Analytical and Clinical Validation of Expressed Variants and Fusions From the Whole Transcriptome of Thyroid FNA Samples

Trevor E. Angell, Lori J. Wirth, Maria E. Cabanillas, Maisie L. Shindo, Edmund S. Cibas, Joshua E. Babiarz, Yangyang Hao, Su Yeon Kim, P. Sean Walsh, Jing Huang, Richard T. Kloos, Giulia C. Kennedy, Steven G. Waguespack

Research output: Contribution to journalArticlepeer-review

19 Scopus citations


Introduction: 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. Methods: 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. 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%. Conclusion: 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.

Original languageEnglish (US)
Article number612
JournalFrontiers in Endocrinology
StatePublished - Sep 11 2019


  • RNA-sequencing
  • atypia of undetermined significance
  • fine-needle aspiration
  • follicular neoplasm
  • molecular diagnostics
  • thyroid cancer
  • thyroid molecular assays
  • transcriptome

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism


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