[High-throughput screening method of KRAS mutations at codons 12 and 13 in formalin-fixed paraffin-embedded tissue specimens of metastatic colorectal cancer].
[High-throughput screening method of KRAS mutations at codons 12 and 13 in formalin-fixed paraffin-embedded tissue specimens of metastatic colorectal cancer].
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转移性结直肠癌福尔马林固定石蜡包埋组织标本KRAS 12、13密码子突变的高通量筛查方法
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发表时间：
2011
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通讯作者：
T. Yoshino
中科院分区：
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作者：
Yoshiyuki Fukushima;Shoko Yanaka;Kazumi Murakami;Y. Abe;T. Koshizaka;Hirotaka Hara;Chihiro Samejima;Y. Kishi;M. Kaneda;T. Yoshino
国外使用治疗性抗EGFR单克隆抗体（西妥昔单抗或帕尼单抗）治疗转移性结直肠癌（mCRC）的临床研究显示，KRAS突变是缓解的阴性预测标志物。因此，日本厚生劳动省于2010年4月批准了KRAS突变检测的医疗报销。抗EGFR单克隆抗体疗法目前被用作mCRC患者的一线治疗。为了推进简单的高通量KRAS突变检测，我们建立了一个高通量筛选系统，用于检测KRAS突变，利用Luminex（xMAP）技术（基于荧光珠的多重分析物分析方法），结合聚合酶链反应-反向序列特异性寡核苷酸方法。在这里，我们评估了我们的系统的基本性能，并证实了其在检测携带突变KRAS基因的质粒DNA和福尔马林固定的石蜡包埋组织中的密码子12和13处的KRAS突变的高特异性和重现性。我们证明了石蜡包埋的结直肠癌组织中的KRAS突变状态，并证实其结果与直接测序法的结果相当。我们的高通量方法在96孔PCR板的一个孔中同时分析多个突变方面具有优势，并将推进临床实验室的KRAS突变检测。
Clinical studies overseas using the therapeutic anti-EGFR monoclonal antibodies, cetuximab or panitumumab against metastatic colorectal cancer(mCRC), have revealed KRAS mutations as a negative predictive marker of response. Accordingly, the Ministry of Health, Labour and Welfare in Japan approved medical reimbursement of the KRAS mutation test in April 2010. Anti-EGFR monoclonal antibody therapies are now used as first-line treatment for patients with mCRC. To advance the simple high-throughput KRAS mutation test, we established a high-throughput screening system for detecting KRAS mutations utilizing Luminex(xMAP)technology(the fluorescent bead-based multiplex analyte profiling method), in combination with the polymerase chain reaction-reverse sequence-specific oligonucleotide method. Here we evaluated the basic performance of our system and confirmed its high specificity and reproducibility in detecting KRAS mutations at codons 12 and 13 in both plasmid DNAs carrying mutant KRAS genes and formalin-fixed paraffin-embedded tissues from mCRC patients. We demonstrated the KRAS mutation status in paraffin-embedded tissues of mCRC and confirmed that the results were comparable to those of the direct sequencing method. Our high-throughput method has an advantage in simultaneous analysis of multiple mutations in one well of 96-well PCR plates, and will advance the KRAS mutation test in clinical laboratories.