Molecular Diagnostics Department, The Institute of Cancer Research and the Royal Marsden NHS Foundation Trust, 15 Cotswold Road, London SM2 5NG, UK.
Br J Cancer. 2012 Jul 10;107(2):345-51. doi: 10.1038/bjc.2012.259. Epub 2012 Jun 19.
KRAS mutation testing is required to select patients with metastatic colorectal cancer (CRC) to receive anti-epidermal growth factor receptor antibodies, but the optimal KRAS mutation test method is uncertain.
We conducted a two-site comparison of two commercial KRAS mutation kits - the cobas KRAS Mutation Test and the Qiagen therascreen KRAS Kit - and Sanger sequencing. A panel of 120 CRC specimens was tested with all three methods. The agreement between the cobas test and each of the other methods was assessed. Specimens with discordant results were subjected to quantitative massively parallel pyrosequencing (MPP). DNA blends were tested to determine detection rates at 5% mutant alleles.
Reproducibility of the cobas test between sites was 98%. Six mutations were detected by cobas that were not detected by Sanger, and five were confirmed by MPP. The cobas test detected eight mutations which were not detected by the therascreen test, and seven were confirmed by MPP. Detection rates with 5% mutant DNA blends were 100% for the cobas and therascreen tests and 19% for Sanger.
The cobas test was reproducible between sites, and detected several mutations that were not detected by the therascreen test or Sanger. Sanger sequencing had poor sensitivity for low levels of mutation.
KRAS 基因突变检测是选择转移性结直肠癌（CRC）患者接受抗表皮生长因子受体抗体治疗的必要条件，但最佳的 KRAS 基因突变检测方法尚不确定。
我们对两种商业 KRAS 基因突变试剂盒 - cobas KRAS Mutation Test 和 Qiagen therascreen KRAS Kit - 与 Sanger 测序进行了双地点比较。使用所有三种方法对 120 个 CRC 标本进行了检测。评估了 cobas 检测与其他两种方法之间的一致性。对具有不一致结果的标本进行了定量大规模平行焦磷酸测序（MPP）。对 DNA 混合物进行了检测，以确定在 5%突变等位基因的检测率。
cobas 检测在两个地点之间的重复性为 98%。cobas 检测到 6 个未被 Sanger 检测到的突变，其中 5 个通过 MPP 得到了证实。cobas 检测到 8 个未被 therascreen 检测到的突变，其中 7 个通过 MPP 得到了证实。含有 5%突变 DNA 混合物的检测率为 cobas 和 therascreen 检测的 100%，而 Sanger 检测的 19%。
cobas 检测在两个地点之间具有可重复性，并且检测到了一些 therascreen 检测或 Sanger 检测不到的突变。Sanger 测序对低水平的突变敏感性较差。
