Simple SummaryEndometrial cancer is a significant and growing health concern worldwide. In this study, researchers aimed to find a safe and practical way of detecting early signs of endometrial cancer, which is crucial for effective treatment and improved patient outcomes. The researchers designed a panel targeting specific genes related to endometrial cancer and tested it using samples from 38 endometrial cancer patients and 208 women with risk factors. The results showed that the panel performed well, producing high-quality data and detecting genetic mutations with high sensitivity. The best sample type for detecting mutations was endometrial biopsy using the Pipelle aspirator, which had the best consistency with surgical tumor specimens. The findings suggest that this targeted panel sequencing method combined with ultra-deep sequencing is a promising tool for the early detection of endometrial cancer and could have significant clinical implications for patients.AbstractEndometrial cancer (EC) is a major gynecological malignancy with rising morbidity and mortality worldwide. The aim of this study was to explore a safe and readily available sample and a sensitive and effective detection method and its biomarkers for early diagnosis of EC, which is critical for patient prognosis. This study designed a panel targeting variants for EC-related genes, assessed its technical performance by comparing it with whole-exon sequencing, and explored the diagnostic potential of endometrial biopsies using the Pipelle aspirator, cervical samples using the Pap brush, and vaginal specimens using the swab from 38 EC patients and 208 women with risk factors for EC by applying targeted panel sequencing (TPS). TPS produced high-quality data (Q30 > 85% and mapping ratios > 99.35%) and was found to have strong consistency with whole-exome sequencing (WES) in detecting pathogenic mutations (92.11%), calculating homologous recombination deficiency (HRD) scores (r = 0.65), and assessing the microsatellite instability (MSI) status of EC (100%). The sensitivity of TPS in detection of EC is slightly better than that of WES (86.84% vs. 84.21%). Of the three types of samples detected using TPS, endometrial biopsy using the Pipelle aspirator had the highest sensitivity in detection of pathogenic mutations (81.87%) and the best consistency with surgical tumor specimens in MSI (85.16%). About 84% of EC patients contained pathogenic mutations inPIK3CA, PTEN, TP53, ARID1A, CTNNB1, KRAS,andMTOR, suggesting that this small gene set can achieve an excellent pathogenic mutation detection rate in Chinese EC patients. The custom panel combined with ultra-deep sequencing serves as a sensitive method for detecting genetic lesions from endometrial biopsy using the Pipelle aspirator.