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

Abstract

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.

Limitations of Detecting Genetic Variants from the RNA Sequencing Data in Tissue and Fine-Needle Aspiration Samples

“…Although sequencing of DNA isolated from cells collected by FNA or tumor tissue sections is a standard approach used for detecting DNA variants, the utility of RNA converted to cDNA has been recently explored as an alternative template for sequencing and detection of DNA mutations (5)(6)(7)(8)(9). Such approach is based on the premise that specific DNA regions encoding genes that are expressed in the tissue of interest are transcribed into mRNA, and DNA mutations located in the coding regions of these genes can be detected by RNA sequencing (RNA-Seq).…”

“…Indeed, several studies have demonstrated that only about half of all genetic variants detectable by sequencing of human tissue and cell line DNA could be captured using RNA-Seq (5)(6)(7)(8). Furthermore, only limited information is available on the completeness of detection of genetic variants using the RNA-Seq data in thyroid nodules and cancer (9).…”

Limitations of preoperative cytology for medullary thyroid cancer: Proposal for improved preoperative diagnosis for optimal initial medullary thyroid carcinoma specific surgery

“…About a quarter of MTC cases are negative for characteristic DNA variants or fusions, 35 so these tests employ alternative or additional strategies to achieve high sensitivity for MTC 36‐39 . These tests can additionally inform on genomic alterations which may help guide targeted therapies should systemic treatment be necessary 36,37,40 . While several molecular tests are currently employed, Afirma and Thyroseq are commonly used options.…”

Preoperative molecular testing in thyroid nodules with Bethesda VI cytology: Clinical experience and review of the literature

“…The second most frequent gene alteration was TERT promoter mutations (26/241 or 11%), highlighting the limitation of molecular strategies that only interrogate exome variants. For example, the Afirma Xpression Atlas, based on transcriptome RNA sequencing, has a reported false negative rate of 26% relative to targeted DNA sequencing and cannot assess variants outside the fraction of exonic sequences that are actually transcribed into mRNA 28 …”