title: Prevalence, tumorigenic role, and biochemical implications of rare BRAF alterations.
abstract: BACKGROUND
  Papillary thyroid carcinoma (PTC) is the most common malignant tumor of the thyroid gland, accounting for 74-80% of all thyroid cancers. The 1799T>A transversion is an activating mutation of the BRAF oncogene that is common in and specific to conventional PTC. We studied the prevalence, tumorigenic role, and biochemical implications of rare BRAF variants in a large cohort of patients.
  
  
  METHODS
  A total of 2131 fine-needle aspiration biopsy samples were collected and subjected to BRAF mutation analysis. BRAF genetic variants were analyzed by Western blot, immunofluorescence, and in silico analysis.
  
  
  RESULTS
  BRAF mutations were found in 50% (347/700) of thyroid cancers (644 PTCs, 22 anaplastic thyroid carcinomas, 34 follicular thyroid carcinomas). They were the classic (c.1799T>A, p.V600E) mutation in 96.8% (336/347) and rare genetic variants in 3.2% (11/347). In all, five infrequent BRAF alterations were detected: (i) c.1795_1797dupACA (p.T599dup); (ii) c.1801A>G (p.K601E); (iii) c.1799_1801delTGA (p.V600_K601>E); (iv) c.1799_1814>A (p.V600_S605>D); and (v) c.1798_1810delinsA (p.V600_W604>R). The last BRAF variant has never been described in the literature. Western blot analysis and immunofluorescence both revealed a variegated reactivity pattern, again emphasizing the peculiar role of every specific BRAF genetic alteration. In silico analysis of the samples studied revealed a stabilization of the "active" geometrical conformation of the B-raf enzyme associated with the activated and productive state of the kinase domain.
  
  
  CONCLUSIONS
  Rare BRAF variants were found in 1.6% of all thyroid malignancies, all clustered around the codon V600, in the binding pocket named A-loop, confirming its crucial role in the enzymatic activation of the B-Raf protein. These mutations were associated mainly with the activation of key effectors in the mitogen-activated protein kinase pathway, but a simultaneous stimulation of the PI3k/Akt cascade was demonstrated in some cases. The rare BRAF variants were not generally associated with an aggressive behavior of the PTC. To our knowledge, this is the largest series of thyroid cancers analyzed to identify and functionally characterize rare BRAF variants.
authors.affiliation: 1 Endocrinology Unit, Department of Medicine, University of Padua , Padua, Italy .
authors.forename: Susi
authors.initials: S
authors.lastname: Barollo
authors.forename: Raffaele
authors.initials: R
authors.lastname: Pezzani
authors.forename: Andrea
authors.initials: A
authors.lastname: Cristiani
authors.forename: Marco
authors.initials: M
authors.lastname: Redaelli
authors.forename: Laura
authors.initials: L
authors.lastname: Zambonin
authors.forename: Beatrice
authors.initials: B
authors.lastname: Rubin
authors.forename: Loris
authors.initials: L
authors.lastname: Bertazza
authors.forename: Mariangela
authors.initials: M
authors.lastname: Zane
authors.forename: Carla
authors.initials: C
authors.lastname: Mucignat-Caretta
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bulfone
authors.forename: Gianmaria
authors.initials: G
authors.lastname: Pennelli
authors.forename: Eric
authors.initials: E
authors.lastname: Casal Ide
authors.forename: Maria Rosa
authors.initials: MR
authors.lastname: Pelizzo
authors.forename: Franco
authors.initials: F
authors.lastname: Mantero
authors.forename: Stefano
authors.initials: S
authors.lastname: Moro
authors.forename: Caterina
authors.initials: C
authors.lastname: Mian
mesh_terms: D018263:Adenocarcinoma, Follicular / Q000235:genetics* / Q000378:metabolism; D000328:Adult; D000368:Aged; D002277:Carcinoma / Q000235:genetics* / Q000378:metabolism; D002291:Carcinoma, Papillary; D020134:Catalytic Domain; D003062:Codon; D015331:Cohort Studies; D005260:Female; D005500:Follow-Up Studies; D056726:Genetic Association Studies; D006801:Humans; D008297:Male; D008875:Middle Aged; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009154:Mutation; D011487:Protein Conformation; D055550:Protein Stability; D048493:Proto-Oncogene Proteins B-raf / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000077273:Thyroid Cancer, Papillary; D065646:Thyroid Carcinoma, Anaplastic / Q000235:genetics* / Q000378:metabolism; D013964:Thyroid Neoplasms / Q000235:genetics* / Q000378:metabolism; D055815:Young Adult
chemical_list: D003062:Codon; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf