Pharmacogenomic Laboratory, CROM - Centro Ricerche Oncologiche di Mercogliano, Avellino, Italy.
Int J Oncol. 2012 Feb;40(2):378-84. doi: 10.3892/ijo.2011.1221. Epub 2011 Oct 4.
Patients with metastatic colorectal carcinoma (mCRC) carrying activating mutations of the KRAS gene do not benefit from treatment with anti-epidermal growth factor receptor (EGFR) monoclonal antibodies. Therefore, KRAS mutation testing of mCRC patients is mandatory in the clinical setting for the choice of the most appropriate therapy. Co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) is a novel modification of the conventional PCR method that selectively amplifies minority alleles from a mixture of wild-type and mutant sequences irrespective of the mutation type or position within the sequence. In this study, we compared the sensitivity of a COLD-PCR method with conventional PCR/sequencing and the real-time PCR-based Therascreen kit to detect KRAS mutations. By using dilutions of KRAS mutant DNA in wild-type DNA from colon cancer cell lines with known KRAS status, we found that Fast COLD-PCR is more sensitive than the conventional PCR method, showing a sensitivity of 2.5% in detecting G>A and G>T mutations. The detection of G>C transversions was not improved by either Fast COLD-PCR or Full COLD-PCR. We next analyzed by COLD-PCR, conventional PCR and Therascreen 52 formalin-fixed paraffin-embedded samples from mCRC patients. Among 36 samples with >30% tumor cells, 8 samples were negative by conventional PCR, Therascreen and Fast COLD-PCR; 20 mutations identified by conventional PCR were confirmed by Therascreen and Fast COLD-PCR; 8 cases undetermined by conventional PCR were all confirmed to carry KRAS G>A or G>T mutations by using either Therascreen or Fast COLD-PCR. Conventional PCR was able to detect only 2 KRAS mutations among 16 samples with <30% tumor cells (12.5%), whereas Therascreen and Fast COLD-PCR identified 6 mutants (37.5%). These data suggest that Fast COLD-PCR has a higher clinical sensitivity as compared with conventional PCR in detecting G>C to A>T changes in the KRAS gene, which represent >90% of the mutations of this oncogene in CRC.
患有转移性结直肠癌（mCRC）且 KRAS 基因发生激活突变的患者不能从抗表皮生长因子受体（EGFR）单克隆抗体治疗中获益。因此，在临床实践中，对 mCRC 患者进行 KRAS 基因突变检测对于选择最合适的治疗方法是强制性的。低温共扩增聚合酶链反应（COLD-PCR）是一种常规 PCR 方法的新变体，可选择性地扩增野生型和突变序列混合物中的少数等位基因，而与突变类型或序列中的位置无关。在这项研究中，我们比较了 COLD-PCR 方法与常规 PCR/测序和基于实时 PCR 的 Therascreen 试剂盒检测 KRAS 突变的敏感性。通过使用来自已知 KRAS 状态的结肠癌细胞系的 KRAS 突变 DNA 稀释液与野生型 DNA 的混合物，我们发现 Fast COLD-PCR 比常规 PCR 方法更敏感，在检测 G>A 和 G>T 突变时，其敏感性为 2.5%。Fast COLD-PCR 和 Full COLD-PCR 均未改善 G>C 颠换的检测。接下来，我们使用 COLD-PCR、常规 PCR 和 Therascreen 分析了 52 例来自 mCRC 患者的福尔马林固定石蜡包埋样本。在 36 个肿瘤细胞>30%的样本中，8 个样本经常规 PCR、Therascreen 和 Fast COLD-PCR 检测均为阴性；20 个经常规 PCR 鉴定的突变经 Therascreen 和 Fast COLD-PCR 得到确认；8 个常规 PCR 未确定的病例均通过 Therascreen 或 Fast COLD-PCR 证实携带 KRAS G>A 或 G>T 突变。常规 PCR 仅能够在肿瘤细胞<30%的 16 个样本中检测到 2 个 KRAS 突变（12.5%），而 Therascreen 和 Fast COLD-PCR 鉴定了 6 个突变体（37.5%）。这些数据表明，Fast COLD-PCR 在检测 KRAS 基因中 G>C 到 A>T 变化方面的临床敏感性高于常规 PCR，这代表 CRC 中该致癌基因的>90%突变。
