title: Young investigator challenge: Can the Ion AmpliSeq Cancer Hotspot Panel v2 be used for next-generation sequencing of thyroid FNA samples?
abstract: BACKGROUND
  Fine-needle aspiration (FNA) cytology is accurate and cost-effective in the evaluation of thyroid nodules. Molecular techniques may contribute to risk stratification in indeterminate cases. Although next-generation sequencing (NGS) is a promising technique for the molecular testing of thyroid FNA specimens, thyroid-specific cancer gene panels are not commercially available. Conversely, the Ion AmpliSeq Cancer Hotspot Panel v2 (CHPv2), which includes the genes most frequently mutated in thyroid neoplasms, is commercially available and may represent an alternative to thyroid-specific panels. To the authors' knowledge to date, CHPv2 has performed well only on "ideal" cytological samples featuring abundant, high-quality DNA and satisfactory postsequencing metrics. The objective of the current study was to extend NGS to less-than-ideal samples, which represent a large percentage of routine clinical specimens.
  
  
  METHODS
  A total of 37 thyroid smears were retrospectively analyzed using CHPv2, regardless of any preanalytical and postsequencing metric thresholds. Specifically, the authors evaluated the performance of CHPv2 on the BRAF, NRAS, HRAS, KRAS, and RET genes. Results were verified by pyrosequencing.
  
  
  RESULTS
  Of the 37 thyroid FNA specimens, 34 (91.8%) were successfully processed. BRAF, NRAS, and RET somatic variants were detected in 22 of these 34 specimens (64.7%). NGS was found to have a high sensitivity (89.4%), specificity (85.7%), and accuracy (88.4%).
  
  
  CONCLUSIONS
  CHPv2 is a valid option for the molecular evaluation of thyroid FNA specimens by NGS. It is interesting to note that this approach is accurate and effective even when applied to routine cytology samples that usually do not have optimal preanalytical and postsequencing requirements. Cancer Cytopathol 2016;124:776-84. © 2016 American Cancer Society.
authors.affiliation: Department of Public Health, University of Naples "Federico II,", Naples, Italy.
authors.forename: Claudio
authors.identifier: http://orcid.org/0000-0002-7479-6457
authors.initials: C
authors.lastname: Bellevicine
authors.affiliation: Department of Public Health, University of Naples "Federico II,", Naples, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Sgariglia
authors.affiliation: Department of Public Health, University of Naples "Federico II,", Naples, Italy.
authors.forename: Umberto
authors.initials: U
authors.lastname: Malapelle
authors.affiliation: Department of Public Health, University of Naples "Federico II,", Naples, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Vigliar
authors.affiliation: Department of Public Health, University of Naples "Federico II,", Naples, Italy.
authors.forename: Mariantonia
authors.initials: M
authors.lastname: Nacchio
authors.affiliation: Department of Advanced Biomedical Sciences, University of Naples "Federico II,", Naples, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Ciancia
authors.affiliation: Department of Oncology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Markus
authors.initials: M
authors.lastname: Eszlinger
authors.affiliation: Department of Endocrinology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.|Department of Oncology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Ralf
authors.initials: R
authors.lastname: Paschke
authors.affiliation: Department of Public Health, University of Naples "Federico II,", Naples, Italy.
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Troncone
mesh_terms: D000483:Alleles; D014408:Biomarkers, Tumor; D044963:Biopsy, Fine-Needle; D004252:DNA Mutational Analysis; D020869:Gene Expression Profiling / Q000379:methods; D005787:Gene Frequency; D005820:Genetic Testing / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation; D012680:Sensitivity and Specificity; D013964:Thyroid Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D016606:Thyroid Nodule / Q000175:diagnosis* / Q000235:genetics*
chemical_list: D014408:Biomarkers, Tumor
keywords: Ion Torrent; limited samples; molecular diagnosis; next-generation sequencing; postsequencing metrics; preanalytical factors; thyroid cancer; thyroid fine-needle aspiration