Detection of EGFR Activating and Resistance Mutations by Droplet Digital PCR in Sputum of EGFR-Mutated NSCLC Patients

Abstract

Background: Proof of the T790M resistance mutation is mandatory if patients with EGFR-mutated non-small cell lung cancer (NSCLC) progress under first- or second-generation tyrosine kinase inhibitor therapy. In addition to rebiopsy, analysis of plasma circulating tumor DNA is used to detect T790M resistance mutation. We studied whether sputum is another feasible specimen for detection of EGFR mutations. Methods: Twenty-eight patients with advanced EGFR-mutated NSCLC were included during stable and/or progressive disease. The initial activating EGFR mutations (exon 19 deletions or L858R mutations) at stable disease and at progressive disease (together with T790M) were assessed in simultaneously collected plasma and sputum samples and detected by droplet digital polymerase chain reaction (ddPCR). Results: Activating EGFR mutations were detected in 47% of the plasma samples and 41% of sputum samples during stable disease, and in 57% of plasma samples and 64% of sputum samples during progressive disease. T790M was detected in 44% of the plasma samples and 66% of the sputum samples at progressive disease. In ddPCR T790M-negative results for both specimens (plasma and sputum), negativity was confirmed by rebiopsy in 5 samples. Concordance rate of plasma and sputum for T790M was 0.86, with a positive percent agreement of 1.0 and a negative percent agreement of 0.80. Conclusions: We demonstrated that EGFR mutation analysis with ddPCR is feasible in sputum samples. Combination of plasma and sputum analyses for detection of T790M in NSCLC patients with progressive disease increases the diagnostic yield compared with molecular plasma analysis alone.

Monitoring cfDNA in Plasma and in Other Liquid Biopsies of Advanced EGFR Mutated NSCLC Patients: A Pilot Study and a Review of the Literature

“…Data showed 83.78% of overall concordance rate between blood and saliva ctDNA (concordance rate was calculated including 10 paired blood and saliva healthy donors’ samples). Another notably result arise from Hackner et al [ 26 ], which investigated the feasibility to use sputum test in the detection of EGFR activating and resistance mutations on 28 NSCLC patient’s cohort using ddPCR. The concordance rate of the EGFR sensitizing mutations status between plasma and sputum samples was 71%, whereas the concordance rate of the T790M was 86%.…”

Multivariate Risk Analysis of RAS, BRAF and EGFR Mutations Allelic Frequency and Coexistence as Colorectal Cancer Predictive Biomarkers

“…Considering that the copy-number of a fresh tumor sample varies with the amount of background wild-type gDNA by comparing with FFPE tumor tissues that have >85% tumor cells, at least two positive droplets for each investigated mutation in a triplicate had to be present for calling a sample positive for a given mutation [ 25 , 26 , 27 ]. The threshold was manually set based on positive control samples for each channel, and the threshold for positivity was ≥ 0.1 mutant copies for 10 3 haploid genomes for all assays.…”

RAS, BRAF and EGFR Related Genetic Mutations as Predictive Biomarkers in Colorectal Cancer

“…Considering that the copy-number of a fresh tumor sample varies with the amount of background wild-type gDNA by comparing with FFPE tumor tissues that have > 85% tumor cells, at least 2 positive droplets for each investigated mutation in a triplicate had to be present for calling a sample mutant positive [36][37][38]. The threshold was manually set based on positive control samples for each channel, and the threshold for positivity was ≥0.1 mutant copies for 10 3 haploid genomes for all assays.…”