Four Different Molecular Assays for BRAF Mutational Testing: A Sensitivity and Specificity Analysis
B Yang, X Zhang, X Ji, M Jakubowski, JL Hunt. Cleveland Clinic, Cleveland, OH
Background: Oncogene mutational testing in tumors is becoming important for clinical management of patients with some carcinomas. But, the sensitivity and specificity of different assays to detect somatic point mutations is not well understood. This study compared sensitivity of four different assays for detecting the BRAF exon 15 oncogene mutation: gene sequencing, allele specific polymerase chain reaction (ASP), the Mutector kit, and pyrosequencing.
Design: DNA was extracted from microdissected tissue fragments using the High Pure Template Preparation Kit (Roche Applied Science, Indianapolis, IN). A dilution series ranging from 100% BRAF mutant to 1% mutant, diluted in normal DNA, and 15 cases of colon cancer were included in the study. For the gene sequencing, PCR for a 224 basepair product was followed by forward and reverse strand cycle sequencing. ASP was done with fluorescently labeled primers (forward wild-type, forward mutant, and reverse mutant) and analysis performed on the ABI 3730. Pyrosequencing (Biotage Inc. Sweden) was performed on biotinylated PCR products (196 basepair product) with biotinylated forward PCR primer and reverse primer. A reverse primer is used for pyrosequencing. The pyrogram provides percentage and ratio between wild type and mutant alleles The Mutector kit (TrimGen, Sparks, MD) was used according the manufacturer's instructions.
Results: Gene sequencing detected samples where the mutant was present in 25% or more of the sample, with the other three assays detected down to 2% mutant. All 15 patient samples were concordant between these methods (6/15 positive for the BRAF mutation).
Conclusions: Traditional gene sequencing is the least sensitive assay, only detecting mutant when it is present in greater than 25% of the sample. Pyrosequencing, ASP, and the Mutector kits all have excellent sensitivities, yielding interpretable positive results when mutant is present in greater than 2% of the sample. All four methods were equally sensitive and specific for microdissected tissue fragments, which are likely to contain high percentage of tumor cells.
Wednesday, March 11, 2009 1:00 PM
Poster Session VI # 228, Wednesday Afternoon