triaging HPV DNA-Positive Women

Abstract

Background: The study presents cross-sectional accuracy of E6 and E7 (E6/E7) mRNA detection and p16/ki67 dual staining, alone or in combination with cytology and human papillomavirus (HPV)16/18 genotyping, as a triage test in HPV DNA-positive women and their impact on cervical intraepithelial neoplasia (CIN2+) overdiagnosis.

Methods: Women aged 25-64 years were recruited. HPV DNA-positive women were triaged with cytology and tested for E6/E7 mRNA and p16/ki67. Cytology positive women were referred to colposcopy, and negatives were randomly assigned to immediate colposcopy or to 1-year HPV retesting. Lesions found within 24 months since recruitment were included. All P values were 2-sided.

Results: 40 509 women were recruited, and 3147 (7.8%) tested HPV DNA positive; 174 CIN2+ were found: sensitivity was 61.0% (95% confidence interval [CI] = 53.6 to 68.0), 94.4% (95% CI = 89.1 to 97.3), and 75.2% (95% CI = 68.1 to 81.6) for cytology, E6/E7 mRNA, and p16/ki67, respectively. Immediate referral was 25.6%, 66.8%, and 28.3%, respectively. Overall referral was 65.3%, 78.3%, and 63.3%, respectively. Cytology or p16/ki67, when combined with HPV16/18 typing, reached higher sensitivity with a small impact on referral. Among the 2306 HPV DNA-positive and cytology-negative women, relative CIN2+ detection in those randomly assigned at 1-year retesting vs immediate colposcopy suggests a -28% CIN2+ regression (95% CI = -57% to +20%); regression was higher in E6/E7 mRNA-negatives (Pinteraction = .29). HPV clearance at 1 year in E6/E7 mRNA and in p16/ki67 negative women was about 2 times higher than in positive women (Pinteraction < .001 for both).

Conclusions: p16/ki67 showed good performance as a triage test. E6/E7 mRNA showed the highest sensitivity, at the price of too high a positivity rate to be efficient for triage. However, when negative, it showed a good prognostic value for clearance and CIN2+ regression.

Figure 1.

Study flowchart reporting recruitment process, randomization, and primary endpoint results. Women were invited for screening by the standard program management system. Participating women were assessed for eligibility and, if eligible, were asked to participate; those willing signed informed consent. All women underwent screening with human papillomavirus (HPV) DNA test with reflex cytology if positive; those included in the study and positive for HPV DNA were also tested for biomarkers (E6/E7 mRNA, p16/ki67). Women with atypical squamous cells of undeterminde significance (ASC-US) or more severe cytology were referred to colposcopy; those negative for intraepithelial lesion or malignancy (NILM) were randomized to immediate colposcopy or to 1-year retesting (the routine protocol in Italy; Supplementary Figure 1, available online). If HPV DNA positive at retesting, women were referred to colposcopy. CIN = cervical intraepithelial neoplasia; colpo = colposcopy.

Figure 2.

Results by randomization arm and biomarker results. Flowchart of human papillomavirus (HPV) DNA-positive/cytology-negative women randomly assigned to immediate colposcopy or 1-year HPV retesting, distinguishing between E6/E7 mRNA-positive and E6/E7 mRNA-negative women (A) and distinguishing between p16/ki67-positive and p16/ki67-negative women (B). Baseline and up to 24 months of results are reported. Among the 2306 HPV-positive/cytology-negative women randomly assigned, 2297 had a valid E6/E7 mRNA test, and 2132 had a valid p16/ki67 test. Dotted arrows show the comparison between cervical intraepithelial neoplasia grade 2 or more severe (CIN2+) detection in the 2 arms; for the 2 biomarkers, the ratio between detection in the 1-year referral arm vs that in the immediate colposcopy arm represents an unbiased estimate of lesion regression in 1 year. Here, it is reported as reduction (negative percentage) of the CIN2+ proportion found in the immediate colposcopy arm. In panel (A), overall CIN2+ detection was 3.6% in the immediate colposcopy referral arm (34 of 951) and 2.6% in the 1-year referral arm (25 of 971), resulting in a 28% reduction (95% CI from a 57% reduction to a 20% increase). CI = confidence interval; colpo = colposcopy; cyto = cytology.