Diagnostic accuracy of the droplet digital PCR POLE mutation test in endometrial cancer: Comparison with Sanger sequencing and NGS

초록

Objectives Recent studies have shown that molecular classification, especially identifying POLE mutations, significantly helps predict endometrial cancer (EC) patient outcomes. Traditional testing methods like Sanger sequencing and next-generation sequencing (NGS) are widely used, but slow and expensive. This study aims to evaluate the diagnostic accuracy of droplet digital PCR (ddPCR) for detecting POLE mutations in EC, comparing it with Sanger sequencing and NGS. Methods We analyzed tumor DNA from 132 patients with endometrial cancer. Initially, POLE mutations were evaluated using Sanger sequencing and ddPCR. Subsequently, 20 samples with discrepant results between these two methods underwent further testing with NGS for final confirmation. The diagnostic accuracy and sensitivity of ddPCR were compared to those of Sanger sequencing, using NGS results as the gold standard. Results Among the 20 discordant samples between ddPCR and Sanger sequencing, ddPCR detected POLE mutations in 15 cases (75%). ddPCR showed perfect agreement with NGS in all 5 targeted pathogenic variants (P286R, V411L, S297F, S459F, and A456P), especially detecting low-frequency mutations missed by Sanger sequencing. Specifically, ddPCR and NGS identified mutations in 11 samples that Sanger sequencing did not detect. Additionally, ddPCR corrected two false-positive results initially reported by Sanger sequencing. All five mutation-negative samples matched across ddPCR and NGS. A correlation analysis between mutation index measured by ddPCR and variant allele frequency from NGS demonstrated a strong linear relationship (r = 0.9588, p < 0.00001), further verifying the consistency between the 2 methods. Conclusions The ddPCR method demonstrated superior sensitivity and specificity for detecting POLE mutations in endometrial cancer, accurately matching NGS results and surpassing traditional Sanger sequencing. With its rapid turnaround and accuracy, ddPCR is highly suitable for routine clinical use, potentially enhancing patient management and outcomes in endometrial cancer.