Complementarity between miRNA expression analysis and DNA methylation analysis in hrHPV-positive cervical scrapes for the detection of cervical disease

Abstract

Cervical screening by high-risk HPV (hrHPV) testing requires additional risk stratification (triage), as most infections are transient and only a subset of hrHPV-positive women harbours clinically relevant disease. Molecular triage markers such as microRNAs (miRNAs) and DNA methylation markers are particularly promising, as they can be objectively tested directly on hrHPV-positive scrapes and cervicovaginal self-samples. Here, we evaluated the marker potential of 10 candidate miRNAs in 209 hrHPV-positive scrapes of women with underlying precancer (cervical intraepithelial neoplasia, grade 2–3 (CIN2-3)), cancer, or without disease (CIN0/1). A predictive miRNA classifier for CIN3 detection was built using logistic regression, which was compared to and combined with DNA methylation marker FAM19A4 . Markers were correlated to histology parameters and hrHPV genotype. A miRNA classifier consisting of miR-149, miR-20a, and miR-93 achieved an area under the curve (AUC) of 0.834 for CIN3 detection, which was not significantly different to that of FAM19A4 methylation (AUC: 0.862, p = 0.591). Combining miRNA and methylation analysis demonstrated complementarity between both marker types (AUC: 0.939). While the miRNA classifier seemed more predictive for CIN2, FAM19A4 methylation was particularly high in HPV16-positive and histologically advanced CIN3, i.e. CIN3 with high lesion volume. The miRNA classifier, FAM19A4 methylation, and the miRNA/methylation combination were highest in cancer-associated scrapes. In conclusion, a panel of three miRNAs is discriminatory for CIN3 in hrHPV-positive scrapes and can complement DNA methylation analysis for the efficient detection of cervical disease. Combined analysis of the two marker types warrants further evaluation as triage strategy in hrHPV-based screening.

Selection of endogenous control and identification of significant microRNA deregulations in cervical cancer

“…A similar study was performed by Nilsen et al., who selected the combination of miR-151-5p, miR-152-3p, and miR-423-3p as an endogenous control in their study of hypoxic miRNA markers in cervical cancer ( 27 ). Partially similar results were obtained by Babion et al., who used small RNA sequencing to select an endogenous control for their studies of miRNA expression in cervical scrapings ( 28 , 29 ). They chose miR-423-3p as an endogenous control, but combined it with RNU24 (small nucleolar RNA).…”

The Role of Methylation of Host and/or Human Papillomavirus (HPV) DNA in Management of Cervical Intraepithelial Neoplasia Grade 2 (CIN2) Lesions

“…The expression of various other types of host and HPV miRNA in plasma and cervical samples has been studied by other research groups. The authors reported conflicting results with regard to the association between miRNA expression and the grade of the lesion [ 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 ].…”

Role of microRNAs as biomarkers of cervical carcinogenesis: a systematic review

“…Most studies enrolled healthy patients (normal controls) through clinical settings that were convenient to investigators. Only three studies recruited normal and healthy participants through community screenings, which generally contributed to higher scoring [ 16 , 17 , 49 ]. Similar mediocre results were achieved during the assessment of cohort studies, with half of the studies scored as good quality [ 44 , 46 ].…”