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

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 distinguish individuals with neoplasia. We investigated whether a quantitative molecular test that measures methylated DNA markers could identify high-risk lesions in the cervix with accuracy. Results Marker discovery was performed in TCGA-CESC Infinium Methylation 450 K Array database and verified in three other public datasets. The panel was technically validated using Quantitative Multiplex-Methylation-Specific PCR in tissue sections ( N = 252) and cervical smears ( N = 244) from the USA, South Africa, and Vietnam. The gene panel consisted of FMN2 , EDNRB , ZNF671 , TBXT , and MOS . Cervical tissue samples from all three countries showed highly significant differential methylation in squamous cell carcinoma (SCC) with a sensitivity of 100% 95% CI 74.12–100.00, and specificity of 91% 95% CI 62.26–99.53 to 96% 95% CI 79.01–99.78, and receiver operating characteristic area under the curve (ROC AUC) = 1.000 95% CI 1.00–1.00 compared to benign cervical tissue, and cervical intraepithelial neoplasia 2/3 with sensitivity of 55% 95% CI 37.77–70.84 to 89% 95% CI 67.20–98.03, specificity of 93% 95% CI 84.07–97.38 to 96% 95% CI 79.01–99.78, and a ROC AUC ranging from 0.793 95% CI 0.68–0.89 to 0.99 95% CI 0.97–1.00 compared to CIN1. In cervical smears, the marker panel detected SCC with a sensitivity of 87% 95% CI 77.45–92.69, specificity 95% 95% CI 88.64–98.18, and ROC AUC = 0.925 95% CI 0.878–0.974 compared to normal, and high-grade squamous intraepithelial lesion (HSIL) at a sensitivity of 70% (95% CI 58.11–80.44), specificity of 94% (95% CI 88.30–97.40), and ROC AUC = 0.884 (95% CI 0.822–0.945) compared to low-grade intraepithelial lesion (LSIL)/normal 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 HSIL in cervical smears with a high level of sensitivity and specificity. Molecular tests with the ability to rapidly detect high-risk HSIL will lead to timely treatment for those in need and prevent 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.