Triage of high-risk HPV-positive women in population-based screening by miRNA expression analysis in cervical scrapes; a feasibility study

Abstract

Background: Primary testing for high-risk HPV (hrHPV) is increasingly implemented in cervical cancer screening programs. Many hrHPV-positive women, however, harbor clinically irrelevant infections, demanding additional disease markers to prevent over-referral and over-treatment. Most promising biomarkers reflect molecular events relevant to the disease process that can be measured objectively in small amounts of clinical material, such as miRNAs. We previously identified eight miRNAs with altered expression in cervical precancer and cancer due to either methylation-mediated silencing or chromosomal alterations. In this study, we evaluated the clinical value of these eight miRNAs on cervical scrapes to triage hrHPV-positive women in cervical screening.

Results: Expression levels of the eight candidate miRNAs in cervical tissue samples (n = 58) and hrHPV-positive cervical scrapes from a screening population (n = 187) and cancer patients (n = 38) were verified by quantitative RT-PCR. In tissue samples, all miRNAs were significantly differentially expressed (p < 0.05) between normal, high-grade precancerous lesions (CIN3), and/or cancer. Expression patterns detected in cervical tissue samples were reflected in cervical scrapes, with five miRNAs showing significantly differential expression between controls and women with CIN3 and cancer. Using logistic regression analysis, a miRNA classifier was built for optimal detection of CIN3 in hrHPV-positive cervical scrapes from the screening population and its performance was evaluated using leave-one-out cross-validation. This miRNA classifier consisted of miR-15b-5p and miR-375 and detected a major subset of CIN3 as well as all carcinomas at a specificity of 70%. The CIN3 detection rate was further improved by combining the two miRNAs with HPV16/18 genotyping. Interestingly, both miRNAs affected the viability of cervical cancer cells in vitro.

Conclusions: This study shows that miRNA expression analysis in cervical scrapes is feasible and enables the early detection of cervical cancer, thus underlining the potential of miRNA expression analysis for triage of hrHPV-positive women in cervical cancer screening.

Keywords: CIN; Cervical cancer; HPV; Scrape; Screening; Triage; miRNA; qRT-PCR.

Fig. 1

Differential expression of selected miRNAs in cervical scrapes. qRT-PCR results were normalized to RNU24 and miR-423, and all values were square root transformed. *p < 0.05, **p < 0.005

Fig. 2

ROC curve analysis of miRNA classifiers for the detection of CIN3. Results obtained from 66 hrHPV-positive scrapes from women without underlying disease and 121 scrapes from women with CIN3 were used to build (a) individual miRNA classifiers and (b) a 2-miRNA classifier. Classifiers were validated by leave-one-out cross-validation

Fig. 3

ROC curve analysis of HPV16/18 genotyping and the 2-miRNA classifier for the detection of CIN3. Results obtained from scrapes with known HPV16/18 genotyping results (65 normal, 108 CIN3) were used to build classifiers for HPV16/18 genotyping (HPV), a new 2-miRNA classifier (miR-15b/375) and the 2-miRNA classifier combined with HPV16/18 genotyping (miR-15b/375/HPV). Classifiers were validated by leave-one-out cross-validation. The model miR-15b/375/HPV is significantly better than the 2-miRNA classifier (p = 0.011)

Fig. 4

Functional effect of our selected marker miRNAs in cervical cancer cell lines. Cell viability of SiHa and CaSki cells upon (a) knockdown of miR-15b-5p and (b) ectopic expression of miR-375. Results are representative of two independent experiments