DNA methylation as a complementary biomarker for HPV diagnostics in cervical lesions analyzed from FFPE material: new possibilities for stratification from pilot study

Persistent high-risk HPV (human papillomavirus) infection is a key factor in cervical cancer, but additional molecular alterations drive lesion progression. DNA methylation has been proposed as a biomarker of transforming HPV infections and lesion progression. We evaluated the feasibility of DNA methylation testing in histologically verified FFPE (formalin-fixed, paraffin-embedded ) cervical tissue and explored associations between methylation status, HPV genotype, lesion grade, and immunohistochemical markers. Fifty-three patients with cervical intraepithelial neoplasia (CIN1–3) were included. FFPE punch biopsy or conization samples were analyzed using the Allplex™ HPV28 assay and GynTect ® DNA methylation testing. Immunohistochemistry for p16 and Ki-67 was performed. Associations between methylation, HPV genotype, lesion grade and Ki-67 expression were analyzed using Fisher’s exact or Fisher–Freeman–Halton test. HPV DNA was detected in 49/53 samples (92.5%), with HPV16 in 26/49 (53.1%). Methylation analysis was successful in 39/53 samples (73.6%), and positive in 16/39 (41%), exclusively in HSIL. Methylation was associated with HPV16 positivity ( p = 0.012) and higher Ki-67 expression ( p = 0.008). p16 expression was largely concordant with HPV status. All LSIL (low-grade squamous intraepithelial lesion) samples with valid methylation results were methylation-negative. This exploratory study demonstrates the technical feasibility of DNA methylation testing and HPV genotyping in histologically verified FFPE cervical tissue. In this cohort, methylation positivity was associated with HSIL (high-grade squamous intraepithelial lesion), HPV16 infection, and increased Ki-67 expression. Further studies with control tissues and validated FFPE-specific protocols are needed.