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...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2015-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4425536?pdf=render |
| _version_ | 1819213187536388096 |
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| author | Kazuko Sakai Junji Tsurutani Takeharu Yamanaka Azusa Yoneshige Akihiko Ito Yosuke Togashi Marco A De Velasco Masato Terashima Yoshihiko Fujita Shuta Tomida Takao Tamura Kazuhiko Nakagawa Kazuto Nishio |
| author_facet | Kazuko Sakai Junji Tsurutani Takeharu Yamanaka Azusa Yoneshige Akihiko Ito Yosuke Togashi Marco A De Velasco Masato Terashima Yoshihiko Fujita Shuta Tomida Takao Tamura Kazuhiko Nakagawa Kazuto Nishio |
| author_sort | Kazuko Sakai |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-23T06:54:53Z |
| format | Article |
| id | doaj.art-19f7edc251154a5fb2fabc94f36b8b4b |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-23T06:54:53Z |
| publishDate | 2015-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-19f7edc251154a5fb2fabc94f36b8b4b2022-12-21T17:56:21ZengPublic Library of Science (PLoS)PLoS ONE1932-62032015-01-01105e012189110.1371/journal.pone.0121891Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing.Kazuko SakaiJunji TsurutaniTakeharu YamanakaAzusa YoneshigeAkihiko ItoYosuke TogashiMarco A De VelascoMasato TerashimaYoshihiko FujitaShuta TomidaTakao TamuraKazuhiko NakagawaKazuto NishioSomatic 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.http://europepmc.org/articles/PMC4425536?pdf=render |
| spellingShingle | Kazuko Sakai Junji Tsurutani Takeharu Yamanaka Azusa Yoneshige Akihiko Ito Yosuke Togashi Marco A De Velasco Masato Terashima Yoshihiko Fujita Shuta Tomida Takao Tamura Kazuhiko Nakagawa Kazuto Nishio Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing. PLoS ONE |
| 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 |
| url | http://europepmc.org/articles/PMC4425536?pdf=render |
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