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Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing
Somatic mutations in KRAS, NRAS, and BRAF genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended RAS and BRAF mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somati...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4425536/ https://www.ncbi.nlm.nih.gov/pubmed/25954997 http://dx.doi.org/10.1371/journal.pone.0121891 |
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author | Sakai, Kazuko Tsurutani, Junji Yamanaka, Takeharu Yoneshige, Azusa Ito, Akihiko Togashi, Yosuke De Velasco, Marco A. Terashima, Masato Fujita, Yoshihiko Tomida, Shuta Tamura, Takao Nakagawa, Kazuhiko Nishio, Kazuto |
author_facet | Sakai, Kazuko Tsurutani, Junji Yamanaka, Takeharu Yoneshige, Azusa Ito, Akihiko Togashi, Yosuke De Velasco, Marco A. Terashima, Masato Fujita, Yoshihiko Tomida, Shuta Tamura, Takao Nakagawa, Kazuhiko Nishio, Kazuto |
author_sort | Sakai, Kazuko |
collection | PubMed |
description | Somatic mutations in KRAS, NRAS, and BRAF genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended RAS and BRAF mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within KRAS, NRAS, and BRAF, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffin-embedded (FFPE) tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate (α = 2 x 10(-5)). The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/100). KRAS mutation concordance between this assay and Scorpion-ARMS was 92% (92/100). DNA obtained from 15 plasma samples was also analyzed. KRAS and BRAF mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant RAS and BRAF mutations using FFPE and liquid samples. |
format | Online Article Text |
id | pubmed-4425536 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-44255362015-05-21 Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing Sakai, Kazuko Tsurutani, Junji Yamanaka, Takeharu Yoneshige, Azusa Ito, Akihiko Togashi, Yosuke De Velasco, Marco A. Terashima, Masato Fujita, Yoshihiko Tomida, Shuta Tamura, Takao Nakagawa, Kazuhiko Nishio, Kazuto PLoS One Research Article Somatic mutations in KRAS, NRAS, and BRAF genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended RAS and BRAF mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within KRAS, NRAS, and BRAF, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffin-embedded (FFPE) tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate (α = 2 x 10(-5)). The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/100). KRAS mutation concordance between this assay and Scorpion-ARMS was 92% (92/100). DNA obtained from 15 plasma samples was also analyzed. KRAS and BRAF mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant RAS and BRAF mutations using FFPE and liquid samples. Public Library of Science 2015-05-08 /pmc/articles/PMC4425536/ /pubmed/25954997 http://dx.doi.org/10.1371/journal.pone.0121891 Text en © 2015 Sakai et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Sakai, Kazuko Tsurutani, Junji Yamanaka, Takeharu Yoneshige, Azusa Ito, Akihiko Togashi, Yosuke De Velasco, Marco A. Terashima, Masato Fujita, Yoshihiko Tomida, Shuta Tamura, Takao Nakagawa, Kazuhiko Nishio, Kazuto Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing |
title | Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing |
title_full | Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing |
title_fullStr | Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing |
title_full_unstemmed | Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing |
title_short | Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing |
title_sort | extended ras and braf mutation analysis using next-generation sequencing |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4425536/ https://www.ncbi.nlm.nih.gov/pubmed/25954997 http://dx.doi.org/10.1371/journal.pone.0121891 |
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