37 Diagnostic utility of molecular and imaging biomarkers 2 test performance from the initial key publication – true cost-effectiveness may be less optimistic [53, 69, 158, 159]. Next generation sequencing To improve the sensitivity of the miRInform® thyroid test, the existing 7-gene mutation panel was expanded to include additional gene mutations, fusions and translocations, and a microRNA gene expression panel. In addition, it adopted promising next generation sequencing (NGS) techniques. NGS enables the simultaneous targeted testing for multiple mutations in large gene panels and is faster, more sensitive and more cost-effective than traditional Sanger sequencing and other PCRbased methods [109, 118, 160]. As NGS only requires a very small amount (5-10 ng) of nucleic acids, remainder material from regular FNAC passes suffices and no additional aspirates are required [118, 160]. The first thyroid-specific NGS-based gene panel was the ThyroSeq® v1, presented in 2013. It detected gene variations in 110 of 145 investigated thyroid cancer tissue samples and 5 of 83 benign specimens. Unfortunately, indeterminate FNAC samples were not analysed separately in this study. Nonetheless, Nikiforova et al. demonstrated that NGS had a very high success rate and could be a promising molecular technique for thyroid FNAC samples [160]. Following the ThyroSeq® v1, the road was paved for further exploration of NGS-based diagnostics. Soon, the ThyroSeq® v2 (CBLPath, Ocala, FL, USA) was developed, with a number of primers for TERT promotor variants added to its panel. It simultaneously tested for point mutations in 13 genes and for 42 types of gene fusion products [118]. The ThyroSeq® v2 was tested on 143 Bethesda IV thyroid nodules. Forty-two genetic alterations were found, most frequently NRAS. Diagnostic accuracy of the ThyroSeq® v2 was 92%, with astonishing 90% sensitivity and 93% specificity [118]. More recently, Nikiforov et al. tested the ThyroSeq® v2.1 – including point mutations in 14 genes and 42 gene fusion transcripts – in 462 Bethesda III nodules. Based on the promising results of the previous study, surgery was withheld for 362 of 431 ThyroSeq®-negative patients. In the 95 patients with available histopathology, the ThyroSeq® v2.1 demonstrated 91% sensitivity and 92% specificity. Additionally, diagnostic accuracy was estimated for malignancy rates varying between 6% and 48%: PPV would range from 42% to 91%, NPV from 92% to 99%. Within reasonable limits the ThyroSeq® v2.1 is highly reliable to rule out malignancy [60]. Le Mercier et al. retrospectively tested a different commercially available 50-gene NGS panel, the Ampliseq™ Cancer Hotspot Panel v2 (ThermoFisher, San Diego, California, USA), which is a tumournonspecific NGS panel for detection of somatic tumour variants. This panel does not include thyroidspecific RET/PTC, PAX8/PPARγ and NTRK1 rearrangements. Albeit the study only assessed 34 FNAC samples, with a 71% sensitivity and 89% specificity in indeterminate thyroid nodules the Ampliseq™ panel seems less accurate than the ThyroSeq® [109].
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