Human papilloma virus (HPV) is an infectious carcinogenic agent. Nearly all cervical cancers are positive for one of the high-risk HPV subtypes. Although the introduction of the HPV vaccines in many countries have shown tremendous positive effects on the incidence of both cervical intraepithelial lesions (CIN) and invasive cancer, the large majority of females worldwide are still not vaccinated. Patients with diagnosed high-grade CIN need a lifelong close monitoring of possible relapse or development of invasive cancer. Different blood-based liquid biopsy approaches have shown great promise as an easily obtainable minimally invasive tool for early detection and monitoring of disease. Among the different liquid biopsy approaches the clinical relevance of cell-free DNA (cfDNA) in cervical cancer has been best investigated. In cervical cancer, the DNA fragments can be of both, human as well as viral origin. Thus, the mutation and methylation status of genes related to carcinogenesis as well as the HPV status can be analysed in plasma from cervical cancer patients. This review describes recent advances in different cfDNA approaches for early detection and monitoring of cervical cancer and its precursor lesions.

Cervical cancer is the fourth most common cancer in women, which is associated in >95% with a high-risk human papillomavirus (HPV) infection. Methylation of specific genes has been closely associated with the progress of cervical high-grade dysplastic lesions to invasive carcinomas. Therefore, DNA methylation has been proposed as a triage for women infected with high-risk HPV. Methylation analyses of cervical cancer tissue have shown that cell adhesion molecule 1 (CADM1) and myelin and lymphocyte protein (MAL) methylation are present in over 90% of all cervical high-grade neoplasias and invasive cervical cancers. Here, we established a liquid biopsy-based assay to detect MAL and CADM1 methylation in cell free (cf)DNA of cervical cancer. Methylation of the target gene was validated on bisulfite converted smear-DNA from cervical dysplasia patients and afterward applied to cfDNA using quantitative real-time PCR. In 52 smears, a combined analysis of CADM1 and/or MAL (CADM1/MAL) showed methylation in 86.5% of the cases. In cfDNA samples of 24 cervical cancer patients, CADM1/MAL methylation was detected in 83.3% of the cases. CADM1/MAL methylation was detected already in 81.8% of stage I-II patients showing the high sensitivity of this liquid biopsy assay. In combination with a specificity of 95.5% towards healthy donors (HD) and an area under the curve (AUC) of 0.872 in the receiver operating characteristic (ROC) analysis, CADM1/MAL cfDNA methylation detection might represent a novel and promising liquid biopsy marker in cervical cancer.

Background: Over 95% of cervical cancers and their precancerous lesions are caused by human papillomavirus (HPV). Molecular screening approaches for HPV detection are gaining promising data over cytologic analysis. Similarly, liquid biopsy-based approaches are becoming a powerful tool for cancer detection and monitoring. Materials and Methods: An HPV panel detecting 24 HPV types was developed suitable for liquid biopsy approaches. Here, HPV measurement was performed with an iPLEX multiplex PCR-based workflow combined with mass spectrometry-based analysis (MassARRAY System, Agena Bioscience). The assay was validated on HPV positive plasmid controls and cell lines. Furthermore, 52 cervical smear samples from HSIL positive women and 40 cfDNA samples (peripheral blood) from cervical cancer patients were screened using this approach. Results: According to already published ring trial results, the HPV types HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 58 and 59 were considered proficient with the required detection level of 50 and 5 international units (IU)/5 µl for HPV 16 and 18, and 500 and 50 HPV genome equivalents (GE)/5µl for the other HPV types (Arroyo Mühr et al., 2022). Further analyses showed a sensitivity of 0.03% for HPV16 and 0.33% HPV18 and 0.33% for the other tested HPV types. HPV DNA was detected in 94% of (49/52) smear samples analyzed. 86.5% were found positive for at least one of the high-risk HPV (hrHPV) types in the study cohort. Two of the three HPV negative samples were truly negative according to clinical data. In 15 samples, multiple HPV types were found, with positivity in up to five different HPV types. For smear samples a sensitivity of 98.14% and a positive predictive value of 0.98 (49/50) were thus obtained. The overall agreement between the MassARRAY and Seegene (from clinical records) HPV tests was 97.2% (κ=0.84). 27 of 40 (67.5%) cfDNA cervical cancer samples were tested positive for any HPV type. The distribution of HPV types showed most infections being due to hrHPV (25/27 plasma HPV positive). HPV positive samples were found in all FIGO stages, with 56.5% positivity in FIGO stage I and 87.5% in FIGO stage III and IV. Conclusions: The HPV assay showed reliable results for detecting a large number of HPV types in a multiplex mass spectrometry-based assay. A high sensitivity was achieved for both, cervical smear samples of HSIL patients as well as cfDNA from blood samples of cervical cancer patients. Citation Format: Johanna Herbst, Vanessa Vohl, Maroje Krajina, Katharina Prieske, Anna Jaeger, Markus Leffers, Leticia Oliveira-Ferrer, Yvonne Goy, Klaus Pantel, Alexander Sartori, Caren Vollmert, Linn Wölber, Katharina Effenberger, Harriet Wikman. Development of a liquid biopsy cfDNA assay for detection of multiple HPV types in cervical neoplasias [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6688.