학술논문
QPOLE : A Quick, Simple, and Cheap Alternative for POLE Sequencing in Endometrial Cancer by Multiplex Genotyping Quantitative Polymerase Chain Reaction.
Document Type
Article
Author
Van den Heerik, Anne Sophie V.M.; Ter Haar, Natalja T.; Vermij, Lisa; Jobsen, Jan J.; Brinkhuis, Mariel; Roothaan, Suzan M.; Leon-Castillo, Alicia; Ortoft, Gitte; Hogdall, Estrid; Hogdall, Claus; Van Wezel, Tom; Lutgens, Ludy C.H.W.; Haverkort, Marie A.D.; Khattra, Jas; McAlpine, Jessica N.; Creutzberg, Carien L.; Smit, Vincent T.H.B.M.; Gilks, C. Blake; Horeweg, Nanda; Bosse, Tjalling
Source
Subject
*POLYMERASE chain reaction
*ENDOMETRIAL cancer
*DNA sequencing
*GENETIC variation
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Language
ISSN
2687-8941
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. QPOLE: a rapid, simple, and cheap method for POLE assessment in endometrial cancer by multiplex quantitative polymerase chain reaction. [ABSTRACT FROM AUTHOR]