High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens

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dc.citation.articleNumber9043en_US
dc.citation.journalTitleScientific Reportsen_US
dc.citation.volumeNumber11en_US
dc.contributor.authorCheng, Lauren Y.en_US
dc.contributor.authorHaydu, Lauren E.en_US
dc.contributor.authorSong, Pingen_US
dc.contributor.authorNie, Jianyien_US
dc.contributor.authorTetzlaff, Michael T.en_US
dc.contributor.authorKwong, Lawrence N.en_US
dc.contributor.authorGershenwald, Jeffrey E.en_US
dc.contributor.authorDavies, Michael A.en_US
dc.contributor.authorZhang, David Yuen_US
dc.contributor.orgSystems, Synthetic, and Physical Biologyen_US
dc.date.accessioned2021-06-07T20:22:44Zen_US
dc.date.available2021-06-07T20:22:44Zen_US
dc.date.issued2021en_US
dc.description.abstractMutations in the BRAF gene at or near the p. V600 locus are informative for therapy selection, but current methods for analyzing FFPE tissue DNA generally have a limit of detection of 5% variant allele frequency (VAF), or are limited to the single variant (V600E). These can result in false negatives for samples with low VAFs due to low tumor content or subclonal heterogeneity, or harbor non-V600 mutations. Here, we show that Sanger sequencing using the NuProbe VarTrace BRAF assay, based on the Blocker Displacement Amplification (BDA) technology, is capable of detecting BRAF V600 mutations down to 0.20% VAF from FFPE lymph node tissue samples. Comparison experiments on adjacent tissue sections using BDA Sanger, immunohistochemistry (IHC), digital droplet PCR (ddPCR), and NGS showed 100% concordance among all 4 methods for samples with BRAF mutations at ≥ 1% VAF, though ddPCR did not distinguish the V600K mutation from the V600E mutation. BDA Sanger, ddPCR, and NGS (with orthogonal confirmation) were also pairwise concordant for lower VAF mutations down to 0.26% VAF, but IHC produced a false negative. Thus, we have shown that Sanger sequencing can be effective for rapid detection and quantitation of multiple low VAF BRAF mutations from FFPE samples. BDA Sanger method also enabled detection and quantitation of less frequent, potentially actionable non-V600 mutations as demonstrated by synthetic samples.en_US
dc.identifier.citationCheng, Lauren Y., Haydu, Lauren E., Song, Ping, et al.. "High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens." <i>Scientific Reports,</i> 11, (2021) Springer Nature: https://doi.org/10.1038/s41598-021-88391-5.en_US
dc.identifier.digitals41598-021-88391-5en_US
dc.identifier.doihttps://doi.org/10.1038/s41598-021-88391-5en_US
dc.identifier.urihttps://hdl.handle.net/1911/110695en_US
dc.language.isoengen_US
dc.publisherSpringer Natureen_US
dc.rightsThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.en_US
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/en_US
dc.titleHigh sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimensen_US
dc.typeJournal articleen_US
dc.type.dcmiTexten_US
dc.type.publicationpublisher versionen_US
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