Mafficini Andrea, Simbolo Michele, Parisi Alice, Rusev Borislav, Luchini Claudio, Cataldo Ivana, Piazzola Elena, Sperandio Nicola, Turri Giona, Franchi Massimo, Tortora Giampaolo, Bovo Chiara, Lawlor Rita T, Scarpa Aldo
ARC-Net Research Centre, University and Hospital Trust of Verona, Verona, Italy.
Department of Pathology & Diagnostics, University and Hospital Trust of Verona, Verona, Italy.
Oncotarget. 2016 Jan 12;7(2):1076-83. doi: 10.18632/oncotarget.6834.
BRCA mutated ovarian cancers respond better to platinum-based therapy and to the recently approved PARP-inhibitors. There is the need for efficient and timely methods to detect both somatic and germline mutations using formalin-fixed paraffin-embedded (FFPE) tissues and commercially available technology. We used a commercial kit exploring all exons and 50bp exon-intron junctions of BRCA1 and BRCA2 genes, and semiconductor next-generation sequencing (NGS) on DNA from 47 FFPE samples of high-grade serous ovarian cancers. Pathogenic mutations were found in 13/47 (28%) cancers: eight in BRCA1 and five in BRCA2. All BRCA1 and two BRCA2 mutations were germline; three BRCA2 mutations were somatic. All mutations were confirmed by Sanger sequencing. To evaluate the performance of the NGS panel, we assessed its capability to detect the 6,953 variants described for BRCA1 and BRCA2 in ClinVar and COSMIC databases using callability analysis. 6,059 (87.1%) variants were identified automatically by the software; 829 (12.0%) required visual verification. The remaining 65 (0.9%) variants were uncallable, and would require 15 Sanger reactions to be resolved. Thus, the sensitivity of the NGS-panel was 99.1%. In conclusion, NGS performed with a commercial kit is highly efficient for detection of germline and somatic mutations in BRCA genes using routine FFPE tissue.
携带BRCA突变的卵巢癌对铂类疗法以及最近获批的PARP抑制剂反应更佳。需要利用福尔马林固定石蜡包埋（FFPE）组织和市售技术，开发高效且及时的方法来检测体细胞和种系突变。我们使用了一种商业试剂盒，对BRCA1和BRCA2基因的所有外显子以及50bp的外显子-内含子连接区进行检测，并对47例高级别浆液性卵巢癌FFPE样本的DNA进行了半导体下一代测序（NGS）。在13/47（28%）的癌症中发现了致病性突变：8例在BRCA1基因，5例在BRCA2基因。所有BRCA1突变和2例BRCA2突变是种系突变；3例BRCA2突变是体细胞突变。所有突变均通过桑格测序法得到证实。为评估NGS检测板的性能，我们使用可调用性分析评估了其检测ClinVar和COSMIC数据库中描述的BRCA1和BRCA2的6953个变异的能力。软件自动识别出6059个（87.1%）变异；829个（12.0%）需要人工核查。其余65个（0.9%）变异无法调用，需要15次桑格反应才能解决。因此，NGS检测板的灵敏度为99.1%。总之，使用商业试剂盒进行的NGS检测对于利用常规FFPE组织检测BRCA基因中的种系和体细胞突变非常高效。
