The Johns Hopkins Medical Institutions, The Sol Goldman Pancreatic Cancer Research Center. David Koch Cancer Research Building-II, Suite 341. 1550 Orleans St. Baltimore, MD 21231, USA.
J Mol Diagn. 2009 Nov;11(6):583-9. doi: 10.2353/jmoldx.2009.090061. Epub 2009 Oct 8.
Patients with biliary tract carcinoma have a poor prognosis. Early detection efforts are urgently needed to ameliorate the dismal prognosis for these patients. Mutations of the KRAS2 gene are one of the most common genetic aberrations in this cancer. In this study, we used LigAmp, an ultrasensitive technology for detecting point mutations, to analyze KRAS2 mutations in patients with a variety of neoplastic and non-neoplastic pancreatobiliary diseases. DNA was isolated from 64 samples, including 44 bile samples and 20 serum samples. Oligonucleotides specific for KRAS2 G35A (GAT, G12D), G35T (GTT, G12V), and G34A (AGT, G12S) mutations were used. KRAS2 mutations were detected in 14 of 16 (87.5%) neoplastic bile samples and in 9 of 28 (32.1%) non-neoplastic bile samples. However, the mutation levels were significantly lower in the non-neoplastic bile (median = 0.4%) compared with those in the neoplastic bile (median = 5.1%). KRAS2 mutations were also detected in 9 of 11 (81.8%) serum samples from patients with biliary tract carcinoma, which was further confirmed by cloning BstN1-refractory PCR products and DNA sequencing. However, KRAS2 mutations were not present in the sera from eight patients with benign pancreatobiliary diseases. These data demonstrate that KRAS2 mutations are detectable in both bile and serum using LigAmp. This technology has the potential for early biliary tract carcinoma detection and possibly for residual disease monitoring post-therapy.
胆道癌患者的预后较差。迫切需要进行早期检测工作，以改善这些患者的惨淡预后。KRAS2 基因突变是这种癌症中最常见的遗传异常之一。在这项研究中，我们使用 LigAmp（一种用于检测点突变的超灵敏技术）来分析各种肿瘤性和非肿瘤性胰胆管疾病患者的 KRAS2 突变。从 64 个样本中提取 DNA，包括 44 个胆汁样本和 20 个血清样本。使用针对 KRAS2 G35A（GAT，G12D）、G35T（GTT，G12V）和 G34A（AGT，G12S）突变的特异性寡核苷酸。在 16 个（87.5%）肿瘤性胆汁样本中的 14 个和 28 个（32.1%）非肿瘤性胆汁样本中的 9 个中检测到 KRAS2 突变。然而，非肿瘤性胆汁中的突变水平明显低于肿瘤性胆汁（中位数=0.4%）。在 11 个（81.8%）胆道癌患者的血清样本中也检测到 KRAS2 突变，这通过克隆 BstN1 抗性 PCR 产物和 DNA 测序进一步得到证实。然而，在 8 名患有良性胰胆管疾病的患者的血清中未发现 KRAS2 突变。这些数据表明，使用 LigAmp 可以在胆汁和血清中检测到 KRAS2 突变。该技术有可能用于早期胆道癌检测，并可能用于治疗后残留疾病监测。
