EGFR T790M Mutation in TKI-Naïve Clinical Samples: Frequency, Tissue Mosaicism, Predictive Value and Awareness on Artifacts

Abstract

Background: This study evaluated the distribution of epidermal growth factor receptor (EGFR) T790M mutations in treatment-naïve tumor and normal samples obtained from cancer patients. Methods: We utilized allele-specific PCR (AS-PCR), digital droplet PCR (ddPCR) and next generation sequencing (NGS) to detect EGFR T790M allele in several collections of tumor and normal human tissues. Results: AS-PCR analysis of treatment-naïve tumor samples revealed somatic T790M mutation in 3/394 (1%) non-small cell lung carcinomas (NSCLC) carrying the tyrosine kinase inhibitor (TKI)-sensitizing EGFR mutation, but in none of 334 NSCLC lacking EGFR exon 19 deletions (ex19del) or L858R substitutions and in none of 235 non-lung tumors. Use of highly sensitive and quantitative assays, such as ddPCR and NGS, produced a high number of T790M-specific signals even in presumably T790M-negative DNA specimens. This background noise was evidently higher in degraded DNA isolated from formalin-fixed paraffin-embedded tissues as compared to high molecular weight DNA. A combination of AS-PCR, ddPCR and NGS revealed mosaic EGFR T790M allele in 2/68 (3%) NSCLC treated with the first-generation TKI. Both these tumors produced evident and durable response to gefitinib. Conclusion: Detection of mosaic EGFR T790M mutation in treatment-naïve samples may be compromised by yet unresolved technical issues and may have limited clinical value.

Different Clinicopathologic and Computed Tomography Imaging Characteristics of Primary and Acquired EGFR T790M Mutations in Patients with Non-Small-Cell Lung Cancer

“…Our study suggests that primary T790M mutation is rare (1.05%) in mainland Chinese patients with EGFR -positive NSCLC detected by routine mutation testing, which is consistent with the results of a previous study from another center in mainland China (1.07% in patients with EGFR -positive NSCLC) 17 and lower than those of a study in Taiwan (25.2% in patients with NSCLC detected by matrix-assisted laser desorption ionization-time of flight mass spectrometry and 2.8% detected by NGS) 15 and another study of non-Asian patients (6.9% in patients with EGFR -positive NSCLC). 32 Although T790M detection rates increase with higher sensitivity methods, 33 , 34 false-positive rates should be considered in clinical molecular testing, especially for samples with low DNA quality and quantity and/or formalin-fixed and paraffin-embedded-derived artifacts. 35 , 36 Here, we used two routine detection methods, ARMS and NGS, to accurately diagnose primary T790M mutation, in line with the current clinical reality.…”

Systematic detection of mosaicism by using digital NGS reveals three new MEN1 mosaicisms

“…NGS offers the possibility of detecting mosaic variants using DNA from blood ( 7 , 8 , 14 , 16 , 17 ). The challenge is to lower the threshold of variant detection without including sequencing artifacts and also to distinguish variants from artifacts ( 17 , 20 , 21 ). UMIs are unique oligonucleotide sequences which are added to DNA prior to any amplification and these differentially label each molecule in the native DNA fragment.…”

Changes in the concentration of EGFR-mutated plasma DNA in the first hours of targeted therapy allow the prediction of tumor response in patients with EGFR-driven lung cancer

“…Some NSCLCs treated by first-or second-generation EGFR TKIs demonstrate emergence of EGFR T790M mutations before clinical disease progression [58,59]. However, treatment-naïve tumors usually do not contain EGFR T790M mutation as base-line [60] therefore our study considered only monitoring of ex19del-and L858R-mutated ctDNA.…”