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

鈥� An ASCO Reading Room selection

July 19, 2023

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

JCO Global Oncology

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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 97.2-99.9), a sensitivity of 95.2% (95% CI 90.7-99.8), and a specificity of 100%. After DNA sequencing of 8.8% equivocal cases, the final sensitivity and specificity were 96.0% (95% CI 92.1-99.8) 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.

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

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JCO Global Oncology

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