Angell Trevor E, Wirth Lori J, Cabanillas Maria E, Shindo Maisie L, Cibas Edmund S, Babiarz Joshua E, Hao Yangyang, Kim Su Yeon, Walsh P Sean, Huang Jing, Kloos Richard T, Kennedy Giulia C, Waguespack Steven G
Division of Endocrinology, Diabetes and Metabolism, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States.
Department of Medicine, Massachusetts General Hospital, Boston, MA, United States.
Front Endocrinol (Lausanne). 2019 Sep 11;10:612. doi: 10.3389/fendo.2019.00612. eCollection 2019.
The Afirma® Xpression Atlas (XA) detects gene variants and fusions in thyroid nodule FNA samples from a curated panel of 511 genes using whole-transcriptome RNA-sequencing. Its intended use is among cytologically indeterminate nodules that are Afirma GSC suspicious, Bethesda V/VI nodules, or known thyroid metastases. Here we report its analytical and clinical validation. DNA and RNA were purified from the same sample across 943 blinded FNAs and compared by multiple methodologies, including whole-transcriptome RNA-seq, targeted RNA-seq, and targeted DNA-seq. An additional 695 blinded FNAs were used to define performance for fusions between whole-transcriptome RNA-seq and targeted RNA-seq. We quantified the reproducibility of the whole-transcriptome RNA-seq assay across laboratories and reagent lots. Finally, variants and fusions were compared to histopathology results. Of variants detected in DNA at 5 or 20% variant allele frequency, 74 and 88% were also detected by XA, respectively. XA variant detection was 89% when compared to an alternative RNA-based detection method. Low levels of expression of the DNA allele carrying the variant, compared with the wild-type allele, was found in some variants not detected by XA. 82% of gene fusions detected in a targeted RNA fusion assay were detected by XA. Conversely, nearly all variants or fusions detected by XA were confirmed by an alternative method. Analytical validation studies demonstrated high intra-plate reproducibility (89%-94%), inter-plate reproducibility (86-91%), and inter-lab accuracy (90%). Multiple variants and fusions previously described across the spectrum of thyroid cancers were identified by XA, including some with approved or investigational targeted therapies. Among 190 Bethesda III/IV nodules, the sensitivity of XA as a standalone test was 49%. When the Afirma Genomic Sequencing Classifier (GSC) is used first among Bethesda III/IV nodules as a rule-out test, XA supplements genomic insight among those that are GSC suspicious. Our data clinically and analytically validate XA for use among GSC suspicious, or Bethesda V/VI nodules. Genomic information provided by XA may inform clinical decision-making with precision medicine insights across a broad range of FNA sample types encountered in the care of patients with thyroid nodules and thyroid cancer.
Afirma® Xpression Atlas（XA）利用全转录组RNA测序，在511个基因的精选面板中检测甲状腺结节细针穿刺抽吸（FNA）样本中的基因变异和融合。其预期用途是在Afirma基因序列分类器（GSC）可疑的细胞学不确定结节、贝塞斯达V/VI级结节或已知的甲状腺转移瘤中使用。在此，我们报告其分析和临床验证情况。从943份盲法FNA样本的同一样本中纯化DNA和RNA，并通过多种方法进行比较，包括全转录组RNA测序、靶向RNA测序和靶向DNA测序。另外695份盲法FNA样本用于确定全转录组RNA测序和靶向RNA测序之间融合的性能。我们对全转录组RNA测序检测在不同实验室和试剂批次间的可重复性进行了量化。最后，将变异和融合与组织病理学结果进行比较。在DNA中检测到的变异等位基因频率为5%或20%的变异中，XA分别检测到了74%和88%。与另一种基于RNA的检测方法相比，XA变异检测率为89%。在一些未被XA检测到的变异中，发现携带变异的DNA等位基因与野生型等位基因相比表达水平较低。在靶向RNA融合检测中检测到的基因融合，82%可被XA检测到。相反，几乎所有被XA检测到的变异或融合都被另一种方法所证实。分析验证研究表明，其板内重复性高（89%-94%）、板间重复性高（86%-91%）以及实验室间准确性高（90%）。XA鉴定出了先前在各类甲状腺癌中描述的多种变异和融合，包括一些有获批或正在研究的靶向治疗方法的变异和融合。在190个贝塞斯达III/IV级结节中，XA作为独立检测方法的敏感性为49%。当在贝塞斯达III/IV级结节中首先使用Afirma基因序列分类器（GSC）作为排除检测时，XA可在GSC可疑的结节中补充基因组信息。我们的数据从临床和分析角度验证了XA在GSC可疑或贝塞斯达V/VI级结节中的应用。XA提供的基因组信息可通过精准医学见解，为甲状腺结节和甲状腺癌患者护理中遇到的广泛FNA样本类型的临床决策提供参考。
