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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\u2009\u2265\u20091% 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.<\/jats:p>","DOI":"10.1038\/s41598-021-88391-5","type":"journal-article","created":{"date-parts":[[2021,4,27]],"date-time":"2021-04-27T06:13:30Z","timestamp":1619504010000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":26,"title":["High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens"],"prefix":"10.1038","volume":"11","author":[{"given":"Lauren Y.","family":"Cheng","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lauren E.","family":"Haydu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ping","family":"Song","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianyi","family":"Nie","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael T.","family":"Tetzlaff","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lawrence N.","family":"Kwong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeffrey E.","family":"Gershenwald","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael A.","family":"Davies","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David Yu","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,4,27]]},"reference":[{"key":"88391_CR1","doi-asserted-by":"publisher","first-page":"7","DOI":"10.3322\/caac.21590","volume":"70","author":"RL Siegel","year":"2020","unstructured":"Siegel, R. L., Miller, K. D. & Jemal, A. Cancer statistics. CA Cancer J. Clin. 70, 7\u201330 (2020).","journal-title":"CA Cancer J. Clin."},{"key":"88391_CR2","doi-asserted-by":"publisher","first-page":"35","DOI":"10.2147\/ITT.S134842","volume":"7","author":"B Domingues","year":"2018","unstructured":"Domingues, B., Lopes, J. M., Soares, P. & Populo, H. Melanoma treatment in review. Immunotargets Ther. 7, 35 (2018).","journal-title":"Immunotargets Ther."},{"issue":"8","key":"88391_CR3","doi-asserted-by":"publisher","first-page":"463","DOI":"10.1038\/nrclinonc.2017.43","volume":"14","author":"JJ Luke","year":"2017","unstructured":"Luke, J. J., Flaherty, K. T., Ribas, A. & Long, G. V. Targeted agents and immunotherapies: Optimizing outcomes in melanoma. Nat. Rev. Clin. Oncol. 14(8), 463 (2017).","journal-title":"Nat. Rev. Clin. 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Improved survival with ipilimumab in patients with metastatic melanoma. N. Engl. J. Med. 363(8), 711\u2013723 (2010).","journal-title":"N. Engl. J. Med."},{"issue":"26","key":"88391_CR7","doi-asserted-by":"publisher","first-page":"2521","DOI":"10.1056\/NEJMoa1503093","volume":"372","author":"C Robert","year":"2015","unstructured":"Robert, C. et al. Pembrolizumab versus ipilimumab in advanced melanoma. N. Engl. J. Med. 372(26), 2521\u20132532 (2015).","journal-title":"N. Engl. J. Med."},{"issue":"1","key":"88391_CR8","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/1479-5876-10-1","volume":"10","author":"PA Ascierto","year":"2012","unstructured":"Ascierto, P. A. et al. The role of BRAF V600 mutation in melanoma. J. Transl. Med. 10(1), 1\u20139 (2012).","journal-title":"J. Transl. Med."},{"issue":"10","key":"88391_CR9","doi-asserted-by":"publisher","first-page":"1239","DOI":"10.1200\/JCO.2010.32.4327","volume":"29","author":"GV Long","year":"2011","unstructured":"Long, G. 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Dabrafenib and trametinib versus dabrafenib and placebo for Val600 BRAF-mutant melanoma: A multicentre, double-blind, phase 3 randomised controlled trial. Lancet 386(9992), 444\u2013451 (2015).","journal-title":"Lancet"},{"issue":"20","key":"88391_CR15","doi-asserted-by":"publisher","first-page":"1867","DOI":"10.1056\/NEJMoa1408868","volume":"371","author":"J Larkin","year":"2014","unstructured":"Larkin, J. et al. Combined vemurafenib and cobimetinib in BRAF-mutated melanoma. N. Engl. J. Med. 371(20), 1867\u20131876 (2014).","journal-title":"N. Engl. J. Med."},{"issue":"5","key":"88391_CR16","doi-asserted-by":"publisher","first-page":"603","DOI":"10.1016\/S1470-2045(18)30142-6","volume":"19","author":"R Dummer","year":"2018","unstructured":"Dummer, R. et al. Encorafenib plus binimetinib versus vemurafenib or encorafenib in patients with BRAF-mutant melanoma (COLUMBUS): A multicentre, open-label, randomised phase 3 trial. 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MTT has advisory board relationships with Novartis, LLC, Myriad Genetics, and Nanostring. LNK reports receiving a commercial research grant from Array Biopharma. JEG has served as a consultant and\/or on an advisory board for Merck, Syndax, Novartis, and Bristol Myers Squibb (unrelated to the content of this work). Sheldon G. Adelson Medical Research Foundation, the AIM at Melanoma Foundation, the NIH\/NCI 2T32CA009666-21, Cancer Fighters of Houston, and philanthropic contributions to the Melanoma Moon Shots Program of MD Anderson. MAD has been a consultant to Roche\/Genentech, Array, Novartis, BMS, GSK, Sanofi-Aventis, Vaccinex and Apexigen, and he has been the PI of funded research grants to his institution by Roche\/Genentech, GSK, Sanofi-Aventis, Merck, Myriad, and Oncothyreon, and is supported by the Dr. Miriam and DYZ is a co-founder, significant equity holder, and consultant of NuProbe Global and of Torus Biosystems, and is a consultant for Avenge Bio. LEH reports no conflict of interest.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"9043"}}