Detection of EGFR mutations in lung adenocarcinoma; comparing cobas 4800 EGFR assay with Sanger bi-directional sequencing

Abstract

Background: Accurate detection of EGFR mutation is crucial in management of patients with lung adenocarcinoma and would qualify them for targeted therapy. Historically Snager bi-directional sequencing (SBS) has been used as the gold standard for EGFR mutational analysis, however there are emerging new assays utilizing targeted real time PCR technology. In this study we compared the performance of ‘Cobas 4800’ (COBAS) against Sanger sequencing.Methods: 480 consecutive formalin fixed paraffin embedded samples of lung adenocarcinoma were simultaneously tested for EGFR mutations by SBS and COBAS. Mutational results were catogorised as positive, negative and invalid. Unweighted Kappa test was utilsed to compare the concordance between two assays.Results: After exclusion of invalid results (n=16), 477 samples from 458 patients (47.2% male, 52.8% female) underwent statistical analysis. There was an excellent observed percentage agreement of 98.3% (kappa value 0.944, SE 0.0194) between the two methods. The combined mutation detection rate (19%) was superior to either SBS (18.4%) or COBAS (18%). EGFR mutation frequency was significantly higher in women (23%) compare to men (12%).Discussion: COBAS assay is a diagnostically robust platform comparable with Sanger with very high analytical sensitivity and short turns around time. COBAS failure is due to lower sensitivity to samples with low DNA quality and its limited primer detection range, while Sanger is mostly affected by its lower analytic sensitivity related to low volume of diagnostic material. Consequently, the higher combined mutation detection rate necessitates a dual testing strategy to guarantee detection of novel mutations and mutations outside the COBAS detection range, and avoiding false negative results due to lower analytical sensitivity of SBS. Background: Accurate detection of EGFR mutation is crucial in management of patients with lung adenocarcinoma and would qualify them for targeted therapy. Historically Snager bi-directional sequencing (SBS) has been used as the gold standard for EGFR mutational analysis, however there are emerging new assays utilizing targeted real time PCR technology. In this study we compared the performance of ‘Cobas 4800’ (COBAS) against Sanger sequencing. Methods: 480 consecutive formalin fixed paraffin embedded samples of lung adenocarcinoma were simultaneously tested for EGFR mutations by SBS and COBAS. Mutational results were catogorised as positive, negative and invalid. Unweighted Kappa test was utilsed to compare the concordance between two assays. Results: After exclusion of invalid results (n=16), 477 samples from 458 patients (47.2% male, 52.8% female) underwent statistical analysis. There was an excellent observed percentage agreement of 98.3% (kappa value 0.944, SE 0.0194) between the two methods. The combined mutation detection rate (19%) was superior to either SBS (18.4%) or COBAS (18%). EGFR mutation frequency was significantly higher in women (23%) compare to men (12%). Discussion: COBAS assay is a diagnostically robust platform comparable with Sanger with very high analytical sensitivity and short turns around time. COBAS failure is due to lower sensitivity to samples with low DNA quality and its limited primer detection range, while Sanger is mostly affected by its lower analytic sensitivity related to low volume of diagnostic material. Consequently, the higher combined mutation detection rate necessitates a dual testing strategy to guarantee detection of novel mutations and mutations outside the COBAS detection range, and avoiding false negative results due to lower analytical sensitivity of SBS.