Development of a sensitive COLD-PCR method for the detection of the EGFR mutations in plasma or serum.

Abstract

e13614 Background: Several variations of the COLD-PCR method have been used for mutation detection. To enrich for all types of substitutions, insertions and deletions in a single assay, we have developed specific Ice COLD-PCR assays for analysis of genes of interest in oncology. Ice COLD-PCR is a method that preferentially amplifies low levels of mutant DNA in a sample containing a vast excess of wild-type DNA. The use of a reference sequence oligonucleotide (RS-oligo) complementary to one of the wild-type strands results in linear amplification of the wild-type sequences but exponential amplification of any the mutant sequences present. Methods: NSCLC tumors harboring the EGFR L858R mutation and the exon 19 deletions respond to anti-EGFR therapy. The methods developed used RS-oligos containing locked nucleic acids (LNA’s) complementary to the wild-type sequence in the L858 mutation or the exon 19 deletion regions. Results: Ice COLD-PCR amplification of control and mutant plasmids containing the exon 19 deletion or the L858 region followed by Sanger sequencing confirmation had a limit of detection (LOD) of 0.1-0.5%. This LOD indicates the methodology shows great promise for detection of low abundance mutations. Ice COLD-PCR with an LNA-RS oligo was also used for detecting exon 19 deletions. Coupled with Sanger sequencing, an LOD of 0.5% mutant DNA was achieved. Thirty-three DNA samples extracted from plasma were tested for both the L858R mutation and exon 19 deletions by Ice COLD-PCR. These samples had previously been analyzed for mutations using SURVEYOR Nuclease digestion. Anaylsis of these samples using the Ice COLD-PCR methodology confirmed previously detected mutations and additional samples with mutations. The detection of these additional mutant samples is due to the greater sensitivity of Ice COLD-PCR. Conclusions: The results demonstrate the utility of using Ice COLD-PCR for the highly sensitive detection of EGFR exon 19 deletions and L858R mutations in DNA derived from serum. Coupled with mutation detection methods such as Sanger sequencing, Ice COLD-PCR provides a new model for highly sensitive, economical testing of EGFR mutations to aid patient treatment decisions and disease monitoring.