Accurate detection of KRAS, NRAS and BRAF mutations in metastatic colorectal cancers by bridged nucleic acid-clamp real-time PCR
Abstract Background Patients with metastatic colorectal cancer can benefit from anti-EGFR therapy, such as cetuximab and panitumumab. However, colorectal cancers harboring constitutive activating mutations in KRAS, NRAS and BRAF genes are not responsive to anti-EGFR therapy. To select patients for a...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2019-11-01
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| Series: | BMC Medical Genomics |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12920-019-0610-8 |
| _version_ | 1819180416689504256 |
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| author | Yuki Nagakubo Yosuke Hirotsu Kenji Amemiya Toshio Oyama Hitoshi Mochizuki Masao Omata |
| author_facet | Yuki Nagakubo Yosuke Hirotsu Kenji Amemiya Toshio Oyama Hitoshi Mochizuki Masao Omata |
| author_sort | Yuki Nagakubo |
| collection | DOAJ |
| description | Abstract Background Patients with metastatic colorectal cancer can benefit from anti-EGFR therapy, such as cetuximab and panitumumab. However, colorectal cancers harboring constitutive activating mutations in KRAS, NRAS and BRAF genes are not responsive to anti-EGFR therapy. To select patients for appropriate treatment, genetic testing of these three genes is routinely performed. Methods We applied bridged nucleic acid-clamp real-time PCR (BNA-clamp PCR) to detect somatic hotspot mutations in KRAS, NRAS and BRAF. PCR products from BNA-clamp PCR were subsequently analyzed Sanger sequencing. We then compared results with those from the PCR–reverse sequence-specific oligonucleotide probe (PCR-rSSO) method, which has been used as in vitro diagnostic test in Japan. To validate the mutation status, we also performed next generation sequencing using all samples. Results In 50 formalin-fixed paraffin-embedded tissues, KRAS mutations were detected at frequencies of 50% (25/50) and 52% (26/50) by PCR-rSSO and BNA-clamp PCR with Sanger sequencing, respectively, and NRAS mutations were detected at 12% (6/50) and 12% (6/50) by PCR-rSSO and BNA-clamp PCR with Sanger sequencing, respectively. The concordance rate for detection of KRAS and NRAS mutations between the two was 94% (47/50). However, there were three discordant results. We validated these three discordant and 47 concordant results by next generation sequencing. All mutations identified by BNA-clamp PCR with Sanger sequencing were also identified by next generation sequencing. BNA-clamp PCR detected BRAF mutations in 6% (3/50) of tumor samples. Conclusions Our results indicate that BNA-clamp PCR with Sanger sequencing detects somatic mutations in KRAS, NRAS and BRAF with high accuracy. |
| first_indexed | 2024-12-22T22:14:00Z |
| format | Article |
| id | doaj.art-71a4ab5820f4475d86cad024e6594a8b |
| institution | Directory Open Access Journal |
| issn | 1755-8794 |
| language | English |
| last_indexed | 2024-12-22T22:14:00Z |
| publishDate | 2019-11-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Medical Genomics |
| spelling | doaj.art-71a4ab5820f4475d86cad024e6594a8b2022-12-21T18:10:49ZengBMCBMC Medical Genomics1755-87942019-11-0112111110.1186/s12920-019-0610-8Accurate detection of KRAS, NRAS and BRAF mutations in metastatic colorectal cancers by bridged nucleic acid-clamp real-time PCRYuki Nagakubo0Yosuke Hirotsu1Kenji Amemiya2Toshio Oyama3Hitoshi Mochizuki4Masao Omata5Division of Genetics and Clinical Laboratory, Yamanashi Central HospitalDivision of Genetics and Clinical Laboratory, Yamanashi Central HospitalDivision of Genetics and Clinical Laboratory, Yamanashi Central HospitalDepartment of Pathology, Yamanashi Central HospitalGenome Analysis Center, Yamanashi Central HospitalDepartment of Gastroenterology, Yamanashi Central HospitalAbstract Background Patients with metastatic colorectal cancer can benefit from anti-EGFR therapy, such as cetuximab and panitumumab. However, colorectal cancers harboring constitutive activating mutations in KRAS, NRAS and BRAF genes are not responsive to anti-EGFR therapy. To select patients for appropriate treatment, genetic testing of these three genes is routinely performed. Methods We applied bridged nucleic acid-clamp real-time PCR (BNA-clamp PCR) to detect somatic hotspot mutations in KRAS, NRAS and BRAF. PCR products from BNA-clamp PCR were subsequently analyzed Sanger sequencing. We then compared results with those from the PCR–reverse sequence-specific oligonucleotide probe (PCR-rSSO) method, which has been used as in vitro diagnostic test in Japan. To validate the mutation status, we also performed next generation sequencing using all samples. Results In 50 formalin-fixed paraffin-embedded tissues, KRAS mutations were detected at frequencies of 50% (25/50) and 52% (26/50) by PCR-rSSO and BNA-clamp PCR with Sanger sequencing, respectively, and NRAS mutations were detected at 12% (6/50) and 12% (6/50) by PCR-rSSO and BNA-clamp PCR with Sanger sequencing, respectively. The concordance rate for detection of KRAS and NRAS mutations between the two was 94% (47/50). However, there were three discordant results. We validated these three discordant and 47 concordant results by next generation sequencing. All mutations identified by BNA-clamp PCR with Sanger sequencing were also identified by next generation sequencing. BNA-clamp PCR detected BRAF mutations in 6% (3/50) of tumor samples. Conclusions Our results indicate that BNA-clamp PCR with Sanger sequencing detects somatic mutations in KRAS, NRAS and BRAF with high accuracy.http://link.springer.com/article/10.1186/s12920-019-0610-8KRASNRASBRAFColorectal cancerBNA |
| spellingShingle | Yuki Nagakubo Yosuke Hirotsu Kenji Amemiya Toshio Oyama Hitoshi Mochizuki Masao Omata Accurate detection of KRAS, NRAS and BRAF mutations in metastatic colorectal cancers by bridged nucleic acid-clamp real-time PCR BMC Medical Genomics KRAS NRAS BRAF Colorectal cancer BNA |
| title | Accurate detection of KRAS, NRAS and BRAF mutations in metastatic colorectal cancers by bridged nucleic acid-clamp real-time PCR |
| title_full | Accurate detection of KRAS, NRAS and BRAF mutations in metastatic colorectal cancers by bridged nucleic acid-clamp real-time PCR |
| title_fullStr | Accurate detection of KRAS, NRAS and BRAF mutations in metastatic colorectal cancers by bridged nucleic acid-clamp real-time PCR |
| title_full_unstemmed | Accurate detection of KRAS, NRAS and BRAF mutations in metastatic colorectal cancers by bridged nucleic acid-clamp real-time PCR |
| title_short | Accurate detection of KRAS, NRAS and BRAF mutations in metastatic colorectal cancers by bridged nucleic acid-clamp real-time PCR |
| title_sort | accurate detection of kras nras and braf mutations in metastatic colorectal cancers by bridged nucleic acid clamp real time pcr |
| topic | KRAS NRAS BRAF Colorectal cancer BNA |
| url | http://link.springer.com/article/10.1186/s12920-019-0610-8 |
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