Comparison between a Luminex-based multiplex kit with a sequence-specific oligonucleotide probe and next-generation...

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). 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. 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. 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.