QPOLE : A Quick, Simple, and Cheap Alternative for POLE Sequencing in Endometrial Cancer by Multiplex Genotyping Quantitative Polymerase Chain Reaction

Detection of 11 pathogenic variants in the gene in endometrial cancer (EC) is critically important to identify women with a good prognosis and reduce overtreatment. Currently, status is determined by DNA sequencing, which can be expensive, relatively time-consuming, and unavailable in hospitals without specialized equipment and personnel. This may hamper the implementation of -testing in clinical practice. To overcome this, we developed and validated a rapid, low-cost hotspot test by a quantitative polymerase chain reaction (qPCR) assay, . Primer and fluorescence-labeled 5'-nuclease probe sequences of the 11 established pathogenic mutations were designed. Three assays, -frequent for the most common mutations and -rare-1 and QPOLE-rare-2 for the rare variants, were developed and optimized using DNA extracted from formalin-fixed paraffin-embedded tumor tissues. The simplicity of the design enables status assessment within 4-6 hours after DNA isolation. An interlaboratory external validation study was performed to determine the practical feasibility of this assay. Cutoffs for wild-type, -mutant, equivocal, and failed results were predefined on the basis of a subset of mutants and wild-types for the internal and external validation. For equivocal cases, additional DNA sequencing is recommended. Performance in 282 EC cases, of which 99 were -mutated, demonstrated an overall accuracy of 98.6% (95% CI, 97.2 to 99.9), a sensitivity of 95.2% (95% CI, 90.7 to 99.8), and a specificity of 100%. After DNA sequencing of 8.8% equivocal cases, the final sensitivity and specificity were 96.0% (95% CI, 92.1 to 99.8) and 100%. External validation confirmed feasibility and accuracy. is a qPCR assay that is a quick, simple, and reliable alternative for DNA sequencing. detects all pathogenic variants in the exonuclease domain of the gene. will make low-cost -testing available for all women with EC around the globe.