Comparison between a Luminex-based multiplex kit with a sequence-specific oligonucleotide probe and next-generation sequencing for the detection of POLE oncogenic mutations in endometrial cancer

Objective: In endometrial cancer, detection of oncogenic mutations in the polymerase epsilon ( POLE ) gene is crucial for accurate staging according to the 2023 International Federation of Gynecology and Obstetrics classification and for minimizing overtreatment. However, POLE sequencing is expensive, time-consuming, and often inaccessible in settings without specialized equipment. We developed a novel multiplex kit for the detection of POLE mutations using a Luminex (xMAP) assay in a single reaction. The aim of this study was to evaluate the accuracy of the multiplex kit for routine clinical samples and compare it with that of conventional next-generation sequencing (NGS). Methods : Hysterectomy specimens and endometrial biopsies were collected at the National Cancer Center Hospital between 1999 and 2023. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissues. Both the Luminex (xMAP)-based multiplex kit and NGS targeting all POLE exons were used. Concordance was assessed using Cohen’s kappa. Results: Of the 502 samples, 432 were hysterectomy specimens and 70 were biopsies. In the surgical samples, both the Luminex (xMAP)-based kit and NGS detected 52 POLE mutations (12.0%) with perfect concordance (κ=1.000). In the biopsies, 33 POLE mutations were identified using both methods, with complete concordance. Notably, the Luminex (xMAP)-based kit successfully analyzed all 28 samples that failed NGS quality control and detected four cases with POLE mutations. Conclusions: The Luminex (xMAP)-based kit demonstrates high concordance with NGS for the detection of POLE mutations. With further external validation, this kit could become a reliable and accessible alternative to NGS.