Sensitive multiplex detection of KRAS codons 12 and 13 mutations in paraffin-embedded tissue specimens
Sensitive multiplex detection of KRAS codons 12 and 13 mutations in paraffin-embedded tissue specimens
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DOI：
10.1136/jcp.2010.081539
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发表时间：
2011-01-01
影响因子：
3.4
通讯作者：
Labourier, Emmanuel
中科院分区：
文献类型：
--
作者：
Laosinchai-Wolf, Walairat;Ye, Fei;Labourier, Emmanuel
背景：在密码子12或13中携带KRAS突变的结直肠癌患者不能从目前的抗表皮生长因子受体（EGFR）单克隆抗体治疗中获益。因此，需要有效和强大的方法进行常规临床检测KRAS突变status.Aims的目的是评估一种新的多重快速检测常见的KRAS突变福尔马林固定石蜡包埋（FFPE）tissues.Methods基因组DNA的多重PCR扩增靶向KRAS密码子12/13区域和内部控制基因的引物。PCR产物进行杂交的液体珠阵列含有目标特异性探针和检测通过粒子流式cytometer.Results分析性能评估与质粒DNA和基因组DNA提取的细胞系或模型FFPE细胞系稀释显示特异性检测7个不同的KRAS突变的检测限相当于1%的肿瘤。在两个独立的研究中心对该检测试剂盒进行了评价，共使用了140份临床标本。在研究中心1，在一组86份存档的KRAS突变状态未知的FFPE组织块中，发现约45%的标本KRAS突变呈阳性。在网站2，每一个7个突变中检测到至少5个独立的标本从一组选定的54个残留的基因组DNA先前测试与ARMS/蝎子实验室开发的test.Conclusions这种新型的单孔检测是一种敏感的工具，兼容的临床实验室工作流程，快速评估KRAS突变的实体瘤标本。其性能和多重形式保证了更广泛的面板，包括EGFR途径中的其他相关突变的发展。
Background Colorectal cancer patients harbouring KRAS mutations in codon 12 or 13 do not benefit from current anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapies. Efficient and robust methods are therefore required for routine clinical testing of KRAS mutation status.Aims To evaluate a novel multiplex assay for the rapid detection of common KRAS mutations in formalin-fixed paraffin-embedded (FFPE) tissues.Methods Genomic DNA was amplified by multiplex PCR using primers targeting the KRAS codon 12/13 region and an internal control gene. PCR products were hybridised on a liquid bead array containing target-specific probes and detected by particle flow cytometry.Results Analytical performance assessed with plasmid DNA and genomic DNA extracted from cell lines or model FFPE cell line dilutions showed specific detection of seven distinct KRAS mutations with a limit of detection equivalent to 1% tumour. The assay was evaluated at two independent sites with a total of 140 clinical specimens. At site 1, about 45% of the specimens from a set of 86 archived FFPE blocks with unknown KRAS mutation status were found positive for a KRAS mutation. At site 2, each of the seven mutations was detected in at least five independent specimens from a selected set of 54 residual genomic DNAs previously tested with an ARMS/Scorpion laboratory-developed test.Conclusions This novel single-well assay is a sensitive tool compatible with the clinical laboratory workflow for the rapid assessment of KRAS mutations in solid tumour specimens. Its performance and multiplex format warrant the development of broader panels including other relevant mutations in the EGFR pathway.