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Highly Sensitive Droplet Digital PCR Method for Detection of de novo EGFR T790M Mutation in Patients with Non-Small
Xun Wang1, Xiao Li1, Haifa Guo1
1Department of Thoracic Surgery, Peking University People's Hospital, Peking University, Beijing 100044, People's Republic of China.
Objective:
The aim of this study was to investigate the allelic relation between de novo T790M and concomitant sensitizing EGFR mutations in EGFR-TKIs naïve NSCLCs and to explore whether the formalin-fixed and paraffin-embedded (FFPE) materials affect the detection of de novo EGFR T790M mutation.
Methods:
Specimens of 300 consecutive EGFR-TKI naïve NSCLCs who received surgical resection between January 2016 and June 2018 were retrospectively investigated. All the snap-frozen tumor tissues from 300 NSCLCs were screened by droplet digital PCR (ddPCR) for the detection of de novo T790M mutation. The allelic relation between de novo T790M mutation and concomitant sensitizing EGFR mutations was also investigated. Furthermore, we assessed de novo T790M mutation in paired FFPE specimens of 50 patients which included tumor tissues and paired normal lung tissues of the pretreatment NSCLCs to investigate whether FFPE materials affect the detection of de novo T790M mutation.
Results:
The de novo T790M mutation was observed in four patients which included one patient of single de novo T790M mutation and three patients of de novo T790M mutation coexisting with L858R mutation. The incidence of de novo T790M in pretreatment NSCLCs who harboring EGFR mutations was 2.9% (4/139). All the de novo T790M mutations were detected in cis with the concomitant L858R mutations for the three NSCLCs. Our ddPCR method demonstrated that the frequency of de novo T790M mutation was ranging from 0.1% to 0.5% among 90% (45/50) of the FFPE tumor samples and 92% (46/50) of the paired FFPE adjacent normal lung samples. The frequency of de novo T790M mutation in the paired snap-frozen samples was all below 0.1%.
Conclusion:
Our study demonstrated that most de novo T790M mutations were detected in cis with concomitant sensitizing mutations for pretreatment NSCLCs. Analytical cut-off of ddPCR assay for FFPE specimens should be validated carefully considering the possibility of FFPE-derived artificial gene mutations.
Insights
This study found that de novo T790M mutations in non-small cell lung cancer (NSCLC) often occur alongside sensitizing EGFR mutations. Formalin-fixed paraffin-embedded (FFPE) samples may affect T790M detection accuracy.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Epidermal growth factor receptor (EGFR) mutations are key drivers in non-small cell lung cancer (NSCLC).
- The T790M mutation is a common resistance mechanism to EGFR tyrosine kinase inhibitors (TKIs).
- De novo T790M mutations, present at diagnosis, can influence treatment strategies.
Purpose of the Study:
- To investigate the allelic relationship between de novo T790M and sensitizing EGFR mutations in EGFR-TKI-naïve NSCLC patients.
- To determine if formalin-fixed and paraffin-embedded (FFPE) materials impact the detection of de novo EGFR T790M mutations.
Main Methods:
- Retrospective analysis of 300 EGFR-TKI-naïve NSCLC surgical specimens.
- Screening for de novo T790M mutation using droplet digital PCR (ddPCR) on snap-frozen tissues.
- Assessment of de novo T790M mutation in paired FFPE tumor and normal lung tissues from 50 patients.
Main Results:
- De novo T790M mutation was found in 2.9% of NSCLCs with EGFR mutations (4/139).
- All detected de novo T790M mutations were found 'in cis' with L858R mutations.
- FFPE samples showed higher frequencies of de novo T790M (0.1%-0.5%) compared to snap-frozen samples (<0.1%), suggesting potential FFPE-artefact.
Conclusions:
- De novo T790M mutations in NSCLC are frequently detected 'in cis' with sensitizing EGFR mutations.
- The analytical cut-off for ddPCR assays using FFPE specimens requires careful validation due to potential artificial gene mutations.
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