Evaluation of 2 Real-Time PCR Assays for In Vitro Diagnostic Use in the Rapid and Multiplex Detection of EGFR Gene Mutations in NSCLC

Abstract

Activating mutations of the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer predict for a favorable clinical response to tyrosine kinase inhibitor therapy. Although Sanger sequencing is a conventional method to detect EGFR gene mutations, multiplex real-time allele-specific polymerase chain reaction (PCR) systems are increasingly used in the routine molecular diagnostic setting. We aim to evaluate 2 proprietary real-time PCR assays (cobas and therascreen) against Sanger sequencing in the detection of EGFR gene mutations. The overall concordance rate between cobas and therascreen assays with Sanger sequencing was 89% and 88%, respectively, and increased to 96% and 98%, respectively, if the mutations not covered were excluded. The cobas assay showed a superior coverage of exon 20 mutations, but L861Q was not targeted. The nature of specimen, DNA integrity, and tumor cell content are factors that affect the assay performance. DNA extracted from cell block and clot of pleural fluid gave rise to 1 invalid call and 1 false-negative result by the cobas assay and 1 missed T790M mutation and 1 false-negative result by the therascreen assay. Both assays are around 5 times more expensive compared with Sanger sequencing in terms of reagent cost. We conclude that both assays prove to be a rapid, simple, and validated method in detecting the most common and clinically significant EGFR gene mutations in non-small cell lung cancer. Although less convenient compared with real-time PCR assays, Sanger sequencing is cheaper in terms of reagent cost and allows the detection of rare or novel EGFR gene mutations.

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

“…Allele-specific real-time PCR has been widely used in detecting EGFR “hotspot” mutations in cancerous tissues [[10], [11], [12], [13], [14], [15]]. Therascreen ® (Qiagen) is a FDA-approved real time PCR in vitro diagnostic (IVD) test that may be used to detect 21 EGFR mutations in exons 18, 19, 20, and 21 against a background of wild type genomic DNA [16].…”

Molecular pathology in lung cancer: a guide to the techniques used in clinical practice

“…A further study comparing 4 targeted methods with Sanger sequencing found that although the targeted methods had similar LODs their efficacy when testing lung cancer samples varies; in this cohort PNA‐LNA Clamp PCR was found to have the highest rates of analytical failure and discordance . Two studies which directly compared Therascreen with COBAS showed both assays to have similar assay performance …”

EGFR Testing in Advanced Non–Small-Cell Lung Cancer, A Mini-Review

“…Compared to Sanger sequencing, 100% sensitivity and specificity have been reported for the Therascreen assay28 , and the Cobas EGFR assay showed equal or superior performance29,30 . However, in another study, these assays performed suboptimally, under calling mutations as compared to Sanger sequencing (89% and 88% concordance rates with Sanger for Cobas and Therascreen assays, respectively)31 . Other technologies are designed to query mutations in several different cancer-associated genes, including EGFR.…”