Validation of Pyrosequencing for the Analysis of KRAS Mutations in Colorectal Cancer
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概要
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The use of antibodies against epidermal growth factor receptor( EGFR) in conjunction with conventionalchemotherapy for metastatic colorectal cancer (CRC) in patients with KRAS wild-type tumors has beenproven to be efficacious. Recently, KRAS testing prior to anti-EGFR therapy has become mandatory formetastatic CRC patients. Although newly developed pyrosequencing is expected to be one of the highthroughput procedures detecting such mutations, the accuracy of the procedure has not been well evaluated.In the present study, we aimed to validate the accuracy, especially the potential for a false-negative result,in detecting KRAS mutations by pyrosequencing using cultured tumor cells. DNA extracted from culturedìNOZî gallbladder cancer cells( known to contain KRAS mutation G12V) at concentrations of 1%, 5%, 10%, and 25%, as well as 2 DNA samples extracted from a resected CRC specimen( known to contain anotherKRAS mutation, G12C) at concentrations of 5% and 25%, were prepared. We analyzed KRAS mutationalstatus and nonexistent and/or nonfunctional mutations of these 6 samples using pyrosequencing. TheKRAS mutation detection rates in the 4 NOZ samples( 1%, 5%, 10%, and 25%) were 0.37%, 2.79%, 5.28%,and 13.85%, respectively. Some artifacts of KRAS mutations unlikely to be present were detected in 1%samples of NOZ at a rate similar to that of the G12V mutation( G12C, 0.29%;G13C, 0.42%). Although theKRAS mutation G12C was detected at rates of 1.26% and 6.49% in samples with 5% and 25% DNA extractedfrom resected CRC specimen, respectively, no other type of KRAS mutation was detected in suchsamples. Pyrosequencing could not detect KRAS mutations correctly in the sample containing 1% DNA.This might cause false negatives. A sample mutated DNA concentration of at least 5% was necessary forprecise analyses by this procedure.
- 2013-03-25
著者
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YAMAGUCHI Takeshi
Department Biochemistry, National Institute of Agrobiological Science
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Otake Yosuke
Department Of Thoracic Surgery Nishi-kobe Medical Center
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Ichikawa Kazuhito
Department Of Surgery Kyowa Central Hospital
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Tomita Shigeki
Department Of Surgical And Molecular Pathology Dokkyo University School Of Medicine
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Fujimori Takahiro
Department Of Geriatric Medicine Osaka University Graduate School Of Medicina
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Saito Yutaka
Endoscopic Division National Cancer Center Hospital
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Okamoto Yosuke
Department Of Cell Physiology Akita University Graduate School Of Medicine
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Akimoto Naohiko
Department of Surgical and Molecular Pathology, Dokkyo Medical University School of Medicine
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Ikematsu Hiroaki
Department of Surgical and Molecular Pathology, Dokkyo Medical University School of Medicine
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Akimoto Naohiko
Department of Surgical and Molecular Pathology, Dokkyo Medical University School of Medicine : Division of Gastroenterology, Department of Internal Medicine, Nippon Medical School
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IKEMATSU Hiroaki
Department of Gastroenterology and Gastrointestinal Oncology, National Cancer Center Hospital East
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Ikematsu Hiroaki
Department of Surgical and Molecular Pathology, Dokkyo Medical University School of Medicine : Department of Gastorointestinal Oncology and Endoscopy, National Cancer Center Hospital
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Otake Yosuke
Department of Surgical and Molecular Pathology, Dokkyo Medical University School of Medicine : Endoscopy Division, National Cancer Center Hospital
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Okamoto Yosuke
Department of Surgical and Molecular Pathology, Dokkyo Medical University School of Medicine : Division of Gastroenterology and Hepatology, Department of Internal Medicine, Toho University, Omori Medical Center
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Tomita Shigeki
Department of Physical Electronics, Tokyo Institute of Technology, 2-12-1-S9-9 O-okayama, Meguro-ku, Tokyo 152-8552, Japan
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Otake Yosuke
Department of Ophthalmology, Keio University School of Medicine
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