Droplet Digital PCR as a Molecular Tool for the Detection of the EGFR T790M Mutation in NSCLC Patients with the EGFR Activating Mutations
S Durgut1, L Salihefendić1, D Pećar1
1Alea Genetic Centre, Health Institute Alea Dr. Kandić, Sarajevo, Bosnia and Herzegovina.
Background:
Almost 50% of NSCLC patients who initially show a successful response to tyrosine kinase inhibitors targeted therapy (TKI therapy) eventually develop acquired EGFR T790M mutation. The T790M secondary mutation can cause resistance to the targeted therapy and disease relapse. Since this mutation can be present at very low frequencies in liquid biopsy samples, droplet digital PCR (ddPCR), due to its high sensitivity, has opened the possibility for minimally invasive monitoring of the disease during TKI targeted therapy.
Materials And Methods:
For this study, a total of 45 plasma samples from NSCLC patients with previously detected EGFR-activating mutations were analyzed. Extracted circulating free DNA was amplified and examined for the presence of T790M mutation using ddPCR technology. For the data analysis, QuantaSoft Software was used.
Results:
Of 45 tested plasma samples, a total of 14 samples were identified as positive for the T790M mutation. The same samples eventually showed the presence of T790M mutation in FFPE. Droplet digital PCR showed its great advantage in high sensitivity detection of rare allele variants. Our ddPCR assay detected T790M mutant allele in frequencies from 0.1%. The average number of droplets generated by ddPCR was 9571.
Conclusion:
Monitoring of the T790M mutation has an important role in the examination of the effects of the prescribed TKI therapy. Since monitoring of potential changes during TKI therapy requires repeated sampling, our results showed that ddPCR technology has made it possible to use liquid biopsy as an adequate minimally invasive alternative for single nucleotide polymorphisms (SNP) detection.
Insights
Droplet digital PCR (ddPCR) effectively detects the acquired EGFR T790M mutation in liquid biopsies, enabling minimally invasive monitoring of non-small cell lung cancer (NSCLC) patients during tyrosine kinase inhibitor therapy.
Area of Science:
- Oncology
- Molecular Diagnostics
- Genetics
Background:
- Acquired EGFR T790M mutation develops in nearly 50% of NSCLC patients on TKI therapy, leading to resistance and relapse.
- This secondary mutation can be present at low frequencies, complicating early detection.
- Minimally invasive monitoring is crucial for managing TKI therapy effectiveness.
Purpose of the Study:
- To evaluate the sensitivity and utility of droplet digital PCR (ddPCR) for detecting the acquired EGFR T790M mutation in liquid biopsies from NSCLC patients.
- To assess the feasibility of using liquid biopsy as a minimally invasive alternative for monitoring TKI therapy response.
Main Methods:
- Analysis of 45 plasma samples from NSCLC patients with known EGFR-activating mutations.
- Circulating free DNA extraction and amplification.
- Detection of T790M mutation using ddPCR technology and QuantaSoft Software for data analysis.
Main Results:
- 14 out of 45 plasma samples tested positive for the T790M mutation.
- ddPCR demonstrated high sensitivity, detecting T790M mutant allele frequencies as low as 0.1%.
- The T790M mutation was subsequently confirmed in FFPE samples from the positive cases.
Conclusions:
- ddPCR is a highly sensitive method for detecting rare allele variants like the T790M mutation in liquid biopsies.
- Liquid biopsy using ddPCR offers a minimally invasive alternative for monitoring NSCLC patients during TKI therapy.
- Monitoring T790M mutation status is vital for assessing TKI therapy efficacy and managing disease progression.
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