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

Abstract Mutations 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...

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Autores principales:	Lauren Y. Cheng, Lauren E. Haydu, Ping Song, Jianyi Nie, Michael T. Tetzlaff, Lawrence N. Kwong, Jeffrey E. Gershenwald, Michael A. Davies, David Yu Zhang
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a31f95e2d618422bb5e0d6e0b871a339
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spelling	oai:doaj.org-article:a31f95e2d618422bb5e0d6e0b871a3392021-12-02T13:41:23ZHigh sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens10.1038/s41598-021-88391-52045-2322https://doaj.org/article/a31f95e2d618422bb5e0d6e0b871a3392021-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-88391-5https://doaj.org/toc/2045-2322Abstract Mutations 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.Lauren Y. ChengLauren E. HayduPing SongJianyi NieMichael T. TetzlaffLawrence N. KwongJeffrey E. GershenwaldMichael A. DaviesDavid Yu ZhangNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-9 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Lauren Y. Cheng Lauren E. Haydu Ping Song Jianyi Nie Michael T. Tetzlaff Lawrence N. Kwong Jeffrey E. Gershenwald Michael A. Davies David Yu Zhang High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens
description	Abstract Mutations 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.
format	article
author	Lauren Y. Cheng Lauren E. Haydu Ping Song Jianyi Nie Michael T. Tetzlaff Lawrence N. Kwong Jeffrey E. Gershenwald Michael A. Davies David Yu Zhang
author_facet	Lauren Y. Cheng Lauren E. Haydu Ping Song Jianyi Nie Michael T. Tetzlaff Lawrence N. Kwong Jeffrey E. Gershenwald Michael A. Davies David Yu Zhang
author_sort	Lauren Y. Cheng
title	High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens
title_short	High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens
title_full	High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens
title_fullStr	High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens
title_full_unstemmed	High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens
title_sort	high sensitivity sanger sequencing detection of braf mutations in metastatic melanoma ffpe tissue specimens
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/a31f95e2d618422bb5e0d6e0b871a339
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High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens

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