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

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.