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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RESULTS

A total of 45 plasma samples were enrolled in this study. All selected plasma samples from NSCLC patients had previously shown the presence of EGFR-activating mutations on FFPE samples, in the following order: del19 (66.66%), L858R (17.77%), G719X (8.88%) and ins20 (4.44%). Out of 45 FFPE samples, one sample was invali- dated for molecular testing for EGFR-activating mutations and only the patient’s plasma sample was enrolled in the study. Out of 45 samples, 14 samples were identified as positive for the T790M mutation, while 31 samples did not show the presence of the T790M mutant allele variant. The same 14 samples eventually showed the presence of T790M mutation in FFPE, while 31 tissue samples were identified as negative. In comparison to tumor tissue re- sults, the sensitivity and specificity for EGFR T790M mutation in plasma samples were 100%. Our ddPCR as say detected T790M mutant allele in frequencies ranging from 0.1%. The number of mutant molecules per µL of tested cfDNA ranged from 0.28 to 209 as depicted in Table 1. The average number of droplets generated by ddPCR was 9571. As can be noticed from Table 1, plasma sample num- ber 4 had an excessive amount of T790M mutant alleles. It is worth noting that the amount of tissue material from this patient was insufficient for valid results. Therefore, the result obtained from this tissue sample was reported as an invalid result and was not considered in the comparison between plasma and tissue samples. In the patient’s plasma sample, 10 038 droplets were generated and the ratio of the number of droplets containing mutated gene variants to the total number of generated droplets was 12.3% (Figure 1). However, in plasma sample 38, which was reported negative for the T790M mutation, one highly differentiated positive droplet was observed. For this sample, a small number of amplified copies were detected. To confirm the result, this plasma sample was run and analyzed twice, obtaining identical results, which indicates the accuracy of the procedure and the validity of the results. Although insufficient to be declared as positive, this result should not be ignored, and it indicates the need for liquid rebiopsy to determine the exact mutational status of the patient. This patient should be monitored for early detection of the T790M to adjust therapy if needed. Out of 14 plasma EGFR T790M positive samples, detected EGFR-activat- ing mutations on FFPE samples were as follows: del19 (76.92%), L858R (15.38%), and G719X (7.69%).

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