Anatomical Pathology, PathWest Laboratory Medicine, QEII Medical Centre, Nedlands, WA, Australia.
Anatomical Pathology, PathWest Laboratory Medicine, QEII Medical Centre, Nedlands, WA, Australia; The University of Western Australia, School of Pathology and Laboratory Medicine, Crawley, WA, Australia; The University of Western Australia, School of Medicine and Pharmacology, Crawley, WA, Australia.
Pathology. 2018 Jun;50(4):389-401. doi: 10.1016/j.pathol.2018.01.005. Epub 2018 May 8.
The introduction of next generation sequencing (NGS) in the routine diagnostic setting is still in the development phase and has been limited by its complexity. Targeted NGS offers an attractive alternative to performing multiple single target assays and is very useful in meeting the increasing clinical demand for testing of multiple genetic aberrations in cancer specimens. To this end, we carried out a blinded validation study on 113 tumours in a diagnostic laboratory and compared mutation results from targeted NGS with those from Sanger sequencing, pyrosequencing, competitive allele specific TaqMan polymerase chain reaction (CAST PCR) and Cobas assays. DNA was extracted from formalin fixed, paraffin embedded (FFPE) tissue samples that included core biopsies, resections and cytology samples from three common and one rare cancer types [non-small cell lung cancer (NSCLC), colorectal cancer (CRC), malignant melanoma (MM) and gastrointestinal stromal tumour (GIST)]. Libraries were prepared using the TruSight Tumour 26 gene panel and NGS was carried out on the MiSeq instrument. Results from NGS were concordant with the mutational status determined by other platforms in 107 of the 113 cases tested (94.7%). The sequencing quality for NGS failed in four of the six false negative cases, while a further two samples gave false negative results because the c-KIT mutations were located outside the range of the NGS panel. One NSCLC sample contained an EGFR mutation previously detected by the Cobas assay. Reanalysis of the NGS data for this sample using a cut-off allele frequency of 1% revealed the mutation had an allele frequency of 2%, which was below the recommended software-determined threshold of 3%. NGS detected 113 additional mutations that were not previously known from analysis by the conventional methods. Twenty-six of these have known clinical importance, 37 have potential clinical significance, while 50 were novel mutations with unknown clinical significance. NGS detected variants using inputs of 10-20 ng of FFPE extracted DNA and from specimens with a tumour cell content less than 50%, for which when possible we recommend microdissection. We conclude that results from targeted NGS are highly concordant with those from other mutation testing platforms. Targeted NGS is suitable for a range of sample types received in the diagnostic pathology laboratory, including those with limited material or with low tumour cell content (TCC). This work has allowed us to determine the quality parameter settings required in order to obtain robust mutation data by NGS.
下一代测序（NGS）在常规诊断中的应用仍处于发展阶段，其复杂性受到限制。靶向 NGS 提供了替代执行多个单靶标检测的有吸引力的方法，并且非常有用，可以满足癌症标本中多个遗传异常检测的不断增长的临床需求。为此，我们在诊断实验室对 113 个肿瘤进行了盲法验证研究，并将靶向 NGS 的突变结果与 Sanger 测序、焦磷酸测序、竞争性等位基因特异性 TaqMan 聚合酶链反应（CAST PCR）和 Cobas 检测的结果进行了比较。从福尔马林固定、石蜡包埋（FFPE）组织样本中提取 DNA，这些样本包括三种常见癌症和一种罕见癌症类型（非小细胞肺癌（NSCLC）、结直肠癌（CRC）、恶性黑色素瘤（MM）和胃肠道间质瘤（GIST））的核心活检、切除术和细胞学样本。使用 TruSight Tumor 26 基因panel 制备文库，并在 MiSeq 仪器上进行 NGS。在 113 例测试病例中，107 例（94.7%）的 NGS 结果与其他平台确定的突变状态一致。在六个假阴性病例中，有四个的测序质量失败，而另外两个样本的假阴性结果是因为 c-KIT 突变位于 NGS 面板范围之外。一个 NSCLC 样本包含先前通过 Cobas 检测到的 EGFR 突变。对该样本的 NGS 数据重新进行分析，使用等位基因频率为 1%的截止值，显示该突变的等位基因频率为 2%，低于推荐的软件确定的 3%的阈值。NGS 检测到了 113 个以前通过常规方法分析不知道的额外突变。其中 26 个具有已知的临床重要性，37 个具有潜在的临床意义，而 50 个是具有未知临床意义的新突变。NGS 检测到的变体使用 10-20ng 的 FFPE 提取 DNA 输入和肿瘤细胞含量小于 50%的样本，对于这些样本，在可能的情况下，我们建议进行微切割。我们得出的结论是，靶向 NGS 的结果与其他突变检测平台的结果高度一致。靶向 NGS 适用于诊断病理学实验室收到的多种类型的样本，包括那些材料有限或肿瘤细胞含量低（TCC）的样本。这项工作使我们能够确定通过 NGS 获得可靠突变数据所需的质量参数设置。
