Sara Bønløkke

Evaluation of targeted next‐generation sequencing for detection of HPV genotypes and sublineages in cervical liquid‐based cytology SurePath samples from the Danish screening program

Abstract The carcinogenicity of HPV genotypes is well established. However, HPV genotypes have sublineages with individual risk profiles, and these are much less described with respect to carcinogenicity. Research to characterize HPV sublineages by next‐generation sequencing (NGS) on screening‐derived liquid‐based cytology (LBC) samples is limited because of the technical and quality assurance challenging nature of sublineage analysis. This study aimed to evaluate the feasibility of detecting HPV sublineages from 14 HPV genotypes in SurePath LBC samples from Danish cervical cancer screening. We included 41 HPV plasmids (the Global HPV LabNet DNA Genotyping Proficiency Panel 2023) to quality assure the NGS approach and 120 SurePath LBC samples from the screening program for proof of concept. Our results of the HPV plasmids showed the correct sublineage for all included HPV genotypes except for HPV68b, where the coverage was inadequate for sublineage analysis. The NGS analysis enabled HPV sublineage analysis in 99.1% (112/113) of HPV‐positive SurePath LBC samples. Sublineages belonging to the A lineage were most frequent for HPV16, 18, 31, 33, 35, 39, 51, 52, 58, 59, and 68, while B‐type sublineages showed the highest frequency in HPV45, 56, and 66. The most diverse sublineage data was obtained for HPV31 with sublineages from the A, B, and C lineages. In conclusion, our method enables the identification of HPV sublineages in SurePath LBC screening samples. This information can be used in future studies to determine the usefulness of HPV sublineage analysis in screening settings for risk stratification and clinical management of HPV‐positive women.

Ultrasensitive next‐generation sequencing–based detection of circulating human papillomavirus DNA for cervical cancer recurrence monitoring

Abstract Background Circulating cell‐free human papillomavirus (ccfHPV) DNA is a promising biomarker for cervical cancer monitoring. This study used next‐generation sequencing (NGS) to assess its prognostic and surveillance value. Methods The authors included 141 cervical cancer patients (International Federation of Gynecology and Obstetrics IA1–IVB) grouped by treatment: primary surgery ( n  = 50), primary surgery + adjuvant oncological ( n  = 22), and primary oncological treatment ( n  = 69). Plasma was collected pretreatment, at early follow‐up (first post‐treatment sample [FPS]), and during longer follow‐up. ccfHPV DNA was detected using an NGS‐based assay, enabling high sensitivity without prior knowledge of human papillomavirus (HPV) genotype. Associations between ccfHPV DNA status and recurrence were assessed using Cox proportional hazards models, and predictive accuracy was evaluated via sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Results Pretreatment ccfHPV DNA positivity was detected in 8.0%, 36.4%, and 82.6% of patients across the groups, and ccfHPV DNA positivity was significantly associated with higher recurrence risk (adjusted hazard ratio [HR], 4.92; 95% confidence interval [CI], 1.29–18.7; p  = .02). At FPS, ccfHPV DNA positivity strongly predicted recurrence (adjusted HR, 19.1; 95% CI, 7.27–50.2), with a PPV of 83% and NPV of 90% for recurrence within 50 months. Among patients who developed recurrence, ccfHPV DNA was detectable before clinical evidence of recurrence in 60.9% of cases with a mean lead time of 144 days. Conclusion ccfHPV DNA detected by NGS is a strong prognostic biomarker for residual disease or recurrence. Its strong performance, already at early follow‐up, supports its integration into individualized follow‐up strategies. Prospective multicenter studies should validate these findings and guide clinical implementation. Trial Registration The study was registered with ClinicalTrials.gov (identifiers: NCT03749720)

Increased Risk of Second Squamous Cell Carcinomas Following Cervical Cancer: A Nationwide Danish Case–Control Study

