Clinical Chemistry
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Clinical Chemistry 53: 62-70, 2007. First published November 16, 2006; 10.1373/clinchem.2006.074831
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(Clinical Chemistry. 2007;53:62-70.)
© 2007 American Association for Clinical Chemistry, Inc.

Cancer Diagnostics

Detection of Epidermal Growth Factor Receptor Variations by Partially Denaturing HPLC

Tan Min Chin1, Diyanah Anuar2, Ross Soo1, Manuel Salto-Tellez2,3,4, Wei Qi Li5, Baidah Ahmad2, Soo Chin Lee1, Boon Cher Goh1, Kazuyuki Kawakami6, Amanda Segal7, Barry Iacopetta5 and Richie Soong2,3,a

Departments of1 Haematology and Oncology and 4 Pathology, National University Hospital, Singapore.
2 Oncology Research Institute and 3 Department of Pathology, National University of Singapore, Singapore.
5 School of Surgery and Pathology, University of Western Australia, Western Australia, Perth, Australia.
6 Department of Surgery, Kanazawa University School of Medicine, Kanazawa, Japan.
7 Department of Anatomical Pathology, Pathwest, Sir Charles Gairdner Hospital, Perth, Western Australia, Australia.

aAddress correspondence to this author at: Oncology Research Institute, National University of Singapore, MD11 Level 5, 10 Medical Dr., Singapore 117597. Fax 65-68739664; e-mail: nmirs{at}nus.edu.sg.

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.




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