# A Novel Self-Competitive Fishing Primer qPCR Approach for Efficient POLE Mutation Detection in Endometrial Cancer Molecular Classification

## TL;DR

A new qPCR method called SCF primer was developed to detect POLE mutations in endometrial cancer more efficiently and affordably than next-generation sequencing.

## Contribution

The novel SCF primer qPCR system offers a rapid and cost-effective alternative to NGS for detecting POLE exonuclease domain mutations in endometrial cancer.

## Key findings

- The SCF qPCR system achieved 1% mutation-detection sensitivity across various mutation points.
- Results from 86 endometrial cancer samples showed complete concordance with NGS analysis for 11 pathogenic POLE mutations.
- The system demonstrated superior specificity compared to the original SuperSelective primer-based qPCR.

## Abstract

This study developed and validated a Self-competitive Fishing (SCF) primer qPCR system as a rapid, cost-effective alternative to next-generation sequencing (NGS) for detecting POLE exonuclease domain mutations (EDMs) in endometrial cancer. The system detects 11 pathogenic POLE EDMs using SuperSelective primers combined with wild-type-blocking oligonucleotides that prevent amplification of wild-type DNA, thereby enhancing mutant DNA detection. The validation process involved comparing specificity using genomic DNA from tumors with known POLE mutations identified by NGS. Sensitivity testing used POLE-mutated DNA diluted in wild-type DNA, while precision was confirmed by analyzing 86 endometrial cancer samples against NGS results. The SCF qPCR system demonstrated superior specificity compared to the original SuperSelective primer-based qPCR, achieving 1% mutation-detection sensitivity across various mutation points. Importantly, results from all endometrial cancer cases showed complete concordance with NGS analysis for the 11 pathogenic POLE-EDM points tested. This cost-effective and efficient SCF primer qPCR system provides an accessible method for routine molecular classification of endometrial cancer in clinical settings, offering a practical alternative to NGS for detecting pathogenic POLE mutations and supporting clinical decision-making.