Investigation of acquired resistance to EGFR-targeted therapies in lung cancer using cDNA microarrays.

University of Southern California, Los Angeles, CA, USA.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2012; 795:233-53. DOI: 10.1007/978-1-61779-337-0_16
Source: PubMed

ABSTRACT Clinical tools to accurately describe, evaluate, and predict an individual's response to cancer therapy are a field-wide priority; in many advanced cancers, only 10-20% of individuals will have a clinical benefit from therapy, yet we treat the entire population. Furthermore, many therapies are initially effective, but lose effectiveness over time. Here we describe methods to derive in vitro models of resistance to EGFR tyrosine kinase inhibitors. We additionally describe approaches to characterize possible mechanisms of resistance by genomic and transcriptomic approaches.

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    ABSTRACT: Mutations in the epidermal growth factor receptor (EGFR) are associated with clinical and radiographic responses to EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Currently available methods of EGFR mutation detection rely on direct DNA sequencing, which requires isolation of DNA from a relatively pure population of tumor cells, cannot be done on small diagnostic specimens, and lack sensitivity. Here we describe the use of a sensitive screening method that overcomes many of these limitations. We screened 178 non-small cell lung cancer specimens for mutations in exons 18 to 21 of EGFR using a DNA endonuclease, SURVEYOR, which cleaves mismatched heteroduplexed DNA. Samples were analyzed by high-performance liquid chromatography on the Transgenomic WAVE HS system. Selected specimens that produced digestion products using SURVEYOR were subsequently reanalyzed by size separation or under partially denaturing conditions, followed by fractionation and sequencing. The specimens included DNA isolated from frozen tumor specimens, dissected formalin-fixed, paraffin-embedded tumor specimens undergoing clinical sequencing, and undissected formalin-fixed, paraffin-embedded specimens. One hundred sixty specimens were independently analyzed using direct DNA sequencing in a blinded fashion. EGFR mutations were detected in 16 of 61 fresh frozen tumor specimens, 24 of 91 dissected formalin-fixed, paraffin-embedded tumor specimens, and 11 of 26 undissected formalin-fixed, paraffin-embedded tumor specimens. Compared with sequencing, the sensitivity and specificity of the present method were 100% and 87%. The positive and negative predictive values were 74% and 100%, respectively. SURVEYOR analysis detected 7 (4%) mutations that were not previously detected by direct sequencing. SURVEYOR analysis provides a rapid method for EGFR mutation screening with 100% sensitivity and negative predictive value. This unbiased scanning technique is superior to direct sequencing when used with undissected formalin-fixed, paraffin-embedded specimens.
    Clinical Cancer Research 03/2006; 12(3 Pt 1):751-8. · 8.19 Impact Factor


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Jun 2, 2014