Systematic Multiplatform Discovery of Methylation Markers Enables Non-Invasive Early Detection of Endometrial Cancer

Endometrial cancer, the most common gynecological malignancy with an annual increase of 1%-3%, lacks suitable noninvasive diagnostic tools, as current methods like hysteroscopy and biopsy are invasive and impractical for routine screening. We conducted a comprehensive, multilayered marker discovery workflow integrating whole-genome bisulfite sequencing and targeted methylation panels in tumor and control tissues, then prioritized and optimized candidates for detection in cervical exfoliated cells. Using a two-stage design, we built and tested a quantitative methylation-specific PCR (qMSP) model in ThinPrep Cytology Test (TCT) samples, with 148 samples for discovery/model construction and an independent cohort of 80 TCT samples for validation. We identified a three-gene methylation panel-ZNF626, GRIA4, and SPDYA-that demonstrated high accuracy for early endometrial cancer detection from cervical cytology. In the validation cohort, the model showed strong performance across menopausal subgroups: in premenopausal women, sensitivity was 90.91% and specificity for benign endometrial disease was 92.59%; in postmenopausal women, sensitivity and specificity were 96.55% and 84.62%, respectively. Notably, the approach achieved a 92.86% detection rate for stage I endometrial cancer. These results support a robust, noninvasive diagnostic strategy that leverages simple cervical cytology sampling to enable early detection, facilitate clinical decision-making, and potentially improve outcomes for patients at risk of endometrial cancer.