Application of ddPCR in detection of the status and abundance of EGFR T790M mutation in the plasma samples of non-small cell lung cancer patients

Abstract

Background/objective: The third-generation epidermal growth factor receptor (EGFR) -tyrosine kinase inhibitor (TKIs), such as osimertinib, designed for targeting the acquired drug-resistant mutation of EGFR T790M, was approved as the first-line therapy for advanced EGFR-mutated non-small cell lung cancer (NSCLC). Thus, detection of the EGFR T790M mutation for NSCLC is crucial. However, tissue samples are often difficult to obtain, especially in patients at advanced stages. This study assessed the performances of droplet digital polymerase chain reaction (ddPCR) and next-generation sequencing (NGS) in detecting EGFR T790M status and abundance in the plasma ctDNA samples of patients with NSCLC. We also explored the association between T790M status and abundance and the response to third-generation EGFR-TKIs.

Methods: A total of 201 plasma samples with matched tissues, 821 plasma samples, and 56 patients who received third-generation EGFR-TKIs with response evaluation were included in this study. ddPCR and NGS were used to detect the mutation status and abundance of T790M in the tissues and/or blood samples.

Results: The results showed that the sensitivity and the specificity of EGFR T790M mutation status detected by ddPCR in plasma samples were 81.82% and 91.85%, respectively, compared with the tissue samples, with a consistency coefficient of 0.740. Among the 821 plasma samples, the positive rates of EGFR T790M detected by ddPCR and NGS were 34.2% (281/821) and 22.5% (185/821), respectively. With NGS results as the reference, the sensitivity and the specificity of ddPCR were 100% and 84.91%, respectively, and the consistency coefficient of the two methods was 0.717. In addition, we found that a higher EGFR T790M abundance was linked to a higher treatment response rate to the third-generation EGFR-TKIs regardless of the classification of the median value of 0.43% (P = 0.016) or average value of 3.16% (P = 0.010).

Conclusion: Taking these data together, this study reveals that ddPCR is an alternatively potent method for the detection of EGFR T790M in the plasma samples of NSCLC patients.

Keywords: EGFR T790M; NGS; ddPCR; plasma; third-generation EGFR-TKIs.

Figure 1

Flow chart of this study.

Figure 2

Bland–Altman agreement plots of T790M abundance detected in plasma and tumor tissues by ddPCR. (A) Mean of the differences of 54 T790M-positive samples’ mutation abundance in both plasma samples and tumor tissue samples; P-value >0.05, no significance. (B) The Bland–Altman bias of differences between plasma samples and tumor tissue samples detected EGFR T790M abundance.

Figure 3

Bland–Altman agreement plots of T790M mutation abundance detected by ddPCR assay and NGS. (A) Mean of the differences of 185 T790M-positive samples’ mutation abundance in both the ddPCR assay and the NGS assay; P-value >0.05, no significance. (B) Bland–Altman bias of differences between ddPCR assay and NGS assays detected EGFR T790M abundance.

Figure 4

T790M mutation abundance of ddPCR T790M-positive but NGS T790M-negative samples. Pie chart showing the T790M abundance distribution of 96 ddPCR T790M-positive but NGS T790M-negative samples.

Figure 5

Relationship between T790M mutation abundances and the response to third-generation EGFR-TKI and the prognosis of non-small cell lung cancer (NSCLC) patients. (A) T790M mutation abundance in the CR+PR and SD+PD groups. (B–D) Kaplan–Meier curves were used to assess the relationship between T790M mutation abundance and the overall survival of patients with NSCLC.