Gisela Helenius

Cervical screening with self-sampling for postmenopausal women with molecular triage using extended genotyping and methylation

With the transition from cytology to human papilloma virus (HPV) testing in cervical cancer screening, it is possible to use self-sampling instead of professionally collected samples. Most studies have included women between 20 and 60 years age. Here we aimed to study postmenopausal women and investigate whether vaginal self-sampling is equally effective as professional sampling for detection of HSIL and the possibility to use a method for molecular triage directly on the screening sample. Postmenopausal women in Örebro county, Sweden, were invited (n = 7835) during 2018-2020 to participate in the study including both professional and self-sampling. In total 2258 women returned both sample types, that were analyzed for HPV followed by triage for cytology, HPV genotyping and methylation and clinical follow-up according to national guidelines. The prevalence of HPV was 3.4 % in the professionally collected samples and 12.6 % in the self-collected. All women with high-grade squamous intraepithelial lesion (HSIL) were HPV-positive in both professionally and self-collected samples. For self-collected samples, we compared different triage strategies. Cytology was the most efficient strategy. Among the molecular triage methods, the combination of methylation and genotyping was most efficient but resulted in twice as many colposcopy referrals as cytology. In conclusion, HPV self-sampling with molecular triage detects HSIL to the same extent as professional screening with cytological triage. The specificity of molecular triage is, however, unacceptably low, and to avoid overtreatment other triage methods following primary self-sampling need to be developed.

Optimization of droplet digital PCR assays for the type-specific detection and quantification of five HPV genotypes, including additional data on viral loads of nine different HPV genotypes in cervical carcinomas

The droplet digital PCR (ddPCR) system enables high-sensitivity detection of nucleic acids and direct absolute quantification of the targets. The aim of this research was to evaluate this system for viral load (VL) analysis of the human papillomavirus (HPV) genotypes HPV31, 35, 39, 51 and 56 measured in number of viral particles per cell. The sample types used for the optimization of the ddPCR assay were formalin-fixed paraffin-embedded (FFPE) tissues and cervical liquid cytology samples. The presently optimized ddPCR assays, together with assays optimized previously for HPV16, 18, 33 and 45, with the same ddPCR method, were used for the VL analysis of cervical tumor samples. Results published previously on the present study cohort showed that women with a cervical tumor containing multiple high-risk HPV genotypes had a worse prognosis compared to women with single-genotype-infected tumors. The VL was therefore analyzed in this study for the same cohort, as a possible explanatory factor to the prognostic differences. The results of the optimization part of the study, with analysis of VL using ddPCR in DNA from varying sample types (FFPE and liquid cytology samples), showed that each of the five assays demonstrated good inter- and intra-assay means with a coefficient of variation (CV) under 8% and 6% respectably. The cohort results showed no difference in VL between tumors with multiple and single HPV infections, and therefore did most likely not constitute a contributing factor for prognostic differences observed previously. However, tumors from women aged 60 years or older or containing certain HPV genotypes and genotype genera were associated with a higher VL.