Use of denaturing high performance liquid chromatography (dHPLC) for detection of EGFR mutations in patients with NSCLC

Abstract

10068 Background: Somatic mutations of the epidermal growth factor receptor (EGFR) gene may predict responsiveness to tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). These mutations are commonly identified using DNA sequencing methods. Although considered the gold standard, this approach is expensive and time-consuming. In addition, a high ratio of tumour to normal tissue DNA is required for optimal results which is not often available in biopsies obtained from these patients. The primary objective of this study was to develop a rapid and sensitive method for screening of EGFR mutations. Materials and Methods: Clinical specimens from 110 NSCLC patients were analysed for EGFR mutations in exons 19 and 21. After DNA extraction and PCR, both dHPLC and direct sequencing were performed and results were compared. Results: Sequencing revealed a total of 7 (6%) mutations: 4 missense mutations in exon 21 and 3 deletion mutations in exon 19. dHPLC showed the presence of genomic alterations in 18 (16%) samples, including the 7 identified by sequencing plus 11 additional samples (10 in exon 19 and one in exon 21). dHPLC fractions were isolated, reamplified, and sequenced to confirm these results. In serial dilution studies, dHPLC was able to detect mutations in samples containing as little as 10% mutated DNA whereas direct sequencing required at least 30%. Conclusions: dHPLC is an efficient and accurate, as well as a a more sensitive method for screening of genomic alterations in exons 19 and 21 of the EGFR gene compared to direct sequencing. This data suggests that dHPLC should be implemented as a screening tool for detection of EGFR mutations. No significant financial relationships to disclose.