Analytic performance studies and clinical reproducibility of a real-time PCRassay for the detection of epidermal growth factor receptor gene mutations informalin-fixed paraffin-embedded tissue specimens of non-small cell lungcancer

Abstract

BackgroundEpidermal growth factor receptor (EGFR) gene mutations identify patients with non-small cell lung cancer (NSCLC) who have a high likelihood of benefiting from treatment with anti-EGFR tyrosine kinase inhibitors. Sanger sequencing is widely used for mutation detection but can be technically challenging, resulting in longer turn-around-time, with limited sensitivity for low levels of mutations. This manuscript details the technical performance verification studies and external clinical reproducibility studies of the cobas EGFR Mutation Test, a rapid multiplex real-time PCR assay designed to detect 41 mutations in exons 18, 19, 20 and 21.MethodsThe assay’s limit of detection was determined using 25 formalin-fixed paraffin-embedded tissue (FFPET)-derived and plasmid DNA blends. Assay performance for a panel of 201 specimens was compared against Sanger sequencing with resolution of discordant specimens by quantitative massively parallel pyrosequencing (MPP). Internal and external reproducibility was assessed using specimens tested in duplicate by different operators, using different reagent lots, instruments and at different sites. The effects on the performance of the cobas EGFR test of endogenous substances and nine therapeutic drugs were evaluated in ten FFPET specimens. Other tests included an evaluation of the effects of necrosis, micro-organisms and homologous DNA sequences on assay performance, and the inclusivity of the assay for less frequent mutations.ResultsA >95% hit rate was obtained in blends with >5% mutant alleles, as determined by MPP analysis, at a total DNA input of 150 ng. The overall percent agreement between Sanger sequencing and the cobas test was 96.7% (negative percent agreement 97.5%; positive percent agreement 95.8%). Assay repeatability was 98% when tested with two operators, instruments, and reagent lots. In the external reproducibility study, the agreement was > 99% across all sites, all operators and all reagent lots for 11/12 tumors tested. Test performance was not compromised by endogenous substances, therapeutic drugs, necrosis up to 85%, and common micro-organisms. All of the assessed less common mutations except one (exon 19 deletion mutation 2236_2248 > AGAC) were detected at a similar DNA input level as that for the corresponding predominant mutation.ConclusionThe cobas EGFR Mutation Test is a sensitive, accurate, rapid, and reproducible assay.

EGFR T790M mutation testing within the osimertinib AURA Phase I study

“…The cobas ® EGFR Mutation Test is a reliable and previously validated method for identifying mutations in exons 18, 19, 20, and 21 of the EGFR gene in NSCLC using FFPE tissue [23]. It has demonstrated superior sensitivity and specificity when compared with conventional Sanger sequencing [27].…”

“…The cobas ® EGFR Mutation Test (Roche Molecular Systems, Inc., Pleasanton, CA, USA) is a sensitive, accurate, rapid and reproducible real-time polymerase chain reaction (PCR) test that identifies 41 EGFR mutations in exons 18,19,20, and 21 of the gene [23]. This EGFR mutation test is registered in Europe for the detection of EGFR gene mutations [24] and has been granted premarket approval for use as the companion diagnostic test for TAGRISSO™ (osimertinib) in the USA [25].…”

Detection of EGFR mutations in patients with non-small cell lung cancer by high resolution melting. Comparison with other methods

“…Six positive cases by EGFR-Cobas assay were negative by direct sequencing and mutation was detected by MPS. O'Donnell et al [28] only found one mutation not detected by EGFR-Cobas in a group of 152 NSCLC biopsies (using MPS as reference method) due to the low burden of tumor DNA (only 1.1% of the total). Summarizing, EGFR-Cobas assay is a method with excellent sensitivity able to detect more than 90% of the mutations described in EGFR.…”

From Traditional Histology to Next-Generation Pathology: A Review of The Workflow for the Characterisation and Molecular Profiling of Non-Small Cell Lung Cancer Samples

“…The use of CB is also suggested because most commercially available kits are validated for FFPE materials. 70 The loss of tumour cells and hypocellularity are the two main disadvantages of CB. Molecular tests can be performed on direct smears, liquid-based preparations, and materials stored on filter papers.…”