Lung Cancer Research Today is a free monthly online journal that collates and summarizes the latest research about Lung Cancer, including details on symptoms, smoking, genetics, treatment, causes. | ||||||||
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Detection of epidermal growth factor receptor variations by partially denaturing HPLC.Chin TM, Anuar D, Soo R, Salto-Tellez M, Li WQ, Ahmad B, Lee SC, Goh BC, Kawakami K, Segal A, Iacopetta B, Soong R Department of Haematology and Oncology, National University Hospital, Singapore. BACKGROUND: Epidermal growth factor receptor gene (EGFR) variants may be useful markers for identifying responders to gefitinib and erlotinib, small-molecule tyrosine kinase inhibitors of EGFR; therefore, sensitive and cost-effective assays are needed to detect EGFR variants in routine clinical samples. We have developed a partially denaturing HPLC (pDHPLC) assay that is superior to direct sequencing with respect to detection limits, costs, and time requirements. METHODS: Primers, temperatures, and buffer conditions were optimized for PCR-pDHPLC analysis of EGFR exons 18-21. We evaluated the detection limits of pDHPLC and direct sequencing by analyzing mixtures of wild-type and variant EGFR DNA and screened 192 lung cancer samples to examine the diversity of pDHPLC-detectable variants. To assess amenability to routine analysis, we tested lung and pleural tissue specimens from 14 lung cancer patients treated with gefitinib. RESULTS: The detection limits for variant alleles were 1:100 for pDHPLC and 1:5 for direct sequencing. pDHPLC analysis detected 26 unique EGFR variants, including the common deletions in exon 19 and substitutions in codons 787 and 858. Direct sequencing could not identify 30% (18 of 60) of the variant amplicons identified by pDHPLC. We identified these 18 amplicons by fraction collection after pDHPLC analysis. Analysis of a limited series of lung biopsy samples detected EGFR variants more frequently in gefitinib responders than in nonresponders. pDHPLC analysis was 56% less expensive and 39% faster than direct sequencing. CONCLUSIONS: pDHPLC-based analysis detects EGFR variations in routine clinical samples with a better detection limit and lower cost and time requirement than direct sequencing. Published 4 January 2007 in Clin Chem, 53(1): 62-70.
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