Shahi Rajendra B, De Brakeleer Sylvia, De Grève Jacques, Geers Caroline, In't Veld Peter, Teugels Erik
*Department of Medical Oncology and Laboratory of Medical and Molecular Oncology, Oncologisch Centrum †Department of Pathology, UZ-Brussel, Vrije Universiteit Brussel, Brussels, Belgium.
Appl Immunohistochem Mol Morphol. 2015 Mar;23(3):163-71. doi: 10.1097/PDM.0000000000000035.
Somatic mutations in the epidermal growth factor receptor-tyrosine kinase (EGFR-TK) domain of non-small cell lung cancer (NSCLC) influence the responsiveness of these tumors to EGFR-TK inhibitors, indicating their usefulness as a predictive molecular marker. However, for mutation analysis, the amount of clinical material available from NSCLC patients is often very limited, suboptimally preserved, and composed of both normal and tumor cells. As a consequence, the total amount of recovered DNA is frequently very limited, with mutant alleles being often strongly underrepresented, and thus requiring highly sensitive methods for the detection of mutations. In the present study, EGFR mutation screening was performed on 210 NSCLC clinical samples by heminested polymerase chain reaction (PCR), followed by denaturing gradient gel electrophoresis (DGGE). Candidate mutations were further characterized by sequencing. Seventeen different types of pathogenic EGFR-TK domain mutations were detected in 55 of the 210 samples (26%). We reanalyzed 149 of the 155 samples in which no mutation was found by real-time PCR for the presence of recurrent exon 21 and exon 19 mutations using peptide nucleic acid probes in the PCR mix to increase sensitivity by mutant allele enrichment. Four additional samples with exon 19 mutations were detected. Thus, it is found that the relatively simple and inexpensive PCR-DGGE assay is already very sensitive for the detection of mutations in clinical samples, including samples with low tumor cellularity (10% or higher tumor cell content), although the sensitivity and speed of the assay can be further increased for a restricted panel of mutations by introducing peptide nucleic acid probes in the DGGE or real-time PCR-based assay.
非小细胞肺癌（NSCLC）表皮生长因子受体-酪氨酸激酶（EGFR-TK）结构域中的体细胞突变会影响这些肿瘤对EGFR-TK抑制剂的反应性，表明其作为预测性分子标志物的有用性。然而，对于突变分析，NSCLC患者可获得的临床材料数量通常非常有限，保存不佳，且由正常细胞和肿瘤细胞组成。因此，回收的DNA总量常常非常有限，突变等位基因往往严重代表性不足，因此需要高度灵敏的方法来检测突变。在本研究中，通过半巢式聚合酶链反应（PCR）对210份NSCLC临床样本进行EGFR突变筛查，随后进行变性梯度凝胶电泳（DGGE）。候选突变通过测序进一步表征。在210份样本中的55份（26%）中检测到17种不同类型的致病性EGFR-TK结构域突变。我们对155份通过实时PCR未发现突变的样本中的149份进行重新分析，在PCR混合物中使用肽核酸探针检测外显子21和外显子19的复发性突变，以通过突变等位基因富集提高灵敏度。又检测到4份具有外显子19突变的样本。因此，发现相对简单且廉价的PCR-DGGE检测方法对于检测临床样本中的突变已经非常灵敏，包括肿瘤细胞含量低（10%或更高肿瘤细胞含量）的样本，尽管通过在DGGE或基于实时PCR的检测中引入肽核酸探针，对于一组受限的突变，该检测方法的灵敏度和速度可以进一步提高。
