title: Break-apart interphase fluorescence in situ hybridization assay in papillary thyroid carcinoma: on the road to optimizing the cut-off level for RET/PTC rearrangements.
abstract: OBJECTIVE
  Chromosomal rearrangements of the RET proto-oncogene is one of the most common molecular events in papillary thyroid carcinoma (PTC). However, their pathogenic role and clinical significance are still debated. This study aimed to investigate the prevalence of RET/PTC rearrangement in a cohort of BRAF WT PTCs by fluorescence in situ hybridization (FISH) and to search a reliable cut-off level in order to distinguish clonal or non-clonal RET changes.
  
  
  METHODS
  Forty BRAF WT PTCs were analyzed by FISH for RET rearrangements. As controls, six BRAFV600E mutated PTCs, 13 follicular adenomas (FA), and ten normal thyroid parenchyma were also analyzed.
  
  
  METHODS
  We performed FISH analysis on formalin-fixed, paraffin-embedded tissue using a commercially available RET break-apart probe. A cut-off level equivalent to 10.2% of aberrant cells was accepted as significant. To validate FISH results, we analyzed the study cohort by qRT-PCR.
  
  
  RESULTS
  Split RET signals above the cut-off level were observed in 25% (10/40) of PTCs, harboring a percentage of positive cells ranging from 12 to 50%, and in one spontaneous FA (1/13, 7.7%). Overall, the data obtained by FISH matched well with qRT-PCR results. Challenging findings were observed in five cases showing a frequency of rearrangement very close to the cut-off.
  
