Improved Detection of the BRAF c.1799T>A (p.V600E) Mutation in Melanoma with a Single Nucleotide Primer Extension Assay
Gabriel C Caponetti, Emilian Racila, Aaron Stence, Jonathan Pruessner, Susan Forde, Jason Hackman, Deqin Ma, Jonathan Heusel, Aaron Bossler. University of Iowa Hospitals and Clinics, Iowa City, IA
Background: BRAF mutations have been identified in approximately 66% of melanomas. The most common BRAF mutation is the c.1799T>A on exon 15 with the resultant p.V600E. Melanoma patients bearing this mutation can significantly benefit from vemurafenib therapy. Sanger sequencing (SS) is the most commonly used test for the detection of this mutation. However, its analytical sensitivity is only 20% in formalin-fixed paraffin-embedded (FFPE) tissue. Therefore, it is crucial to identify a more sensitive method. In addition, melanin is known to inhibit Taq polymerase activity. The aims of this study were to determine the analytical sensitivity of a Single Nucleotide Primer Extension (SNPE) assay for the detection of the BRAF c.1799T>A mutation in melanoma and assess the impact of endogenous melanin on the performance of both assays.
Design: Genomic DNA (gDNA) was extracted from FFPE tissue of 21 melanoma cases. The SNPE assay was performed using the ABI PRISM® SNaPshot® kit (Applied Biosystems, Carlsbad, CA) in parallel with bi-directional SS. The analytical sensitivity of the SNPE assay was determined using purified plasmids containing the BRAF c.1799T>A (p.V600E) mutation at limiting dilutions admixed with 25 ng of wild type gDNA. The percentage of melanin content in the cases of melanoma was assessed by histological review.
Results: The analytical sensitivity of the SNPE assay was as low as 2%. In 24% (n=5) of the cases, the SS reaction failed but the SNPE was able to identify the mutation. Only one SNPE reaction failed. In cases with initial failure of the SS reaction, the average melanin content was 38% (ranging from 1% to 90%). A significantly lower melanin content (0% to 40%, average 15%) was seen in cases with successful sequencing. By diluting the DNA or re-sampling of an area of tumor with less melanin, we were able to successfully perform SS in all the cases that had initially failed.
Conclusions: Although the number of cases analyzed in this series is small, our findings suggest that the prevalence of the BRAF c.1799T>A (p.V600E) mutation in melanoma can be underestimated due the intrinsic limitations of SS and its susceptibility to interference by melanin. The SNPE assay is a much more sensitive alternative for the identification of this mutation and less susceptible to interference by melanin. The development of a multiplexed SNPE assay for the identification of other BRAF mutations appears promising and is currently being evaluated.
Monday, March 19, 2012 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 312, Monday Morning