Abstract 4219: A novel high-sensitive assay for detection of EGFR T790M mutation using high resolution melting analysis with mutant-enriched COLD PCR.
Authors: Hashida Shinsuke , Soh Junichi , Toyooka Shinichi , Tada Ryuhei , Shien Kazuhiko , Furukawa Masashi , Yamamoto Hiromasa , Asano Hiroaki , Tsukuda Kazunori , Miyoshi Shinichiro 
Published: August 2014

Abstract
[Background] Epidermal growth factor receptor (EGFR) mutation is a predictive marker for clinical outcome of EGFR-tyrosine kinase inhibitors (TKIs) treatment in non-small lung cancer (NSCLC) patients. Approximately 80 % of NSCLC patients with EGFR mutation show good responsiveness to EGFR-TKI treatment. However, resistance acquisition to EGFR-TKIs occurs in most cases and a half of EGFR-TKIs resistances are caused by EGFR T790M mutation. We and others have previously reported that high sensitive assays such as mutant-enriched PCR could detect minor T790M mutant allele in NSCLC tumors even before EGFR-TKI treatment, suggesting early detection of T790M mutation may enable to propose subsequent therapy such as next generation EGFR-TKIs. In this study, we developed a novel high-sensitive assay for detection of T790M mutation using high resolution melting (HRM) analysis combined with mutant-enriched and COLD PCRs, and evaluated its sensitivity and frequency of T790M mutations in NSCLCs.
[Material and Method] We determined EGFR mutational status in 50 NSCLCs that were surgically resected at our institute. EGFR exon 19 deletions and L858R mutations were determined using mutant-enriched PCR assays. EGFR T790M mutation was examined by HRM analysis with or without mutant-enriched PCR and/or COLD PCR. A mutant-enriched PCR enhanced mutant alleles by treating conventional PCR product with a restriction enzyme that specifically digested wild-type alleles. A COLD PCR enhanced mutant alleles by decreasing denature temperature utilizing difference of melting temperature between mutant and wild-type amplicons. The sensitivity of each assay was determined using variable proportion of mixture DNAs of human bronchial epithelial cell with wild-type EGFR and NCI-H1975 with EGFR L858R and T790M mutations.
[Result] EGFR exon 19 deletion or L858R mutation was found in 13 out of 50 NSCLCs. Conventional HRM, COLD HRM, mutant-enriched HRM, and mutant-enriched COLD HRM could detect one T790M mutant allele out of 10, 100, 1000, and 10000 wild-type alleles, respectively. Although conventional HRM and direct sequencing could not detect any T790M mutation in 50 NSCLCs, mutant-enriched COLD HRM found T790M mutation in one NSCLC patient harboring L858R mutation.
[Conclusion] Mutant-enriched COLD HRM assay is very sensitive to detect minor T790M allele among vast amount of wild-type alleles, suggesting that this assay may predict T790M-related EGFR-TKIs resistance.
Citation Format: Shinsuke Hashida, Junichi Soh, Shinichi Toyooka, Ryuhei Tada, Kazuhiko Shien, Masashi Furukawa, Hiromasa Yamamoto, Hiroaki Asano, Kazunori Tsukuda, Shinichiro Miyoshi. A novel high-sensitive assay for detection of EGFR T790M mutation using high resolution melting analysis with mutant-enriched COLD PCR. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4219. doi:10.1158/1538-7445.AM2013-4219