Published in:
Open Access 01-12-2013 | Technical advance
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
Published in: BMC Cancer | Issue 1/2013
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Background
Epidermal growth factor receptor (EGFR) gene mutations identify patients withnon-small cell lung cancer (NSCLC) who have a high likelihood of benefitingfrom treatment with anti-EGFR tyrosine kinase inhibitors. Sanger sequencingis widely used for mutation detection but can be technically challenging,resulting in longer turn-around-time, with limited sensitivity for lowlevels of mutations. This manuscript details the technical performanceverification studies and external clinical reproducibility studies of thecobas EGFR Mutation Test, a rapid multiplex real-time PCR assay designed todetect 41 mutations in exons 18, 19, 20 and 21.
Methods
The assay’s limit of detection was determined using 25 formalin-fixedparaffin-embedded tissue (FFPET)-derived and plasmid DNA blends. Assayperformance for a panel of 201 specimens was compared against Sangersequencing with resolution of discordant specimens by quantitative massivelyparallel pyrosequencing (MPP). Internal and external reproducibility wasassessed using specimens tested in duplicate by different operators, usingdifferent reagent lots, instruments and at different sites. The effects onthe performance of the cobas EGFR test of endogenous substances and ninetherapeutic drugs were evaluated in ten FFPET specimens. Other testsincluded an evaluation of the effects of necrosis, micro-organisms andhomologous DNA sequences on assay performance, and the inclusivity of theassay for less frequent mutations.
Results
A >95% hit rate was obtained in blends with >5% mutant alleles, asdetermined by MPP analysis, at a total DNA input of 150 ng. The overallpercent agreement between Sanger sequencing and the cobas test was 96.7%(negative percent agreement 97.5%; positive percent agreement 95.8%). Assayrepeatability was 98% when tested with two operators, instruments, andreagent lots. In the external reproducibility study, the agreementwas > 99% across all sites, all operators and all reagentlots for 11/12 tumors tested. Test performance was not compromised byendogenous substances, therapeutic drugs, necrosis up to 85%, and commonmicro-organisms. All of the assessed less common mutations except one (exon19 deletion mutation 2236_2248 > AGAC) were detected at asimilar DNA input level as that for the corresponding predominantmutation.
Conclusion
The cobas EGFR Mutation Test is a sensitive, accurate, rapid, andreproducible assay.