Detection of clinically significant mutations in the epidermal growth factor receptor missed by direct sequencing using a highly sensitive DNA endonuclease
| Autor: | Alison J. Holmes, Yanan Kuang, M. Taron, Giuseppe Giaccone, Victoria A. Joshi, Pasi A. Jänne, M. Gallegos Ruiz, Andrew M. Rogers, Ana M. Borras, Robert J. Distel |
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| Rok vydání: | 2006 |
| Předmět: | |
| Zdroj: | Journal of Clinical Oncology. 24:10054-10054 |
| ISSN: | 1527-7755 0732-183X |
| DOI: | 10.1200/jco.2006.24.18_suppl.10054 |
| Popis: | 10054 Background: Mutations in the epidermal growth factor receptor (EGFR) are associated with sensitivity and resistance to EGFR inhibitors gefitinib and erlotinib in patients with non-small cell lung carcinoma (NSCLC). Direct sequencing is currently used for mutation detection but sensitivity is limited and requires dissection to obtain a relatively pure population of tumor cells. We examined a DNA endonuclease, SURVEYOR, which cleaves mismatched heteroduplexed DNA, as a more sensitive method for EGFR mutation screening and compared it to direct sequencing. Methods: EGFR exons 18–21 from tumor DNA were amplified using PCR, digested with SURVEYOR, and the products analyzed by HPLC. Specimens that produced digestion products were re-analyzed by size separation or by denaturing HPLC followed by fractionation and sequencing. Tumor specimens from 191 NSCLC patients were analyzed: 61 frozen tumors specimens; 91 dissected formalin fixed paraffin embedded (FFPE) and 39 un-dissected FFPE tumor specimens from patients treated with gefitinib or erlotinib in whom clinical outcome was available. 173 specimens were independently analyzed by direct sequencing. Results: We detected 48 EGFR mutations by sequencing and 61 using SURVEYOR. All EGFR mutations identified by sequencing, including those using un-dissected tumor specimens, were detected by SURVEYOR and none were missed (sensitivity: 100%, negative predictive value: 100%). 13 mutations were detected by SURVEYOR not detected by sequencing. This included 5 mutations (4 exon 19 deletions; 1 L858R) in 7 (71%) patients who clinically had a PR to gefitinib or erlotinib but who were wild type by sequencing. In 4 patients, 2 with clinical acquired resistance to gefitinib, a T790M mutation was found which was undetected by sequencing. Conclusions: SURVEYOR analysis is a more sensitive method for EGFR mutation detection than direct sequencing. It can be used to detect EGFR mutations from un-dissected tumor specimens and can detect clinically significant activating or resistance associated EGFR mutations not detected by direct sequencing. [Table: see text] |
| Databáze: | OpenAIRE |
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