Real-time PCR and targeted next-generation sequencing in the detection of low level EGFR mutations: Instructive case analyses

Abstract

Background Allele specific real-time PCR and next-generation sequencing (NGS) are widely used to detect somatic mutation in non-small cell lung cancer (NSCLC). Both methods commonly use formalin-fixed paraffin-embedded (FFPE) tissues as diagnostic materials. Real-time PCR has the advantage of being easy to use and more tolerant of variable DNA quality, but has limited multiplex capability. NGS, in contrast, allows simultaneous analysis of many genomic loci while revealing the exact sequence changes; it is, however, more technically demanding and more expensive to employed. A challenge for both platforms is the varied limit of detection (LoD) for target genomic loci, even within the same gene. The variability of detection sensitivity may be problematic if well-known actionable somatic mutations are missed. Cases We compared LoDs between real-time PCR and targeted NGS tests for some commonly observed EGFR mutations in NSCLC specimens. Conclusions The FDA-approved real-time PCR test was superior to the NGS in detecting low level EGFR exon 19 deletion (near 1% variant allele fraction (VAF)). The cancer hotspot NGS detects low level EGFR c.2369C > T, p.T790M (2–5% VAF) better than the FDA-approved real-time PCR method. We conclude that the real-time PCR and hotspot NGS methods have complementary strengths in accurately determining clinically important EGFR mutations in NSCLC.

Application of liquid biopsy-based targeted capture sequencing analysis to improve the precision treatment of non-small cell lung cancer by tyrosine kinase inhibitors

“…It was observed that the NGS-based assay showed high sensitivity in testing tumour biomarker genes in plasma cfDNA as gene allele variant fraction of less than 0.1% and the copy number of gene allele variant of less than 1 copy/mL plasma were also successfully detected (figures 1 and 3). This confirms other studies in which the deep sequencing approach showed higher detection sensitivity in testing tumour biomarker gene variants in cfDNA than qPCR and MassArray-based assays 30 32 33…”

Comprehensive Approach to Distinguish Patients with Solid Tumors from Healthy Controls by Combining Androgen Receptor Mutation p.H875Y with Cell-Free DNA Methylation and Circulating miRNAs

“…Considering this, we speculate that there is no literature concerning other tumors until now because other studies that analyzed this specific AR mutation focused primarily on breast and prostate cancer. Besides, we used a qPCR-based method to detect cfDNA mutations, which is shown to have a better sensitivity to detect low allele fraction variants than sequencing [ 31 ]. Still, the underlying mechanisms of the involvement of AR p.H875Y mutation in the carcinogenesis of these cancer types should be investigated.…”

Non-Small Cell Lung Cancer Harboring Concurrent EGFR Genomic Alterations: A Systematic Review and Critical Appraisal of the Double Dilemma

“…Moreover, this interesting analysis pointed out the urgent need of further investigations in order to clarify the mechanism of differential responses to TKIs. In the study by Zhang et al [54], patients with a sensitive EGFR alteration such as 19Del/p.L858R/p.L861Q, plus a p.T790M de novo or an exon 20 insertion exhibited the worst clinical outcomes [116], [118]. This could be clarified as they were treated with first-and second-generation EGFR-TKIs.…”

“…Notably, the presence of coexisting genetic alteration might likely justify resistance to TKIs treatment [116]. Particularly, different clinical trials assessed that the concurrent presence of mutation provides a worse prognosis in EGFR-positive NSCLC patients treated with first-, second-, and third-generation TKIs.…”