Detection of the EGFR G719S Mutation in Non-small Cell Lung Cancer Using Droplet Digital PCR

Abstract

Objectives: The main objectives of the study were (1) to set-up a droplet digital PCR (ddPCR) assay for the non-invasive detection of G719S EGFR mutation in NSCLC patients; (2) to determine the limits of detection of the ddPCR assay for G719S mutation and (3) to compare COBAS® and ddPCR System for G719S quantification in plasma. Materials and Methods: Blood samples were collected from 22 patients diagnosed with advanced NSCLC. Then, plasma ctDNA was extracted with the Qiagen Circulating Nucleic Acids kit and quantified by QuantiFluor® dsDNA System. The mutational study of EGFR was carried out by digital droplet PCR (ddPCR) with the QX200 Droplet Digital PCR System with specific probes and primers. Results: We observed the lowest percentage of G719S mutant allele could be detected in a wildtype background was 0.058%. In the specificity analysis, low levels of G719S mutation were detected in healthy volunteers with a peak of 21.65 mutant copies per milliliter of plasma and 6.35 MAFs. In those patients whose tissue biopsy was positive for G719S mutation, mutant alleles could also be detected in plasma using both ddPCR and COBAS® System. Finally, when mutational status was studied using both genotyping techniques, higher mutant copies/ml and higher mutant allele fraction (MAF) correlated with higher Semiquantitative Index obtained by COBAS®. Conclusions: Although tissue biopsies cannot be replaced due to the large amount of information they provide regarding tumor type and structure, liquid biopsy and ddPCR represents a new promising strategy for genetic analysis of tumors from plasma samples. In the present study, G719S mutation was detected in a highly sensitive manner, allowing its monitorization with a non-invasive technique.

Keywords: EGFR; G719S; ddPCR = droplet digital PCR; liquid biopsy; lung cancer; non-small cell lung cancer.

Figure 1

Validation assays for G719S mutation detection in plasma samples. (A) Temperature gradient to determine the optimum annealing temperature; mutant positive events (top) and wild-type positive events (bottom) across the thermal gradient (57–67°C). (B) Two-fold dilution series of mutant DNA into wild-type DNA: concentration is shown in copies per microliter for mutant (blue) and wild-type (green) events and fractional abundance in percentage (orange). Error bars show 95% CI.

Figure 2

Detection of G719S in a healthy population using ddPCR. Concentration is represented in copies per milliliter of plasma in both healthy (∙) and patient (♦) groups; where dashed line represents a candidate threshold for positive results with high sensitivity.

Figure 3

Summary of results obtained by COBAS and ddPCR and comparative evaluation for G719S detection in plasma. SQI, Semiquantitative Index obtained by COBAS® 4800 System. *For sample LB010 SQI was not available. **For sample LB020 no mutation was detected in plasma using COBAS (SQI).