ABSTRACT Introduction This nationwide case–control study investigated the risk of second primary cancers among cervical cancer (CC) survivors compared to cancer‐free women. Methods Women aged ≥ 18 diagnosed with CC from 1987 to 2012 were identified via the Danish Cancer Registry (DCR) and matched 1:5 by age and residence to cancer‐free controls. A subpopulation with histologically confirmed squamous cell carcinoma of the cervix (SCC‐C) was defined using the Danish Pathology Register. Adjusted sub‐hazard ratios (aSHR) for subsequent primary cancers were estimated. Results The study included 10,728 CC cases and 53,597 matched controls, including 7910 SCC‐C cases and 39,358 controls. Over a median follow‐up of 14.2 years, CC survivors had a modest but statistically significant reduction in the overall risk of second primary cancers (aSHR: 0.87; 95% CI, 0.81–0.93), including lower risks of colorectal cancer (aSHR: 0.77; 95% CI, 0.63–0.93), breast cancer (aSHR: 0.76; 95% CI, 0.67–0.87), and malignant melanoma (aSHR: 0.59; 95% CI, 0.42–0.84). In contrast, the risk of lung cancer was increased (aSHR: 1.33; 95% CI, 1.15–1.54). Among SCC‐C survivors, the risk of second SCC was increased at both HPV‐related sites (aSHR: 1.78; 95% CI, 1.18–2.68) and non‐HPV sites (aSHR: 2.59; 95% CI, 1.92–3.47), primarily due to a marked increased risk of lung SCC (aSHR: 2.88; 95% CI, 1.97–4.20). Discussion Although the overall risk of second primary cancers was lower among CC survivors compared to controls, the increased incidence of second SCCs, particularly lung SCC, highlights the need for targeted long‐term surveillance strategies.

The Diagnostic Value of Circulating Cell-Free HPV DNA in Plasma from Cervical Cancer Patients

Circulating cell-free HPV DNA (ccfHPV DNA) may serve as a marker for cervical cancer. In this study, we used digital droplet PCR (ddPCR) to detect and quantify ccfHPV DNA in plasma from patients with HPV16- or HPV18-associated cervical cancer. Blood samples from 60 patients diagnosed with cervical cancer (FIGO IA1-IVA) at Aarhus or Odense University Hospital (June 2018 to March 2020) were collected prior to treatment, and patients were subdivided into an early stage (n = 30) and a late-stage subgroup (n = 30) according to disease stage. Furthermore, blood samples from eight women with HPV16- or 18-associated premalignant conditions (CIN3), and 15 healthy controls were collected. ddPCR was used to analyze plasma from all participants. ccfHPV DNA was detected in 19 late-stage patients (63.33%), 3 early stage patients (10.00%), and none of the CIN3 patients or controls. Quantitative evaluation showed significant correlations between ccfHPV DNA level and stage, tumor score, and tumor size. Thus, our results indicate that ccfHPV DNA may not be a useful marker for early detection of cervical cancer. However, for patients with advanced stage cervical cancer, ccfHPV DNA level represents a promising tool to establish tumor burden, making it useful for establishing treatment response and monitoring the disease.

Circulating cell‐free HPV DNA is a strong marker for disease severity in cervical cancer

For cervical cancer (CC), circulating cell‐free HPV DNA (ccfHPV) may establish disease severity. Furthermore, HPV integration has been correlated to viral load and survival. In this study, pre‐treatment plasma from 139 CC cases (50 primary surgery patients, 22 primary surgery + adjuvant oncological therapy patients, and 67 primary oncological therapy patients) was collected (2018–2020). Furthermore, plasma from 25 cervical intraepithelial neoplasia grade 3 patients and 15 healthy women (negative controls) were collected. Two next‐generation sequencing (NGS) panels were used to establish ccfHPV presence and human papillomavirus type 16 (HPV16) integration status. ccfHPV was detected in four primary surgery (8.0%), eight primary surgery + adjuvant oncology (36.4%), and 54 primary oncology (80.6%) patients. For primary oncology patients with HPV16‐related cancer ( n  = 37), more ccfHPV neg than ccfHPV pos patients had HPV16 integration ( P  = 0.04), and in patients with HPV16 integration ( n  = 13), ccfHPV pos patients had higher disease stages than ccfHPV neg patients ( P  = 0.05). In summary, ccfHPV presence is related to disease severity and may add to the debated Sedlis criteria used for identifying patients for adjuvant oncological therapy. However, ccfHPV detection is influenced by HPV integration status and disease stage, and these factors need to be considered in ccfHPV neg patients.

HPV in Blood Samples From Cervical Cancer Patients.

By means of digital droplet PCR (ddPCR) anf targeted Next Generation Sequencing (NGS), this study examines blood samples from patients newly diagnosed with cervical cancer to investigate whether it is possible to measure the presence and amount of HPV DNA in these blood samples. The first blood sample is taken at time of diagnosis, and follow-up blood samples are collected during treatment- and follow-up visits. We expect to find a correlation between the disease stage and the viral load and also a decline in viral load after treatment. Furthermore, we hope that this method may serve as a way of detecting disease recurrence earlier than what is possible today.