Background: Major screening abnormalities in pre-colposcopic stage are tests results that imply direct referral to colposcopy (and/or expedited treatment) without performing additional high-grade squamous intraepithelial lesions or worse (HSIL+) risk selection testing. Currently, both clinically validated HSIL+ risk selection tests, reflex cytology and reflex p16/Ki67 dual staining (DS), are being compared for use in primary HPV-based screening to avoid possible overtreatment, but there is still no sufficient data available for their performance. Methods: Among 30,066 liquid-based cervical cancer screening tests results, a group of 332 women was selected with available HRHPV tests results with 16/18 limited genotyping, liquid-based cytology, DS, and histology results from standardized colposcopy with biopsy. In HPV 16/18+ cases, three triage approaches were retrospectively analyzed. Predictive values for detection of HSIL+ were calculated and number of colposcopies required in each strategy. Results: Both triage models with DS used (reflex cytology followed by DS, and reflex DS alone in all cases) had significantly higher PPV for HSIL+ than strategy with reflex cytology alone (44.2%/45.7% vs. 28.3%; p<0.0001). In models with DS, less colposcopies were required (95/92 vs. 152) and less colposcopies were needed per HSIL+ detection (2,26/2,19 vs. 3,54). Only 1 HSIL+ case was missed in both triage models with DS incorporation. Conclusions: p16/Ki67 dual-stain may be an effective, alone or combined with cytology, triage test to detect HSIL+ in patients with major screening abnormalities in primary HPV-based cervical cancer screening. Performing cytology as the first triage test improves the strategy by enabling referrals to expedited treatment in selected cases.

Background: The introduction of primary HPV cervical cancer screening requires the implementation of an appropriate triage strategy that will be effective in detecting high-grade cervical disease without losing diagnostic specificity. Methods: From the 30.066 screening tests results, a total of 1086 with available high-risk human papillomavirus (HRHPV) with limited genotyping, cytology and p16/Ki67 dual-stain were selected. Two triage strategies for primary HPV screening were analyzed retrospectively based on the study group. Performance characteristics for p16/Ki67 and cytology triage in detection of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and grade 3 or worse (CIN3+) were calculated, detected in colposcopic biopsy. Results: In HPV16/18-positive cases, primary HPV with p16/Ki67 triage was significantly more specific than cytology (53.1%/16.8% for CIN2+; p<0.0001; 45.9%/17.0% for CIN3+; p<0.0001), with yielded sensitivity (95.7%/84.8% for CIN2+; p=0.0955; 100.0%/87.5% for CIN3+; p=0.0832). In other HRHPV-positive cases (N16/N18), p16/Ki67 triage was also significantly higher specific (51.3%/15.3% for CIN2+; p<0.0001; 44.5%/16.5% for CIN3+; p<0.0001), with sensitivity (92.3%/74.4% for CIN2+; p=0.0522; 90.9%/81.8% for CIN3+; p=0.5637). Diagnostic predictive values were significantly higher for p16/Ki67 triage with the highest PPV in HPV16/18-positive cases for CIN2+ (45.4%; 95% CI: 35.2-55.8; p<0.0001) and very high NPV in all HPV-positive cases regardless of detected genotype (96.3%-100.0%). The risk (1-NPV) for CIN3+ in HRHPV16/18-positive/p16/Ki67-negative women was 0.0%. Conclusions: Superior diagnostic performance compared to cytology for detecting cervical cancer precursors indicates that p16/Ki67 dual-immunostain may be a highly effective tool of triage in primary HPV screening with limited HPV 16/18 genotyping in the secondary cervical cancer prevention.