To date, the frequency of EGFR T790M in TKI-naïve patients remains unclear, ranging from 2% to 80% depending on the sensitivity and specificity of the methods. In this study we aimed to identify the frequency of EGFR T790M in NSCLCs before TKI treatment, comparing the detection rate of three highly sensitive molecular methods.
Among 1100 NSCLCs (adenocarcinomas at stage IIIB or IV), we identified 130 NSCLCs with EGFR TKI-sensitive mutations, by MALDI-TOF mass spectrometry (MALDI-TOF MS) and Myriapod® Lung Status kit. The diagnostic performance in detecting de-novo EGFR T790M in these 130 tumors was evaluated comparing three methods, namely MALDI-TOF MS, Real Time AS-PCR (Easy®EGFR kit) and ddPCR™ (QX200 Droplet Digital PCR, PrimePCR™ Assays). Sensitivity and specificity of each method were defined by using a DNA reference standard set (Horizon). Limit of blank (LOB) of AS-PCR and ddPCR were determined by measuring replicates of 16 wild-type EGFR DNA samples obtained from peripheral blood lymphocytes and from formalin-fixed paraffin embedded (FFPE) normal lung tissues.
Comparison of the three methods was possible for 91 of the 130 NSCLCs. Overall, we identified a total of 16 de-novo T790M in the analyzable tumors (18%). In detail, 4 cases were identified by MALDI-TOF MS and confirmed by AS-PCR and ddPCR. Two T790M-mutated cases were additionally detected by AS-PCR. ddPCR confirmed EGFR T790M in all these tumors and additionally identified 10 mutated cases. Most of mutated cases showed a mutant-allele frequency between 5% and 0.1%. Titration experiments using a DNA reference standard set demonstrated higher sensitivity of ddPCR (0.1%) than AS-PCR (1%) and MALDI-TOF MS (5%). Analysis of wild-type EGFR DNA from FFPE samples was crucial for the determination of LOB of ddPCR in order to maximize sensitivity, avoiding loss of specificity.
In this study, 18% of TKI-naïve NSCLCs show EGFR T790M mutation together with an EGFR activating mutation. Most of mutated cases showed a mutant-allele frequency between 5% and 0.1%. ddPCR is a robust method enabling the detection of mutant-allele frequencies as low as 0.1%. However, a careful preliminary evaluation of the specificity of this test is mandatory, especially when FFPE tissues are investigated.
Clinical trial identification
Legal entity responsible for the study
Asst-Sette Laghi Ospedale di Circolo Varese
All authors have declared no conflicts of interest.