Department of Laboratory Medicine and Pathology, Divisions of Laboratory Genetics and Experimental Pathology, Mayo Clinic, Rochester, Minnesota.
Department of Health Sciences Research, Division of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, Minnesota.
J Mol Diagn. 2018 Jul;20(4):495-511. doi: 10.1016/j.jmoldx.2018.03.007. Epub 2018 Jun 19.
We assessed the performance characteristics of an RNA sequencing (RNA-Seq) assay designed to detect gene fusions in 571 genes to help manage patients with cancer. Polyadenylated RNA was converted to cDNA, which was then used to prepare next-generation sequencing libraries that were sequenced on an Illumina HiSeq 2500 instrument and analyzed with an in-house developed bioinformatic pipeline. The assay identified 38 of 41 gene fusions detected by another method, such as fluorescence in situ hybridization or RT-PCR, for a sensitivity of 93%. No false-positive gene fusions were identified in 15 normal tissue specimens and 10 tumor specimens that were negative for fusions by RNA sequencing or Mate Pair NGS (100% specificity). The assay also identified 22 fusions in 17 tumor specimens that had not been detected by other methods. Eighteen of the 22 fusions had not previously been described. Good intra-assay and interassay reproducibility was observed with complete concordance for the presence or absence of gene fusions in replicates. The analytical sensitivity of the assay was tested by diluting RNA isolated from gene fusion-positive cases with fusion-negative RNA. Gene fusions were generally detectable down to 12.5% dilutions for most fusions and as little as 3% for some fusions. This assay can help identify fusions in patients with cancer; these patients may in turn benefit from both US Food and Drug Administration-approved and investigational targeted therapies.
我们评估了一种 RNA 测序 (RNA-Seq) 检测方法的性能特征，该方法旨在检测 571 个基因中的基因融合，以帮助管理癌症患者。多聚腺苷酸化的 RNA 被转化为 cDNA，然后用于制备下一代测序文库，这些文库在 Illumina HiSeq 2500 仪器上进行测序，并使用内部开发的生物信息学管道进行分析。该检测方法在另一种方法（例如荧光原位杂交或 RT-PCR）检测到的 41 个基因融合中的 38 个，敏感性为 93%。在 15 个正常组织标本和 10 个经 RNA 测序或 Mate Pair NGS 检测为融合阴性的肿瘤标本中未发现假阳性基因融合（特异性为 100%）。该检测方法还在 17 个未被其他方法检测到的肿瘤标本中鉴定出 22 个融合。这 22 个融合中的 18 个以前没有被描述过。在存在或不存在基因融合的重复检测中，观察到良好的室内和室间重现性，完全一致。通过用融合阴性 RNA 稀释来自基因融合阳性病例的 RNA 来测试该检测方法的分析灵敏度。对于大多数融合，基因融合通常可检测到 12.5%的稀释度，对于一些融合，可检测到低至 3%的稀释度。该检测方法可以帮助鉴定癌症患者的融合；这些患者可能会反过来受益于美国食品和药物管理局批准的和正在研究的靶向治疗。
