title: Frequent silencing of RASSF1A via promoter methylation in follicular thyroid hyperplasia: a potential early epigenetic susceptibility event in thyroid carcinogenesis.
abstract: OBJECTIVE
  Follicular thyroid hyperplasia (FTH) refers to enlargement of the thyroid gland due to cellular hyperplasia. It is frequently encountered in clinical practice in nontoxic uninodular or multinodular goiter. The genetic and epigenetic events associated with the origin and malignant potential of FTH are poorly understood.
  
  
  OBJECTIVE
  To analyze FTH samples for known recurrent genetic and epigenetic driver events in thyroid neoplasms such as activating mutations in proto-oncogenes BRAF and NRAS and promoter hypermethylation of tumor suppressor genes CDKN2A, PTEN, and RASSF1A.
  
  
  METHODS
  Clinical characteristics and thyroid specimens were prospectively obtained from 43 patients who underwent thyroid surgery at Yale-New Haven Hospital.
  
  
  METHODS
  Presence of BRAF(V600E) and NRAS codon 61 mutations were assessed in FTH. Methylation status of CDKN2A, PTEN, and RASSF1A gene promoters in FTH, follicular thyroid adenoma, and follicular thyroid carcinoma was quantified. Regulation of RASSF1A messenger RNA (mRNA) and protein expression and its potential neoplastic role in FTH were examined.
  
  
  RESULTS
  An exploratory cohort of FTH (n = 10) was negative for BRAF(V600E) and NRAS codon 61 mutations. In contrast, epigenetic analysis displayed significant promoter hypermethylation of the tumor-suppressor gene RASSF1A in 6 FTH samples (60%) compared with their adjacent normal tissue (P = .01). The overall genome CpG methylation and promoter methylation of PTEN and CDKN2A were unaffected in the lesions. Further analysis of an expanded cohort of patients with FTH (n = 23), follicular thyroid adenoma (n = 10), and follicular thyroid carcinoma (n = 10) showed RASSF1A promoter hypermethylation in 14 (61%), 9 (90%), and 7 (70%), respectively (P < .001). The overall hypermethylation level in FTH showed a statistically significant inverse correlation with RASSF1A mRNA expression (P = .005). Immunohistochemistry demonstrated minimal or no protein expression in most FTH samples studied. To explore the potential neoplastic contribution of RASSF1A downregulation, we analyzed the expression pattern of thyroid proliferation markers Ki-67 and NF-κB in representative samples. Although Ki-67 expression was undetectable, similar to normal tissue, FTH samples expressed high levels of NF-κB, similar to the expression levels in thyroid tumors.
  
  
  CONCLUSIONS
  We demonstrate silencing of tumor suppressor RASSF1A in a subset of FTH in the absence of other known thyroid cancer-associated genetic and epigenetic changes. Silencing of RASSF1A and concurrent NF-κB activation demonstrate that a subset of FTH shares epigenetic changes and downstream signaling events associated with malignant lesions, suggesting that FTH may have the potential to be a premalignant lesion.
authors.affiliation: Yale Endocrine Neoplasia Laboratory, Department of Surgery, Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Taylor C
authors.initials: TC
authors.lastname: Brown
authors.affiliation: Yale Endocrine Neoplasia Laboratory, Department of Surgery, Yale University School of Medicine, New Haven, Connecticut2Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.
authors.forename: C Christofer
authors.initials: CC
authors.lastname: Juhlin
authors.affiliation: Yale Endocrine Neoplasia Laboratory, Department of Surgery, Yale University School of Medicine, New Haven, Connecticut.
authors.forename: James M
authors.initials: JM
authors.lastname: Healy
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Manju L
authors.initials: ML
authors.lastname: Prasad
authors.affiliation: Yale Endocrine Neoplasia Laboratory, Department of Surgery, Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Reju
authors.initials: R
authors.lastname: Korah
authors.affiliation: Yale Endocrine Neoplasia Laboratory, Department of Surgery, Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Tobias
authors.initials: T
authors.lastname: Carling
mesh_terms: D018263:Adenocarcinoma, Follicular / Q000235:genetics* / Q000473:pathology; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D019175:DNA Methylation; D044127:Epigenesis, Genetic / Q000235:genetics*; D020558:GTP Phosphohydrolases; D020022:Genetic Predisposition to Disease; D006801:Humans; D006965:Hyperplasia / Q000235:genetics; D008565:Membrane Proteins; D009154:Mutation; D011230:Precancerous Conditions / Q000235:genetics*; D011401:Promoter Regions, Genetic; D048493:Proto-Oncogene Proteins B-raf / Q000737:chemistry / Q000235:genetics; D037941:Silencer Elements, Transcriptional / Q000235:genetics; D013961:Thyroid Gland / Q000473:pathology*; D013964:Thyroid Neoplasms / Q000235:genetics* / Q000473:pathology; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: D008565:Membrane Proteins; C411762:RASSF1 protein, human; D025521:Tumor Suppressor Proteins; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human