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首页> 外文期刊>Pharmacogenomics >Detection of KRAS mutations in colorectal carcinoma patients with an integrated PCR/sequencing and real-time PCR approach.
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Detection of KRAS mutations in colorectal carcinoma patients with an integrated PCR/sequencing and real-time PCR approach.

机译：使用集成的PCR /测序和实时PCR方法检测大肠癌患者的KRAS突变。

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AIMS: Patients with metastatic colorectal carcinoma (mCRC) carrying activating mutations of the KRAS gene do not benefit from treatment with anti-EGF receptor monoclonal antibodies. Therefore, KRAS mutation testing of mCRC patients is mandatory in the clinical setting to aid in the choice of appropriate therapy. MATERIALS & METHODS: We developed a cost-effective approach for the determination of KRAS mutations in codons 12 and 13 in clinical practice based on a sensitive PCR/sequencing technique and the commercially available real-time PCR-based Therascreen kit (DxS Ltd). RESULTS & CONCLUSION: The PCR/sequencing test was able to detect 10% mutant DNA in a background of wild-type DNA. By using this assay, we determined the mutational status of KRAS in 527 out of 540 (97.6%) formalin-fixed paraffin-embedded tissues from mCRC patients. PCR/sequencing was not conclusive in 13 cases, in which low-intensity peaks suggestive of potential mutations were identified. The DxS assay, which showed a sensitivity of 1%, identified mutations in 11 out of 13 inconclusive cases. Interestingly, five of these 11 cases showed high levels of DNA fragmentation. No significant difference was found in the ability of PCR/sequencing and DxS to identify KRAS mutations within 160 cases with more than 30% tumor cells. However, in 24 samples with less than 30% tumor cells, DxS showed an higher sensitivity. In conclusion, our findings suggest that PCR/sequencing can be used for mutational analysis of the majority of tumor samples that have more than 30% tumor cell content, whereas more sensitive and expensive tests should be reserved for inconclusive cases and for samples with a low amount of tumor cells.
机译：目的：患有转移性结直肠癌（mCRC）且携带KRAS基因激活突变的患者无法从抗EGF受体单克隆抗体的治疗中受益。因此，在临床环境中必须对mCRC患者进行KRAS突变测试，以帮助选择适当的治疗方法。材料与方法：我们基于灵敏的PCR /测序技术和市售的基于实时PCR的Therascreen试剂盒（DxS Ltd），开发了一种经济有效的方法，可在临床实践中确定12位和13位密码子的KRAS突变。结果与结论：PCR /测序测试能够在野生型DNA背景中检测到10％的突变DNA。通过使用该分析，我们确定了来自mCRC患者的540个（97.6％）福尔马林固定石蜡包埋的组织中的527个中KRAS的突变状态。在13例病例中，PCR /测序尚无定论，在这些病例中发现了提示潜在突变的低强度峰。 DxS分析显示1％的敏感性，在13例不确定的病例中有11例鉴定出突变。有趣的是，这11例病例中有5例显示出高水平的DNA片段化。在160例肿瘤细胞超过30％的病例中，PCR /测序和DxS识别KRAS突变的能力没有发现显着差异。但是，在24个肿瘤细胞少于30％的样品中，DxS显示出更高的灵敏度。总之，我们的研究结果表明，PCR /测序可用于大多数肿瘤细胞含量超过30％的肿瘤样品的突变分析，而对于不确定的病例和低水平的样品，应保留更为敏感和昂贵的检测方法肿瘤细胞数量。

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