title: RNA sequencing identifies multiple fusion transcripts, differentially expressed genes, and reduced expression of immune function genes in BRAF (V600E) mutant vs BRAF wild-type papillary thyroid carcinoma.
abstract: BACKGROUND
  The BRAF V600E mutation (BRAF-MUT) confers an aggressive phenotype in papillary thyroid carcinoma, but unidentified additional genomic abnormalities may be required for full phenotypic expression.
  
  
  OBJECTIVE
  RNA sequencing (RNA-Seq) was performed to identify genes differentially expressed between BRAF-MUT and BRAF wild-type (BRAF-WT) tumors and to correlate changes to patient clinical status.
  
  
  METHODS
  BRAF-MUT and BRAF-WT tumors were identified in patients with T1N0 and T2-3N1 tumors evaluated in a referral medical center. Gene expression levels were determined (RNA-Seq) and fusion transcripts were detected. Multiplexed capture/detection and digital counting of mRNA transcripts (nCounter, NanoString Technologies) validated RNA-Seq data for immune system-related genes.
  
  
  METHODS
  BRAF-MUT patients included nine women, three men; nine were TNM stage I and three were stage III. Three (25%) had tumor infiltrating lymphocytes. BRAF-WT included five women, three men; all were stage I, and five (62.5%) had tumor infiltrating lymphocytes.
  
  
  RESULTS
  RNA-Seq identified 560 of 13 085 genes differentially expressed between BRAF-MUT and BRAF-WT tumors. Approximately 10% of these genes were related to MetaCore immune function pathways; 51 were underexpressed in BRAF-MUT tumors, whereas 4 (HLAG, CXCL14, TIMP1, IL1RAP) were overexpressed. The four most differentially overexpressed immune genes in BRAF-WT tumors (IL1B; CCL19; CCL21; CXCR4) correlated with lymphocyte infiltration. nCounter confirmed the RNA-Seq expression level data. Eleven different high-confidence fusion transcripts were detected (four interchromosomal; seven intrachromosomal) in 13 of 20 tumors. All in-frame fusions were validated by RT-PCR.
  
  
  CONCLUSIONS
  BRAF-MUT papillary thyroid cancers have reduced expression of immune/inflammatory response genes compared with BRAF-WT tumors and correlate with lymphocyte infiltration. In contrast, HLA-G and CXCL14 are overexpressed in BRAF-MUT tumors. Sixty-five percent of tumors had between one and three fusion transcripts. Functional studies will be required to determine the potential role of these newly identified genomic abnormalities in contributing to the aggressiveness of BRAF-MUT and BRAF-WT tumors.
authors.affiliation: Department of Medicine (R.C.S.), Division of Endocrinology and Metabolism, Mayo Clinic, Jacksonville, Florida 32224; Department of Otorhinolaryngology-Head and Neck Surgery (A.M.C., J.D.C.), Mayo Clinic, Jacksonville, Florida 32224; Department of Health Sciences Research (Y.W.A., D.J.S.), Mayo Clinic, Jacksonville, Florida 32224; Department of Medicine, Division of Endocrinology (H.V.R., N.L.E., B.M.), Mayo Clinic, Rochester, Minnesota 55905; Department of Laboratory Medicine and Pathology (K.W.C., S.K.G.), Division of Clinical Biochemistry and Immunology, Mayo Clinic, Rochester, Minnesota 55905; Department of Laboratory Medicine and Pathology (M.R.), Division of Anatomic Pathology, Mayo Clinic, Rochester, Minnesota 55905; Department of Cancer Biology (B.N., J.M.C., J.A.C., E.A.T.), Mayo Clinic, Jacksonville, Florida 32224; and Department of Biochemistry and Molecular Biology (N.L.E.), Mayo Clinic, Rochester, Minnesota 55905.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Smallridge
authors.forename: Ana-Maria
authors.initials: AM
authors.lastname: Chindris
authors.forename: Yan W
authors.initials: YW
authors.lastname: Asmann
authors.forename: John D
authors.initials: JD
authors.lastname: Casler
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Serie
authors.forename: Honey V
authors.initials: HV
authors.lastname: Reddi
authors.forename: Kendall W
authors.initials: KW
authors.lastname: Cradic
authors.forename: Michael
authors.initials: M
authors.lastname: Rivera
authors.forename: Stefan K
authors.initials: SK
authors.lastname: Grebe
authors.forename: Brian M
authors.initials: BM
authors.lastname: Necela
authors.forename: Norman L
authors.initials: NL
authors.lastname: Eberhardt
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Carr
authors.forename: Bryan
authors.initials: B
authors.lastname: McIver
authors.forename: John A
authors.initials: JA
authors.lastname: Copland
authors.forename: E Aubrey
authors.initials: EA
authors.lastname: Thompson
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002291:Carcinoma, Papillary / Q000235:genetics* / Q000473:pathology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D017423:Sequence Analysis, RNA; D013964:Thyroid Neoplasms / Q000235:genetics* / Q000473:pathology
chemical_list: D048493:Proto-Oncogene Proteins B-raf