Comparative analysis of QS3D versus droplet digital PCR for quantitative measures of EGFR T790M mutation from identical plasma

Qiaomei Guo; Lin Wang; Xiaohui Liang; Mingna Zhao; Xia Huang; Wanxing Xu; Jiatao Lou; Lihua Qiao

doi:10.1016/j.heliyon.2022.e11339

Comparative analysis of QS3D versus droplet digital PCR for quantitative measures of EGFR T790M mutation from identical plasma

Heliyon. 2022 Oct 29;8(11):e11339. doi: 10.1016/j.heliyon.2022.e11339. eCollection 2022 Nov.

Authors

Qiaomei Guo^{1

2}, Lin Wang¹, Xiaohui Liang¹, Mingna Zhao¹, Xia Huang², Wanxing Xu¹, Jiatao Lou¹, Lihua Qiao¹

Affiliations

¹ Department of Laboratory Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, No. 100 Haining Road, Hongkou District, Shanghai, China.
² Department of Laboratory Medicine, Shanghai Chest Hospital, Shanghai Jiao Tong University, No. 241 Huai Hai Road (W), Shanghai, China.

Abstract

Objectives: The capacity of QuantStudio™ 3D (QS3D) and droplet digital PCR (dPCR) for the detection of plasma Epidermal Growth Factor Receptor (EGFR) mutations have been widely reported. Few comparative studies on the quantitative test of the identical DNA material, however, are carried out. Here we compared the performance of the two methods in detecting EGFR T790M mutation in cell-free DNA (cfDNA) from the same lung cancer patients.

Methods: We recruited 72 non-small cell lung cancer (NSCLC) patients who initially respond to tyrosine kinase inhibitor treatment but subsequently developed resistance. Two tubes of 10mL anticoagulant blood were collected and cfDNA was isolated from plasma. Identical cfDNA samples were analyzed for T790M mutation using QS3D and droplet dPCR in parallel.

Results: T790M mutation was detected in 15 and 21 cfDNA samples by QS3D and droplet digital PCR, respectively. The 6 discordant samples showed low mutation abundance (∼0.1%) and the discrepancy is caused by the stricter threshold settings for QS3D dPCR. The overall agreement between the two methods was 91.7% (66/72). The median allele frequencies for QS3D dPCR and droplet dPCR to detect T790M mutation was 2.01% and 2.62%, respectively. There was no significance in mutation abundance detected by both methods. Both methods are highly correlated with allele frequencies and copy numbers in T790M wild type and mutant, with R² of 0.98, 0.92 and 0.95, respectively.

Conclusion: Our study demonstrated that QS3D dPCR are highly consistent with droplet PCR for quantitative determination of EGFR T790M mutation in plasma cfDNA.

Keywords: Concordance; Digital PCR; Mutation abundance; T790M; cfDNA.