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

Anne Sophie V M Van den Heerik, Natalja T Ter Haar, Lisa Vermij, Jan J Jobsen, Mariel Brinkhuis, Suzan M Roothaan, Alicia Leon-Castillo, Gitte Ortoft, Estrid Hogdall, Claus Hogdall, Tom Van Wezel, Ludy C H W Lutgens, Marie A D Haverkort, Jas Khattra, Jessica N McAlpine, Carien L Creutzberg, Vincent T H B M Smit, C Blake Gilks, Nanda Horeweg, Tjalling Bosse*

*Corresponding author af dette arbejde

Abstract

PURPOSE: Detection of 11 pathogenic variants in the POLE gene in endometrial cancer (EC) is critically important to identify women with a good prognosis and reduce overtreatment. Currently, POLE 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 POLE-testing in clinical practice. To overcome this, we developed and validated a rapid, low-cost POLE hotspot test by a quantitative polymerase chain reaction (qPCR) assay, QPOLE.

MATERIALS AND METHODS: Primer and fluorescence-labeled 5'-nuclease probe sequences of the 11 established pathogenic POLE mutations were designed. Three assays, QPOLE-frequent for the most common mutations and QPOLE-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 POLE status assessment within 4-6 hours after DNA isolation. An interlaboratory external validation study was performed to determine the practical feasibility of this assay.

RESULTS: Cutoffs for POLE wild-type, POLE-mutant, equivocal, and failed results were predefined on the basis of a subset of POLE mutants and POLE 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 POLE-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.

CONCLUSION: QPOLE is a qPCR assay that is a quick, simple, and reliable alternative for DNA sequencing. QPOLE detects all pathogenic variants in the exonuclease domain of the POLE gene. QPOLE will make low-cost POLE-testing available for all women with EC around the globe.

OriginalsprogEngelsk
Artikelnummere2200384
TidsskriftJCO global oncology
Vol/bind9
ISSN2687-8941
DOI
StatusUdgivet - maj 2023

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