Methylation test versus dual staining in the triage of HR-HPV positive women – pilot data from cervical cancer screening

Czech version

Authors: M. Bartošová ¹; I. Kinkorová Luňáčková ¹; J. Bouda ² ; K. Černá ^1,3 ; N. Janovská ⁴ ; J. Martínková Němcová ^1,3,5
Authors‘ workplace: Bioptická laboratoř, Plzeň ¹; Gynekologicko-porodnická klinika LF UK a FN Plzeň ²; Šiklův ústav patologie, FN Plzeň ³; Gynekologicko-porodnická klinika 3. LF UK a FNKV, Praha ⁴; Biomedicínské centrum, Lékařská fakulta v Plzni ⁵
Published in: Ceska Gynekol 2026; 91(5): 357-362
Category: Original Article
doi: https://doi.org/10.48095/cccg2026357

Overview

Introduction: Introduction of high-risk human papillomavirus (HR-HPV) testing into cervical cancer screening increases the detection of high-grade cervical lesions, but also identifies women with transient HR-HPV infections who are at a low risk of progression. To improve risk stratification, biomarkers that can be assessed from the same cervical sample used for HPV testing may be employed, particularly p16/Ki-67 dual-stain cytology and host-cell DNA methylation analysis. Dual staining identifies cells showing HPV-induced transforming activity, but may not fully capture the biological variability of either the host or the HPV infection. Methylation testing reflects epigenetic changes associated with persistent HR-HPV infection and biologically advanced cervical lesions that may therefore provide a more direct indication of the short-term risk of lesion progression.

Objective: To compare performance of p16/Ki-67 dual staining and DNA methylation analysis for risk stratification of HR--HPV-positive women.

Methods: The study included 44 HR-HPV-positive women (median age, 35 years) participating in the Czech cervical cancer screening program who underwent cervical biopsy or conization. Histologically confirmed HSIL/AIS was diagnosed in 52% of cases. Results of both triage methods were evaluated according to age, HPV genotype, and histological findings.

Results: The two methods showed concordant results in approximately 60% of cases, with the highest concordance observed in women older than 35 years. Among women aged 35 years or younger, discordant results were more frequent, most commonly consisting of positive dual staining combined with a negative methylation test. High concordance between the two methods was observed in HPV16-associated lesions, whereas among infections with other HR-HPV genotypes, positive dual staining accompanied by a negative methylation result was more common.

Conclusion: These findings suggest that methylation testing may be particularly useful for the triage of younger HR-HPV-positive women, potentially helping to reduce unnecessary referrals and invasive procedures without compromising the detection of clinically relevant cervical lesions. The findings of this pilot study are currently being validated in a larger prospective study conducted in collaboration with a specialized colposcopy clinic.

Keywords:

DNA methylation – cervical cancer screening – HPV testing – p16/Ki-67 dual staining – triage of HR-HPV-positive women

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