Description
The therascreen EGFR Plus RGQ PCR Kit is an in vitro diagnostic real-time PCR test intended for the qualitative detection and identification of 42 mutations in exons 18, 19, 20 and 21 of the epidermal growth factor receptor (EGFR) gene in DNA samples extracted from plasma or formalin-fixed paraffin-embedded (FFPE) tumor tissue taken from non-small cell lung cancer (NSCLC) patients. Furthermore, the kit also indicated for the semi-quantitative measurement of mutations in exons 18, 20 and 21 of the EGFR gene in DNA samples extracted from human plasma as an aid in the clinical management of NSCLC patients. The test is intended to be used as an aid in selecting patients with NSCLC for therapy with an EGFR tyrosine kinase inhibitor (TKI).
When compared to the analytical reference method, there was a high level of concordance between therascreen EGFR Plus RGQ PCR Kit and the analytical accuracy method(s). The reference and discrepancy resolution methods used were: PNA qPCR, Sanger bi-directional sequencing; next-generation sequencing, therascreen EGFR RGQ PCR Kit V2 (cat. no. 874111) and therascreen EGFR Plasma RGQ PCR Kit (cat. no. 870311). Results were analyzed to assess the positive percent agreement (PPA), negative percent agreement (NPA) and overall percent agreement (OPA) regarding EGFR mutation status (mutant; MT or wild-type; WT) and EGFR target (mutation identification) for FFPE and plasma samples between therascreen EGFR Plus RGQ PCR Kit and respective reference method and following discrepancy resolution method. FFPE samples In the study 170 FFPE samples were tested, of which 48 gave valid interpretable results (148 sample status and 155 target status). When the results of the therascreen EGFR Plus RGQ PCR Kit were compared to the results of the respective reference method, four EGFR sample status (MT or WT) showed discordance. Following discrepancy resolution method analysis, the number of discordant samples statuses (MT or WT) decreased to one discrepant, false-negative sample status. The PPA, NPA and OPA with the corresponding two-sided 95% confidence intervals (CI) are summarized in Table 1 and Table 2. Table 1. Analysis of agreement of overall mutation status per sample: therascreen EGFR Plus RGQ PCR Kit and reference method comparison for FFPE samples Lower limit 95% CI Upper lim it 95% CI OPA 97.30% 93.22% 99.26% PPA (sensitivity) 93.65% 84.53% 98.24% NPA (specificity) 100.00% 95.75% 100.00% Table 2. Analysis of agreement of overall mutation status per sample after discordant investigation for FFPE samples Lower limit 95% CI Upper limit 95% CI OPA 99.32% 96.29% 99.98% PPA (sensitivity) 98.33% 91.06% 99.96% NPA (specificity) 100.00% 95.89% 100.00% Plasma samples In the study, 106 plasma samples were tested and 106 gave valid interpretable results (106 sample status and 121 target status). When the results of therascreen EGFR Plus RGQ PCR Kit were compared to the results of the respective reference method, nine EGFR sample status (MT or WT) showed discordance. Following discrepancy resolution method analysis, the number of discordant samples status (MT or WT) decreased to three discrepant, one false-negative, and two false-positive sample status. The PPA, NPA, and OPA with the corresponding two-sided 95% confidence intervals (CI) are summarized in Table 3 and Table 4. Table 3. Analysis of agreement of overall mutation status per sample: therascreen EGFR Plus RGQ PCR Kit and reference method comparison for plasma samples Lower limit 95% CI Upper limit 95% CI OPA 91.51% 84.49% 96.04% PPA (sensitivity) 87.27% 75.52% 94.73% NPA (specificity) 96.08% 86.54% 99.52% Table 4. Analysis of agreement of overall mutation status per sample after discordant investigation for plasma samples Lower limit 95% CI Upper limit 95% CI OPA 97.17% 91.95% 99.41% PPA (sensitivity) 97.96% 89.15% 99.95% NPA (specificity) 96.49% 89.89% 99.57%
The therascreen EGFR Plus RGQ PCR Kit comprises four triplex assays, each containing all the oligonucleotides (primers, probes and oligonucleotide clamp, where applicable) necessary to amplify and detect the targeted EGFR mutations. In each assay, two target mutations and one internal amplification control are assayed to determine the presence or absence of mutant DNA and to identify reaction failures due to suboptimal DNA input, or the presence of inhibitory substances in the sample matrix. Allele-specific technology allows accurate and highly reproducible detection of mutations. DNA is selectively amplified using ARMS primers, probes and PCR clamps, with sensitive signal detection using the Rotor-Gene Q MDx 5plex HRM instrument. Result reporting is fully automated using Rotor-Gene AssayManager v2.1 software. If both the positive and no template controls are valid and the sample internal controls are valid, the EGFR mutation status will be displayed in the software.
Flexibility is key to the therascreen EGFR Plus RGQ PCR System – with a choice of sample types and DNA extraction methods before real-time PCR. DNA extraction from FFPE tissue or plasma samples can be performed manually or automated on the QIAsymphony SP, for walkaway sample processing. DNA is extracted from FFPE tissue samples manually using the QIAamp DSP DNA FFPE Tissue Kit, while automated processing uses the QIAsymphony DSP DNA Mini Kit. Plasma samples are processed manually using the QIAamp DSP Circulating Nucleic Acid Kit, while the QIAsymphony DSP Circulating DNA Kit offers automated DNA purification. Following DNA extraction and quantification, sensitive real-time PCR is performed on the Rotor-Gene Q MDx 5plex HRM instrument. Automated data analysis displays qualitative results in Rotor-Gene AssayManager software, informing the system operator if one or more of the 42 mutations detected by the kit are present. The assay can be completed in ~8 hours, providing next-day results and informing earlier treatment decisions.
