Balkan Journal of Medical Genetics

DROPLET DIGITAL PCR AS A MOLECULAR TOOL FOR THE DETECTION OF THE EGFR T790M MUTATION IN NSCLC PATIENTS WITH THE EGFR ACTIVATING MUTATIONS
Durgut S, Salihefendić L, Pećar D, Čeko I, Mulahuseinović N, Izmirlija M, Konjhodžić R
*Corresponding Author: Selma Durgut, MD, ALEA Genetic Center, Olovska 67, 71000 Sarajevo, Bosnia and Herzegovina; Mob.: +38761904549, Email: selma.durgut@agc.ba
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MATERIALS AND METHODS

For this study, a total of 45 blood samples were col- lected from NSCLC patients who had previously tested positive for EGFR-activating mutations. The samples were collected at the Alea Genetic Center in Sarajevo. The selec- tion of blood samples was based on identified EGFR muta- tions from lung adenocarcinoma tissue samples, and during this study they were tested for the presence of T790M mutation. The Alea Genetic Center did not perform the analysis for EGFR-activating mutations, and patients were selected according to their pathohistological and molecular testing results from different institutions. In this study, we concentrated solely on T790M mutation, activating EGFR mutations were determined by laboratories that tested the tissue samples. Blood plasma was separated from the blood, with 1-4 ml taken from each sample and stored at a temperature of -20˚C. The circulating free DNA (cfDNA) was extracted from these plasma samples using QIAamp ® Circulating Nucleic Acid Kit, following the manufacturer’s instruc- tions [14] . To process plasma samples faster and more effi- ciently, the QIAvac 24 Plus with the QIAvac Connecting System and QIAGEN Vacuum Pump was used according to the manufacturer’s instructions [15] . Extracted cfDNA was quantified using Qubit 3 Fluorometer ® and Qubit ® dsDNA HS assay kit [16] . Starting DNA concentration for ddPCR reaction was 15 ng for every sample. Using Bio-Rad QX200 Droplet Digital PCR technology, all 45 samples were tested for a specific EGFR T790M mutation and its corresponding wild-type amplicons. The commercial assay used for T790M mutation and wild-type amplicon detection was PrimePCR™ ddPCR™ Mutation Detection Assay Kit: EGFR WT for p.T790M, and EGFR p.T790M. The multiplex assay was 20X concentrated and ddPCR supermix for probes was 2X concentrated. In order to portion each sample into droplets, they were placed into a QX200 droplet generator. Droplets were then transferred to a 96-well plate and placed into Bio-Rad T100 thermal cycler. The amplification was performed according to the manufacturer’s instructions [17] . After DNA amplification, the droplet-containing plate was positioned in the QX200 droplet reader, allowing for the analysis of each droplet individually through a two-color system (FAM and HEX/ VIC) [13] . For the analysis of the results, QuantaSoft Soft- ware was used. When processing the results, wild-type and mutant T790M alleles were expressed as a number of FAM (mu- tant) and HEX (wild-type) droplets. The frequency of mutant alleles was determined by calculating the ratio of mutant droplets to the sum of mutant and wild-type droplets. Since ddPCR enables absolute quantification, mutant cfDNA templates were expressed as a number of copies per µl of tested DNA. The ddPCR threshold for positive mutant alleles was set at two or more positive droplets considering the added amount of DNA and gener- ated number of droplets [18,19] .

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