title: The combination of four molecular markers improves thyroid cancer cytologic diagnosis and patient management.
abstract: BACKGROUND
  Papillary thyroid cancer is the most common endocrine malignancy. The most sensitive and specific diagnostic tool for thyroid nodule diagnosis is fine-needle aspiration (FNA) biopsy with cytological evaluation. Nevertheless, FNA biopsy is not always decisive leading to "indeterminate" or "suspicious" diagnoses in 10%-30% of cases. BRAF V600E detection is currently used as molecular test to improve the diagnosis of thyroid nodules, yet it lacks sensitivity. The aim of the present study was to identify novel molecular markers/computational models to improve the discrimination between benign and malignant thyroid lesions.
  
  
  METHODS
  We collected 118 pre-operative thyroid FNA samples. All 118 FNA samples were characterized for the presence of the BRAF V600E mutation (exon15) by pyrosequencing and further assessed for mRNA expression of four genes (KIT, TC1, miR-222, miR-146b) by quantitative polymerase chain reaction. Computational models (Bayesian Neural Network Classifier, discriminant analysis) were built, and their ability to discriminate benign and malignant tumors were tested. Receiver operating characteristic (ROC) analysis was performed and principal component analysis was used for visualization purposes.
  
  
  RESULTS
  In total, 36/70 malignant samples carried the V600E mutation, while all 48 benign samples were wild type for BRAF exon15. The Bayesian neural network (BNN) and discriminant analysis, including the mRNA expression of the four genes (KIT, TC1, miR-222, miR-146b) showed a very strong predictive value (94.12% and 92.16%, respectively) in discriminating malignant from benign patients. The discriminant analysis showed a correct classification of 100% of the samples in the malignant group, and 95% by BNN. KIT and miR-146b showed the highest diagnostic accuracy of the ROC curve, with area under the curve values of 0.973 for KIT and 0.931 for miR-146b.
  
  
  CONCLUSIONS
  The four genes model proposed in this study proved to be highly discriminative of the malignant status compared with BRAF assessment alone. Its implementation in clinical practice can help in identifying malignant/benign nodules that would otherwise remain suspicious.
authors.affiliation: Division of Surgical, Molecular, and Ultrastructural Pathology, University of Pisa and Pisa University Hospital, Via Roma 57, Pisa, 56100, Italy. panebiancof@upmc.edu.|Department of Pathology, University of Pittsburgh School of Medicine, 200 Lothrop St, Pittsburgh, PA, 15261, USA. panebiancof@upmc.edu.
authors.forename: Federica
authors.initials: F
authors.lastname: Panebianco
authors.affiliation: Division of Surgical, Molecular, and Ultrastructural Pathology, University of Pisa and Pisa University Hospital, Via Roma 57, Pisa, 56100, Italy. c.mazzanti@fondazionepisascienza.org.|Pisa Science Foundation, Via Panfilo Castaldi 2, Pisa, 5612, Italy. c.mazzanti@fondazionepisascienza.org.
authors.forename: Chiara
authors.initials: C
authors.lastname: Mazzanti
authors.affiliation: Sidra Medical and Research Center, Research Branch, Division of Translational Medicine, Al Corniche Street, PO 26999, Doha, Qatar. stomei@sidra.org.
authors.forename: Sara
authors.initials: S
authors.lastname: Tomei
authors.affiliation: Pisa Science Foundation, Via Panfilo Castaldi 2, Pisa, 5612, Italy. p.aretini@fondazionepisascienza.org.
authors.forename: Paolo
authors.initials: P
authors.lastname: Aretini
authors.affiliation: Pisa Science Foundation, Via Panfilo Castaldi 2, Pisa, 5612, Italy. sara.franceschi@fondazionepisascienza.org.
authors.forename: Sara
authors.initials: S
authors.lastname: Franceschi
authors.affiliation: Pisa Science Foundation, Via Panfilo Castaldi 2, Pisa, 5612, Italy. f.lessi@fondazionepisascienza.org.
authors.forename: Francesca
authors.initials: F
authors.lastname: Lessi
authors.affiliation: Section of Cytopathology, University of Pisa and Pisa University Hospital, Via Roma 57, Pisa, 56100, Italy. g.dicoscio@ao-pisa.toscana.it.
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Di Coscio
authors.affiliation: Division of Surgical, Molecular, and Ultrastructural Pathology, University of Pisa and Pisa University Hospital, Via Roma 57, Pisa, 56100, Italy. generoso.bevilacqua@med.unipi.it.|Section of Cytopathology, University of Pisa and Pisa University Hospital, Via Roma 57, Pisa, 56100, Italy. generoso.bevilacqua@med.unipi.it.
authors.forename: Generoso
authors.initials: G
authors.lastname: Bevilacqua
authors.affiliation: Division of Surgical, Molecular, and Ultrastructural Pathology, University of Pisa and Pisa University Hospital, Via Roma 57, Pisa, 56100, Italy. i.marchetti@ao-pisa.toscana.it.|Section of Cytopathology, University of Pisa and Pisa University Hospital, Via Roma 57, Pisa, 56100, Italy. i.marchetti@ao-pisa.toscana.it.
authors.forename: Ivo
authors.initials: I
authors.lastname: Marchetti
mesh_terms: D000368:Aged; D001499:Bayes Theorem; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D044963:Biopsy, Fine-Needle; D005260:Female; D020869:Gene Expression Profiling; D005819:Genetic Markers; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D025341:Principal Component Analysis; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics* / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D000077278:RNA, Mitochondrial; D012372:ROC Curve; D012680:Sensitivity and Specificity; D013964:Thyroid Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; D005819:Genetic Markers; D035683:MicroRNAs; D009363:Neoplasm Proteins; D048493:Proto-Oncogene Proteins B-raf; D019009:Proto-Oncogene Proteins c-kit; D012313:RNA; D000077278:RNA, Mitochondrial; C542542:MIRN146 microRNA, human; C525710:MIR222, human; C418741:TCIM protein, human