e23251 Background: The cobas EGFR Mutation Test v2 (cobas EGFR test) is a real-time PCR test for the qualitative detection of 42 mutations in exons 18, 19, 20, and 21 of the epidermal growth factor receptor (EGFR) gene in DNA derived from formalin-fixed paraffin-embedded (FFPET) tumor tissue and human plasma from non-small cell lung cancer (NSCLC) patients. The test is also indicated for the semi-quantitative measurement of these EGFR mutations in plasma, allowing for serial measurements as an aid in management of NSCLC patients. Methods: The assay’s limit of detection (LoD) was determined using 31 FFPET derived DNA blends and 27 contrived, healthy donor plasma sample blends with intact (21) or sheared (6) cell line DNA. Assay performance for 118 FFPET and 74 plasma NSCLC specimens was compared between the cobas EGFR test and Next Generation Sequencing (NGS). The FFPET specimens were also compared with v1 and v2 of the cobas EGFR test. Internal repeatability was assessed in duplicate with different operators, reagent lots, and instruments across multiple days using FFPET specimens and contrived healthy donor plasma samples with intact cell line DNA. Linearity was established testing contrived healthy donor plasma sample blends with intact cell line DNA. Results: FFPET: LoD for detecting EGFR mutations of FFPET samples is ≤ 5% mutation level using the standard input of 50 ng per reaction well. The cobas EGFR test v2, when compared to the original test (v1), had positive (PPA), negative (NPA), and overall (OPA) percent agreement of 95%, 100%, and 97% respectively. The cobas EGFR test v2, when compared to NGS, had PPA of 87%, NPA of 100%, and OPA of 93%. Two internal repeatability studies had call accuracies of 96.3% and 99.2%. Plasma: LoD for detecting EGFR mutations in plasma samples is ≤ 100 cp/mL. The cobas EGFR test v2, when compared to NGS had PPA of 80.0%, NPA of 94.9%, and OPA of 87.8%. An internal repeatability study had a call accuracy of 99.1%. The cobas EGFR test v2 with plasma samples is linear between 100 and 10,000 cp/mL. Conclusions: The cobas EGFR Mutation Test v2 is a sensitive, accurate, rapid, and reproducible assay capable of testing DNA isolated from either FFPET or plasma from NSCLC patients.

Background: Epidermal 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.Methods: The 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.Results: A >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.Conclusion: The cobas EGFR Mutation Test is a sensitive, accurate, rapid, and reproducible assay.

Aim To conduct a methods correlation study of three different assays for the detection of mutations at EGFR gene in human formalin-fixed paraffin-embedded tumour (FFPET) specimens of non-small cell lung carcinomas (NSCLC). Methods We conducted a 2-site method comparison study of two european conformity (CE) in vitro diagnostic (IVD)-marked assays, the cobas EGFR Mutation Test and the Therascreen EGFR29 Mutation Kit, and 2× bidirectional Sanger sequencing. We blind-tested 124 NSCLC FFPET specimens with all three methods; the cobas test was performed at both sites. Positive (PPA) and negative percent agreements (NPA) were determined for the cobas test versus each of the other two methods. Specimens yielding discordant test results between methods were further tested using quantitative massively parallel pyrosequencing (MPP). Results PPA between cobas and Sanger was 98.8%; NPA was 79.3%. Overall there were seven discordant results. MPP confirmed an exon 19 deletion in two cases and L858R mutation in four cases. PPA between cobas and Therascreen was 98.9% and NPA was 100%. There was one discordant result. Reproducibility of the cobas test between the two sites was 99.2%. Conclusions The invalid rates for the cobas test and Therascreen were lower than Sanger sequencing. The cobas and Therascreen assays showed a high degree of concordance, and both were more sensitive for the detection of exon 19 deletion and L858R mutations than Sanger. The cobas test was highly reproducible between the two testing sites, used the least amount of DNA input and was the only test with automated results reporting.

EGFR mutations identify patients with NSCLC who have a high likelihood of benefiting from first-line treatment with anti-EGFR tyrosine kinase inhibitors. Sanger sequencing is widely used for mutation detection but can be time-consuming, and has limited sensitivity for low levels of mutations. We describe the analytic performance of the cobas EGFR Mutation Test, a multiplex, 3-reaction real-time PCR assay designed to detect 41 mutations in exons 18, 19, 20 and 21. The mutations detected and the mutation report calls are listed below: The amount of DNA required for the assay (150 ng) can typically be isolated from a single 5-micron FFPET section, and the test can be performed in 95% hit rate was obtained in blends with >5% mutant alleles for L858R and exon 19 deletions as determined by 454 sequencing (quantitative massively parallel pyrosequencing) at a total DNA input of 150ng, or 50 ng per PCR amplification. The cobas test was compared to 2x bidirectional Sanger sequencing using a set of 152 NSCLC FFPET specimens. The overall percent agreement (OPA) between the 2 methods was 96.7% (negative agreement - NPA 97.5%; positive agreement - PPA 95.8%). Specimens with discordant cobas EGFR Test and Sanger results were analyzed by 454 and a revised agreement analysis was performed based on the composite results of the 3 assays. The revised OPA was 98.7% (NPA 98.8%; PPA 98.6%). The call repeatability of a panel of NSCLC FFPET specimens was 98% when tested with two operators, instruments, and reagent lots. Necrotic tissue, hemoglobin, triglycerides, and common respiratory organisms did not interfere with the assay. The test showed no cross reactivity with the corresponding exon sequences from the HER2, HER3, and HER4 genes. These analytic studies demonstrate that the cobas EGFR Mutation Test is a sensitive, accurate and reproducible assay. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1707. doi:1538-7445.AM2012-1707