Standard PCR Sequencing Underestimates KRAS and BRAF Mutations in Advanced Colorectal Carcinomas: Comparison with LNA-Modified PCR Sequencing and Sequenom Mass Spectrometry Genotyping
ME Arcila, CY Lau, K Nafa, M Ladanyi. Memorial Sloan Kettering Cancer Center, New York
Background: KRAS and BRAF mutations are associated with primary resistance to epidermal growth factor receptor (EGFR) inhibitors and, together, are identified in about 50% of patients with advanced colorectal carcinoma. Mutation screening can therefore be used to identify a large proportion of patients who are unlikely to respond to this therapy. Detecting mutations in this setting can be challenging, particularly when testing volume is high and based on samples with a low proportion of tumor cells which, if below 25%, may not be reliably detected by standard sequencing. To address these issues, we designed and evaluated two assays - a locked nucleic acid (LNA)-based PCR-sequencing assay, to allow the detection of low levels of mutant DNA and a Sequenom MALDI-TOF mass spectrometry-based genotyping assay, suitable for large scale mutation screening.
Design: Clinical cases consecutively tested for KRAS exon 2 and BRAF exon 15 mutations by standard sequencing (with microdissection as needed) were retested with modified PCR and sequencing, using standard primers and a 10-mer LNA oligonucleotide designed to suppress amplification of non-mutant DNA. Samples were also tested using a Sequenom assay targeting the most common mutations in both genes.
Results: We tested 308 tumor samples by all 3 methods. Initial testing using standard sequencing detected 121 KRAS (39%) and 10 BRAF mutations, for a combined 42.5%. Retesting with the LNA-based method and the Sequenom assay detected 20 (141/308, 46%) and 4 (125/308, 41%) additional KRAS mutants, respectively. One additional BRAF mutant was detected by the Sequenom assay only, for a combined KRAS/BRAF proportion of 49.4% for the two new assays. The analytical sensitivity of the LNA-modified method, based on serial dilutions of mutant and normal control DNA, is below 0.3% for both KRAS and BRAF. The analytical sensitivity of the Sequenom assay ranged from 1-10% depending on the mutation tested.
Conclusions: The technical sensitivity of the LNA-PCR sequencing assay is well beyond that of standard PCR-sequencing and is particularly useful in the testing of specimens not amenable to pre-dissection due to a diffuse infiltrative pattern without discrete tumor nests. The Sequenom assay has an intermediate sensitivity but offers significant advantages for large scale mutation screening. Standard sequencing (even with microdissection) may miss approximately 14% (49.4%-42.5% /49.4%) of patients resistant to EGFR antibody therapy.
Monday, March 22, 2010 8:15 AM
Platform Session: Section G 1, Monday Morning