  
  CONCLUSIONS
  FISH approach represents a powerful tool to estimate the ratio between broken and non-broken RET tumor cells. Establishing a precise FISH cut-off may be useful in the interpretation of the presence of RET rearrangement, primarily when this strategy is used for cytological evaluation or for targeted therapy.
authors.affiliation: Department of Pathology and DiagnosticsARC-NET Research CentreUniversity of Verona, Policlinico GB Rossi, Piazzale LA Scuro, 10, Piastra Odontoiatrica (II floor), 37134 Verona, ItalyDepartment of Internal MedicineEndocrinology, and Metabolism and Biochemistry, University of Siena, Siena, ItalyDepartment of Surgery and OncologyUniversity of Verona, Verona, ItalyDivision of SurgicalMolecular and Ultrastructural, Section of Cytopathology, University Hospital of Pisa, Pisa, ItalyNuclear Medicine UnitUniversity Hospital of Verona, Verona, Italy chiara.colato@ospedaleuniverona.it.
authors.forename: Chiara
authors.initials: C
authors.lastname: Colato
authors.affiliation: Department of Pathology and DiagnosticsARC-NET Research CentreUniversity of Verona, Policlinico GB Rossi, Piazzale LA Scuro, 10, Piastra Odontoiatrica (II floor), 37134 Verona, ItalyDepartment of Internal MedicineEndocrinology, and Metabolism and Biochemistry, University of Siena, Siena, ItalyDepartment of Surgery and OncologyUniversity of Verona, Verona, ItalyDivision of SurgicalMolecular and Ultrastructural, Section of Cytopathology, University Hospital of Pisa, Pisa, ItalyNuclear Medicine UnitUniversity Hospital of Verona, Verona, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: Vicentini
authors.affiliation: Department of Pathology and DiagnosticsARC-NET Research CentreUniversity of Verona, Policlinico GB Rossi, Piazzale LA Scuro, 10, Piastra Odontoiatrica (II floor), 37134 Verona, ItalyDepartment of Internal MedicineEndocrinology, and Metabolism and Biochemistry, University of Siena, Siena, ItalyDepartment of Surgery and OncologyUniversity of Verona, Verona, ItalyDivision of SurgicalMolecular and Ultrastructural, Section of Cytopathology, University Hospital of Pisa, Pisa, ItalyNuclear Medicine UnitUniversity Hospital of Verona, Verona, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Cantara
authors.affiliation: Department of Pathology and DiagnosticsARC-NET Research CentreUniversity of Verona, Policlinico GB Rossi, Piazzale LA Scuro, 10, Piastra Odontoiatrica (II floor), 37134 Verona, ItalyDepartment of Internal MedicineEndocrinology, and Metabolism and Biochemistry, University of Siena, Siena, ItalyDepartment of Surgery and OncologyUniversity of Verona, Verona, ItalyDivision of SurgicalMolecular and Ultrastructural, Section of Cytopathology, University Hospital of Pisa, Pisa, ItalyNuclear Medicine UnitUniversity Hospital of Verona, Verona, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Pedron
authors.affiliation: Department of Pathology and DiagnosticsARC-NET Research CentreUniversity of Verona, Policlinico GB Rossi, Piazzale LA Scuro, 10, Piastra Odontoiatrica (II floor), 37134 Verona, ItalyDepartment of Internal MedicineEndocrinology, and Metabolism and Biochemistry, University of Siena, Siena, ItalyDepartment of Surgery and OncologyUniversity of Verona, Verona, ItalyDivision of SurgicalMolecular and Ultrastructural, Section of Cytopathology, University Hospital of Pisa, Pisa, ItalyNuclear Medicine UnitUniversity Hospital of Verona, Verona, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Brazzarola
authors.affiliation: Department of Pathology and DiagnosticsARC-NET Research CentreUniversity of Verona, Policlinico GB Rossi, Piazzale LA Scuro, 10, Piastra Odontoiatrica (II floor), 37134 Verona, ItalyDepartment of Internal MedicineEndocrinology, and Metabolism and Biochemistry, University of Siena, Siena, ItalyDepartment of Surgery and OncologyUniversity of Verona, Verona, ItalyDivision of SurgicalMolecular and Ultrastructural, Section of Cytopathology, University Hospital of Pisa, Pisa, ItalyNuclear Medicine UnitUniversity Hospital of Verona, Verona, Italy.
authors.forename: Ivo
authors.initials: I
authors.lastname: Marchetti
authors.affiliation: Department of Pathology and DiagnosticsARC-NET Research CentreUniversity of Verona, Policlinico GB Rossi, Piazzale LA Scuro, 10, Piastra Odontoiatrica (II floor), 37134 Verona, ItalyDepartment of Internal MedicineEndocrinology, and Metabolism and Biochemistry, University of Siena, Siena, ItalyDepartment of Surgery and OncologyUniversity of Verona, Verona, ItalyDivision of SurgicalMolecular and Ultrastructural, Section of Cytopathology, University Hospital of Pisa, Pisa, ItalyNuclear Medicine UnitUniversity Hospital of Verona, Verona, Italy.
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Di Coscio
authors.affiliation: Department of Pathology and DiagnosticsARC-NET Research CentreUniversity of Verona, Policlinico GB Rossi, Piazzale LA Scuro, 10, Piastra Odontoiatrica (II floor), 37134 Verona, ItalyDepartment of Internal MedicineEndocrinology, and Metabolism and Biochemistry, University of Siena, Siena, ItalyDepartment of Surgery and OncologyUniversity of Verona, Verona, ItalyDivision of SurgicalMolecular and Ultrastructural, Section of Cytopathology, University Hospital of Pisa, Pisa, ItalyNuclear Medicine UnitUniversity Hospital of Verona, Verona, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Chilosi
authors.affiliation: Department of Pathology and DiagnosticsARC-NET Research CentreUniversity of Verona, Policlinico GB Rossi, Piazzale LA Scuro, 10, Piastra Odontoiatrica (II floor), 37134 Verona, ItalyDepartment of Internal MedicineEndocrinology, and Metabolism and Biochemistry, University of Siena, Siena, ItalyDepartment of Surgery and OncologyUniversity of Verona, Verona, ItalyDivision of SurgicalMolecular and Ultrastructural, Section of Cytopathology, University Hospital of Pisa, Pisa, ItalyNuclear Medicine UnitUniversity Hospital of Verona, Verona, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Brunelli
authors.affiliation: Department of Pathology and DiagnosticsARC-NET Research CentreUniversity of Verona, Policlinico GB Rossi, Piazzale LA Scuro, 10, Piastra Odontoiatrica (II floor), 37134 Verona, ItalyDepartment of Internal MedicineEndocrinology, and Metabolism and Biochemistry, University of Siena, Siena, ItalyDepartment of Surgery and OncologyUniversity of Verona, Verona, ItalyDivision of SurgicalMolecular and Ultrastructural, Section of Cytopathology, University Hospital of Pisa, Pisa, ItalyNuclear Medicine UnitUniversity Hospital of Verona, Verona, Italy.
authors.forename: Furio
authors.initials: F
authors.lastname: Pacini
authors.affiliation: Department of Pathology and DiagnosticsARC-NET Research CentreUniversity of Verona, Policlinico GB Rossi, Piazzale LA Scuro, 10, Piastra Odontoiatrica (II floor), 37134 Verona, ItalyDepartment of Internal MedicineEndocrinology, and Metabolism and Biochemistry, University of Siena, Siena, ItalyDepartment of Surgery and OncologyUniversity of Verona, Verona, ItalyDivision of SurgicalMolecular and Ultrastructural, Section of Cytopathology, University Hospital of Pisa, Pisa, ItalyNuclear Medicine UnitUniversity Hospital of Verona, Verona, Italy Department of Pathology and DiagnosticsARC-NET Research CentreUniversity of Verona, Policlinico GB Rossi, Piazzale LA Scuro, 10, Piastra Odontoiatrica (II floor), 37134 Verona, ItalyDepartment of Internal MedicineEndocrinology, and Metabolism and Biochemistry, University of Siena, Siena, ItalyDepartment of Surgery and OncologyUniversity of Verona, Verona, ItalyDivision of SurgicalMolecular and Ultrastructural, Section of Cytopathology, University Hospital of Pisa, Pisa, ItalyNuclear Medicine UnitUniversity Hospital of Verona, Verona, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Ferdeghini
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002277:Carcinoma / Q000235:genetics* / Q000473:pathology; D002291:Carcinoma, Papillary; D002648:Child; D015331:Cohort Studies; D005260:Female; D015321:Gene Rearrangement; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008297:Male; D008875:Middle Aged; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D000090063:Proto-Oncogene Mas; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics*; D011933:Reagent Kits, Diagnostic; D012015:Reference Standards; D000077273:Thyroid Cancer, Papillary; D013964:Thyroid Neoplasms / Q000235:genetics* / Q000473:pathology; D016707:Tissue Fixation; D055815:Young Adult
chemical_list: C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011933:Reagent Kits, Diagnostic; D051096:Proto-Oncogene Proteins c-ret