Detection of epidermal growth factor receptor variations by partially denaturing HPLC.
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.
Chin
Tan Min
TM
Department of Haematology and Oncology, National University Hospital, Singapore.
Anuar
Diyanah
D
Soo
Ross
R
Salto-Tellez
Manuel
M
Li
Wei Qi
WQ
Ahmad
Baidah
B
Lee
Soo Chin
SC
Goh
Boon Cher
BC
Kawakami
Kazuyuki
K
Segal
Amanda
A
Iacopetta
Barry
B
Soong
Richie
R
D000970:Antineoplastic Agents / Q000627:therapeutic use; D001706:Biopsy; D002851:Chromatography, High Pressure Liquid / Q000191:economics; D003062:Codon; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics*; D005091:Exons; D000077156:Gefitinib; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000191:economics; D011799:Quinazolines / Q000627:therapeutic use; D012680:Sensitivity and Specificity
D000970:Antineoplastic Agents; D003062:Codon; D011799:Quinazolines; D066246:ErbB Receptors; D000077156:Gefitinib