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.
Highlights
Cancer is the second leading cause of death in developed countries [1] and lung cancer is the leading cause of cancer death in Europe
Gly719 with Ser (G719S) droplet digital PCR (ddPCR) assay was tested across an annealing temperature gradient to optimize thermocycling conditions
Using 22 mutant copies/mL as threshold for a positive result and Mutant-allele fraction (MAF) of 6.5%, five of 22 patients included in the study were real G719S plasmapositive patients
Summary
Cancer is the second leading cause of death in developed countries [1] and lung cancer is the leading cause of cancer death in Europe. Less frequent mutations have been found like the mutation within the phosphate-binding loop (P-loop) that comprises part of the ATP-binding pocket which replaces Gly719 with Ser (G719S) [6, 7]. All of these mutations produce a gain of function. NSCLC cells become dependent on this aberrant signaling and inhibition with tyrosine kinase inhibitors (TKIs) specific for EGFR like erlotinib and gefitinib, among others, drive to cell death through intrinsic apoptosis [8, 9] achieving long survival rates over 2 years in some cases. TKIs effects are limited because of resistance occurrence due to several mechanisms, one of which being secondary resistance mutations in EGFR (normally T790M mutation) [10, 11]
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