High-performance methylated DNA markers for the detection of cervical lesions at risk of malignant progression in low- and middle-income countries

Abstract

Abstract Background Cervical cancer remains a leading cause of death, particularly in developing countries. WHO screening guidelines recommend Human Papilloma Virus (HPV) detection as a means to identify women at risk of developing cervical cancer. While HPV testing identifies those at risk, it does not specifically recognize individuals with neoplasia. We investigated whether a molecular test that quantitatively measures methylated DNA markers could identify high-risk lesions in the cervix with accuracy. Results Marker discovery was performed in TCGA-CESC Infinium Methylation 450K Array database, and the selected 5-gene panel was verified in three other public datasets. The panel was technically validated using Quantitative Multiplex-Methylation Specific PCR (QM-MSP) in tissue sections (N = 293) and cervical smears (N = 244) from the U. S., S. Africa, and Vietnam. The gene panel consisted of FMN2, EDNRB, ZNF671, TBXT, and MOS. Cervical tissue samples from U.S., South Africa, and Vietnam showed highly significant differential methylation in squamous cell carcinoma (SCC) with 100% sensitivity, 91–93% specificity, and a Receiver Operating Characteristic Area under the curve (ROC AUC) = 1.000 [CI 1.000 to 1.000], and cervical intraepithelial neoplasia 2/3 (CIN2/3) with 55–100% sensitivity, 91–96% specificity, and a ROC AUC ranging from 0.793 [CI 0.681 to 0.905] to 1.000 [CI 1.000 to 1.000] compared to normal. In cervical smears, the marker panel detected SCC at 87% sensitivity, 95% specificity, and ROC AUC = 0.925 [CI 0.878 to 0.974], and high-grade intraepithelial lesion (HSIL) at 74% sensitivity, 95% specificity, and a ROC AUC = 0.907 [CI 0.851 to 0.964] in an analysis of pooled data from the three countries. Similar to HPV-positive, HPV-negative cervical carcinomas were frequently hypermethylated for these markers. Conclusions This 5-marker panel detected SCC and CIN2/3 in cervical smears with a high level of sensitivity and specificity. Molecular tests with the ability to rapidly detect high-risk CIN3 + lesions will lead to timely treatment for those in need while preventing unnecessary procedures in women with low-risk lesions throughout the world. Validation of these markers in prospectively collected cervical smear cells followed by the development of a hypermethylated marker-based cervical cancer detection test is warranted.