Center of Predictive Molecular Medicine, University-Foundation, Chieti, Italy.
PLoS One. 2012;7(7):e42164. doi: 10.1371/journal.pone.0042164. Epub 2012 Jul 27.
Microdeletions at exon 19 are the most frequent genetic alterations affecting the Epidermal Growth Factor Receptor (EGFR) gene in non-small cell lung cancer (NSCLC) and they are strongly associated with response to treatment with tyrosine kinase inhibitors. A series of 116 NSCLC DNA samples investigated by Sanger Sequencing (SS), including 106 samples carrying exon 19 EGFR deletions and 10 without deletions (control samples), were subjected to deep next generation sequencing (NGS). All samples with deletions at SS showed deletions with NGS. No deletions were seen in control cases. In 93 (88%) cases, deletions detected by NGS were exactly corresponding to those identified by SS. In 13 cases (12%) NGS resolved deletions not accurately characterized by SS. In 21 (20%) cases the NGS showed presence of complex (double/multiple) frameshift deletions producing a net in-frame change. In 5 of these cases the SS could not define the exact sequence of mutant alleles, in the other 16 cases the results obtained by SS were conventionally considered as deletions plus insertions. Different interpretative hypotheses for complex mutations are discussed. In 46 (43%) tumors deep NGS showed, for the first time to our knowledge, subpopulations of DNA molecules carrying EGFR deletions different from the main one. Each of these subpopulations accounted for 0.1% to 17% of the genomic DNA in the different tumors investigated. Our findings suggest that a region in exon 19 is highly unstable in a large proportion of patients carrying EGFR deletions. As a corollary to this study, NGS data were compared with those obtained by immunohistochemistry using the 6B6 anti-mutant EGFR antibody. The immunoreaction was E746-A750del specific. In conclusion, NGS analysis of EGFR exon 19 in NSCLCs allowed us to formulate a new interpretative hypothesis for complex mutations and revealed the presence of subpopulations of deletions with potential pathogenetic and clinical impact.
外显子 19 的微缺失是影响非小细胞肺癌 (NSCLC) 表皮生长因子受体 (EGFR) 基因的最常见遗传改变，它们与酪氨酸激酶抑制剂治疗反应密切相关。通过 Sanger 测序 (SS) 研究了一系列 116 例 NSCLC DNA 样本，包括 106 例携带外显子 19 EGFR 缺失的样本和 10 例无缺失的对照样本 (对照样本)，并进行了深度下一代测序 (NGS)。SS 显示所有缺失的样本均有缺失，而对照样本无缺失。在 93 例 (88%) 病例中，NGS 检测到的缺失与 SS 鉴定的缺失完全对应。在 13 例 (12%) 病例中，NGS 解决了 SS 无法准确识别的缺失。在 21 例 (20%) 病例中，NGS 显示存在复杂 (双/多重) 移码缺失，导致净框内改变。在其中 5 例中，SS 无法确定突变等位基因的精确序列，在其他 16 例中，SS 得到的结果通常被认为是缺失加插入。对于复杂突变，讨论了不同的解释性假说。在 46 例 (43%) 肿瘤中，NGS 首次发现，携带 EGFR 缺失的 DNA 分子亚群与主要缺失不同。在不同研究的肿瘤中，这些亚群中的每一个都占基因组 DNA 的 0.1% 至 17%。我们的发现表明，在携带 EGFR 缺失的大部分患者中，外显子 19 中的一个区域高度不稳定。作为这项研究的推论，将 NGS 数据与使用 6B6 抗突变 EGFR 抗体进行的免疫组化数据进行了比较。免疫反应是 E746-A750del 特异性的。总之，对非小细胞肺癌 EGFR 外显子 19 的 NGS 分析使我们能够为复杂突变提出新的解释性假说，并揭示了具有潜在发病和临床影响的缺失亚群的存在。
