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

Abstract

Detection of 11 pathogenic variants in the <i>POLE</i> gene in endometrial cancer (EC) is critically important to identify women with a good prognosis and reduce overtreatment. Currently, <i>POLE</i> 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 <i>POLE</i>-testing in clinical practice. To overcome this, we developed and validated a rapid, low-cost <i>POLE</i> hotspot test by a quantitative polymerase chain reaction (qPCR) assay, <i>QPOLE</i>. Primer and fluorescence-labeled 5'-nuclease probe sequences of the 11 established pathogenic <i>POLE</i> mutations were designed. Three assays, <i>QPOLE</i>-frequent for the most common mutations and <i>QPOLE</i>-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 <i>POLE</i> 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 <i>POLE</i> wild-type, <i>POLE</i>-mutant, equivocal, and failed results were predefined on the basis of a subset of <i>POLE</i> mutants and <i>POLE</i> 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 <i>POLE</i>-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. <i>QPOLE</i> is a qPCR assay that is a quick, simple, and reliable alternative for DNA sequencing. <i>QPOLE</i> detects all pathogenic variants in the exonuclease domain of the <i>POLE</i> gene. <i>QPOLE</i> will make low-cost <i>POLE</i>-testing available for all women with EC around the globe.