Simultaneous detection of BRCA mutations and large genomic rearrangements in germline DNA and FFPE tumor samples.
Simultaneous detection of BRCA mutations and large genomic rearrangements in germline DNA and FFPE tumor samples.
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DOI：
10.18632/oncotarget.11259
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发表时间：
2016-09-20
期刊：
影响因子：
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通讯作者：
Haracska L
中科院分区：
文献类型：
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作者：
Enyedi MZ;Jaksa G;Pintér L;Sükösd F;Gyuris Z;Hajdu A;Határvölgyi E;Priskin K;Haracska L
乳腺癌和卵巢癌的发生与不同种系和体细胞改变导致的BRCA1和BRCA2基因失活密切相关，其诊断对于肿瘤靶向治疗具有重要意义，因为最近批准的PARP抑制剂在治疗BRCA缺陷型肿瘤中显示出高效。这就提出了对新诊断方法的需求，这些方法不仅能够对种系 DNA 进行 BRCA 基因综合突变分析，而且还能够对福尔马林固定石蜡包埋 (FFPE) 肿瘤样本中的 BRCA 基因进行综合突变分析。在这里，我们描述了这种基于下一代测序和用于数据分析的新生物信息学软件的方法的开发。该诊断方法最初是在 Illumina MiSeq NGS 平台上使用种系突变干细胞系开发的，然后也适用于 Ion Torrent PGM NGS 平台。我们还研究了 NGS 覆盖数据在检测拷贝数变异和外显子缺失方面的可用性，以替代传统的 MLPA 技术。最后，我们在乳腺癌和卵巢癌患者的 FFPE 样本上测试了开发的工作流程。我们的方法满足生殖系和 FFPE 样本中乳腺癌和卵巢癌基因诊断的敏感性和特异性要求。
The development of breast and ovarian cancer is strongly connected to the inactivation of the BRCA1 and BRCA2 genes by different germline and somatic alterations, and their diagnosis has great significance in targeted tumor therapy, since recently approved PARP inhibitors show high efficiency in the treatment of BRCA-deficient tumors. This raises the need for new diagnostic methods that are capable of performing an integrative mutation analysis of the BRCA genes not only from germline DNA but also from formalin-fixed and paraffin-embedded (FFPE) tumor samples. Here we describe the development of such a methodology based on next-generation sequencing and a new bioinformatics software for data analysis. The diagnostic method was initially developed on an Illumina MiSeq NGS platform using germline-mutated stem cell lines and then adapted for the Ion Torrent PGM NGS platform as well. We also investigated the usability of NGS coverage data for the detection of copy number variations and exon deletions as a replacement of the conventional MLPA technique. Finally, we tested the developed workflow on FFPE samples from breast and ovarian cancer patients. Our method meets the sensitivity and specificity requirements for the genetic diagnosis of breast and ovarian cancers both from germline and FFPE samples.