Optimizing a gene panel for enhanced detection of hotspot mutations in endometrial cancer using SiMSen-seq

Abstract

Endometrial cancer is the sixth most common cancer among women globally, with over 420,000 cases reported in 2022. Current diagnostic techniques lack sufficient specificity or sensitivity, emphasizing the need for new methods to improve early detection and clinical outcomes. One promising approach is analysis of circulating tumor DNA (ctDNA) in liquid biopsies. This project aimed to optimize a gene panel intended for ctDNA-based mutation detection in endometrial cancer using SiMSen-seq, a next generation sequencing method. The project focused on enhancing the performance and coverage of the panel through complementary assay design and incorporation of additional target sites. We designed and validated six new assays targeting mutations in five of the most frequently mutated genes in endometrial cancer (ARID1A, CHD4, PIK3CA, PIK3R1 and PTEN) for potential incorporation into the panel. Validation was performed primarily using real-time quantitative PCR and automated electrophoresis, and confirmed compatibility of the new assays with the existing multiplexes, with little observed interference. Based on mutation data from the Catalogue of Somatic Mutations in Cancer, a hypothetical detection coverage was calculated. Estimated, the updated panel would detect an additional 2.6% of mutation-positive endometrial tissue samples, increasing the total coverage to 70.4%. Future efforts should focus on validation using sequencing to confirm individual assay performance and diagnostic utility.