Approximately 50% of patients with non-small-cell lung cancer (NSCLC) experience clinical benefit with gefitinib (IRESSA) treatment, in terms of an objective response or stable disease, often associated with symptom improvement. Recent evidence suggests that mutations in the tyrosine-kinase domain of the epidermal growth factor receptor (EGFR) gene may explain some of the objective responses to gefitinib in NSCLC (Lynch et al. N Engl J Med 2004;350:2129-2139; Paez et al. Science 2004;304:1497-1500). Screening for mutations is complex and takes around 4 weeks. The detection of mutations or variations in DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor samples is limited by low recovery of DNA, which can be partially degraded. Analysis is further complicated by the presence of contaminating host DNA. We have evaluated 3 methods for the sensitive detection of acquired mutations within the EGFR gene in DNA extracted from FFPE tumor samples: DNA sequencing, Amplification Refractory Mutation System (ARMS), and heteroduplex Cel 1 digestion with denaturing high-performance liquid chromatography (dHPLC) analysis. DNA was isolated from 35 lung adenocarcinomas from female patients and the quantity and quality was assessed by quantitative polymerase chain reaction (PCR). We successfully detected mutations using all 3 methods. Two mutations were found in the same samples using all 3 methods and a further 5 mutations were identified in other samples using the combination of heteroduplex Cel 1 digestion and dHPLC analysis. We found that each of the techniques had advantages and limitations. Novel mutations can be identified by DNA sequencing or dHPLC analysis. However, DNA sequencing was the least sensitive of the methods and additional analysis of dHPLC positives is required to characterize mutations. ARMS is the most sensitive method, but the system cannot detect novel mutations. Once an ARMS assay has been developed, results can be obtained within 2 days, as opposed to 2 weeks for the other 2 methods. We found that using all 3 methods in combination with quantitative PCR reduced the limitations of each method and ensured that all variations present in the samples could be identified and validated. IRESSA is a trademark of the AstraZeneca group of companies

[Proc Amer Assoc Cancer Res, Volume 46, 2005]