Fig. 1. Study group selection – flowchart. Abbreviations: LBC, liquid-based cytology; HRHPV, 14 high-risk types human papillomavirus test; DS, p16/Ki67 dual staining test; HPV 16/18+, human papillomavirus types 16 and/or 18 positive results; HPV HR12+, human papillomavirus 12 high-risk types other than types 16 and 18 positive results.
Major screening abnormalities were defined in this study as screening abnormalities with potentially higher HSIL/CIN2+ risk that justifies a direct referral to colposcopy of HPV 16/18+ patients without prior triage testing, identified through primary HPV-based screening with limited genotyping. Management in these cases was according to ASCCP 2019 Risk-Based Management Consensus Guidelines [14-16].
HRHPV testing
Detection of HRHPV was performed using the Abbott RealTime High Risk HPV molecular in vitro PCR test (Abbott Molecular, Des Plaines, IL, USA), in adherence to the manufacturer’s instructions. This test phenotypes 12 types of high-risk HPV DNA (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68), as well as genotypes HPV 16 and 18 (limited genotyping). Test results were classified as HPV 16/18+ if either of these types was detected, and as HPV HR12+ if one or more of the other twelve non-16 and non-18 HRHPV types were detected. All screening samples were collected using the Cervex-Brush device (Rovers Medical Devices, Oss, Netherlands), and all tests (HRHPV, LBC, and DS) were performed in the same laboratory.
Liquid-based cytology
Liquid-based SurePath slides were prepared by an external laboratory using the automatic PrepStain Slide system (Becton Dickinson, Franklin Lakes, NJ, USA) following the manufacturer’s protocol. Residual cervical samples were stored for three months under specific conditions, enabling additional tests including DS triage to be performed. This approach facilitated comprehensive cervical cancer diagnostics from a single sample collection. A gynecological cytopathologist evaluated all cytology samples according to the Bethesda 2014 guidelines, being aware of the HRHPV status of the samples. Quality and control procedures aligned with benchmarks from US laboratories accredited by the College of American Pathologists [29].
p16/Ki67 dual-stain testing
The CINtec PLUS detection kit (Roche, MTM AG laboratories, Munich, Germany) was utilized for dual immunocytochemical staining with p16 and Ki67 proteins. This staining was performed in an automated BenchMark XT system (Ventana Medical Systems, Inc., Oro Valley, AZ, USA) following the manufacturer’s protocol. DS was conducted using residual cellular samples from the original SurePath vials stored in the laboratory after cytology and/or HPV testing. A qualified gynecological pathologist, specially trained and certified, assessed the DS based on established definitions [30].
Colposcopy protocol
Patients with DS-positive results, ASC-US or LSIL HRHPV-positive results, or those with cytologic ASC-H or worse, regardless of previous HRHPV status or with unknown HRHPV status, were referred for a colposcopy with biopsy. The protocol adhered to Polish recommendations, with extension of the 2012 ASCCP guidelines and 2015 ASCCP interim guidelines [31-34]. The requirements of minimal colposcopy protocol included endocervical sampling (curettage and brushing), direct biopsies for all abnormal colposcopic findings or those suggestive for invasion, or additional random biopsies as per Polish protocols. All histologic diagnoses of cervical biopsies and endocervical sampling were reviewed by a gynecological pathologist according to the LAST 2012/WHO 2014 terminology [35,36] with more details available in a series of related studies [26-29].
Triage models analyzed
Three HSIL/CIN2+ risk triage models for HPV 16/18+ cases in primary HPV-based cervical cancer screening were compared and analyzed. A reflex cytology was evaluated in all positive cases in model 1 (M1), in model 1A (M1A) a DS was assessed as the second triage test after NILM, ASC-US or LSIL results (reflex cytology in all positive cases, as in M1) and in model 2 (M2) a reflex DS in all HPV 16/18+ cases was analyzed. The proposed management in the analyzed triage models was presented in Figure 2.