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.

Keywords: Epidermal growth factor receptor; FDA; Fine needle aspiration; Formalin-fixed paraffin-embedded tissue; Next-generation sequencing; Non-small cell lung cancer; therascreen®.

Fig. 1

The identification of EGFR c.2573T > G (p.Leu858Arg) sequence change at 3% allele fraction by the cancer hotspot NGS (upper panel) and the therascreen^® (lower panel) tests.

Fig. 2

An EGFR exon 19 deletion was accurately scored using the therascreen^® assay (upper panel). Manual review of the cancer hotspot NGS data revealed this deletion at 0.8% allele fraction (lower panel).

Fig. 3

(A) A suspected EGFR T790M variant (Ct = 36.96) was not scored within the therascreen^® reference range (upper panel). Alternatively, cancer hotspot NGS accurately called this variant at 5% allele fraction (lower panel). (B) Another example of a late EGFR T790M therascreen^® curve (Ct = 32.96) resulted in the deltaCt not within the reference range (upper panel). Cancer hotspot NGS identified this variant at 2% allele fraction (lower panel). Note: the EGFR exon 19 deletions are not shown in the NGS pileup results.

Fig. 4

A complex EGFR exon 19 sequence change. The therascreen^® result indicated an EGFR exon 19 deletion (upper panel). Cancer hotspot NGS revealed this EGFR exon 19 sequence change consists of a 10-base and two individual single-base deletions (lower panel).

Fig. 5

An unusual EGFR exon 19 sequence change was determined as deletion by therascreen^® (upper panel). The cancer hotspot NGS revealed the sequence change involves two consecutive missense variants (lower panel).