Behn M, Qun S, Pankow W, Havemann K, Schuermann M
Abteilung Hämatologie/Onkologie, Philipps-Universität Marburg, Germany.
Clin Cancer Res. 1998 Feb;4(2):361-71.
RFLP-mediated PCR has been successfully applied as a reliable tool in the detection of ras mutations in many cancers and provides a basis for "mutant-enriched PCR" protocols. We have, therefore, modified this technique to the sensitive detection of K-ras codon 12 and also p53 "hot spot" mutations, which, frequently in lung cancer, affect codons at the positions 157, 175, 245, 248, 249, and 273. With a high sensitivity of 1 mutant allele in 10(4) normal alleles, our enrichment assay allows the detection of oncogene alleles when only a few tumor cells are present within a normal cell population. Brush cytology material obtained from the tumor site of 20 patients with endoscopically apparent bronchial carcinoma was compared to macroscopically normal mucosa taken from the contralateral bronchus ("control" cytology). We found K-ras codon 12 mutations in 5 cases (25%) and p53 mutations in 13 cases (65%) in the tumor-derived cell material but, with the exception of two cases, not in cell material taken from the control cytology. Seventy-five % of the samples analyzed showed that at least one of the two oncogenes was affected. In several cases, two p53 lesions were detected concomitantly. The majority of the mutations could be reconfirmed by an alternative approach exploiting changes in the genomic RFLP pattern induced by these mutations and were also demonstrated in separate diagnostic biopsies taken. Thus, we conclude that the established enriched PCR protocol ensures a high sensitivity and preserved specificity for the diagnosis of oncogene lesions associated with lung cancer. Because conventional techniques normally yield a lower incidence of corresponding ras and p53 mutations, we think that both the high rate and the heterogeneity of p53 mutations found in some cases are, indeed, related to the increased sensitivity of this new enriched PCR technique.
RFLP介导的PCR已成功应用于检测多种癌症中的ras突变，是一种可靠的工具，并为“突变富集PCR”方案提供了基础。因此，我们对该技术进行了改良，用于灵敏检测K-ras密码子12以及p53“热点”突变，这些突变在肺癌中经常影响第157、175、245、248、249和273位密码子。我们的富集检测法灵敏度高达10⁴个正常等位基因中有1个突变等位基因，当正常细胞群体中仅存在少数肿瘤细胞时，就能检测到癌基因等位基因。将20例经内镜检查确诊为支气管癌患者肿瘤部位获取的刷检细胞学材料，与取自对侧支气管的宏观正常黏膜（“对照”细胞学）进行比较。我们在肿瘤来源的细胞材料中发现5例（25%）存在K-ras密码子12突变，13例（65%）存在p53突变，但除2例之外，对照细胞学获取的细胞材料中未发现此类突变。75%的分析样本显示，两种癌基因中至少有一种受到影响。在几例样本中，同时检测到两个p53病变。多数突变可通过利用这些突变引起的基因组RFLP模式变化的另一种方法再次得到证实，并且在单独采集的诊断性活检中也得到了证实。因此，我们得出结论，既定的富集PCR方案对诊断与肺癌相关的癌基因病变具有高灵敏度和良好的特异性。由于传统技术通常检测到的相应ras和p53突变发生率较低，我们认为在某些病例中发现的p53突变的高发生率和异质性确实与这种新型富集PCR技术的更高灵敏度有关。
